Chair of Medical Biology, Microbiology, Virology,
and Immunology
Shigella. Vibrio
Lecturer Prof. S.I. Klymnyuk
Causative agents of Shigellosis
The causative agent of dysentery was described in 1888
by A. Chantemesse and in 1891 by A. Grigoryev and F.
Widal. In 1898 this organism was studied in detail by
K. Shiga in Japan and in 1900-1901 by V. Kruse in
Germany (Shiga bacillus).
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 coli-typhoid-paratyphoid
group. Some strains of Flexner bacilli are found to possess
cilia.
Shigellae Biochemical Properties
slowly
–
–
–
+
slowly
–
–
+
–
Catalase
S. sonnei – D
–
+
+
–
Indole
production
S. boydii – C
–
+
+
+
succrose
S. fiexneri – B
+
+
+
+
succrose
–
–
–
+
mannite
S. dysenteriae – A
glucose
Fermentation of carbohydrates
lactose
Subgroup
–
+
–
+
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.
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.
Antigens
Antigenic formula
Subgroup
A. Does not
ferment mannite
Species and
serotype
Type
antigen
Group
antigens
S.dysenteria
1-12
B. Ferments
S. flexneri
mannite as a rule 1, 2, 3, 4, 5,
6, X variant
Y variant
C. Ferments
S. boydii,
mannite as a rule 1-18
D. Ferments
mannite, slowly
lactose and
saccharose
Subserotypes
S. sonnei
1a, 1b,
2a, 2b,
3a, 3b,
4a, 4b
I, II, III, 2, 3, 4,
IV, V, VI 6, 7, 8
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.
It has been known for many years that 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.
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.
For the serological diagnosis of dysentery 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 10th-12th day
of the disease. To distinguish between patients with
subclinical forms of the disease and Shigella carriers,
identify immunoglobulins of the G class.
Treatment and Control of Shigellosis
Intravenous replacement of fluids and electrolytes plus
antibiotic therapy are used for severe cases of
shigellosis. Ampicillin frequently is not effective, and
alternative
therapies
include
sulfamethoxazole/
trimethoprim and, with increasing sulfamethoxazole/
trimethoprim resistance, the quinolone antibiotics such
as nalidixic acid and ciprofloxacin.
In the Far East, India, and Brazil where shigellosis is
more common than in the United States, multiple
antibiotic resistance because of the acquisition of
plasmids has become common. Shigellosis also is
common in Latin America.
The injection of killed vaccines is worthless, because humoral
IgG does not seem to be involved in immunity to the localized
intestinal infection.
Live vaccines that cannot grow in the absence of streptomycin
(ie, streptomycin-dependent vaccines) have been developed and
used in clinical trials, but success has been equivocal.
Summary
SHIGELLA
1. Genus characteristics
a. Slender, gram-negative rod; non lactose-fermenting
(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
e.
Toxin production limited to a few strains
f.
All have O antigens-four groups (A-D)
g.
Differentiating species
i. S. dysenteriae - no mannitol fermentation
ii. S. boydii - C antigen group
iii. S. flexneri - B antigen group
iv. S. sonnei-orniltine decarbexylase production
h. Specimens
i. Rectal swab from colonic ulcer is best for culture.
ii. Fecal specimen - must be immediately innoculated onto
transport media or culture media.
Sensitive to acids present in feces.
2. Epidemiology
a. Species
i. S. sonnei most frequent isolate in U.S.
ii. S. dysenteriae and S. boydii most frequently isolated in
developing countries - cause more severe disease course.
iii. S. flexneri more commonly isolated from homosexual
men
b. Humans and higher primates are only natural reservoir.
c. High communicability (< 200 bacilli needed to produce
disease)
d. May be spread by fomites;”food, fingers, feces, and
files”.
3. Clinical manifestations
a. Desease - bacillary dysentery (shigellosis), characterized
by painful, frequent, low-volume stools; feces contain blood
and mucous. Bacteremia is rare since organisms usually do
not enter bloodstream. Polymorphonuclear leukocyles
present in stool.
b. Carriers
i. One to four weeks after the disease; carrier state may be
set up if organism is not cleared.
ii. Long-term; recurrent bouts of disease
4. Therapy and prevention
a. Hydration and electrolyte replacement
b. Ampicillin - decreases duration of symptoms and
carrier state; tetracycline; trimethoprimsulfamelhoxazole
c. Prevention through personal hygiene, proper garbage
disposal and water purification.
