MASARYK UNIVERSITY
Faculty of Science
Department of Experimental Biology
FUNCTIONAL ROLE OF SULFATE-REDUCING BACTERIA
IN THE DEVELOPMENT OF BOWEL DISEASES
IN HUMAN AND ANIMALS
Habilitation thesis
Mgr. Ivan Kushkevych, Ph.D., Dr.Sc.
Brno 2019
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„The method is the first and the main thing... From the method depends on the
severity of the study. The method holds the fate of the study in hand.“
I. P. Pavlov (1849 – 1936)
The proposed mechanism of pathogenesis of inflammatory bowel disease at elevated levels
of sulfate-reducing bacteria (the scheme is modified from Fava and Danese, 2011)
Acknowledgements
I would like at the beginning to say thank you to all members of the Department of
Experimental Biology of the Faculty of Science at Masaryk University as well as
colleagues from other internal and external workplaces, which have cooperated
with me in this research and helped me in obtaining and publishing the result. I
would like to thank to Prof. Jan Šmarda and Assoc. Prof. Monika Vítězová for
their support and help. Special thanks to former bachelors and master students who
have communicated to obtain the results published and described in this
habilitation thesis. Finally, I thanks to colleagues from the Faculty of Pharmacy at
VFU Brno which are co-authors of my publications.
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ABSTRACT
This habilitation thesis is focused on the study of physiological, biochemical and pathogenic
properties of sulfate-reducing bacteria (SRB) isolated from human and animal feces. As a
consequence of this work, a conception of the functional role of SRB in the intestinal microbiota
based on the ability of these microorganisms in the process of dissimilatory sulfate reduction to
produce toxic metabolites that are a prerequisite for colon disease was developed. The intestinal
SRB, isolated from healthy and individuals with ulcerative colitis, were not differ statistically
based on its physiological and biochemical properties. The ratio of dominant SRB, Desulfovibrio
and Desulfomicrobium genera, was 93:7% in the feces of healthy people and 99:1% in the feces
of people with ulcerative colitis. The main criteria for assessing the development and progression
of an inflammatory process involving the intestinal SRB is: a change in microbial and colonic
pH, bacterial enzyme activity, sulfate reduction, SRB growth rate and sulfate reduction intensity,
hydrogen sulfide and acetate concentration in the feces. Prevention of this process can be
accomplished by inhibiting two enzymes of the dissimilatory sulfate reduction process,
particularly sulfite reductase and lactate dehydrogenase, which are the most sensitive side in
intestinal SRB metabolism pathway.
The experimental results described in this work may be useful for a more detailed study of an
inflammatory bowel disease with using a therapeutic strategy. Determining the number of SRBs
and the hydrogen sulfide and acetate concentration allowed the development of basic criteria for
assessing the aggressiveness of these bacteria, the toxic products of their metabolism to the
intestinal mucosa as well as assessing the level of risk of the disease and the course of the
inflammatory process. The work can be scientifically beneficial mainly because the results
described are important for the study of ulcerative colitis in animal models, especially for the
study of mechanisms of action of antimicrobials with prophylactic or therapeutic target of
specific components involved in the pathogenesis of the disease. The study of the stability of
selected strains of bacteria on antimicrobial and newly synthesized substances and also their
influence on the physiological and biochemical properties of SRB allows the search for new and
promising drugs. Such studies are promising in developing methods of preventing bowel disease.
Key words: sulfate-reducing bacteria, Desulfovibrio piger, Desulfomicrobium orale, hydrogen
sulfide, toxicity, bowel disease.
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CONTENTS
Introduction…………………………………………………………………………………..… 5
1. The scientific novelty and conceptions of the researches………………............................. 7
2. Diversity of microbial communities in the intestine……………………………..………….. 12
3. Biochemical characteristics of Desulfovibrio piger Vib-7
and Desulfomicrobium orale Rod-9………………………………...……………………..…21
4. Physiological characteristics bacteria Desulfovibrio piger Vib-7…………….….…………..24
5. Etiological role of sulfate-reducing bacteria in the development
of colon diseases……………………………………………………………..………….……27
6. Summarizing………………………………………………………………………….……… 29
Conclusions…………………………………………………………….………………...……..34
Publication list by the habilitation thesis…………………………………….…………..…... 36
A curriculum vitae of the author……………………………………………….………………..39
Attachment manuscripts by the theme of habilitation
Attachment 1………………………………………………………………………………..41
Attachment 2………………………………………………………………………………..51
Attachment 3………………………………………………………………………………..59
Attachment 4………………………………………………………………………………..65
Attachment 5……………………………………………………………………………..…74
Attachment 6……………………………………………………………………………..…80
Attachment 7…………………………………………………….……………………….…86
Attachment 8…………………………………………………..……………………………96
Attachment 9……………………………………………….……………………..…….…102
Attachment 10…………………………………………..…………………………………108
Attachment 11………………………………………..……………………………………115
Attachment 12………………………………………..……………………………………122
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INTRODUCTION
Sulfate-reducing bacteria (SRB) are anaerobic microorganisms that metabolize sulfates as the
terminal acceptor of electrons to hydrogen sulfide in the process of „dissimilatory sulfate reduction“
(or dissimilatory sulfate respiration). For this process, SRB uses exogenous electron donors, such as
organic compounds and molecular hydrogen (Postgate, 1984). Depending on the SRB genera,
organic compounds are oxidized incompletely to acetate (acetogenic SRB) or completely to carbon
dioxide (Barton & Hamilton, 2010).
SRB are widespread in anaerobic areas of soils, wetlands, marine and fresh water, they are
available in microbiocenosis large intestine of humans and animals (Postgate, 1984; Barton &
Hamilton, 2010). Acetogenic SRB and their final products of metabolism (including, hydrogen
sulfide and acetate) are often found in the feces of people with bloody diarrhea (Pitcher et al., 1996,
1998) and the mono- and polymicrobial infections of the gastrointestinal tract (McDougall et al.,
1997; Loubinoux et al., 2003; Rowan et al., 2009). It is believed that SRB can cause frequent
defecation, weight loss, increased intestinal permeability (Pitcher et al., 2000; Cummings et al.,
2003; Rowan et al., 2009). The species and quantitative composition of the SRB on the surface of
the intestinal mucosa are differs from those microorganisms in the lumen (Macfarlane et al., 1999;
Gibson et al., 1991). Such genera of SRB, Desulfovibrio, Desulfomicrobium, Lawsonia, Bilophila,
are the most isolated from the intestines of healthy and sick people and animals (Gibson et al.,
1991; Barton & Hamilton, 2010; Brenner et al., 2005).
In recent years, the number of cases of inflammatory bowel disease, including ulcerative
colitis is growing, the cause of the occurrence of which is still unknown. This disease is mainly
distributed in people aged 15 to 30 years, although there is evidence of cases of disease in people
aged 50–70 years, which can be in potential risk group. Frequent cases of the disease recorded in
both developed and underdeveloped countries (Lakatos et al., 2006; O'Connor et al., 2011; Low et
al., 2013). Ulcerative colitis suffer not only people but also animals: horses (Scott, et al., 2014) and
cows (Braun et al., 2015), pigs (Thomson, 2009; White, 2016), dogs (Cerquetella et al., 2010), cats
(Heilmann & Suchodolski, 2015), rodents (mice and rats) (Lee et al., 2014; Dugani et al., 2016).
The functional role of SRB in the development of inflammatory bowel disease, including ulcerative
colitis and etiology of these diseases is less explored, the mechanisms of the disease has not been
fully elucidated.
The fundamental basis of the thesis is research approach based on identifying the basic
mechanisms of inflammatory bowel disease, based on a study of the functional role of SRB; to
develop the basic evaluation criteria of aggressiveness of these microorganisms, and toxicity of
their products of metabolism for the intestinal mucosa. This work is focused on theoretical and
experimental study of mechanisms of inflammatory bowel disease under the influence of SRB and
based on a comprehensive study of their physiological and biochemical characteristics,
implementation of kinetic analysis of enzyme involved in process of dissimilatory sulfate reduction.
The aim of this work was to create and justify the functional role of acetogenic sulfatereducing
bacteria in the development of bowel disease in humans and animals based on the
physiological and biochemical properties of its dominant genera in the gut.
To achieve of this purpose, the following tasks were:
1. Investigation of the composition of intestinal microbial communities in healthy people and
patients with ulcerative colitis, isolate and identify by morphological, physiological,
biochemical and genetic characteristics sulfate-reducing bacteria;
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2. Determinatoin of the biochemical characteristics of the isolated bacteria, in particular
kinetic analysis of main enzymes involved in the process of the transporting substances,
dissimilatory sulfate reduction, oxidation of lactate to acetate, and antioxidant protection;
3. Investigation of the physiological characteristics of SRB, parameters of biomass
accumulation and process dissimilation sulphate under the influence of different values of
pH, concentration of hydrogen sulfide, electron acceptors and donors, and to carry out the
kinetic analysis of these parameters;
4. The creation a model of ulcerative colitis in rats using isolated strains of sulfate-reducing
bacteria;
5. Molecular and genetic construction of SRB which can utilize hydrogen sulfide and its
metabolizing into cysteine;
6. Identification of the main criteria for assessing the aggressiveness of SRB, toxicity of their
metabolic products for the colon mucosa, and establishment of functional role of bacteria
in the disease development.
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1. THE SCIENTIFIC NOVELTY AND CONCEPTIONS OF THE RESEARCHES
The conception of the basic developmental mechanisms of inflammatory bowel diseases,
including ulcerative colitis, was created based on the physiological and biochemical characteristics
of acetogenic SRB. The main provisions of the conception are:
 Acetogenic representatives of the Desulfovibrio and Desulfomicrobium genera are
dominant among SRB in the intestines of healthy people and patients with colitis.
Increasing the number of SRB and concentration of the main metabolic products
(hydrogen sulfide and acetate) in the consumption of exogenous electron acceptor
(sulfate) causes changes in the structure of the microbiota of the intestine, including
decreasing of the number of autochthonous representatives of bacterial genera:
Bifidobacterium and Lactobacillus, increasing the number of elective and opportunistic
bacteria genera: Bacteroides, Escherichia, Clostridium and Proteus, changing the ratio of
intestinal microbiota groups, sustainability index and frequency of detection.
 Genetically constructed strain of D. piger Vib-7, containing the CysK gene, able to
metabolize hydrogen sulfide to non-toxic cysteine, which contributes to reduce of the
toxic effects of this compound on the colon mucosa. Using of this strain was
demonstrated the influence of dissimilatory (not assimilatory) process of sulfate reduction
on the occurrence of inflammation in the intestines of animals.
 Inflammatory bowel disease can develop by such mechanism: the increased number of
acetogenic SRB, due to exogenous acceptors (sulfate), activates and accelerates the
process of dissimilatory sulfate reduction, accumulation of hydrogen sulfide and acetate.
Acetate causes lowering pH in intestinal lumen to 6.8, showing a synergistic effect and
increases the negative impact of toxic hydrogen sulfide on the mucous membrane and the
colon cells, leading to changes in species diversity of autochthonous microflora,
contributes to the destruction of the protective mucous layer of intestinal cells with
subsequent development of inflammatory process, the withering away of the epithelial
cells and the occurrence of ulcerative colitis.
The number of Lactobacillus and Bifidobacterium bacterial genus in the feces of people with
ulcerative colitis was reduced by 6–7 orders, but the numbers of facultative (Bacteroides,
Escherichia, Enterococcus genera) and conventionally pathogenic (Clostridium and Proteus genera)
microflora were increased by 1–4 orders. The number of acetogenic SRB, Desulfovibrio and
Desulfomicrobium, genera were also significantly (by 5 orders) increased. The ratio of these
bacterial genera was 93:7% in healthy people and 99:1% in people with UC. The isolated and
described dominant SRB in the large intestine by morphological, physiological, biochemical and
genetic characteristics were identified as the Desulfovibrio and Desulfomicrobium genera. Based on
statistical analysis methods, including cluster, the affinity of SRB isolated from healthy people and
patients with UC was shown.
Using the model selected strains, D. piger Vib-7 and D. orale Rod-9, the physiological
characteristics of growth and process of dissimilatory sulfate reduction in these bacteria were
determined. The main enzymes and kinetic parameters of transporting substances, process of
dissimilatory sulfate reduction, metabolize of lactate to acetate in the SRB cells were characterized.
The highest enzymatic activities was determined at temperature (+35 °C) and pH (pH 7.0–8.5).
These data is consistent with the the colon condition which is optimal for the growth of these
microorganisms. Hydrogen sulfide affects not only the representatives of the autochthonous
intestinal flora, but also completely inhibit the growth of its producers (D. piger Vib-7) and their
process of sulfate reduction in concentration of 7 mM H2S. Based on the simulation of the electron
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acceptor and donor on the SRB growth, optimal concentrations (10.5 mM and 53.4 mM,
respectively) were determined. Under these conditions, their biomass was increased in 2.5 times and
the maximum amount (6.06±0.59 mM) of hydrogen sulfide was accumelated. It can be one of the
prerequisites of inflammation process.
The model of ulcerative colitis in rats using acetogenic SRB was created based on study of
their physiological and biochemical characteristics. It is possible to define the role of these
microorganisms and possible mechanisms of the colon diseases, assess the aggressiveness of SRB
and toxicity of final products of their metabolism on the formation of ulcers. Final SRB metabolites
cause changes in the colon lumen conditions, such as pH and reducing the amount of lactic acid
microorganisms. Acetate produced by SRB shows synergistic effect and enhances the negative
effect of hydrogen sulfide on the mucous membrane, which leads to inflammation followed its
complications. According to the model of ulcerative colitis in rats using a strain of genetically
constructed D. piger Vib-7M containing gene CysK, able to metabolize hydrogen sulfide to nontoxic
compounds (cysteine). A decisive role in the inflammatory process plays SRB with excessive
production of hydrogen sulfide, which has toxic effects on epithelial cells and leads to the formation
of ulcers.
The practical significance of the researches. The described physiological and biochemical
properties of metabolic processes of acetogenic SRB from intestine of animals and humans allow to
assess their ability to induce inflammatory bowel disease. Data on changes in the number of SRB,
their proportion in the feces of humans, and production of hydrogen sulfide can be used for
predicting of the course of bowel inflammation. The indicators for determining of the
aggressiveness of SRB were proposed based on toxicity of their metabolic products for the
intestinal mucosa. New scientific approaches and criteria for evaluating of bowel inflammation
progress, allowing determination of the level of risk of the disease with the aim of early diagnosis
and timely predicting were proposed. The results of the work are recommended for developing
prevention methods and using of antimicrobial compounds for the treatment of intestinal diseases.
A collection of SRB strains isolated from human intestine was created and complemented by
genetically constructed D. piger Vib-7M strain which produce of cysteine as the end product of
sulfate reduction but non-toxic hydrogen sulfide.
The practical value of the results is implementated and applicated at the Laboratory of
Anaerobic Microorganisms at the Department of Experimental Biology of Faculty of Science at
Masaryk University. The study is used in pedagogical process in teaching courses „General
Microbiology”, „Bacterial Physiology”, „Human Physiology”, „Human Pharmacology and
Toxicology”, „Veterinary Microbiology”, „Molecular Biology”.
Personal contribution of the author. The habilitation thesis is an independent work of the
author. Based on the results obtained in the study of physiological and biochemical characteristics
of sulfate-reducing bacteria, the concept and hypotheses by the topic of the dissertation were
created. The author himself made information retrieval and conducted an analytical review of the
literature. The methodology was substantiated and the main experimental studies and the statistical
processing, analysis and summarizing of the results were carried by author personally as well as the
articles for printing were prepared. SRB strains were isolated from human intestine, their
physiological and biochemical properties were studied, and the cluster, correlation, crosscorrelation
analysis and the analysis of variance of parameters of dissimilatory sulfate reduction
process in SRB, kinetic analysis of the process, were carried. Based on isolated bacteria, a model of
ulcerative colitis in rats was created by the author.
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The discussion of the directions of the research and the main results of the research was
carried out jointly with a scientific consultant Assoc. Prof. Monika Vítězová, Ph.D. The modeling
of physiological and biochemical parameters of SRB was carried out jointly with Dr. Mark Bolis
from Mario Negri Institute of pharmacological research (Milan, Italy). Together with Assoc. Prof.
Milan Bartoš , Ph.D., the molecular and genetic studies and identification of SRB were carried out,
and described in joint scientific publications.
Approbations the results of the habilitation thesis. The results of the habilitation thesis
were presented at the international scientific conferences „Youth and Progress of Biology” (Lviv,
Ukraine 2012); International Medical Congress of Students and Young Scientists (Ternopil,
Ukraine 2011, 2012); 73 University Scientific Conference „Achievements of Modern Medicine”
(Danylo Halytsky Lviv National Medical University, Lviv, Ukraine 2012); International Congress
of Medical Sciences (Sofia, Bulgaria 2014; 2015); Czech-Slovak International Conference
„Květinův den” (Brno, Czech Republic, 2013, 2014, 2015); International scientific conference
„Actual problems of Biophysics” (Lviv, 2014); International scientific-practical conference
„Current problems of modern biology, animal husbandry and veterinary medicine” (Lviv, Ukraine
2014); XIII National Scientific Conference of Young Scientists „Young scientists in solving current
problems of biology, animal husbandry and veterinary medicine” (Lviv, Ukraine 2014),
Proceedings of International PhD Students Conference – MendelNet (Brno, 2017, 2018). The
results of the thesis are described in 30 scientific papers in foreign scientific journals registered in
the scientific-metric database (Web of Science, Scopus, PubMed, Google Scholar, etc.).
The main methods used in this thesis. SRB strains isolated from human and animal feces
were used in this work. Isolated strains have been identified based on their physiological and
biochemical properties as described in the paper by Kushkevych (2013) as well as on the basis of
sequential analysis of the 16S rRNA gene (Kushkevych et al., 2014). The strains have been kept in
a collection of microorganisms in the Laboratory of Anaerobic Microorganisms at the Department
of Experimental Biology at Masaryk University in Brno.
The ecological-trophic groups of microorganisms of intestinal microflora were determined in
the stool samples of the people. Determination of the number of microorganisms was carried out by
ten-fold dilutions with followed screening on elective nutrient media, according to conventional
methods (Egorov, 1976; Gerhardt, 1984). To determine the number of SRB and the isolation of
their pure cultures, isolated colonies were multiple replanting in the appropriate modified selective
liquid and agar Kravtsov-Sorokin medium (Sorokin, 1966). Pure cultures of microorganisms were
isolated by Koch method. Cultivation was for 10 days at a temperature of +37°C under the fixed
anaerobic conditions using boxes with oxygen absorbing of generators (Genbox anaer Biomerieux,
France). Phenotypic studies of pure SRB cultures were carried out by generally known methods
(Postgate, 1984; Rozanova et al. 1989; Stebbings et al., 2002). Electron microscope examination of
SRB cell morphology was conducted by Reynolds method (Reynolds, 1963). Samples were
microscopic using transmission electronic microscope (PEM-100). The concentration of sulfate ions
was determined by turbidometric method (Kolmert et al. 2000), sulfide ions by spectrophotometric
methods (Cline 1969), and lactate level by the dehydratation reaction using Lactate Assay Kit
(Sigma-Aldrich, USA). Accumulation of acetate during the growth of SRB was determined using a
commercial reagent of Acetate Assay Kit (Sigma-Aldrich Co. LLC, USA).
To determine the phylogenetic identity of SRB, the molecular and genetic identification using
analysis of 16S rRNA gene full sequences was carried out. Isolation and purification of DNA
carried out using biomass of three daily cultures of SRB by using „QIAmp DNA Mini Kit
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(QIAGEN, Germany)”. Amplification of 16S rRNA gene fragments was performed by
W.G. Weisburg et al. (1991) and D.H. Pershing (2011) using pairs of universal primer: 8FPL
(5 ́-AGT-TTG-ATC-CTG-GCT-CAG-3 ́), 1492RPL (5 ́-GGT-TAC-CTT-GTT-ACG-ACT-T-3 ́),
8FPL (5 ́-AGT-TTG-ATC-CTG-GCT-CAG-3 ́) and 806R (5 ́-GGA-CTA-CCA-GGG-TAT-CTAAT-3
́) (Eurofins Scientific, Luxembourg). To purification of amplicons, the “MinElute Gel
Extraction Kit” (QIAGEN) was used. Sequencing was performed using the “Genetic Analyzer”
(Life Technologies Corporation, USA) and reagents “BigDye Terminator v3.1 Cycle Sequencing
Kit” (Applied Biosystems, USA). Analysis of the obtained sequences of 16S rRNA gene with
homologous nucleotide sequences of the genes deposited in database GenBank, was performed
using BLASTN program (www.ncbi.nlm.nih.gov/nblast). The sequences of gene fragments of 16S
rRNA SRB were deposited in the GenBank database as the numbers: KT881309, KT989311 –
KT989316.
To determine the enzymatic characteristics of intestinal SRB, the cell-free extracts were
obtained. The concentration of protein in the cell-free extracts was determined by Lowry method
(Lowry 1951). Enzymatic activity was determined by following methods: ATPase (Flynn et al.
2001; Rathbun & Betlach 1969), ATP sulfurylase (Ravilious et al. 2013), APS reductase (Peck et al.
1965), sulfite reductase, desulfoviridin, desulforubridin (Kobayashi et al. 1972; 1974; Lee et al.
1973), pyrophosphatase (Akagi & Campbell 1963), hydrogenase (Yu & Wolin 1969), lactate
dehydrogenase using reagents “Cytotoxity Assay Kit” (Roche Diagnostics GmbH, Germany),
pyruvate:ferredoxin oxidoreductase (Pieulle et al. 1995; Zeikus et al. 1977), phosphotransacetylase
(Shimizu et al., 1969), acetate kinase (Rose et al. 1954; Mannens et al. 1988). Catalase activity was
determined spectrophotometrically (Biospectrophotometer, Eppendorf, USA) by the reaction of
decomposition of hydrogen peroxide (Luck 1963; Goldblith & Proctor 1950), superoxide dismutase
by inhibiting reduction of nitroblue tetrazol (Beavchamp & Fridovich 1971). One unit (U) of
enzyme activity was such amount of enzyme, which forms 1 micromol of primary product per
minute.
A kinetic analysis of enzymatic reactions was performed in the standard incubation medium
with changed the physical and chemical conditions of such parameters: incubation time, substrate
concentration, temperature and pH. The following kinetic parameters of enzymatic reactions: initial
(immediate) reaction rate (V0), maximum reaction rate (Vmax), the maximum amount of the reaction
product (Pmax) and the reaction time (half saturation period, τ) were determined. Amount of reaction
product was calculated stoichiometric as described by Keleti (1988). Kinetic parameters: Michaelis
constant (Km) and maximum rate of reaction of the product accumulation was identified in
Lineweaver–Burk coordinates (Lineweaver & Burk 1934). To analyze of the kinetic mechanisms of
substrate consumption, the initial (instantaneous) reaction rate under standard conditions with
different concentration substrate was determined.
To investigate the role of SRB in the development of colon diseases and creating a model of
ulcerative colitis, in total 45 laboratory rats were used. The animals were divided into three groups
(15 animals in each group): the first group was used as control; second and third experimental group
for creating a model of ulcerative colitis. The control group of animals received a standard certified
feed for laboratory rats (PK-120-1, Laboratorsnab). Animal of second group received standard feed
and 1 ml modified Kravtsov-Sorokin medium every day. The third group received standard animal
feed, 1 ml suspension of Desulfovibrio piger Vib-7 and Desulfomicrobium orale Rod-9 (ratio 1:1)
bacterial strains in Kravtsov-Sorokin medium (bacterial concentration was 3 mg/mL). Quantitative
and qualitative analysis of the intestinal microflora of experimental animals was determined after 5,
10, 15, 20, 25 days from the start of the experiment. The identification of isolated cultures of
microorganisms was carried out by morphological, tinctirial, cultural, biochemical properties using
11
test systems API 20E (bioMerieux, France). After incubation, the number of microbial colonies was
counted and expressed as an index log10 CFU/gram of wet weight of the sample. Microflora of the
lumen colon was evaluated by an Sustainability Index (SI, %) and frequency detection (Pi) (Odum
1986; Klymiuk 1995). The release and concentration of H2S of 1 g wet weight faeces calculated as
was described in the papers (Ohge et al. 2003; Levitt et al. 2003). Acetate concentration in the
samples was determined colorimetrically using reagents of Acetate Colorimetric Assay Kit (SigmaAldrich,
USA).
For molecular and genetic construction of SRB strain, which would be able to cysteine
synthesis, CysK gene from E. coli K-12 genome was cloned and transformed in SRB cells by
modified method (Hanahan 1983; Seidman 1994). For cloning of the gene, the commercial reagents
“HotStar Master Mix Taq polymerase” (QIAGEN), UDG-glycosylase (New England Biolabs) and a
pair of primers: direct CysK-F (5’-CGAGGCAGATCTTAG-3’) and reverse CysK-R (5’ –
CGAGGCAGATCTCTCGA-GTAG-3’) (Generi-Biotech). Regime of thermal cycling: 2 min
(+37°C), initial denaturation (15 min, +95°C), 35 cycles (10 s, +95°C), 20 s
(+ 50°C) and 30 s (+72°C), 5 min (+72°C) and final cooling (to +10°C). PCR products analyzed by
electrophoresis in 1.5% PAA gel with using ethidium bromide (Sigma-Aldrich, USA) and field
strength of 5 V/cm. As molecular weight marker was used 100 bp ladder (New England Biolabs,
USA). Isolation and purification of DNA fragments from agarose carried out by centrifugation
(14,000 g) through aerosol filters and using “MinElute Gel Extraction Kit” (QIAGEN, USA). PCR
products transferred to SRB by using reagents “AccepTorTM Vector Kit” (Novagen, USA).
Plasmid DNA of SRB was isolated using reagents “QIAprep Spin Miniprep Kit” (QIAGEN). The
presence of prebuilt CysK gene in the plasmid was confirmed by PCR using “MaximaTM
Probe
qPCR Master Mix 2X” (Fermentas, Lithuania) and a pair of universal primers: direct M13F20
(5’-CTAACGACGGCCAG-3’) and reverse M13R (5’-CAGGAAACAGCTATGAC-3’) (Applied
Biosystems, USA). The sequence of the cloned CysK gene in the plasmid confirmed by sequencing.
The presence of the CysK gene was also determined by activity of O-acetylserine(thiol)lyase
(Fimmel & Loughlin 1977) and cysteine concentration in the medium which was determined as the
previously described in the paper (Gaitonde 1967).
The basic statistical parameters including arithmetic mean (M) and standard error (m) of the
arithmetic mean (M ± m) were calculated. To assess of the reliability of the differences between the
statistical characteristics of alternative of data, ratio Student’s coefficient was calculated (Bailey
1995). All research results processed by variation statistics methods. The difference of the
parameter of the reliability of P≤0.05 was considered as an authentically. The value of the statistical
reliability of the parameters using Fisher test (F-test) was tested. Statistical analysis of the results
was carried out by cluster, multivariate variance, correlation and cross-correlation analysis using of
software packages: Statistica 6.1 (www.statistica.software.informer.com) and Origin 7.0
(www.originlab.com). The absolute value of the linear correlation coefficient (r) was calculated by
the method of Pearson. Cross-correlation graphical data converted to digital using the software
package GRAPH DIGITIZER (www.getdata-graph-digitizer.com). Obtaining equations of
functions approximation of the experimental data was performed by least squares (Chen &
Popovich 2002). The share impact on the accumulation of SRB biomass (η2
, %), Fisher’s
coefficients (F practical, F critical) and the reliability of the impact (Lapach et al. 2001). The
obtained time series of the process of sulfate reduction was determined using a STATGRARNICS
software package (www.statgraphics.com).
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2. DIVERSITY OF MICROBIAL COMMUNITIES IN THE INTESTINE
The study of intestinal microbial communities in healthy people and patients with UC showed
the changes in the ratio of main genera of autochthonous and facultative microbiocenosis (Fig. 1).
The number of representatives of the autochthonous bacterial flora, particularly Lactobacillus and
Bifidobacterium genera, in patients with UC were by 6–7 orders lower, and facultative and
conditionally pathogenic bacteria, including genera Bacteroides, Escherichia, Enterococcus, and
Proteus were by 1–4 orders higher compared with microflora of healthy people. The content of
bacteria capable to sulfate reduction (particularly, Clostridium and SRB) in stool samples of
patients with UC was greater, respectively, by 4 and 5 orders, compared with samples of healthy.
The number of methanogens microorganisms was also decreased by 2 orders (Fig. 1A), which is
consistent with the literature data on the substrate competition of these microbial groups (Gibson et
al. 1993).
The different ratio in the members of the dominant SRB morphotypes in the feces of healthy
people and patients with UC was determined. Two morphotypes of SRB colonies were detected:
large colonies I (ø 2–3 mm vibrios cell shape) and small colonies II (ø to 1 mm, short rods). In the
feces of healthy people, the vibrios SRB (93%) were dominated, while amount of SRB of rod shape
was only 7%. In patients with colitis, the ratio (in %) of these morphotypes was 99:1. The
concentration of hydrogen sulfide in the feces of persons with UC was 2 times higher, compared
with healthy and dependent on the number of SRB. The culture of SRB isolated from patients with
UC produced by 1.5–2 times higher of this metabolite than isolates from healthy (Fig. 1B). It is
consistent with the literature (Florin et al., 1990).
Fig. 1. Changes in the number of the main microbial genera in the intestine of healthy people and patients
with UC (A), the ratio of SRB morphotypes isolates and accumulation of hydrogen sulfide (B).
It is known that hydrogen sulfide is in the ionized, non-volatile and volatile state (S,
HS-
and
H2S) (Roediger et al., 1993; Levitt et al., 1999). M. D. Levitt et al. found that 95% of total sulfide in
the intestine is absorbed, and only 5% in feces is bound form with other compounds (Levitt et al.,
1999; 2002). H2S concentration in feces is largely dependent on its ability to interact with other
substances than stand by bacteria in the intestinal lumen (Ohge et al., 2003). Some researchers
reported that total number of SRB was not increased in the feces of people with UC, but the ratio of
their species was only changed. They were aggressive and intense growing in the culture medium.
The level of hydrogen sulfide production in feces was increased by 28% (Willis et al., 1997).
13
Since we have established a significant amount of SRB and high concentrations of H2S in the
faeces of patients with UC, 157 SRB colonies were selected and analyzed, including 79% (124
colonies) belonged to morphotype I and 21% (33 colonies) belonged to morphotype II. Isolates
from the patients with colitis and healthy people did not differ in morphology and size of their cells.
For further phenotypic identification, 20 isolates of bacteria were selected, including 10 of
them from patients with UC and 10 from healthy people. All bacteria were catalase positive and
able to reduction of nitrate to nitrite, but not form indole, and also produced a significant amount of
hydrogen sulfide. As a source of carbon and electron donor they metabolized lactate, pyruvate,
malate, citrate, ethanol and glucose and not used formate, propionate, methanol, glycerin, oleate,
stearate and benzoate. Growth of the isolates was observed in the medium with H2 and CO2 +
acetate in the presence. Intestinal SRB assimilated organic acids, alcohols and some amino acids
(alanine, aspartate, glutamate). The type of their growth was hemolitoheterotrophic. Maximum
biomass of SRB (3.89±0.35 g/l) and their production of hydrogen sulfide (3.23±0.29 mM) was
determined on the 6th
day of cultivation (time of achievement of stationary growth phase) at +35 °C
and pH 7.0–8.0, which correspond to conditions of existence of SRB in the gut. SRB actively
dissimilate of sulfate and accumulate of hydrogen sulfide produced, while lactate oxidized
uncomplete to acetate. Based on morphological, cultural, physiological and biochemical research
(by Bergey's Manual of Determinative Bacteriology, 1994) isolated vibrios shaped of SRB (Vib-1,
Vib-2, Vib-3, Vib-4, Vib-5, Vib-6, Vib-7, Vib-8, Vib-9, Vib-10) was previously identified as the
Desulfovibrio genus, and short rod shaped of SRB (Rod-1, Rod-2, Rod-3, Rod-4, Rod-5, Rod-6,
Rod-7, Rod-8, Rod-9, Rod-10) as the Desulfomicrobium genus.
The length of lag phase, generation time (Td) and the maximum growth rate (µmax) strains
isolated from patients and healthy people was calculated (Table 1). As seen from the results, the
generation time was less (1.73±0.15 h) in the Desulfovibrio strains rom patients with UC compared
with species of the Desulomicrobium genus (2.17±0.23 h). The maximum growth rate in the
desulfovibrios was also higher (0.058±0.007 h-1
). It may indicate the intense growth of these
bacteria in the gut. As for the Desulfomicrobium bacterial strains, they are able to multiply faster in
healthy people, the generation time of microorganisms was 1.93±0.17 h and maximum growth rate
was 0.052±0.004 h-1
.
Table 1. The kinetic parameters of growth of the strains of Desulfovibrio and Desulfomicrobium
genus isolated from healthy people and patients with UC
SRB strains
Duration of
lag phase
(hour)
Time of
generation
Td (hour)
Maximal speed of the
growth
µmax (hour-1
)
Desulfovibrio isolated from
patients with UC 5.21±0.49 1.73±0.15 0.058±0.007
healthy people 5.46±0.51 1.77±0.19 0.056±0.009
Desulfomicrobium isolated from
patients with UC 4.02±0.35 2.17±0.23 0.046±0.002
healthy people 5.08±0.46 1.93±0.17 0.052±0.004
The kinetic parameters of growth of SRB from patients and healthy people did not differ
statistically and consistent with the literature, where were also not found physiological differences
between intestinal SRB isolated from patients with UC and healthy people (Levine et al., 1998).
Based on the sequences of 16S rRNA gene analysis, the detailed identification of SRB was carried
14
out. The nucleotide similarity of 20 studied SRB with strains deposited in GenBank, determined by
the results of the analysis and presented some of them in Table 2.
For bacteria of Desulfovibrio was found that the nucleotide sequence of 7 strains (Vib-1,
Vib-2, Vib-3, Vib-4, Vib-7, Vib-8, Vib-10) had homology of 99% with the nucleotide sequence of
the strain of Desulfovibrio piger ATCC 29098 deposited in the GenBank database by accession
number NR 041778.1. As for the strains of Desulfovibrio sp. Vib-5, Vib-6 and Vib-9, these
sequences data are not allowed to determine their species membership, but only on their genera
level, because nucleotide sequences have homology of 95–96% to referent strain D. piger ATCC
29098, that indicates they probable belong to other species. The nucleotide sequence of 16S rRNA
gene of the strain Rod-9 of Desulfomicrobium genus was 99% homologous to Desulfomicrobium
orale strain NY677 (AJ251629.1) deposited in the GenBank database. For the other 9 strains of
Desulfomicrobium genus, their species membership could not determine according to the sequences
analysis, because a percentage of homology to the representatives of the D. orale species was only
96–98%, they were only identified to genus (data not shown in table). The obtained nucleotide
sequence fragments of 16S rRNA gene of identified Desulfovibrio piger cultures been deposited in
the GenBank database under the corresponding numbers (KT989311), Vib-2 (KT989312), Vib-3
(KT989313), Vib-4 (KT989314), Vib-7 (KT881309), Vib-8 (KT989315), Vib-10 (KT989316).
Table 2. The results of sequence analysis of 16S rRNA gene of SRB strains isolated from human
intestine
Strains and
access number
in GenBank
Length of the
gene fragment
(bp)
Reference strains in GenBank
and accession number
Identity
(%)
Vib-7
KT881309
1370
D. piger ATCC 29098 NR 041778.1
D. fairfieldensis ATCC700045
D. desulfuricans Essex 6 NR 104990.1
99
96
95
Vib-10 KT989316 734
D. piger ATCC 29098 NR 041778.1
D. desulfuricans Essex 6 NR 104990.1
D. legallii strain H1 NR 108301.1
99
95
94
Vib-1 KT989311
Vib-2 KT989312
Vib-3 KT989313
Vib-4 KT989314
Vib-8 KT989315
742
742
743
736
748
D. piger ATCC 29098 NR 041778.1
D. desulfuricans Essex 6 NR 104990.1
D. legallii strain H1 NR 108301.1
D. intestinalis KMS2 NR 026413.1
99
94–95
93–94
93–94
Vib-5 748
D. piger ATCC 29098 NR 041778.1
D. desulfuricans Essex 6 NR 104990.1
D. intestinalis KMS2 NR 026413.1
96
95
93
Vib-6
Vib-9
722
742
D. piger ATCC 29098 NR 041778.1
D. piger ATCC 29098 NR 041778.1
95
96
Rod-9 1470
D. orale NY677 AJ251629.1
D. orale DSM 12838 CP014230.1
D. orale JCM 17150 NR 113205.1
99
99
99
Based on the results of sequence analysis of 16S rRNA gene, the phylogenetic analysis was
carried out and dendrogram showing the affinity of the D. piger Vib-7 and
15
D. orale Rod-9 strains with other members of the Desulfovibrio and Desulfomicrobium genera was
built (Fig. 2). As shown in the dendrogram, bacteria D. piger Vib-7 belonged to one cluster with D.
piger ATCC 29098, D. desulfuricans Essex 6, D. fairfieldensis ATCC700045, D. intestinalis DSM
11275, which were also isolated from the intestine of humans and animals. As for bacteria
Desulfomicrobium orale Rod-9, they form a single cluster with group D. orale strains.
From the literature it is known that D. piger belong to SRB, which are the commensal
microflora in human intestine (Gibson et al., 1991, 1993; Loubinoux et al., 2003). In general,
bacteria of Desulfovibrio genera are most common among members of SRB in the feces of people
with inflammatory bowel disease (Tee et al., 1996; McDougall et al., 1997; Cummings et al.,
2003). Isolated and identified in this study bacteria D. piger were similar by the genetic,
physiological and biochemical characteristics to Desulfomonas pigra, which was isolated by W.E.
Moore in 1976 from feces for the first time (Moore et al., 1976) and subsequently reclassified as D.
piger (Loubinoux et al., 2002). A typical strain isolated from human faeces is D. piger, which is
represented in various collections ATCC 29098, HAQ-6, EBA23-28 (Brenner et al., 2005). As for
bacteria Desulfomicrobium orale, they often isolated from oral cavity of people with gum problems,
gum bleeding and periodontal disease (Langendijk et al., 2001).
Fig. 2. Dendrogram of phylogenetic analysis of relationship sequences of 16S rRNA gene of Desulfovibrio
and Desulfomicrobium genera with SRB isolated from intestine.
Comment: The dendrogram was constructed by Neighbor Joining methods, the scale indicates the genetic distance
between the species.
Thus, according to the phylogenetic identification on the basis of sequences analysis of 16S
rRNA gene of 20 strains of SRB isolated from human intestine was found that the nucleotide
sequence of SRB strains had 99 % homology with sequences strain of Desulfovibrio piger ATCC
29098 (NR 041778.1) and Desulfomicrobium sp. strain Rod-9 belongs to the representatives of the
D. orale NY677 species (AJ251629.1).
16
To study the role of SRB in causing ulcerative colitis, the determined parameters of
dissimilatory sulfate reduction, including bacterial growth, their consumption of sulfate and lactate,
production of hydrogen sulfide and acetate were used as well as a statistical study of the
multidimensional dataset was carried out. Using method of cluster analysis, the SRB strains isolated
from patients with colitis and healthy people were combined in groups by their physiological and
biochemical parameters of dissimilatory sulfate reduction process.
As seen from the obtained results of cluster analysis (Fig. 3), strains of the SRB isolated from
patients with UC formed two separate clusters, which were distant by Euclidean distance
(I and III) as well as formed by bacteria Desulfovibrio piger (Vib-1, Vib-2) and representatives of
the Desulfomicrobium genus (Rod): Rod-1, 2, 5. It can be explained by the fact that the SRB strains
belonging to different genera.
Fig. 3. Dendrogram of SRB affinity based on
physiological and biochemical
parameters of dissimilatory sulfate
reduction.
Comment: red color on the correlogram indicates
the SRB strains isolated from samples of
patients with colitis and black color from
healthy people. Roman numerals indicate
the grouping of bacterial isolates in
clusters.
The cluster II, which consists of separate not
clearly defined subclusters, included strains of
bacteria Rod and Vib, both from healthy people
and patients with UC. Conventionally, this
cluster might call “transitional”, the ratio of
bacteria isolates from healthy people and patients
in the cluster was 9:5, respectively.
Based on a detailed cluster analysis for each
of physiological and biochemical parameters
separately, the ratio of SRB isolated from
patients with colitis and healthy people and their
distribution in clusters was established. The
differences between bacteria of different genera
were in the fact that determining factors of the
process of dissimilatory sulfate reduction for
Desulfovibrio genus, which led to the emergence
of symptoms of colitis, were parameters such as:
biomass accumulation and production of
hydrogen sulfide and acetate. For the
Desulfomicrobium genus, all parameters has
influenced, although most clear division by the
symptoms of the bowel inflammation led
parameters such as production of acetate and
hydrogen sulfide, dividing bacteria to separate
two clusters, which included those that were
mainly isolated from healthy people and patients
with colitis.
These data contribute to a detailed understanding of the degree of affinity between intestinal SRB.
The defining parameters playing a significant role in colitis development are products dissimilatory
sulfate reduction in the gut. This, in turn, is the result of revenues intestinal agents (sulfates and
volatile organic acids, including lactate), which lead to the emergence the colitis symptoms.
To establish the role of final products of SRB metabolism and patterns of development
symptom of colitis under their actions, and the statistical relationship between the variables of
dissimilatory sulfate reduction, the correlation and cross-correlation analysis were carried out
(Bailey, 1995, Chen & Popovich, 2002). Analyzing the parameters of biomass accumulation and
products of sulfate reduction was found positive cross-correlation (Fig. 4).
17
Fig. 4. The cross-correlograms based on parameters of SRB biomass accumulation and final products of
sulfate reduction.
Comment: red color (here and thereafter) on the correlogram indicates the SRB strains isolated from
samples of patients with colitis and black color from healthy people.
The correlation coefficients of parameters of dissimilatory sulfate reduction for all SRB
strains varied in time and closely related in magnitude and sign between the parameters of biomass
and hydrogen sulfide production. Based on these data, it can be argued that the mechanisms and rate
of hydrogen sulfide production in the intestine by studied bacterial isolates are interrelated, but not
identical, because the landslides in biomass accumulation values on 3–4 days were found in
representatives of Desulfomicrobium genus isolated from patients with UC. Temporary slight shift
of some correlation coefficients for SRB from patients with UC, depicted on cross-correlogram in
different directions, indicate significant stable cross-correlation relationships between parameters of
sulfate dissimilation.
Based on the cross-correlation between the biomass and the amount of acetate was found
differences compared with the previous couple of parameters: the value of biomass and hydrogen
sulfide. Time landslides and changes of correlation coefficients for values of biomass in both
bacterial genera were oserved at 3–5 days, while for acetate at 1–3 days. The production of acetate
was significantly different from the production of hydrogen sulfide in time. A similar pattern was
18
observed at 2–3 days for all SRB strains of Desulfomicrobium genus isolated from patients with
colitis. Significant advance of values of acetate production and biomass accumulation of D. piger
Vib-4 strain isolated from patient with colitis compared to other strains on 1–4 day was oserved.
Obviously, such advance in acetate production compared to the biomass accumulation of SRB
isolated from patients with UC can be caused by the action of acetate on the bacterial growth
because during the accumulation of acetate occur a decrease in pH, which can affect the growth of
SRB.
Cross-correlation analysis was conducted to establish time changes between parameters of
consumption of lactate and sulfate, production of acetate and sulfide in the studied SRB strains from
healthy people and patients with UC (Fig. 5).
Fig. 5. The cross-correlograms based on parameters of electron donor/acceptor consumption and final
products of sulfate reduction.
For both genera of SRB, a positive correlation between the studied parameters was confirmed.
The final stage of the dissimilatory sulfate reduction described by changes in indicators of reaction
products is similar in strains of both genera. There no significant differences between the
parameters of linear correlations in SRB from healthy people and patients with UC. The correlation
parameters of final products of sulfate reduction (acetate and hydrogen sulfide) on the cross-
19
correlograms showing a significant landslide of correlation coefficients on 1–3 days for mostly
representatives of Desulfovibrio isolated patients. The landslides correlation coefficients for
bacteria of Desulfomicrobium were different. It may be due, on the one hand, the non-linear nature
of changes of the parameters or instability period, and on the other hand, the periodic presence of
other components. Another explanation of landslides may be different time limits of metabolite
production in dissimilatory sulfate reduction process: the difference between the amount of
hydrogen sulfide and acetate varies during 1–2 days. Due to the synergistic interaction, acetate
enhances the action of hydrogen sulfide on the intestinal cells, and exactly this factor can cause the
disease development.
A cross-correlation analysis of the dynamics of joint metabolism process in intastinal SRB
isolated from healthy people and patients with colitis given more opportunity to understand time
mechanisms of sulfate reduction process by species of different Desulfovibrio and
Desulfomicrobium genera. The determined analogy in time between the dynamics of energy
metabolism and process of dissimilatory sulfate reduction affirmed the universality of the process in
the studied bacteria. From the literature it is known that the relationship between time changes of
these parameters may occur periodically (Chen & Popovich, 2002).
Based on these data we can generalize and assert that the mechanism sulfate reduction process
in different instestinal SRB genera is almost identical. Differences in the sulfate reduction were
observed for parameters of production of acetate and hydrogen sulfide. Consequently, we can assert
that the decisive role in causing symptoms of colitis belongs to this metabolite. They can manifest
their combined or synergistic effect (potentiating effect) on the intestinal cells. On the basis of the
synergistic action of acetate may occur such processes as: the effect on the mechanisms of hydrogen
sulfide binding, its distribution in the lumen, and withdrawal or absorption in different intestinal
parts. It can affect on the biochemical and physiological parameters of intestinal cells and cause the
changes microbiocenosis in general. The acceleration of the absorption or increase permeability
histohematogenous barriers to hydrogen sulfide under the influence acetate can cause inhibition of
the enzymes, including rhodanese (also known as thiosulfate thiotransferase or thiosulfate cyanide
transsulfurase) that can render high concentrations of this compound (Rowan et al., 2009). Synergic
interaction of these substances may be as the result of acetate and hydrogen sulfide effect on various
functionally important biological substrates, membrane and cytoplasmic receptors, and ionophores
(Lodish et al., 2000). The potentiation (synergy) of acetate and hydrogen sulfide can increase the
expressiveness level of their side effects with the further development of inflammation and its
complications (Roediger et al., 1993; Florin et al., 1991; Aslam et al., 1992).
Besides microbiological component, particularly studied above parameters of dissimilatory
sulfate reduction in SRB, the physical and chemical parameters affect also on intestinal
inflammatory processes. To investigate the influence of physical and chemical factors (temperature
and pH) on the biomass accumulation, dispersion analysis, which allow for the impact of these
independent factors, was carried out (Liubischev, 1986). To exclude from consideration the impact
of the natural increase in the number of bacteria in the growth (caused by the time factor), all effects
of stimulation was assessed on the regression line. Using this method of analysis is appropriate for
small samples and carried out with using a sample dispersion, which is called dispersion
measurements (Bailey, 1995).
To establish the total impact of physical and chemical factors (temperature and pH, and other
uncontrollable (unaccounted) factor) on the growth of strains of the Desulfovibrio and
Desulfomicrobium genera isolated from healthy people and patients with UC, dispersion analysis,
which allowed to take into account the percentage of these factors in one system, was carried out
(Table 3).
20
Table 3. Two-factor dispersion analysis of temperature and pH influence on SRB biomass
accumulation
Factor of influence
Indicators of analysis of variance
Strains of
Desulfovibrio genus
Strains of
Desulfomicrobium genus
UC Healthy UC Healthy
Share of influence
(η2
, %)
t 60.58 61.35 45.3 49.03
pH 38.39 37.18 51.21 48.28
Not included factors (%) 1.03 1.47 3.45 2.69
Fisher coefficient
(F practical)
t 235.60 167.11 52.58 72.87
pH 149.30 101.29 59.39 71.77
Comments: Fisher coefficient (F critical) for all parameters is 3, the reliability of influence (p) is 0.99.
Two-factor dispersion analysis of influence of temperature and pH on biomass accumulation
of bacterial strains, Desulfovibrio and Desulfomicrobium genera, gave an opportunity to identify the
proportion of these factors. As shown in the Table 3, share of the influence of temperature and pH,
respectively, were 60:38% for bacteria of the Desulfovibrio genus and 50:50% for
Desulfomicrobium genus. Unaccounted growth factors (biomass accumulation) of
Desulfomicrobium were in 2.5 times more compared to the strains of the Desulfovibrio genus. The
obtained data show the significance of the differences between the averages of variable factors. It is
known that during the development of bowel inflammation, the temperature increases, which in turn
obviously leads to a more intensive growth of the Desulfovibrio genus (share effect of temperature
was 60%), that is why they are often found as dominant at various inflammatory processes. On the
basis of dispersion analysis, the level of variability rate of growth (biomass accumulation) was
determined that are not due to changes in its level caused by the presence or absence of
experimental influences. The start of the inflammatory process caused by any other factors
exogenous or endogenous nature contributes to the bacterial growth of dominant Desulfovibrio
genus in the gut and leads to intensive production of hydrogen sulfide. The changes in pH are
probably due to an increase in the production of acetate in SRB. Such conditions may provide
progression from further complications.
21
3. BIOCHEMICAL CHARACTERISTICS OF DESULFOVIBRIO PIGER VIB-7
AND DESULFOMICROBIUM ORALE ROD-9
In the previous chapter, the influence of basic metabolites of dissimilatory sulfate reduction
by SRB from human intestine on the emergence of inflammatory processes was studied. The
differences in the sulfate reduction to hydrogen sulfide and metabolize of lactate to acetate was
found. For a detailed study of the kinetics of these changes, two model SRB strains, D. piger Vib-7
and D. orale Rod-9, was selected, because they are typical representatives of SRB which are often
isolated from human intestine. These microorganisms exhibit high metabolic activity, the most
biomass accumulation, production of hydrogen sulfide and acetate. The process of dissimilatory
sulfate reduction is very complicated and a large number of enzymes are involved (Phartiyal et al.,
2006; Czechowski et al., 1990; Furdui et al., 2000; Nicolet et al., 1999 and others). However, no
information on the enzymatic properties of SRB, including the Desulfovibrio and Desulfomicrobium
genera, isolated from the intestine.
