Microbes and Men Martin Krsek Masaryk University Brno, Faculty of Medicine Department of Public Health Microbiota x microbiome What is the main role of microbes?! Degradation Nutrient cycling Photosynthesis Interaction with plants and animals Keeping live alive/running Numbers represent the number of organisms per gram of homogenized tissue or fluid or per square centimeter of skin surface. How many microbial cells are on/in our body? Skin – 1012, mouth – 1010, intestine - 1014 How many cells are in our body? How many cells are in our body? 5 billion (109) – 200 million trillion (1018) ? Mean weight - 70 trillion cells (1012) Mean volume – 15 trillion cells (1012) Perhaps 37.2 trillion cells (1012) The total ratio of human to bacterial cells is • • 1 : 1-10 • • • So what are we?! Significance of the Normal Flora The normal flora influences the anatomy, physiology, susceptibility to pathogens, and morbidity of the host. The normal microbial flora is relatively stable, with specific genera populating various body regions during particular periods in an individual's life. Three developmental changes in humans: weaning, the eruption of the teeth, and the onset and cessation of ovarian functions Even though most elements of the normal microbial flora inhabiting the human skin, nails, eyes, oropharynx, genitalia, and gastrointestinal tract are harmless in healthy individuals, these organisms frequently cause disease in compromised hosts. Significance of the Normal Flora Germ-free animals (intestinal atonia) -alimentary lamina propria is underdeveloped, little or no immunoglobulin is present in sera or secretions, intestinal motility is reduced, and the intestinal epithelial cell renewal rate is approximately one-half that of normal animals - they live longer but … Animals treated with streptomycin infected with streptomycin-resistant Salmonella. -106 cells x fewer than 10 to establish a gastrointestinal infection -fermentation products (acetic and butyric acids) produced by the normal flora inhibited Salmonella growth in the gastrointestinal tract. The adult human - 2 square meters of skin- permanent contact with environment Skin regions = geographic regions of Earth: the desert of the forearm, the cool woods of the scalp, and the tropical forest of the armpit. - greasy, sweaty (head, neck,…) – production of sebum – Propionbacterium spp. - wet regions – Corynebacterium spp. - dry regions – the highest diversity – Staphylococcus spp. large differences in the microflora - significance for defense different microbial communities also in different layers of the skin Normal Flora of Skin Normal Flora of Skin Most bacteria on the skin are sequestered in sweat glands. Gram-positive cocci: Micrococcus sp. Staphylococcus epidermidis - secretion of antimicrobial substances – defense - but also life threatening disease in hospitals - catheters and surgery S. epidermis – secretion of lipoteichoic acids - prevention of release of inflammatory cytokins from skin cells S.epidermis – on skin of axenic mouse – support of immunity (T-cells) Skin microbiota and immunity Corynebacteria Propionibacterium sp. – considered non-pathogenic - staphylococci and propionibacteria produce SCFA from fats - inhibit the growth of fungi and yeast on the skin. but, if Propionibacterium acnes becomes trapped in hair follicle, pores and glands, it may cause inflammation and acne P. acnes – strains on healthy skin – genes for thiopeptides – inhibition of G+ bacteria (Staphylococcus epidermis) - strains of P. acnes responsible for acne do not produce thiopeptides - also micromycetes – Malassezia spp. - mainly on legs Various microbes influence different part of immune system. Some microbes produce antimicrobial substances, other induce release of cytokins connected with illnesses. Skin probiotics? Return to the original status? •Skin microorganisms have adapted to utilize the sparse nutrients available on the skin • •Many cutaneous microorganisms can produce molecules that inhibit the colonization of other microorganisms or alter their behaviour •Skin microorganisms have important roles in educating the innate and adaptive arms of the cutaneous immune system . The skin