Chair of Medical Biology, Microbiology,
Virology, and Immunology
PATHOGENIC
ENTEROBACTERIACEAE
Classification of the Enterobacteriaceae
Genera
Escherichia
Edwardsiella
Shigella
Salmonella
Citrobacter
Enterobacter
Serratia
Providencia
Yersinia
Klebsiella
Hafnia
Proteus
Morganella
Erwinia
Shigella
a. Slender, gram-negative rod; non lactosefermenting (except for S. sonnei)
b. In contrast to E. coli: no H2S production, no
lysine decarboxylation, no acetate
utilization
c. Invasive (key to pathogenesis)
d. In contrast to Salmonella: non-motile; no gas
from glucose fermentation; no H2S production
e. Toxin production limited to a few strains
f. All have O antigens-four groups (A-D)
g. Differentiating species ( S. dysenteriae - no
mannitol fermentation; S. boydii - C antigen group;
S. flexneri - B antigen group; S. sonnei-orniltine
decarbexylase production)
h. Specimens
i. Rectal swab from colonic ulcer is best for culture.
j. Fecal specimen - must be immediately
innoculated onto transport media or culture media.
k. Sensitive to acids present in feces.
Morphology.
Morphologically
dysentery
bacilli
correspond
to
the
organisms of the family
Enterobacteriaceae.
Dysentery bacilli have
no flagella and this is
one of the differential
characters
between
these organisms and
bacteria of the colityphoid-paratyphoid
group.
Dysentery bacilli
Intracellular
Shigella
Cultivation.
Colonies of dysentery bacilli on Ploskirev's medium
Colonies of Salmonella on Mac-Conkey medium
Fermentative properties.
None of the species of dysentery bacilli
liquefy gelatin nor produce hydrogen sulphide.
They ferment glucose, with acid formation,
with the exception of the Newcastle subspecies
which sometimes produce both acid and gas
during this reaction. With the exception of the
Sonne bacilli, none of them ferment lactose.
Shigellae Biochemical Properties
Subgroup
Fermentation of
Catalase
+
–
–
–
–
–
S. fiexneri – B
–
–
+
+
+
+
+
+
+
+
+
–
–
–
+
–
+
–
+
–
+
S. boydii – C
S. sonnei – D
slowly
succrose
succrose
–
glucose
S. dysenteriae
–A
lactose
mannite
Indole
production
carbohydrates
slowly
Test for determination of motility and producing hydrogen sulphide
1. S. flexneri – nonmotile, no produce hydrogen sulphide;
2. Enterobacter cloacea – motile, no produce hydrogen sulphide;
3. Proteus mirabilis – motile, produce hydrogen sulphide.
Toxin production.
S. dysenteriae produce thermolabile
exotoxin which displays marked tropism to the
nervous system and intestinal mucous
membrane. This toxin may be found in old
meat broth cultures, lysates of a 24-hour-old
agar culture, and in desiccated bacterial cells.
An intravenous injection of small doses of
the exotoxin is fatal to rabbits and white mice.
Such an injection produces diarrhoea, paralysis
of the hind limbs, and collapse.
The dysentery exotoxin causes the
production of a corresponding antitoxin. The
remaining types of dysentery bacilli produce no
soluble toxins. They contain endotoxins, which
are of a gluco-lipo-protein nature, and occur in
the smooth but not in the rough variants.
Thermolabile
substances
exerting
a
neurotropic effect were revealed in some S.
sonnei strains. They were extracted from old
cultures by treating the latter with trichloracetic
acid.
Antigenic structure.
Dysentery bacilli are subdivided into 4
subgroups within which serovars may be
distinguished. The antigenic structure of
shigellae is associated with somatic Oantigens and surface K-antigens.
Antigens
Antigenic formula
Subgroup
Species and Subseserotype
rotypes
A. Does not
ferment mannite
S.dysenteria
B. Ferments
mannite as a rule
S. flexneri
1, 2, 3, 4, 5,
6, X variant
Y variant
C. Ferments
mannite as a rule
S. boydii,
1-18
D. Ferments
mannite, slowly
lactose and
saccharose
S. sonnei
Type
antigen
Group
antigen
s
I, II, III,
IV, V, VI
2, 3, 4,
6, 7, 8
1-12
1a,
2a,
3a,
4a,
1b,
2b,
3b,
4b
Classification.