Vibrio Cholerae
The causative agents of cholera are the classical
Vibrio cholera biovars discovered by R. Koch in
1883 and the El Tor vibrio biovar isolated from the
cadaver of a pilgrim on the Sinai peninsula by
Gotschlich in 1906. Vibrio cholerae biovar Proteus
(N. Gamaleya, 1888) and Vibrio cholerae biovar
albensis were discovered" later. V. cholerae was
described by F. Pacini in 1854.
Classification. Vibrio cholerae belongs to family
Vibrionaceae, genus Vibrio consisting of 5 species. The
species Vibrio cholerae is subdivided into four biological
variants: biovar cholerae, biovar El Tor, biovar
Proteus, and biovar albensis.
Biovar cholerae and biovar El Tor of Vibrio cholerae are
the causative agents of human cholera. Biovar Proteus of
Vibrio cholerae causes diarrhoea in birds and
gastroenteritis in humans; biovar albensis of Vibrio
cholerae was revealed in fresh water and in human faeces
and bile.
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.
Vibrio cholerae: 1-pure culture; 2- flagellate
vibrios
Cultivation. Cholera vibrios are facultative (anaerobes).
The optimum growth temperature is 37° C, and growth is
arrested below 14 °C and above 42° C. The organisms grow
readily on alkaline media at pH 6.0-9.0, and on solid media
the colonies are transparent with a light-blue hue, forming
domes with smooth edges.
Fermentative properties
sacharose
mannose
arabinose
Sheep erythrocyte
hemolysis
Lysis by specific O-1
subgroup phages
Agglutination by O-1
cholera serum
Sensitivity to
polymixin B
Vibrio
cholerae
biovar cholerae
A
A
–
–
+
+
+
Vibrio
cholerae
biovar El Tor
A
A
–
+
+
+
–
Vibrio
cholerae
biovar Proteus
A
A
–
+
–
–
–
Vibrio
cholerae
biovar albensis
А
–
–
–
–
–
–
Fermentation
within 24 hrs
Vibrio
Toxin production. The cholera vibrio produces an
exotoxin (cholerogen) which is marked by an
enterotoxic effect and plays an important role in the
pathogenesis of cholera; the endotoxin also exerts a
powerful toxic effect. The cholera vibrios produce
fibrinolysin, hyaluronidase, collagenase, mucinase,
lecithinase, neuraminidase, and proteinases.
Remember that non-O1 and non-O139 strains of
V.cholerae also cause a wide spectrum of infections,
ranging from mild diarrhea to one indistinguishable
from classic cholera. Some of these serotypes are
known to produce a choleratoxin that is identical to
that of the classic biotypes, whereas other products a
heat-stable enterotoxin analogous to the ST of E. coli.
Antigenic structure. The cholera vibrios have
thermostable O-antigens (somatic) and thermolabile Hantigens (flagellar). The O-antigen possesses species
and type specificity, the H-antigen is common for the
genus Vibrio. The cholerae vibrios, El Tor biovars and
biovars cholera belong to the O-1 subgroup.
Pathogenesis and diseases in man.
Cholera is
undoubtedly the most dramatic of the water-borne
diseases. As far as is known, cholera was confined to
India for the almost 2000 years between its first
description by Hindu physicians in 400 b c and its spread
to Arabia, Persia, Turkey, and Southern Russia in the
early1800s. There were six major pandemics of cholera
during the 1800s covering the entire world, killing
millions wherever it struck.
Three phases can be distinguished in the development of
the disease.
1. Cholera enteritis (choleric diarrhoea) which lasts 1 or 2
days.
2. Cholera gastroenteritis. 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
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 35.5-34 °C. As a
result of blood concentration cardiac activity is drastically
weakened and urination is suppressed.
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 (Fig. 3).
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 reinoculated 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
Treatment. The mortality rate of cholera can be reduced
to less than1% by the adequate replacement of fluids and
electrolytes. 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. 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.
SUMMURY
I. GENUS VIBRIO
A. GENERAL CHARACTERISTICS
1. From family Vibnonaceae
2 Naturally occur in water-marine and fresh waters; some
occur in cold-blooded animals.
3. Oxidase-positive-differentiates Vibrios from
Enterobacteriaceae.
4. Characteristic comma shape
B VIBRIO CHOLERAE
1 Species characteristics
a. Comma-shaped when first isolated
b. Aerobic
c. Unipolar flagellum-motile
d. Primary isolation-simple media, MacConke/s agar,
tellurite taurocholate gelatin agar (TTGA),
thiosulfate citrate bile salts agar (TCBS)
e. Sensitive to 2,4-diamme-6,7 d isopropyi pteridine
(0/129), useful in distinguishing from other
gram-negative, oxidase-posilive bacilli (i.e., Aeromonas)
f. Prefer alkaline environment.