microbiota of a healthy adult remains stable over time, despite environmental perturbations •Some skin diseases are associated with an altered microbial state; reversion of this dysbiosis may help prevent and/or treat the disease Skin microbiota - conclusion Skin commensal interactions with Staphylococcus aureus. Skin microbial communities are shaped by interactions between organisms and with the host. In the skin, many interactions between commensals and Staphylococcus aureus have been identified. Antibiotics produced by coagulase-negative Staphylococcus and specifically by Staphylococcus lugdunensis prohibit colonization of S. aureus. Also, Staphylococcus epidermidis can inhibit S. aureus biofilm formation with production of the serine protease glutamyl endopeptidase (Esp). Moreover, when Esp-expressing S. epidermidis induces keratinocytes to produce antimicrobial peptides via immune cell signalling, S. aureus is effectively killed. In addition, Staphylococcus hominis-produced lantibiotics synergize with human antimicrobial peptide LL-37 to decrease S. aureus colonization. In contrast to inhibiting S. aureus, Propionibacterium acnes produces a small molecule, coproporphyrin III, that promotes S. aureus aggregation and biofilm formation. Normal Flora of the Conjunctiva Historicaly - small numbers of Staphylococcus epidermidis and certain coryneforms (Propionibacterium acnes) -blinking wipes the conjunctiva every few seconds -lachrymal secretions (tears) also contain bactericidal substances including lysozyme - - -Neisseria gonorrhoeae and Chlamydia trachomatis are thought to be able to specifically attach to the conjunctival epithelium Today – rich microbiota including viruses Conjunctiva and cornea – about 12 slightly different bacterial genera 1/3 of them – impossible to classify Normal Flora of the Respiratory Tract upper respiratory tract (nasopharynx), mainly nares (nostrils) heavily colonized - Staphylococcus epidermidis and corynebacteria - 20% of the general population - Staphylococcus aureus (MRSA) - healthy sinuses sterile - pharynx (throat) - streptococci and various Gram-negative cocci - sometimes pathogens (Streptococcus pneumoniae, Streptococcus pyogenes, Haemophilus influenzae and Neisseria meningitidis) lower respiratory tract (trachea, bronchi, and pulmonary tissues) nearly free of microorganisms (cleansing action of the ciliated epithelium, coughing, sneezing, swallowing) - islands of microbes – Streptococcus spp., Prevotella spp., Veillonella spp. - damaged epithelium - H. influenzae or S. pneumoniae descending from the nasopharynx Normal Flora of the Urogenital Tract Urine - sterile, microorganisms have problems gaining access and becoming established. Anterior urethra - Staphylococcus epidermidis, Enterococcus faecalis, alpha-hemolytic streptococci, some enteric bacteria (e.g. E. coli, Proteus) and corynebacteria (contaminants) Vagina - various microbiota depending on race , age - highly individual - colonized after birth with corynebacteria, staphylococci, streptococci, E. coli, and a lactic acid bacterium historically named "Doderlein's bacillus" (Lactobacillus acidophilus). During reproductive life the vaginal epithelium contains glycogen. Doderlein's bacillus predominates, being able to metabolize the glycogen to lactic acid. The lactic acid and other products of metabolism inhibit colonization by all except this lactobacillus and a select number of lactic acid bacteria. The resulting low pH of the vaginal epithelium prevents establishment by most other bacteria as well as the potentially-pathogenic yeast, Candida albicans. This is a striking example of the protective effect of the normal bacterial flora for their human host. Vagina - most of the women - dominate one of the 4 Lactobacillus spp. strains - sometimes other anaerobic bacteria - but they also produce lactic acid - dominance of Lactobacillus spp. 80% Asian women and 90% white (pH 4.4 and 4.2) 60% Hispanic and Black (pH 5.0 and 4.7) - possible association with premature birth on average 10% of premature births in an area dominated by black and Hispanic populations it is 20% - fast changes of microbiota (even 24 hours) Vagina Maternal microbiota