In this chapter, the main stages of intestinal SRB metabolism and related enzymes involved in
the dissimilatory sulfate reduction, including in the process of transporting substances, dissimilatory
sulfate reduction to hydrogen sulfide, oxidation of lactate to acetate, were characterized (Table 4).
The maximum value of enzymatic activity in D. piger Vib-7 and D. orale Rod-9 strains was
determined at incubation temperature +35ºC and pH in range 8.0–8.5 that was consistent with the
conditions of the human colon and, obviously, caused by adaptive mechanisms of SRB. The initial
and maximum speed of enzymatic reactions, the maximum amount of reaction products were by
1–2 orders higher in bacteria D. piger Vib-7 compared to D. orale Rod-9. This indicates a more
intense metabolism in bacteria of the Desulfovibrio genus, because they are often found as the
dominant representatives in the intestine of humans and animals.
Transportation of sulfate ions and organic compounds to the cell cytoplasm of SRB occurs by
the mechanism of active transport with using ATP energy (Barton & Hamilton, 2010), that is, as an
electron donor and acceptor can compete by these systems. Constants of inhibition of ATPase by
ouabain were 22.9±4.6 and 15.8±2.6 mM, respectively, for D. piger Vib-7 and
D. orale Rod-9. Obviously, the kinetic parameters ATPase of SRB depend on activity of ATP
sulfurylase that competes with the enzyme for ATP in transportation process of sulfate. Equally
important transport function belongs to hydrogenases, which is involved in the transfer of hydrogen
through the plasma membrane in dissimilatory sulfate reduction process. The high activity of this
enzyme and its maximum rate was found in both SRB strains. These parameters were the highest
compared with other enzymatic activity of sulfate dissimilation process, which may be due to
excessive formation of hydrogen in the cytoplasm.
It should be noted that the value of inorganic pyrophosphatase activity in D. piger Vib-7 and
D. orale Rod-9 were, respectively, 24.272.47 and 8.160.82 U×mg protein-1
, and the maximum
rate of reaction was 3.864.24 and 13.741.32 µmole×min-1
×mg-1
. Obviously, this enzyme is
involved not only in sulfate reduction but also in exchange of ATP, since, as already mentioned, for
ATPase of studied microorganisms is also detected high value of activities and the maximum rates
of the reactions.
Thus, transport processes in the SRB cells were quite active and although competition by
ATPase interrelated by substrate specificity, which enters the cell, and rate of its metabolism.
22
Table 4. Enzymatic characteristics of the intestinal SRB strains
The kinetic characteristics of enzymes
(EC number)
SRB strains
D. piger Vib-7 D. orale Rod-9
Transportation of substances
ATPase
(EC 3.6.1.3)
А (U×mg-1
of protein) 16.111.87 7.310.98
V0 (µmole×min-1
×mg-1
) 15.951.58 10.690.93
Vmax (µmole×min-1
×mg-1
) 36.102.87 16.641.73
Km (mM) 2.240.21 2.060.18
Hydrogenases
(EC 1.12.99.6)
А (U×mg-1
of protein) 1421.4123.7 568.745.6
V0 (µmole×min-1
×mg-1
) 205.6718.91 58.165.38
Vmax (µmole×min-1
×mg-1
) 2500219 1111107
Km (mM) 86473 66962
Reduction of sulfate to hydrogen sulfide
ATP sulfurylase
(EC 2.7.7.4)
А (U×mg-1
of protein) 2.260.231 0.980.0082
V0 (µmole×min-1
×mg-1
) 5.480.57 4.120.38
Vmax (µmole×min-1
×mg-1
) 4.870.55 2.110.22
Km (mM) 1.980.21 1.070.12
APS reductase
(EC 1.8.99.2)
А (U×mg-1
of protein) 0.340.029 0.110.012
V0 (µmole×min-1
×mg-1
) 0.6750.062 0.2310.022
Vmax (µmole×min-1
×mg-1
) 0.8620.084 0.2820.027
Km (mM) 4.330.47 3.570.32
Sulfite reductase
(EC 1.8.99.1)
А (U×mg-1
of protein) 0.0320.0026 0.0280.0022
V0 (µmole×min-1
×mg-1
) 0.3510.033 0.1380.012
Vmax (µmole×min-1
×mg-1
) 0.0670.0053 0.0450.0039
Km (mM) 3.530.334 3.860.341
Oxidation of lactate to acetate
Lactate dehydrogenase
(EC 1.1.1.27)
А (U×mg-1
of protein) 0.4720.037 0.1530.014
V0 (µmole×min-1
×mg-1
) 0.1140.012 0.0260.022
Vmax (µmole×min-1
×mg-1
) 1.200.11 0.650.053
Km (mM) 0.830.07 1.540.14
Pyruvate:ferredoxin
oxidoreductase
(EC 1.2.7.1)
А (U×mg-1
of protein) 1.240.127 0.480.051
V0 (µmole×min-1
×mg-1
) 4.150.43 1.370.12
Vmax (µmole×min-1
×mg-1
) 2.540.261 0.890.092
Km (mM) 2.720.283 2.550.245
Phosphotransacetylase
(EC 2.3.1.8)
А (U×mg-1
of protein) 1.190.122 0.370.041
V0 (µmole×min-1
×mg-1
) 5.680.58 2.140.23
Vmax (µmole×min-1
×mg-1
) 2.730.31 0.980.089
Km (mM) 3.360.35 5.970.62
Acetate kinase
(EC 2.7.2.1)
А (U×mg-1
of protein) 1.520.163 0.460.044
V0 (µmole×min-1
×mg-1
) 6.160.63 1.390.14
Vmax (µmole×min-1
×mg-1
) 3.120.32 1.030.098
Km (mM) 2.540.26 2.680.25
23
To determine, which one of metabolic processes are proactive: dissimilatory sulfate reduction
to hydrogen sulfide or lactate to acetate and which enzymes of these reactions would be “sensitive
area”, the enzymatic activities of these two processes were determined and the kinetic analysis of
the enzymatic reactions was carried out (Fig. 6).
Fig. 6. The comparison of processes of sulfate reduction and lactate oxidation.
According to the dynamics of the maximum rate (Vmax) and the time ( , half saturation
period) of enzymatic reactions, the intestinal SRB can oxidize of lactate and produce of acetate
faster (total process time is 20–27 min) compared with dissimilatory sulfate reduction and
accumulation of hydrogen sulfide (the process is 51–58 min). It is confirmed in previous calculated
kinetic parameters of consumption of acceptor/donor of electrons and the production of the final
products of intestinal SRB metabolism. Maximum rates of consumption of sulfate and lactate,
production of hydrogen sulfide and acetate by bacteria D. piger Vib-7, respectively, were
0.315±0.028, 1.082±0.103, 0.510±0.043, 1.193±0.117 h-1
. A similar pattern was found for the strain
of bacteria D. orale Rod-9. The sensitive area of sulfate reduction was dissimilatory sulfite
reductase, which slowed this process almost in 10 times. For the oxidation of lactate to acetate, the
“sensitive area” was observed at the initial stage of the process: lactate dehydrogenase. The
determined low activity sulfite reductase consistent with the literature (Kobayashi et al., 1972,
1974; Ogata et al., 1981; Czechowski et al., 1990).
Thus, based on studied enzymatic parameters was found that the production of acetate is
2 times faster compared to hydrogen sulfide and the consumption of lactate compared to sulfate.
The study of compounds, which can inhibit sulfite reductase and lactate dehydrogenase in SRB, is
promising to create methods and drugs to prevent and reduce the risk of bowel inflammation.
Accordingly, SRB, dissimilating sulfate and oxidizing lactate, will not be able to metabolize and
produce high concentrations of hydrogen sulfide and acetate. The perspective is to create methods
for diagnosis of ulcerative colitis based on the determination of the main activity of these enzymes.
Comprehensive study of enzymes of dissimilatory sulfate reduction and sulfide accumulation
enables the formation of a holistic understanding involvement of these systems of SRB in
maintaining ion homeostasis and their effects on the intestinal cells.
24
4. PHYSIOLOGICAL CHARACTERISTICS BACTERIA
DESULFOVIBRIO PIGER VIB-7
Since based on biochemical studies was found high kinetic parameters of studied enzymes in
D. piger Vib-7 strain and given the fact that the properties of intestinal SRB are still unexplored,
this chapter describes of sulfate dissimilation under different conditions of cultivation. The
influence of medium pH, different concentrations of sulfide, electron donor and acceptor in this
complex and multistage process was studied.
Optimal parameters of D. piger Vib-7 growth, their consumption of sulfate and lactate,
accumulation of hydrogen sulfide and acetate depending on the acidity of the medium was studied
(Kushkevych, 2014). The maximum values of the process were determined by the optimal pH 7.0-
8.0, which is consistent with the results of the activity of enzymes described in the previous chapter.
Under these conditions, the highest growth rate (0.047–0.050 h-1
), the production of hydrogen
sulfide (0.035–0.036 h-1
) and acetate (0.102–0.104 h-1
) were detected (Table 5). Decrease of pH < 7
or increase of pH > 9 caused inhibition of sulfate reduction process. The pH in the colon humans is
8–9, which apparently causes intensive growth of microorganisms and therefore their production of
sulfide. Decrease of pH to 6–7 is critical for colon, because neutral or slightly acidic pH can cause
of development of conditional pathogens (Nugent et al., 2001).
Table 5. Kinetics of D. piger Vib-7 bacterial growth under the effect of pH medium and hydrogen
sulfide
Effect
Duration of
lag phase
(hour)
Time of
generation Td
(hour)
Maximal speed of accumulation
µmax (hour-1
)
Biomass Hydrogen sulfide Acetate
pH medium
4 47±4.5 27±2.3 0.020±0.0019 0.001±0.0001 0.065±0.0055
5 14±1.3 3±0.28 0.026±0.0023 0.010±0.0015 0.092±0.0088
6 5.8±0.4 2±0.22 0.038±0.0034 0.024±0.0021 0.094±0.0091
7 6.6±0.5 1.8±0.15 0.047±0.0045 0.036±0.0033 0.102±0.0098
8 6.2±0.5 1.7±0.12 0.050±0.0049 0.035±0.0029 0.104±0.0095
9 6.7±0.6 2±0.23 0.039±0.0033 0.032±0.0025 0.089±0.0081
10 24±2.3 4±0.37 0.014±0.0011 0.0083±0.0009 0.070±0.0062
Hydrogen sulfide (mM)
0 6.58±0.61 1.79±0.14 0.49±0.042 0.034±0.0036 0.103±0.009
1 6.65±0.65 2.37±0.22 0.37±0.036 0.025±0.0022 0.093±0.0088
2 7.72±0.73 3.31±0.29 0.25±0.022 0.013±0.0011 0.079±0.0072
3 8.05±0.76 6.20±0.57 0.11±0.001 0.004±0.0001 0.064±0.0057
4 10±1.11 15.21±1.55 0.01±0.0001 0.001±0.0001 0.052±0.0046
5 15±1.23 15.29±1.61 0.01±0.0001 0 0.031±0.0025
No less important factor that influenced on the D. piger Vib-7 growth and their sulfate
reduction is hydrogen sulfide. Despite the fact that D. piger Vib-7 is a producer of sulfide, but its
25
additional adding in the medium in a concentration of 1 mM caused inhibition of bacterial growth
and reduced of sulfide and acetate production in the bacteria by 21% compared with the control.
The maximum growth rate under these conditions was also decreased by 25% compared with the
control medium without introducing hydrogen sulfide. Increasing concentrations of hydrogen
sulfide to 6 mM caused a significant decrease in the maximum rate of growth and the production of
final products of intestinal SRB metabolism (Table 5). Complete inhibition of bacterial growth and
sulfate reduction process was determined at the concentration of 7.0 mM hydrogen sulfide. It is
known that the concentration of free (unbound) sulfide in the lumen of the human colon is in the
micromolar range and sulfide ions in millimolar concentrations (1.0–2.4 mM), because it is bound
with a large number of feces components (Blachier et al., 2010). Hydrogen sulfide is cytotoxic for
intestinal cells of mice in the concentration range of 0.2–1.0 mM (Deplancke et al., 2003) and
human intestines in 0.3–3.4 mM (Florin et al., 1991; Pochart et al., 1992; Magee et al., 2000).
However, the mechanisms of protection of intestinal cells under prolonged exposure to this toxic
substance are still not fully elucidated.
The integrity of the gut epithelial layer is preserved through detoxification mechanisms
(Picton et al., 2002) or permanent removal of damaged cells (Attene-Ramos et al., 2006). Thus,
hydrogen sulfide was toxic not only to gut microflora and its cells, but also for its producers, as
described in the literature (Beauchamp et al., 1984). The effect of electron acceptor and donor on
the growth rate and the dissimolatory sulfate reduction was determined (Table 6). It helped clarify
which one of these paramters will play a more significant role in the development of diseases.
Table 6. Kinetics of D. piger Vib-7 growth under the effect of electron acceptor and donor
Electron acceptor Electron donor
Sulfate
(mM)
Lag-
phase
(hour)
Generation
time
Td (hour)
µmax
(hour-1
)
Lactate
(mM)
Lag-phase
(hour)
Generation
time
Td (hour)
µmax
(hour-1
)
0.87 38.2±3.5 16.5±1.5 0.009±0.0001 4.45 36.6±3.7 14.5±1.35 0.009±0.008
1.75 5.9±0.46 4.3±0.44 0.02±0.001 8.9 7.1±0.66 3.6±0.33 0.03±0.001
3.5 6.4±0.62 1.8±0.15 0.05±0.004 17.3 6.4±0.60 1.8±0.12 0.05±0.004
7.0 7.4±0.73 1.1±0.10 0.08±0.007 35.6 4.9±0.43 1.1±0.10 0.08±0.007
10.5 3.3±0.31 1.3±0.12 0.06±0.005 53.4 3.1±0.29 1.3±0.11 0.07±0.005
17.5 5.5±0.59 1.6±0.14 0.05±0.005 89.0 5.4±0.51 1.5±0.13 0.06±0.004
The greatest amount of hydrogen sulfide (6.06±0.59 mM) at the presence of 10.5 mM sulfate
and acetate (21.10±2.16 mM) at the presence of 53.4 mM lactate was accumulated in the cultivation
medium. Under these conditions, the highest growth rate (0.080 h-1
) was observed at the electron
acceptor concentration of 7 mM and electron donor of 35.6 mM. Obviously, sulfate and lactate
determined concentrations lead to stimulating the SRB growth in the gut, their production of sulfide
and acetate, which will increase the risk of inflammation.
The intensity of the sulfate reduction process may also depend on the number of SRB. The
increase in SRB biomass to 1 mg/ml led to a decrease in the length of the exponential growth phase
and the process of dissimilation sulfate. At the introduction of 1.0 mg/ml of the cell, the growth
phase duration (te) was 12 hours. Under these conditions, the greatest value of absolute growth rate
(0.020±0.002 mg/ml×h) was detected; while the maximum intensity of the sulfate reduction process
achieved at the point (te) after 12 hour of cultivation (Fig. 7).
26
The highest specific growth rate of D. piger Vib-7 and parameters of sulfate dissimilation
(µmax) was detected in the exponential growth phase in a range of critical points of the growth from
Т1 to Те. Increasing the number of SRB in the range of 0.5–1.0 mg/ml (х0) led to an increase in the
absolute growth rate of D. piger Vib-7 and stimulate of the sulfate reduction process. Reducing the
duration of these processes (te) indicates the intensive production of hydrogen sulfide and,
accordingly, the occurrence of sudden flares of inflammation. In general, 1 mg of SRB cells in 1 ml
was titer 106
CFU/ml. It is obviously critical to the emergence of symptoms of colitis.
Fig. 7. Kinetics of D. piger Vib-7 growth depending on number of their cells.
Thus, the optimum parameters, providing the greatest rate of SRB growth are:
pH 7.0–8.0 (rates of growth 0.047–0.050 h-1
, production of hydrogen sulfide 0.036 h-1
, and acetate
0.102–0.104 h-1
), 7.0 mM electron acceptor and 35.6 mM electron donor. The intensive
accumulation of hydrogen sulfide in concentrations of 1 mM inhibited the maximum growth rate by
24%, and sulfate reduction process in SRB by 26%. Increasing the number of SRB in the range of
0.5–1.0 mg/ml led to a reduction lag- and exponential growth phase; stimulate growth and rate of
the dissimilatory sulfate reduction process. Determination of the kinetics of SRB growth and their
sulfate reduction process are particular importance for predicting the risk of occurrence of
symptoms and duration of the bowel disease.
27
5. ETIOLOGICAL ROLE OF SULFATE-REDUCING BACTERIA
IN THE DEVELOPMENT OF COLON DISEASES
Inflammatory bowel disease, including ulcerative colitis, is complex multifactorial diseases
mostly unknown etiology. The development of the pathology is detailed studied on animal models
of inflammation, inducing it’s by chemical substances such as dextran sulfate, acetic acid,
carrageenan, sodium lignosulfonate or amylopectin sulfate (Nell et al., 2010; Barnett et al., 2011;
Perse et al., 2012; Low et al., 2013). In this chapter, using SRB, ulcerative colitis in rats (in vivo)
was experimentally induced and assessed ulceration level in different parts of the intestine. The
determined concentrations of hydrogen sulfide and acetate in feces samples taken from different
parts of the intestine of rats correlated with the level of ulceration. Analysis of the microflora of the
distal intestine of rats was carried out. The microflora of the first (control) group of animals during
twenty-five days was not significantly changed. The significant changes in microbiota in animals of
the second and third group on 25 day of experiment were observed. Under these conditions, the
number of microorganisms, their ratio, index and incidence of sustainability was significantly
changed. Number microbial genera Bifidobacterium and Lactobacillus was decreased by 90%,
while the number of bacterial genera of Clostridium (71%), Escherichia (79–94%), Proteus (63%),
Klebsiella (80%), Staphylococcus (43%) and SRB (95%) were increased for a specified number of
percents. It is obvious that these microorganisms play an important role in the development of
inflammatory bowel disease. Dysbiosis of intestinal microflora may indicate the initial stages of
inflammatory bowel disease and other pathological processes.
The concentration of sulfide and acetate were increasing directly proportional to the duration
of the experiment. Increase in the concentration of these compounds by 32 and 65%, and 35% and
69%, respectively, in the second and third group of animals compared to controls was observed at
the 5th
day. On the 25th
day, the levels of hydrogen sulfide and acetate in the colon was increased by
88 and 95%, respectively, (2nd
group of animals) and by 93 and 97% (3rd
group of animals). A large
number of ulcers (78±7.50%) in the descending colon, compared with other parts of the colon of
rats, were detected in this period in the experimental animals. The highest level of ulceration was
found in animals of third group compared with the second group. Obviously, SRB showed high
aggressiveness on the colon of rats, compared with their own microflora, causing intensive
development of inflammatory bowel disease including ulcerative colitis.
The genetically-modified strain of D. piger Vib-7M, containing CysK gene transformed from
E.coli, was constructed. It is able to convert toxic hydrogen sulfide to cysteine through activity of
O-acetylserine(thiol)lyase (97.56 U×mg of protein-1
), which uses it as a substrate. This bacterial
strain is able to utilize its own produced and exogenous hydrogen sulfide, released by other SRB.
The mutant strain of SRB accumulate biomass by 36% more than wild type and 5.380.52 mM of
cysteine at 84th hour of cultivation. Using this strain, assimilatory sulfate reduction way was
modeled.
Daily adding 1 ml of D. piger Vib-7M (3 mg/ml) suspension in the medium in the diet to rats
with colitis helped restore the diversity of the autochthonous microbiota (Fig. 8). The number of
lactic acid bacteria of Lactobacillus and Bifidobacterium genera was increased by 93%,
accordingly, the number of representatives of facultative and conditionally pathogenic microflora,
including Bacteroides, Escherichia, Enterococcus and Proteus was decreased. The number of SRB
which able to produce hydrogen sulfide was significantly (98%) decreased. It can be caused by
competition for the acceptor. Indeed, as already noted, a mutant strain capable with higher speeds
(1.5 times) using sulfate, compared with strains that are in the intestine of animals with colitis.
28
Fig. 8. The microbiota of large intestine in rats under conditions of ulcerative colitis induced by SRB and
after the introduction of the D. piger Vib-7M strain being able to utilize of hydrogen sulfide.
The titer of these bacteria on the 25th day amounted to 3.8×105
CFU/g of feces, and hydrogen
sulfide levels decreased by 96% compared to the control group animals that were not treated. Using
this D. piger Vib-7M strain experimentally was confirmed that the development of inflammatory
diseases involved bacteria which are able to dissimilatory sulfate reduction but not assimilatory.
Thus, based on studied SRB strains, a model of intestinal inflammation in rats was created
and proved their functional role in the development of the disease. Introduction SRB suspension in
the gastrointestinal tract of rats causes changes in the structure of its microbiota. The highest
accumulation levels of hydrogen sulfide, which is toxic for cells, and acetate was found in the
descending colon. It is evidenced most frequent cases of inflammation in these areas are the large
intestine of animals and humans. Using SRB constructed strain that can metabolize hydrogen
sulfide to cysteine, competing with other SRB by the substrate, experimentally proved and justified
that the main role in the inflammatory process belongs to SRB which exercising dissimilatory
sulfate reduction.
29
6. SUMMARIZING
Inflammatory bowel diseases, including ulcerative colitis, are complex and multifactorial.
People with infections of the gastrointestinal tract often show an increased number of SRB.
The physiological and biochemical characteristics of SRB the under different conditions,
consumption of sulfate and lactate, production of hydrogen sulfide and acetate, functional role of
these microorganisms in disease development were studied. It was shown that ulcerative colitis in
humans is caused by changing the qualitative and quantitative levels of the intestinal microbiota
representatives. In people with ulcerative colitis is reduced by 6–7 orders of the bacterial genus of
Lactobacillus and Bifidobacterium and increased by 1–4 orders of representatives of optional and
conditional pathogenic microflora. The increased by 5 orders of the SRB number in the intestine of
people with colitis was detected. The ratio of SRB genera, Desulfovibrio and Desulfomicrobium, is
93:7% in healthy and 99:1% in people with colitis. The dominant of SRB in the intestine of healthy
and people with UC are members of the Desulfovibrio genus. It is known that these bacteria often
form a microbial biofilm on the mucosa of the colon of humans and animals (Macfarlane et al.,
2007; Gibson et al., 1991).
Based on cluster analysis of physiological and biochemical parameters, the differences
between SRB isolated from patients with UC and healthy people were investigated. These
differences were in the fact that for bacteria of the Desulfovibrio genus determining factors of
dissimilatory sulfate reduction process, which led to the emergence of symptoms of colitis, were
biomass accumulation, production of hydrogen sulfide and acetate. For species of the
Desulfomicrobium genus were all influential parameters, although most significant division on
grounds of illness led parameters such as: production of acetate and hydrogen sulfide. Thus, we can
argue that the defining parameters, playing a significant role in causing colitis, are products
dissimilatory sulfate reduction (hydrogen sulfide and acetate). This is the result of revenues sulfate
in large intestine and volatile organic acids, including lactate, which lead to the strengthening of the
process and the emergence of symptoms of colitis.
A cross-correlation analysis of the dynamics of metabolic processes in studied SRB isolated
from patients with colitis and healthy was carried out and has allowed more understand the time
mechanisms of the sulfate reduction process in the species of different genera of Desulfovibrio and
Desulfomicrobium. The analogy was observed between the dynamics in time of energy metabolism
and process of dissimilatory sulfate reduction. It was provided an opportunity to confirm the
universality of the process in the bacterial strains. As a result of joint action of hydrogen sulfide and
acetate on intestinal cells can occur potentiation effect (synergistic effect) than separately effects of
these substances. The basis of the synergistic action of acetate may be processes such as: effect on
the mechanisms binding of hydrogen sulfide, its distribution in the bowel lumen and deactivation or
absorption in different parts of the intestine. During the development of bowel inflammation,
increasing temperature, it in turn, obviously, leads to a more intensive growth of the Desulfovibrio
genus (share effect of temperature is 60%). The launch of inflammation process caused by any other
factors exogenous or endogenous nature and contributing to an increase in growth rates among the
dominant SRB of Desulfovibrio, leads to an intense production of hydrogen sulfide and increase the
pH causes the intensive accumulation of acetate. Consequently, these conditions can provide of the
progression of the disease with its further complications.
Sulfate-reducing bacteria isolated from feces of patients with UC and healthy people were not
statistically different by their physiological and biochemical properties. Among the studied bacteria,
the greatest concentration of hydrogen sulfide (3.23 ± 0.29 and 3.14 ± 0.27 mM) and acetate (15.87
± 1.46 and 16.24 ± 1.54 mM) was producted by D. piger Vib-7 and D. orale Rod-9. Therefore, they
30
were selected as model objects for studying of biochemical and physiological characteristics as well
as their etiological role in the development of inflammatory bowel disease.
The maximum of enzymatic activities for both strains was observed at a temperature of +35ºC
and pH 7.0–8.5. Obviously, such conditions ensure their intensive development in the gut. The
initial and maximum speed of enzymatic reactions in D. piger Vib-7 was in 2 times higher than in
D. orale Rod-9. Thus, bacteria of Desulfovibrio, dissimilating sulfate and oxidizing lactate faster,
can be more aggressive and play a pathogenic role in the development of intestinal diseases. Based
on the research of enzymatic parameters, the advance in the production of acetate compared to
hydrogen sulfide was detected. The inhibition of two enzymes of SRB, sulfite reductase and lactate
dehydrogenase, is a promising in development of the method for preventing and reducing of the risk
of inflammation in the intestine.
Given the fact that the kinetic parameters of enzymes of sulfate reduction are higher in
bacteria D. piger Vib-7, they were selected to study of their physiological characteristics under
different conditions. The highest growth rate (0.047–0.050 h-1
), production of hydrogen sulfide
(0.035–0.036 h-1
) and acetate (0.102–0.104 h-1
) at optimum pH 7.0–8.0 was determined. It is
consistent with the results of enzymatic activities of studied SRB. However, intensive production of
hydrogen sulfide and its accumulation in 1 mM concentration can inhibit by 24% of maximum
growth rate and by 26% of sulfate reduction in SRB. Increasing the number of SRB in the range of
0.5–1.0 mg/ml led to a reduction lag- and exponential growth phases; stimulate growth and speed of
the process of dissimilatory sulfate reduction. All these factors are involved in development of
inflammatory processes and their subsequent complications and the occurrence of UC.
Injection of sulfate-containing medium or SRB suspension in the gastrointestinal tract caused
significant changes in the structure and sustainability of intestinal microbiota of rats. It corresponds
to research of intestinal microflora of people with colitis. Number of bacterial genera of
Lactobacillus and Bifidobacterium were decreased by 93–95%, but the number of bacteria
Clostridium (73%), Escherichia (96%), Staphylococcus (45%), Proteus (67%) genera and SRB
(97%) were increased. Perhaps, SRB with other intestinal microbial groups can compete by
substrates, particularly volatile organic acids, and metabolic products of normal microflora as well
as show the impact on obligate microflora (Lactobacillus and Bifidobacterium) and inhibit their
development under effect of hydrogen sulfide. The highest accumulation levels of hydrogen sulfide
and acetate found in the descending colon, where they grow in direct proportion to the duration of
dosing sulfate-containing medium or suspension of SRB. These data are consistent with the results
of bowel ulceration in third animal group, where the ulceration was significantly higher than in the
second group. Obviously, SRB contribution in the development of the disease is much greater than
their induced own endogenous of SRB in rats.
On the basis of these results, the generalized scheme mechanisms of inflammation
development involving SRB with the subsequent emergence of ulcerative colitis and bacteremia
was created (Fig. 9). The increased number of SRB (×108
) in the intestine causes competition with
methanogens by molecular hydrogen as evidenced their decrease number by 66%. Under these
conditions, SRB intensely attached to epithelial cells, forming a biofilm together with Bacteroides
and Clostridium. These studies are consistent with data from the literature on the formation of
resistant biofilms formed by SRB (Macfarlane et al., 2007; Newton et al., 1998).
31
Fig. 9. The scheme of mechanisms of inflammation process development involving SRB with the
subsequent emergence of ulcerative colitis and bacteremia.
Dissimilatory sulfate reduction process and production of aggressive metabolites for
microorganisms (acetate: 97% and hydrogen sulfide: 68%) leads to a change in pH from slightly
alkaline (pH 8–9) to neutral or even slightly acid (pH 6.8–7.2) in the large intestine. It can cause
changes in microbiota and, accordingly, the growth of facultative and opportunistic
microorganisms. Under these conditions, after reduced number of obligate microflora, SRB firmly
attached to the mucosal cells and begin to rapidly destroy the protective mucous layer of the
intestine. In the current biofilm clostridia, which are resistant to SRB and are also able to produce
32
hydrogen sulfide. Clostridia in this biofilm can metabolize more complex organic compounds,
including mucins layer containing polysaccharides, to volatile organic compounds and release
sulfated residues, which SRB reduce to hydrogen sulfide and acetate.
Violation of the intestinal barrier layer by SRB and the loss of the epithelial cells ability to
utilize of aggressive toxic metabolites leads to inflammation of the further formation of ulcers.
Acetate is produced faster but may be partially neutralized in the lumen intestine, enabling of SRB
to produce of sufficient amount of hydrogen sulfide, because the alkalinity of the colon allows quite
a long time to maintain homeostasis of intestinal lumen. Under these conditions, the intestinal
lumen, obviously, decreases the concentration required chelating compounds to hydrogen sulfide,
which leads to a lesser extent its binding and neutralization. Accordingly, permanently attached
SRB have toxic affect on cells of the intestine and thereby destroying the epithelium, causing
inflammation of the appearance of ulcers.
The hypothetical mechanisms of colitis development under the influence of SRB and their
dissimilatory sulfate reduction was confirmed by constructed mutant strain, which was capable to
metabolize sulfate and hydrogen sulfide to cysteine.
Stability studies of mutant strain D. piger Vib-7M that can convert hydrogen sulfide to
cysteine and the likelihood of its reversion to wild type show that the genetically modified strain
was stable to reversion (3% revertants). Mutant strain competing for substrate with other intestinal
SRB and actively growing faster, metabolizing sulfate and lactate, can convert hydrogen sulfide to
untoxic cysteine. It was recommended for use in the study of inflammatory diseases of created
animal models.
It was proved that in the development of colitis is required exactly free hydrogen sulfide in a
high concentration. Constructed mutant strain allowed modeling “assimilation sulfate reduction”,
which are also capable of many members of the intestinal microflora (Clostridium, Escherichia,
Salmonella) and prove that their number does not affect the formation of ulcers. The effect of these
microorganisms is based on the production of secondary toxins that can not be produced for some
time and wait for the time of their induction. Hydrogen sulfide is the primary metabolite of SRB
and formed in the sulfate reduction, therefore, the development of inflammation depends on the
intensity of dissimilatory sulfate reduction. Using “cysteine” mutant SRB strain, experimentally
proved and justified that the main role in the inflammatory process belongs to SRB, which are
present in the gut, scilicet, the appearance of inflammatory process causing bacteria, which capable
to dissimilatory sulfate reduction, but not assimilatory. Constructed mutant strain is a valuable and
indispensable to study the factors involved in the pathogenesis of inflammatory bowel disease, and
essential for use in therapeutic research.
A new created animal model of inflammatory diseases based on SRB is scientifically valuable
in the research stages of the disease with its subsequent complications. Specificity of the
pathogenesis of inflammatory bowel disease is complex, and the etiology of ulcerative colitis can be
even caused affect of genetic, microbiological, environmental and other factors. These results are
important for the prevention of diseases of the gastrointestinal tract, since, for the first time, the
detailed information of the etiological role of SRB in causing intestinal diseases is described, which
are valuable for their diagnosis.
Sulfate-reducing bacteria are probably involved in the occurrence of pathological states such
as: increased intestinal permeability, frequent bowel movements, weight loss, and general malaise
development. Based on the number of SRB, production of hydrogen sulfide and acetate, their
aggressiveness and toxicity of metabolic products for intestinal mucosa was assessed. The described
results are new and made it possible to create models of colitis involving SRB, based on which was
developed the main evaluation criteria of progress inflammation: changes in the composition of
33
microflora and pH of the colon, changes in enzymes of dissimilatory sulfate reduction, changes in
the rate of SRB growth and intensity of sulfate reduction, changes in the concentration of hydrogen
sulfide and acetate in faeces. Understanding the role of SRB and their contribution to the
development of diseases, it is necessary to adjust the amount and value of SRB in the large intestine
and, consequently, decrease the production of sulfide and acetate. The described experimental
results are important for further careful study of the mechanisms of inflammatory bowel disease
with the use of appropriate therapeutic strategies, for studying the mechanisms of action of
antimicrobial drugs with a prophylactic or therapeutic purpose of specific components involved in
the pathogenesis of the disease. The study of selected strains of bacteria resistance to antimicrobial
and new synthesized chemotherapy drugs based on the research of patterns of course process of
occurrence of ulcerative colitis with considering the complex of physiological, biochemical,
enzymatic characteristics of SRB, enables the search for new effective and harmless to the patient's
drug-inhibitors. Such research is also perspective to develop methods for diagnosis and prevention
of inflammatory bowel disease.
34
CONCLUSIONS
1. Based on experimental and theoretical approaches and studies of physiological and biochemical
characteristics of dominant intestinal acetogenic sulfate-reducing bacteria, the concept of their
functional role in the development of diseases of the large intestine in animals and human, was
created.
2. The changes of hunam intestinal microbiota, including bacteria of Bifidobacterium and
Lactobacillus genus, were demonstated. Their number in people with ulcerative colitis was
reduced (2.49±0.22 and 3.47±0.33 log10CFU/g of feces). Species of the facultative and
conditionally pathogenic microflora in the intestines of these patients was increased by 1–4
orders and SRB was also increased by 5 orders, compared to the healthy.
3. On the basis of morphological, physiological, biochemical, molecular and genetic features, the
isolated bacteria were identified as species of Desulfovibrio and Desulfomicrobium genera.
SRB from the intestine of healthy people and with UC statistically did not differ on
physiological and biochemical properties. The dominated genera among SRB were
Desulfovibrio (93%) and Desulfomicrobium (7%) in feces of healthy people and their ratio in
patients with colitis was 99:1%, respectively.
4. The determining factors of dissimilatory sulfate reduction process, which lead to the
appearance of symptoms of colitis was the SRB biomass accumulation, their production of
hydrogen sulfide and acetate, most important of which are the final products. Share the
influence of temperature and pH on the growth of the Desulfovibrio genus was, respectively, 60
and 38% and leads to the launch of the inflammatory process. The affiliation to genera of SRB
was slightly affects on the differences in the mechanisms dissimilatory sulfate reduction and
the emergence of symptoms of colitis.
5. Based on the determined enzymatic parameters of strains of D. piger Vib-7 and D. orale Rod-9,
the advancing production of acetate in 4 times compared with hydrogen sulfide (time advance
was 19:5 min) was determined for the first time. It leads to potentiation of the effect and
increases the aggressiveness of these metabolites. Sensitive area of the sulfate dissimilation
process is sulfite reductase and lactate dehydrogenase for lactate oxidation.
6. The optimal parameters of the environment, providing the greatest rate of growth of SRB
isolated from the intestine are: 7.0–8.0 pH, concentrations of 7.0 mM sulfate and 35.6 mM
lactate. Hydrogen sulfide in concentrations of 1 mM inhibits the maximum growth rate (by
24%) and the process sulfate reduction (by 26%). Increased SRB biomass (to 0.5–1.0 mg/ml)
leads to reduction of lag-phase and exponential phase by 12 hours, to stimulate growth speed
and the process of dissimilatory sulfate reduction.
7. For the first time, a model of ulcerative colitis in rats using strains of D. piger Vib-7 and D.
orale Rod-9 was created and their etiological role in the development of disease was
demonstrated. Introduction suspension of the strains in the gastrointestinal tract changes the
structure of the microbiota, sustainability index and their frequency detection. An increase in
the production of aggressive metabolites: acetate (97%), hydrogen sulfide (68%) compared
with control was detected in experimental animals. It changes the parameters of pH in the colon
of rats from 8–9 to 6.8–7.2.
35
8. In intestine of rads with UC, the decrease in number of the Bifidobacterium and Lactobacillus
(93–95%), Peptococcus (81%) genera and increase bacteria of the Clostridium (73%),
Escherichia (96%), Proteus (67%), Klebsiella (86%), Staphylococcus (45%) genera and SRB
(97%) were detected. It is consistent with data on intestinal microflora people with UC.
Increased levels of hydrogen sulfide and acetate, respectively, 88 and 95% and ulceration
(78%) are found in the descending colon of animals with colitis.
9. The mutant strain of SRB D. piger Vib-7M, containing CysK gene, which encodes the enzyme
O-acetylserine(thiol)lyase, and is able to metabolize toxic hydrogen sulfide to cysteine, was
constructed. Daily adding suspensions of D. piger Vib-7M in ration of rats with colitis
contributes the restore the population of lactic acid bacteria, reducing the number of
representatives of facultative and conditional pathogens and SRB, which able to produce
hydrogen sulfide. The concentration of hydrogen sulfide in the feces is reduced by 95% and the
number of ulcers by 72%, compared to animals that did not receive in the diet of the mutant
strain.
10. The main criteria for evaluating the development and course of inflammatory process involving
SRB is changes: composition of the microflora and pH of the large intestine, the activities of
enzymes of dissymilatory sulfate reduction, growth rate of SRB and intensity of sulfate
reduction, the concentration of hydrogen sulfide and acetate in feces. Prevent of this process
can be achieved by inhibiting two enzymes sulfate reducrion process, sulfite reductase and
lactate dehydrogenase, which is most sensitive areas in metabolism of studied SRB.
36
PUBLICATION LIST BY THE HABILITATION THESIS
Book:
Kushkevych I. Intestinal Sulfate-Reducing Bacteria (ed. Larry Barton), MUNIPRESS, 2017. 320 p.
ISBN 978-80-210-8614-2.
Publications in the Journals indexed in Scopus and Web of Science with IF:
1. Kushkevych I., Kollar P., Suchy P., Parak K., Pauk K., Imramovsky A. Activity of selected
salicylamides against intestinal sulfate-reducing bacteria. Neuroendocrinol Lett, 2015, 36, 106-
113.
2. Kushkevych I., Fafula R., Parak T., Bartos M. Activity of Na+
/K+
-activated Mg2+
-dependent
ATP hydrolase in the cell-free extracts of the sulfate-reducing bacteria Desulfovibrio piger Vib-
7 and Desulfomicrobium sp. Rod-9. Acta Vet Brno, 2015, 84, 3-12.
3. Kushkevych I.V. Activity and kinetic properties of phosphotransacetylase from intestinal
sulfate-reducing bacteria. Acta Biochemica Polonica, 2015, 62, 103-108.
4. Kushkevych I.V. Kinetic Properties of Pyruvate Ferredoxin Oxidoreductase of Intestinal
Sulfate-Reducing Bacteria Desulfovibrio piger Vib-7 and Desulfomicrobium sp. Rod-9. Polish
J Microbiol, 2015, 64, 107-114.
5. Kushkevych I., Kollar P., Ferreira A.L., Palma D. Antimicrobial effect of salicylamide
derivatives against intestinal sulfate-reducing bacteria. J Appl Biomed, 2016, 14, 125-130.
6. Kushkevych I., Vítězová M., Fedrová M., Vochyanová Z., Paráková L., Hošek J. Kinetic
properties of growth of intestinal sulphate-reducing bacteria isolated from healthy mice and
mice with ulcerative colitis. Acta Vet Brno, 2017, 86, 405-411.
7. Kushkevych I., Vítězová M., Vítěz T., Bartoš M. Production of biogas: relationship between
methanogenic and sulfate-reducing microorganisms. Open Life Sciences, 2017, 12, 82-91.
8. Kushkevych I., Vítězová M., Vítěz T., Kováč J., Kaucká P., Jesionek W., Bartos M., Barton L.
A new combination of substrates: biogas production and diversity of the methanogenic
microorganisms. Open Life Sciences, 2018, 13, 119-128.
9. Kushkevych I., Dordević D., Vítězová M., Kollár P., Cross-correlation analysis of the
Desulfovibrio growth parameters of intestinal species isolated from people with colitis.
Biologia, 2018, 73, 1137-1143.
10. Kushkevych I., Kos J., Kollar P., Kralova K., Jampilek J. Activity of ring-substituted 8hydroxyquinoline-2-carboxanilides
against intestinal sulfate-reducing bacteria Desulfovibrio
piger. Medicinal Chemistry Research, 2018, 27, 278-284.
11. Kushkevych I., Kováč J., Vítězová M., Vítěz T., Bartoš M. The diversity of sulfate-reducing
bacteria in the seven bioreactors. Arch Microbiol, 2018, 200, 945-950.
12. Kushkevych I., Vítězová M., Kos J., Kollár P., Jampílek J. Effect of selected 8hydroxyquinoline-2-carboxanilides
on viability and sulfate metabolism of Desulfovibrio piger.
J. App Biomed., 2018, 16, 241-246.
13. Kováč J., Vítězová M., Kushkevych I. Metabolic activity of sulfate-reducing bacteria from
rodents with colitis. Open Med, 2018, 13, 344-349.
14. Kováč J., Kushkevych I. New modification of cultivation medium for isolation and growth of
intestinal sulfate-reducing bacteria. Proceeding of International PhD Students Conference
MendelNet, 2017, 702-707.
15. Černý M., Vítězová M., Vítěz T., Bartoš M., Kushkevych I. Variation in the Distribution of
Hydrogen Producers from the Clostridiales Order in Biogas Reactors Depending on Different
Input Substrates, 2018, Energies, 11(12), 3270.
37
16. Kushkevych I., Dordević D., Kollár P. Analysis of physiological parameters of Desulfovibrio
strains from individuals with colitis. Open Life Sciences, 2018, 13, 481-488.
17. Kushkevych I., Dordević D., Vítězová M. Analysis of pH dose-dependent growth of sulfatereducing
bacteria. Open Med, 2018, 13, 381-389.
18. Kushkevych I., Dordević D., Vítězová M. Toxicity of hydrogen sulfide toward sulfate-reducing
bacteria Desulfovibrio piger Vib-7. Archives of Microbiology, 2019, 201, 1-9.
Publications in the Journals indexed in Scopus:
19. Kushkevych I.V., Hnatush S.O., Mutenko H.V. Glutathione level of Desulfovibrio
desulfuricans ІМV К-6 under the influence of heavy metal salts. Ukr Biochem J., 2011, 83(6),
105-110.
20. Kushkevych I.V. Acetate kinase Activity and Kinetic Properties of the Enzyme in
Desulfovibrio piger Vib-7 and Desulfomicrobium sp. Rod-9 Intestinal Bacterial Strains. The
Open Microbiol J., 2014, 8, 138-143.
21. Kushkevych I., Bolis M., Bartos M. Model-based characterization of the kinetic parameters of
dissimilatory sulfate reduction under the effect of different initial density of Desulfovibrio piger
Vib-7 bacterial cells. The Open Microbiol J., 2015, 9, 55-69.
Other in the list indexed on Web of Science
Publications in the Journals indexed in other databases:
22. Kushkevych I.V. Sulfate-reducing bacteria of the human intestine. I. Dissimilatory sulfate
reduction. Stud Biol. 2012, 6, No 2, 149-180 (article in Ukrainian).
23. Kushkevych I.V. Sulfate-reducing bacteria of the human intestine. II. The role in the diseases
development. Stud Biol. 2012. 6, No 2, 221-250 (article in Ukrainian)
24. Kushkevych I.V. Dissimilatory sulfate reduction by various Desulfovibrio sp. strains of the
human intestine. Microbiol and Biotech, 2013, 350-63.
25. Kushkevych I.V., Moroz O.M. Growth of various strains of sulfate-reducing bacteria of human
large intestine. Stud Biol., 2012. 6, No 3, 115-124.
26. Kushkevych I.V., Beno Y.J. Cluster and Cross-correlation Analysis of some Physiological
Parameters by Various Desulfovibrio sp. and Desulfomicrobium sp. Bacterial Strains of the
Human Intestine. SOJ Microbiol & Inf Dis., 2013, 1, 1-9.
27. Kushkevych I.V. Growth of the Desulfomicrobium sp. strains, their sulfate- and lactate usage,
production of sulfide and acetate by the strains isolated from the human large intestine.
Microbiol. Discov., 2014, 2, 1-8.
28. Kushkevych I.V. Identification of sulfate-reducing bacteria strains of the human large intestine.
Stud Biol., 2013, 7, No 3, 115-124.
29. Kushkevych I.V., Bartos M., Bartosova L. Sequence analysis of the 16S rRNA gene of sulfatereducing
bacteria isolated from human intestine. Int J Curr Microbiol Appl Sci., 2014, 3, No 2,
239-248.
30. Kushkevych I.V. Effect of hydrogen sulfide at differential concentrations on the process of
dissimilatory sulfate reduction by the bacteria Desulfovibrio piger. Sci notes of Ternopol Nat
Ped Univ. Series Biol., 2013, 4, No 57, 74-80.
31. Kushkevych I.V. Dose-dependent effect of electron acceptor and donor on dissimilatory sulfate
reduction by bacteria Desulfovibrio piger Vib-7 of human intestine. Stud Biol., 2014, 8, No 1,
103-116.
38
32. Kushkevych I.V., Antonyak H.L. New Desulfovibrio sp. strains isolated from human intestine
and their dissimilatory sulfate reduction. Int Cong Med Sci. (Sofia, Bulgaria). Abstract, 2014,
P. 22.
33. Kushkevych I.V. Antonyak H.L. Activity of periplasmic hydrogenase of the intestinal sulfatereducing
bacteria. The Animal Biol J., 2014, 16, No 2, 35-41.
34. Kushkevych I.V. Dissimilatory sulfate reduction in bacterium Desulfovibrio piger Vib-7 under
the effect of medium with differential acidity. American J Microbiol & Biotechnol. 2014, 1, No
2, 49-55.
35. Kushkevych I.V. The effect of hydrogen sulfide on the dissimilatory sulfate reduction of the
intestinal bacteria Desulfovibrio piger. Int Cong Med Sci. (Sofia, Bulgaria). Abstract, 2015, P.
80.
36. Kushkevych I.V., Fafula R.V. Dissimilatory sulfite reductase in cell-free extracts of intestinal
sulfate-reducing bacteria. Stud Biol., 2014, 8, No 2, 101-112.
37. Kushkevych I.V., Fafula R.V., Antonyak H.L. Catalase Activity of Sulfate-Reducing Bacteria
Desulfovibrio piger Vib-7 and Desulfomicrobium sp. Rod-9 Isolated from Human Large
Intestine. Microbes and Health J., 2014, 3, No 1, 15-20.
38. Kushkevych I.V., Antonyak H. L., Fafula R. V. Superoxide dismutase activity of the sulfatereducing
bacteria Desulfovibrio piger Vib-7 and Desulfomicrobium sp. Rod-9. Microbiol and
Biotechnol., 2014, 4, No 28, 26-35.
39. Kushkevych I.V. Kinetic characteristics of pyrophosphatase of the sulfate-reducing bacteria
from human intestine. Sci J. “Visnyk of Lviv Uni. Biol Series”, 2014, 68, 158-166.
40. Kushkevych I.V. Lactate dehydrogenase activity in cell-free extracts of sulfate-reducing
bacteria Desulfovibrio piger Vib-7 and Desulfomicrobium sp. Rod-9. Sci J “Visnyk of L’viv
Uni. Biol Series”, 2014, 67, 243-251.
41. Kushkevych I.V., Hnatush S.О., Mutenko H.V. The activity of catalase and superoxide
dismutase in bacteria Desulfovibro desulfuricans Ya-11 under the influence of heavy metals.
Visn Dnipro Univ. Series: Biology. Ecology, 2010, 10, No. 2, 25-33 (article in Ukrainian)
42. Kushkevych I.V. Activity and kinetic properties of adenosine 5′-phosphosulfate reductase in
the intestinal sulfate-reducing bacteria. Microbiol and Biotechnol., 2014, 2, No 26, 54-63.
43. Kushkevych I.V. Antonyak H.L., Bartos M. Kinetic properties of dissimilatory adenosine
triphosphate sulfurylase of intestinal sulfate-reducing bacteria. Ukr Biochem J., 2014, 86, N 6,
129-138.
44. Kushkevych I. Molecular cloning CysK gene from Escherichia coli genome, its transferring in
the intestinal sulfate-reducing bacteria and the expression analysis of Oacetylserine(thiol)lyase.
Microbes and Health, 2015, 4, No 1, 19-24.
45. Kushkevych I. V. Etiological role of sulfate-reducing bacteria in the development of
inflammatory bowel diseases and ulcerative colitis. American J Inf Dis & Microbiol., 2014, 2,
No 3, 63-73.
39
CURRICULUM VITAE
Mgr. Ivan Kushkevych, Ph.D., Dr.Sc.
Personal Data:
Birth date/place: 28th
June 1984 / L’viv region, Ukraine
Workplace: Department of Experimental Biology (Section of Microbiology), Faculty of Science at Masaryk
University (MU), Kamenice 753/5, 625 00 Brno, Czech Republic
Phone: +420 549 49 5315
e-mail: kushkevych@mail.muni.cz
URL for web site: https://www.researchgate.net/profile/Ivan_Kushkevych
University Degrees:
2006 Master study programme at Microbiology Department, Biology Faculty, Ivan Franko L’viv National
University (LNU), Ukraine (diploma with honours).
2010 Postgraduate study program at LNU. Ph.D. thesis in Microbiology: “Influence of heavy metals salts
on physiological and biochemical characteristics of sulfur cycle bacteria”.
2016 Doctor of Biological Sciences (Habilitated Doctor in Microbiology): “The functional role of sulfatereducing
bacteria in the development of bowel diseases in human and animals”. Zabolotny Institute
of Microbiology and Virology of NAS of Ukraine.
2017 Position of Associate Professor at Department of Experimental Biology at MU.
Employment Positions:
2017 – so far Position of Associate Professor at Department of Experimental Biology (Section of
Microbiology) at MU.
