TRACING THE CRIMINAL
                                                                                         Part Four:

                                                                             Enterobacteria (& Co.)

                                                                    Microbiological institute shows

A poem for beginning…

We can check a healthy hen.

Everything OK. But then

Pigeons start to fly to it

Infect others, that‘s their hit.

Then the egg is used for ice

Looking very, very nice.

But salmonellas can hide

invisible, still inside.

And then youngsters eat the ice…

…and their speed increases twice!

Survey of topics

Clinical description: Enterobacteriaceae

Story one

n  Miss Theresa ate a cake with egg cream after her lunch. In the afternoon, she started to have
problems. She visited a doctor and he made the anal swab. Several days later, officers of public
health administration called her. Finally, although she did not believe, that cake was not guilty.

Who is guilty now, then?

n  Bacterial criminal is Salmonella enterica serovar Enteritidis, briefly Salmonella Enteritidis

n  Cream cake cannot be the criminal! Incubation time does not correspond.

n  Another cake, consumed two days before, was later proven to be the infection source.

n  As to humans, probably someone in the „Hysterical Maid Cafee“ failed to do something. Public
health officers do their monitoring just now.

Salmonella



Salmonella endocarditis

Primary pathogens among enterobacteria

n  Enterobacteriaceae is the clinically most important family among Gram– rods (not only
clinically)

n  The worst pathogens perform systemic infections: it is Yersinia pestis (plague) and so named
anthropopatogenous serovars of Salmonella (serovar Typhi, Paratyphi A, Paratyphi B and C) – typhoid
fever

n  Obligatory pathogens causing usually intestinal infections only are important, too. Even here,
sometimes a systemic infection may occur.

n  We are speaking about genera Salmonella, Shigella and Yersinia

Plague (Yersinia pestis)

Story Two

n  Mrs. Wet is a pacient of a urological clinic.

n  She has repeated problems with urination. After three children, he has dammaged muscles of
pelvic region, she was treated even for urine incontinence. The doctors warned her, thet she is in
an evevated risk of infection. And really – now, she got infected again.

Who is guilty now?

Urinary bladder with adhered escherichiae







Survey of enterobacteria

Action of Klebsiella

Clinical description: Campylobacter, Helicobacter and Vibrionaceae

Story three

n  Student Francis often visits fast-foods. Especially, he likes chicken meat.

n  Thus, even public health officers were not able to source the food responsible for his diarrhoic
problems. Francis thougth about salmonelosis. But it was not this. Salmonella comes mostly from
eggs, this one rather from chicken meat.

Criminal‘s name

n  Campylobacter jejuni, Gram-negative curved rod. It does not belong in family of enterobacteria,
but the infection is simillar that caused by Salmonella

n  Number of cases in Czechia is about the same as that of salmonelosis. Formerly it was not so
common, but maybe only the diagnostic was not so much developed.

Campylobacters

Impotant notice: stool sampling

n  Unlike parasitology and virology, where a bit of stool is necessary, in bacteriology we are used
to send an anal swab (but a bit of stool is not a mistake).

n  Todays method of sampling is use of a cotton swab with transport medium (usually Amies). This is
especielly because of Campylobacter – Salmonella would survive even on a swab without transport
medium.

Story Four

n  Mr. Acid has a problem: pyrosis.

n  He comes very often to gastroenterology, and a fibroscope is often present in his esofagus.

n  At last fibroscopy, the doctors took two samples endoscopically – one for histological, another
for microbiological examination.

n  Bothe methods showed the same: it is there.

Only half-criminal…

n  Peptic (= gastric / duodenal) ulcus is caused by more causes. Such diseases are called
multifactorial diseases.

n  The part of Helicobacter pylori on ulceral disease is still discussed, not only among GPs, but
even among specialists. Even healthy persons may have a helicobacter in their stomach.
Nevertheless, certain and not negligeable role of this pathogen is sure.