vagina - Lactobacillus newborn - Actinobacteria, Proteobacteria, Bacteroides postpartum placenta - E. coli, Bacteroides, Prevotella tannerae, Neisseria lactamica - species composition - the most similar to oral cavity - metabolic pathways - metabolism of cofactors and vitamins breast milk - microbial soup - Streptococcus spp., Staphylococcus spp., Serratia spp., Corynebacterium spp., Lactobacillus spp., but highly individual - mother handover microbes from the child's environment - training of immune systém - prebiotic human milk oligosaccharides (HMOs) that preferentially feed beneficial gut bacteria during pregnancy and breastfeeding – changes in maternal microbe – important even for mother‘s health Breast milk contains prebiotic human milk oligosaccharides (HMOs) that preferentially feed beneficial gut bacteria, including Bifidobacterium [13]. HMOs are unconjugated glycans with a lactose core varying in chain length from 3 to 15 carbohydrates (glucose, galactose, fucose, N-acetylglucosamine (GlcNAc), and N-acetylneuraminic acid (NeuAc) or sialic acid) [14, 15]. Maternal microbiota Mastitis - disappearance of Lactobacillus spp., dominance of one strain of pathogenic bacteria Adding lactobacilli to food - the same bacterium has been shown in milk after a three-week cure – disappearance of problems - better than antibiotics? Breast - their own microbiota even at non breast feeding woman - meaning for cancer? 1676 - Anthony van Leewenhoek - "animacules" 1890 - W.D. Miller - The Microorganisms of the Human Mouse 2016 - Jakubovics - structure of dental plaque 500-600 (700) of different bacterial species (30% non-cultivated) streptococci, lactobacilli, staphylococci, corynebacteria and anaerobic bacteria (bacteroides) a common development with us for millions of years - use of fire, agriculture, processed food (sugar), - antimicrobial therapy age-dependent: - after birth - Streptococcus salivarius (98%) - tooth eruption - S. mutans (lactic acid) and S. sanguis - around puberty - Bacteroides and Spirochetes Normal Flora of the Oral Cavity Normal Flora of the Oral Cavity Three Habitats - mucous membranes of the cheeks, gums, hard palate - Firmicutes, Streptococcus sp. - throat, tonsils, back of the tongue, saliva – Veillonella spp., Neisseria spp.and Leptotrichia spp. - dental plaque – Capnocytophaga spp., Actinomyces spp., Rothia spp. and Corynebacterium spp. Mouth - warm, moisture, nutrients - saliva, crevicular fluid of gums - proteins, glycoproteins - immunoglobulin A, lactoferrin, lactoperoxidase, lysozyme, staterin, histatin - result is a balanced microbiota - these substances together with other (even microbial) form a biofilm allowing microbes to hold and at the same time protect teeth against acid attack Biofilm in general Normal Flora of the Oral Cavity -at first, the physical process - covering the surfaces with a film of dissolved substances - then (minutes) - reversible attachment - irreversible - oral bacteria - various adhesions - interactions with molecules and receptors on other bacteria - Streptococcus spp., Actinomycetes spp., Veillonella spp., Porphyromonas spp., Fusobacterium spp., Spirochetes spp., Candida spp. Normal Flora of the Oral Cavity Biofilm - the coadhesion of late colonizers to previously attached ones - increased diversity and biofilm biomass - the importance of bacterial polymers (glucans, fructans, heteropolymers) - matrix - nutrients, water, enzymes - gradient - pH, O2, nutrients - regulation of gene expression - different from the same planktonic microbes - facilitated horizontal gene transfer - quorum sensing peptides increased the frequency of transformation in S. mutans 10-600x - increased resistance to antimicrobial agents Normal Flora of the Oral Cavity Viruses - pathogenic - mumps, rabies, hepatitis, HIV, respiratory infections - most but bacteriophages - lytic, lysogenic - ....... Fungi - 85 genera were detected - Candida spp., Cladosporium spp., Aureobasidium spp., Saccharomycetales spp., Aspergillus spp., Fusarium spp., and Cryptococcus spp. - C. albicans - its presence allows the growth of anaerobes in aerobic conditions Archea - minor component - metanogens - especially people with