2015 – 2016 Assistant Professor at Department of Experimental Biology at MU.
2013 – 2015 Postdoc at Department of Human Pharmacology and Toxicology at University of Veterinary
and Pharmaceutical Sciences (UVPS) Brno, Czech Republic.
2011 – 2012 Lector at Department of Microbiology, Virology and Immunology at Danylo Halytsky Lviv
National Medical University, Ukraine.
2007 – 2010 Assistant Professor at Microbiology Department at LNU, Ukraine.
Research Stays:
2018 Internship in Laboratory of Pathogenic Microorganisms of Fishes at the Department of Microbiology
and Ecology at University of Valencia, Spain (1 month).
2016 Visiting prof. Galina O. Iutynska, Dr.Sc., Corresponding Member of the NAS of Ukraine, Zabolotny
Institute of Microbiology and Virology of NAS of Ukraine (1 week).
2013 Research Internship in the Laboratory of Molecular Biology at the Department of Molecular
Biochemistry and Pharmacology at Institute of Institute pharmacological research Mario Negri,
Milan, Italy (3 months). Detailed information on website: http://www.vfu.cz/informace-ouniverzite/vita-universitatis/pdf/vu_2014_2.pdf
(page 19).
2008 ІІІ Summer School «Mоlecular міcrobiology and biotechnology», Оdessa, Ukraine (2 weeks).
Pedagogical Activities:
From 2016 Practical courses: General Microbiology and Bacterial Physiology. Supervisor of Microbiology
Diploma Thesis and Bachelor Thesis at the Faculty of Science at MU.
From 2014: Lector of 2 subjects at the Faculty of Pharmacy at UVPS (General Biology and Microbiology
and practical classes for both Czech and foreign students).
From 2013: Supervisor of 4 master program students at the Department of Human Pharmacology and
Toxicology, Faculty of Pharmacy at UVPS.
From 2007: Supervisor of 7 Microbiology Diploma Thesis and 3 Bachelor Thesis (10 of them have
successfully defended) at the Microbiology Department LNU, Ukraine.
40
Scientific and Research Activities:
 Molecular microbiology – especially biochemistry of sulfate-reducing bacteria (SRB) and their enzymes
of dissimilatory sulfate reduction under the effect various compounds.
o High level of knowledge – applicant graduated in Microbiology and awarded by Certificate in
molecular biology (Italy, 2013).
 Physiology and Biochemistry of intestinal SRB – especially activity of their enzymes.
o Advanced level of knowledge – applicant established this research direction at Department of
Experimental Biology, Faculty of Science at MU in 2016.
Commissions of Trust
From 2014: Editorial board member and reviewer of three international journals: American Journal of
Infectious Diseases and Microbiology, American Journal of Microbiology and Biotechnology,
International Journal of Biological Sciences and Applications.
Ad-hoc referee of more than 3 scientific journals (incl. Archives of Medical Science).
Publications, Citations:
Total publications 57 (https://www.researchgate.net/profile/Ivan_Kushkevych)
Total citations without self-citations (Web of Science): 41
H-index: 8
Author or co-author of at least 35 conference papers, active attendance at 16 conferences.
Research monographs, chapters in collective volumes and any translations thereof:
Ivan Kushkevych. Intestinal Sulfate-Reducing Bacteria (ed. Barton, L.L.), MUNIPRESS, 2017. 320 p.
ISBN 978-80-210-8614-2.
Education:
2017 Position of Associate Professor at Department of Experimental Biology at MU.
2016 Doctor of Biological Sciences (Habilitated Doctor in Microbiology).
2013 Postdoc at Department of Human Pharmacology and Toxicology at UVPS Brno (3 years).
2010 Philosophy Doctor in Microbiology at LNU, Ukraine.
2006 Master’s degree in Microbiology at LNU, Ukraine.
Pedagogical Activities:
Microbiology, Physiology of Microorganisms (practical classes), supervisor of diploma thesis.
Scientific and Research Activities:
 Anti-inflammatory activity of chemical compounds, animal models of ulcerative colitis.
 Internal grant projects UVPS Brno (“Pharmaco-toxicological evaluation of newly synthesized (isolated)
compounds as an integration tool for pre-clinical disciplines at VFU Brno” Project reg. No.
CZ.1.07/2.3.00/30.00 (04/2013–04/2015)), and grant projects of West-Ukrainian BioMedical Research
Center for young biomedical researchers in Ukraine (06/2007–06/2008).
 Active attendance at conferences (ICMS, Sofia, Bulgaria, 2014, 2015).
ATTACHMENT 1
Pages 41 – 50
_____________________________________________________________________________
Activity of Na+
/K+
-activated Mg2+
-dependent ATP-hydrolase in the cell-free extracts of the
sulfate-reducing bacteria Desulfovibrio piger Vib-7 and Desulfomicrobium sp. Rod-9
Ivan Kushkevych, Roman Fafula, Tomáš Parák, Milan Bartoš
Acta Veterinaria Brno, 2015, 84: 3-12, ISSN 0001-7213
_____________________________________________________________________________
Activity of Na+
/K+
-activated Mg2+
-dependent ATP-hydrolase in the cell-free extracts
of the sulfate-reducing bacteria Desulfovibrio piger Vib-7
and Desulfomicrobium sp. Rod-9
Ivan Kushkevych1
, Roman Fafula2
, Tomáš Parák1
, Milan Bartoš1
1
University of Veterinary and Pharmaceutical Sciences Brno, Faculty of Pharmacy,
Department of Human Pharmacology and Toxicology, Brno, Czech Republic
2
Danylo Halytsky Lviv National Medical University, Faculty of Pharmacy, Lviv, Ukraine
Received March 28, 2014
Accepted October 22, 2014
Abstract
The aim of our work was to study Na+
/K+
-activated Mg2+
-dependent ATPase activity in cellfree
extracts of the sulfate-reducing bacteria Desulfovibrio piger Vib-7 and Desulfomicrobium sp.
Rod-9 isolated from the human large intestine, and to carry out the kinetic analysis of the enzyme
reaction. The maximum ATPase activity for both bacterial strains at +35 ºC was determined. The
highest activities of the studied enzyme in the cell-free extracts of D. piger Vib-7 at pH 7.0 and
Desulfomicrobium sp. Rod-9 at pH 6.5 were measured. Based on experimental data, the analysis
of kinetic properties of the ATP-hydrolase reaction by the studied bacteria was carried out. The
enzyme activity, initial (instantaneous) reaction rate (V0
) and maximum rate of the ATPase
reaction (Vmax
) was significantly higher in D. piger Vib-7 cells than in Desulfomicrobium sp. Rod-
9. Michaelis constants (Km
) of the enzyme reaction for both bacterial strains were determined.
ATPase activity, hydrogen sulfide, intestinal microbiocenosis, bowel diseases, ulcerative colitis
Sulfate-reducing bacteria carry out the dissimilatory sulfate reduction during anaerobic
respiration (Barton and Hamilton 2007). The final product of the sulfate reduction in
the human intestine is hydrogen sulfide which is carcinogenic to its cells, and can cause
inhibition of cytochrome oxidase, colonocytes oxidation of butyrate, destruction of
epithelial cells, development of ulcers, and inflammation with subsequent development
of colon cancer (Pitcher and Cummings 1996; Gibson et al. 1991; Cummings et
al. 2003). The transport of sulfate ions and organic compounds in the cytoplasm of the
bacterial cells occurs through active transport using ATP energy (Barton and Hamilton
2007). In this regard, it is very important to study the mechanisms of sulfate ions transport,
enzymatic activity and kinetic properties of other ATP-dependent enzymes of sulfatereducing
bacteria from human intestine.
Plentiful data are available on the functions of biological membranes including the
integral membrane protein ATP-dependent systems of transport ions (Lodish et al. 2000;
Yuan et al. 2005; Tian et al. 2006). However, the ATPase activity of the sulfate-reducing
bacteria Desulfovibrio piger and Desulfomicrobium sp. isolated from the human large
intestine has not been studied yet. A comprehensive study of the functioning and role of
Na+
/K+
-pump as a system of energy-dependent transport of different ions in the regulation
of the dissimilatory sulfate reduction and accumulation of hydrogen sulfide will enable to
form a holistic view on the participation of these systems in maintaining ion homeostasis
of the sulfate-reducing bacteria cells.
The aim of our work was to study Na+
/K+
-activated Mg2+
-dependent ATPase activity
in cell-free extracts of the sulfate-reducing bacteria Desulfovibrio piger Vib-7 and
Desulfomicrobium sp. Rod-9 isolated from the human large intestine, and to carry out the
kinetic analysis of the enzyme reaction.
ACTA VET. BRNO 2015, 84: 3–12; doi:10.2754/avb201585010003
Address for correspondence:
Dr. Ivan Kushkevych, M.Sc., Ph.D.,
Department of Human Pharmacology and Toxicology, Faculty of Pharmacy
University of Veterinary and Pharmaceutical Sciences Brno
Palackého tř. 1/3, 612 42 Brno, Czech Republic
Phone: +420 732 215 046
Fax:  +420 541 240 605
E-mail: ivan.kushkevych@gmail.com
http://actavet.vfu.cz/
Materials and Methods
Bacterial strains
Objects of the study were sulfate-reducing bacteria Desulfovibrio piger Vib-7 and Desulfomicrobium sp. Rod-9
isolated from the human large intestine (Kushkevych 2013; Kushkevych et al. 2014). The strains have been
kept in the collection of microorganisms at the Biotechnology Laboratory of the Faculty of Pharmacy, University
of Veterinary and Pharmaceutical Sciences Brno (Czech Republic).
Preparation of cell-free extracts
Cell-free extracts were prepared from the exponential phase of growth. The bacteria were grown anaerobically
in nutrition-modified Kravtsov-Sorokin’s liquid medium (Kushkevych 2013). The cold extraction buffer
(50 mM potassium phosphate buffer, pH 7.5, 10-5
M EDTA (ethylenediaminetetraacetic acid) was added to
centrifuged sedimented cells to bind heavy metal ions. A total of 10-5
M PMSF (phenylmethylsulfonyl fluoride)
for the inhibition of proteases, which is effective at pH above 7.0, was added. After this procedure, a suspension
of cells (150–200 mg/ml, which correspond to optical density 0.569–0.761) was obtained. The cells were
homogenized using the ultrasonic disintegrator (Soniprep 150 Plus MSE Limited, United Kingdom) at 22 kHz
for 5 min at 0 ºC. The soluble extracts constituted by the supernatant were used as the source of the enzyme. The
soluble fraction was displaced into centrifugal tubes and cell-free extracts were separated from the cells fragments
by centrifugation for 30 min at 14,000 × g and at 4 ºC. Pure supernatant was then used as cell-free extracts. The
spinned cells fragments were used as sedimentary fraction. Protein concentration in the cell-free extracts was
determined by the Lowry method (Lowry et al. 1951).
Measuring ATPase activity
The total Na+
/K+
-ATPase enzymatic activity of bacterial cell-free extracts was measured in the incubation
medium (volume 1 ml) of the following composition: 120 mM NaCl, 30 mM KCl, 5 mM MgCl2
, 1.5 mM ATP,
1 mM EGTA, 20 mM Hepes-Tris-buffer (pH 7.4), 0.1 μM thapsigargin (selective inhibitor of Ca2+
/Mg2+
-ATPase
E (C)) (Flynn et al. 2001). Calcium chelators EGTA were added for the binding of endogenous Ca2+
ions in
the incubation medium. The enzymatic reaction was initiated by adding bacterial cell-free extracts (100 µl) in
the incubation medium (the amount of the protein in the sample did not exceed 50–150 mg/ml). The incubation
period was 1–20 min. The enzymatic reaction was stopped by adding 1 ml of cooled stop solution of the following
composition: 1.5 M sodium acetate, 3.7% formaldehyde, 14% ethanol, 5% trichloroacetic acid (pH 4.3). The
suspension was centrifuged (1,600 × g, 10 min) and the concentration of inorganic phosphorus (Pi
) was measured
spectrophotometrically (Biospectrophotometer, Eppendorf, USA) in the obtained supernatant without protein.
The amount of reaction products were determined by Rathbun and Betlach (1969) and expressed as µmol Pi
/
min·mg-1
protein. Magnesium-ATPase activity of the bacterial cell-free extracts was tested in the same incubation
medium, in the presence of 1 mM ouabain (selective inhibitor of Na+
/K+
-ATPase). The Na+
/K+
-ATPase activity
was calculated by the difference between the value of total ATPase and Mg2+
-ATPase activity. The standard
incubation medium (without the adding of cell-free extract) of non-enzymatic hydrolysis of ATP was used in the
experiments as a control. A mixture of bacterial cells in saline for measuring the amount of endogenous Pi
in the
bacterial suspension was also used as a control.
Kinetic analysis
The study of kinetic properties of enzymatic reactions Na+
/K+
-activated Mg2+
-dependent ATP-hydrolase
reaction was performed in a standard incubation medium, which was modified by physical and chemical
characteristics or composition of the respective components (incubation time, protein bacterial mixture in the
sample, the concentration of ATP, Na+
(K+
), Mg2+
, ouabain). All experiments to study the properties of the Na+
/K+
ATPase
reaction were performed using the initial rate V0
(linear accumulation of product (Pi
) in time). The kinetic
indicators characterizing the reaction release Рі
during Na+
/K+
-activated Mg2+
-dependent of ATP hydrolysis were
the initial (instantaneous) reaction rate (V0), maximum rate of the Na+
/K+
-ATPase reaction (Vmax
), maximum
amount (plateau) of the reaction product (Pmax
) and the characteristic reaction time (time half saturation) τ were
determined. The kinetic indicators that characterize the Na+
/K+
-activated Mg2+
-dependent ATP-hydrolase reaction
(the constant activation of ions Mg2+
, K+
(KMg
2+
, KK
+
), Michaelis constant (KmАТР) and maximal rate of ATP
hydrolysis by the Mg2+
, K+
(VMg
2+
, VK
+
) and ATP (VАТР) were determined by the Lineweaver-Burk plot (Keleti
1988). The obtained concentration dependence of the rate of enzymatic reaction on the studied reagents (ATP
and Mg2+
, Na+
(K+
)) was constructed in the coordinates {1/V on 1/S}, where S is the concentration of the reagent
(АТР, Mg2+
or Na+
(K+
)), and V is the rate of enzymatic hydrolysis of ATP at a concentration of АТР, Mg2+
or Na+
(K+
), respectively. In determining the effectiveness of the ouabain on ATPase activity (the constant of inhibition
(I0,5
) and Hill coefficient (nH)), the linearized curves of the concentration dependence were constructed in Hill
coordinates {lg[(A0–A)/A]; lg[I]} according to the empirical Hill’s equation: lg[(A0
–A)/A] = –nH
lg I0.5
+ nH
lg
[I], where A0
and A are the specific activity of the enzyme in the absence and in the presence of ouabain in a
concentration I in medium.
Statistical analysis
Kinetic and statistical calculations of the results were carried out using the software MS Office and Origin
computer programs. The research results were treated by methods of variation statistics using Student’s t-test.
4
The significance of the calculated indicators of line was tested by Fisher’s F-test. The accurate approximation
was when P ≤ 0.05 (Bailey 1995).
Results
ATPase activity and the effect of temperature and pH
The activity of Na+
/K+
-ATPase was studied in different fractions (Table 1). Our results
revealed a high enzyme activity in all fractions (16.11 ± 1.87, 15.89 ± 1.72, 28.27 ± 2.53
U·mg-1
protein for cell-free extracts, soluble and sedimentary, respectively), which were
obtained from D. piger Vib-7 cells. The activity was significantly lower (P < 0.01) in the
fractions obtained from Desulfomicrobium sp. Rod-9.
The activity of the enzyme in cell-free extracts under the effect of temperatures and pH
in the medium incubation was determined (Fig. 1). The Na+
/K+
-ATPase activity exhibited
bell-shaped curves as a function of temperature and pH. A peak of the activity for both
bacterial strains was determined at +35 ºC. An increase or decrease in temperature of the
incubation led to a decrease of the activity of the enzyme. Similar peaks of the activity were
also determined in extracts of D. piger Vib-7 at pH 7.0 and Desulfomicrobium sp. Rod-9
at pH 6.5.
Kinetic analysis of the release of inorganic phosphorus in the ATP hydrolysis
To study the characteristics and mechanism of Na+
/K+
-ATPase, the initial (instantaneous)
reaction rate (V0
), maximum (plateau) amount formation of product reaction (Pmax
) and
reaction time (τ) was defined. To calculate the kinetic indicators of ATP hydrolysis which is
5
Table 1. Na+
/K+
-ATPase activity in different fractions.
Fractions	Na+
/K+
-ATPase activity (U·mg-1
protein)	
	 Desulfovibrio piger Vib-7	 Desulfomicrobium sp. Rod-9
Cell-free extract	 16.11 ± 1.87	 7.31 ± 0.98
Individual fractions:		
soluble	 15.89 ± 1.72	 6.92 ± 0.86
sedimentary	 28.27 ± 2.53	 15.25 ± 1.62
Fig. 1. Effect of temperature (A) and pH (B) on the Na+
/K+
-ATPase activity in the cell-free extracts
catalyzed by Na+
/K+
-ATPase, the dynamics of the product accumulation of ATP hydrolysis
reaction was studied. For that, bacterial suspensions were incubated in a standard incubation
medium for different periods of time (1–20 min). Our experimental data imply that the kinetic
curves of ATP hydrolysis in both bacterial strains have tendency to saturation (Fig. 2).
The kinetic of ATP hydrolysis catalyzed in the bacterial cells was consistent with the
zero-order reaction in the range 0–5 min (the graph of dependence of P on the incubation
time is almost linear in this time interval). Therefore, the duration of incubation of bacterial
cell extracts and accordingly the ATP hydrolysis was 5 min in subsequent experiments.
The amount of Рі
released by the ATPase from D. piger Vib-7 was higher compared to
Desulfomicrobium sp. Rod-9 in the entire range of time factor. The main kinetic properties
of the ATP hydrolysis reaction were calculated by the linearization of the data in the
{P/t; P} coordinates (Fig. 2B).
Kinetic indicators of ATP hydrolysis in the cell-free extracts were significantly
different (P > 0.05). Values of initial reaction rate for the enzyme reaction by the
maximum amount of the reaction product (Pmax) were calculated. The V0
(15.95
± 1.58 µmol Pi
/min·mg-1
protein) for D. piger Vib-7 was 1.6 greater compared to
Desulfomicrobium sp. Rod-9 (10.69 ± 0.93 µmol Pi
/min·mg-1
protein). Pmax was 62.02
± 4.21 and 38.01 ± 2.81 µmol Pi
·mg-1
protein for D. piger Vib-7 and Desulfomicrobium
sp. Rod-9, respectively. Based on these data, we assume that the transport of Na+
and
K+
ions through the membrane is slower and less active in Desulfomicrobium sp.
Rod-9 cells compared to D. piger Vib-7. However, they are characterized by almost the
same reaction time, which was 3.89 ± 0.34 and 3.56 ± 2.28 min for D. piger Vib-7 and
Desulfomicrobium sp. Rod-9, respectively.
Initial rate of ATP hydrolysis depending on protein concentration
in incubation mixture
The dependence of the Na+
/K+
-ATPase activity on the protein concentration was
studied in a range of concentrations from 15 to 150 mg protein/ml. A gradual increase in
the concentration of protein in the bacterial cell-free extracts of the incubation medium
increased the initial (instantaneous) rate of enzyme reaction; the V0
reached its a maximum
value at 115 µg/ml for extract of D. piger Vib-7 and 75 µg/ml for Desulfomicrobium sp.
Rod-9 (Fig. 3).
6
Fig. 2. Dynamics of inorganic phosphorus (Pi
) release in the process of ATP hydrolysis (A) and linearization
curves of inorganic phosphorus (Рі
) accumulation in {P/t; P} coordinates (B) (M ± m, n = 5, R2
> 0.95; P < 0.02).
Similarly, the dependency of Рі
accumulation depending on the protein content of
bacterial cells in the incubation medium had the same character for both strains. However,
the data imply that the initial reaction rate was significantly different (P > 0.05).
Kinetic analysis of ATP hydrolytic activity depending on ATP concentration
According to the results obtained for the extracts by ATP at concentrations ranging from
0.25 to 2.5 mM (at a constant concentration of 5 mM of Mg2+
), there was observed a
monotonic increase in enzymatic activity of Na+
/K+
-ATPase to maximum values under
substrate concentrations over 1.75 mM and after that activity was maintained on an
unchanged (plateau) level (Fig. 4).
7
Fig. 4. ATP effect on the Na+
/K+
-ATPase activity (A) and the linearization of the concentration curves (B), which
are shown in Fig. 4A, in the Lineweaver-Burk plot, where V is the rate of enzyme reaction (M ± m, n = 5; R2
>
0.95; F < 0.005).
Fig. 3. Initial rate of ATP hydrolysis depending on protein concentration (M ± m, n = 5)
The enzyme activity in the D. piger Vib-7 was higher compared to the Desulfomicrobium
sp. Rod-9 within the range of ATP concentrations. To elucidate the possible mechanism of
changes of the enzymatic ATPase activity, the kinetic indicators of ATP hydrolysis were
defined. The maximum rate of ATP hydrolysis was 36.10 ± 2.87 and 16.64 ± 1.73 µmol Pi
/
min·mg-1
protein for D. piger Vib-7 and Desulfomicrobium sp., respectively. The value for
D. piger Vib-7 was × 2 greater than for Desulfomicrobium sp. (P < 0.001). However, the
Michaelis affinity constant (Km
) of the ATPase to ATP in the extract from D. piger Vib-7
(2.24 ± 0.21 mM) was not significantly different from Desulfomicrobium sp. Rod-9 (2.06
± 0.18 mM). Thus, according to the obtained kinetic indicators of the ATP hydrolysis for
both bacterial strains, we concluded that the activity of Na+
/K+
-ATPase, V0
and Vmax
was
significantly higher (P < 0.001) in D. piger Vib-7 cells than Desulfomicrobium sp. Rod-9.
Kinetic analysis of ATP hydrolytic activity depending on Mg2+
ion
concentration
Experiments focusing on the effect of Mg2+
ions on the ATPase activity were performed
in a range of MgCl2
concentrations from 0 to 7 mM (at constant ATP concentration of 1.5
mM) (Fig. 5).
Enzyme activity depended on the concentration of Mg2+
ions in the incubation medium,
and the activity curves had a typical bell-shape. Results presented in Fig. 5A indicate that
the maximum value of activity was observed at 4.5 mM MgCl2
for both bacterial strains.
As shown in Fig. 5B, the curves of dose-dependent of Mg2+
ions effect on the ATPase
activity in {1/V, 1/[Mg2+
]} coordinates differed by the tangent slope. The Vmax
(20.75 ± 2.3
and 10.33 ± 1.22 µmol Pi
/min·mg-1
protein for D. piger Vib-7 and Desulfomicrobium sp.
Rod-9, respectively) were significantly different from each other (P < 0.001). The apparent
activation constant of Mg2+
ions was 2.65 ± 0.21 mM for D. piger Vib-7 and 3.04 ± 0.29
mM for Desulfomicrobium sp. Rod-9.
Kinetic analysis of ATP hydrolytic activity depending on Na+
and K+
ion
concentration
To study the effect of different concentrations of Na+
and K+
ions on the specific enzymatic
activity of the Na+
/K+
-ATPase, NaCl was changed by isotonic KCl in the incubation medium
8
Fig. 5. Mg2+
ions effect on the Na+
/K+
-ATPase activity (A) and linearization of concentration curves (B), which
are shown in Fig. 5A, in the Lineweaver-Burk plot, where V is the rate of the enzyme reaction (M ± m, n = 5;
R2
> 0.9; P < 0.001).
(the isotonic conditions of Na+
+ K+
ions equal 150 mM). ATPase activity depended on
concentration of Na+
and K+
ions and had a typical dome shape (Fig. 6).
The optimal ion concentration ratio for the functioning of the enzyme is 125 Na+
: 30
K+
in incubation medium for both strains. In the absence of one of the ion species in the
incubation medium, the Na+
/K+
-ATPase activity was not observed. The maximum rate of
ATP hydrolysis (12.24 ± 1.29 and 4.66 ± 0.38 µmol Pi
/min·mg-1
protein for D. piger Vib-
7 and Desulfomicrobium sp. Rod-9, respectively) was determined by Na+
ions. The Vmax
in both strains was significantly different (P < 0.001). However, the apparent constants
of activation of Na+
and K+
ions were close (15.12 ± 1.4 and 12.39 ± 1.25 mM) in both
strains.
Thus, based on calculation of the data received from the kinetic indicators, we concluded
that the enzyme activity is lower in the Desulfomicrobium sp. Rod-9 than D. piger Vib-
7 strain. Perhaps, it is through reduced indicators of the enzyme rate. It can be assumed
that the decrease in values of Vmax
in the Desulfomicrobium sp. Rod-9 can be a speciesspecific
trait, which could be contributed to a lower Na+
/K+
electrochemical gradient of the
cytoplasmic membrane of the Desulfomicrobium sp. Rod-9 and a decrease in the number
of the transported units (a decrease of their expression in the membrane) or a decrease in
the enzyme rate. Perhaps, the decreased value of the apparent activation constants KNa
+
in
the Desulfomicrobium sp. Rod- 9 indicated the affinity increase of the Na+
/K+
-ATPase to
sodium ions.
Analysis of the inhibition effect of ouabain on the ATP hydrolytic activity
As shown in Fig. 7, inhibition by ouabain is the same for both cell-free extracts. To
clarify the indicators of the inhibition of Na+
/K+
-ATPase by ouabain, the linearization of the
concentration curves in Hill’s coordinates was implemented. Values of apparent inhibition
constants (I0.5
) and Hill coefficient (nH
) did not differ significantly. The I0.5
was 22.9 ± 4.6
µM and 15.8 ± 2.6 µM as well as nH
was 1.1 ± 0.12 and 0.99 ± 0.08, for D. piger Vib-7
and Desulfomicrobium sp. Rod-9, respectively. However, it should be noted that inhibition
coefficient does not match to the apparent inhibition constant, which is determined when
achieving a stationary level of inhibition of the enzyme.
9
Fig. 6. Effect of Na+
and K+
ions (isotonic conditions of Na+
+ K+
is 150 mM) on the Na+
/K+
-ATPase activity (A)
and linearization of concentration curves (B), which are shown in Fig. 6A, in the Lineweaver-Burk plot, where V
is the rate of the enzyme reaction (M ± m, n = 5; R2
> 0.9; P < 0.001).
Discussion
Except for ATP, magnesium ions are necessary for the functioning of Na+
/K+
-ATPase,
where ions act as cofactors. The Mg2+
ions form Mg-АТР chelate complex, which is a
substrate of enzymatic reaction. The Mg2+
ions interact with the phosphate group of ATP
and polarize them, and thus facilitate the nucleophilic attack on the terminal γ-phosphate.
The Mg2+
ions can also bind to the regulatory center of Na+
/K+
-ATPase (Yuan et al. 2005;
Tian et al. 2006).
Na+
/K+
-activated Mg2+
-dependent ATPase combines transport, hydrolytic and
receptor functions and specifically interacts with exogenous inhibitors in particular
with glycosides or their endogenous counterparts (Lodish et al. 2000; Yuan et al.
2005; Tian et al. 2006). Ouabain is a steroid compound, which is a highly selective
inhibitor of Na+
/K+
-ATPase. Ouabain enzyme binds to the outside of the cytoplasmic
membrane. It is supposed that ouabain blocks an enzyme in the conformation of the
P-E2
, which, in turn, prevents the further course of the catalytic cycle of the enzyme.
In experimental conditions (insufficient time for binding ouabain in unsteady mode,
antagonism of K+
ions at its high concentration in samples) the coefficient inhibition
does not match the apparent inhibition constant, determined in achieving the stationary
level of inhibition the enzyme. Inhibition indicators characterize the highly sensitive
phenotype of the Na+
/K+
-ATPase to ouabain under these experimental conditions of
inhibition. Ouabain is defined by the similarity of patterns of the receptor sites, which
is typical for all isoenzymes Na+
/K+
-ATPase (Yuan et al. 2005; Tian et al. 2006).
Perhaps, Na+
/K+
-ATPase activity of D. piger Vib-7 and Desulfomicrobium sp. Rod-9
can have the same receptors, which are characterized by sensitivity to inhibition.
The reduction of sulfate ions to hydrogen sulfide occurs as a result of the formation
of many intermediate compounds. The sulfate reduction enzymes are located in the
cytoplasm and peripheral plasma. The initial stages of the sulfate reduction include
uptake of sulfate ions in the bacterial cells. The sulfate ions can be transported into
the cells simultaneously with protons and some sulfate-reducing bacteria can absorb
sulfate from the flow of sodium ions (Barton and Hamilton 2007). It is known that
the center of the ATP hydrolysis is located on the cytoplasmic surface of the membrane
(Yuan et al. 2005; Tian et al. 2006).
Guarraia and Peck (1971) determined the ATPase activity in both the soluble and
10
Fig. 7. Ouabain inhibited effect on the Na+
/K+
-ATPase activity (A) and linearization of concentration curves (B)
in Hill’s coordinates (M ± m, n = 5; R2
> 0.9; P < 0.001).
particulate fractions of the anaerobic sulfate-reducing bacterium, Desulfovibrio gigas. As
the soluble ATPase was labile to storage, only the particulate enzyme was studied in detail.
ATPase was stimulated by both Ca2+
and Mg2+
, but the magnitude of stimulation depended
on pH. In the presence of Ca2+
the optimum pH was 6.5, whereas, in the presence of Mg2+
the pH optimum was 8.0. However, under optimum conditions, the activity was the same
with both Mg2+
and Ca2+
.
Taking into consideration all of the obtained results, according to change of the hydrolase
activity, the transport of Na+
and K+
ions by Na+
/K+
-ATPase in Desulfomicrobium sp.
Rod-9 is slower and less intense compared to the D. piger Vib-7 strain. However, it is
characterized by almost the same capacity. The Na+
/K+
-ATPase activity, V0
and Vmax
were
significantly higher in the D. piger Vib- 7 cells than in Desulfomicrobium sp. Rod-9.
The affinity of Na+
/K+
-ATPase to ATP is also the lowest in the Desulfomicrobium sp.
Rod-9 compared to D. piger Vib-7. At the same time, the Mg2+
-binding site of Na+
/K+
ATPase
of the studied bacterial strains is native. Increasing adenosine triphosphate in
the concentration range from 0.25 to 2.5 mM caused a monotonic increase in enzymatic
activity to maximum values and after that activity was maintained on an unchanged
(plateau) level. The increase in the affinity of enzyme activity to Na+
ions in the cell-free
bacterial extracts and maintaining the receptor properties of Na+
/K+
-ATPase to ouabain
was observed.
The above described experimental data can be used for further clarification of the
mechanisms of ion exchange membrane of D. piger Vib-7 and Desulfomicrobium sp. Rod-
9 as well as to study the transport mechanisms. According to these data, we can assume
that the transports of sulfate ions into D. piger Vib-7 cells are much more intensive, and
can lead, in turn, to an increase of the intensity of sulfate reduction and the accumulation
of hydrogen sulfide, which is toxic for epithelial cells of the intestine and can cause bowel
diseases, in particular ulcerative colitis both in humans and animals.
Acknowledgements
This study was supported by University of Veterinary and Pharmaceutical Sciences Brno (project OPVK
“Pharmaco-toxicological evaluation of newly synthesized (isolated) compounds as an integration tool for preclinical
disciplines at UVPS Brno” CZ.1.07/2.3.00/30.0053).
References
Bailey NTJ 1995: Statistical Methods in Biology. Cambridge University Press, Cambridge, 252 p.
Barton LL, Hamilton WA 2007: Sulphate-reducing Bacteria. Environmental and Engineered Systems. Cambridge
University Press, Cambridge, 553 p.
Cummings JH, Macfarlane GT, Macfarlane S 2003: Intestinal bacteria and ulcerative colitis. Curr Issues Intest
Microbiol 4: 9-20
Flynn ERM, Bradley KN, Muir TC, McCarron JG 2001: Functionally separate intracellular Ca2+
stores in smooth
muscle. J Biol Chem 276: 36411-36418
Gibson GR, Cummings JH, Macfarlane GT 1991: Growth and activities of sulphate-reducing bacteria in gut
contents of health subjects and patients with ulcerative colitis. FEMS Microbiol Ecol 86: 103-112
Guarraia LJ, Peck HD 1971: Dinitrophenol-stimulated adenosine triphosphatase activity in extracts of
Desulfovibrio gigas. J Bacteriol 106: 890-895
Keleti T 1988: Basic Enzyme Kinetics. Akademiai Kiado, Budapest, 422 p.
Kushkevych IV 2013: Identification of sulfate-reducing bacteria strains of human large intestine. Studia Biologica
7: 115-132
Kushkevych IV, Bartos M, Bartosova L 2014: Sequence analysis of the 16S rRNA gene of sulfate-reducing
bacteria isolated from human intestine. Int J Curr Microbiol Appl Sci 3: 239-248
Lodish H, Berk A, Zipursky SL, Matsudaira P, Baltimore D, Darnell J 2000: Molecular Cell Biology. Freeman
and Company, New York, USA, 1184 p.
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ 1951: Protein determination with the Folin phenol reagent.
J Biol Chem 193: 265-275
Pitcher MC, Cummings JH 1996: Hydrogen sulphide: a bacterial toxin in ulcerative colitis? Gut 39: 1-4
Rathbun W, Betlach V 1969: Estimation of enzymically produced orthophosphate in the presence of cysteine and
adenosine triphosphate. Anal Biochem 28: 436-447
11
Tian J, Cai T, Yuan Z 2006: Binding of Src to Na+
/K+
-ATPase forms a functional signaling complex. Mol Biol
Cell 17: 317-326
Yuan Z, Cai T, Tian J, Ivanov AV, Giovannucci DR, Xie Z 2005: Na/K-ATPase tethers phospholipase C and IP3
receptor into a calcium-regulatory complex. Mol Biol Cell 16: 4034-4045
12
ATTACHMENT 2
Pages 51 – 58
_____________________________________________________________________________
Activity of selected salicylamides against intestinal sulfate-reducing bacteria
Kushkevych I., Kollar P., Suchy P., Parak T., Pauk K., Imramovsky A.
Neuro Enocrinology Letters, 2015, 36(1):106-113, ISSN: 0172-780X
_____________________________________________________________________________
To cite this article: Neuroendocrinol Lett 2015;36(Suppl. 1):106–113
ORIGINALARTICLE Neuroendocrinology Letters Volume 36 Suppl. 1 2015
ISSN: 0172-780X; ISSN-L: 0172-780X; Electronic/Online ISSN: 2354-4716
Web of Knowledge / Web of Science: Neuroendocrinol Lett
Pub Med / Medline: Neuro Endocrinol Lett
Activity of selected salicylamides against
intestinal sulfate-reducing bacteria
Ivan Kushkevych1, Peter Kollar1, Pavel Suchy1, Tomas Parak1,
Karel Pauk2, Ales Imramovsky2
1 Department of Human Pharmacology and Toxicology, Faculty of Pharmacy, University of Veterinary
and Pharmaceutical Sciences Brno, Brno, Czech Republic
2 Institute of Organic Chemistry and Technology, Faculty of Chemical Technology, University of
Pardubice, Pardubice, Czech Republic
Correspondence to: Dr. Ivan Kushkevych, MSc., PhD.
Department of Human Pharmacology and Toxicology
Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences Brno
Palackeho tr. 1/3, 61242 Brno, Czech Republic.
tel: +420-732215046; fax: +420-541240605; e-mail: ivan.kushkevych@gmail.com
Submitted: 2015-07-18 Accepted: 2015-09-09 Published online: 2015-10-15
Key words: salicylamides; Desulfovibrio piger; bowel diseases; ulcerative colitis; sulfatereducing
bacteria
NeuroendocrinolLett2015;36(Suppl.1):106–113 PMID:26757109 NEL360915A12 ©2015NeuroendocrinologyLetters•www.nel.edu
Abstract OBJECTIVES: The aim of our work was to evaluate effect of selected salicylamides
on cell viability of sulfate-reducing bacterium Desulfovibrio piger Vib-7 isolated
from the human large intestine, as well as to assess antimicrobial activity and
biological properties of these compounds.
METHODS: Microbiological, biochemical, biophysical methods, and statistical
processing of the results were used.
RESULTS: An antimicrobial activity and biological properties of salicylamides
against intestinal sulfate-reducing bacteria was studied. Primary in vitro screening
of the synthesized selected salicylamides was performed against D. piger Vib-7.
Adding 0.37–1.10 μmol.L–1 (N-(4-bromophenyl)-5-chloro-2-hydroxybenzamide,
5-chloro-2-hydroxy-N-[4-(trifluoromethyl)phenyl]benzamide, 5-chloro-N-(3,4dichlorophenyl)-2-hydroxybenzamide,
5-chloro-2-hydroxy-N-(4-nitrophenyl)
benzamide and 4-chloro-N-(3,4-dichlorophenyl)-2-hydroxybenzamide) caused
decrease in biomass accumulation by 8–53, 64–66, 49–50, 82–90, 43–46% compared
to control, respectively. The studied compounds completely inhibited the
growth of D. piger Vib-7 under the effect of 30 μmol.L–1. Moreover, addition
of the compounds in the culture medium inhibited the process of dissimilation
sulfate dose dependently. Treatment with salicylamides led to the bacterial growth
inhibition which correlated with the level of inhibition of sulfate reduction. The
data on relative survival of D. piger Vib-7 cells and cytotoxicity of salicylamides
are consistent to our research in previous series of the biomass accumulation
experiments.
CONCLUSIONS: A significant cytotoxic activity under the influence of salicylamides
was determined. These results are consistent with a data on bacterial
growth and inhibition process of dissimilation sulfate. The strongest cytotoxic
effect of the derivatives was observed in compounds of 5-chloro-2-hydroxy-N-[4(trifluoromethyl)phenyl]benzamide
and 5-chloro-2-hydroxy-N-(4-nitrophenyl)
benzamide which showed low survival and high toxicity rates.
107Neuroendocrinology Letters Vol. 36 Suppl. 1 2015 • Article available online: http://node.nel.edu
Intestinal sulfate-reducing bacteria
Abbreviations
CFU - colony-forming unit
DMSO - dimethyl sulfoxide
IC50 - inhibitory concentration
KS - Kravtsov-Sorokin medium
MIC - minimal inhibitory concentration
OD - optical density
PBS - phosphate-buffered saline
SRB - sulfate-reducing bacteria
UC - ulcerative colitis
INTRODUCTION
The increased number of sulfate-reducing bacteria
(SRB) and intense process of dissimilatory sulfate reduction
in the gut is thought to be a significant risk factor
of inflammatory bowel diseases in both humans and
animals (Gibson et al. 1991, 1993; Kushkevych 2012a).
These bacteria are often found in persons with rheumatic
diseases, and with ankylosing spondylitis, etc.
(Barton & Hamilton 2010). There is also an assumption
that SRB can be responsible for some forms of cancer of
the rectum through the formation of hydrogen sulfide,
which affects the metabolism of intestinal cells and give
rise to various inflammatory bowel diseases. The species
of Desulfovibrio genus can cause bloody diarrhea,
weight loss, anorexia, epithelial hyperplasia, abscesses
and inflammatory infiltrates in animals and humans
(Loubinoux et al. 2000, 2002). The increased number of
SRB was found in feces from people with ulcerative colitis
in comparison with healthy individuals (Cummings
et al. 2003). The injection of these bacteria in hamster
intestine caused infection clinically similar to human
colitis (Pitcher et al. 1996; Cummings et al. 2003).
Ulcerative colitis (UC) is a chronic inflammatory
disease of the colon that affects up to 12 per 100,000
people in Western countries, mostly between 15 and
30 years of age (Rowan et al. 2009). The treatment of
mild to moderate UC includes, in a first approach,
sulfasalazine and mainly 5-aminosalicylate containing
drugs which type and dosage depend on the location
and severity of the disease. Other options of treatment
include corticosteroids and immunosuppressants (for
moderate to severe UC, with a high mortality disease)
or probiotics (for improving the microbial balance)
(Cummings et al. 2003; Kushkevych 2012b). Despite
the bacterial nature of the disease, antibiotics failed in
the treatment of UC so far. However, new antibacterial
compounds with high specific effect against SRB could
yield better efficiency in the treatment of this disease.
At present, the effects of salicylamides on the intestinal
sulfate-reducing bacteria D. piger have never been wellcharacterized
and have not been studied yet.
The aim of our work was to evaluate effect of selected
salicylamides on cell viability of sulfate-reducing bacterium
D. piger Vib-7 isolated from the human large
intestine, as well as to assess antimicrobial activity and
biological properties of these compounds.
MATERIALS AND METHODS
Tested compounds
The studied salicylamides 1–5 were synthesized by means
of microwave-assisted synthesis described in literature.
The compounds were isolated and fully characterized
(melting point, elemental analysis, infrared as well as 1H
and 13C NMR spectroscopy) (Pauk et al. 2013). The compounds
were kept in microtubes dissolved in dimethyl
sulfoxide (DMSO) solution. The quantity of DMSO necessary
to dissolve each compound was calculated previously
so that the concentration of the component would
be 30 mmol.L–1. Afterwards it was diluted 4 times in a
proportion 1:3 so that there would be 5 different concentrations
of chemical compound: 0.37; 1.1; 3.3; 10 and
30mmol.L–1. The maximum concentration of DMSO in
the assays never exceeded 0.1%.
Bacterial culture
The sulfate-reducing bacteria D. piger Vib-7 were
isolated from the healthy human large intestine as
described previously (Kushkevych 2013; Kushkevych
et al. 2014). The strain has been kept in the collection
of microorganisms at the Department of Molecular
Biology and Pharmaceutical Biotechnology, Faculty of
Pharmacy at the University of Veterinary and Pharmaceutical
Sciences Brno (Czech Republic).
Bacterial cultivation
The bacteria were grown for 72 hours at 37°C under
anaerobic conditions in nutrition modified KravtsovSorokin’s
(KS) liquid medium (Kushkevych & Moroz
2012). Before bacterial passage in the medium,
0.05ml.L–1 of sterile solution of Na2S×9H2O (1%)
was added. The sterile 10 mol.L–1 solution of NaOH
(0.9ml.L–1) in the medium was used to provide the
final pH7.2. The medium was heated in boiling water
for 30 min in order to obtain an oxygen-free medium,
and cooled to 30°C. The tubes were brim-filled with
medium and closed to provide anaerobic conditions.
Assay of bacterial cell concentration
About 1 mL of liquid medium without Mohr’s salt
(blank) was transferred into a plastic cuvette and taken
to a biophotometer (Eppendorf®) for taring. Subsequently,
1 mL of bacterial suspension was transferred
into another cuvette and taken again to the biophotometer
for measuring at OD340. Before SRB were used for
the experiments, optical density (OD340) was always
measured to assure there was approximately the same
amount of bacteria in each experiment. If it was necessary
proper dilutions were made in order to obtain the
desired concentration of the bacteria.
The best concentration of D. piger Vib-7 was assessed
to be 5×105 CFU.mL–1. Based on our previous work a
correlation between OD340 and the amount of cells in
the solutions measured in the biophotometer was concluded
as:
108 Copyright © 2015 Neuroendocrinology Letters ISSN 0172–780X • www.nel.edu
Ivan Kushkevych, Peter Kollar, Pavel Suchy, Tomas Parak, Karel Pauk, Ales Imramovsky
y = 1.0×109 χ – 6.0×106,
where y means the bacterial concentration and χ
means the OD340 measured (Figure 1).
The biomass of the D. piger Vib-7 cells was calculated
by the formula:
K
nE
C
×
= ,
where C – bacterial biomass (mg.mL–1); E – extinction;
n – dilution factor, times; К – coefficient of conversion,
obtained gravimetrically.
Assay of sulfate, lactate sulfide and
acetate in cultivation medium
The sulfate ion concentration in the medium was determined
by the turbidimetric method after it had been
precipitated by barium chloride. To stabilize the suspension,
glycerol was used (Kolmert et al. 2000). Lactate
concentration was measured through the dehydrogenation
reaction using Lactate Assay Kit (Sigma-Aldrich,
Catalog Number MAK064). Sulfide concentration in
the culture medium was assayed by the spectrophotometric
method as was described (Cline 1969). Accumulation
of acetate ions in process of bacterial growth in
the medium was determined using Acetate Assay Kit
(Colorimetric, Catalog Number KA3764).
Tratment of bacterial culture
Bacterial culture of stationary phase of growth was
centrifuged for 3 min at a rotation speed of 3,500 rpm.
Supernatant was removed and replaced by new and
fresh KS liquid medium where the bacterial precipitate
was diluted. The bacterial suspension was mixed and
OD340 was measured. Numbers and viabilities of the
bacterial cells were determined by counting with a haemocytometer
after staining with erythrosine B [0.1%
erythrosine B (w·v−1) in phosphate-buffered saline
(PBS), pH7.2–7.4]. Unstained cells were considered to
be viable.
The bacterial suspension (initial concentration
0.5mg.mL–1) was poured to the top in microtubes
(280 μL) which contained samples + solvent control
(DMSO) + sample control + blanks; it was prepared 3
times each sample as well as the controls so that the
average of the results did not had a great discrepancy
from the results independently (except in the 3 blanks).
The sample controls contained only bacterial suspension
and medium (free of tested compounds); and for
the 3 blanks only medium.
Calculations were made to assess how much bacterial
solution should be in each, based on the OD. Determination
of biomass and concentrations of sulfate,
lactate, acetate and sulfide in the culture medium under
the treatment of 0.37; 1.1; 3.3; 10 and 30 μmol.L–1 compounds
after 12, 24, 36, 48, 60, 72 hours was carried
out. During experiments, bacteria were grown at 37°C
under anaerobic conditions.
Analysis of viability of D. piger Vib-7
and cytotoxicity of the compounds
The bacterial suspension (5×104 cells·well−1 in 200 μL
culture medium) was filled to the top in 96-well plates
in triplicate in the KS liquid medium (without Mohr’s
salt), treated with 0.37; 1.1; 3.3; 10 and 30 μmol.L–1
compounds and incubated at 37 °C. Relative survival of
D. piger Vib-7 cells and cytotoxicity of the compounds
was determined by 36th hour of cultivation using a
WST-1 assay kit (Roche Diagnostics, Mannheim, Germany)
according to the manufacturer’s instructions
(Zídek et al. 2014). The relative survival rate was calculated
by the following equation:
ABlankAControl
ABlank )(ASample
−
− ,
Fig. 1. The correlation between optical density and the amount of bacterial cells.
109Neuroendocrinology Letters Vol. 36 Suppl. 1 2015 • Article available online: http://node.nel.edu
Intestinal sulfate-reducing bacteria
and multiplied by 100 for the result in percentage. The
relative cytotoxicity rate was determined as described
previously (Tengler et al. 2013). All data were evaluated
using GraphPad Prism 5.00 software (GraphPad Software,
San Diego, CA, USA, http://www.graphpad.com).
Statistical analysis
The statistical calculations of the results were carried
out using the software MS Office and Origin computer
programs. The research results were treated by methods
of variation statistics using Student t-test. Statistical
significance was tested using the one-way analysis
of variance with Dunnett’s test and Tukey post-test
for comparisons between the means, and differences
between two conditions were retained for p≤0.05. Statistical
significance was determined at levels of p<0.05,
p<0.01, and p<0.001 (Bailey 1995).
RESULTS
The structure of the discussed salicylamides, their
molecular weight and position of the lateral group are
shown in Table 1.
The effect of the selected salicylamides in increasing
concentrations on the growth of sulfate-reducing bacteria
D. piger Vib-7 was studied. As shown in Figure 2,
the highest level of bacterial biomass accumulation
was found in the control KS medium (compounds-free
group). Maximum biomass (3.88±0.36mg.mL–1) of the
bacteria D. piger Vib-7 on the 60th hour of cultivation
was accumulated. Exponential growth phase started
from the beginning of bacterial cells seeding in the
medium and continued until 48th hour, subsequently,
the stationary growth phase started. Treatment with
salicylamides in the concentration range between 0.37
to 30 μmol.L–1 led to decrease in biomass accumulation
(Figure 2).
Under the effect of 0.37–1.1 μmol.L–1 1–5 compounds,
the growth on the 48th hour of cultivation
was inhibited by 8–53, 64–66, 49–50, 82–90, 43–46%
compared to control, respectively. The highest level of
growth inhibition was observed at higher concentrations
(3.3–10 μmol.L–1) of the compounds. Treatment
with 30 μmol.L–1 of studied compounds completely
inhibited the growth of D. piger Vib-7. Most likely, this
concentration of salicylamides was the most toxic for
bacterial cells, as a complete lack of the growth and statistically
significant reduction in the initial biomass of
the cells (initial seeding) was observed. Based on these
data, we can assume that these compounds in concentration
of 30 μmol.L–1 can cause the lysis of bacterial
cells.
Sulfate-reducing bacteria consume the sulfate ions
as electron acceptor in the process of dissimilatory sulfate
reduction (Kushkevych 2012a, 2012b). The final
metabolite of such a process is hydrogen sulfide, which
accumulates in the medium. These bacteria also need
organic compounds as electron donor (Kushkevych
& Moroz 2012). In our previous studies, it was shown
that the bacteria D. piger Vib-7 consumed sulfate and
accumulated hydrogen sulfide in concentration of
2.31±0.21mmol.L–1. They use lactate as electron donor
which oxides to acetate (Kushkevych 2013).
The effect of different concentration of salicylamides
on the process of dissimilatory sulfate reduction in D.
piger Vib-7 cells on the 36th of cultivation was studied.
As shown in Figure 3, the addition of the compounds in
the culture medium inhibits the process of dissimilation
sulfate directly proportional to the increase in concentrations
(0.37–30 μmol.L–1).
Under these conditions, the utilization of sulfate and
lactate was inhibited, hence the level of accumulation
of hydrogen sulfide and acetate was reduced. These
data are consistent with our research in previous series
of the experiments. The percentage inhibition of sulfate
reduction process correlates with the percentage
of bacterial growth inhibition under the salicylamides
treatment.