How can it survive at low pH?

n  It adapts its microenvironment – in alcalizes it, by splitting urea

n  Urea is split into acid carbon dioxide, that is breathed out, and alcalic ammonia, that remains
and alcalizes the environment:



NH[2]-CO-NH[2] + 3 H[2]O à CO[2] + 2 NH[4]OH







Story Five

n  Mr. Exotic was on his exotic holiday. He was used to drink water from local sources

n  So, no surprise, he was attacked by a diarrhoea.

n  This one, neverhteless, was more heavy than usually. It was watery and very profound

n  Oral ingestion of water was insufitient. Only infusion supplementation of missing liquids
brought a help to him.

Vibrionaceae

n  Vibrio cholerae causes cholera, a profound diarhoeic disease in subtropic and tropic countries

n  Other members of genus Vibrio may accidentally perform either diarrhoea, too, or wound
infections. They are called „halophilic vibrios“, as they prefere elevated NaCl concentrations

n  Aeromonas, the second important genus, also causes wound infections, e. g. at preparing meals
from fishes and seafood.

Pictures of bacteria

Photographs from criminal database: Salmonella

Proteus mirabilis, P. vulgaris (below)

Proteus according to P. Ondrovčík



Klebsiella & Escherichia

Escherichia



Vibrio cholerae



Diagnostics of Enterobacteriaceae

Enterobacteria – methods

n  Direct

n  Microscopy – not very important in real diagnostic, but we will use it in our practicals

n  Culture – many various media

n  Biochemical identification – very important

n  Antigen analysis – Salmonella, Shigella, EPEC

n  Indirect methods

n  Widal reaction in typhoid fever, antibodies agains yersinia

Diferencial diagnostics

n  Gram stain differenciates Gram - rods from other bacteria

n  Endo agar (I): among clinically important, only enterobacteria, Vibrionaceae and Gram negative
non fermenters do grow

n  Non fermenters may be differenciated by not fermenting glucose (Hajna medium remains red, no
change of colour).

n  Vibrionaceae are oxidase positive, unlike enterobacteria

Gram– rods: group differenciation of the three groups able to grow on Endo agar

n  Enterobacteria are oxidase negative and glucose fermenters

n  Vibrionaceae are glucose fermenters, too, but allways oxidase positive

n  Gram– non-fermenters never fermentate glucose. Oxidase may be positive or negative. They are
sometimes cocobacilli.

Diagnostic algoritm

Mutual differenciation of Enterobacteriaceae

n  Endo agar (II): orientation differeciation of obligatory pathogens (usually L-) and opportune
pathogens (usually L+, L = lactose)

n  More media: XLD, MAL, DC, WB and more for Salmonella, CIN for Yersinia, etc.

n  Biochemical tests: Hajna medium, MIU test, Švejcar plate, ENTEROtests etc.

n  Antigen analysis usually using slide agglutination

Diagnostics of Campylobacter, Helicobacter and Vibrio will be discussed at tasks 7, 8 and 9.

Lactose splitting

Lactose positive bacteria have dark red surroundings on Endo agar. Lactose negative bacteria have
pale colonies on the same medium.

Culture characteristics of several enterobacteria

n  On XLD agar:

n  Salmonella has pale colonies with black centre (the centre is like a yolk in a fried egg)

n  other bacteria do not grow at all, or grow in colonies of different morphology

n  On MAL agar the results are simillar to those on XLD agar (slightly different colours of some
colonies etc.)

n  On CIN agar Yersinia would grow in tiny, dark pink colonies. If no bacteria do grow on the
medium, then no one of your strains is a Yersinia.

Salmonella on MAL agar

Biochemical testing of enterobacteria

n  For biochemical testing of Enterobacteriaceae, we use various tests. In Czechia, the most common
ones are EnteroTest 16 and EnteroTest 24. We will use the first of them.

n  The first reaction is ONPG test (a test tube with reagent strip, like in VPT test in StaphyTest
and StreptoTest). First row in your panel is 2^nd to 9^th reaction, second row is 10^th to 17^th
reaction.