periodontitis Normal Flora of the Oral Cavity Bacteria Expanded Human Oral Microbiome Database (eHOMD) - November 22, 2017 - 772 prokaryotic species (70% cultivated) - 16S rDNA profiling - 6 groups: Firmicutes, Actinobacteria, Proteobacteria, Fusobacteria, Bacteroids and Spirochaetes = 96% - the discovery of ultra-small bacteria (not just in the oral cavity) - reduced genome, absence of many biosynthetic and metabolic pathways including the electron transport chain - obligatory symbionts? Normal Flora of the Oral Cavity Dental plaque - bacteria living side by side use different nutrients, produce different substances: - synergism, induction of response in target cells and competition - the same substance - different functions – according to situation, concentration - H2O2 - inhibition, but in sub-lethal concentrations signaling function - likewise organic acids, bacteriocins - antagonism, synergism - bacteriocins – induction of cell competence, DNA release -the mere presence inhibits colonization of GIT by potential pathogens -nitrate metabolism and cardiovascular disease - ¼ of the received nitrate returns to the oral cavity (saliva ...) - the microbiota converts nitrates into nitrites - into the blood and transformed into NO - NO - vital to vascular health - flexibility, elasticity – blood pressure reduction - but also erections, relaxation of the muscles in the digestive system - transport of food - the use of antibacterial mouthwash significantly reduced the intake of nitrite, eliminating the effect of nitrite on decrease of blood pressure - also connection with other systemic diseases: - metastatic infections of heart, brain, spleen, pancreas, liver, and bone - - arthrosclerosis, cardiovascular diseases, stroke, - respiratory diseases, meningitis, pneumonia, diabetes The role of normal oral cavity microflora The role of normal oral cavity microflora Negative effects -dental caries, gingivitis, periodontitis - UK - 46% of 15-year-old children - decay in permanent teeth - 45% of adults - perionditis Tooth decay -dissolving the teeth structure by acid from sugars, reducing buffering capacity of saliva and thus reducing pH in mouth -change of oral microbiota in the benefit of acidophilic species - Streptococcus mutans and lactobacilli - further acid production S. mutans is not the only one – Bifidobacterium spp., Propionibacterium spp., Scardovia spp. Some bacteria, on the contrary, increase the pH (urea, arginine - ammonia) - the same obligatory anaerobes are enriched in both inflammatory and tumorous tissues The role of normal oral cavity microflora gingivitis -90% of adults - dental plaque - primary colonizers - G + aerobic and fac. anaerobic bacteria (streptococci and Actinomyces) - mature plaque - G- anaerobic – Fusobacterium spp., Treponema spp., Synergistetes spp. - regular teeth brushing- plaque "does not mature" - neglected hygiene - endotoxins and other enzymes into the gums – irritation and inflammation - loss of gum and tooth connections - periodontal pocket - anaerobic colonization - host reaction (protease formation) makes the situation even worse - teeth release - Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, Anaeroblobus geminatus, Eubacterium saphenum, Allocus filipactor, Porphyromonas endodontalis, Prevotella denticola and others The role of normal oral cavity microflora Healthy mouth - most bacteria in symbiosis with the host Disease - increased occurrence of cariogenic and periodontopathic bacteria - disturbance of balance - dysbiotic status Dental plaque and dental diseases Dental plaque and dental diseases Non-specific Plaque Hypothesis (19th Century) -dental infections caused by non-specific increase of all bacteria - remove the maximum Specific plaque hypothesis -diseases caused by a few species, their removal will solve the problem Ecological Plaque Hypothesis (1980) -significant changes in the plaque environment change relationships between bacteria -enrichment of some species. -disease prevention not only by inhibiting pathogens but also by changing conditions of the environment. Normal Flora of the Oral Cavity Dentures - more aerobes, yeast and lactobacilli - on denture - Candida albicans - under