Tab. 1. Structure of the discussed salicylamides (Pauk etal.2013); and minimal inhibitory concentration (MIC) and inhibitory concentration
(IC50) of compounds 1–5 and ciprofloxacin standard against intestinal sulfate-reducing bacteria D. piger Vib-7.
R2
R1
OH
O
N
H
1
2
3
4
5
Comp. R1 R2 MW Name
MIC
(μmol.L–1)
IC50
(μmol.L–1)
1 5-Cl 4-Br 326.57 N-(4-bromophenyl)-5-chloro-2-hydroxybenzamide <0.37 >1.1
2 5-Cl 4-CF3 315.67 5-chloro-2-hydroxy-N-[4-(trifluoromethyl)phenyl]benzamide <0.16 >0.28
3 5-Cl 3,4-Cl 316.57 5-chloro-N-(3,4-dichlorophenyl)-2-hydroxybenzamide <0.25 >0.37
4 5-Cl 4-NO2 292.68 5-chloro-2-hydroxy-N-(4-nitrophenyl)benzamide <0.27 >0.32
5 4-Cl 3,4-Cl 316.57 4-chloro-N-(3,4-dichlorophenyl)-2-hydroxybenzamide <0.24 >1.1
CPX – – – ciprofloxacin >41 >68
110 Copyright © 2015 Neuroendocrinology Letters ISSN 0172–780X • www.nel.edu
Ivan Kushkevych, Peter Kollar, Pavel Suchy, Tomas Parak, Karel Pauk, Ales Imramovsky
Fig. 2. Growth of D. piger Vib-7 cells under the effect of salicylamides in indicated concentrations.
The relative survival of D. piger Vib-7 cells and
cytotoxicity of salicylamides using WST-1 method was
studied. In this series of experiments, compounds 1–5
exerted cytotoxicity against these bacteria already in
low (0.37–1.1 μmol.L–1) concentrations (Figure 4).
The data on relative survival of D. piger Vib-7 cells
and relative cytotoxicity under the salicylamides effect
were in concert with the previously obtained results of
bacterial growth and inhibition level of dissililatory sulfate
reduction process. Treatment with all tested com-
111Neuroendocrinology Letters Vol. 36 Suppl. 1 2015 • Article available online: http://node.nel.edu
Intestinal sulfate-reducing bacteria
Fig. 3. Sulfate reduction of D. piger Vib-7 cells under the treatment of indicated doses of salicylamides.
Fig. 4. Relative survival of D. piger Vib-7 cells and cytotoxicity of salicylamides.
112 Copyright © 2015 Neuroendocrinology Letters ISSN 0172–780X • www.nel.edu
Ivan Kushkevych, Peter Kollar, Pavel Suchy, Tomas Parak, Karel Pauk, Ales Imramovsky
pounds led to a significant dose-dependent cytotoxicity,
with the strongest effect observed in compounds 4 and 5.
Since some of salicylamides (such as 4, 2, or 3) have a
pronounced antimicrobial effect against D. piger Vib-7
(combination of low survival and high toxicity rates)
they could be considered as promising agents against
the growth of this type of bacteria.
Based on obtained results, the minimal inhibitory
concentration (MIC) and inhibitory concentration
(IC50) of salicylamides against intestinal sulfate-reducing
bacteria were established. As shown in Table 1, the
MIC for all compounds was lesser 0.37 μmol.L–1.
Regarding IC50, for the compounds 1 and 5, it was
over 1.1 μmol.L–1. On the other hand, for the compounds
3 and 4 the IC50 was over 0.37 and 0.32 μmol.L–1
respectively. Concerning IC50, only for the compound 2
was over 0.28 μmol.L–1, while all other compounds had
shown IC50 over 30 μmol.L–1.
DISCUSSION
Sulfate-reducing bacteria Desulfovibrio genus belongs
to the intestinal microbiota of humans and animals
(Kushkevych 2012b). They are anaerobic microorganisms,
dissimilating sulfate as an electron acceptor and
organic compounds as an electron donor and carbon
source in the process of “dissimilatory sulfate reduction”
(also known as “dissimilatory anaerobic sulfate
respiration”) (Kushkevych 2012a). Lactate is the most
common substrate used by the species belonging to
the intestinal sulfate-reducing bacteria. The species of
Desulfovibrio oxidize lactate incompletely to acetate.
Lactate oxidation to acetate occurs together with the
concurrent reduction of sulfate to sulfide (Barton &
Hamilton 2010). The presence of lactate and sulfate in
the human intestine contributes to the intensive bacteria
growth and the accumulation of their final metabolic
products, acetate and hydrogen sulfide, which are
toxic, mutagenic and cancerogenic to epithelial intestinal
cells (Pitcher et al. 1996; Rowan et al. 2009). There
is also an assumption that sulfate-reducing bacteria can
cause some forms of cancer of the rectum through the
formation of hydrogen sulfide.
In the light of our results it can be stated that
almost all of salicylamides in concentrations above
10 μmol.L–1 have an interesting antimicrobial activity
against Desulfovibrio piger. This activity was concentration-dependent,
with the strongest effect in
30μmol.L–1 concentrations. Tested compounds can be
considered as good alternative for the treatment of colitis
or colorectal cancer although it should be taken in
account that these compounds can be aggressive also
to commensally bacteria and even to other parts of the
human body. This should be a concern to be clarified in
the near future with complementary assays.
Based on all obtained results in our study we
can conclude that compounds 1–5 in concentration
0.37–1.1μmol.L–1 inhibited the growth on the 48th
hour of cultivation by 8–53, 64–66, 49–50, 82–90,
43–46% compared to control, respectively. The highest
level of growth inhibition was observed at higher
concentrations (3.3–10 μmol.L–1) of the compounds.
These results are consistent with a data on inhibition
process of dissimilation sulfate. A significant cytotoxic
activity under the influence of salicylamides was determined.
The strongest cytotoxic effect was observed in
compounds 4 and 5. Derivatives 4, 2, and 3 showed low
survival and high toxicity rates, and thus are interesting
for further studies.
Understanding the role of sulfate-reducing bacteria
in colonic conditions would be enhanced by the ability
to inhibit the number of the sulfate-reducing bacteria
and/or reduce the production of sulfide and acetate.
This would help to clarify the factors influencing sulfide
production in the human and animal colon.
ACKNOWLEDGEMENTS
The authors express their gratitude to Dr. Mark Scott
(University of Mary Washington, Fredericksburg,
Virginia, USA) for his reading and editing of the
manuscript. This study was supported by University
of Veterinary and Pharmaceutical Sciences Brno
(project OPVK „Pharmaco-toxicological evaluation
of newly synthesized (isolated) compounds as an integration
tool for pre-clinical disciplines at VFU Brno”
CZ.1.07/2.3.00/30.0053). The authors wish to acknowledge
the institutional support of the Faculty of Chemical
Technology, University of Pardubice funded by the
Ministry of Education, Youth and Sports of the Czech
Republic. The authors thank also Dr. Josef Jampilek
(Department of Chemical Drugs, Faculty of Pharmacy,
University of Veterinary and Pharmaceutical Sciences
Brno, Czech Republic) for discussion related to properties
of salicylamide derivatives.
REFERENCES
1 Bailey NTJ (1995). Statistical Methods in Biology. Cambridge
University Press.
2 Barton LL, Hamilton WA (2010). Sulphate-reducing bacteria.
Environmental and Engineered Systems. Cambridge University
Press.
3 Cline JD (1969). Spectrophotometric determination of hydrogen
sulfide in natural water. Limnol and Ocean. 14: 454–458.
4 Cummings JH, Macfarlane GT, Macfarlane S (2003). Intestinal
bacteria and ulcerative colitis. Curr Issues Intest Microbiol. 4:
9–20.
5 Gibson GR, Cummings JH, Macfarlane GT (1991). Growth and
activities of sulphate-reducing bacteria in gut contents of health
subjects and patients with ulcerative colitis. FEMS Microbiol
Ecol. 86: 103–112.
6 Gibson GR, Macfarlane S, Macfarlane GT (1993). Metabolic interactions
involving sulphate-reducing and methanogenic bacteria
in the human large intestine. FEMS Microbiol Ecol. 12: 117–125.
7 Kolmert A, Wikstrom P, Hallberg KB (2000). A fast and simple
turbidimetric method for the determination of sulfate in sulfatereducing
bacterial cultures. J Microbiol Methods. 41: 179–184.
8 Kushkevych IV (2012a). Sulfate-reducing bacteria of the human
intestine. I. Dissimilatory sulfate reduction. Stud Biol. 6: 149–180.
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9 Kushkevych IV (2012b). Sulfate-reducing bacteria of the human
intestine. II. The role in the diseases development. Stud Biol 6:
221–250.
10 Kushkevych IV (2013). Identification of sulfate-reducing bacteria
strains of human large intestine. Stud Biol. 7: 115–124.
11 Kushkevych IV, Bartos M, Bartosova L (2014). Sequence analysis
of the 16S rRNA gene of sulfate-reducing bacteria isolated from
human intestine. Int J Curr Microbiol Appl Sci. 3: 239–248.
12 Kushkevych IV, Moroz OM (2012). Growth of various strains of
sulfate-reducing bacteria of human large intestine. Stud Biol. 6:
115–124.
13 Loubinoux J, Bronowicji JP, Pereira IA (2002). Sulphate-reducing
bacteria in human feces and their association with inflammatory
diseases. FEMS Microbiol Ecol. 40: 107–112.
14 Loubinoux J, Mory F, Pereira IA, Le Faou AE (2000). Bacteremia
caused by a strain of Desulfovibrio related to the provisionally
named Desulfovibrio fairfieldensis. J Clin Microbiol. 38: 931–934.
15 Loubinoux J., Valente FMA, Pereira IAC (2002). Reclassification of
the only species of the genus Desulfomonas, Desulfomonas pigra,
as Desulfovibrio piger comb. nov. Int J of System and Evol Microbiol.
52: 1305–1308.
16 Pauk K, Zadražilová I, Imramovský I, Vinšová J, Pokorná M,
Masaříková M, (2013). New derivatives of salicylamides: preparation
and antimicrobial activity against various bacterial species.
Bioorg & Med Chem. 21: 6574–6581.
17 Pitcher MC, Cummings JH (1996). Hydrogen sulphide: a bacterial
toxin in ulcerative colitis? Gut. 39: 1–4.
18 Rowan FE, Docherty NG, Coffey JC, O’Connell PR (2009). Sulphate-reducing
bacteria and hydrogen sulphide in the aetiology
of ulcerative colitis. British J Surgery. 96: 151–158.
19 Tengler J, Kapustikova I, Pesko M, Govender R, Keltosova S,
Mokry P, et al. (2013). Synthesis and biological evaluation of
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ATTACHMENT 3
Pages 59 – 64
_____________________________________________________________________________
Activity and kinetic properties of phosphotransacetylase
from intestinal sulfate-reducing bacteria
Kushkevych I. V.
Acta Biochemica Polonica, 2015, 62, 103-108, ISSN: 0001-527X
_____________________________________________________________________________
Regular paper
Activity and kinetic properties of phosphotransacetylase from
intestinal sulfate-reducing bacteria
Ivan V. Kushkevych*
Laboratory of Molecular Biology and Clinical Biochemistry, Institute of Animal Biology of NAAS of Ukraine, Lviv, Ukraine
Phosphotransacetylase activity and the kinetic properties
of the enzyme from intestinal sulfate-reducing bacteria
Desulfovibrio piger and Desulfomicrobium sp. has
never been well-characterized and has not been studied
yet. In this paper, the specific activity of phosphotransacetylase
and the kinetic properties of the enzyme in cellfree
extracts of both D. piger Vib-7 and Desulfomicrobium
sp. Rod-9 intestinal bacterial strains were presented at
the first time. The microbiological, biochemical, biophysical
and statistical methods in this work were used. The
optimal temperature and pH for enzyme reaction was
determined. Analysis of the kinetic properties of the
studied enzyme was carried out. Initial (instantaneous)
reaction velocity (V0), maximum amount of the product
of reaction (Pmax), the reaction time (half saturation period,
τ) and maximum velocity of the phosphotransacetylase
reaction (Vmax) were defined. Michaelis constants
(Km) of the enzyme reaction (3.36 ± 0.35 mM for D. piger
Vib-7, 5.97 ± 0.62 mM for Desulfomicrobium sp. Rod-9)
were calculated. The studies of the phosphotransacetylase
in the process of dissimilatory sulfate reduction and
kinetic properties of this enzyme in intestinal sulfate-reducing
bacteria, their production of acetate in detail can
be perspective for clarification of their etiological role in
the development of the humans and animals bowel diseases.
These studies might help in predicting the development
of diseases of the gastrointestinal tract, by providing
further details on the etiology of bowel diseases
which are very important for the clinical diagnosis of
these disease types.
Key words: sulfate-reducing bacteria, phosphotransacetylase, kinetic
analysis, inflammatory bowel diseases
INTRODUCTION
Dissimilatory sulfate-reducing bacteria reduce inorganic
sulfate or other oxidized sulfur forms to sulfide
(Barton & Hamilton, 2010). This bacteria are heterotrophs
and therefore, require an organic carbon source. In
the case of Desulfovibrio and Desulfomicrobium genera, this
carbon source can be supplied by simple organic molecules
such as lactate, pyruvate, and malate. These are
subsequently oxidized to acetate with the concurrent reduction
of sulfate to sulfide (Rowan et al., 2009; Kushkevych,
2012a).
The process of organic compounds oxidation is a
complex and multistage that provides the bacterial cells
with energy (Kushkevych, 2012b). The lactate is the
most common substrate used by the species belonging
to the sulfate-reducing bacteria (Barton & Hamilton,
2010). This compound is oxidized to acetate via pyruvate
(Sadana, 1954; Kushkevych, 2012a).
In our previous researches, we have demonstrated
that lactate was oxidized incompletely to acetate by the
intestinal sulfate-reducing bacteria D. piger Vib-7 and
Desulfomicrobium sp. Rod-9 (Kushkevych, 2013). Lactate
oxidation to acetate occurs with the intermediate compounds
formation: pyruvate, acetyl-CoA and atsetylfosfat
(Kushkevych, 2012a).
One important step in this degradative pathway involves
the transfer of an acetyl group from acetyl-S-CoA to
orthophosphate to form acetyl-PO4. The acetyltransferase
catalyzing this reaction is phosphotransacetylase (acetyl-S-CoA:
orthophosphate acetyltransferase, EC 2.3.18)
(Kushkevych, 2012a):
In the presence of sulfate, lactate in human intestine
contributes to the intensive bacteria growth and the accumulation
of their final metabolism product, hydrogen
sulfide, which is toxic, mutagenic and cancerogenic to
epithelial intestinal cells (Pitcher & Cummings, 2003;
Rowan et al., 2009; Gibson et al., 1991; Kushkevych,
2012a). The increased number of the sulfate-reducing
bacteria and intensity of dissimilatory sulfate reduction
in the gut can cause inflammatory bowel diseases of humans
and animals (Cummings et al., 2003; Gibson et al.,
1991; Loubinoux et al., 2000; Kushkevych, 2012b).
As far as it is aware, phosphotransacetylase from intestinal
sulfate-reducing bacteria D. piger and Desulfomicrobium
has never been well-characterized. In literature,
there are some data on phosphotransacetylase in various
organisms as well as in the sulfate-reducing bacteria isolated
from environment (Goldman, 1958; Reichenbecher
& Schink 1997; Robinson & Sagers, 1972; Sadana, 1954;
Shimizu, et al., 1969). However, the data on activity and
the kinetic properties of this enzyme from intestinal sulfate-reducing
bacteria Desulfovibrio piger and Desulfomicrobium
sp. has not been reported yet.
*
e-mail: ivan.kushkevych@gmail.com
Abbreviations: EDTA, ethylenediaminetetraacetate; SRB, sulfatereducing
bacteria
Received: 24 June, 2014; revised: 02 December, 2014; accepted: 02
February, 2015; available on-line: 18 March, 2015
Vol. 62, No 1/2015
103–108
http://dx.doi.org/10.18388/abp.2014_845
104											 2015I. V. Kushkevych and others
The aim of this work was to study phosphotransacetylase
activity in cell-free extracts of intestinal sulfate-reducing
bacteria D. piger Vib-7 and Desulfomicrobium sp.
Rod-9 and to carry out the kinetic analysis of enzymatic
reaction.
The aim was accomplished using microbiological, biochemical,
biophysical methods, and statistical processing
of the results; the obtained data were compared with
those from the literature.
MATERIALS AND METHODS
Objects of the study were sulfate-reducing bacteria
Desulfovibrio piger Vib-7 and Desulfomicrobium sp. Rod-9
isolated from the healthy human large intestine and identified
by the sequence analysis of the 16S rRNA gene
(Kushkevych, 2013; Kushkevych et al., 2014).
Bacterial growth and cultivation. Bacteria were
grown in a nutrition-modified Kravtsov-Sorokin’s liquid
medium (Kushkevych & Moroz, 2012). Before seeding
bacteria in the medium, 0.05 ml/l of sterile solution of
Na2S×9H2O (1%) was added. A sterile 10N solution of
NaOH (0.9 ml/l) in the medium was used to provide
the final pH 7.2. The medium was heated in boiling water
for 30 min in order to obtain an oxygen-free medium,
and then cooled to 30°C. The bacteria were grown
for 72 hours at 37°C under anaerobic conditions. The
tubes were brim-filled with medium and closed to provide
anaerobic conditions.
Obtaining cell-free extracts. Cells were harvested at
the beginning of the stationary phase, centrifuged and
suspended in 100 ml of 50 mM Tris(hydroxymethyl)
aminomethane (Tris)-hydrochloride, pH 7.0 (henceforth
referred to as Tris buffer), containing 1 mM ethylenediaminetetraacetate
(EDTA). A suspension of cells (150–
200 mg/ml) was obtained and homogenized using the
ultrasonic disintegrator at 22 kHz for 5 minutes at 0°C
to obtain cell-free extracts. The homogenate was centrifuged
for 20 min at 16 000 g to remove the cell debris.
The pellet was then used as the sedimentary fraction,
and the supernatant obtained was termed the soluble
fraction. The supernatant fluid and a Tris buffer wash
of the pellet were subjected to a second centrifugation at
16 000 g for 40 min (Robinson & Sagers, 1972). The soluble
extract constituted by the supernatant was used as
the source of the enzyme. A pure supernatant, containing
the soluble fraction, was then used as a cell-free extract.
Protein concentration in the cell-free extracts was
determined by the Lowry method (Lowry et al., 1951).
Assays for phosphotransacetylase activity. The
phosphotransacetylase activity was assayed by measuring
acetyl-P arsenolyzed in the presence of CoA as
described previously in paper (Shimizu et al., 1969). A
reaction mixture, containing 6 µmoles of acetyl-P, 15.8
nmoles of CoA (5 Lipmann units), 5 µmoles of cysteine,
20 µmoles of Tris-HC1 (pH 8.0), 50 µmoles of
potassium arsenate (pH 8.0) and the enzyme in a final
volume of 1 ml was incubated at 25°C for 12 min.
The enzyme was diluted 10–250 times with 50 mM
Tris-HC1 (pH 8.0) before incubation, and 50 µl of the
solution were added to the mixture. One unit of phosphotransacetylase
is defined as the amount of the enzyme
which catalyzes the decomposition of µmole of
acetyl-P under the specified conditions. Specific enzyme
activity was expressed as U × mg-1 protein. The specific
activity of the studied enzyme in the cell-free extracts
of both bacterial strains under the effect of different
temperature (20, 25, 30, 35, 40, 45°C) and pH
(5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0) in the
incubation medium was measured.
Kinetic analysis. Kinetic analysis of the enzyme
reaction was performed in a standard incubation medium
(as it was described above) with modified physical
and chemical characteristics of the respective parameters
(the incubation time, substrate concentration,
temperature and pH). The kinetic parameters characterizing
the phosphotransacetylase reaction are the
initial (instantaneous) reaction velocity (V0), maximum
velocity of the reaction (Vmax), maximum amount of
the reaction product (Pmax) and characteristic reaction
time (time half saturation) τ were determined. The
amount of the reaction product was calculated stoichiometrically.
The kinetic parameters characterizing
phosphotransacetylase reactions such as Michaelis
constant (Km) and maximum reaction velocity of substrate
decomposition were determined by Lineweaver-Burk
plot (Keleti, 1988). For analysis of the substrate
kinetic mechanism of phosphotransacetylase,
initial velocities were measured under standard assay
conditions with different substrate concentrations.
The resulting data were also analyzed by global curve
fitting in SigmaPlot (Systat Software, Inc.) to model
the kinetic data for rapid equilibrium rate equations
describing ordered sequential, V=(Vmax [A] [B])/(KA
KB+KB [A]+[A] [B]), and random sequential, V=(Vmax
[A] [B])/(α KA KB+KB [A]+KA [B]+[A] [B]), kinetic
mechanisms, where V is the initial velocity, Vmax is the
maximum velocity, KA and KB are the Km values for
substrates A and B, respectively, and α is the interaction
factor if the binding of one substrate changes the
dissociation constant for the other (Segal, 1975).
Statistical analysis. Kinetic and statistical calculations
of the results were carried out using the software
MS Office and Origin computer programs. The
research results were treated by the methods of variation
statistics using Student’s t-test. The equation of
the straight line that the best approximates the experimental
data was calculated by the method of least
squares. The absolute value of the correlation coefficient
r was from 0.90 to 0.98. The significance of
the calculated parameters of line was tested by the
Fisher’s F-test. The accurate approximation was when
P ≤ 0.05 (Bailey, 1995).
RESULTS AND DISCUSSION
Specific activity of phosphotransacetylase, an important
enzyme in the process of organic compounds
oxidation in sulfate-reducing bacteria, was measured
in different fractions obtained from D. piger Vib-7 and
Desulfomicrobium sp. Rod-9 cells (Table 1). Results of our
study showed that the highest specific activity of the enzyme
was detected in cell-free extracts (1.19 ± 0.122 and
0.37 ± 0.041 U × mg–1 protein for D. piger Vib-7 and Desulfomicrobium
sp. Rod-9, respectively). The slightly lower
values of activity of phosphotransacetylase were determined
in the soluble fraction compared to the cell-free
extracts. Its values designated 0.87 ± 0.091 U × mg–1 protein
for D. piger Vib-7 and 0.32 ± 0.036 U × mg–1 protein
for Desulfomicrobium sp. Rod-9. The enzyme activity in
sedimentary fraction was not observed.
The effect of temperature and pH of the reaction
mixture on phosphotransacetylase activity in the cellfree
extracts of the sulfate-reducing bacteria was studied
(Fig. 1). The maximum specific activity for both bacterial
strains was determined at 30…35ºC. The highest enzyme
Vol. 62 						 105Activity and kinetic properties of phosphotransacetylase
activity of phosphotransacetylase for D. piger Vib-7 and
Desulfomicrobium sp. Rod-9 was measured at pH 7.5…8.5.
The enzyme activity exhibited typical bell-shaped curves
as a function of temperature and pH.
Thus, temperature and pH optimum of this enzyme
was 30…35ºC and pH 7.5…8.5, respectively. An increase
or decrease in temperature and pH led to a decrease
of the activity of studied enzyme in the cell-free
bacterial extracts of the sulfate-reducing bacteria.
To study the characteristics and mechanism of phosphotransacetylase
reaction, the initial (instantaneous) reaction
velocity (V0), maximum velocity of the reaction
(Vmax), maximum amount of reaction product (Pmax) and
reaction time (τ) were defined. Dynamics of reaction
product accumulation was studied for investigation of
the kinetic parameters of phosphotransacetylase (Fig. 2).
Experimental data showed that the kinetic curves of
phosphotransacetylase activity have tendency to saturation
(Fig. 2A). Analysis of the results allows to reach
the conclusion that the kinetics of phosphotransacetylase
activity in the sulfate-reducing bacteria was consistent
to the zero-order reaction in the range of 0–3 min
(the graph of the dependence of product formation on
the incubation time was almost linear in this interval of
time). Therefore, the duration of the incubation of bacterial
cells extracts was 5 min in subsequent experiments.
Amount of product of phosphotransacetylase reaction
in the D. piger Vib-7 was higher (15.43 ± 1.61 µmol × mg–1
protein) compared to the Desulfomicrobium sp. Rod-9
(4.56 ± 0.47 µmol × mg–1 protein) in the entire range of
time factor. The basic kinetic properties of the reaction
in the sulfate-reducing bacteria were calculated by linearization
of the data in the {P/t; P} coordinates (Fig. 2B,
Table 2).
The kinetic parameters of phosphotransacetylase from
both D. piger Vib-7 and Desulfomicrobium sp. Rod-9 were
significantly different. Values of initial (instantaneous)
reaction velocity (V0) for the enzyme was calculated by
the maximal amount of the product reaction (Pmax). As
shown in Table 2, V0 for phosphotransacetylase reaction
was slightly higher (5.68 ± 0.58 µmol × min–1 × mg–1
protein) in D. piger Vib-7 compared to Desulfomicrobium
sp. Rod-9 (2.14 ± 0.23 µmol×min–1×mg–1 protein). In
this case, the values of the reaction time (τ) were more
similar for the studied enzyme in both D. piger Vib-7 and
Desulfomicrobium sp. Rod-9 strains. Based on these data,
there is an assumption that the D. piger Vib-7 can consume
lactate ion much faster in their cells than a Desulfomicrobium
sp. Rod-9. Moreover, this hypothetical assumption
can be also confirmed by obtained data on maximal
velocities of accumulation of the final reaction products,
where Vmax for enzyme reaction in D. piger Vib-7 were
also more intensively compared to Desulfomicrobium sp.
Rod-9 (Table 3).
The kinetic analysis of phosphotransacetylase activity
dependence on concentration of substrate (acetyl-CoA)
was carried out. The increasing of acetyl-CoA concentrations
from 0.5 to 5.0 mM caused a monotonic rise of
the studied enzyme activity and the activity was maintained
on unchanged level (plateau) under substrate concentrations
over 3.0 mM. (Fig. 2C). Clearly, the enzyme
was saturated with substrate and the higher concentrations
(3.0–5.0 mM acetyl-CoA) did not affect its activity,
so the activity was maintained on unchanged (plateau)
level.
Curves of the dependence {1/V; 1/[S]} were distinguished
by the tangent slope and intersect the vertical
axis in one point (Fig. 2D). The basic kinetic parameters
Figure 1. The effect of temperature (A) and pH (B) on the phosphotransacetylase activity in the cell-free extracts of the intestinal
sulfate-reducing bacteria
Table 1. Phosphotransacetylase activity in different fractions obtained from the bacterial cells
Sulfate-reducing bacteria
Specific activity of the enzyme (U × mg-1 protein)
Cell-free extract
Individual fractions
Soluble Sedimentary
Desulfovibrio piger Vib-7 1.19 ± 0.122 0.87 ± 0.091 0
Desulfomicrobium sp. Rod-9 0.37 ± 0.041*** 0.32 ± 0.036** 0
Comment: The assays were carried out at a protein concentration of 41.17 mg/ml (for D. piger Vib-7) and 38.12 mg/ml (for Desulfomicrobium sp.
Rod-9). Enzyme activity was determined after 10 min incubation. Statistical significance of the values M ±m, n = 5; **P < 0.01, ***P < 0.001, compared
to D. piger Vib-7 strain.
106											 2015I. V. Kushkevych and others
of phosphotransacetylase activity in D. piger Vib-7 and
Desulfomicrobium sp. Rod-9 were identified by linearization
of the data in the Lineweaver-Burk plot (Table 3).
Calculation of the kinetic parameters of enzyme
activity indicates that the maximum velocities (Vmax) of
acetyl-CoA in the D. piger Vib-7 and Desulfomicrobium
sp. Rod-9 were significantly different from each other
(P < 0.001). The values of Km were also quite different
for acetyl-CoA (3.36 ± 0.35, 5.97 ± 0.62 mM) in both
D.  piger Vib-7 and Desulfomicrobium sp. Rod-9 strains, re-
spectively.
The described results of the phosphotransacetylase activity
and the kinetic properties of the enzyme in cellfree
extracts of D. piger Vib-7 and Desulfomicrobium sp.
Rod-9 strains are new and have never reported in the
literature before. These obtained studies were differed
significantly from previously described by Sadana (1954).
A soluble enzyme system from Desulfovibrio desufuricans
which catalyses the conversion of two moles of pyruvate
to one mole of acetyl phosphate, one mole of ethyl alcohol,
and two moles of CO2 was described. The system
required inorganic phosfate for pyruvate dissimilation.
Pyrophosphate and arsenate could replace inorganic
phosphate. The reaction was most rapid at pH 6.4. The
optimum phosphate concentration was 12 M. The requirement
of phosphate for the metabolism of pyruvate by
the bacterial extract suggests the formation of acetyl coensyme
A as an intermediate which is converted to acetyl
phosphate and coenzyme A in the presence of inorganic
phosphate by transacetylase. The effect is not due
to hydrolysis of pyrophosphate to inorganic phosphate
since the extracts show no appreciable pyrophosphatase
activity (Sadana, 1954).
The phosphotransacetylase activity was also studied
in crude extracts of Escherichia coli K-12 by Goldman in
1958. A little later, in 1969, Shimizu et al. have obtained
and purified the phosphotransacetylase from crude extracts
of Escherichia coli B with the use of ammonium
Table 2. Kinetic parameters of the phosphotransacetylase from intestinal sulfate-reducing bacteria
Kinetic parameters
Sulfate-reducing bacteria
Desulfovibrio piger Vib-7 Desulfomicrobium sp. Rod-9
V0 (µmol × min–1 × mg–1 protein) 5.68 ± 0.58 2.14 ± 0.23**
Pmax (µmol × mg–1 protein) 15.43 ± 1.61 4.56 ± 0.47***
τ (min) 2.72 ± 0.29 2.12 ± 0.22
Comment: V0 is initial (instantaneous) reaction velocity; Pmax is maximum amount (plateau) of the product of reaction; τ is the reaction time (half
saturation period). Statistical significance of the values M ± m, n = 5; **P < 0.01, ***P < 0.001, compared to the D. piger Vib-7 strain.
Figure 2. Kinetic parameters of phosphotransacetylase activity in D. piger Vib-7 and Desulfomicrobium sp. Rod-9
(A) dynamics of product accumulation (M ± m, n = 5); (B) linearization of curves of product accumulation in {P/t; P} coordinates (n = 5;
R2  > 0.95; F < 0.02); (C) the effect of different concentrations of substrate (Acetyl-coenzyme A) on the enzyme activity (M ± m, n = 5); (D)
linearization of concentration curves, which are shown in Fig. 2C, in the Lineweaver-Burk plot, where V is velocity of the enzyme reaction
and [Acetyl-coenzyme A] is substrate concentration (n = 5; R2 > 0.95; F < 0.005).
Vol. 62 						 107Activity and kinetic properties of phosphotransacetylase
sulfate as a stabilizer. The purified enzyme was homogeneous
in ultracentrifugal analysis and molecular weight
was tentatively estimated from the s value. Km values of
this enzyme were 3×10–3 and 4×10–3 M for acetyl phosphate
and 3.2×10–4 M for CoA (Shimizu et al., 1969).
The values of calculated Km of acetyl-CoA for phosphotransacetylase
reaction in the cell-free extracts of D. piger
Vib-7 and Desulfomicrobium sp. Rod-9 were slightly different
to those previously described by Shimizu et al. (1969)
Robinson and Sagers (1972) have measured the activity
of phosphotransacetylase from Clostridium acidiurici. Authors
showed that the phosphotransacetylase has two properties
not observed for this enzyme in other bacteria: it required
a divalent metal for activity, and it was not subject to uncoupling
by arsenate. The enzyme was obtained in highly
purified form, with a specific activity 500-fold higher than
crude extracts (Robinson & Sagers, 1972).
The described data were also different from results
described by Reichenbecher and Schink (1997) for phosphotransacetylase
activity from the Desulfovibrio inopinatus.
Authors demonstrated that the activity was 44 mU (mg
protein)–1 in extracts of ethanol-grown cells, while it was
below 3 mU (mg protein)–1 after growth with hydroxyhydroquinone
or lactate (Reichenbecher and Schink,1997).
Lawrence and co-authors (2006) have reported structural
and functional studies suggesting a catalytic mechanism
for the phosphotransacetylase from Methanosarcina thermophila.
Two crystal structures of phosphotransacetylase from
the methanogenic archaeon M. thermophila in complex with
the substrate CoA revealed one CoA (CoA1) bound in the
proposed active site cleft and an additional CoA (CoA2)
bound at the periphery of the cleft. Kinetic and calorimetric
analyses of site-specific replacement variants indicated
that there are catalytic roles for Ser309 and Arg310, which
are proximal to the reactive sulfhydryl of CoA1. The reaction
is hypothesized to proceed through base-catalyzed
abstraction of the thiol proton of CoA by the adjacent
and invariant residue Asp316, followed by nucleophilic attack
of the thiolate anion of CoA on the carbonyl carbon
of acetyl phosphate. Authors have proposed the mechanism
of the reaction catalyzed by phosphotransacetylase
from M. thermophila (Lawrence et al., 2006):
Perhaps, the proposed mechanism for phosphotransacetylase
from the methanogenic archaeon M. thermophila
and the description given by Lawrence and co-authors
(2006) may be similar to the studied enzymes from
D.  piger Vib-7 and Desulfomicrobium sp. Rod-9. However,
to confirm this hypothetical assumption, the enzyme
should be purified from cell-free extracts to study its
structure and properties in detail.
CONCLUSIONS
The phosphotransacetylase, an important enzyme in
process of dissimilatory sulfate reduction and lactate oxidation
in sulfate-reducing bacteria, carries out the central
step in oxidative decarboxylation of acetyl-CoA to ace-
tyl-P.
The enzyme activity, V0 and Vmax were significantly
higher in the D. piger Vib-7 cells than Desulfomicrobium
sp. Rod-9. However, Michaelis constants for acetyl-CoA
were quite higher (5.97 ± 0.62 mM) in Desulfomicrobium
sp. Rod-9 strain compared to D. piger Vib-7. The maximum
enzyme activity for both strains was determined
at +30…35ºC and at pH 7.5…8.5. The kinetic parameters
of enzyme reaction are depended on the substrate
concentration.
The studies of the phosphotransacetylase in the process
of dissimilatory sulfate reduction and kinetic properties
of this enzyme in the D. piger Vib-7 and Desulfomicrobium
sp. Rod-9 intestinal strains, their production of
acetate and hydrogen sulfide in detail can be perspective
for clarification of their etiological role in the development
of the humans and animals bowel diseases. Data
on the activity and kinetic properties of this enzyme in
the strains can be useful to predict the velocity of the
accumulation of the final products of metabolism of
these bacteria, hydrogen sulfide and acetate, which are
formed in the process of dissimilatory sulfate reduction.
Assessing rate of formation of these dangerous products
in the gut, we are able to predict their toxicity and occurrence
of bowel diseases.
Acknowledgements
The author expresses his gratitude to Dr. Roman
Fafula, M.Sc., Ph.D. from Biophysics Department of
Faculty of Pharmacy, Danylo Halytsky Lviv National
Medical University (Ukraine) for his assistance in performing
the kinetic analysis and critical reading of the
manuscript.
REFERENCES
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Barton LL, Hamilton WA (2010) Sulphate-Reducing Bacteria. Environmental
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and Ulcerative Colitis. Curr Issues Intest Microbiol 4: 9–20.
Gibson GR, Cummings JH, Macfarlane GT (1991) Growth and activities
of sulphate-reducing bacteria in gut contents of health subTable
3. Kinetic parameters of phosphotransacetylase reaction
Kinetic parameters
Sulfate-reducing bacteria
Desulfovibrio piger Vib-7 Desulfomicrobium sp. Rod-9
Vmax
Acetyl-CoA (µmol × min-1 × mg-1 protein) 2.73 ± 0.31 0.98 ± 0.089***
Km
Acetyl-CoA (mM) 3.36 ± 0.35 5.97 ± 0.62*
Comment: Vmax is maximum velocity of the enzyme reaction; Km is Michaelis constant which was determined by substrate (acetyl-CoA). Statistical
significance of the values M ± m, n = 5; *P < 0.05, ***P < 0.001, compared to the D. piger Vib-7 strain.
Scheme of the mechanism of the phosphotransacetylase reaction
(by Lawrence et al., 2006, modified)
108											 2015I. V. Kushkevych and others
jects and patients with ulcerative colitis. FEMS Microbiol Ecol 86:
103–112.
Goldman DS (1958) Purification of phosphotransacetylase from Escherichia
coli K-12. Biochim Biophys Acta 28: 436–437.
Keleti T (1988) Basic Enzyme Kinetics. Akademiai Kiado, 422 p.
Kushkevych IV (2012a) Sulfate-reducing bacteria of the human intestine.
I. Dissimilatory sulfate reduction. Sci Int J Biol Studies/Studia
Biol 6: 149–180.
Kushkevych IV (2012b) Sulfate-reducing bacteria of the human intestine.
II. The role in the diseases development. Sci Int J Biol Studies/
Studia Biol 6: 221–250.
Kushkevych IV, Moroz OM (2012) Growth of various strains of sulfate-reducing
bacteria of human large intestine. Sci Int J Biol Studies/
Studia Biol 6: 115–124.
Kushkevych IV (2013) Identification of sulfate-reducing bacteria strains
of human large intestine. Sci Int J Biol Studies/Studia Biol 7: 115–124.
Kushkevych IV, Bartos M, Bartosova L (2014) Sequence analysis of
the 16S rRNA gene of sulfate-reducing bacteria isolated from human
intestine. Int J Curr Microbiol Appl Sci 3: 239–248.
Lawrence SH, Luther KB, Schindelin H, Ferry JG (2006) Structural
and functional studies suggest a catalytic mechanism for the phosphotransacetylase
from Methanosarcina thermophila. J Bacteriol 188:
1143–1154.
Loubinoux J, Mory F, Pereira IA, Le Faou AE (2000) Bacteremia
caused by a strain of Desulfovibrio related to the provisionally named
Desulfovibrio fairfieldensis. J Clin Microbiol 38: 931–934.
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein determination
with the Folin phenol reagent. J Biol Chem 193: 265–275.
Pitcher MC, Cummings JH (1996) Hydrogen sulphide: a bacterial toxin
in ulcerative colitis? Gut 39: 1–4.
Reichenbecher W, Schink B (1997) Desulfovibrio inopinatus, sp. nov., a
new sulfate-reducing bakterium that degrades hydroxyhydroquinone
(1,2,4-trihydroxybenzene). Arch Microbiol 168: 338–344.
Robinson JR and Sagers RD (1972) Phosphotransacetylase from Clostridium
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Rowan FE, Docherty NG, Coffey JC, O’Connell PR (2009) Sulphatereducing
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Segal IH (1975) Enzyme kinetics: behavior and analysis of rapid equilibrium
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ATTACHMENT 4
Pages 65 – 73
_____________________________________________________________________________
Kinetic Properties of Pyruvate Ferredoxin Oxidoreductase of Intestinal Sulfate-Reducing
Bacteria Desulfovibrio piger Vib-7 and Desulfomicrobium sp. Rod-9.
Kushkevych I. V.
Polish Journal of Microbiology, 2015, 64(2), 107-14, ISSN: 1733-1331
_____________________________________________________________________________
Polish Journal of Microbiology
2015, Vol. 64, No 2, 107–114
ORIGINAL PAPER
*  Corresponding author: I.V. Kushevych, Institute of Animal Biology of NAAS of Ukraine, Lviv, Ukrain; e-mail: ivan.kushkevych@
gmail.com
Introduction
Intestinal sulfate-reducing bacteria are often isolated
from the gut of healthy humans and persons with ulcerative
colitis and inflammatory bowel diseases (Gibson
et al., 1991; Barton and Hamilton, 2010). A  greater
number of these bacteria is found mainly in sick people
(Cummings et al., 2003; Gibson et al., 1991). In the presence
of sulfate, lactate in human intestine contributes to
the intensive bacteria growth and the accumulation of
their final metabolism product, hydrogen sulfide, which
is toxic, mutagenic and cancerogenic to epithelial intestinal
cells (Pitcher and Cummings, 2003; Gibson et al.,
1991; Kushkevych, 2012a). The increased number of
sulfate-reducing bacteria and the intensity of dissimilatory
sulfate reduction in the gut can cause inflammatory
bowel diseases of humans and animals (Cummings
et al., 2003; Gibson et al., 1991; Kushkevych, 2012b).
Lactate is the most common substrate used by the
species belonging to the sulfate-reducing bacteria
(Kushkevych, 2012a). This compound is oxidized to
acetate via pyruvate. The type of enzyme present in
these microorganisms appears to be a pyruvate-ferredoxin
oxidoreductase, as can be deduced from the low
potential electron carriers, ferredoxin and flavodoxin,
which serve as electron acceptors for the enzyme
(Akagi, 1967; Hatchikian et al., 1979; Guerlesquin et al.,
1980). In strict anaerobes microorganisms, pyruvate
is oxidatively decarboxylated by pyruvate oxidoreductase
(EC 1.2.7.1). Pyruvate ferredoxin oxidoreductase
catalyzes the oxidative decarboxylation of pyruvate to
acetyl-CoA and CO2
(Akagi, 1967; Barton and Hamilton,
2010; Kushkevych, 2012a).
The reaction of this enzyme has been most extensively
studied in the forward (oxidative decarboxylation)
direction beginning with a series of seminal studies
published in 1971 by Raeburn and Rabinowitz which
have isolated and characterized pyruvate-ferredoxin
oxidoreductase. They have also demonstrated that
low potential electron donors, like reduced ferredoxin,
can drive the reductive carboxylation of acetyl-CoA
(Raeburn and Rabinowitz, 1971).
Kinetic Properties of Pyruvate Ferredoxin Oxidoreductase
of Intestinal Sulfate-Reducing Bacteria Desulfovibrio piger Vib-7
and Desulfomicrobium sp. Rod-9
IVAN V. KUSHKEVYCH*
Institute of Animal Biology of NAAS of Ukraine, Lviv, Ukraine
Submitted 18 June 2014, revised 27 March 2015, accepted 13 April 2015
A b s t r a c t
Intestinal sulfate-reducing bacteria reduce sulfate ions to hydrogen sulfide causing inflammatory bowel diseases of humans and animals.
The bacteria consume lactate as electron donor which is oxidized to acetate via pyruvate in process of the dissimilatory sulfate reduction.
Pyruvate-ferredoxin oxidoreductase activity and the kinetic properties of the enzyme from intestinal sulfate-reducing bacteria Desulfovibrio
piger and Desulfomicrobium sp. have never been well-characterized and have not been yet studied. In this paper we present for the first time
the specific activity of pyruvate-ferredoxin oxidoreductase and the kinetic properties of the enzyme in cell-free extracts of both D. piger
Vib-7 and Desulfomicrobium sp. Rod-9 intestinal bacterial strains. Microbiological, biochemical, biophysical and statistical methods were
used in this work. The optimal temperature (+35°C) and pH 8.5 for enzyme reaction were determined. The spectral analysis of the purified
pyruvate-ferredoxin oxidoreductase from the cell-free extracts was demonstrated. Analysis of the kinetic properties of the studied
enzyme was carried out. Initial (instantaneous) reaction velocity (V0
), maximum amount of the product of reaction (Pmax
), the reaction
time (half saturation period) and maximum velocity of the pyruvate-ferredoxin oxidoreductase reaction (Vmax
) were defined. Michaelis
constants (Km
) of the enzyme reaction were calculated for both intestinal bacterial strains. The studies of the kinetic enzyme properties in
the intestinal sulfate-reducing bacteria strains in detail can be prospects for clarifying the etiological role of these bacteria in the development
of inflammatory bowel diseases.
K e y w o r d s:  kinetic analysis, inflammatory bowel diseases, pyruvate ferredoxin oxidoreductase, sulfate-reducing bacteria
Kushkevych I.V. 2108
As far as we are aware, pyruvate-ferredoxin oxidoreductase
from intestinal sulfate-reducing bacteria
D. piger and Desulfomicrobium sp. has never been wellcharacterized.
In the literature there are a lot of data on
pyruvate-ferredoxin oxidoreductase in various organisms
as well as in sulfate-reducing bacteria isolated from
environment (Akagi, 1967; Barton and Hamilton, 2010;
Hatchikian et al., 1979; Furdui et al., 2000; Garczarek
et al., 2007; Guerlesquin et al., 1980; Zeikus et al., 1977;
Raeburn and Rabinowitz, 1971; Uyeda and Rabinowitz,
1971; Ma et al., 1997; Meinecke, et al., 1989; Pieulle et al.,
1995). However, data on the activity of this enzyme from
intestinal sulfate-reducing bacteria D. piger and Desulfomicrobium
sp. have not yet been reported.
The aim of this work was to study pyruvate-ferredoxin
oxidoreductase activity in cell-free extracts of
intestinal sulfate-reducing bacteria D. piger Vib-7 and
Desulfomicrobium sp. Rod-9 and to carry out the kinetic
analysis of enzymatic reaction.
The aim was accomplished using microbiological,
biochemical, biophysical methods, and statistical processing
of the results; the obtained data were compared
with those from the literature.
Experimental
Materials and Methods
The objects of the study were sulfate-reducing bacteria
D. piger Vib-7 and Desulfomicrobium sp. Rod-9 isolated
from the human large intestine and identified by
sequence analysis of the 16S rRNA gene (Kushkevych,
2013; Kushkevych et al., 2014).
Bacterial growth and cultivation. Bacteria were
grown in a nutrition-modified Kravtsov-Sorokin’s
liquid medium (Kushkevych, 2013). Before seeding
bacteria in the medium, 0.05 ml/l of sterile solution of
Na2
S × 9H2
O (1%) was added. A sterile 10 N solution of
NaOH (0.9 ml/l) in the medium was used to provide the
final pH 7.2. The medium was heated in boiling water
for 30 min in order to obtain an oxygen-free medium,
and then cooled to +30°C. The bacteria were grown
for 72 hours at +37°C under anaerobic conditions. The
tubes were brim-filled with medium and closed to provide
anaerobic conditions.
Obtaining cell-free extracts. Cells were harvested
at the beginning of the stationary phase, suspended in
10 mM Tris-HCl buffer in a 1/1 ratio (w/v) at pH 7.6,
and disrupted using a Manton-Gaulin press at 9000 psi.
The extract was centrifuged at 15,000 g for 1 h; the pellet
was then used as sedimentary fraction, and the
supernatant obtained was termed the soluble fraction
(Gavel et al., 1998). The soluble extract constituted by
the supernatant was used as the source of the enzyme.
This extract was subjected to further centrifugation at
180,000 g for 1 h to eliminate the membrane fraction.
A pure supernatant, containing the soluble fraction,
was then used as cell-free extract.
Protein concentration in the cell-free extracts was
determined by the Lowry method (Lowry et al., 1951).
Assays for pyruvate-ferredoxin oxidoreductase
activity. The pyruvate-ferredoxin oxidoreductase was
assayed and purified as described in paper (Pieulle
et al., 1995). The enzyme activity was routinely determined
spectrophotometrically by following the reduction
of methyl viologen as previously described (Zeikus
et al., 1977). All enzyme assays were performed under
anaerobic conditions at +35°C using serum-stoppered
cuvettes. Samples of enzyme were made anaerobic by
flushing the solution with argon as previously reported
(Fernandez et al., 1985). The reaction mixture containing
50 µmol Tris-HCl (pH 8.5), 10 µmol sodium pyruvate,
0.1 µmol sodium coenzyme A, 2 µmol methyl
viologen and 16 µmol dithioerythritol, in a final volume
of 1.0 ml, was bubbled with argon for 20 min and
the cell was then incubated at +30°C. The reaction was
started by injection of pyruvate-ferredoxin oxidoreductase
into the assay cuvette using a gastight syringe and
the absorbance at 604 nm was followed. Rates of methyl
viologen reduction were calculated using an absorption
coefficient of 13.6 mM–1
 × cm–1
. A regenerating system
was used to determine the Km
for coenzyme A as previously
described (Meinecke et al., 1989). One unit of
enzyme activity was defined as the amount of enzyme,
which catalyzes the oxidation of 1 µmol of pyruvate or
the reduction of 2 µmol of methyl viologen per min
under the specified conditions. Specific enzyme activity
was expressed as U × mg–1
protein. Michaelis constant
(Km
) for pyruvate-ferredoxin oxidoreductase reaction
has been determined by substrate (pyruvate and coenzyme
A). In order to maintain the concentration of oxidized
ferredoxin, a recycling system consisting of spinach
ferredoxin-NADP reductase (5 µg/assay) (Sigma)
and NADP+
(5 mM) was used. The overall rate was
measured by the appearance of NADPH. The activity
of the studied enzyme in the cell-free extracts of both
bacterial strains at different temperature (from +20°C
to +45°C) and pH (in the range from 5.0 to 10.0) in
the incubation medium was measured. Spectral analysis
of the purified enzyme was carried out as previously
described (Pieulle et al., 1995).
Kinetic analysis. Kinetic analysis of the enzyme
reaction was performed in a standard incubation
medium (as it was described above) with modified
physical and chemical characteristics of the respective
parameters (incubation time, substrate concentration,
temperature and pH). The kinetic parameters characterizing
the pyruvate-ferredoxin oxidoreductase reaction
are the initial (instantaneous) reaction velocity (V0
),
Kinetic properties of pyruvate ferredoxin oxidoreductase2 109
maximum velocity of the reaction (Vmax
), maximum
amount of the reaction product (Pmax
) and characteristic
reaction time (time half saturation) were determined.
The amount of the reaction product was calculated stoichiometrically.
The kinetic parameters characterizing
pyruvate-ferredoxin oxidoreductase reactions such
as Michaelis constant (Km
) and maximum reaction
velocity of substrate decomposition were determined
by Lineweaver-Burk plot (Keleti, 1988). For analysis of
the substrate kinetic mechanism of pyruvate-ferredoxin
oxidoreductase, initial velocities were measured under
standard assay conditions with different substrate concentrations.