Dysentery bacilli are differentiated on the
basis of the whole complex of antigenic and
biochemical properties. S. sonnei have four
fermentative types which differ in the activity
of ramnose and xylose and in sensitivity to
phages and colicins.
Epidemiology and Pathogenesis of
Shigellosis.
Humans seem to be the only natural hosts for the
shigellae, becoming infected after the ingestion of
contaminated food or water. Unlike Salmonella, the
shigellae remain localized in the intestinal epithelial
cells, and the debilitating effects of shigellosis are
mostly attributed to the loss of fluids, electrolytes, and
nutrients and to the ulceration that occurs in the colon
wall.
Pathogenesis
of shigellosis in humans
Shigella dysenteriae type 1 secreted one or more
exotoxins (called Shiga toxins), which would cause
death when injected into experimental animals and
fluid accumulation when placed in ligated segments
of rabbit ileum.
The mechanism whereby Shiga toxin causes fluid
secretion is thought to occur by blocking fluid
absorption in the intestine. In this model, Shiga toxin
kills absorptive epithelial cells, and the diarrhea
results from an inhibition of absorption rather than
from active secretion.
To cause intestinal disease, shigellae must
invade the epithelial cells lining of the
intestine. After escaping from the phagocytic
vacuole, they multiply within the epithelial
cells. Thus, Shigella virulence requires that the
organisms invade epithelial cells, multiply
intracellularly, and spread from cell to cell by
way of finger-like projections to expand the
focus of infection, leading to ulceration and
destruction of the epithelial layer of the colon.
Gross pathology of shigellosis
Histopathology of acute
colitis following peroral
infection with shigellae.
Immunity.
Immunity acquired after dysentery is specific
and type-specific but relatively weak and of a
short duration. For this reason the disease may
recur many times and, in some cases, may
become chronic. This is probably explained by
the fact that Shigella organisms share an antigen
with human tissues.
Laboratory diagnosis.
Reliable results of laboratory examination
depend, to a large extent, on correct sampling
of stool specimens and its immediate
inoculation onto a selective differential
medium. The procedure should be carried out
at the patient's bedside, and the plate sent to the
laboratory.
Rules the correct procedure of material collection :
carry out bacteriological examination of faeces before
aetiotropic therapy has been initiated;
 collect faecal samples (mucus, mucosal admixtures)
from the bedpan and with swabs (loops) directly from
the rectum (the presence in the bedpan of even the
traces of disinfectants affects the results of
examination);
 inoculate without delay the collected material onto
enrichment media, place them into an incubator or store
them in preserving medium in the cold;
send the material to the laboratory as soon as
possible.
Bacteriological examination.
Faecal samples are streaked onto plates with Ploskirev's
medium and onto a selenite medium containing phenol
derivatives, beta-galactosides, which retard the growth of
the attendant flora, in particular E. coli. The inoculated
cultures are placed into a 37 °C incubator for 1S-24 hrs. The
nature of tile colonies is examined on the second day.
Colourless lactose-negative colonies are subcultured to
Olkenitsky's medium or to an agar slant to enrich for pure
cultures. On the third day, examine the nature of the growth
on Olkenitsky's medium for changes in the colour of the
medium column without gas formation. Subculture the
material to Hiss' media with malonate, arabinose, rhamnose,
xylose, dulcite, salicine, and phenylalanine. Read the results
indicative of biochemical activity on the following day.
Shigellae ferment carbohydrates with the formation of acid
To determine the species of Shigellae, one can employ the
following tests:
1.The agglutination test is performed first with a mixture
of sera containing those species, and variants of Shigellae
that are prevalent in a given area, and then the slide
agglutination test with monoreceptor species sera.
2. The coagglutination test which allows to determine the
specificity of the causative agent by a positive reaction with
protein A of staphylococci coated with specific antibodies.
On a suspected colony put a drop of specific sensitized
protein A of Staphylococcus aureus, then rock the dish and
15 min later examine it microscopically for the appearance
of the agglutinate (these tests may also be carried out on the
second day of the investigation with the material from
lactose-negative colonies).