denture - a more acidic environment, fewer saliva - again C. albicans Tooth decay - vaccination, genetically modified S. mutans -BUT – S. mutants can be replaced by other bacteria - S. pneumoniae - -benefits of microbes - vitamins, inducing low levels of circulating and secretory antibodies, antagonism against nonindigenous species (fatty acids, peroxides and bacteriocins) -at the same time – diseases: abscesses, dental caries, gingivitis, periodontal disease but also abscesses of alveolar bone, lung, brain, or the extremities (Bacteroides melaninogenicus) - Microflora of the oral cavity and tooth brushing -cleaning teeth for several thousand years before Christ - but regular use of brushes and pastes only after World War II - various teeth cleaning techniques - manual x electric brush - choice of paste -- ads - sterile oral cavity ?! 2-3x higher risk of periodontitis in diabetics - an increased level of glycated hemoglobin increases the risk of diabetic complications including periodontitis With periodontitis, the risk of developing diabetes is up to 1/3 higher - mediators of inflammation and oral pathogens (P. gingivalis) and their toxins can penetrate the blood into the body and induce inflammation, which is a risk factor for the development of insulin resistance and diabetes Periodontitis and Diabetes Periodontitis and preterm birth Directly -periodontal pathogens through the bloodstream to the placenta and into the amniotic fluid, locally initiate inflammation and this can lead to the initiation of preterm labor Indirectly -inflammatory markers and toxins from the oral cavity through the blood to the placenta and liver -induction of the production of other pro-inflammatory molecules, the increased level of which is associated with the initiation of labor Periodontitis and rheumatoid arthritis Periodontitis can impair the effect of anti-RA treatment Treatment of RA often also improves the periodontal condition Reverse addiction controversial and many other examples of contexts Normal Flora of the Gastrointestinal Tract Birth - bacteria enter with the first food - breastfed babies - bifidobacteria (90%), Enterobacteriaceae and enterococci - artificial nutrition - bifidobacteria do not dominate - switching to cow's milk or solid food - bifidobacteria plus enteric bacteria, bacteroides, enterococci, lactobacilli and clostridia - human milk - a growth factor supporting the growth of bifidobacteria - bifidobacteria prevent colonization by non-indigenous or pathogenic species - evidence of "tissue tropism" and specific adherence Breast milk and its importance for the health of the child Normal Flora of the Gastrointestinal Tract -age, diet, cultural conditions, use of antibiotics, … -esophagus – none bacteria -stomach – pH - acid-tolerant lactobacilli -Helicobacter pylori - gastric ulcers, gastric and duodenal cancer -urease – urea – ammonium -maybe also positive functions - protection against infant diarrhea and esophageal disease. - normalflorapylori.gif Normal Flora of the Gastrointestinal Tract – cont. -small intestine - high flow rates - lactobacilli and Enterococcus faecalis - 105 - 107 -distal part (108/ml) - additional species - E. coli and relatives and Bacteroides -digestion mainly by human enzymes, microbes could compete for essential nutrients - -large intestine (colon) - similar microbes as in feces - 1011/ml or even more – 1013 -bacteria - about 35-50% of the colon contents - 2 lbs -coliforms, enterococci, clostridia and lactobacilli -predominant anaerobic Bacteroides and anaerobic lactic acid bacteria Bifidobacterium (Bifidobacterium bifidum) - significant numbers of anaerobic methanogens Normal Flora of the Gastrointestinal Tract – cont. Role of bacteria in GI tract -protection from infection by alien microbes -contribution to nutrition and digestion (polysaccharides – acetate, butyrate and propionate - source of carbon and energy for mucosal cells of the colon) - production of vitamins -stimulation of development and activity of the immunological tissues - -BUT ALSO: -production of carcinogenic metabolites (colon cancer) -alterations in the GI flora (poor nutrition, antibiotics) -shifts in populations and colonization