The resulting data were also analyzed by
global curve fitting in SigmaPlot (Systat Software, Inc.)
to model the kinetic data for rapid equilibrium rate
equations describing ordered sequential, V=(Vmax
[A]
[B])/(KA
KB
+KB
[A]+[A] [B]), and random sequential,
V=(Vmax
[A] [B])/(α KA
KB
+KB
[A]+KA
[B]+[A] [B]),
kinetic mechanisms, where V is the initial velocity, Vmax
is the maximum velocity, KA
and KB
are the Km
values
for substrates A and B, respectively, and α is the interaction
factor if the binding of one substrate changes the
dissociation constant for the other (Segal, 1975).
Statistical analysis. Kinetic and statistical calculations
of the results were carried out using the software
MS Office and Origin computer programs. The research
results were treated by the methods of variation statistics
using Student t-test. The equation of the straight
line that the best approximates the experimental data
was calculated by the method of least squares. The absolute
value of the correlation coefficient r was from 0.90
to 0.98. The significance of the calculated parameters of
line was tested by Fisher’s F-test. The accurate approximation
was when P ≤ 0.05 (Bailey, 1995).
Results and Discussion
Specific activity of pyruvate-ferredoxin oxidoreductase,
an important enzyme in the process of organic
compounds oxidation in sulfate-reducing bacteria, was
measured in different fractions obtained from D. piger
Vib-7 and Desulfomicrobium sp. Rod-9 cells (Table I).
Results of our study showed that the highest specific
activity of the enzyme was detected in cell-free
extracts (1.24 ± 0.127 and 0.48 ± 0.051 U × mg–1
protein
for D. piger Vib-7 and Desulfomicrobium sp. Rod-9,
respectively). The slightly lower values of activity of
pyruvate-ferredoxin oxidoreductase were determined
in the soluble fraction compared to cell-free extracts.
Its values designated 1.11 ± 0.114 U × mg–1
protein for
D. piger Vib-7 and 0.37 ± 0.033 U × mg–1
protein for
Desulfomicrobium sp. Rod-9. The enzyme activity in
sedimentary fraction was not observed.
The effect of temperature and pH of the reaction
mixture on pyruvate-ferredoxin oxidoreductase activity
in the cell-free extracts of the sulfate-reducing bacteria
was studied (Fig. 1). The maximum specific activity for
both bacterial strains was determined at +35°C. The
highest enzyme activity of pyruvate-ferredoxin oxidoreductase
for D. piger Vib-7 and Desulfomicrobium sp.
Rod-9 was measured at pH 8.5.
Thus, temperature and pH optimum of this enzyme
was +35°C and pH 8.5, respectively. An increase or
decrease in temperature and pH led to a decrease of
the activity of studied enzyme in the cell-free bacterial
extracts of the sulfate-reducing bacteria. The enzyme
activity exhibited typical bell-shaped curves as a function
of temperature and pH.
Next task of this study was to carry out a spectral
analysis of the purified pyruvate-ferredoxin oxidoreductase
from the cell-free extracts of D. piger Vib-7 and
Desulfomicrobium sp. Rod-9. The absorption maxima
were 317 and 423, 316 and 425 nm for pyruvate-ferredoxin
oxidoreductase from D. piger Vib-7 and Desul­fomicrobium
sp. Rod-9, respectively (Fig. 2). Ten-minute
incubation of the enzyme with 0.75 mM sodium pyruvate
led to a slight decrease in absorption maxima.
The same peaks of absorption as without addition of
sodium pyruvate were observed. However, the significant
decrease in absorption spectra after the addition
of 0.75 mM sodium pyruvate and 0.1 mM coenzyme A
Desulfovibrio piger Vib-7	 1.24 ± 0.127	 1.11 ± 0.114	 0
Desulfomicrobium sp. Rod-9	 0.48 ± 0.051**	
0.37 ± 0.033***	 0
Table I
Pyruvate-ferredoxin oxidoreductase activity in different fractions obtained
from the bacterial cells
Comment: The assays were carried out at a protein concentration of 43.57 µg/ml (for D. piger
Vib-7) and 41.94 µg/ml (for Desulfomicrobium sp. Rod-9). Enzyme activity was determined
after 20 min incubation. Statistical significance of the values M ± m, n = 5; **P < 0.01,
***P < 0.001, compared to D. piger Vib-7 strain
Sulfate-reducing bacteria
Specific activity of the enzyme (U×mg–1
protein)
Cell-free extract
Individual fractions
Soluble Sedimentary
Kushkevych I.V. 2110
in the incubation medium was registered. The absorption
peaks was no observed (Fig. 2A). The spectroscopic
analyses of oxidized and reduced pyruvate-ferredoxin
oxidoreductase from D. piger Vib-7 and Desulfomicrobium
sp. Rod-9 strains were also carried out (Fig. 2B).
Similar data on the absorption spectra of pyruvateferredoxin
oxidoreductase from Desulfovibrio africanus
were obtained by Pieulle et al. (1995). The authors
described the ultraviolet-visible spectrum of studied
enzyme which was typical of an iron-sulfur protein with
a broad absorbance band around 400 nm and a shoulder
in the 315 nm region (Pieulle et al., 1995). Iron
and acid-labile sulfide content, as well as the absorption
coefficient at 400 nm suggest the presence of six
[4Fe-4S] clusters per molecule of enzyme. The absorption
band at 400 nm was partially bleached after addition
of dithionite; this indicates only partial reduction
of the protein, if one considers that full reduction of
iron-sulfur clusters should lead to about 50% decrease
of the absorption band. Pyruvate reduced the enzyme
slightly, whereas pyruvate and CoASH produced a more
pronounced reduction of the protein than that obtained
with dithionite (Pieulle et al., 1995).
To study the characteristics and mechanism of
pyruvate-ferredoxin oxidoreductase reaction, the initial
(instantaneous) reaction velocity (V0
), maximum
velocity of the reaction (Vmax
), maximum amount of
reaction product (Pmax
) and reaction time (τ) were
defined. Dynamics of reaction product accumulation
was studied for investigation of the kinetic parameters
of pyruvate-ferredoxin oxidoreductase (Fig. 3).
Experimental data showed that the kinetic curves
of pyruvate-ferredoxin oxidoreductase activity have
a saturation tendency (Fig. 3A). Analysis of the results
allows to reach the conclusion that the kinetics of
pyruvate-ferredoxin oxidoreductase activity in the
Fig. 1.  The effect of temperature (A) and pH (B) on the pyruvate-ferredoxin oxidoreductase activity in the cell-free extracts
of the sulfate-reducing bacteria
Fig. 2.  Absorption spectra of pyruvate-ferredoxin oxidoreductase from D. piger Vib-7 and Desulfomicrobium sp. Rod-9. The serum-stoppered
cuvette contains 3 μM of pure enzyme in 50 mM Tris-HC1 (pH 8.5) under argon at +35°C, final volume, 1 ml (A). The spectra
were recorded in a final volume of 1 ml in a serum-stoppered cuvette of path length 1 cm under argon. Spectrum of the oxidized enzyme
and spectrum of the reduced enzyme after injection of 2 μl dithionite (150 mM) (B)
Kinetic properties of pyruvate ferredoxin oxidoreductase2 111
Fig. 3. Kinetic parameters of pyruvate-ferredoxin oxidoreductase activity in D. piger Vib-7 and Desulfomicrobium sp. Rod-9: A – dynamics
of product accumulation (M ± m, n = 5); B – linearization of curves of product accumulation in {P/t; P} coordinates (n = 5; R2
 > 0.95;
F < 0.02); C, E – the effect of different concentrations of substrate (pyruvate and coenzyme A) on the enzyme activity (M ± m, n = 5);
D, F – linearization of concentration curves, which are shown in fig. 3C, E, in the Lineweaver-Burk plot, where V is velocity of the
enzyme reaction and [Pyruvate] or [Coenzyme A] is substrate concentration (n = 5; R2
 > 0.9; F < 0.005)
sulfate-reducing bacteria was consistent to the zeroorder
reaction in the range of 0–10 min (the graph of
the dependence of product formation on the incubation
time was almost linear in this interval of time).
Therefore the duration of the incubation of bacterial
cells extracts was 10 min in subsequent experiments.
The amount of the product of pyruvate-ferredoxin
oxidoreductase reaction in the D. piger Vib-7 was the
higher (36.28 ± 3.59 µmol × mg–1
protein) compared
to the Desulfomicrobium sp. Rod-9 (14.95 ± 1.48 µmol
× mg–1
protein) in the entire range of time factor.
The basic kinetic properties of the reaction in the
Kushkevych I.V. 2112
sulfate-reducing bacteria were calculated by linearization
of the data in the {P/t; P} coordinates (Fig. 3B,
Table II).
The kinetic parameters of pyruvate-ferredoxin oxidoreductase
from both D. piger Vib-7 and Desulfomicrobium
sp. Rod-9 were significantly different. Values
of initial (instantaneous) reaction velocity (V0
) for
the enzyme was calculated by the maximal amount of
the product reaction (Pmax
). As shown in Table II, V0
for pyruvate-ferredoxin oxidoreductase reaction was
slightly higher (4.15 ± 0.43 µmol × min–1
× mg–1
protein)
in D. piger Vib-7 compared to Desulfomicrobium
sp. Rod-9 (1.37 ± 0.12 µmol × min–1
× mg–1
protein).
In this case, the values of the reaction time (τ) were
more similar for the studied enzyme in both D. piger
Vib-7 and Desulfomicrobium sp. Rod-9 strains. Based
on these data, it may be assumed that the D. piger Vib-7
can consume lactate ion much faster in their cells than
a Desulfomicrobium sp. Rod-9. Moreover, this hypothetical
assumption can be also confirmed by obtained
data on maximal velocities of accumulation of the final
reaction products, where Vmax
for enzyme reaction in
D. piger Vib-7 were also more intensively compared to
Desulfomicrobium sp. Rod-9 (Table III).
The kinetic analysis of pyruvate-ferredoxin oxidoreductase
reaction depending on concentration of substrate
(pyruvate and coenzyme A) was carried out. The
increasing pyruvate concentrations from 0.5 to 5.0 mM
and coenzyme A concentrations from 0.1 to 1.0 µM
caused a monotonic rise of the studied enzyme activity
and the activity was maintained on unchanged level
(plateau) under substrate concentrations over 5.0 mM
and 1.0 µM, respectively. (Fig. 3C, E). Curves of the
dependence {1/V; 1/[S]} were distinguished by the tangent
slope and intersect the vertical axis in one point
(Fig. 3D, F). The basic kinetic parameters of pyruvateferredoxin
oxidoreductase activity in D. piger Vib-7 and
Desulfomicrobium sp. Rod-9 were identified by linearization
of the data in the Lineweaver-Burk plot (Table III).
Calculation of the kinetic parameters of enzyme
activity indicates that the maximum velocities (Vmax
)
of pyruvate and coenzyme A in the D. piger Vib-7 and
Desulfomicrobium sp. Rod-9 were significantly different
from each other. However, it was observed a correlative
relationship between Vmax
Pyruvate
and Vmax
CoA
in
both intestinal bacterial strains. Michaelis constants
(Km
) of pyruvate-ferredoxin oxidoreductase reaction
were identified for pyruvate and coenzyme A. The values
of Km
were quite similar for pyruvate (2.72 ± 0.283,
2.55 ± 0.245 mM) and coenzyme A (0.54 ± 0.052,
0.42 ± 0.044 μM) in both D. piger Vib-7 and Desulfomicrobium
sp. Rod-9 strains, respectively.
The obtained parameters of pyruvate-ferredoxin
oxidoreductase reaction in D. piger Vib-7 are consistent
with previously described data by Pieulle et al. for
the activity of pyruvate-ferredoxin oxidoreductase
from D. africanus. The apparent Km
for pyruvate and
coenzyme A were also 2.5 mM and 0.5 μM, respectively
and the Vmax
values were 10240 min–1
and 5890 min–1
,
respectively. The apparent Km
for methyl viologen was
found to be 0.5 mM in the presence of 10 mM and
0.1 mM of pyruvate and CoASH, respectively. Kinetics
studies done with the enzyme and a slight decrease in
the affinity for pyruvate and in the catalytic activity
(Km
of 5.5 mM and Vmax
of 4810 min–1
) were reported
(Pieulle et al., 1995).
Furdui and Ragsdale (2000) have described the
pyruvate-ferredoxin oxidoreductase from the Clostridium
thermoaceticum. The Michaelis-Menten parameters
for pyruvate synthesis by the enzyme were:
Vmax
1.6 unit/mg, Km
Acetyl-CoA
9 μM. The intracellular concentrations
of acetyl-CoA, CoASH, and pyruvate were
also measured (Furdui and Ragsdale, 2000).
Pyruvate-ferredoxin oxidoreductatse, an important
enzyme in process of dissimilatory sulfate reduction and
organic compounds oxidation in sulfate-reducing bacteria,
carries out the central step in oxidative decarboxylation
of pyruvate to acetyl-CoA (Kushkevych, 2012a):
Comment: V0
is initial (instantaneous) reaction velocity; Pmax
is maximum
amount (plateau) of the product of reaction; τ is the reaction time
(half saturation period). Statistical significance of the values M ± m,
n = 5; **P < 0.01, ***P < 0.001, compared to the D. piger Vib-7 strain.
V0
(µmol × min–1
 × mg–1
protein)	  4.15 ± 0.43	  1.37 ± 0.12***
Pmax
(µmol × mg–1
protein)	 36.28 ± 3.59	 14.95 ± 1.48**
τ (min)	 8.74 ± 0.88	10.89 ± 1.11
Table II
Kinetic parameters of the pyruvate-ferredoxin oxidoreductase
from intestinal sulfate-reducing bacteria
Kinetic parameters
Sulfate-reducing bacteria
Desulfovibrio
piger Vib-7
Desulfomicrobium
sp. Rod-9
Vmax
Pyruvate
µmol × min–1
 × mg–1
protein)	
2.54 ± 0.261	 0.89 ± 0.092***
Km
Pyruvate
(mM)	 2.72 ± 0.283	 2.55 ± 0.245
Vmax
CoA
(µmol×min–1
×mg–1
protein)	
2.51 ± 0.248	 0.81 ± 0.076***
Km
CoA
(μM)	 0.54 ± 0.052	0.42 ± 0.044
Table III
Kinetic parameters of pyruvate-ferredoxin oxidoreductase
reaction
Comment: Vmax
is maximum velocity of the enzyme reaction; Km
is Michaelis constant which was determined by substrate (pyruvate
and coenzyme A). Statistical significance of the values M ± m, n = 5;
***P < 0.001, compared to the D. piger Vib-7 strain.
Kinetic parameters
Sulfate-reducing bacteria
Desulfovibrio
piger Vib-7
Desulfomicrobium
sp. Rod-9
Kinetic properties of pyruvate ferredoxin oxidoreductase2 113
Garczarek et al. (2007) have purified this enzyme
from Desulfovibrio vulgaris Hildenborough as part of
a systematic characterization of as many multiprotein
complexes as possible for this organism (Garczarek
et al., 2007).
Thus, based on the obtained studies results and
according to the kinetic parameters of pyruvateferredoxin
oxidoreductatse reaction for both bacterial
strains, we have concluded that the enzyme activity,
V0
and Vmax
were significantly higher in the D. piger
Vib-7 cells than Desulfomicrobium sp. Rod-9. However,
Michaelis constants were quite similar for pyruvate
(2.72 ± 0.283, 2.55 ± 0.245 mM) and coenzyme A
(0.54 ± 0.052, 0.42 ± 0.044 μM) in both bacterial strains.
The maximum enzyme activity for both strains was
determined at +35°C and at pH 8.5. These data correspond
to conditions which are present in the human
large intestine from where the bacterial strains were
isolated. Perhaps such conditions favor intensive development
of the D. piger and Desulfomicrobium sp. bacterial
strains in the gut. The kinetic parameters of enzyme
reaction are depended on the substrate concentration.
The studies of the pyruvate-ferredoxin oxidoreductatse
in the process of dissimilatory sulfate reduction and
kinetic properties of this enzyme in the D. piger Vib-7
and Desulfomicrobium sp. Rod-9 intestinal strains,
their production of acetate in detail can be a perspective
for clarification of their etiological role in the development
of the humans and animals bowel diseases. These
studies might help in predicting the development of
diseases of the gastrointestinal tract, by providing further
details on the etiology of bowel diseases, which
are very important for the clinical diagnosis of these
disease types.
Acknowledgements
The author expresses his gratitude to Dr. Roman Fafula, M. Sc.,
Ph.D. from Biophysics Department of Faculty of Pharmacy, Danylo
Halytsky Lviv National Medical University (Ukraine) for his assistance
in performing the kinetic analysis and critical reading of the
manuscript.
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ATTACHMENT 5
Pages 74 – 79
_____________________________________________________________________________
Antimicrobial effect of salicylamide derivatives against intestinal sulfate-reducing bacteria
Ivan Kushkevych, PeterKollar, Ana Luisa Ferreira, DiogoPalma, Aida Duarte,
Maria Manuel Lopes, Milan Bartos, Karel Pauk, Ales Imramovsky, Josef Jampilek
Journal of Applied Biomedicine, 2016, 14, 125-130, ISSN: 1214-021X
_____________________________________________________________________________
Original Research Article
Antimicrobial effect of salicylamide derivatives
against intestinal sulfate-reducing bacteria
Ivan Kushkevych a,b,*, Peter Kollar a
, Ana Luisa Ferreira c
, Diogo Palma c
,
Aida Duarte c
, Maria Manuel Lopes c
, Milan Bartos b
, Karel Pauk d
,
Ales Imramovsky d
, Josef Jampilek e,**
a
Department of Human Pharmacology and Toxicology, Faculty of Pharmacy, University of Veterinary and
Pharmaceutical Sciences Brno, Czech Republic
b
Department of Molecular Biology and Pharmaceutical Biotechnology, Faculty of Pharmacy, University of Veterinary
and Pharmaceutical Sciences Brno, Czech Republic
c
Department of Microbiology and Immunology, Faculty of Pharmacy, University of Lisbon, Portugal
d
Institute of Organic Chemistry and Technology, Faculty of Chemical Technology, University of Pardubice, Czech
Republic
e
Department of Chemical Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences Brno,
Czech Republic
j o u r n a l o f a p p l i e d b i o m e d i c i n e 1 4 ( 2 0 1 6 ) 1 2 5 – 1 3 0
a r t i c l e i n f o
Article history:
Received 28 November 2015
Received in revised form
26 January 2016
Accepted 28 January 2016
Available online 8 February 2016
Keywords:
Sulfate-reducing bacteria
Desulfovibrio piger
Desulfomicrobium sp.
Salicylamides
Bowel disease
Lipophilicity
Structure–activity relationships
a b s t r a c t
Sulfate-reducing bacteria (SRB) are most likely involved in both the initiation and maintenance
of inflammatory bowel disease (IBD); unfortunately present antibacterial chemotherapeutics
used in the treatment of IBD have been ineffective. Thus, the antimicrobial activity
of salicylamide derivatives against two different genera of intestinal SRB, Desulfovibrio and
Desulfomicrobium, was investigated. Six 2-(phenylcarbamoyl)phenyl N-[(benzyloxy)carbonyl]
alkanoates and three 2-hydroxy-N-[(2S)-1-oxo-1-(phenylamino)alkan-2-yl]benzamides
showed MIC values in the range from 0.22 to 0.35 mM against Desulfovibrio piger Vib-7 and
in the range from 0.27 to 8.52 mM against Desulfomicrobium sp. Rod-9, while MIC values of
ciprofloxacin were 41.2 mM and 39.3 mM. The highest potency against the two strains was
observed for 4-chloro-N-{(2S)-1-[(3,4-dichlorophenyl)amino]-3-methyl-1-oxobutan-2-yl}-2hydroxybenzamide
(MIC 0.22 mM and 0.27 mM). 4-Chloro-2-[(4-nitrophenyl)carbamoyl]phenyl
(2S)-2-{[(benzyloxy)carbonyl]amino}-3-methylbutanoate showed high activity against D.
piger Vib-7 (MIC = 0.26 mM), while 4-chloro-2-[(4-methylphenyl)carbamoyl]phenyl (2S)-2[(tert-butoxycarbonyl)amino]-3-(1H-indol-2-yl)propanoate
expressed high activity against
Desulfomicrobium sp. Rod-9 (MIC = 0.31 mM). Structure–activity relationships are discussed.
# 2016 Faculty of Health and Social Studies, University of South Bohemia in Ceske
Budejovice. Published by Elsevier Sp. z o.o. All rights reserved.
* Corresponding author at: Department of Human Pharmacology and Toxicology, Faculty of Pharmacy, University of Veterinary and
Pharmaceutical Sciences Brno, Palackeho 1, 61242 Brno, Czech Republic.
** Corresponding author at: Department of Chemical Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences
Brno, Palackeho 1, 61242 –>Brno–>, –>Czech Republic–>.
E-mail addresses: ivan.kushkevych@gmail.com (I. Kushkevych), josef.jampilek@gmail.com (J. Jampilek).
Available online at www.sciencedirect.com
ScienceDirect
journal homepage: http://www.elsevier.com/locate/jab
1214-021X/$ – see front matter # 2016 Faculty of Health and Social Studies, University of South Bohemia in Ceske Budejovice. Published
by Elsevier Sp. z o.o. All rights reserved.
http://dx.doi.org/10.1016/j.jab.2016.01.005
Introduction
Ulcerative colitis (UC) is one of the two major forms of
idiopathic inflammatory bowel disease (IBD) (Cummings et al.,
2003). Both acute and chronic forms of the illness affect the
colon and rectum and can be a highly disabling condition
(Barton and Hamilton, 2010). This disease is more common in
North America and Western Europe with the increasing
incidence in Asia. The reported incidence is 1.2–20.3 cases
per 100,000 persons per year, and the prevalence is 7.6–245
cases per 100,000 per year (Feuerstein and Cheifetz, 2014).
Ulcerative colitis usually has a relapsing/remitting pattern and
current medical approaches focus on treating active disease to
address symptoms, to improve the quality of life and
thereafter to maintain remission. Bloody diarrhoea, an urgent
need to defecate and abdominal pain are the main symptoms
of active disease or relapse. The treatment chosen for active
disease depends not only on clinical severity, but also on the
extent of disease and the person's preference (Loubinoux et al.,
2000, 2002a,b; Kornbluth and Sachar, 2010). Conventional drug
therapy for UC involves the use of 5-aminosalicylates (the
mainstay of treatment for mild to moderate disease), corticosteroids
(for patients who failed 5-aminosalicylates therapy
and for acute episodes), azathioprine/6-mercaptopurine,
cyclosporine and anti-tumour necrosis factor therapy (Lissner
and Siegmund, 2013).
Several reports suggested the possible involvement of
sulfate-reducing bacteria (SRB), a group of phylogenetically
diverse anaerobic microorganisms, in both the initiation and
maintenance of the disease (Loubinoux et al., 2000, 2002a,b;
Zinkevich and Beech, 2000; Cummings et al., 2003). SRB such as
Desulfovibrio and Desulfomicrobium genera, are normal inhabitants
of the human and animal large intestine, capable of
dissimilatory sulfate reduction (Gibson et al., 1991, 1993;
Kushkevych, 2012a,b; Kushkevych and Moroz, 2012). Most of
the SRB utilize sulfate or other sulfur compounds such as
thiosulfate, sulfite and sulfur as terminal electron acceptors.
The main product of SRB metabolism, hydrogen sulfide, is a
compound that may act through inhibition of butyrate
oxidation, the main energy source for colonocytes. In addition
it is cytotoxic, mutagenic and cancerogenic to epithelial
intestinal cells. All these properties of hydrogen sulfide lead
to the damage of the epithelial barrier function resulting in
inflammatory responses characteristic for IBD (Pitcher and
Cummings, 1996; Zinkevich and Beech, 2000). Therefore the
association between SRB and inflammatory bowel diseases,
such as ulcerative colitis, was hypothesized (Loubinoux et al.,
2002a,b; Rowan et al., 2009; Kushkevych, 2014). Unlike Crohn's
disease, ulcerative colitis occurs only in the large bowel, where
bacteria amount is greater than in the rest of the gut and also
where the rate of passage of material is characterized by slow
movement of digestive materials (Cummings et al., 2003).
An antibiotic for animal colitis, in order to be effective,
should have activity against gut anaerobes. Such antibiotics
that specifically target Gram-negative facultative species are
not successful in IBD (Cummings et al., 2003). The benefits of
antibiotic therapy in UC are mediated by different mechanisms
such as decreasing the concentration of luminal
bacteria, altering the composition of gut microflora, decreasing
bacterial tissue invasion and decreasing bacterial translocation
and systemic dissemination. Antibiotics have been
prescribed for UC, however they have been largely ineffective.
Therefore, it is necessary to study new antibacterial compounds
in order to improve the treatment and discover
alternative therapeutics (Garud and Peppercorn, 2009).
In the previous studies it was found that salicylamide-like
compounds can be considered as promising antimicrobial
agents (Vinsova et al., 2007; Imramovsky et al., 2009a,b, 2011;
Pauketal.,2013;Zadrazilova etal.,2015a,b).Therefore thisstudy
focused on the investigation of the antimicrobial activity of
selected derivatives of 2-(phenylcarbamoyl)phenyl N-[(benzyloxy)carbonyl]alkanoates
and 2-hydroxy-N-[(2S)-1-oxo-1-(phenylamino)alkan-2-yl]benzamides
against two different genera
of SRB, Desulfovibrio and Desulfomicrobium, is a follow-up paper to
the previous contributions. The investigated salicylamide
derivatives showed high potency against different bacterial
strains as was published recently (Pauk et al., 2013; Zadrazilova
et al., 2015a). Both SRB are Gram-negative strictly anaerobe
genera. Desulfovibrio piger is usually considered as a commensal
bacterium in humans. More recently, D. piger has attracted more
interest as it was found to be the most prevalent species of SRB
in faeces of patients with inflammatory bowel disease (Holt
et al., 1994; Barton and Hamilton, 2010).
Materials and methods
Tested compounds
The discussed salicylamide derivatives (see Table 1) were
synthesized previously (Pauk et al., 2013) by means of
microwave-assisted synthesis and rearrangement described
in literature (Imramovsky et al., 2006, 2009a, 2010, 2011; Pauk
et al., 2013). The compounds were fully characterized by
melting point, CHN analyses, IR and NMR spectroscopy (Pauk
et al., 2013).
In vitro antibacterial susceptibility testing
The synthesized compounds were evaluated for in vitro
antibacterial activity against the intestinal sulfate-reducing
bacteria D. piger Vib-7 and Desulfomicrobium sp. Rod-9 that were
isolated from the healthy human large intestine as described
previously (Kushkevych, 2013; Kushkevych et al., 2014). The
strains have been kept in the collection of microorganisms at
the Department of Molecular Biology and Pharmaceutical
Biotechnology of the Faculty of Pharmacy at the University of
Veterinary and Pharmaceutical Sciences Brno (Czech Republic).
Ciprofloxacin (Sigma–Aldrich) was used as the standard.
Prior to testing, each strain was passaged onto nutrition
modified Kravtsov-Sorokin's (KS) agar medium (Kushkevych
and Moroz, 2012). Bacterial inocula were prepared by suspending
a small portion of bacterial colony in sterile KS liquid
medium (pH 7.5). The cell density was adjusted to 0.5
McFarland units using a densitometer (Densi-La-Meter, LIAP,
Latvia). The final inoculum was made to a 1:20 dilution of
the suspension with KS liquid medium. Before bacterial
passage in the medium, 10 mL/L of sterile Mohr's salt solution
[(NH4)SO4Fe(SO4)2Á6H2O] (10%) for detecting colonies of the
j o u r n a l o f a p p l i e d b i o m e d i c i n e 1 4 ( 2 0 1 6 ) 1 2 5 – 1 3 0126
sulfate-reducing bacteria was added. As a result, FeS was
formed by the bacterial cells that caused black coloured
colonies. The compounds were dissolved in DMSO (Sigma),
and the final concentration of DMSO in the KS liquid medium
did not exceed 0.1% of the total solution composition. The final
concentrations of the evaluated compounds ranged from 100
to 0.05 mM. The medium dilution micro-method modified
according to NCCLS guidelines (CLSI, 2012, 2014) in KS medium
was used to determine the minimum inhibitory concentration
(MIC). Drug-free controls, sterility controls and controls
consisted of KS medium and DMSO alone were included. Petri
plates were introduced into an anaerobic box with oxygen
uptake generators (GENbox anaer, France) for anaerobiosis.
The determination of results was performed visually after 72 h
of static incubation in the darkness at 37 8C under anaerobic
conditions. The MICs were defined as the lowest concentration
of the compound at which no visible bacterial growth was
observed. The results are summarized in Table 1.
Results
The studied compounds can be divided into two groups based
on their chemical structure: Group I includes N-protected
amino esters of N-phenylsalicylamides 1a–f, and Group II
includes compounds 2a–c that can be named as diamides,
since they contain two amidic moieties. The activity of both
groups of salicylamide derivatives against sulfate-reducing
bacteria D. piger Vib-7 and Desulfomicrobium sp. Rod-9 were
compared with the effect of ciprofloxacin as a clinically used
drug. The in vitro antibacterial activity of compounds was
expressed as the minimum inhibitory concentration (MIC) that
is defined for bacteria as a 90% (IC90) or greater reduction of
growth in comparison with the control. The MIC/IC90 value is
routinely and widely used in bacterial assays, being a standard
detection limit according to the Clinical and Laboratory
Standards Institute (CLSI, 2012, 2014). In the case of potency
against D. piger all the compounds showed a narrow range of
the MICs from 0.22 to 0.35 mM, while the activity against
Desulfomicrobium sp. ranged from 0.27 to 8.52 mM. Nevertheless,
the potency of all discussed compounds was much higher
against both genera than that of ciprofloxacin (MIC 41.2 mM or
39.3 mM). All the results are listed in Table 1.
Lipophilicity is the most frequent physicochemical parameter
employed in structure–activity relationship analysis. In a
number of studies examining the biological activity of potential
drugs, the relationship between lipophilicity and/or other
descriptors (e.g., electronic parameters or molar volume of
substituents) and their potency have been investigated. In the
present study the calculated lipophilicity (Clog P values), see
Table 1, was found as the main parameter influencing general
antibacterial potency. Clog P value is the logarithm of n-octanol/
water partition coefficient based on established chemical
interactions.
Within Group I, 4-chloro-2-[(4-nitrophenyl)carbamoyl]phenyl
(2S)-2-{[(benzyloxy)carbonyl]amino}-3-methylbutanoate
(1d) showed the highest activity (MIC = 0.26 mM) against D.
piger, while compound 1c (R1
= 4-Cl, R2
= 4-Br, R3
= -CH2-cHx)
demonstrated the lowest activity (MIC = 0.35 mM). Within
Group II 4-chloro-N-{(2S)-1-[(3,4-dichlorophenyl)amino]-3methyl-1-oxobutan-2-yl}-2-hydroxybenzamide
(2b) was found
to be the most effective compound with MIC = 0.22 mM. Fig. 1
illustrates the dependence of the antibacterial activity against
D. piger expressed as log(1/MIC) of all the tested compounds on
the lipophilicity expressed as Clog P. Activity within Group I
decreases with the lipophilicity increase almost linearly
Table 1 – Structures of tested compounds 1a–f and 2a–c (Pauk et al., 2013); calculated values of Clog P; and in vitro
antibacterial activity (MIC) of salicylamides compared to ciprofloxacin (CPX) standard against intestinal sulfate-reducing
bacteria.
OH
N
H
H
N
R3O
O
R2R1
2
1
O
N
H
O
O
H
N O
R3 O
R2
R1 2
1
3
4
5
6
3
4
5
6
1 2
Comp. R1
R2
R3
Clog Pa
MIC [mM]
DVP DMS
1a 4-Cl 4-CH3 (S)-Bn 6.5605 0.31 0.34
1b 4-Cl 4-CH3 (R)-CH2-indolyl 6.0835 0.28 0.31
1c 4-Cl 4-Br (S)-CH2-cHx 8.2553 0.35 0.39
1d 4-Cl 4-NO2 (S)-iso-Pr 5.7077 0.26 6.98
1e 5-Cl 4-Br (S)-iso-Pr 6.5333 0.29 0.33
1f 5-Cl 3,4-Cl (S)-Bn 7.5008 0.33 0.37
2a 4-Cl 4-Br (S)-iso-Pr 5.7186 0.27 8.52
2b 4-Cl 3,4-Cl (S)-iso-Pr 6.2517 0.22 0.27
2c 4-Cl 3,4-Cl (S)-Bn 6.7417 0.24 0.33
CPX – – – – 41.2 39.3
a
ChemBioDraw ver. 13 (CambridgeSoft – PerkinElmer, Cambridge, MA, USA).
DVP = Desulfovibrio piger Vib-7, DMS = Desulfomicrobium sp. Rod-9.
j o u r n a l o f a p p l i e d b i o m e d i c i n e 1 4 ( 2 0 1 6 ) 1 2 5 – 1 3 0 127
(r = 0.9659, n = 6), while for Group II biphasic course can be
found with the lipophilicity optimum Clog P = 6.25 (compound
2b, R1
= 4-Cl, R2
= 3,4-Cl, R3
= iso-Pr). It can be stated that
diamides of Group II of comparable lipophilicity in the range of
Clog P 6.2517–6.7417 can be considered more potent against D.
piger than esters of Group I.
Based on the obtained MIC values of compounds from
Groups I and II, it can be stated that there is no significant
difference between the potency of both groups against
Desulfomicrobium sp. Rod-9, see Table 1 and Fig. 2, where the
dependence of the antibacterial activity against Desulfomicrobium
sp. expressed as log(1/MIC) on lipophilicity is
illustrated. Compounds 2b (R1
= 4-Cl, R2
= 3,4-Cl, R3
= iso-Pr)
and 4-chloro-N-{(2S)-1-[(3,4-dichlorophenyl)amino]-1-oxo-3phenylpropan-2-yl}-2-hydroxybenzamide
(2c) showed the
highest potency (MIC = 0.27 mM and MIC = 0.33 mM) within
Group II. Within Group I, 4-chloro-2-[(4-methylphenyl)
carbamoyl]phenyl (2S)-2-[(tert-butoxycarbonyl)amino]-3(1H-indol-2-yl)propanoate
(1b), 5-chloro-2-[(4-bromophenyl)carbamoyl]phenyl
(2S)-2-{[(benzyloxy)carbonyl]amino}-
3-methylbutanoate (1e) and 4-chloro-2-[(4-methylphenyl)
carbamoyl]phenyl (2S)-2-{[(benzyloxy)carbonyl]amino}-3phenylpropanoate
(1a) demonstrated similar efficiency in
the MIC range from 0.31 to 0.34 mM. In general, based on the
observation from Fig. 2, it can be stated that the antibacterial
activity against Desulfomicrobium sp. is connected with
the lipophilicity in the range of Clog P approx. from 6.1 to
6.8. The biphasic dependences of activity on lipophilicity
can be found for both groups of salicylamide derivatives.
The lowest activity was determined for the least
lipophilic compounds 1d (R1
= 4-Cl, R2
= 4-NO2, R3
= iso-Pr;
MIC = 6.98 mM, Clog P = 5.7077) and 2a (R1
= 4-Cl, R2
= 4-Br,
R3
= iso-Pr; MIC = 8.52 mM, Clog P = 6.7417), from which the
activity sharply increased to compound 2b (r = 0.9731, n = 4),
where an optimum lipophilicity can be found (Clog P = 6.25).
With a subsequent increase of the lipophilicity, the activity
slightly decreased (r = 0.8508, n = 7) to compounds 1f
(R1
= 5-Cl, R2
= 3,4-Cl, R3
= Bn; MIC = 0.37 mM, Clog P = 7.5008)
and 1c (R1
= 4-Cl, R2
= 4-Br, R3
= -CH2-cHx; MIC = 0.39 mM,
Clog P = 8.26) with the highest lipophilicity, see Fig. 2.
Discussion
Thus, based on the MIC values, it can be concluded that
diamide 2b is the most potent compound against both SRB
genera. From Group I, ester of N-phenylsalicylamides 1d is the
most potent against D. piger Vib-7, while compounds 1b, 1e and
1a showed an acceptable activity against Desulfomicrobium sp.
Rod-9. Based on these results and the limited number of the
compounds it is not possible to assess if R1
substitution in C(4)
or C(5) is more advantageous. Rather electron-withdrawing R2
substituent and less lipophilic (isopropyl, benzyl) R3
substituent
seem to be also more favourable for higher activity.
Compound 2b is much more potent than any compound from
Group I. These results are opposite to the results of the recently
published study (Pauk et al., 2013), where just N-protected
amino esters of N-phenylsalicylamides showed high activity
against Staphylococcus aureus, methicillin-resistant Staphylococcus
aureus, Clostridium perfringens, Pasteurella multocida and,
overall, were more effective than diamides. However, these
microorganisms were more resistant to the effect of the
compounds compared with D. piger Vib-7 and Desulfomicrobium
sp. Rod-9. On the other hand, it is important to note that
diamides were found to have bactericidal effect against three
clinical isolates of methicillin-resistant Staphylococcus aureus
(MRSA) and S. aureus ATCC 29213 as the reference and quality
control strain (Zadrazilova et al., 2015a). None of above
discussed compounds did not show any effect against typical
Gram-negative bacterial strains, such as Escherichia coli, Pseudomonas
aeruginosa, Salmonella, Proteus or Helicobacter (Zadrazilova,
2013; Zadrazilova et al., 2015a,b).
Salicylanilides show broad spectrum of antimicrobial
activities, because due to their structure they can affect a
wide range of targets, although exact mechanisms have not
been elucidated. For example, they inhibit the two-component
regulatory systems of bacteria, bind to protein kinase epidermal
growth factor receptor, inhibit interleukin-12p40 production,
inhibit bacterial enzymes, such as transglycosylases,
D-alanine-D-alanine ligase, isocitrate lyase and methionine
aminopeptidase or destruct the cellular proton gradient due to
Fig. 1 – Dependence of antibacterial effect of tested
compounds 1a–f (Group I) and 2a–c (Group II) against
Desulfovibrio piger Vib-7 expressed as log(1/MIC [M]) on
lipophilicity.
Fig. 2 – Dependence of antibacterial effect of tested
compounds 1a–f (Group I) and 2a–c (Group II) against
Desulfomicrobium sp. Rod-9 expressed as log(1/MIC [M]) on
lipophilicity.
j o u r n a l o f a p p l i e d b i o m e d i c i n e 1 4 ( 2 0 1 6 ) 1 2 5 – 1 3 0128
their function as proton shuttles (Zadrazilova, 2013; Zadrazilova
et al., 2015a,b). Nevertheless, such sensitivity of studied
intestinal SRB to salicylamides derivatives can be caused by
the peculiarities of their metabolism including their physiological,
biochemical, cytological and ecological properties
(Barton and Hamilton, 2010; Kushkevych, 2012a,b).
Despite the fact that both evaluated bacteria belong to the
same group and even physiological subgroup, it is known that
Desulfovibrio species is different from Desulfomicrobium in
biochemical and physiological properties. Genera Desulfovibrio
and Desulfomicrobium have the same basic structure of bacterial
cell wall (peptidoglycan), but Desulfovibrio have such phenotypic
features as the presence of desulfoviridin, cytochrome c3 and
menaquinone MK-6. Desulfomicrobium contains desulforubidin,
not desulfoviridin, and also the structure of cytochrome is
different. The activity, structure and properties of vital enzymes
of Desulfovibrio and Desulfomicrobium genera differ (Holt et al.,
1994; Brenner et al., 2005; Barton and Hamilton, 2010).
Based on above-mentioned facts, the inhibitory effect of
salicylamides 1a–f and 2a–c can be obviously caused by their
action on the process of dissimilatory sulfate reduction in both
Desulfovibrio and Desulfomicrobium genera and their growth and
productionofhydrogensulfide.However,thedifferenceinthese
parameters may be due to different mechanisms of sulfate
transport, the presence in their cells of different transport
systems or, even, enzymes providing this process. Relative
survival in D. piger Vib-7 or Desulfomicrobium sp. Rod-9 cells and
antimicrobial effect under the influence of salicylamides
derivatives 1a–f and 2a–c is different. It is due to the chemical
structure of these compounds (diamides were described as
bactericidal agents) and the genus characteristics of Desulfovibrio
and Desulfomicrobium.
Conclusions
Compounds of Group 1 1a–f (2-(phenylcarbamoyl) phenyl
N-[(benzyloxy)carbonyl]alkanoates) and Group II 2a–c (2-hy-
droxy-N-[(2S)-1-oxo-1-(phenylamino)alkan-2-yl]benzamides)
were found to inhibit the intestinal bacterial growth of D. piger
Vib-7 and Desulfomicrobium sp. Rod-9. 4-Chloro-N-{(2S)-1-[(3,4-
dichlorophenyl)amino]-3-methyl-1-oxobutan-2-yl}-2-hydroxybenzamide
(2b) demonstrated the highest potency against both
strains:MIC = 0.22 mMagainstD.pigerandMIC = 0.27 mMagainst
Desulfomicrobium sp. 4-Chloro-2-[(4-nitrophenyl)carbamoyl]
phenyl (2S)-2-{[(benzyloxy)carbonyl]amino}-3-methylbutanoate
(1d) showed high activity against D. piger Vib-7
(MIC = 0.26 mM) and 4-chloro-2-[(4-methylphenyl)carbamoyl]
phenyl (2S)-2-[(tert-butoxycarbonyl)amino]-3-(1H-indol-2-yl)
propanoate (1b) showed high activity against Desulfomicrobium
sp. Rod-9 (MIC = 0.31 mM). Lipophilicity was recognized as a
significant parameter affecting biological activities. It is not
possible to assess if R1
substitution in C(4) or C(5) is more
advantageous, but in general for both SRB strains rather
electron-withdrawing R2
substituent and less lipophilic (isopropyl
or benzyl) R3
substituent are also more favourable for higher
activity. Based on the results it can be hypothesized
that salicylamide derivatives interact with enzymatic systems
of the bacteria affecting vital cell functions, and diamide 2b
could probably serve as bactericidal active agent. Therefore,
salicylamide derivatives seem to be promising candidates of
potential agents with activity against sulfate-reducing bacteria.
Conflict of interest
The authors report no conflict of interests. The authors alone
are responsible for the content and writing of the paper.
Acknowledgements
This study was supported by IGA VFU Brno 44/2014/FaF and by
project CZ.1.07/2.3.00/30.0053 at VFU Brno. The authors wish
to acknowledge the institutional support of the Faculty
of Chemical Technology, University of Pardubice funded by
the Ministry of Education, Youth and Sports of the Czech
Republic.
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and their derivatives against MRSA. (Rigorous thesis)
Department of Chemical Drugs, Faculty of Pharmacy and
Department of Infectious Diseases and Microbiology, Faculty
of Veterinary Medicine, University of Veterinary and
Pharmaceutical Sciences Brno, Czech Republic.
Zadrazilova, I., Pospisilova, S., Pauk, K., Imramovsky, A.,
Vinsova, J., Cizek, A., Jampilek, J., 2015a. In vitro bactericidal
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against MRSA.
Biomed. Res. Int. 1–8 Article ID 349534.
Zadrazilova, I., Pospisilova, S., Masarikova, M., Imramovsky, A.,
Monreal-Ferriz, J., Vinsova, J., Cizek, A., Jampilek, J., 2015b.
Salicylanilide carbamates: promising antibacterial agents
with high in vitro activity against methicillin-resistant
Staphylococcus aureus (MRSA). Eur. J. Pharm. Sci. 77, 197–207.
Zinkevich, V., Beech, I.B., 2000. Screening of sulfate-reducing
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j o u r n a l o f a p p l i e d b i o m e d i c i n e 1 4 ( 2 0 1 6 ) 1 2 5 – 1 3 0130
ATTACHMENT 6
Pages 80 – 85
_____________________________________________________________________________
Kinetic properties of growth of intestinal sulphate-reducing bacteria isolated
from healthy mice and mice with ulcerative colitis
Ivan Kushkevych, Monika Vítězová, Pavla Fedrová, Zora Vochyanová,
Lenka Paráková, Jan Hošek
Acta Veterinaria Brno, 2015, 86, 405-411, ISSN 0001-7213
_____________________________________________________________________________
Kinetic properties of growth of intestinal sulphate-reducing bacteria
isolated from healthy mice and mice with ulcerative colitis
Ivan Kushkevych1,2
, Monika Vítězová1
, Pavla Fedrová1
, Zora Vochyanová3
,
Lenka Paráková2
, Jan Hošek3
1
Masaryk University, Faculty of Science, Department of Experimental Biology,
Section of Microbiology, Brno, Czech Republic
2
University of Veterinary and Pharmaceutical Sciences Brno, Faculty of Pharmacy,
Department of Human Pharmacology and Toxicology, Brno, Czech Republic
3
University of Veterinary and Pharmaceutical Sciences Brno, Faculty of Pharmacy,
Department of Molecular Biology and Pharmaceutical Biotechnology, Brno, Czech Republic
Received January 13, 2017
Accepted December 19, 2017
Abstract
Inflammatory bowel disease including ulcerative colitis are complex multifactorial diseases of
unknown aetiology. Sulphate-reducing bacteria are often associated with the occurrence of the
disease. The physiological properties of intestinal sulphate-reducing bacteria including kinetic
characteristic of their growth have never been reported. The aim of this research was to evaluate
the presence of sulphate-reducing bacteria isolated from the intestines of mice, study their growth,
calculate and compare the kinetic growth properties on the model of dextran sulphate sodium
induced ulcerative colitis in the mice. The number of viable intestinal sulphate-reducing bacteria
from the bowel lumen of mice with ulcerative colitis was higher (P > 0.05) by 22% at 12 h of
cultivation compared with cultures of sulphate-reducing bacteria from the bowel lumen of healthy
mice. The sulphate-reducing bacteria from mice with colitis also had a slightly higher generation
time (14.29 h) and exponential growth phase (22.24 h) compared with cultures from healthy
mice. The time of lag-phase was 2 × shorter (P > 0.01) in the cultures of sulphate-reducing
bacteria from mice with ulcerative colitis. The described research is new and important for the
prediction of the sulphate-reducing bacteria number in the gut and their rate of dissimilatory
sulphate reduction. The kinetic characteristic of their growth is important for further clarification
of the mechanisms of sulphate reduction and accumulation of hydrogen sulphide, which is toxic
for epithelial cells of the intestine and can cause bowel diseases both in humans and animals, in
particular ulcerative colitis.
Intestinal microbiota, growth rate, hydrogen sulphide, bowel diseases
Sulphate-reducing bacteria (SRB) are widespread in anaerobic areas of soils, wetlands,
fresh and marine waters and available in the microbiota of the large intestine of humans
and animals (Barton and Hamilton 2010). These microorganisms metabolize sulphate
as an electron acceptor to hydrogen sulphide. The sulphate dissimilation process is called
the “dissimilatory sulphate reduction” or “sulphate respiration” (Kushkevych 2016a,b).
For this process, SRB needs exogenous electron donors, including organic compounds
or molecular hydrogen. Dependent on SRB genera, organic compounds are oxidized
incompletely to acetate (acetogenic SRB) or completely to carbon (IV) oxide (Barton and
Hamilton 2010).
The intensity of sulphate reduction in SRB and, accordingly, the accumulation of hydrogen
sulphide in high or toxic concentrations in the intestines can lead to the development of
various diseases (Kushkevych 2014a). Hydrogen sulphide is the final product in the
sulphate reduction process of SRB metabolism. At high concentrations, this final metabolite
ACTA VET. BRNO 2017, 86: 405–411; https://doi.org/10.2754/avb201786040405
Address for correspondence:
Ivan Kushkevych, M.Sc., Ph.D., Dr.Sc., Assoc. Prof.
Section of Microbiology
Department of Experimental Biology, Masaryk University
Kamenice 753/5, 625 00 Brno, Czech Republic
Phone: +420 549 49 5315
Fax:  +420 541 211 214
E-mail: kushkevych@mail.muni.cz
http://actavet.vfu.cz/
is toxic and carcinogenic for the intestinal cell and can cause inhibition of cytochrome
oxidase, colonocytes oxidation of butyrate, destruction of epithelial cells, and development
of ulcers and inflammation with subsequent development of ulcerative colitis (Pitcher et al.
1996; Gibson et al. 1991; Cummings et al. 2003; Kushkevych 2015c,d).
Acetogenic SRB and their final products of metabolism (including hydrogen sulphide and
acetate) are often found in the faeces of people with bloody diarrhoea (Pitcher et al. 1996)
and the mono and polymicrobial infections of the gastrointestinal tract (McDougall et al.
1997). It is believed that SRB can cause frequent defecation, weight loss, and increased
intestinal permeability (Kushkevych 2016b). The species and quantitative composition
of the SRB on the surface of the intestinal mucosa are different from those microorganisms
in the lumen (Macfarlane et al. 2000, 2007; Zinkevich et al. 2000; Kushkevych 2015e).
Such genera of SRB, Desulfovibrio, Desulfomicrobium, Desulfobacter, Desulfobulbus,
Desulfotomaculum, Lawsonia and Bilophila, are the most isolated from the intestines
of healthy and ill humans and animals (Gibson et al. 1991, 1993; Kushkevych 2016b).
The number of cases of inflammatory bowel disease (IBD) including ulcerative colitis
(UC) is growing. The cause of the occurrence is still unknown (Cummings et al. 2003).
This disease is mostly observed in the human population of the age group of 15–30 years;
although there is clear evidence of this disease occurring in the human population of the
age group of 50–70 years, which can be labelled as a potential risk group. Frequent cases
of this disease are recorded in both developed and underdeveloped countries (Garud and
Peppercorn 2009). Ulcerative colitis is diagnosed not only in people but also in animals;
e.g., horses, cows, pigs, dogs, cats, and rodents (mice and rats). However, the functional
role of SRB in the development of inflammatory bowel disease including ulcerative colitis
and their participation in the mechanisms of the disease have never been studied properly.
The physiological properties of intestinal SRB including the kinetic characteristic of the
SRB growth (doubling time, absolute and relative (specific) growth rate, the maximum
growth rate and time of lag-phase) and their comparison between healthy mice and mice
with induced ulcerative colitis, have never been reported.
The aim of our research was to induce ulcerative colitis in mice using dextran sulphate
sodium, accumulate the SRB cultures from the intestines of healthy mice and of mice with
ulcerative colitis, isolate and identify SRB cultures to study their growth, calculate and
compare the kinetic growth properties.