Antigen analysis

n  Antigen analysis is used in some situations only, so not very commonly.

n  There are two main situations like this:

n  In obligatory pathogens (Salmonella, Shigella, Yersinia) to make the diagnose more sure, and for
epidemiological reasons

n  In intestinal isolates E. coli in case of suspicion for EPEC or STEC (but usually not the other
groups of E. coli)

n  Both cases are demonstrated by examples

E. coli agglutination for EPEC

n  We try to detect any of 12 main serovars belonging to EPEC

n  If nonavalent serum (I, II, III) is positive

n  we continue with three trivalent sera (I, II and III)

n  when one of them is positive, we continue with corresponding monovalent sera

n  If trivalent serum IV is positive, we continue with monovalent sera belonging the „IV“ group

n  Understand: there exist hundreds of serovars in E. coli species. So, the result „E. coli, EPEC
excluded“ means „it is one of remaining cca 200 serovars“

Salmonella agglutination

n  At agglutination of any motile enterobacterium, we evalate two types of antigens: body = O
antigens, and flagellar =  H antigens (exceptionally also capsullar K antigens).

n  So, each Salmonella, too, has its specific antigenic structure. E. g. Salmonella of serovar
Enteritidis has body (O) antigens type 9, 12 and flagellar (H) antigen type m.

n  So, if we have a Salmonella Enteritidis, we should find presence of agglutination both for body
and flagellar antigens.

Tests of atb susceptibility

n  We do not perform atb tests for stool strains. (Usually, use of antibiotics in bacterial
diarrhoea leads paradoxically to longer time of presence of the pathogen in stool; dietary
treatment and probiotics in reconvalescence are then recomended rather than anitibotics.)

n  So, usually we test it in UTI origin strains, therefore also some of antibiotics are anti-UTI
(like nitrofurantoin)

Susceptibility zones table – example

Diagnostics of Campylobacter, Helicobacter and Vibrionaceae

Diagnostics of Campylobacter

n  Campylobacter does not grow on common media; together with its typical morphology (curved rod)
the diagnostic is possible

n  Look at culture results of campylobacter and write down its description

n  Look at oxidase tests result (the test will be performed by your teacher as a demonstration)

Some notes to Campylobacter diagnostics

n  They need four special conditions:

n  Their special black medium – its name CCDA is not used commonly, so it is simply „Campylobacter
medium“

n  Temperature elevated to 42 °C. (Primarily, it is a bird pathogen, and bird body temperature is
elevated if compared with others)

[n    ]Elevated pCO[2]

n  Prolonged culture period – not 24, but 48 hrs

Urease test in diagnostics of Helicobacter

n  Helicobacter, too, does not grow on common media. It requires its own special medium, and it
needs cca five days until any growth is visible.

n  Urea splitting is one of very specific test for Helicobacter. Unlike other biochemical tests
used in microbiology, it can be used directly with the specimen (gastric tissue) and not with a
strain. In task 8, you will see a difference between positive and negative result.



Urea breath test

n  The patient gets a mixture with urea with heavy carbon isotope (^13C) or radioactive carbon
isotope (^14C)

n  In healthy person urea comes to the bowel and comes out of the body with stool

n  In presence of helicobacter, it is split in the stomach and labelled CO[2] is found in the air.
The more CO[2], the more helicobacter

Vibrionaceae – diagnostics

n  Similar to Enterobacteriaceae, but they are oxidase positive.

n  Microscopically: motile, curved rods

n  We use special media like alcalic pepton water or TCBS (Thioglykolate, cystein, bile salts)

n  We use simillar biochemical tests as for enterobacteria

n  But we have to choose „oxidase positive“ in computer system (there is no codebook for them)

Differential dg. ofVibrionaceae

n  In microscopy, Vibrio is a curved rod (see next picture, draw it to your laboratory report)

n  For culture, we use TCBS medium (a solid medium) and alcalic pepton water (a liquid medium)

n  For biochemical identification, we use the same Enterotest 16 as for enterobacteriaceae, but we
need another codebook (or another programme in PC)

n  By antigen analysis, we can find the major serotypes of Vibrio cholerae: O1 and O139.

n  More precise differentiation inside O1 serotype (into biotypes Classic and El Tor) would require
more biochemical tests



The End