by nonresidents -gastrointestinal disease normalfloracolon.jpg Villi Lymphatic tissue (GALT) in the submucosa Absorption (both passive and active) Prevents bacterial translocation and penetration of toxins Physiological component (mucus, bacteria and their products) Immunological component (slgA, serum IgA and IgG, RES) Abnormal microbiota can increase intestinal permeability Bacterial components including LPS can enter the blood The concentration of circulating pro-inflammatory cytokines will increase Barrier function of the intestine Intestine 200 m2 – in contact with the outside environment The greatest concentration of microbes The largest immune organ - 70% of immune cells After the brain (100 billion) the largest reservoir of neutrons (500 mil) - comparable to the spinal cord - the "second brain„ Nervus vagus – connection between the brain and the gut Neurotransmitters are produced here by bacteria (serotonin) SCFA Gut – Brain Axis Two-way communication between the central and enteric nervous systems, connecting the emotional and cognitive centers of the brain with peripheral gut functions. Recent research advances have described the importance of the gut microbiota in influencing these interactions. This interaction between the microbiota and GBA appears to be bidirectional, namely through signaling from the gut microbiota to the brain and from the brain to the gut microbiota via neural, endocrine, immune and humoral connections. Viruses and our microbiota -majority of known viruses are pathogenic - because we learned just about them (diseases) - but many viruses infect us without any disease symptoms - molecular methods - many other viruses discovered - originally 2 human polyomaviruses - today 13 - infect us during childhood and then waiting -Koch's postulates - a problem with viruses (cultivation in pure culture) -majority of viruses are neither completely pathogenic nor harmless - according to the situation Viruses and our microbiota Benefits of viruses - childhood infections stimulate the development of the immune system - protection against later infection and exaggerated reaction – allergies - comensal virus may provide protection against a pathogenic virus - pegivirus C (related to hepatitis C, Zika virus, dengue fever) mitigates the effects of HIV - ¾ billion people are infected - viruses prefer fast dividing cells - cancerous - spontaneous regression - about 8% of our genome are retroviral DNA sequences - some of their functions are essential for host survival and development Mycobiota - 2015 - only 269 out of 6,000 results for microbes mentioned fungi - mykobiome - only 55 results - oral mycobiota of HIV patients (C. albicans) - more severe symptoms of hepatitis B correlate with a number of species of Candida spp. and Saccharomyces spp. - the excessive presence of Candida tropicalis exacerbates the symptoms of inflammatory bowel disease (IBD) - undisturbed microbial community - fungi harmless or even beneficial - after disruption - possible problems Mycobiota -oral cavity - not just Candida spp. and Saccharomyces spp. - study on 20 volunteers - 101 fungi identified, each with 9-23 species - Cladosporium spp. (asthma), Aureobasidium spp.(transplant), Aspergillus spp. (devastating infections), Fusarium spp. (difficult infections), Cryptococcus spp. (meningitis in HIV) - elsewhere on the body of Malassezia spp. - skin diseases - lungs - Aspergillus spp. in healthy people, Candida spp. in patients (cystic fibrosis, cardiovascular diseases, ...) - inflammatory bowel disease - other mycobiota than in healthy people - obesity - more Ascomycota spp. representatives, but overall reduced diversity - Mucor spp. - slim people (correlation with obesity) Do we all have the same microbes? Differences: digestive tract, vagina (species and metabolic diversity) Similarities: microflora of the mouth and skin - changes in time, but less variable in different individuals Human Microbiome Project - launched in 2008 - five-year project - total budget of $115 million -(Human Genome Project - launched in 1990 and was declared complete in 2003) -oral, skin, vaginal, gut, and nasal/lung – from 300 healthy subjects -how changes in