Materials and Methods
Manipulation with animals
Male C57Bl/6 mice (20 g ± 2 g) were obtained from the Animal Breeding Facility of Masaryk University (Brno,
Czech Republic). They were kept under standard conditions (22 ± 2 °C, 50 ± 10% relative humidity) and alternating
12 h light/dark cycles. The animals had access to a standard diet and drinking water ad libitum. Manipulations with
the animals were carried out according to the bioethical rules as per the principles of the “European Convention for
the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes” adopted in Strasbourg
in 1986. The study was also approved by the “Commission for the Protection of Animals against Cruelty” and the
Ethics Committee of the University of Veterinary and Pharmaceutical Sciences in Brno, Czech Republic.
In total, 6 animals in two groups (4 + 2 animals in the first and second group, respectively) were randomly
separated and used in this experiment. In the dextran sulphate sodium (DSS) group (n = 4), colitis was induced
by administering 5% (w/v) DSS (MP Biomedicals, Illkirch-Graffenstaden, France; MW 36.000–50.000 Da) in
drinking water for 7 days. The mice in the intact group (n = 2) received drinking water only. On the last day
of experiment, the animals were killed by decapitation under isoflurane anaesthesia. The isolated distal colonic
segments were selected for the analysis of qualitative and quantitative composition of intestinal microflora of both
groups of the animals.
Accumulation of intestinal SRB
In total, 100 mg of faeces were taken from healthy mice and mice with UC. A 5 mm-piece of the intestine was
also taken for studying SRB which can be in biofilms from both groups of animals. All samples were suspended
in tubes (volume 1.5 ml) with pre-heated (36.6 °C) liquid modified Postgateʼs medium I (Postgate 1984)
of the following composition (g/l): Na2
SO4
(4.0), KH2
PO4
(0.3), K2
HPO4
(0.5), (NH4
)2
SO4
(0.2), NH4
Cl
406
(1.0), CaCl2
×6H2
O (0.06), MgSO4
×7H2
O (1.0), C3
H6
O3
(6 ml), yeast extract (1.0), Na3
C6
H5
O7
×2H2
O (0.3).
Additional solutions (g/l) were prepared separately using deionized water: sodium pyruvate (2 g in 100 ml
of H2
O, 100 ml), NaHCO3
(2 g in 50 ml of H2
O, 50 ml), Na2
S2
O4
(30 mg in 1 ml of H2
O, 1 ml), Mohrʼs salt
solution ((NH4)SO4
Fe(SO4
)2
×6H2
O) (10%) 10 ml, Na2
S×9H2
O (1%) 3 µl. The medium and solutions were
sterilized separately. Each of these 5 solutions was added to the medium. To adjust the pH value on 8.5 sterile 10N
solution of NaOH was used. The redox and anaerobic conditions were controlled by resazurin sodium (Oxoid,
BR 0055B) as an indicator. In addition, reduced FeS and Na2
S contained in the medium provided the necessary
redox conditions for SRB cultures. The discoloration of resazurin sodium (redox potential of discoloration
Eh = -100 mV) confirmed the decrease of the redox potential. The SRB suspensions were cultivated at 36.6 °C
under anaerobic conditions for 48 h. The tubes were filled with medium and closed by a rubber plug to provide
anaerobic conditions. The positive cultures of SRB created a black sediment because Mohr’s salt solution was
added to the medium. As a result, FeS was formed by the bacterial cells that caused the black sediment.
Isolation and identification of intestinal SRB pure cultures
For isolation of the SRB pure cultures, each positive suspension with SRB was diluted to 10-12
in a series
of tubes containing the liquid medium I. To obtain colonies for isolation, the same Postgateʼs agar (12 g/l) medium I
was prepared. To prevent solidification, the medium I in Erlenmeyer flask (500 ml) was placed in water bath tempered
to 45 °C. To obtain isolated SRB colonies, 20 ml of agar medium I + 100 µl of each dilution suspension were mixed
and consequently poured to Petri dishes. After cooling down, the dishes were placed into an anaerobic box with oxygen
uptake generators (GENbox anaer, bioMérieux, France) for anaerobiosis. The cultivation was carried out in incubator ES
120 (Nüve, Turkey) operated at 36.6 ± 0.5 °C. Black colonies were visible on the agar medium after 24 h of cultivation.
Isolated colonies were taken in the liquid medium I and grown for another 24 h. To make sure the samples were
pure culture, this process was repeated × 2–3. The identification of SRB was carried out as described previously
(Kushkevych 2013; Kushkevych et al. 2014b). The SRB isolates were identified as Desulfovibrio genus.
Additional tests of the identification (including morphological characteristics, physiological and biochemical
properties) showed that no other genera were present. The obtained SRB cultures were kept in the Laboratory
of Anaerobic Microorganisms of the Section of Microbiology and Molecular Biotechnology at the Department
of Experimental Biology at the Masaryk University (Brno, Czech Republic).
Testing of bacterial growth by the Bioscreen method
For the testing of bacterial growth, the suspensions of SRB and pure culture of Desulfovibrio genus isolated
from 2 healthy mice (5 isolates from the faeces + 5 isolates from the intestinal surface which were in biofilm) as
well as from 4 mice with UC (5 isolates from the faeces + 5 isolates from the intestinal surface).
Grown SRB in the liquid medium I without Mohr’s salt solution were diluted in the liquid fresh medium
I (transmittance was 90 at λ = 620 nm). Consequently, 320 µl of each suspension obtained were pipetted to the
wells (total volume 350 µl) of multiwell plates. To provide anaerobic condition, sterile paraffin oil (30 µl) was
added to each well. As a control, the medium without any sample (320 µl) + 30 µl of sterile paraffin oil was used.
For statistical evaluation, each sample was pipetted into 5 wells. For the study of bacterial growth, the Bioscreen
C (Oy Growth Curves Ab Ltd., Finland) was used. Bioscreen was set at a temperature of 36.6 °C, measuring
at a 30-min interval within the 24 h of measurement.
Calculation of growth indices and statistical analysis
The growth indices of SRB were characterized by the following basic constants (Widdel 2010): generation
time (G), absolute growth rate (R), relative (specific) rate (µ), the maximum growth rate (µmax
) and duration
of the lag-phase (L).
Generation time (G) was defined as the time (t) per generation of one bacterial cell (N = number of SRB cells
in the interval time), in practice, it is the duration of the cell cycle of one cell:
where t0
is initial time (t0
= 0) and N0
is initial number of bacterial cells (N0
= 5.69 log10
CFU·ml-1
). The absolute
growth rate (or doubling rate) was calculated by the number of divisions (generations) per unit time. The absolute
growth rate (R) was defined as the number of cells formed per time:
The relative (specific) growth rate (μ) was determined by the absolute growth rate related to the population size
(number cells formed per time and existing number of cells, N):
Maximum growth rate was considered as the maximum value of relative (specific) growth rate. Time of lagphase
was calculated by formula: L= tk
–te
, where tk
was the duration of the experiment, and te
was the duration of
the exponential growth phase:
407
0
00 loglog
3.2
lnln1
tt
NN
t
NN
Ndt
dN
−
−
⋅=
−
=⋅=µ
dt
dN InIn
0loglog
2log
1 0
NN
tt
R
G
−
−
⋅==
0
0loglog
2log
1
tt
NN
t
N
R
−
−
⋅==
)log(log
2log
1loglog
2log
1
0
0
NNG
R
NN
te −⋅⋅=
−
⋅=
Kinetic and statistical calculations of the results were carried out using the software MS Office and Origin
computer programs. Using the experimental data, the basic statistical parameters (mean: M, standard error:
m, M ± m) were calculated. The research results were treated by methods of variation statistics using Student’s t-test.
The significance of the calculated indicators of line was tested by Fisher’s F-test. The accurate approximation was when
P ≤ 0.05 (Bailey 1995).
Results
The first stage of our research was to compare the kinetic properties of SRB isolated
from the lumen (faeces) of both experimental groups. The number of viable intestinal SRB
from the bowel lumen of mice with UC was higher (P > 0.05) by 22% at 12 h of cultivation
compared to cultures of SRB from the bowel lumen of healthy mice (Fig. 1A). These data
correlate with specific maximal growth rate (µmax
) which was also higher (P > 0.05) by
13% in SRB from mice with UC. The specific maximal growth rate of the SRB from both
groups was achieved at 12–12.5 h (Fig. 1B). These microorganisms also had a slightly
higher generation time (14.29 h) and exponential growth phase (22.24 h) compared to
the cultures from healthy mice (Table 1). It should be noted that the time of the lag-phase
was × 2 shorter (P > 0.01) in the cultures of SRB from the faeces of mice with UC. That
may indicate adaptive capacities of the SRB and their participation in the development
of UC. The intestinal SRB from the faeces of healthy mice and mice with UC achieved
the exponential phase after an adaptation period (3.49 and 1.76 h, respectively) in the lag
phase. The stationary phase was achieved after 20.51 and 22.24 h, respectively (Table 1).
Sulphate-reducing bacteria can be found not only in the faeces or in the intestinal lumen
but they can also be in interaction with other intestinal microorganisms in the biofilms.
These biofilms are often formed by the SRB with species of Clostridium, Bacteroides
or Escherichia genera. However, the features of the formation of these biofilms and the
interaction of SRB with other bacterial genera are still unexplored.
Cultures of SRB from the intestinal surface of the biofilm of healthy mice and of mice with
UC were accumulated in the medium and the kinetic properties of their growth were studied
(Fig. 1C). The growth of SRB cultures from the bowel surface of both groups of mice was
almost the same until 4.5 h of cultivation. The level of growth of SRB from the biofilm of mice
with UC was increased (P > 0.05) within the time interval of 4.5–12.5 h and after that they
were released from the biofilm of healthy mice. The specific maximal growth rate was higher
(P > 0.05) by 26% in SRB from healthy mice compared to mice with UC (Table 1). However,
maximal growth rate of SRB from the biofilm of healthy mice was achieved earlier at 1.5 h,
whereas µmax
of SRB from the biofilm of mice with UC was achieved later at 9.5 h of cultivation
(Fig. 1D). These results correlated with specific means and absolute growth rates which were
also higher (P > 0.05) by 17–18% in SRB from healthy mice. The generation time (12.72 h) and
408
Suspension	 te	
Lag-phase	 G	 Absolute rate
	 Specific rate
of SRB from	 (h)	 (h)	 (h)	 (number of cells/hour)
	 (number of cells/hour)
	 Average	 µmax
	 healthy	 20.51	 3.49	 13.13	 0.076 ± 0.023	 0.052 ± 0.016	 0.063 ± 0.011
	 UC	 22.24	 1.76	 14.29	 0.069 ± 0.007	 0.048 ± 0.005	 0.071 ± 0.002
	 healthy	 19.74	 4.26	 12.72	 0.079 ± 0.009	 0.054 ± 0.062	 0.081 ± 0.014
	 UC	 21.21	 2.79	 15.48	 0.065 ± 0.023	 0.045 ± 0.014	 0.060 ± 0.005
Table 1. Kinetic properties of SRB cultures isolated from mice.
Comment: te
is duration of the exponential growth phase (hours), G is generation time (hours), µmax
is maximum growth rate,
SRB: sulphate-reducing bacteria, UC: mice with ulcerative colitis
FaecesSurface
(biofilm)
exponential growth phase (19.74 h) of these microorganisms were shorter (P > 0.05) compared
to the cultures of SRB from mice with UC. It should be noted that the time of the lag-phase in
SRB from surface was × 1.5 shorter (P > 0.05) in the cultures of SRB from mice with UC. It
correlated with the lag-phase in the SRB isolated from the bowel lumen of mice with UC. The
stationary phase of both SRB cultures was achieved after 20h of cultivation.
Discussion
Inflammatory bowel disease including ulcerative colitis are complex multifactorial
diseases of unknown aetiology (Cummings et al. 2003). However, the aetiological
role of the sulphate-reducing bacteria Desulfovibrio genus in the development of these
inflammatory diseases was demonstrated in our previous research (Kushkevych 2014a).
Ulcerative colitis was experimentally induced in the animals by application of sulphatereducing
bacteria. The initiation of the animals’ own potential intestinal microflora of
sulphate-reducing bacteria was observed in those animals which obtained a dose of
sulphate containing medium. The changes in the colonic microbiota were observed in
the animals which received a dose of SRB suspension. The bacteria belonging to the
normal colonic microbiota were associated with the aetiology of inflammatory bowel
disease and ulcerative colitis. The concentration of sulphide and acetate in faeces from
different sections of the large intestine was determined. The level of ulcerations in the
second and third group of sick animals under the specific conditions was demonstrated
(Kushkevych 2014a).
409
Fig. 1. Physiological indices of sulphate-reducing bacteria isolated from mice: bacterial growth (A, C) and
specific growth rate (B, D), the arrows indicate the maximum rate of the growth and CFU is colony-forming units
(M ± m, n = 5)
The increasing number of viable intestinal SRB was also described previously in several
researches published by Gibson et al. (1991, 1993), Kushkevych (2015a), Cummings
et al. (2003), and Pitcher et al. (1996). The increasing specific maximal growth rate
(µmax
) of SRB from mice with UC can lead to intensive sulphate reduction (Kushkevych
2014c). The increased concentration of sulphate in the intestine can lead to an increase in
the specific maximal growth rate of the SRB and the production of hydrogen sulphide and
acetate in high concentrations.
The reduction of sulphate ions to hydrogen sulphide occurs as a result of the formation
of many intermediate compounds (Postgate 1984). The sulphate reduction enzymes are
located in the cytoplasm and peripheral plasma. The initial stages of sulphate reduction
include the uptake of sulphate ions in the bacterial cells.The sulphate ions can be transported
into the cells simultaneously with protons and some sulphate-reducing bacteria can absorb
sulphate from the flow of sodium ions (Barton and Hamilton 2007). It is known that the
centre of adenosine triphosphate (ATP) hydrolysis is located on the cytoplasmic surface
of the membrane. In another one of our studies, the ATPase activity in cell-free extracts
of the sulphate-reducing bacteria isolated from the human large intestine was demonstrated.
The maximum ATPase activity for SRB strains at +35 °C and at pH 7.0 was described
(Kushkevych et al. 2015).
The described research is novel and important to predict the SRB number in the gut
and their speed of dissimilatory sulphate reduction. The kinetic characteristic of their
growth is important for further clarification of the mechanisms of sulphate reduction and
accumulation of hydrogen sulphide, which is toxic for epithelial cells of the intestine
and can cause bowel disease both in humans and animals, in particular ulcerative colitis.
These results could be particularly useful for the study of IBD and its therapeutic strategy.
These data are also indispensable for application into mechanistic details that will facilitate
better preclinical drug/therapy design to target specific components involved in the disease
pathogenesis.
Acknowledgements
This study was supported by the University of Veterinary and Pharmaceutical Sciences Brno (project OPVK
“Pharmaco-toxicological evaluation of newly synthesized (isolated) compounds as an integration tool for preclinical
disciplines at VFU Brno” CZ.1.07/2.3.00/30.0053). This project was also supported by the grant of the
Ministry of Health of the Czech Republic No. 16-27522A (to Z. V. and J. H.) and by the Grant Agency of the
Masaryk University (MUNI/A/0906/2016).
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Kushkevych I, Fafula R, Parák T, Bartoš M 2015b: Activity of Na+
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in the cell-free extracts of the sulfate-reducing bacteria Desulfovibrio piger Vib-7 and Desulfomicrobium sp.
Rod-9. Acta Vet Brno 84: 3-12
Kushkevych I, Kollar P, Suchy P. et al. 2015c: Activity of selected salicylamides against intestinal sulfatereducing
bacteria. Neuroendocrinol Lett 36:106-113
Kushkevych I 2015d: Kinetic Properties of Pyruvate Ferredoxin Oxidoreductase of Intestinal Sulfate-Reducing
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Kushkevych I, Kollar P, Ferreira A, Palma D, Duarte A, Lopes M, Bartos M, Pauk K, Imramovsky A, Jampilek
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ATTACHMENT 7
Pages 86 – 95
_____________________________________________________________________________
Production of biogas: relationship between methanogenic and sulfate-reducing microorganisms
Ivan Kushkevych, Monika Vítězová, Tomáš Vítěz, Milan Bartoš
Open Life Sciences, 2017, 12, 82-91, ISSN 2391-5412
_____________________________________________________________________________
Open Life Sci. 2017; 12: 82–91
Keywords: biogas, methane production, methanogenic
microorganisms, sulfate-reducing bacteria
1 Introduction
Bioenergy production from agricultural, municipal, and
industrial waste is efficiently accomplished through
anaerobic digestion to biogas. Biogas produced usually
contains 55 – 70% of methane (CH4
) and 30 – 45% of
carbon dioxide (CO2
) and it is stored in gas holders and
subsequently used as a potential source of energy [1-4].
Anaerobic fermentation is a natural process in which
microorganisms convert biodegradable substrate into
biogas. It occurs in marshes, wetlands, river sediments,
and also in the digestive tract of ruminants [5,6]. The
microorganisms are also active in landfills where they
are responsible for biodegradable waste decomposition
[2,5,7].Biogasisthefinalproductofanaerobicmetabolism.
The process occurs in an anaerobic environment through
the consecutive biochemical breakdown of polymers to
methane and carbon dioxide [8, 9]. This is a result of the
metabolism of different microorganisms which include
fermentative microbes (acidogens); hydrogen-producing,
acetate-forming microbes (acetogens); and methaneproducing
microbes (methanogens) [10-13].
Recently, the interest in anaerobic fermentation is
mainly focused on its use in the economic recovery of
biogas from industrial and agricultural surpluses [14-16].
On the basis of homologous sequence analysis of 16S
rRNAs, methanogens have been classified into one of the
three primary kingdoms of living organisms: the Archaea
[10,14,17].RecombinantDNAtechnologyisoneofthemost
powerful techniques for characterizing the biochemical
and genetic regulation of methanogenesis [18]. This
necessitates the selection of genetic markers, an efficient
genetic transformation system, and a vector system for
genetic recombination as prerequisites [19]. Anaerobic
methane generation systems are known as methane
bioreactors. The production of methane and growth of
methanogenic microorganisms in the bioreactors depend
DOI 10.1515/biol-2017-0009
Received November 11, 2016; accepted March 1, 2017
Abstract: Theproductionofhigh-qualitymethanedepends
on many factors, including temperature, pH, substrate,
composition and relationship of the microorganisms. The
qualitative and quantitative composition of methanogenic
and sulfate-reducing microorganisms and their
relationship in the experimental bioreactors has never
been studied. The aim of this research was to characterize,
for the first time, the diversity of the methanogenic
microorganisms and sulfate-reducing bacteria, and study
their relationship and biogas production in experimental
bioreactors. Amplification of 16S rRNA gene fragments
was carried out. Purified amplicons were paired-end
sequenced on an Illumina Mi-Seq platform. The dominant
morphotypes of these microorganisms in the bioreactor
were homologous (99%) by the sequences of 16S rRNA
gene to the Methanosarcina, Thermogymnomonas,
Methanoculleus genera and Archaeon deposited in
GenBank. Three dominant genera of sulfate-reducing
bacteria, Desulfomicrobium, Desulfobulbus and
Desulfovibrio, were detected in the bioreactor. The
phylogenetic trees showing their genetic relationship
were constructed. The diversity and number of the genera,
production of methane, hydrogen sulfide and hydrogen in
the bioreactor was investigated. This research is important
for understanding the relationship between methanogenic
microbial populations and other bacterial physiological
groups, their substrate competition and, in turn, can be
helpful for controlling methanogenesis in bioreactors.
© 2017 Ivan Kushkevych et al., published by De Gruyter Open.
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License.
*Corresponding author: Ivan Kushkevych, Department of
Experimental Biology, Faculty of Science, Masaryk University,
Kamenice 753/5, 62500 Brno, Czech Republic;
E-mail: ivan.kushkevych@gmail.com
Monika Vítězová, Milan Bartoš, Section of Microbiology and Molecular
Biotechnology, Department of Experimental Biology, Faculty of
Science, Masaryk University, Brno, Czech Republic
Tomáš Vítěz, Department of Agricultural, Food and Environmental
Engineering, Faculty of Agronomy, Mendel University in Brno, Brno,
Czech Republic
Ivan Kushkevych*, Monika Vítězová, Tomáš Vítěz, Milan Bartoš
Production of biogas: relationship between
methanogenic and sulfate-reducing microorganisms
Research Article									 Open Access
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Production of biogas: relationship between methanogenic and sulfate-reducing microorganisms   83
KGaA Germany). Concentration of CH4
, CO2
, H2
S and H2
in
the biogas produced was determined. Quantity of biogas
produced was measured by the gasometer PREMGAS BK
G4 (Elster, Germany) and converted to standard conditions
(T0
= 273 K, p0
= 101325 Pa).
2.2 Determination of the physical and
chemical characteristics
The physical and chemical characteristics of fermenter
content at each bioreactor, including temperature, pH,
redox potential, dry matter content, organic dry matter
content, biogas composition, sulfate content and acetate
content were determined. DM content was determined by
oven drying at 105±5°C followed by cooling in a desiccator
and weighing until a constant weight. EcoCELL 111 (BMT
Medical Technology Ltd., Czech Republic) was used
in accordance with the Czech Standard Method  (CSN
EN 14346 2007) [23]. ODM content was determined by
incineration of the samples in a muffle furnace at 550°C ±
5°C in accordance with the Czech Standard Method (CSN
EN 15169 2007) [24], using a muffle furnace LMH 11/12
(LAC, Ltd., Czech Republic). The pH, and redox potential
were determined by using pH/Cond meter 3320 (WTW
GmbH,Germany)inaccordancewiththestandard(CSNEN
12176 1999) [25]. Temperature of samples was determined
by using the high accuracy PT100 RTD thermometer
HH804U (OMEGAEngineering,INC.,USA).Acetatecontent
was determined using capillary electrophoresis Ionosep
2005 (RECMAN, Czech Republic). For determination of
sulfate concentration, the spectrophotometer DR 3800
(Hach Lange GmbH, Germany) was used.
2.3 Isolation of DNA from collected samples
For DNA isolation samples from all 6 reactors were taken.
These samples were combined and then examined.
The QIAamp Fast DNA Stool Mini Kit (QIAGEN GmbH,
Germany) is designed for rapid purification of total DNA
from stool samples and was used for DNA extraction from
biogas digester samples. DNA extraction was performed
in accordance to the manufacturers’ instructions, with
minor adjustments as described below. Briefly, 100 mg of
sample was washed with 1.4 mL of ASL buffer (QIAGEN
GmbH, Germany), heated at 95°C for 10 minutes. After
centrifugation, an InhibitEX tablet was put in the
supernatant to remove impurities and PCR inhibitors.
After thorough centrifugation, 200 μL of the supernatant
was added to 15 μL of proteinase K, and 200 μL of buffer
on many factors, including temperature, pH, substrate
quality, composition of specific groups of microorganisms
and their accumulation of the toxic metabolic products.
One of such final products of sulfate-reducing bacteria
metabolism is hydrogen sulfide produced in the process
of dissimilatory sulfate reduction [6, 7, 20-22].
It is known that hydrogen sulfide is toxic for living
organisms and can inhibit enzymes of different groups of
microorganisms [6, 20]. The sulfate-reducing bacteria can
compete with the methanogens for substrate components,
in this case for molecular hydrogen, and produce hydrogen
sulfide in high concentration in bioreactors. It, in turn,
can inhibit the growth of methanogenic microorganisms
and their process of methanogenesis.
Recent progress in the molecular biology of
methanogens is reviewed, new digesters are described
and improvements in the operation of various types of
bioreactors are also discussed. However, the prevalence
of the methanogenic populations and sulfate-reducing
bacteria in methane bioreactors, their diversity and
relationship has never been studied properly.
The aim of this research was to characterize the
diversity of the methanogenic microorganisms and
sulfate-reducing bacteria using amplification of gene
fragments and Illumina sequencing, and to study their
relationship and production biogas in the experimental
bioreactors.
2 Materials and Methods
2.1 Batch anaerobic fermentation tests
A total of six laboratory batch fermenters of volume 120
liters each were used. Fermenters were equipped with a
low speed agitator (operated at 60 rpm, 1 min agitation
time, 60 min rest), temperature control, monitoring of
pH, and sampling valve. On the first day of experiments,
all fermenters were filled up with inoculum substrate
and maize silage, in ratio 35:65% (v/v). Inoculum and
maize silage was collected at a full scale biogas plant
in Čejč, Czech Republic. This biogas plant processes
as feedstock maize silage and liquid pig manure and
it is operated in the mesophilic temperature regime
(39°C). Inoculum characteristics were pH=7.3±0.1; DM
3.84%±0.3%; ODM 72.71%±0.4%. For maize silage, dry
matter (DM) was 39.15%±0.5 and organic dry matter (ODM)
was 99.21%±0.6%. Fermenters were set to 39°C ± 0.1°C.
The hydraulic retention time was 26 days. During the
fermentation, the quality of biogas was determined daily
using X-am®
7000 gas analyzer (Drägerwerk AG & Co.
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84   I. Kushkevych, et al.
using the experimental data. For estimation of the validity
of difference between the statistical characteristics of the
data, Student’s index was calculated. The difference was
valid when P<0.05 [31, 32].
3 Results
The physical and chemical characteristics of samples
taken on the 14th day from the experimental bioreactors,
operated at temperature 39°C±0.5, were pH (7.2±0.1),
redox (-5.6±0.2), total solids (3.76%±0.3), volatile solids
(70.85%±0.9). Biogas composition during the tests is
shown in figure 1. Concentration of acetic acid ranged
from 460 to 490 mg/L and concentration of sulfate ranged
from 305 to 333 mg/L, while the sulfate concentration in
fermenters on the first day of experiment ranged from
380 to 450 mg/L. The results of our research show that
methane was intensively produced for the first 12 days,
after this time, the methane production achieved plateau
and was almost unchanged (Fig. 1). This can be explained
by the fact that the methanogenic microorganisms can
achieve the stationary growth phase after 12 days. As
growth slowed, methane production (concentration)
achieved a stable level (46.2–49.3%vol
) during the 15–26
day period. An interesting pattern was observed in the
production of hydrogen and hydrogen sulfide. The level
of hydrogen rapidly grew the first 5 days and after that it
was reduced. This can be explained by the fact that both
hydrogenotrophic methanogens and sulfate-reducing
bacteria consume this simple molecule. This is also
evident when isolated microorganisms are examined
and compared. Both acetotrophic and hydrogenotrophic
metanogens were isolated. Sulfate-reducing bacteria
can use hydrogen as an electron donor in the process of
AL (QIAGEN GmbH, Germany) was added. The mixture
was heated to 70°C for 10 minutes and 200 μL of absolute
ethanol was added. The sample mixture was then passed
through the QIAamp kit column, followed by two washes
with buffers AW1 and AW2 (QIAGEN GmbH, Germany). The
DNA was eluted in a volume of 200 μL of elution buffer.
2.4 Amplification of gene fragments and
Illumina sequencing
Amplification of 16S rRNA gene fragments was carried out
using universal primers for amplification of the V3 and V4
variable regions of the 16S rRNA [26]. The primers were
flanked by molecular barcodes for sample identification.
The PCR reaction was prepared using Maxima™ Probe
qPCR Master Mix (Thermo Fisher Scientific, USA) with
cycling conditions as follows: 95°C for 10 min. followed
by 30 cycles of incubation at 94°C for 30 s, 60°C for 30 s
and 72°C for 120 s, and a final extension step at 72°C for
2 min. PCR products were visualized using electrophoresis
on 1.5% agarose gels and purified from the gel using the
QIAquick Gel Extraction Kit (QIAGEN GmbH, Germany).
DNA was quantified using the Quant-iTPicoGreen dsDNA
Assay (Thermo Fisher Scientific, USA) and equimolar
amounts of the PCR products were pooled together.
Purified amplicons were paired-end sequenced on an
Illumina Mi-Seq platform. QIIME data analysis package
was used for 16S rRNA data analysis [27]. Quality filtering
on raw sequences was performed in accordance to base
quality score distributions, average base content per read
and GC distribution in the reads. Chimeras and reads that
did not cluster with other sequences were removed. The
obtained sequences with qual scores higher than 20 were
shortened to the same length of 350 bp and classified
with RDP Seqmatch with an operational taxonomic
unit (OTU) discrimination level set to 97%. The relative
abundance of the taxonomic groups was calculated to the
microorganisms detected in this study.
Sequences were compared using the BLAST feature
of the National Center for Biotechnology Information
(NCBI) [28]. The sequences were uploaded to Mega7
for comparative genomic analyses [29]. Alignments of
sequences were performed in Mega7 using Clustal W
and clustering was performed by the neighbor-joining
method [30].
The research results were analyzed using software
packages Statistica12 (www.statistica.software.informer.
com) and Origin7.0 (www.originlab.com). The basic
statistic parameters (arithmetic average (M) and standard
error (m) of the arithmetic average, M ± m) were calculated
Fig. 1: Methane, hydrogen and hydrogen sulfide production in the
biogas generated during fermentation test
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Production of biogas: relationship between methanogenic and sulfate-reducing microorganisms   85
KY172640, KY172645, KY172824, KY172823, KY126837. The
obtained sequences were compared with reference strains
using nucleotide Blast:Search. The sequences of the 16S
rRNA gene of the methanogens were homologous (98–
99%) to genera of Methanosarcina, Thermogymnomonas,
Methanoculleus, and Archaeon (Table 1). The sequences
of sulfate-reducing bacteria were homologous (98–99%)
to the genera of Desulfomicrobium, Desulfobulbus and
Desulfovibrio (Table 2). It should be noted that most
described sequences of the methanogenic strains and
sulfate-reducing bacteria in GenBank are identified only
to the domain, kingdom, family or genus and, in some
cases, to species. Most of them are uncultured (www.ncbi.
nlm.nih.gov/genbank).
Based on all of the 16S rRNA gene sequences of
methanogens and sulfate-reducing bacteria from the
bioreactor, a phylogenetic tree demonstrating their
genetic relationship was built (Fig. 2, 3). The detected
genera of methanogens were homologous with
Methanosarcina mazei strain GS14-2aM, uncultured
dissimilatory sulfate reduction. The final product of this
process is hydrogen sulfide. The highest concentration
of hydrogen sulfide was achieved on the 14th day, which
can confirm the high number of sulfate-reducing bacteria
(1050 OUT·mL-1
) in the bioreactor. Different studies
have found both unionized and total hydrogen sulfide
concentrations important in inhibition of the SRB bacteria
and methanogens. However, our results do not confirm
the inhibitory effect of H2
S at maximal concentration
390 ppm, as evident by the biogas production trend.
The distribution of main genera in the bioreactor was
investigated using amplification of 16S rRNA gene and
Illumina sequencing. To clarify the genetic relationship
of the methanogenic and sulfate-reducing populations of
microorganismsinthebioreactor,sampleof16SrRNAgene
sequences were compared with sequences of different
strains from GenBank. The genomic sequences of the
microorganisms are available in GenBank under accession
no. KY172649, KY172662, KY172650, KY194790, KY172816,
KY172822, KY172821, KY172646, KY172643, KY172641,
Table 1: The results of sequence analysis of the 16S rRNA gene of methanogenic microorganisms
Detected sequences and their
accession number in GenBank
(length of the gene fragment )
Reference strains in GenBank Accession number Identity (%)
Sequence 1
KY172649
(424 bp)
Methanosarcina mazei strain GS14-2aM 16S rRNA gene, partial
sequence
KX826992.1 99
Methanosarcina sp. 1H1 gene for 16S rRNA, partial sequence LC170394.1 99
Uncultured Methanosarcina sp. clone T6190SA18-18 16S rRNA
gene, partial sequence
KU355742.1 99
Sequence 2
KY172662
(422 bp)
Uncultured archaeon clone g10-56 16S rRNA gene, partial
sequence
JX576125.1 98
Uncultured Thermoplasmata archaeon clone g8-4 16S rRNA gene,
partial sequence
JX576112.1 98
Uncultured Methanomassiliicoccus sp. clone LZNG25 16S rRNA
gene, partial sequence
JX456453.1 98
Sequence 3
KY172650
(422 bp)
Uncultured Thermogymnomonas sp. partial 16S rRNA gene,
isolate OTU_11
LT624815.1 99
Uncultured Thermogymnomonas sp. partial 16S rRNA gene,
isolate OTU_4
LT624808.1 99
Uncultured archaeon partial 16S rRNA gene, clone AKA055 LN874207.1 99
Sequence 4
KY194790
(423 bp)
Uncultured Thermoplasmatales archaeon partial 16S rRNA gene,
isolate OTU_9
LT624813.1 99
Uncultured Thermoplasmatales archaeon partial 16S rRNA gene,
isolate OTU_6
LT624810.1 99
Uncultured archaeon clone WWA-D10 16S rRNA gene, partial
sequence
KM870439.1 99
Sequence 5
KY172816
(420 bp)
Methanoculleus sp. strain Biowerk_c-HAW 16S rRNA gene, partial
sequence
KX619406.1 99
Methanoculleus bourgensis isolate BA1 genome assembly,
chromosome: I
LT549891.1 99
Methanoculleus sp. MAB1 isolate MAB1 genome assembly,
chromosome: chrI
LT158599.1 99
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86   I. Kushkevych, et al.
Table 2: The results of sequence analysis of the 16S rRNA gene of sulfate-reducing bacteria
Detected sequences and
their accession number
in GenBank (length of the
gene fragment )
Reference strains in GenBank Accession number Identity (%)
Sequence 1
KY172822
(465 bp)
Desulfovibrio desulfuricans strain E4 16S rRNA, partial sequence KJ459863.1 99
Desulfovibrio desulfuricans strain E2 16S rRNA, partial sequence KJ459861.1 99
Uncultured bacterium partial 16S rRNA gene, clone 48h8 HG531899.1 99
Sequence 2
KY172821
(465 bp)
Uncultured Desulfovibrio sp. clone MFC-2-L19 16S rRNA gene, partial
sequence
JX944554.1 99
Uncultured bacterium clone MFC4P_127 16S rRNA gene, partial sequence JF309175.1 99
Desulfovibrio simplex strain JCM 16812 16S rRNA gene, partial sequence NR_113296.1 99
Sequence 3
KY172646
(466 bp)
Desulfobulbus propionicus strain DSM 2032 16S rRNA gene, complete
sequence
NR_074930.1 99
Desulfobulbus propionicus DSM 2032, complete genome CP002364.1 99
Uncultured bacterium clone B01 16S rRNA gene, partial sequence EU136253.1 99
Sequence 4
KY172643
(466 bp)
Desulfobulbus sp. canine oral taxon 078 clone OC011 16S rRNA gene,
partial sequence
JN713241.1 96
Uncultured Desulfobulbus sp. partial 16S rRNA gene, isolate OTU 265 LT625152.1 96
Uncultured Desulfobulbus sp. partial 16S rRNA gene, isolate OTU 199 LT625082.1 96
Sequence 5
KY172641
(466 bp)
Uncultured Desulfomicrobium sp. gene for 16S rRNA, partial sequence,
clone:3CP(-)_3
AB908615.1 96
Uncultured Desulfomicrobium sp. gene for 16S rRNA, partial sequence,
clone:3CP(-)_2
AB908614.1 96
Uncultured Desulfomicrobium sp. gene for 16S rRNA, partial sequence,
clone: 3CP(+)_10
AB908535.1 96
Sequence 6
KY172640
(466 bp)
Desulfobulbus sp. Prop6 16S rRNA gene, partial sequence KU845305.1 99
Uncultured bacterium clone 02d01 16S rRNA gene, partial sequence GQ138500.1 99
Uncultured bacterium clone 04c04 16S rRNA gene, partial sequence GQ134634.1 99
Sequence 7
KY172645
(466 bp)
Uncultured Desulfobulbus sp. partial 16S rRNA gene, isolate OTU 265 LT625152.1 94
Uncultured Desulfobulbus sp. partial 16S rRNA gene, isolate OTU 199 LT625082.1 94
Desulfobulbus sp. oral taxon 041 clone WWP_SS1_G06 16S rRNA gene,
partial sequence
GU398208.1 94
Sequence 8
KY172824
(466 bp)
Uncultured sulfate-reducing bacterium clone 2R1V11 16S rRNA gene,
partial sequence
EF592786.1 93
Uncultured delta proteobacterium clone 2R1U31 16S rRNA gene, partial
sequence
EU104788.1 93
Uncultured sulfate-reducing bacterium clone 2R1V05 16S rRNA gene,
partial sequence
EF592783.1 92
Sequence 9
KY172823
(443 bp)
Uncultured Desulfovibrionales bacterium clone Flu2 26 16S rRNA gene,
partial sequence
JQ701289.1 84
Desulfovibrio sp. S4 gene for 16S rRNA, partial sequence LC186051.1 83
Uncultured Desulfovibrio sp. gene for 16S rRNA, partial sequence, clone:
LR333B-40
LC001349.1 83
Sequence 10
KY126837
(465 bp)
Uncultured Desulfovibrio sp. clone MFC-2-L19 16S rRNA gene, partial
sequence
JX944554.1 95
Desulfovibrio simplex strain JCM 16812 16S rRNA gene, partial sequence NR_113296.1 95
Desulfovibrio intestinalis partial 16S rRNA gene, strain JG-G12 AJ295680.1 95
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Production of biogas: relationship between methanogenic and sulfate-reducing microorganisms   87
DSM 2032, Desulfobulbus sp. clone OC011, uncultured
Desulfomicrobium sp. clone: 3CP(-)_3, Desulfobulbus sp.
Prop6, uncultured Desulfobulbus sp. isolate OTU 265,
uncultured sulfate-reducing bacterium clone 2R1V11,
uncultured Desulfovibrionales bacterium clone Flu2_26
(Fig. 3).
Percentage ratio of methanogenic and sulfatereducing
microorganisms was calculated by OUT·mL-1
archaeon clone g10-56, uncultured Thermogymnomonas
sp. isolate OTU_11, uncultured Thermoplasmatales
archaeon isolate OTU_9, Methanoculleus sp. strain
Biowerk_c-HAW (Fig. 2). Another phylogenetic tree was
built for 16S rRNA gene sequences of sulfate-reducing
bacteria which were homologous with Desulfovibrio
desulfuricans strain E4 16S, uncultured Desulfovibrio
sp. clone MFC-2-L19, Desulfobulbus propionicus strain
Fig. 2: Phylogenetic tree of relationship sequences of 16S rRNA gene of the methanogenic populations in methane bioreactor
Fig. 3: Phylogenetic tree of relationship sequences of 16S rRNA gene of the sulfate-reducing bacteria in methane bioreactor
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88   I. Kushkevych, et al.
12 days and correlates with the accumulation of hydrogen
sulfide. The highest concentration of hydrogen sulfide
(390 ppm) was detected on the 14th day and it correlates
with the titer of sulfate-reducing bacteria (1050 OUT·mL-1
).
The sulfate-reducing bacteria (SRB) are a heterogeneous
group of microorganisms which use sulfate as an electron
acceptor in the process of dissimilatory sulfate reduction
[35, 36]. The final product of this process is hydrogen
sulfide [6, 20, 22, 37]. For sulfate reduction, SRB need
exogenous electron donors, such as: organic compounds
(e.g., lactate, propionate, butyrate, ethanol, etc.) and
molecular hydrogen. Organic compounds for SRB can
be simultaneously electron donors and carbon sources
and oxidized completely (to CO2
) or incompletely (to
acetate and CO2
) [6, 7]. SRB, oxidizing organic compounds
incompletely, belong to the group called “Acetogenic
sulfate-reducing bacteria” [7]. Detected SRB, Desulfovibrio
(48%), Desulfomicrobium (39.8%), and Desulfobulbus
(12%) genera, are acetogenic microorganisms which
oxidize organic compounds, incompletely, to acetate and
CO2
. Produced acetate is consumed by methanogens e.g.,
species of the Methanosarcina genus, which are dominant
microorganisms (62%) in the bioreactor (see Fig. 4). Our
results demonstrated the presence of acetotrophic and
hydrogenotrophic methanogenic archaea. The species
of the Methanosarcina genus can form multicellular
colonies and are anaerobic methanogens. They are
widespread in the rumen of cows, sheep, goats, deer,
and the large intestine of humans [7]. Recently, there
has been a study on M. barkeri, because this species
has the enzyme methylamine methyltransferase, which
catabolizes methylamines leading to methane production.
Methanosarcina sp. possess all three known pathways
for methanogenesis, and can utilize a broad spectrum of
substrates,includinghydrogen.Alltheothermethanogens
can utilize no more than two methanogenic substrates and
possess a single pathway for methanogenesis [38]. It also
has a number of distinct morphological forms, including
single cells with and without a cell envelope, as well as
multicellular packets and lamina [39].
The other dominant genus of microbial methanogens
in the studied bioreactor was Thermogymnomonas
(16%). The species of this genus were also isolated as a
novel thermoacidophilic, cell wall-less archaeon from
a solfataric field in Ohwaku-dani, Hakone, Japan. The
cells were irregular cocci, sometimes lobed, club-shaped
or catenated, and were highly variable in size, ranging
from 0.8 to 8.0 µm in diameter [33]. Itoh et al. (2007)
identified this strain as Thermogymnomonas acidicola.
The strain grew at temperatures in the range 38–68°C
(optimally at 60°C) and at pH 1.8–4.0 (optimally at around
determined from Illumina sequencing (Fig. 4). Results
indicate that three genera of both physiological groups
of microorganisms were detected in the experimental
bioreactors. The dominant genus of methanogens
was Methanosarcina, which was 62% of all detected
methanogens. Two other genera, Thermogymnomonas
and Methanoculleus, were 16 and 2%, respectively, as
well as other Archaeons which were not identified to the
genus (20%). For sulfate-reducing bacteria, three genera
were identified: Desulfomicrobium, Desulfobulbus and
Desulfovibrio in percentage ratios of 48, 39.8, and 12%,
respectively. Other sulfate-reducing bacteria (0.19%) were
not identified to the genus.
Fig. 4: Qualitative and percentage composition of methanogenic and
sulfate-reducing microorganisms
4 Discussion
Methane is the final product of anaerobic metabolism
carried out by communities of hydrolytic bacteria and
fungi, acid-producing intermediary organisms, and
finally, methanogenic microorganisms [17]. Methaneproducing
communities are very stable and resilient, but
they are also complex and largely undefined. The results
of our studies are consistent to other research described in
literature [5, 14, 33, 34]. Production of methane depends
on many factors, including physical (temperature) and
chemical (pH), type of substrate, its concentration and
accessibilityformicroorganisms,compositionandtheratio
of the microorganisms and their metabolic compounds
in the bioreactors. Therefore, bioreactors are a complex
fermentative system which includes various oxidations
and reduction processes with changes in redox potential.
Our research demonstrates that the intensive production
of methane in the experimental bioreactor lasts the first
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Production of biogas: relationship between methanogenic and sulfate-reducing microorganisms   89
and produce hydrogen sulfide in high concentrations
in the bioreactors. This, in turn, can inhibit the growth
of methanogenic microorganisms and their process
of methanogenesis. It can cause unbalance of other
microbial communities and instability of the fermentation
process. However, this was not tested in our experiments
at a maximum concentration of H2
S (390 ppm). The study
of new isolates is still important for selecting the optimal
conditions for methane production process.
Acknowledgements: This study was supported by
Masaryk University (project TAČR GAMA – internal project
CTT MU “Technology for qualitative biogas treatment”
Registration Project ID: 51047). The authors wish to
acknowledge the institutional support of the Faculty of
AgriSciences, Mendel University in Brno funded by the
Ministry of Education, Youth and Sports of the Czech
Republic. The authors express their gratitude to Igor
Starunko, Head of Editorial Office of Studia Biologica
pH 3.0). Strain IC-189T was an obligate anaerobic and
heterotrophic microorganism, requiring yeast extract
for growth. Yeast extract, glucose and mannose served
as carbon and energy sources. Therefore, strain IC-189T
represents a novel genus (order Thermoplasmatales) and
species [33].
The Methanoculleus genus was found in the lowest
number among all detected methanogens. The species
of the genus were frequently described as playing an
important role in different biogas reactor systems [34,
40, 41]. Methanoculleus bourgensis was always detected
as the dominant in biogas systems. The prevalence of
M. bourgensis in reactors performing syntrophic acetate
oxidation under high ammonium concentrations [42,
43, 44], indicates the importance of this methanogen in
corresponding communities. Isolation and/or cultivation
of M. bourgensis, together with acetate-oxidizing
bacteria [45] such as Clostridium ultunense [46], led to
the assumption that syntrophic association may play an
important role for members of the Methanoculleus genus
[42, 47]. The 16S rRNA gene sequence analysis classified
the isolate as a member of the species M. bourgensis with
99% sequence identity to the 16S rRNA gene of strain
MS2T [48, 49]. Genomic DNA of strain BA1 was isolated
and sequenced, applying the paired-end protocol on an
Illumina MiSeq system [34, 48]. In our studies, using
Illumina sequencing, the sequences belonging to this
genus were the most often detected in all bioreactors.
The detected genera of methanogens and SRB
described can compete by molecular hydrogen (Fig. 5).
HydrogenandCO2
canbeusedbythemethanogensfortheir
growth and methane production and, simultaneously,
SRB can also use H2
as a simple electron donor. Therefore,
competition for molecular hydrogen may occur between
methanogens and SRB. Accordingly, decrease of H2
was observed after the 6th
day. Perhaps, the highest
consumption of hydrogen by both microorganism groups
occurred during this period because afterwards hydrogen
levels stabilized. However, SRB produce hydrogen sulfide
which can be toxic for methanogens, and may inhibit
methanogenesis.
5 Conclusions
We conclude that studies of the diversity of methanogenic
microorganisms under the influence of various factors
in the bioreactors require further understanding of the
processofbiogasproduction.Thesulfate-reducingbacteria
can compete with the hydrogenotrophic methanogens for
substrate components, in this case molecular hydrogen,
Fig. 5: The hypothetical scheme of the relationship between methanogenic
and sulfate-reducing microorganisms
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90   I. Kushkevych, et al.
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Conflict of interest: Authors state no conflict of interest
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ATTACHMENT 8
Pages 96 – 101
_____________________________________________________________________________
Effect of selected 8-hydroxyquinoline-2-carboxanilides on viability
and sulfate metabolism of Desulfovibrio piger.
Ivan Kushkevych, Monika Vítězová, Jiří Kos, Peter Kollár, Josef Jampílek
Journal of Applied Biomedicine, 2018, 16(3), 241-246, ISSN 1214-021X
_____________________________________________________________________________
Original research article
Effect of selected 8-hydroxyquinoline-2-carboxanilides on viability and
sulfate metabolism of Desulfovibrio piger
Ivan Kushkevycha,
*, Monika Vítezováa
, Jirí Kosb
, Peter Kollárc
, Josef Jampílekb,
**
a
Masaryk University, Faculty of Science, Department of Experimental Biology, Brno, Czech Republic
b
Comenius University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Bratislava, Slovak Republic
c
University of Veterinary and Pharmaceutical Sciences, Faculty of Pharmacy, Department of Human Pharmacology and Toxicology, Brno, Czech Republic
A R T I C L E I N F O
Article history:
Received 3 August 2017
Received in revised form 5 January 2018
Accepted 15 January 2018
Available online 10 February 2018
Keywords:
Sulfate-reducing bacteria
Dissimilatory sulfate reduction
Inflammatory bowel disease
8-hydroxyquinolines
Structure-activity relationships
A B S T R A C T
An increased number of sulfate-reducing bacteria is often isolated from faeces of patients with
gastrointestinal diseases, which can be the cause of the development of bowel inflammation. Frequent
use of antibiotics causes the resistance of intestinal microorganisms and ineffective treatment of these
diseases. The antimicrobial activity and biological properties of the selected ring-substituted
8-hydroxyquinoline-2-carboxanilides against Desulfovibrio piger Vib-7 were studied. The addition of
these compounds in the cultivation medium inhibited the bacterial growth and the process of sulfate
reduction dose-dependently. A significant cytotoxic activity under the influence of ring-substituted
8-hydroxyquinoline-2-carboxanilides was determined. The strongest cytotoxic effect of the derivatives
was observed for compounds 8-hydroxy-N-(3-methoxyphenyl)quinoline-2-carboxamide and
8-hydroxy-N-(3-trifluoromethylphenyl)quinoline-2-carboxamide that caused a low survival of D. piger
Vib-7 in concentration 17 mM and high toxicity rates.
© 2018 Faculty of Health and Social Sciences, University of South Bohemia in Ceske Budejovice. Published
by Elsevier Sp. z o.o. All rights reserved.
Introduction
A high number of sulfate-reducing bacteria (SRB) and the
intense process of dissimilatory sulfate reduction in the gut are
thought to be significant risk factors of inflammatory bowel
diseases in both humans and animals (Gibson et al., 1991, 1993;
Kushkevych, 2016, Kushkevych et al., 2016). These bacteria are
often found in patients with rheumatic diseases, ankylosing
spondylitis, etc. (Barton and Hamilton, 2010). The species of
Desulfovibrio genus can cause bloody diarrhea, weight loss,
anorexia, epithelial hyperplasia, abscesses and inflammatory
infiltrates in animals and humans (Loubinoux et al., 2000,
2002a,b). There is also an assumption that SRB can be responsible
for some forms of cancer of the rectum through the formation of
hydrogen sulfide that affects the metabolism of intestinal cells.
An increased number of SRB was found in faeces from people
with ulcerative colitis in comparison with healthy individuals
(Cummings et al., 2003). The injection of these bacteria in
hamster intestine caused an infection clinically similar to human
colitis (Cummings et al., 2003; Pitcher and Cummings, 1996).
Using the model of laboratory rats, the etiological role of the
cultures of SRB, Desulfovibrio piger Vib-7 and Desulfomicrobium sp.
Rod-9, in the development of inflammatory processes in the
intestine was examined. It was found that the introduction of pure
cultures of SRB led to a high production of hydrogen sulfide and
acetate in the intestine, and clinical manifestations similar to nonspecific
intestinal inflammation in humans appeared (Kushkevych,
2014).