3. Direct and indirect immunofluorescence test.
IFT: Salmonella enterica serovar Typhimurium inside (green)
and on the surface (blue) of human intestinal epithelial cells.
Actin is labelled in red.
4. The indirect haemagglutination (IHA) test with
erythrocyte diagnosticums with the titre of 1:160 and
higher is performed. The test. is repeated after at least
seven days. Diagnostically important is a four-fold rise in
the antibody litre, which can be elicited from the 10th12th day of the disease. To distinguish between patients
with subclinical forms of the disease and Shigella carriers,
identify immunoglobulins of the G class.
5. ELISA. For the epidemiological purpose the phagovar
and colicinovar of Shigellae are also identified.
6. To determine whether the isolated cultures belong to
the genus Shigella, perform the keratoconjunctival test on
guinea pigs. In contrast to causal organisms of other
intestinal infections, the dysentery Shigellae cause marked
keratitis.
7. An allergic test consisting in intracutaneous injection
of 0.1 ml of dysenterin is applied in the diagnosis of
dysentery in adults and children. Hyperaemia and a papule
2 to 3.5 cm in diameter develop at the site of the injection
in 24 hours in a person who has dysentery. The test is
strictly specific.
8. An allergy intracutaneous test with Tsuverkalov's
dysenterine is of supplementary significance. It becomes
positive in dysentery patients beginning with the fourth day
of the disease. The result is read in 24 hrs by the size of the
formed papula. The test is considered markedly positive in
the presence of oedema and skin hyperaemia 35 mm or
more in diameter, moderately positive if this diameter is
20-34 mm, doubtful if there is no papula and the diameter
of skin hyperaemia measures 10-15 mm, and negative if
the hyperaemic area is less than 10 mm.
9. The nature of the isolated culture may be determined
in some cases by its lysis by a polyvalent dysentery phage
Treatment of Shigellosis
Intravenous
electrolytes;
replacement
of
fluids
and
antibiotic therapy (ampicillin frequently is not
effective, and alternative therapies include
sulfamethoxazole / trimethoprim and, the
quinolone antibiotics such as nalidixic acid and
ciprofloxacin)
Dysentery control is ensured by a complex of general and
specific measures; (1) early and a completely effective
clinical, epidemiological, and laboratory diagnosis; (2)
hospitalization of patients or their isolation at home with
observance of the required regimen; (3) thorough
disinfection of sources of the disease; (4) adequate
treatment of patients with highly effective antibiotics and
use of chemotherapy and immunotherapy; (5) control of
disease centres with employment of prophylaxis measures;
(6) surveillance over foci and the application of
prophylactic measures there; (7) treatment with a phage of
all persons who were in contact with the sick individuals;
(8) observance of sanitary and hygienic regimens in
children's institutions, at home and at places of work, in
food industry establishments, at catering establishments, in
food stores.
Vibrio Cholerae
Morphology. Cholera
vibrios are shaped like
a comma or a curved
rod measuring 1-5
mcm in length and 0.3
mcm in breadth
They are very actively
motile, monotrichous,
nonsporeforming,
noncapsulated,
and
Gram-negative.
Gram’s stain
Scanning electron
micrograph V. cholerae
Cultivation.
Colonies of V. cholerae on bismuth-sulphit-agar
Colonies of V. cholerae on blood agar
Fermentative properties.
The cholera vibrio liquefies coagulated serum and
gelatin; it forms indole and ammonia, reduces
nitrates to nitrites, breaks down urea, ferments
glucose, levulose, galactose, maltose, saccharose,
mannose, mannite, starch, and glycerine (slowly)
with acid formation but does not ferment lactose in
the first 48 hours, and always coagulates milk. The
cholera vibrio possesses lysin and ornithine
decarboxylases and oxidase activity. B. Heiberg
differentiated vibrios into biochemical types
according to their property of fermenting mannose,
arabinose, and saccharose.