the human microbiome are associated with human health or disease -BUT – what was here first ?!?!?!?! -many more (specialized) studies of human microbiome are underway: -American Gut Project, MetaHIT, MyMicrobes, Human Longevity, Earth Microbiome project, Human Food Project RESULTS: -dramatic variation between individuals as far as species composition is concerned -BUT when classified by function - very similar -each individual´s microbiome is fairly stable over time -main problem of interpreting results – public databases - - Importance of human microbiota -up to 2015 – about 19 000 papers published (5,000 in 2015 alone) -majority is descriptive – identification of which microbes are there -but what they are doing there? -how they cooperate in between them and with human host? -human microbiome includes also viruses, archaea, fungi, single-cell eukaryotes…. -human genome -20 000-25 000 protein coding genes -human microbiogenome - 2-20 millions protein coding genes -all microbes constantly excrete/secrete small and large molecules and vesicles containing RNA, DNA, proteins - communication with neighbours and host – many of these molecules and vesicles can penetrate gut wall to bloodstream -microbiome´s metagenome can change much faster than human genome – fast response -microbiome = sophisticated additional organ (or set of organs) Can we change our microbiota? Diet (e.g. fiber) Antibiotics Probiotics Postbiotics (metabolites of probiotics) Prebiotics Symbiotics (probiotics and probiotics) Parabiotics (lysates, components and products of bacteria) Recombinant bacteria Fecal transplant Phage therapy Manipulation of human microbiota -4th century Chinese medical literature mentions faecal transplant to treat food poisoning and severe diarrhea -Bedouins – camel faeces for bacterial dysentery - - -example of successful manipulation of our microbiota is faecal transplant (stool bank - OpenBiome) for treatment of infections caused by Clostridium difficile But first WE MUST KNOW our microbiota, only then we can think about targeted manipulation Holobiont Establishment of human microbiota - fetus in the womb is not sterile - vaginal microflora varies during pregnancy (80% Lactobacillus sp.) - newborn - Actinobacteria, Proteobacteria, Bacteroides - microbiota of the placenta - a non-pathogenic comensal microbiota: Firmicutes, Tenericutes, Proteobacteria, Bacteroides and Fusobacteria (E. coli, Prevotella tannerae, Neisseria lactamica) - similar to the oral microflora - further colonization during delivery, then breastfeeding (milk is not sterile), from the mother's skin - milk: Streptococcus spp., Staphylococcus spp., Serratia spp., Corynebacterium spp. depends on mother's environment (preparation ...) - also baby is exploring the environment by mouth - around the second year of life a certain balance is achieved C.difficile - anaerobic gram-positive spore-forming bacteria - transmitted by fecal-oral route - colonizes the large intestine - release of exotoxins (TcdA, TcdB) causes colitis -typically after recent antibiotic exposure (penicillins, cephalosporins, clindamycin, fluoroquinolones) Presentation: Abdominal pain Fevers Diarrhea, can be bloody Dehydration Clostridium difficile Diagnosis: Stool testing for toxin Sigmoidoscopy Oral antibiotics to treat CDI Metronidazole, Vancomycin à ~70-75% effective Fidaxomicin for Vancomycin failure à 90% effective Clostridium difficile – cont. Clostridium difficile – cont. Fecal transplant Donor contraindication: Donors should not: Have had any antibiotic exposure in the past six months Be immunocompromised Have had any tattooing or body piercing in past six months Have any history of incarceration Have recently traveled to endemic areas Have any chronic GI disorders, such as inflammatory bowel disease Faecal transplant cont. Donor screened for: hepatitis A, B, C HIV syphilis C.difficile Giardia, E.coli, Salmonella, Shigella, H.pylori, Campylobacter infections Recipient Before procedure: -stop any antibiotic therapy two days before the procedure - -a liquid diet followed by laxative preparation the night before scheduled procedure Faecal transplant cont. Faecal transplant cont. - Fresh stool collection -less than 6 hours prior to procedure   -volume 20 ml to 30ml mix 250 ml of sterile 0.9% normal saline with stool in a blender Faecal transplant cont. Faecal transplant cont. Risk of aspiration if not delivered deep into upper small intestine Risk of acquiring infection from donors – rare Risk of complications from sedation and endoscopy – bleeding, perforation, transmission of other infections: 1/1000-1/10,000 -Probiotics – 100 years history – Mutaflor - -MUTAFLOR® IS A PROBIOTIC THAT COMPRISES AS ITS ACTIVE SUBSTANCE A VIABLE NON-PATHOGENIC STRAIN OF ESCHERICHIA COLI (E.COLI) -FIRST ISOLATED IN 1917 BY PROFESSOR ALFRED NISSLE FROM THE FAECES OF A SOLDIER DURING THE FIRST WORLD WAR WHO AS HE REMARKED, “IN CONTRAST TO THE LARGE MAJORITY OF HIS COMRADES, HAD SUFFERED NEITHER FROM DYSENTERY NOR FROM ANY OTHER INTESTINAL DISEASES”. THEREAFTER, APPROPRIATELY NAMED ESCHERICHIA COLI STRAIN NISSLE 1917. -USED TO relief and management of chronic constipation - Characteristics of Escherichia coli strain Nissle 1917 - the ability to colonize - antagonistic activity – Inhibition of growth and or killing of pathogens - anti-invasive – prevention of colonisation in the gut by pathogens - synthesis of endogenous antimicrobial peptides-defensins - mucosal integrity – contributing to luminal metabolism and stability of intestinal milieu-enhanced epithelial barrier function - anti-inflammatory and immunomodulatory effects -stimulation of colonic mucosa - -Adminitered to Hitler by his doctor Theodor Morell during second world war - - - Manipulation of human microbiota -prebiotics – non-digestible part of our food -cellulose, hemicellulose, lignin -but also polyphenols (resveratrol – red wine), minerals -vitamins - riboflavin -lactating mothers produce oligosaccharides that are not digested - bait for pathogens? - supporting beneficial bacteria (able to digest them)? -microbiome influences/modifies efficiency administered medications Manipulation of human microbiota Steinert 2016 Experiments with riboflavin: - Riboflavin increased the incidence of Faecalibacterium prausnitzii - this bacterium is responsible for production of anti-inflammatory peptides - for example IBD (inflammatory bowel disease) is characterized by low numbers of F. prausnitzii Prebiotika – pokr. Importance of human microbiota - disturbance to human microbiota is connected to many diseases: -inflammatory bowel disease (ulcerative colitis and Crohn's disease) - diabetes - obesity - microbes influence signal pathways/communication between digestive system and brain -neural, endocrine, immune signals - - - 2014 – 1,9 billion overweight people (600 mil. obese) - GM of obese people shows special metabolism of carbohydrates and fats - GM of obese people can digest substrates which human enzymes cannot - result is gain of energy, SCFAs – they influence metabolism of glucose, cholesterol, and fats in human tissues - western diet (too much fats) reduce Bacteroides and promote Firmicutes - GM activate bile acids and regulate metabolism (absorption of lipids) - germ free mouse on diet rich in fats do not demonstrate obesity (urine, feaces) - main thing is efficiency of utilization of energy from food Human microbiota and obesity Worldwide obesity has nearly tripled since 1975. In 2016, more than 1.9 billion adults, 18 years and older, were overweight. Of these over 650 million were obese. 39% of adults aged 18 years and over were overweight in 2016, and 13% were obese. Most of the world's population live in countries where overweight and obesity kills more people than underweight. 41 million children under the age of 5 were overweight or obese in 2016. Over 340 million children and adolescents aged 5-19 were overweight or obese in 2016. Obesity is preventable. Facts about obesity Human microbiota and obesity Obesity is not only problem of weight, but is connected with higher risk of cancer (uterus, breast, cervix, large gut, rectum, throat, kidney, prostate, pancreas, etc.) Obesity is connected with ready-to-cook meals (preserve chemicals, emulators) Obesity is connected with higher incidence of Firmicutes and Actinobacteria and lower incidence of Bacteroides Thank you for your attention