Ulcerative colitis (UC) is a chronic inflammatory disease of the
colon that affects up to 12 per 100,000 people in Western
countries, mostly between 15 and 30 years of age (Rowan et al.,
2009). The treatment of mild to moderate UC includes, in the first
instance, sulfasalazine and mainly 5-aminosalicylate containing
drugs, the type and dosage of which depend on the location and
severity of the disease. Other options of treatment include
corticosteroids and immunosuppressants (for moderate to severe
UC, with a high mortality) or probiotics (for improving the
microbial balance) (Cummings et al., 2003; Kushkevych, 2016).
Despite the bacterial nature of the disease, antibiotics have failed
* Author for correspondence: Masaryk University, Faculty of Science, Department
of Experimental Biology, Kamenice 753/5, 625 00 Brno, Czech Republic.
** Author for correspondence: Comenius University, Faculty of Pharmacy,
department of Pharmaceutical Chemistry, Odbojarov 10, 832 32 Bratislava, Slovak
Republic.
E-mail addresses: kushkevych@mail.muni.cz (I. Kushkevych),
josef.jampilek@gmail.com (J. Jampílek).
https://doi.org/10.1016/j.jab.2018.01.004
1214-021X/© 2018 Faculty of Health and Social Sciences, University of South Bohemia in Ceske Budejovice. Published by Elsevier Sp. z o.o. All rights reserved.
Journal of Applied Biomedicine 16 (2018) 241–246
Contents lists available at ScienceDirect
Journal of Applied Biomedicine
journal homepage: www.elsevier.com/locate/jab
in the treatment of UC so far. However, new antibacterial
compounds with high specific effect against SRB could yield
better efficiency in the treatment of this disease.
Quinoline-based compounds have a wide range of promising
biological properties (Cieslik et al., 2012; Jampilek, 2017; Jampilek
et al., 2016, Kos et al., 2015; Mucaji et al., 2017; Musiol et al., 2006,
2007, 2008, 2010); therefore a special attention is paid to them at
research and designing of new drugs (Cieslik et al., 2012; Jampilek
et al., 2016; Kos et al., 2015; Musiol et al., 2006, 2007; Ranjith et al.,
2017). This simple scaffold possesses unique physicochemical
properties and provides a possibility of a great number of targeted
modifications. Ring-substituted 8-hydroxyquinoline-2-carboxanilides
were recently prepared (Kos et al., 2015) and published as
compounds with noteworthy biological activities (Jampilek et al.,
2016; Kos et al., 2015) based on the presence of an amide group and
a hydroxy moiety in the quinoline scaffold (Gonec et al., 2012;
Jampilek et al., 2016; Kos et al., 2015). Thus, in the context of the
above-mentioned facts, the aim of this work was to evaluate
viability of intestinal sulfate-reducing bacteria Desulfovibrio piger
and parameters of their dissimilatory sulfate reduction (production
of hydrogen sulfide and acetate as well as sulfate and lactate
consumption) under effect of selected 8-hydroxyquinoline-2carboxanilides
(Kos et al., 2015).
Materials and methods
Tested compounds
The studied ring-substituted 8-hydroxyquinoline-2-carboxanilides
were synthesized by means of microwave-assisted synthesis
described recently. The compounds were isolated and fully
characterized (melting point, elemental analysis, infrared as well
as 1
H and 13
C NMR spectroscopy) (Kos et al., 2015). The compounds
were kept in microtubes dissolved in dimethyl sulfoxide (DMSO)
solution. The quantity of DMSO necessary to dissolve each
compound was calculated previously to achieve the concentration
of the component 30 mM. Afterwards it was diluted 4-fold in a
proportion 1:3, and 5 different concentrations of the chemical
compound – 5, 10, 15, 20, 25, 30 and 35 mM – were obtained. The
maximum concentration of DMSO in the assays never exceeded
0.1%.
Bacterial culture and cultivation
The sulfate-reducing bacteria D. piger Vib-7 (GenBank:
KT881309.1) were isolated from the healthy human large
intestine as described previously (Kushkevych, 2013; Kushkevych
et al., 2014). The strain has been kept in the collection of
microorganisms at the Department of Experimental Biology,
Faculty of Science at the Masaryk University (Brno, Czech
Republic). The bacteria were grown for 36 h at 37 
C under
anaerobic conditions in nutrition modified Postgate’s liquid
medium (Postgate, 1984). Before bacterial passage in the
medium, 0.05 mM of sterile solution of Na2S Â 9H2O (1%) was
added. The sterile 10 ml solution of NaOH (0.9 mM) in the
medium was used to provide the final pH 7.2. The medium was
heated in boiling water for 30 min in order to obtain an oxygenfree
medium and cooled to 30 
C. The tubes were brim-filled
with medium and closed to provide anaerobic conditions.
Assay of bacterial cell concentration
The best concentration of D. piger Vib-7 was assessed to be
5 Â105
CFU/ml. Based on our previous work, the correlation
between OD340 and the amount of cells in the solutions measured
in the biophotometer was determined as y = 1.0 Â 109
x – 6.0 Â 106
,
where y means the bacterial concentration and x means the OD340
measured as was described in our previous paper (Kushkevych
et al., 2015a).
Treatment of bacterial culture
The bacterial culture of the stationary phase of growth was
centrifuged for 3 min at a rotation speed of 3500 rpm. Supernatant
was removed and replaced by a fresh liquid medium, where the
bacterial precipitate was diluted. The bacterial suspension was
mixed, and OD340 was measured. Numbers and viabilities of the
bacterial cells were determined by counting with a haemocytometer
after staining with erythrosine B [0.1% erythrosine B (w/v)
in phosphate-buffered saline (PBS), pH 7.2–7.4]. Unstained cells
were considered to be viable.
The bacterial suspension (initial concentration 0.5 mg/ml) was
poured in microtubes (350 ml) that contained samples + solvent
control (DMSO) + sample control + blanks; each sample as well as
the controls were prepared in triplicate, so that the average of the
results did not had a great discrepancy from the results
independently (except in 3 blanks). The sample controls contained
only bacterial suspension and medium (free of tested compounds);
and for the 3 blanks only medium. Calculations were made to
assess how much bacterial solution should be in each, based on the
OD. The determination of biomass and concentrations of sulfate,
lactate, acetate and sulfide in the culture medium under the
treatment of 5, 10, 15, 20, 25, 30 and 35 mM compounds after 36 h
was carried out. During experiments, bacteria were grown at 37 
C
under anaerobic conditions.
Analysis of viability of D. piger Vib-7 and cytotoxicity of compounds
The bacterial suspension (5 Â104
cells/well in 300 ml culture
medium) was filled in 100-well plates in triplicate in the Postgate’s
liquid medium (without Mohr’s salt), treated with 5, 10, 15, 20, 25,
30 and 35 mM compounds and incubated at +37 
C. The relative
survival of D. piger Vib-7 cells and the cytotoxicity of the
compounds were determined at the 36th hour of cultivation using
a WST-1 assay kit (Roche Diagnostics, Mannheim, Germany)
according to the manufacturer’s instructions. The relative survival
rate was calculated by the following equation: (ASample – ABlank)/
(AControl – ABlank), and multiplied by 100 for the result in percentage.
The relative toxicity rate was determined as described previously
(Kos et al., 2015). All data were evaluated using GraphPad Prism
5.00 software (GraphPad Software, San Diego, CA, USA, http://
www.graphpad.com).
Assay of sulfate, lactate, sulfide and acetate in cultivation medium
The sulfate ion concentration in the medium was determined by
the turbidimetric method after it had been precipitated by barium
chloride. To stabilize the suspension, glycerol was used (Kolmert
et al., 2000). Lactate concentration was measured through the
dehydrogenation reaction using Lactate Assay Kit (Sigma-Aldrich,
Catalog Number MAK064). Sulfide concentration in the culture
medium was assayed by the spectrophotometric method as was
described (Cline,1969). Accumulation of acetate ions in the process
of bacterial growth in the medium was determined using Acetate
Assay Kit (Colorimetric, Catalog Number KA3764).
Statistical analysis
The statistical calculations of the results were carried out using
the software MS Office and Origin program. The research results
were treated by methods of variation statistics using Student t-test.
242 I. Kushkevych et al. / J. Appl. Biomed. 16 (2018) 241–246
Statistical significance was tested using the one-way analysis of
variance with Dunnett’s test and Tukey post-test for comparisons
between the means, and differences between two conditions were
retained for P 0.05. Statistical significance was determined at
levels of P < 0.05, P < 0.01, and P < 0.001 (Bailey, 1995).
Results
The structures of the selected and discussed ring-substituted
8-hydroxyquinoline-2-carboxanilides are shown in Table 1.
A synthetic pathway and characterization were published by
Kos et al. (2015).
The relative survival of D. piger Vib-7 cells and cytotoxicity
of ring-substituted 8-hydroxyquinoline-2-carboxanilides were
studied. In this series of experiments, compounds 1–7 exerted
cytotoxicity against these bacteria already in low (5–35 mM)
concentrations (Fig. 1).
Treatment with all tested compounds led to a significant
dose-dependent activity with the strongest effect observed in
compounds 3 (R = 3-OCH3) and 6 (R = 3-CF3). Since some of
ring-substituted 8-hydroxyquinoline-2-carboxanilides (such as 3
or 6) have a pronounced antimicrobial effect against D. piger Vib-7
(combination of low survival and high toxicity rates), they could be
considered as promising agents against the growth of this type of
bacteria.
The effect of different concentrations of ring-substituted
8-hydroxyquinoline-2-carboxanilides on the process of dissimilatory
sulfate reduction in D. piger Vib-7 cells at the 36th h of
cultivation was studied. As shown in Figs. 2 and 3, the addition
of the compounds in the culture medium inhibits the process of
dissimilation of sulfate directly proportional to the increase in
concentrations (5–35 mM). Under these conditions, the utilization
of sulfate and lactate was inhibited; hence the level of
accumulation of hydrogen sulfide and acetate was reduced.
These data are consistent with our research in previous series of
the experiments. The percentage inhibition of sulfate reduction
process correlates with the percentage of bacterial growth
inhibition under the ring-substituted 8-hydroxyquinoline-2carboxanilides
treatment.
Based on the obtained results, the MIC and IC50 values of
ring-substituted 8-hydroxyquinoline-2-carboxanilides against
intestinal sulfate-reducing bacteria were established. As shown
in Table 1, the MICs of all the compounds were 33 mM. The
least potent compounds among the tested compounds for the
bacterial strain were 2 (R = 2-OCH3) and 5 (R = 3-Br), the MICs of
which were 28 and 33 mM, respectively. The similar MICs were
determined for compounds 1 (R = H) and 4 (R = 3-CH3). It can be
supposed that these compounds show the same cytotoxicity
effect on sulfate-reducing bacteria D. piger Vib-7 cells. IC50 of
compounds 4, 6 and 7 was 10 mM. Compounds 2 and 5 had IC50
only 17 and 18 mM, which is consistent with the MICs of these
compounds.
Discussion and conclusion
Sulfate-reducing bacteria of Desulfovibrio genus belong to the
intestinal microbiota of humans and animals (Kushkevych, 2012,
2013). They are anaerobic microorganisms, dissimilating sulfate as
an electron acceptor and organic compounds as an electron donor
and carbon source in the process of “dissimilatory sulfate
reduction” (also known as “dissimilatory anaerobic sulfate
respiration”) (Kushkevych, 2016). Lactate is the most common
substrate used by the species belonging to the intestinal sulfatereducing
bacteria. The species of Desulfovibrio oxidize lactate
incompletely to acetate. Lactate oxidation to acetate occurs
together with the concurrent reduction of sulfate to sulfide
(Barton and Hamilton, 2010). The presence of lactate and sulfate in
the human intestine contributes to the intensive bacteria growth
and the accumulation of their final metabolic products, acetate and
hydrogen sulfide, that are toxic, mutagenic and cancerogenic to
epithelial intestinal cells (Pitcher and Cummings, 1996; Rowan
et al., 2009). There is also an assumption that sulfate-reducing
bacteria can cause some forms of cancer of the rectum through the
Table 1
Structures of ring-substituted 8-hydroxyquinoline-2-carboxanilides and in vitro
antibacterial activity against Desulfovibrio piger Vib-7 (minimal inhibition
concentration: MIC, half maximal inhibitory concentration: IC50, minimal
bactericidal concentration: MBC) of the compounds in comparison with ciprofloxacin
(CPX).
Comp. R [mM]
MIC IC50 MBC
1 H 23 12 25
2 2-OCH3 28 17 28
3 3-OCH3 17 11 20
4 3-CH3 23 10 23
5 3-Br 33 18 35
6 3-CF3 17 10 20
7 4-CF3 20 10 22
CPX – 45 28 45
Fig.1. Relative survival of D. piger Vib-7 cells (a) and toxicity (b) of ring-substituted
8-hydroxyquinoline-2-carboxanilides.
I. Kushkevych et al. / J. Appl. Biomed. 16 (2018) 241–246 243
formation of hydrogen sulfide. In our previous studies, it was
shown that bacteria D. piger Vib-7 consumed sulfate and
accumulated hydrogen sulfide in concentration of
2.31 Æ0.21 mM (Kushkevych, 2013).
Based on all the obtained results in this study, it can be
concluded that compounds 1–7 in concentrations 5–35 mM
inhibited the growth and, accordingly, the process of dissimilatory
sulfate reduction. This antimicrobial activity was concentrationdependent,
with the strongest effect in 30 mM concentration.
Similar effect was observed in our previous research for the activity
of selected salicylamides against intestinal sulfate-reducing
bacteria (Kushkevych et al., 2015a, 2016). Derivatives 1, 2, 4, 5
and 7 showed cytotoxic affect at concentrations higher than
17 mM. The highest level of inhibition of this process and high
activity rates were observed at concentration 17 mM of compounds
3 and 6 and thus are interesting for further studies. The cytotoxic
effect of these compounds can be due to the inhibition of the
enzymes of dissimilatory sulfate reduction (Kushkevych, 2015a,b;
Kushkevych et al., 2015b), including sulfite reductase that is
susceptible for various factors in intestinal sulfate-reducing
bacteria D. piger Vib-7 (Kushkevych and Fafula, 2014). The tested
compounds can be considered good alternatives for the treatment
of colitis or colorectal cancer although it should be taken in account
that these compounds can be aggressive also to commensally
bacteria and even to other parts of the human body. This should be
a concern to be clarified in the near future with complementary
assays.
Despite the fact that evaluated bacteria Desulfovibrio species
are heterogeneous group of microorganisms which are widespread
in anaerobic areas of soils, wetlands, marine and fresh
water, they are available in microbiota of large intestine of
humans and animals. It is known that intestinal Desulfovibrio
species are different from other SRB by their biochemical and
physiological properties (Barton and Hamilton, 2010; Brenner
et al., 2005; Holt et al., 1994). So, the mechanisms of the effect of
studied compounds on these bacteria can also differ. These
compounds may influence the synthesis of the bacterial cell wall
(peptidoglycan), ribosomes, enzymes or other phenotypic
features such as inhibition the synthesis of desulfoviridin,
cytochrome c3 and menaquinone MK-6. Effect of studied
compounds may depend on their activity, structure and
properties.
Fig. 2. Sulfate reduction of D. piger Vib-7 cells under the treatment of indicated doses of ring-substituted 8-hydroxyquinoline-2-carboxanilides: sulfate dissimilation (a),
hydrogen sulfide production (b).
244 I. Kushkevych et al. / J. Appl. Biomed. 16 (2018) 241–246
The studied compounds are able to inhibit the number of
sulfate-reducing bacteria and/or reduce the production of sulfide
and acetate. This would help to clarify the factors influencing
sulfide production in the human and animal colon.
Conflict of interests
The authors declare that they have no conflict of interests
regarding the publication of this paper.
Acknowledgements
This study was supported by CZ.1.07/2.3.00/30.0053, APVV-
0516-12 and by Sanofi-Aventis Pharma Slovakia, s.r.o.
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ATTACHMENT 9
Pages 102 – 107
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The diversity of sulfate-reducing bacteria in the seven bioreactors
Kushkevych I., Kováč J., Vítězová M., Vítěz T., Bartoš M.
Archives of Microbiology, 2018, 200, 945-950, ISSN 0302-8933
_____________________________________________________________________________
Vol.:(0123456789)1 3
Archives of Microbiology (2018) 200:945–950
https://doi.org/10.1007/s00203-018-1510-6
ORIGINAL PAPER
The diversity of sulfate-reducing bacteria in the seven bioreactors
Ivan Kushkevych1
   · Jozef Kováč1
 · Monika Vítězová1
 · Tomáš Vítěz2
 · Milan Bartoš1
Received: 23 January 2018 / Revised: 27 March 2018 / Accepted: 29 March 2018 / Published online: 2 April 2018
© Springer-Verlag GmbH Germany, part of Springer Nature 2018
Abstract
Anaerobic technology has a wide scope of application in different areas such as manufacturing, food industry, and agriculture.
Nowadays, it is mainly used to produce electrical and thermal energy from crop processing, solid waste treatment
or wastewater treatment. More intensively, trend nowadays is usage of this technology biodegradable and biomass waste
processing and biomethane or hydrogen production. In this paper, the diversities of sulfate-reducing bacteria (SRB) under
different imputed raw material to the bioreactors were characterized. These diversities at the beginning of sampling and after
cultivation were compared. Desulfovibrio, Desulfobulbus, and Desulfomicrobium genus as dominant among sulfate reducers
in the bioreactors were detected. The Desulfobulbus species were dominant among other SRB genera before cultivation, but
these bacteria were detected only in three out of the seven bioreactors after cultivation dominant.
Keywords  Sulfate-reducing bacteria · Hydrogen sulfide · Bioreactors · Biogas
Introduction
Biogas is a mixture of ­CH4 and ­CO2 and other gases. This
valuable product is result of conversion of the organic mass
in the waste by anaerobic microorganisms in methanogenic
bioreactors (McCarty and Smith 1986). Formation of biogas
is mainly occurred in wetlands, marshes, and in the gastrointestinal
system of ruminants (Krich et al. 2005). Landfilled
food and wastes and another biomass may be degraded
by anaerobic microorganisms in landfills. Potential source
of renewable energy can be used through collected biogas
(Wilkie 2008).
For feed of biogas bioreactor, various substrates are used.
This substrate may differ by composition of organic and
inorganic compounds. Sulfate-reducing bacteria (SRB) may
out-compete methanogens for substrates (e.g., ­H2, ­CO2, and
acetate) by growth in sulfate-rich maize silage or sludge in
anaerobic environments, such as biogas bioreactors. Organic
compound and molecular hydrogen is universal electron
donor in dissimilatory sulfate reduction process as well
as for methane formation. SRB consume hydrogen below
a minimum threshold than methanogens in their hydrogen
metabolism (Lovley 1985; Lovley and Ferry 1985; Kushkevych
et al. 2017a).
Intensive consumption of substrate by SRB during sulfate
reduction may cause a problem of a high concentration of
hydrogen sulfide. This final product of SRB metabolism is
toxic and can cause corrosion of metallic materials in cogeneration
units of biogas plants (Koschorreck 2008). Therefore,
a negative effect on microbial cells due to the precipitation
of essential trace metals as metal sulfides can occur.
In any case, hydrogen sulfide toxicity is unlikely to be decoupled
from competition between SRB and methanogens,
and due to their more favorable growth and thermodynamic
properties, SRB are considered to out-compete other anaerobes
in the presence of excess sulfate. The diversity of SRB
depending on various ratios and composition of substrates
in operated bioreactors has never been studied yet. Such new
data may be useful for regulation and controlling of the processes
of methane production and for more clearly understanding
of the mechanisms of substrates’ transformation.
The aim of this research was to compare the diversity of
sulfate-reducing bacteria populations in operated bioreactors
with different input ratios of the initial substrate and to
Communicated by Harald Huber.
*	 Ivan Kushkevych
	 ivan.kushkevych@gmail.com; kushkevych@mail.muni.cz
1
	 Department of Experimental Biology, Faculty of Science,
Masaryk University, Kamenice 753/5, 62500 Brno,
Czech Republic
2
	 Department of Agricultural, Food and Environmental
Engineering, Faculty of AgriSciences, Mendel University
in Brno, Zemedelska 1, 613 00 Brno, Czech Republic
946	 Archives of Microbiology (2018) 200:945–950
1 3
investigate the ability of SRB genera to grow in vitro and the
change in its percentage ratio before and after cultivation as
well as to create phylogenetic tree of SRB relationships in
each bioreactor under these conditions.
Materials and methods
The three samples were collected from each different biogas
plant reactors of volume ranged from 2500 to 3500 m3
operated
at about 40 ± 4 °C temperature depending on bioreactor
and processing low solid suspension (liquid fermentation).
Organic load rate was 3.5–5.5 kg org. mass/m3
per fermenter
and feed intervals were 80–100 kg/kWhel.
The biogas plant reactors are located at Modřice, Bratčice,
Pánov, Úvalno, Horní Benešov, Rusín, and Loděnice (Czech
Republic). Hydraulic retention time (HRT) ranged from 18
to 20 days for one-stage biogas plant at Modřice and 25–35
days for primary reactor for six remaining biogas plants.
The samples were taken directly from the sampling valve
of the primary reactors into sterile-sampling vessels. After
sampling, the samples were stored in thermos container and
transported to the laboratory for further analysis. Each of the
reactors processed a different type of substrate.
The type of substrate in bioreactor
The type of substrate is shown as the ratio (w/w) of fresh
input substrate. Modřice (1): primary sludge, biological
sludge (50:50), Bratčice (2): maize silage, whole crop
silage, poultry litter (63:31:6), Pánov (3): maize silage, poultry
litter (92:8), Úvalno (4): maize silage, sugar beet pulp,
whole crop silage, cattle manure (44:44:6:6), Horní Benešov
(5): maize silage, sugar beet pulp, whole crop silage, cattle
manure, grass silage (29:39:12:15:5), Rusín (6): maize
silage, sugar beet pulp (70 30), Loděnice (7): maize silage,
sugar beet pulp (75:25).
Isolation of DNA and amplification of 16S rRNA gene
fragments by Illumina sequencing
For DNA isolation, three samples were taken from all seven
reactors and homogenized before sequencing. The QIAamp
Fast DNA Stool Mini Kit (QIAGEN GmbH, Germany) is
designed for rapid purification of total DNA from stool samples
and was used for DNA extraction from biogas digester
samples. DNA extraction was performed in accordance to
the manufacturers’ instructions, with minor adjustments as
described below. Briefly, 100 mg of sample was washed
with 1.4 mL of ASL buffer (QIAGEN GmbH, Germany),
heated at 95 °C for 10 min. After centrifugation, an InhibitEX
tablet was put in the supernatant to remove impurities
and PCR inhibitors. After thorough centrifugation, 200
µL of the supernatant was added to 15 µL of proteinase K,
and 200 µL of buffer AL (QIAGEN GmbH, Germany) was
added. The mixture was heated to 70 °C for 10 min and 200
µL of absolute ethanol was added. The sample mixture was
then passed through the QIAamp kit column, followed by
two washes with buffers AW1 and AW2 (QIAGEN GmbH,
Germany). The DNA was eluted in a volume of 200 µL of
elution buffer.
Amplification of 16S rRNA gene fragments was carried
out using universal primers for amplification of the V3 and
V4 variable regions of the 16S rRNA (Nossa et al. 2010).
The primers were flanked by molecular barcodes for sample
identification. The PCR reaction was prepared using
Maxima™ Probe qPCR Master Mix (Thermo Fisher Scientific,
USA) with cycling conditions as follows: 95 °C for
10 min. followed by 30 cycles of incubation at 94 °C for
30 s, 60 °C for 30 s and 72 °C for 120 s, and a final extension
step at 72 °C for 2 min. PCR products were visualized
using electrophoresis on 1.5% agarose gels and purified from
the gel using the QIAquick Gel Extraction Kit (QIAGEN
GmbH, Germany). DNA was quantified using the QuantiTPicoGreen
dsDNA Assay (Thermo Fisher Scientific, USA)
and equimolar amounts of the PCR products were pooled
together.
Purified amplicons were paired-end sequenced (10 nM
sample DNA in 10 µl) on an Illumina Mi-Seq platform.
QIIME data analysis package was used for 16S rRNA data
analysis (Caporaso et al. 2010). Quality filtering on raw
sequences was performed in accordance to base quality score
distributions, average base content per read and GC distribution
in the reads. Chimeras and reads that did not cluster
with other sequences were removed. The obtained sequences
with qual scores higher than 20 were shortened to the same
length of 350 bp and classified with RDP Seqmatch with an
operational taxonomic unit (OTU) discrimination level set
to 97%. The relative abundance of the taxonomic groups
was calculated to the microorganisms detected in this study.
Sequences were compared using the BLAST feature of
the National Center for Biotechnology Information (NCBI)
(Altschul et al. 1990). The sequences were uploaded to
Mega7 for comparative genomic analyses (Kearse et al.
2012). Alignments of sequences were performed in Mega7
using Clustal W and clustering was performed by the neighbor-joining
method (Larkin et al. 2007).
The sequences of their 16S rRNA gene and sequences
of the strains from GenBank were compared to clarify the
genetic relations of the SRB populations in the specific
bioreactor. The genomic sequences of SRB are available in
GenBank under accession no. MF991912.1, MF991913.1,
KY285258, KY285259, MF991911.1, MF991932.1,
KY285260, KY285261, KY290639, KY290609, KY290608,
KY290610, KY290611, KY290612, KY290614, KY290617,
KY290615, KY290636, KY290646, KY290647, KY290648,
947Archives of Microbiology (2018) 200:945–950	
1 3
KY290649, KY290651, KY290652, KY290653, KY290677,
KY297012, KY295933, MF991933.1, KY295934,
KY297013, MF991934.1, KY305420, KY305433,
KY305431, KY305471, KY305532, KY305531,
MF989223.1, KY305678, KY305658, KY305659,
KY305660, KY305668, KY305679, KY305739,
KY305738, KY305863, MF988750.1, KY305876,
KY306668, KY306669, MF991936.1, KY306671,
KY306670, MF988711.1, KY306672, KY306673,
KY306674, KY306677, KY306676, KY306675,
KY306678, MF991939.1, MF991938.1, MF988725.1.
To compare the genera of SRB diversity and their ability
to growth in vitro, the same samples from each bioreactor
were collected in liquid Postgate’s medium C (Posgate,
1984) and cultivated under anaerobic conditions for 72 h.
After cultivation, samples were sequenced as described
above. The research results were analyzed using software
package Origin7.0 (http://www.origi​n-lab.com).
Results and discussion
The main genera of SRB were investigated in different
bioreactors. The most widespread genera were identified
as Desulfovibrio, Desulfobulbus, Desulfomicrobium,
and other SRB (Fig. 1). The most ratios of SRB genera,
except the other ones, were detected in the bioreactor from
Loděnice where two types of the substrate (maize silage,
sugar beet pulp in the ratio 75:25) were used. On the other
hand, other genera of SRB were detected only in the bioreactor
located at wastewater treatment plant Modřice (12%)
processed primary sludge and biological sludge (50:50)
and in the bioreactor at Rusín (6%) processed maize silage,
sugar beet pulp at ratio (70:30). However, Desulfobulbus
genus was detected as a dominant genus of SRB in all
bioreactors.
Two genera, Desulfovibrio and Desulfobulbus were both
dominant SRB in cultivations from bioreactors at Modřice
and Rusín. Also, single dominant Desulfovibrio genus
was detected after sample cultivation from Úvalno (89%),
Horní Benešov (87%), and Loděnice (81%). Although,
the greatest percentage of Desulfobulbus genus isolated
from Bratčice (98%) and Pánov (97%) after cultivation was
observed and correlated with the same samples (without
cultivation) isolated directly from bioreactors.
Phylogenetic tree of these isolates from each bioreactor
was built (Fig. 2). Also, the phylogenetic tree of the samples
after cultivation was built (Fig. 3) to observe diversity
and ability to be cultivated of these bacteria. It should
be mentioned that the most described sequences of SRB
strains in GenBank are identified only to the domain, kingdom,
family or genus, and in some cases, to species (http://
www.ncbi.nlm.nih.gov/genba​nk). It is clearly visible that
the greatest diversity of SRB is detected in a bioreactor
from wastewater treatment plant located at Modřice.
Methane is the product of anaerobic metabolism carried
out by communities of hydrolytic bacteria and fungi,
acid-producing intermediary organisms, and finally,
methanogenic Archaea (Zeikus 1977). Methane-producing
communities are very stable and resilient, but they
are also complex and largely undefined. The process of
methanogenesis often collapses due to the high concentration
of hydrogen sulfide produced by SRB (Weijma 2001).
Methanogenic microorganisms are sensitive to this final
Fig. 1  Percentage ratio of sulfate-reducing bacteria genera in all analyzed bioreactors (a), diversity of sulfate-reducing bacteria populations
observed after cultivation (b)
948	 Archives of Microbiology (2018) 200:945–950
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metabolite of SRB and can compete with them for molecular
hydrogen as the electron donor (Oremland and Polcin
1982).
SRB are a group of microorganisms which uses sulfate
as an electron acceptor in their process of dissimilatory sulfate
reduction (Postgate 1984; Barton and Hamilton 2010;
Kushkevych 2015a, b; Kushkevych et al. 2015a, b, 2016,
2017b). Hydrogen sulfide is the final product of this process
(Kushkevych 2016; Kushkevych et al. 2018). SRB need for
sulfate reduction exogenous electron donors, such as lactate,
propionate, butyrate, ethanol, and even molecular hydrogen.
Organic compounds for SRB can be simultaneously electron
donors and carbon sources and oxidized completely
(to ­CO2) or incompletely (to acetate and ­CO2) (Barton and
Hamilton 2010; Kushkevych 2016). SRB, oxidizing organic
compounds incompletely, belong to the group called “Acetogenic
sulfate-reducing bacteria” (Barton and Hamilton
2010). Detected SRB, Desulfovibrio, Desulfomicrobium
and Desulfobulbus genera, are acetogenic microorganisms
which oxidize organic compounds, incompletely, to acetate
and ­CO2. There was a wide diversity of SRB in the bioreactors.
The bacterial populations found in all bioreactors examined
were heterogeneous, encompassing an array of different
genera and strains of acetogenic sulfate-reducing bacteria.
In our studies, using Illumina sequencing, the sequences
of Desulfobulbus sp. were the most often detected in all bioreactors.
This genus of SRB was frequently described in the
other literature (Zellner et al. 1989; Lien et al. 1998). Zellner
Fig. 2  Phylogenetic tree of sulfate-reducing bacteria relationships separately in each bioreactor: Modřice (a), Bratčice (b), Pánov (c), Úvalno (d),
Horní Benešov (e), Rusín (f), and Loděnice (g)
949Archives of Microbiology (2018) 200:945–950	
1 3
et al. (1989) which was mainly focused on Desulfobulbus
propionicus has also observed a variety of SRB are similar
to this genus. In our research, we have also observed widely
diversity of SRB related to a genus of Desulfobulbus propionicus
which is visible in our phylogenetic tree (Fig. 2).
Another dominant genus of microbial acetogenic SRB in
the studied bioreactors was Desulfovibrio sp. which is the
most widespread and known genus of the SRB (van Houten
et al. 2009; Wawer et al. 1997). It was dominant genus only
in three bioreactors after cultivation. But we can state that
this genus of SRB is the most cultivable genus from all bioreactors
compared to Desulfobulbus which was dominant
only before cultivation (Fig. 1).
SRB with the lowest percentage in all samples was Desulfomicrobium
genus. Despite its low percentage, this genus
plays an important role in the environment with high heavy
metal concentrations. This genus is mostly cultured at low
temperature and shows heavy metal tolerant properties (Azabou
et al. 2007).
The SRB populations depend on the different types and
initial amount of substrate ratio in the bioreactors. Three
dominant morphotypes of these bacteria in the bioreactors
were homologous (99%) by the sequences of 16S rRNA gene
to the Desulfovibrio, Desulfobulbus and Desulfomicrobium
genera deposited in GenBank. Their genetic relationship can
be shown by the phylogenetic trees and by comparison of the
sequences 16S rRNA gene of these bacteria from methane
bioreactors to the sequences of the strains from GenBank.
Conclusions
To conclude our research, changes of SRB genera profile,
depending on ratios of substrates on the beginning of sampling
and their ability to grow in vitro, were determined.
Before cultivation, Desulfobulbus species was dominant
(46–86%) in all biogas plants. The profile and number of this
genus was changed after the cultivation and its biomass was
increased by 97% and 98% in the samples from Bratčice and
Pánov. Under these conditions, the most viable to grow were
Desulfovibrio species (82–89%) detected in samples from
Úvalno, Horní Benešov and Loděnice. It can be stated that
agricultural biogas stations showed a comparable diversity
of SRB, regardless of feedstock. On the contrary, the biogas
treatment plant showed a higher diversity of SRB, due to a
different type of substrate.
Acknowledgements  This study was supported by Grant Agency of
the Masaryk University (MUNI/A/0906/2017). The authors wish to
acknowledge the institutional support of the Faculty of AgriSciences,
Mendel University in Brno funded by the Ministry of Education, Youth
and Sports of the Czech Republic.
Compliance with ethical standards 
Conflict of interest  The authors declare that they have no conflict of
interest.
Human and animal rights  This article does not contain any studies
with human participants or animals performed by any of the authors.
Fig. 3  Phylogenetic tree of sulfate-reducing bacteria relationships separately after cultivation from each bioreactor: Modřice (a), Bratčice (b),
Pánov (c), Úvalno (d), Horní Benešov (e), Rusín (f), and Loděnice (g)
950	 Archives of Microbiology (2018) 200:945–950
1 3
Informed consent  Informed consent was obtained from all individual
participants included in the study.
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ATTACHMENT 10
Pages 108 – 114
_____________________________________________________________________________
Activity of ring-substituted 8-hydroxyquinoline-2-carboxanilides
against intestinal sulfate-reducing bacteria Desulfovibrio piger
Ivan Kushkevych, Jiri Kos, Peter Kollar, Katarina Kralova, Josef Jampilek
Medicinal Chemistry Research, 2018, 27(1), 278-284, ISSN 1054-2523
_____________________________________________________________________________
Med Chem Res (2018) 27:278–284
DOI 10.1007/s00044-017-2067-7
MEDICINAL
CHEMISTRY
RESEARCH
ORIGINAL RESEARCH
Activity of ring-substituted 8-hydroxyquinoline-2-carboxanilides
against intestinal sulfate-reducing bacteria Desulfovibrio piger
Ivan Kushkevych1 ●
Jiri Kos2 ●
Peter Kollar3 ●
Katarina Kralova4 ●
Josef Jampilek 2
Received: 30 June 2017 / Accepted: 5 September 2017 / Published online: 15 September 2017
© Springer Science+Business Media, LLC 2017
Abstract Desulfovibrio genus is dominant among sulfatereducing
bacteria (SRB) in the large intestine of healthy
people and animals. It is mostly isolated from patients with
inflammatory bowel disease (IBD) and can be involved in
the disease initiation. Primary in vitro screening of 8hydroxyquinoline-2-carboxanilides
was performed against
Desulfovibrio piger Vib-7 representing SRB. The most
effective compounds with MIC90/MBC values in the range
of 17–23 µM/20–23 µM, respectively, were substituted in
C’(3) by CF3, OCH3, CH3 and in C’(4) by CF3. Their activity
was twofold higher than that of ciprofloxacin. These compounds
did not express any significant cytotoxic effect on
THP-1 cells up to the tested concentration of 30 µM. The
antibacterial efficacy of the most active C’(3)-substituted
compounds practically did not change with increasing
compound lipophilicity, indicating that this position of
substitution is favorable for significant antimicrobial effect,
while the antibacterial activity of most of C’(2) and C’(4)substituted
derivatives decreased linearly with increasing
compound lipophilicity. In addition, the dependence of
activity on electronic Hammett’s σ parameter of the substituent
R was quasi-parabolic for the most effective C’(3)substituted
compounds.
Keywords 8-Hydroxyquinolines ●
Sulfate-reducing
bacteria ●
Lipophilicity ●
Electronic parameter ●
Structure–activity relationships
Introduction
The species of Desulfovibrio genus are dominant among
sulfate-reducing bacteria (SRB) and common inhabitants of
the human and animal large intestine, capable of dissimilatory
sulfate reduction (Gibson et al. 1991, 1993; Kushkevych
2015a, b; Kushkevych et al. 2015a, b; Wegmann
et al. 2017). These microorganisms are the most isolated
from patients with inflammatory bowel disease (IBD) and
can be involved in the disease initiation caused by their
main metabolite hydrogen sulfide, which is an inhibitor of
butyrate oxidation in colonocytes. In addition, it is cytotoxic,
mutagenic and cancerogenic to epithelial intestinal
cells, which leads to the damage of the epithelial barrier
function, resulting in inflammatory responses characteristic
for IBD (Kushkevych et al. 2014; Pitcher and Cummings
1996; Zinkevich and Beech 2000). Therefore, the association
between SRB and IBD, such as ulcerative colitis (UC),
was hypothesized (Zinkevich and Beech 2000; Rowan et al.
2009; Loubinoux et al. 2002; Kushkevych 2014; Cummings
et al. 2003). Over 1 million residents in the USA and 2.5
million in Europe are estimated to have IBD, with substantial
costs for health care, whereas these estimates do not
* Ivan Kushkevych
ivan.kushkevych@gmail.com
* Josef Jampilek
josef.jampilek@gmail.com
1
Department of Experimental Biology, Faculty of Science, Masaryk
University, Kamenice 753/5, 625 00 Brno, Czech Republic
2
Department of Pharmaceutical Chemistry, Faculty of Pharmacy,
Comenius University, Odbojarov 10, 832 32 Bratislava, Slovakia
3
Department of Human Pharmacology and Toxicology, Faculty of
Pharmacy, University of Veterinary and Pharmaceutical Sciences,
Palackeho 1, 612 42 Brno, Czech Republic
4
Institute of Chemistry, Faculty of Natural Sciences, Comenius
University, Ilkovicova 6, 842 15 Bratislava, Slovakia
factor in the ‘real’ price of IBD, which can impede career
aspirations, instil social stigma and impair quality of life in
patients (Kaplan 2015). Unlike Crohn’s disease, UC occurs
only in the large bowel, where bacteria amount is greater
than in the rest of the gut and also where the rate of passage
of material is characterized by slow movement of digestive
materials. Both acute and chronic forms of UC affect the
colon and rectum and can be a highly uncomfortable condition
(Cummings et al. 2003). UC usually has a relapsing/
remitting pattern and current medical approaches focus on
treating active disease to address symptoms, to improve the
quality of life and thereafter to maintain remission. Diarrhea
accompanied with blood, an urgent need to defecate and
abdominal pain are the main symptoms of active disease or
relapse. The reported incidence is 1.2 to 20.3 cases per
100,000 persons per year, and the prevalence is 7.6 to 245
cases per 100,000 per year (Feuerstein and Cheifetz 2014;
Cesar da Silva et al. 2014).
The benefits of antibiotic therapy in UC are mediated by
different mechanisms, such as decreasing the concentration
of luminal bacteria, altering the composition of gut microflora,
decreasing bacterial tissue invasion and decreasing
bacterial translocation and systemic dissemination. Antibiotics
have been prescribed for UC, however, they have
been largely ineffective (Cummings et al. 2003; Garud and
Peppercorn 2009). For example, the study of the in vitro
activities of rifaximin and comparator agents against 536
anaerobic intestinal bacteria performed by Finegold et al.
showed that the overall MICs90 of rifaximin for 90% the
tested strains were 338 μM, an activity equivalent to those
of teicoplanin and vancomycin (Finegold et al. 2009).
Nakao et al. (2009) tested the antimicrobial susceptibilities
of 23 strains of Desulfovibrio spp. and found that they were
susceptible to sulbactam-ampicillin, meropenem, clindamycin,
and metronidazole with MIC90 corresponding to
17, 10, 0.45, and 1.46 μM, respectively. On the other hand,
Lozniewski et al. (2001) tested the antimicrobial susceptibilities
of 16 clinical isolates of Desulfovibrio spp. and
found that these isolates were resistant to piperacillintazobactam,
cefoxitin and cefotetan with MIC90 corresponding
to 495, >600 and 111 μM, respectively.
Therefore, it is necessary to study new antibacterial agents
in order to improve the treatment and discover alternative
therapeutics.
This paper follows our recently published articles dealing
with the spectrum of biological activities of hydroxyquinoline-based
compounds (Jampilek et al. 2005; Musiol
et al. 2006, 2007, 2008, 2010; Mrozek-Wilczkiewicz et al.
2010; Gonec et al. 2012; Cieslik et al. 2012, 2015;
Kos et al. 2015a; Jampilek et al. 2016). This study is
focused on the investigation of the efficacy and searching
for the structure–activity relationships within a series of
8-hydroxyquinoline-2-carboxanilides (Kos et al. 2015a)
against Desulfovibrio piger Vib-7 representing SRB. D.
piger is a Gram-negative strict anaerobe that is usually
considered as a commensal bacterium in humans. More
recently, it has attracted more interest as it was found to be
the most prevalent species of SRB in feces of patients with
IBDs (Kushkevych 2014; Barton and Hamilton 2010; Holt
et al. 1994).
Material and methods
Synthesis
The discussed 8-hydroxyquinoline-2-carboxanilides 1–8c
(see Table 1) were synthesized previously (Kos et al. 2015a)
by means of microwave-assisted synthesis. The compounds
were fully characterized by melting point, infrared, nuclear
magnetic resonance, and high-resolution mass spectrometry
(Kos et al. 2015a).
In vitro antibacterial susceptibility testing
The synthesized compounds were evaluated for in vitro
antibacterial activity against the intestinal SRB Desulfovibrio
piger Vib-7 (Genbank: KT881309.1) that were isolated
from the healthy human large intestine as described previously
(Kushkevych 2013; Kushkevych et al. 2014). The
strain has been kept in the collection of microorganisms at
the Department of Molecular Biology and Pharmaceutical
Biotechnology of the Faculty of Pharmacy at the University
of Veterinary and Pharmaceutical Sciences Brno (Czech
Republic). Ciprofloxacin (Sigma-Aldrich) was used as the
standard. Prior to testing, the strain at exponential phase
growth was passaged onto nutrition modified KravtsovSorokin’s
(KS) agar medium (Kushkevych and Moroz
2012). Bacterial inocula were prepared by suspending a
small portion of bacterial colony in sterile KS liquid medium
(pH 7.5). Before bacterial passage in the medium, 10
mL/L of sterile Mohr’s salt solution [(NH4)SO4Fe
(SO4)2·6H2O] (10%) for visual detecting colonies of the
SRB was added. A culture sample (10 mL) was centrifuged
at 15,000 rpm/20 min using a bench top centrifuge (Model
CR 4-12, Jouan Inc., Winchester, VA, USA). Following
removal of the supernatant, the pellet was washed in fresh
liquid KS and re-suspended in fresh supplemented KS (10
mL). The turbidity was adjusted to match McFarland standard
No. 1 (5 × 106
cfu) with KS using a densitometer
(Densi-La-Meter, LIAP, Latvia). The final inoculum was
made to a 1:20 dilution of the suspension with KS liquid
medium. The antimicrobial susceptibility of SRB was
investigated in a 96-well plate format. In these experiments,
sterile KS (300 µL) was added to all outer-perimeter wells
of the plates to minimize evaporation of the medium in the
Med Chem Res (2018) 27:278–284 279
test wells during incubation. Sample wells were composed
of 100 µL of test compound dilution and 100 µL of the
bacterial stock being tested against. The compounds were
dissolved in dimethyl sulfoxide (DMSO, Sigma), and the
final concentration of DMSO in the KS liquid medium did
not exceed 0.1% of the total solution composition. Dilutions
of each compound were prepared in triplicate. The
final concentrations of the evaluated compounds ranged
from 100 to 0.05 μM. Plates were sealed with parafilm,
introduced into an anaerobic box with oxygen uptake
generators (GENbox anaer, France) for anaerobiosis.
The determination of results was performed visually after
72 h of static incubation in the darkness at 37 °C under
anaerobic conditions. In the process of bacterial growth,
hydrogen sulfide was formed and interacted with Fe2+
from
Mohr’s salt. As a result, FeS was formed by the bacterial
cells that caused black colored colonies that was interpreted
as a presence of bacterial growth. The medium dilution
micro-method modified according to NCCLS guidelines
(CLSI 2012, 2014) in KS medium was used to determine
the minimum inhibitory concentration (MIC90), the inhibitory
concentration (IC50) and minimum bactericidal concentration
(MBC). Drug-free controls, sterility controls and
controls consisted of KS medium and DMSO alone were
included. The MICs were defined as the lowest concentration
of the compound at which no visible bacterial growth
was observed. The IC50 values were defined as the compound
concentration causing 50% inhibition of bacterial
growth and the MBCs were defined as the lowest concentration
of the compound at which an antimicrobial agent
kills a bacteria (CLSI 2012, 2014). The results are summarized
in Table 1.
Results and discussion
In the previous studies it was confirmed that 2-substituted 8hydroxyquinolines
are promising antimicrobial agents
(Jampilek et al. 2005; Musiol et al. 2006, 2010; Darby and
Nathan 2010; Gonec et al. 2012; Cieslik et al. 2012; 2015;
Kos et al. 2015a). Based on these observations, 8hydroxyquinoline-2-carboxanilides
prepared according to
Scheme 1 and described recently (Kos et al. 2015a) were
tested against Desulfovibrio piger Vib-7. The testing was
performed according to Kushkevych et al. (2016).
The antibacterial potency of ring-substituted 8hydroxyquinoline-2-carboxanilides
was expressed as the
MICs, IC50 values and MBCs, see Table 1. The activity of
the most potent compounds 7b (R = 3-CF3; MIC = 17 µM,
IC50 = 10 µM, MBC = 20 µM), 7c (R = 4-CF3; MIC = 20
µM, IC50 = 10 µM, MBC = 22 µM) and 2b (R = 3-OCH3;
MIC = 17 µM, IC50 = 11 µM, MBC = 20 µM) was twofold
higher that of the clinically used drug ciprofloxacin (MIC =
MBC = 45 µM) that was used as the standard. Also other
compounds such as 3b (R = 3-CH3; MBC = 23 µM), 1
(R = H; MBC = 25 µM), 2a (R = 2-OCH3; MBC = 28
µM), and 5b (R = 3-Br; MBC = 35 µM) were effective in
killing D. piger Vib-7. It is also important to note that no
significant cytotoxic effect of these compounds on THP-1
cells up to tested concentration 30 µM was observed (Kos
et al. 2015a).
The dependence of log(1/IC50) on compound lipophilicity
expressed by log k (Kos et al. 2015a) is shown in
Fig. 1a. After exclusion of three derivatives: 4-OCH3 (2c),
which precipitated from testing solution due to limited
Table 1 Experimentally determined values of lipophilicity log k,
predicted electronic Hammett’s σ parameters of substituents R, in vitro
antibacterial activity against Desulfovibrio piger Vib-7 (MIC, IC50,
MBC) of the compounds in comparison with ciprofloxacin (CPX)
standard
Compounds R1
log ka
σb
MIC90
[μM]
IC50
[μM]
MBC
[μM]
1 H 0.7600 0 23 12 25
2a 2-OCH3 0.7935 −0.28 28 17 28
2b 3-OCH3 0.8164 0.12 17 11 20
2c 4-OCH3 0.7129 −0.27 557 380 557
3a 2-CH3 0.6944 −0.17 44 15 48
3b 3-CH3 0.9686 −0.07 23 10 23
3c 4-CH3 0.9521 −0.17 75 40 80
4a 2-F 0.6806 0.06 90 50 95
4b 3-F 0.9420 0.34 45 20 50
4c 4-F 0.8598 0.06 60 28 60
5a 2-Cl 0.9566 0.22 120 95 123
5b 3-Cl 1.1718 0.37 50 20 55
5c 4-Cl 1.1543 0.23 480 330 486
6a 2-Br 1.0536 0.22 150 103 150
6b 3-Br 1.2357 0.39 33 18 35
6c 4-Br 1.2347 0.23 337 225 340
7a 2-CF3 0.9147 0.51 48 35 50
7b 3-CF3 1.3206 0.43 17 10 20
7c 4-CF3 1.3653 0.51 20 10 22
8a 2-NO2 1.1277 0.77 197 134 202
8b 3-NO2 0.9845 0.71 237 218 240
8c 4-NO2 1.0495 0.78 223 165 225
CPX – – – 45 28 45
a
Experimental procedure described in Kos et al. (2015a)
b
Predicted using sw. ACD/Percepta ver. 2012
280 Med Chem Res (2018) 27:278–284
aqueous solubility, and 2-F (4a) and 3-NO2 (8b), showing
lower activity than expected, the compounds can be divided
into two groups. The antibacterial activity of nine derivatives
with R = 2-CH3 (3a), 2-OCH3 (2a), 3-OCH3 (2a), 3-F
(4b), 3-CH3 (3b), 3-Cl (5b), 3-Br (6b), 3-CF3 (7b), and 4CF3
(7c) expressed by IC50 value was comparable with that
of unsubstituted derivative 1 (R = H; IC50 = 12 µM) and
varied in the range from 10 µM (3-CF3 and 4-CF3, 3-CH3)
to 20 µM (3-F and 3-Cl), while the antibacterial activity of
other 12 tested (C’(2), C’(4)-substituted derivatives and (1)
compounds decreased linearly with increasing compound
lipophilicity from log k = 0.6944 (2-CH3, 3a; IC50 = 15
µM) to log k = 1.2347 (4-Br, 6c; IC50 = 225 µM) (r =
−0.9477, n = 12). Thus, from seven C’(3)-substituted
compounds, six derivatives belonged to the set of the
above-mentioned most active compounds, the antibacterial
activity of which practically did not change with increasing
compound lipophilicity, indicating that this position of
substitution is favorable for significant antimicrobial effect.
The dependence of log(1/IC50) on the Hammett’s σ
constants of the R substituent is shown in Fig. 1b. Except
for derivatives with R = 4-OCH3 (2c) and 4-CH3 (3c) as
well as derivatives with F, Cl, and Br substituents in positions
C’(2) and C’(4), i.e., 4a, 4c, 5a, 5c, 6a, and 6c, for the
remaining 14 compounds quasi-parabolic dependence of
log(1/IC50) on σ constants was estimated. However, it could
be mentioned that within a narrow range of σ from 0.06 to
0.22/0.23, the activity of C’(4) halogen-substituted compounds
decreased more sharply with increasing σ values
than that of C’(2)-substituted ones.