sacharose
mannose
arabinose
Sheep erythrocyte
hemolysis
Lysis by specific O-1
subgroup phages
Agglutination by O-1
cholera serum
Sensitivity to polymixin
B
Fermentatio
n
within 24
hrs
Vibrio cholerae
biovar cholerae
A
A
–
–
+
+
+
Vibrio cholerae
biovar El Tor
A
A
–
+
+
+
–
Vibrio cholerae
biovar Proteus
A
A
–
+
–
–
–
Vibrio cholerae
biovar albensis
А
–
–
–
–
–
–
Vibrio
Toxin production.
an exotoxin (cholerogen) which is
marked by an enterotoxic effect
the endotoxin also exerts a powerful
toxic effect fibrinolysin
hyaluronidase
collagenase
mucinase
lecithinase
neuraminidase
proteinases
Mechanism of action of cholera enterotoxin according to Finkelstein. Cholera toxin
approaches target cell surface. B subunits bind to oligosaccharide of GM1 ganglioside.
Conformational alteration of holotoxin occurs, allowing the presentation of the A
subunit to cell surface. The A subunit enters the cell. The disulfide bond of the A subunit
is reduced by intracellular glutathione, freeing A1 and A2. NAD is hydrolyzed by A1,
yielding ADP-ribose and nicotinamide. One of the G proteins of adenylate cyclase is
ADP-ribosylated, inhibiting the action of GTPase and locking adenylate cyclase in the
"on" mode.
Cholera toxin activates the adenylate cyclase enzyme in
cells of the intestinal mucosa leading to increased levels of
intracellular cAMP, and the secretion of H20, Na+, K+, Cl-,
and HCO3- into the lumen of the small intestine.
Antigenic Determinants of Vibrio cholerae
Pathogenesis and diseases in man.
Cholera is undoubtedly
the most dramatic of the
water-borne diseases.
The cholera vibrios are
transmitted from sick
persons and carriers by
food, water, flies, and
contaminated hands.
Cholera is characterized by a short incubation
period of several hours to up to 6 days (in a
disease caused by the El Tor vibrio it lasts three to
five days), and the disease symptoms include
general weakness, vomiting, and a frequent loose
stool. The stools resemble rice-water and contain
enormous numbers of torn-off intestinal epithelial
cells and cholera vibrios. The major symptom of
cholera is a severe diarrhea in which a patient may
lose as much as 10 to 20 L or more of liquid per
day. Death, which may occur in as many as 60% of
untreated patients, results from severe dehydration
and loss of electrolytes.
Phases in the development of the disease:
1. Cholera enteritis (choleric diarrhoea) which lasts 1
or 2 days. In some cases the infectious process
terminates in this period and the patient recovers.
2. Cholera gastroenteritis is the second phase of the
disease. Profuse diarrhoea and continuous vomiting
lead to dehydration of the patient's body and this
results in lowering of body temperature, decrease in
the amount of urine excreted, drastic decrease in the
number of mineral and protein substance, and the
appearance of convulsions. The presence of cholera
vibrios is revealed guite frequently in the vomit and
particularly in the stools which have the appearance of
rice water.
3. Cholera algid which is characterized by severe
symptoms. The skin becomes wrinkled due to the
loss of water, cyanosis appears, and the voice
becomes husky and is sometimes lost
completely. The body temperature falls to 35.534° C. As a result of blood concentration cardiac
activity is drastically weakened and urination is
suppressed.
Immunity
acquired after cholera is high-grade but of short
duration and is of an anti-infectious
(antibacterial and antitoxic) character. It is
associated mainly with the presence of
antibodies (lysins, agglutinins, and opsonins).
The cholera vibrios rapidly undergo lysis under
the influence of immune sera which contain
bacteriolysins.
Laboratory diagnosis.
A strict regimen is established in the
laboratory. Examinations are carried out in
accordance with the general rules observed for
particularly hazardous diseases.
Test specimens are collected from stools,
vomit, organs obtained at autopsy, water,
objects contaminated by patient's stools, and,
in some cases, from foodstuffs. Certain rules
are observed when the material is collected
and transported to the laboratory, and
examination is made in the following stages.
1. Stool smears stained by a water solution of
fuchsin are examined microscopically. In the
smears, the cholera vibrios occur in groups similar
to shoals of fish.