Similar trends as for the dependence of log(1/IC50) on
lipophilicity or on Hammett’s σ constants can be found also
for the dependences of the activity expressed as MIC or
MBC values on both parameters; therefore, these dependences
are not illustrated.
Summarizing, it can be concluded that generally for high
antibacterial activity against D. piger, halogen substituent in
the C’(3) position is favorable, whereby the highest antibacterial
activity was exhibited by derivatives with R =
3-CF3 and 4-CF3, i.e., substituents that are known to promote
electrostatic interactions with targets and improve the
cellular membrane permeability of small molecules but also
compounds with low lipophilicity (2-CH3, 3-CH3, 2-OCH3,
3-OCH3).
The estimated MIC values for D. piger (Table 1) are
comparable with those estimated for the antimycobacterial
activity of the tested compounds against Mycobacterium
tuberculosis H37Ra ATCC 25177, M. avium complex
CIT19/06 (clinical isolate) and M. avium subsp. paratuberculosis
CIT03 (clinical isolate) (Kos et al. 2015a). It
was found that with the exception of compounds with R =
4-OCH3 (2c), 4-Cl (5c), 4-Br (6c), neither lipophilicity nor
electronic properties of the R substituent nor the position of
substitution exhibited any significant effect on the antitubercular
activity against M. tuberculosis, and the 2-, 3- and
4-CF3 (7a–c)-substituted derivatives belonged to the most
active compounds with MIC = 24 µM. There were no any
significant differences between antimycobacterial activities
against M. tuberculosis and M. avium subsp. paratuberculosis.
On the other hand, C’(2) and especially C’(3)Scheme
1 Synthesis of ringsubstituted
8-hydroxyquinoline-
2-carboxanilides 1–8c: Reagents
and conditions: a PCl3,
chlorobenzene, microwaveassisted
synthesis (Kos et al.
2015a)
Fig. 1 Dependence of antibacterial activity against Desulfovibrio
piger Vib-7 expressed as log(1/IC50) [M] of tested compounds on
lipophilicity expressed as log k (a) and on Hammett’s σ constants of R
substituent (b). (Compounds not included in individual SAR discussions
are marked by empty symbol.)
Med Chem Res (2018) 27:278–284 281
substituted derivatives expressed higher antimycobacterial
activity against M. avium complex than C’(4)-substituted
ones, and antimycobacterial activity slightly increased with
increasing lipophilicity (log k values) and electronwithdrawing
effect of R substituents; the C’(2)-substituted
compounds with log k > 0.8 and Hammett’s σ constants of R
substituents > 0.1 and C’(4)-substituted compounds with log
k < 0.8 were found to be completely ineffective. However,
the activity of compounds with potency against all three
strains was achieved by the substitution of the C’(3) position
of aniline. The significant bacterial/antimycobacterial
activity of the studied compounds bearing an 8-hydroxyquinoline
fragment and an amide moiety in their molecule is
caused by the fact that these function groups are able to
interact with a number of enzymes/receptors via hydrogen
bonds and in this manner to affect the biological response
(Pattabiraman and Bode 2011; Lavecchia and Di Giovanni
2013; Zumla et al. 2013). The significant contribution of the
hydroxyl moiety in C(8) of quinoline to the antimycobacterial
activity was reported by Gonec et al. (2012),
who observed that its absence in quinoline-2-carboxanilides
resulted in a decrease of antimycobacterial effect, while
strengthening of antimycobacterial potency due to the presence
of the hydroxyl moiety was observed with 1-hydroxynaphthalene-2-carboxanilides
(Gonec et al. 2013) and 6hydroxy-naphthalene-2-carboxanilides
(Kos et al. 2015b)
as compared to naphthalene-2-carboxanilides (Gonec et al.
2012).
Ring-substituted 8-hydroxyquinoline-2-carboxanilides
were previously tested also as photosystem II (PS II)
inhibitors (Jampilek et al 2016). The inhibition of photosynthetic
electron transport (PET) in spinach chloroplasts
by these compounds significantly depended on the position
of substitution, and the inhibitory activity of C’(3)-substituted
compounds was by one or two orders higher
than that of C’(2) and C’(4)-substituted derivatives. For the
most active compounds, the following IC50 values were
observed: 2.7 µM (3-CH3, 3b), 2.3 µM (3-F, 4b), 3.6 µM
(3-Cl, 5b), and 3.4 µM (3-Br, 6b). However, it could be
mentioned that the dependence of the PET-inhibiting
activity on the lipophilicity of the compounds expressed
by log k was quasi-parabolic for C’(3)-substituted derivatives,
while for C’(2) ones a slight increase and for
C’(4) derivatives a sharp decrease of the activity were
observed with increasing lipophilicity. Consequently,
it could be assumed that for targeting the site of action
in the photosynthetic apparatus suggested on the acceptor
side of photosystem II between P680 and plastoquinone
QB substitution in the C’(3) position is the most
favorable.
Previously tested six 2-(phenylcarbamoyl)phenyl N[(benzyloxy)carbonyl]
alkanoates and three 2-hydroxy-N[(2
S)-1-oxo-1-(phenylamino)alkan-2-yl]benzamides showed
MIC values in the range from 0.22 to 0.35 μM against
D. piger Vib-7 and in the range from 0.27 to 8.52 μM
against Desulfomicrobium sp. Rod-9, while the MIC values
of ciprofloxacin were 41.2 μM and 39.3 μM, respectively.
Lipophilicity was recognized as a significant parameter
affecting biological activities, and higher activity for both
SRB was observed rather with electron-withdrawing R2
substituent and less lipophilic (isopropyl or benzyl) R3
substituent, and it was supposed that these derivatives
interact with enzymatic systems of the bacteria affecting
vital cell functions (Kushkevych 2015a, b; Kushkevych
et al. 2015a, b; Kushkevych et al. 2016). This is in agreement
with the presented results concerning the antibacterial
activity of ring-substituted 8-hydroxyquinoline-2carboxanilides
against D. piger Vib-7. Thus, these results
confirmed that the investigated compounds showed high
efficiency not only against the aerobic microorganisms, but
also against the anaerobic microorganism.
Conclusion
Primary in vitro screening of a prepared series of ringsubstituted
8-hydroxyquinoline-2-carboxanilides was performed
against Desulfovibrio piger Vib-7. The most
effective compounds with MIC/MBC values in the range
of 17–23 µM/20–23 µM, respectively, were as follows:
8-hydroxy-N-(3-trifluoromethylphenyl)- (7b), 8-hydroxy-N(3-methoxyphenyl)-
(2b) 8-hydroxy-N-(3-methylphenyl)(3b),
and 8-hydroxy-N-(4-trifluoromethylphenyl)quinoline-
2-carboxamide (7c). Their activity was twofold higher
than that of ciprofloxacin. The antibacterial efficacy of the
most active C’(3)-substituted compounds practically did
not change with increasing compound lipophilicity, indicating
that this position of substitution is favorable for
significant antimicrobial effect, while the antibacterial
activity of most of C’(2) and C’(4)-substituted derivatives
decreased linearly with increasing compound lipophilicity.
In addition, the dependence of activity on electronic Hammett’s
σ parameter of the substituent R was quasi-parabolic
for the most effective C’(3)-substituted compounds. The
most potent compounds did not express any significant
cytotoxic effect on THP-1 cells up to the tested concentration
of 30 µM.
Acknowledgements This study was supported by CZ.1.07/2.3.00/
30.0053, APVV-0516-12, IGA VFU Brno 318/2017/FaF, and also by
SANOFI-AVENTIS Pharma Slovakia, s.r.o.
Compliance with ethical standards
Conflict of interest The authors declare that they have no competing
interests.
282 Med Chem Res (2018) 27:278–284
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ATTACHMENT 11
Pages 115 – 121
_____________________________________________________________________________
Cross-correlation analysis of the Desulfovibrio growth parameters of intestinal species
isolated from people with colitis
Ivan Kushkevych, Dani Dordević, Monika Vítězová, Peter Kollár
Biologia, 2018, 73(11), 1137–1143, ISSN 0006-3088
_____________________________________________________________________________
ORIGINAL ARTICLE
Cross-correlation analysis of the Desulfovibrio growth parameters
of intestinal species isolated from people with colitis
Ivan Kushkevych1,2
& Dani Dordević3
& Monika Vítězová1
& Peter Kollár2
Received: 2 May 2018 /Accepted: 22 August 2018
# Institute of Molecular Biology, Slovak Academy of Sciences 2018
Abstract
Sulfate-reducing bacteria can be involved in inflammatory bowel disease. Cross-correlation parameters of their metabolic process
in the gut have never been reported before. The aim of the research was to statistically (cross-correlation) evaluate the parameters
of growth (biomass) of Desulfovibrio species from colitis people and healthy as well as to investigate the change in dissimilatory
sulfate reduction of these bacterial strains. The microbiological, biochemical, chemical, and statistical methods were used in the
research. The cross-correlation analysis indicates that the strains isolated from people with colitis shifted to the right side of Yaxe
by biomass accumulation, sulfate consumption, lactate oxidation as well as hydrogen sulfide and acetate production, compared
with the strains isolated from healthy individuals. Different percentages were observed in shifting to the right side of Y axe:
biomass accumulation 26%, sulfate consumption 1.5%, sulfide production 5%, lactate oxidation 3% and acetate production 12%.
The biomass accumulation of intestinal sulfate-reducing bacteria and their hydrogen sulfide production are the main factors in
inflammatory bowel disease development, including ulcerative colitis. Acetate production showed lesser influence while sulfate
consumption and lactate oxidation are negligible factors in the inflammatory bowel disease.
Keywords Bowel diseases . Ulcerative colitis . Hydrogen sulfide . Sulfate consumption
Abbreviations
SRB sulfate-reducing bacteria
UC ulcerative colitis
IBD inflammatory bowel disease
DSR dissimilatory sulfate reduction
OD optical density
PCA principal component analysis
Introduction
Inflammatory bowel diseases (IBD) in animals and humans is
highly correlated with the presence of increased number of
sulfate reducing bacteria (SRB); consequently also by their
sulfate reduction process in the gut (Gibson et al. 1991,
1993; Kushkevych et al. 2015a; Kováč et al. 2018). This bacterial
group is present very often in individuals diagnosed with
other diseases, including rheumatic diseases and with ankylosing
spondylitis (Barton and Hamilton 2010; Cummings
et al. 2003; Pitcher and Cummings 1996). SRB consume sulfate
as electron acceptor and convert it in metabolic pathways
to final products, hydrogen sulfide. Hydrogen sulfide, which
is toxic and beside the support of IBD development (including
UC and Crohn’s disease), it can also cause high risk for neurodegenerative
illness (DNA damage and mutation, enzyme
inhibition and mitochondrial respiration inhibition) (AtteneRamos
et al. 2006; Kováč and Kushkevych 2017;
Kushkevych et al. 2015b, 2017b, 2018c, d).
Exogenic electron donors are necessary to provide sulfate
reduction process, including organic compounds (e.g. lactate,
propionate, ethanol, butyrate, etc.), which can play a role as
the source of carbon and energy (Kushkevych et al. 2015a, b).
* Ivan Kushkevych
kushkevych@mail.muni.cz
1
Department of Experimental Biology, Faculty of Science, Masaryk
University, Kamenice 753/5, 625 00 Brno, Czech Republic
2
Department of Human Pharmacology and Toxicology, Faculty of
Pharmacy, University of Veterinary and Pharmaceutical Sciences
Brno, Brno, Czech Republic
3
Department of Plant Origin Foodstuffs Hygiene and Technology,
Faculty of Veterinary Hygiene and Ecology, University of Veterinary
and Pharmaceutical Sciences Brno, Brno, Czech Republic
Biologia
https://doi.org/10.2478/s11756-018-0118-2
Lactate is a universal electron donor for this bacterial group.
These organic compounds are oxidized by SRB, depending on
genera, to acetate (incompletely) or carbon (IV) oxide and
water (completely). Among SRB species, Desulfovibrio genus
is the most often detected in feces from the patients with
bloody diarrhea, weight loss, anorexia, epithelial hyperplasia
and abscesses (both is animals and humans) (Loubinoux et al.
2000, 2002a, b; Kushkevych et al. 2017a, 2018a, d).
There is a constant increase of people diagnosed with ulcerative
colitis. According to statistical results IBD (including
UC) rose up to 12 per 100,000 people in Western countries
(Low et al. 2013; Rowan et al. 2009). The prevalence in
Europe and North America is around 20 per 100,000 individuals,
while in Asia and the Middle East is around 6. The
majority of UC patients are between ages of 15 and 30 years;
another group with higher prevalence are individuals aged 50
to 70 years. The influence of gender has not been observed in
ulcerative risk. Economic and health care burdens are increasing
due to the growing UC prevalence since the severity of
these inflammations can be overviewed by the fact that they
very often lead to severe flares that require hospitalization in
up to 25% of patients (Low et al. 2013; Feuerstein et al. 2016).
Ulcerative colitis development represents complex molecular
etiology including genetic, microbial, environmental and other
still unknown factors. Many studies including animal and
human samples have been conducted for better understanding
of IBD, though many its segments have not been explained
yet (Low et al. 2013; Steenland et al. 2018).
Medication and dosage in medical treatment affect location
and severity of UC disease (Gibson et al. 1993; Pitcher and
Cummings 1996; Kushkevych et al. 2016, 2018d).
Intestinal SRB are the part of the whole gut system, especially
due to their interaction with other microorganisms. Their
process of sulfate reduction is biosystem with autoregulation
whose survival and development are largely dependent on the
fluctuations of different parameters (Brynjarsdottir et al. 2003).
The subordination of the biological clock rhythm serves as a
determining factor in the regulation of oscillatory dynamics of
these processes, during the simultanious separation of bacterial
cells (accompanied by the fluctuations of biophysical and metabolic
parameters) (Chen and Popovich 2002). In previous
studies, ten various Desulfovibrio sp. strains (colitis: n = 5;
healthy: n = 5) were isolated from the human intestine and
identified. The growth of these bacteria, their use of sulfate
and lactate as well as the production of hydrogen sulfide and
acetate were also studied (Kushkevych 2013).
The novelty of the study is the comparison of sulfate reduction
parameters (sulfate and lactate consumption, production of
hydrogen sulfide and acetate, as well as growth of the SRB
strains isolated from healthy and people with UC evaluated
by cross-correlation method have never been presented yet.
The aim of the research was to statistically (crosscorrelation)
evaluate the parameters of growth (biomass) of
Desulfovibrio species from colitis people and healthy as well
as to investigate the change in dissimilatory sulfate reduction
of these bacterial strains.
Materials and methods
Bacterial culture and cultivation In total, 10 of sulfatereducing
bacteria Desulfovibrio strains, five of them isolated
from the healthy human large intestine and five another isolated
from people with colitis, were as described previously
(Kushkevych 2013). The strains have been kept in the collection
of microorganisms at the Department of Experimental
Biology, Faculty of Science at the Masaryk University
(Brno, Czech Republic). The bacteria were grown for 36 h
at 37 °C under anaerobic conditions in modified Postgate’s
liquid medium (Kováč and Kushkevych 2017; Postgate
1984). The redox and anaerobic conditions were controlled
by sodium resazurin (Oxoid, BR 0055B) as an indicator. In
addition reduced FeS and Na2S contained in the medium provided
the necessary redox conditions for SRB cultures. The
discoloration of sodium resazurin (redox potential of discoloration
Eh = −100 mV) confirmed the decrease of redox
potential.
Assay of bacterial biomass About 1 mL of liquid medium
without Mohr’s salt was transferred into a plastic cuvette and
taken to a biophotometer (Eppendorf BioPhotometer®D30)
for taring. Subsequently, 1 mL of bacterial suspension was
transferred into another cuvette and taken again to the
biophotom eter for measuring at OD λ = 340. Before SRB
were used for the experiments, optical density (OD340) was
always measured to assure approximately the same amount of
bacteria in each experiment (Kushkevych et al. 2015b).
Assay of sulfate, lactate sulfide and acetate The sulfate ion
concentration in the medium was determined by the turbidimetric
method after it had been precipitated by barium chloride
as was described in the paper (Kolmert et al. 2000).
Lactate concentration was measured through the dehydrogenation
reaction using Lactate Assay Kit (Sigma-Aldrich,
Catalog Number MAK064). Sulfide concentration in the culture
medium was assayed by the spectrophotometric method
as was described (Cline 1969). Accumulation of acetate ions
in the process of bacterial growth in the medium was determined
using Acetate Assay Kit (Colorimetric, Catalog
Number KA3764).
All analyses were done during 6 days period; the analysis
(biomass determination, sulfate consumption, lactate oxidation,
and production sulfide and acetate) were performed each day.
Statistical analysis The correlation analysis in time has been
carried out for the study of temporal correlations between the
Biologia
growth of bacteria and their metabolic processes. The values
of the defined function parameters, namely the bacterial
growth, the sulfate consumption, lactate oxidation as well as
hydrogen sulfide and acetate production by the Desulfovibrio
strains were used in the correlation study. Overall differences
of indicated above parameters were checked by principal component
analysis (PCA), using SPSS 20 statistical software
(IBM Corporation, Armonk, USA). Cross-correlation plots
were built by software package Origin7.0 (www.origin-lab.
com). Using the experimental data, the basic statistical
parameters (mean: M, standard error: m, M ± m) were
calculated. The accurate approximation was when p ≤ 0.05
(Bailey 1995).
Results
Cross-correlation correlograms that corresponds with data
present in Table 1 are shown in Figs. 1, 2 and 3. All figures
show noticeable differences between healthy and colitis objects,
though the most noticeable difference is observed in
biomass accumulation (Fig. 1) and the smallest in acetate production
(Fig. 3). The differences between analyzed physiological
and biochemical properties of bacterial strains isolated
from feces can be seen through curve time shifts of colitis
samples in comparison with curves indicating results obtained
from healthy individuals. The positive time shifts of colitis
patients are observed in all figures, though differences in results
are also obtained. More significantly expressed positive
correlations for colitis samples can be observed in results for
biomass, sulfide and sulfate; quite low positive correlations
were found for lactate and acetate (Figs. 1, 2 and 3). Crosscorrelation
data between sulfate reduction parameters in
Desulfovibrio isolates from healthy objects and people with
colitis are shown in Table 1. From the Table 1 can be observed
that noticeable (p < 0.05) differences exist between healthy
and colitis objects in all measured parameters (biomass accumulation,
sulfate and lactate consumption, sulfide and acetate
production). The differences in overall cross-correlation factors
are not very well noticeable (the differences are clearly
observable in Fig. 4), but differences between the results in the
middle of cross-correlation factors (the results over zero,
where stability is assumed) between healthy and colitis objects
in biomass accumulation was 26%, sulfate consumption
1.5%, sulfide production 5%, lactate oxidation 3% and acetate
production 12%. The confirmation of these results is shown
Fig. 4 by principal component analysis (PCA). According to
Eigen values 3 groups are formed in PCA graph, showing
differences between healthy and colitis individuals. The most
remarkable differences are confirmed with separate PCA
group differentiating biomass, sulfate and sulfide results of
colitis samples from other samples.
Discussion
The SRB are wide-spread in the nature and sulfate rich environment,
especially in different bioreactors where they interact with
methanogenic microorganisms (Kushkevych et al. 2018b, c).
Sulfate in food may play an important role in human metabolism.
Free sulfate ions affect large bowel metabolism where it is
reduced to hydrogen sulfide (the substance that is considered
Table 1 Data on cross-correlations between sulfate reduction parameters in Desulfovibrio isolates from healthy objects and people with colitis
Series Pair/Lag Biomass Sulfate Sulfide Lactate Acetate
Healthy Colitis Healthy Colitis Healthy Colitis Healthy Colitis Healthy Colitis
-7 −0.29 −0.21 −0.28 −0.21 −0.30 −0.26 −0.34 −0.31 −0.40 −0.39
−6 −0.33 −0.22 −0.28 −0.22 −0.35 −0.27 −0.36 −0.34 −0.47 −0.46
−5 −0.28 −0.23 −0.23 −0.23 −0.29 −0.27 −0.28 −0.27 −0.38 −0.40
−4 −0.18 −0.18 −0.14 −0.18 −0.17 −0.19 −0.15 −0.15 −0.13 −0.19
−3 −0.01 −0.09 0.04 −0.09 0.03 −0.05 0.08 −0.01 0.17 0.10
−2 0.29 0.04 0.22 0.04 0.21 0.13 0.32 0.20 0.47 0.42
−1 0.64 0.34 0.53 0.34 0.55 0.40 0.65 0.50 0.72 0.73
0 0.96 0.92 0.97 0.92 0.97 0.95 0.97 0.97 0.95 0.99
1 0.50 0.55 0.42 0.55 0.57 0.59 0.52 0.62 0.61 0.68
2 0.14 0.28 0.08 0.28 0.27 0.29 0.14 0.27 0.27 0.35
3 −0.09 0.09 −0.08 0.09 0.03 0.10 −0.08 0.03 −0.07 0.00
4 −0.22 −0.16 −0.19 −0.16 −0.17 −0.12 −0.21 −0.13 −0.29 −0.25
5 −0.28 −0.24 −0.24 −0.24 −0.28 −0.28 −0.30 −0.26 −0.38 −0.39
6 −0.29 −0.27 −0.24 −0.27 −0.34 −0.30 −0.30 −0.35 −0.37 −0.42
7 −0.25 −0.27 −0.23 −0.27 −0.33 −0.32 −0.27 −0.33 −0.30 −0.35
Biologia
potentially toxic to colonic epithelium. Sulfate concentrations
are found in more than 200 individual foods (in concentrations:
>10 μmol/g or 1 mg/g), including breads, soya flour, some dried
fruits, some brassicas, and some sausages. Beers, ciders and
wines are also considered sulfate rich sources (in concentrations:
>2.5 μmol/mL or 0.25 mg/mL). The sulfate content of beer is
commented in epidemiological observations which linked ingestion
of beer with colorectal cancer (Florin et al. 1993).
The intestinal SRB metabolize sulfate free ions as an electron
acceptor to hydrogen sulfide. This process of sulfate dissimilation
is called the Bdissimilatory sulfate reduction^ or
Bsulfate respiration^ (Kushkevych 2017a). For this process,
SRB need organic compounds or molecular hydrogen which
are exogenous electron donors. These organic compounds can
be oxidized incompletely to acetate (acetogenic SRB, in particular
Desulfovibrio species) or completely to CO2 (Barton
and Hamilton 2010). The sulfate reduction intensity by SRB
and, accordingly, hydrogen sulfide production in high
concentrations in the intestine can lead to the development
of various diseases (Kushkevych et al. 2015a).
The cross-correlation method represents a generalization of
standard linear correlation analysis. The strength of the correlation
is measured by the correlation coefficient. Crosscorrelation
analysis is the most commonly used as multiple
time series analysis. Cross-correlation gives a correlation between
two time series or two waveforms. One series observations
are correlated with other series observations (Bourke
1996). The cross-correlation data sets of two time-series test
involves many calculations of the coefficient r by timeshifting
the one data set relative to the other data set. The sift
is called Blag^, while the lag time is simply the sampling
period of the two time-series data sets (Bourke 1996). As
can be seen from correlograms (Figs. 1, 2 and 3) and
Table 1, cross-correlation values close to 1 represent the strong
relationship between two waveforms and weak relationship
indicate correlation values close to zero. Negative correlation
values, below zero, are emphasizing a lack of stability in the
relationship (Chen and Popovich 2002; Bourke 1996).
Different landslides of some processes are determined by a
positive correlation coefficient value (Chen and Popovich
2002). The cross-correlation analysis is built from the
correlograms for Desulfovibrio sp. strains. A correlogram is
a sequence of values of the correlation coefficients.
It is well known that Desulfovibrio genus are the most often
detected in people with IBD, including ulcer active colitis
(Kushkevych 2015a, b; Kushkevych et al. 2015a, b). The
presence of sulfate and lactate in the human intestine influences
bacterial growth and the accumulation of their final
metabolic products: acetate and hydrogen sulfide. Hydrogen
sulfide is toxic, mutagenic same as cancerogenic for epithelial
intestinal cells (Pitcher and Cummings 1996; Rowan et al.
2009). This final metabolite in high concentrations is also
carcinogenic for intestinal cells and can cause inhibition of
cytochrome oxidase, colonocytes oxidation of butyrate, destruction
of epithelial cells, and development of ulcers and
Fig. 1 Cross-correlation correlogram showing the bacterial growth
(biomass accumulation) of the Desulfovibrio strains isolated from
healthy people and with colitis. *each point represents one day
measurement during six days period
Fig. 2 Cross-correlation correlograms showing sulfate consumption and hydrogen sulfide production with time by bacterial Desulfovibrio strains,
isolated from healthy and people with colitis. *each point represents one day measurement during six days period
Biologia
inflammation with subsequent development of ulcerative colitis
(Pitcher and Cummings 1996; Gibson et al. 1991;
Cummings et al. 2003; Kushkevych 2015c, d).
The illness development is caused by the increase amount
of intestinal SRB and their production of hydrogen sulfide.
The shift of the Desulfovibrio growth (biomass) and parameters
of sulfate reduction to right side of Y axe (shown in Figs.
1, 2 and 3) can be a main cause disturbance of relationship of
microbial populations in the gut. Consequently, the shift can
indicate the development of IBD, including UC. On the other
side, acetate produced by intestinal SRB can interact with
hydrogen sulfide can increase its effect (synergic effect), leading
to increased IBD risk. Attene-Ramos et al. (2006) published
the research in which the authors are emphasizing that
Na2S is able to provoke genomic DNA damage in colonic
cells (Attene-Ramos et al. 2006). A consensus and better understanding
of the metabolic pathways involved in H2S metabolism
in colonocytes is still needed. Imbalance between the
concentration of free sulfide in the large intestine mucosa and
the metabolisms capacity of epithelial cells results in a loss of
normal oxidative cell capacity. Endogenously produced H2S
plays a role in the neuromodulation of chloride secretion, in
intestinal contractibility control, and on the large intestine
nociception. Though, it is still not confirmed that H2S is acting
as a pro- or antinociceptive agent toward the large intestine.
Mammalian cells use very simple sulfur-containing gas molecule
due to the reminiscence of an ancestral function
(Griesbeck et al. 2002).
Hence, the role of intestinal SRB in colonic conditions is
important in better understanding of their ability to inhibit the
Fig. 3 Cross-correlation correlograms showing lactate consumption and acetate production with time by Desulfovibrio bacterial strains isolated from
healthy and people with colitis
Fig. 4 Principal component
analysis (PCA) of crosscorrelation
data between sulfate
reduction parameters in
Desulfovibrio isolates (data
referring to Table 1). *each point
represents one day measurement
during six days period
Biologia
production of hydrogen sulfide and acetate. Overall, all stated
issues are emphasizing the factors influencing sulfide production
in the human and animal colon.
Conclusions
Biomass accumulation of intestinal SRB and their hydrogen
sulfide production are the main factors in IBD development,
including UC. The statement is in accordance with observation
from correlograms obtained by cross-correlation. Acetate
production showed lesser influence. It should be also not
underestimated the possibility of acetate production synergy
with H2S. According to gained results, sulfate consumption
and lactate oxidation are negligible factors in IBD.
Acknowledgements This study was supported by University of
Veterinary and Pharmaceutical Sciences Brno (project IGA VFU 303/
2018/FaF).
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Human and animal rights This article does not contain any studies with
human participants or animals performed by any of the authors.
Informed consent Informed consent was obtained from all individual
participants included in the study.
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ATTACHMENT 12
Pages 122 – 130
_____________________________________________________________________________
Toxicity of hydrogen sulfide toward sulfate-reducing bacteria Desulfovibrio piger Vib-7.
Ivan Kushkevych, Dani Dordević, Monika Vítězová
Archives of Microbiology, 2019, 201, 1-9, ISSN 0302-8933
_____________________________________________________________________________
Vol.:(0123456789)1 3
Archives of Microbiology
https://doi.org/10.1007/s00203-019-01625-z
ORIGINALPAPER
Toxicity of hydrogen sulfide toward sulfate-reducing bacteria
Desulfovibrio piger Vib-7
Ivan Kushkevych1
   · Dani Dordević2
 · Monika Vítězová1
Received: 22 November 2018 / Revised: 16 January 2019 / Accepted: 25 January 2019
© Springer-Verlag GmbH Germany, part of Springer Nature 2019
Abstract
Sulfate-reducing bacteria (SRB) belonging to the intestinal microbiota are the main producers of hydrogen sulfide and their
increasing amount due to the accumulation of this compound in the bowel are involved in the initiation and maintenance
of inflammatory bowel disease. The purpose of this experiment is to study the relative toxicity of hydrogen sulfide and
survival of Desulfovibrio piger Vib-7 through monitoring: sulfate reduction parameters (sulfate consumption, hydrogen
sulfide production, lactate consumption and acetate production) and kinetic parameters of these processes. The research is
highlighting the survival of intestinal SRB, D. piger Vib-7 under the influence of different hydrogen sulfide concentrations
(1–7 mM). The highest toxicity of ­H2S was measured in the presence of concentrations higher than 6 mM, where growing
was stopped, though metabolic activities were not 100% inhibited. These findings are confirmed by cross correlation and
principal component analysis that clearly supported the above mentioned results. The kinetic parameters of bacterial growth
and sulfate reduction were inhibited proportionally with increasing ­H2S concentration. The presence of 5 mM ­H2S resulted
in two times longer lag phase and generation time was eight times longer. Maximum rate of growth and hydrogen production
was stopped under 4 mM, emphasizing the ­H2S toxicity concentrations to be < 4 mM, even for sulfide producing bacteria
such as Desulfovibrio. The results are confirming ­H2S concentrations toxicity toward Desulfovibrio, especially the study
novelty should be emphasized where it was found that the exact ­H2S limits (> 4 mM) toward this bacterial strain inhabiting
humans and animals intestine.
Keywords  Hydrogen sulfide · Toxicity · Sulfate-reducing bacteria · Ulcerative colitis
Abbreviations
SRB	Sulfate-reducing bacteria
UC	Ulcerative colitis
IBD	Inflammatory bowel disease
DSR	Dissimilatory sulfate reduction
OD	Optical density
PCA	Principal component analysis
Introduction
The main product of the sulfate-reducing bacteria metabolism
is hydrogen sulfide (Barton and Hamilton 2010; Kushkevych
et al. 2015a, b; Kushkevych 2015a, b). Hydrogen
sulfide can be also created endogenously by trans-sulfurization
and it is present in low (non-toxic) concentrations in
the brain, heart, blood vessels, genitourinary and gastrointestinal
tract (Pitcher and Cummings 1996; Attene-Ramos
et al. 2006). Butyrate oxidation, which is the main source of
energy for intestine colonocytes of humans and animals is
inhibited by hydrogen sulfide higher concentrations (Cummings
et al. 2003; Rowan et al. 2009). Intestinal ulceration
in rats is caused by perfusion of colon by low concentrations
of hydrogen sulfide. The literature data are indicating
a connection between the sulfate reducing bacteria and the
Communicated by Erko Stackebrandt.
*	 Ivan Kushkevych
	ivan.kushkevych@gmail.com
	 Dani Dordević
	dani_dordevic@yahoo.com
	 Monika Vítězová
	vitezova@sci.muni.cz
1
	 Department of Experimental Biology, Faculty of Science,
Masaryk University, Kamenice 753/5, 625 00 Brno,
Czech Republic
2
	 Department of Plant Origin Foodstuffs Hygiene
and Technology, Faculty of Veterinary Hygiene and Ecology,
University of Veterinary and Pharmaceutical Sciences, Brno,
Czech Republic
	 Archives of Microbiology
1 3
development of the inflammatory bowel diseases. It should
be stressed out that the important factor is also the intensity
of the hydrogen sulfide concentration produced in the
colon lumen by these bacteria (Gibson et al. 1991, 1993;
Loubinoux et al. 2000, 2002a, b; Griesbeck et al. 2002a,
b; Kushkevych et al. 2016, 2018a, b, c, d, e; Kováč et al.
2018). ­H2S production is higher in the distal intestine than
in the proximal part (Attene-Ramos et al. 2006). The dense
biofilms around ulcer are formed by sulfate reducing bacteria
(Gibson et al. 1991, 1993; Pitcher and Cummings 1996;
Loubinoux et al. 2000, 2002a, b; Griesbeck et al. 2002a, b;
Cummings et al. 2003; Attene-Ramos et al. 2006; Rowan
et al. 2009; Barton and Hamilton 2010; Kushkevych et al.
2015a, 2016, 2018a, b, c, d, e; Kováč et al. 2018). The fringe
between the ulcer and the colonies of the bacteria depends
on the following factors: immune status of the organism
(host), the intestinal lumen pH and the availability of sulfate
(Loubinoux et al. 2000, 2002a). The permeability of
the epithelial barrier of the oral mucosa cells is increased
by ­H2S (Pitcher and Cummings 1996; Rowan et al. 2009;
Blachier et al. 2010).
There are literature data on the effect of hydrogen sulfide
on various microorganisms, including yeast, fungi, other
environmental or intestinal bacteria (Beauchamp et al.
1984). However, there are no sufficient numbers of researchers
focused on the influence of this compound toward its
producers (e.g., SRB of Desulfovibrio genus). Since the
sulfate-reducing bacteria are producers of hydrogen sulfide,
it is very interesting to investigate their sensitivity to this
highly toxic compound.
The aim of this work was to study the relative toxicity of
hydrogen sulfide and survival of Desulfovibrio piger Vib-7
through monitoring: sulfate reduction parameters (sulfate
consumption, hydrogen sulfide production, lactate consumption
and acetate production) and kinetic parameters of these
processes.
Materials and methods
Bacterial culture and cultivation
The objective of the study was the sulfate-reducing bacteria
of the D. piger strain Vib-7 isolated from the human
large intestine. The isolated strain was identified based on
physiological and biochemical properties as described in
the paper (Kushkevych 2013) and sequence analysis of 16S
rRNA gene (Kushkevych et al. 2014). The GenBank accession
number: KT881309.1. The strain was kept in the collection
of microorganisms at the Laboratory of Anaerobic
Microorganisms of Department of Experimental Biology at
Masaryk University (Brno, Czech Republic).
Bacteria were grown in modified liquid Postgate’s C
medium with following composition (Postgate 1984) (g/l):
­Na2SO4 (0.5), ­KH2PO4 (0.3), ­K2HPO4 (0.5), ­(NH4)2SO4
(0.2), ­NH4Cl (1.0), ­CaCl2 × 6H2O (0.06), ­MgSO4 × 7H2O
(0.1), ­C3H5O3Na (2.0), yeast extract (1.0), ­FeSO4 × 7H2O
(0.004), ­C6H5O7Na3 × 2H2O (0.3). The medium was heated
in boiling water for 30 min to obtain an oxygen-free medium,
and cooled to + 37 °C temperature. The final optimal pH 7.5
was provided by a sterile 1 M solution of NaOH (0.9 ml/l).
The bacteria were grown for 72 h at 37 °C under anaerobic
conditions. The tubes with strain were brim-filled with
medium and closed to provide anaerobic conditions (Kováč
and Kushkevych 2017).
Inoculation hydrogen sulfide and its determination
A sterile solution of ­Na2S × 9H2O with different concentration
was added before bacterial seeding the liquid medium.
The final concentration of hydrogen sulfide in the medium
was 1.0; 2.0; 3.0; 4.0; 5.0; 6.0 and 7.0 mM. The D. piger
Vib-7 growth (biomass) and their process of dissimilatory
sulfate reduction (consumption of sulfate and lactate, production
of hydrogen sulfide and acetate) under the effect of
additional introduction of ­H2S were studied. The medium
without ­Na2S × 9H2O served as a control.
Bacterial biomass determination
About 1 mL of liquid medium without Mohr’s salt was transferred
into a plastic cuvette and taken to a biophotometer
(Eppendorf ­BioPhotometer®
D30) for taring. Subsequently,
1 mL of bacterial suspension was transferred into another
cuvette and taken again to the biophotometer for measuring
at OD λ = 340. Before SRB were used for the experiments,
optical density ­(OD340) was always measured to assure
approximately the same amount of bacteria in each experiment
(Kushkevych et al. 2017a).
Sulfate determination
The content of sulfate in the medium was determined by
turbidimetric method right after seeding and after 24 h cultivation.
The calibration curve has been constructed with
the same process. Calibration solutions, has been prepared
in distilled water at concentrations of a 2, 4, 8, 16, 24, 32,
40 and 48 µM sodium sulfate. A suspension of 40 mg/L
­BaCl2 has been prepared in 0.12 M HCl. The resulting solution
was mixed with glycerol in a 1:1 ratio. To the 1 mL of
sample supernatant after centrifugation at 5000×g at 23 °C
was added to 10 mL of prepared ­BaCl2:glycerol solution
and carefully stirred. The mixture has been let to stand for
10 min and right after that the absorbance has been measured
at 520 nm (Spectronic Genesys 5). As a control, the
Archives of Microbiology	
1 3
measurement was repeated in the same manner using a cultivation
medium (Kolmert et al. 2000).
Hydrogen sulfide determination
Hydrogen sulfide was measured spectrophotometrically right
after seeding and after 72 h of cultivation. Calibration solutions
were prepared in distilled water at concentrations of
12.5, 25, 50, and 100 µM sodium sulfide. The calibration
curve has been constructed with the same process. 1 mL of
the sample was added to 10 mL of 5 g/L aqueous solution
of zinc acetate. Right after, 2 mL of 0.75 g/mL p-aminodimethylaniline
in a solution of sulfuric acid (2 M) was added.
The mixture could stand for 5 min at room temperature.
After that, 0.5 mL of 12 g/L solution of ferric chloride dissolved
in 15 mM sulfuric acid was added. After standing
another 5 min at room temperature, the mixture was centrifuged
5000×g at 23 °C. The absorbance of the mixture was
determined to measure hydrogen sulfide at a wavelength of
665 nm by a spectrophotometer (Cecil Aquarius CE 7200
Double Beam Spectrophotometer) (Cline 1969).
Lactate and acetate determination
The measurement was repeated in the same manner using
a cultivation medium and it served as the control sample
(Bailey and Pluth 2013). Measurements of lactate concentration
using Lactate Assay Kit (Sigma-Aldrich, Catalog Number
MAK064) were carried out. Accumulation of acetate
ions in the process of bacterial growth in the medium was
determined using Acetate Assay Kit (Colorimetric, Catalog
Number KA3764).
Statistical analysis
Using the experimental data, the basic statistical parameters
(M—mean, m—standard error, M ± m) have been calculated.
The accurate approximation was when p ≤ 0.05 (Bailey
1995). Statistical significance was measured with the use of
principal component analysis (PCA) that gave overall differences
among compared groups. Statistical analysis was
done by SPSS 20 statistical software (IBM Corporation,
Armonk, USA). Plots were built by software package Origin7.0
(http://www.origi​n-lab.com).
Results and discussion
Relative toxicity of hydrogen sulfide and survival parameters
of D. piger Vib-7 (bacterial growth and sulfate reduction
parameters: sulfate consumption, hydrogen sulfide production,
lactate consumption, and acetate production) are shown
in Figs. 1 and 2.
The addition of sulfide ions in concentrations of 1 mM
to the growth media resulted in 21% relative toxicity and
78% survival of bacterial cells. The increase of sulfide ion
concentrations from 2 to 3 mM increased relative toxicity
to about 50%, same as the decrease of bacterial survival
­(IC50 > 2 mM) (Fig. 1). The observed trend in Fig. 1 corresponds
to data dissimilatory sulfate reduction process
(Fig. 2). The concentrations (> 3 mM) of sulfide ions lead
to more significant trend in relative toxicity growth and
decrease in bacterial survival, while the highest added
(6–7 mM) concentrations possessed toxicity that almost
totally reduced bacterial survival (Figs. 1, 2). However,
the process of sulfate consumption (93%), lactate oxidation
(94%), hydrogen sulfide production (91%) and acetate
accumulation (91%) were not totally stopped under
high sulfide ion concentrations (6–7 mM) that is indicating
cell division inhibition, but not the end of metabolic
processes.
The influence of hydrogen sulfide on D. piger Vib-7
growth and their sulfate reduction parameters was carried
out by cluster analysis shown in Fig. 3. The cluster
analysis grouped important metabolic parameters: growth
and sulfate consumption belong to one cluster, which is
connected with hydrogen sulfide production; the process
of sulfate reduction to hydrogen sulfide needs exogenous
electron donor (lactate) that is oxidized to acetate. The
gained cluster analysis unambiguously supports this
process.
Cross-correlation correlograms are shown in Figs. 4 and
5. The results are indicating both negative (between: biomass
and sulfate, biomass and lactate, sulfate and sulfide, lactate
and acetate) and positive (between: biomass and sulfide,
biomass and acetate) correlations. The low sulfide concentrations
(1–3 mM) did not affect significantly coefficients of
correlation, measured at the time. On the contrary, higher
Fig. 1  Relative toxicity of hydrogen sulfide and survival of D. piger
Vib-7
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sulfide concentrations (from 4 to 5 mM) influenced significantly
(p < 0.05) all measured parameters, beside lactate and
acetate, in comparison with control samples (Fig. 4).
Reciprocal results are shown in Fig. 5, with the positive
(between: sulfate and lactate, sulfide and acetate) and negative
correlations (between: sulfide and lactate, sulfate and
acetate), in comparison with the control samples. Sulfide
concentrations affected most significantly (p < 0.05) sulfate
consumption and lactate oxidation, same as sulfate
consumption and acetate production. Contrarily, different
­H2S concentrations did not have an impact on sulfide
and lactate, though slight influence was noticed between
sulfide and acetate (Fig. 5).
The results gained by cross correlation analysis were
grouped by principal component analysis (PCA) as shown
in Fig. 6. PCA analyses were done, including all measured
parameters, but also separately for each parameter. PCA
analysis that included all measured parameters made two
clusters: the first cluster is formed by 1 mM, 2 mM and
3 mM; the second cluster is formed from 4 mM and 5 mM.
These groups are clearly showing significant (p < 0.05)
influence of sulfide when it is added, especially at higher
concentrations (> 4 mM), compared with control samples
(Fig. 6).
Fig. 2  Relative toxicity of hydrogen sulfide for sulfate reduction parameters: sulfate consumption (a), hydrogen sulfide production (b), lactate
consumption (c), and acetate production (d)
Fig. 3  Cluster analysis of D. piger Vib-7 growth and sulfate reduction
parameters under effect of hydrogen sulfide
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Kinetic parameters (lag phase, generation time, and the
maximum rate of the bacterial growth, hydrogen sulfide
and acetate production) of D. piger Vib-7 affected by the
hydrogen sulfide addition, during 72 h of cultivation were
calculated and presented in Table 1. Higher ­H2S concentrations
had high impact on all calculated parameters. The
time of lag phase was two times longer in the presence of
5 mM ­H2S, while generation time (time of division, Td)
was eight times longer. The maximum rate of the growth,
hydrogen sulfide and acetate was calculated in control
samples; the presence of ­H2S resulted in smaller maximum
Fig. 4  Cross-correlation analysis between biomass accumulation and sulfate consumption, sulfide production, lactate oxidation and acetate accumulation
as well as between sulfate and sulfide, same as between lactate and acetate
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rate parameters where almost the absence of rates were
observed in the presence of 4 mM and 5 mM.
Relative toxicity of hydrogen sulfide is obtained by our
research, where it was found that the external ­H2S addition
inhibited the growth of D. piger Vib-7 and their process of
sulfate reduction, including sulfate consumption, hydrogen
sulfide production, lactate consumption, and acetate production,
corresponding with bacteria survival. These findings
were confirmed by cluster analysis, cross-correlation and
principal component analysis, same as with kinetic parameters
of D. piger Vib-7 growth under the effect of hydrogen
sulfide during 72 h of cultivation.
Intestinal bacteria are sensitive to hydrogen sulfide, same
as SRB, D. piger Vib-7, which are its producers, but at the
same time their limiting factor for growing is higher ­H2S
concentrations. The lumen of the human large intestine is
the point where hydrogen sulfide is present in millimolar
concentrations (1.0–2.4 mM) (Blachier et al. 2010). Nevertheless,
because of a large capacity of fecal components to
bind the sulfide the concentration of free (unbound) sulfide
is in the micromolar range (Pitcher and Cummings 1996;
Attene-Ramos et al. 2006). Hydrogen sulfide can be formed
by endogenous sulfur-containing compounds, including
amino acids. The excessive concentrations of ­H2S can
severely inhibit cytochrome c oxidase, the terminal oxidase
of the mitochondrial electron transport chain, and mitochondrial
oxygen consumption. Colonocytes are able to metabolize
­H2S and that represents an important feature for their
resistance toward an excessive concentration of free luminal
sulfide (Blachier et al. 2010). Prolonged excessive concentration
of sulfide in the luminal content of the large intestine
and detoxifying catalytic activities in the large intestine
epithelium in the colon carcinogenesis have been according
to the literature data scarcely investigated. The isolated
bacteria D. piger Vib-7 can be considered as one of the very
promising points for further studies. ­Na2S at concentrations
of 500 mol/L (and above) provoked genomic DNA damage
in HT-29-Cl.16E colonic cells, according to Attene-Ramos
Fig. 5  Cross-correlation analysis between sulfate consumption and lactate oxidation, acetate production as well as sulfide and lactate, same as
between sulfide and acetate
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Fig. 6  Principal component analysis of the D. piger Vib-7 growth and the parameters of sulfate reduction under effect of hydrogen sulfide
Table 1  Kinetic parameters of
D. piger Vib-7 growth under
effect of hydrogen sulfide
during 72 h of cultivation
H2S (mM) Lag phase (h) Generation time Td (h) Maximum rate of production (µmax, ­h−1
)
Growth (biomass) Hydrogen sulfide Acetate
0 6.58 ± 0.61 1.79 ± 0.14 0.049 ± 0.0042 0.034 ± 0.0036 0.103 ± 0.009
1 6.65 ± 0.65 2.37 ± 0.22 0.037 ± 0.0036 0.025 ± 0.0022 0.093 ± 0.0088
2 7.72 ± 0.73 3.31 ± 0.29 0.025 ± 0.0022 0.013 ± 0.0011 0.079 ± 0.0072
3 8.05 ± 0.76 6.20 ± 0.57 0.011 ± 0,0001 0.004 ± 0.0001 0.064 ± 0,0057
4 10 ± 1.11 15.21 ± 1.55 0.001 ± 0.0001 0.001 ± 0.0001 0.052 ± 0.0046
5 15 ± 1.23 15.29 ± 1.61 0.001 ± 0.0001 0 0.031 ± 0.0025
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et al. (2006); the authors in the research used the assay in
which DNA repair was inhibited.
From the available data considering the effects of ­H2S
on the large intestine mucosa, it appears that this compound
coming from the microbiota metabolic activity can
interfere with the colonic epithelial cell metabolism. Both
beneficial and deleterious effects of luminal sulfide are possible
toward this intestinal epithelium. The generalization
schema reflecting effect of ­H2S on SRB and other groups of
the microorganisms is shown in Fig. 7. On the other side,
hydrogen sulfide can be produced by other microorganisms,
such as Clostridium species, that can decompose more complex
organic compounds (Černý et al. 2018). Consequently,
formed compounds can be more readily used by Desulfovibrio
bacteria strains as electron donors. Sulfides can possess
advantage and disadvantage properties in dependence with:
sulfide concentration inside the colonic lumen, substrate
availability from exogenous (alimentary) and endogenous
origins, metabolic capacity for the microbiota to produce
­H2S, percentages of sulfide in free and bound forms, capacities
of colonic epithelial cells to detoxicate and to use sulfide
as an energy source, availability of anaerobic metabolic
pathway (i.e., glycolysis) and capacity of colonic epithelial
cells to rapidly adapt to an excess in luminal sulfide production
(Attene-Ramos et al. 2006; Blachier et al. 2010).
When sulfide is present in excess, sulfide quinone oxidoreductase,
sulfur dioxygenase, and rhodanese are likely to play
an important role in the detoxication process, though consensus
is still needed regarding the metabolic pathways involved.
This assumption can be considered according to the present
literature data. Endogenously produced ­H2S is an important
factor in the neuromodulation of chloride secretion. According
to the present literature findings, it is not known if ­H2S is a proor
antinociceptive agent in the large intestine. The property of
mammalian cells to metabolise sulfur containing gas molecule
is an indication that is linked with bacteria and marine animals
living in an environment rich in sulfuric compounds (Grieshaber
and Völkel 1998; Griesbeck et al. 2002a, b). On the
other hand, for the production of ­H2S by SRB can be under the
influence of sulfate concentrations in the intestine, present in
the human diet. The concentration of sulfate in diets depends
on food types (Florin et al. 1993). Consequently, sulfidogenic
SRB can be a competitor with other microorganisms, especially
with methanogens (Kushkevych et al. 2017b, 2018a, b,
c, d, e). This kind of competition can inhibit methanogenesis
that leads to more suitable conditions for SRB growth. Higher
counts of SRB and their ­H2S production can also be an important
factor in the development of inflammatory bowel disease
(Kushkevych et al. 2018a, b, c, d, e).
Conclusions
Accumulation of hydrogen sulfide in the intestine depends on
its production by intestinal SRB which is the main producer
of this compound. Hydrogen sulfide represents an important
factor for the growth of SRB and their sulfate and lactate consumption.
The highest relative toxicity of hydrogen sulfide and
survival of D. piger Vib-7 was achieved at its concentrations
ranging from 6 to 7 mM. Despite the fact, that the SRB can be
capable to produce hydrogen sulfide, high concentrations are
toxic to its producers. These findings are confirming ­H2S concentrations
toxicity toward Desulfovibrio. The gained results
emphasized H2S (> 4 mM) limits toward this bacterial strain
inhabiting humans and animals intestine.
Acknowledgements  This study was supported by Grant Agency of the
Masaryk University (MUNI/A/0902/2018).
Author contributions  IK, DD, MV wrote the article. All authors contributed
to the conception, design and critically revised the manuscript.
Compliance with ethical standards 
Conflict of interest  The authors declare that they have no conflict of
interest.
Human and animal rights  This article does not contain any studies
with human participants or animals performed by any of the authors.
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