2. A stool sample is inoculated into 1 per cent
peptone water and alkaline agar. After 6 hours
incubation at 37°C the cholera vibrios form a thin
pellicle in the peptone water, which adheres to the
glass. The pellicle smears are Gram stained, and
the culture is examined for motility. A slide
agglutination reaction is performed with specific
agglutinating O-serum diluted in a ratio of 1 in
100.
Vibrio cholerae (stool smear)
The organisms are then transferred from the
peptone water onto alkaline agar for isolation of
the pure culture. If the first generation of the
vibrios in peptone water is not visible, a drop
taken from the surface layer is re-inoculated into
another tube of peptone water. In some cases
with such re-inoculations, an increase in the
number of vibrios is achieved.
The vibrio culture grown on solid media is
examined for motility and agglutinable
properties. Then it is subcultured on an agar
slant to obtain the pure culture.
3. The organism is identified finally by its
agglutination reaction with specific O-serum,
determination of its fermentative properties
(fermentation of mannose, saccharose, and
arabinose), and its susceptibility to phagolysis.
Colonies of Vibrio cholerae of font varying opacity
(increasing from top right, left bottom right) pseudocoloured
to accentuate differences in gray-scale intensity. Of varying
opacity (increasing from top left to top right, to bottom left to
bottom right) pseudocoloured to accentuate differences in
grey-scale intensity.
The following procedures are undertaken for rapid
diagnosis: (1) dark field microscopy of the stool; (2) stool
culture by the method of tampons incubated for 16-18
hours in an enrichment medium with repeated dark field
microscopy; (3) agglutination reaction by the method of
fluorescent antibodies; (4) bacterial diagnosis by isolation
of cholera vibrios (the faecal mass is seeded as a thin
layer into a dish containing non-inhibiting nutrient agar
and grown for 4-5 hours, the vibrio colonies are detected
with a stereoscopic microscope, and the culture is tested
by the agglutination reaction with O-serum on glass; (5)
since neuraminidase is discharged by the cholera vibrios
and enters the intestine, a test for this enzyme is
considered expedient as a means of early diagnosis (it is
demonstrated in 66-76 per cent of patients, in 50-68 per
cent of vibrio carriers, and occasionally in healthy
individuals).
Treatment.
 antibiotics of the tetracycline group (tetracycline,
sigmamycin), amphenicol, and streptomycin are
prescribed at first intravenously and then by mouth.
 pathogenetic therapy is very important: control of
dehydration, hypoproteinaemia, metabolic disorders,
and the consequences of toxicosis, acidosis in
particular, by infusion of saline (sodium and
potassium) solutions, infusion of plasma or dry serum,
glucose, the use of warm bath, administration of drugs
which improve the tone of the heart and vessels.
Prophylaxis.
Cholera patients and vibrio carriers are the source
of the disease. Individuals remain carriers of the El
Tor vibrio for a lengthy period of time, for several
years. Vibrios of this biotype are widely
distributed in countries with a low sanitary level.
They survive in water reservoirs for a long time
and have been found in the bodies of frogs and
oysters. Infection may occur from bathing in
contaminated water and fishing for and eating
shrimps, oysters, and fish infected with El Tor
vibrio.The following measures are applied in a
cholera focus:
1. detection of the first cases with cholera,
careful registration of all sick individuals,
immediate information of health protection
organs;
2. isolation and hospitalization, according to
special rules, of all sick individuals and
carriers, observation and laboratory testing of
all contacts;
3. concurrent and final disinfection in
departments for cholera patients and in the
focus;
4. protection of sources of water supply,
stricter sanitary control over catering
establishments, control of flies; in view of the
possibility of El Tor vibrio reproducing in
water reservoirs under favourable conditions
(temperature, the presence of nutrient
substrates) systematic bacteriological control
over water reservoirs has become necessary,
especially in places of mass rest and recreation
of the population in the summer;
5. strict observance of individual hygiene;
boiling or proper chlorination of water,
decontamination of dishes, hand washing;
6. specific prophylaxis: immunization with the
cholera monovaccine containing 8 thousand
million microbial bodies per 1 ml or with
the cholera anatoxin. Chemoprophylaxis
with oral tetracycline is conducted for
persons who were in contact with the sick
individual or for patients with suspected
cholera.