2. Serogroups-based on O antigen type
a. V.cholerae O-1
i. Agglutinate antisera against O-1 antigen.
ii. Classic epidemic and pandemic Asiatic cholera; most virulent
serogroup
iii. Produce disease via enterotoxin (heat-labile toxin produciogsecretory diarrhea via cyclic AMP).
iv. Subdivided biochemically into Cholerae and El Tor biotypes
b. Atypical V. cholerae O-1
i. Agglutinate antisera against O-1 antigen.
ii. Do not cause human disease.
in. Do not elaborate classic cholera enlerotoxin, '
c. Non O-1 V cholerae
i. Fail to agglutinate O-1 antisera.
ii. Biochemically and genetically indistinguishable from the O-1
group
iii. Cholera-like diarrhea; rareirr extrainistinal infection
3. Determinants of pathogenicity
a. Adherence to small intestine epithelium - non-invasive
infection
b. Pathogenicily related to host response to (he enterotoxin
(choleragen); humans only host that has pathogenic
response
c. Two major enterotoxin subunits
i. A subunit-A1 peptide promotes activation of adenylate
cyclose
ii.  subunit - binds toxin to small intestinal ganglioside
receptor
d. Principal effect is increased intracettular cyclic AMP
resulting in electrolyte/fluid secretion into small intestine
lumen.
4. Clinical manifestations: Cholera
a Incubation of 1-4 days
b Severe, watery ("rice water") diarrhea with loss of
sodium, chloride, potassium, and bicarbonate
c Associated with nausea, vomiting and abdominal cramps
d Metabolic acidosis
e. Hypovolemic shock
f. Mortality rate without treatment 25-50%
5. Therapy and prevention
a. Rapid rehydration and electrolyte replacement
b.
i. Intravenous in severe cases
ii. Oral therapy in milder cases
Tetracycine (oral)
i. Reduces number of organisms.
ii. Decreases degree of stool output
iii. Helps eliminate chronic carrier slate
iv. Resistant forms recognized in Africa-plasmid
mediated
v. Not indicated for prophylaxis
c.
Present vaccine does not afford adequate protection
d.
Gastric acidity affords some protection.
e.
Infection does provide non-lasting immunity to
reinfection
C. VIBRIO PARAHEMOLYTICUS
1. Species characteristics
a.
Resembles V. cholerae structurally.
b.
Halophilic, requiring at least 2 % NaCl for growth
(in contrast to V.cholerae which does not grow in saline)
c
O and K antigens useful for serologic typing
d.
Isolates causing diarrhea produce hemolysm
(Kanawaga positive)
2. Clinical manifestations
a.
Most cases of diarrhea attributed to mgestion of raw
or improperly handled seafood
b.
Incubation period 12-24 hours
c.
Explosive watery diairhea, headache, abdominal
cramps, fever, and vomiting. Note: Diarrhea may be bloody.
d.
Cloudy swelling, fatty infiltration of liver
e.
Septicemia (particularly with underlying liver
disease)
Localized wound infections from contamination of sea
water
3. Therapy and prevention
a.
Mild disease usually self-limiting; no therapy
required. Usually subsides within 2-4 days
b.
For severe case, fluid and electrolyte replacements,
antibiotics
c.
Organisms usually sensitive to chloramphemcol,
tetracychne, and
cephalosporins
d.
Adequate refrigeration of raw and cooked seafood
aid in prevention
D OTHER VIBRIO INFECTIONS
1. V. alginotyticus
a. Affects wounds, eyes and ears in person with injuries or seawater
contact.
b. Septicemia - unusual
c. No known intestinal disease
2. V. vulnificus
a. Wound infection with intense pustules or bullae
b. Septicemja
c. Occasionally produce enteritis.
3. V.fluvialis,V.hollisae
a. Severe diarrhea in children in Bangladesh
b. Abdominal pain, lever, bloody mucus in stool
4 V.mimicus - diarrneal illness from uncooked seafood, especially
oysters
OTHER GENERA FROM FAMILY VIBRIONACEAE
A. GENUS AEROMONAS
1. Genus characteristics
a. Free-living, gram-negative rods located in fresh water and rarely
marine life
b. A. hydrophila is most important species
c. Oxidase-posilive (differentiates from gram-negative enteric's).
Produce large zone of hemolysis on blood agar.
d Single polar flagellum - motile
e. Resistance to compound O129 and lack of growth in NaCI solution
differentiates from Vibrios
2 Clinical manifestations
a Causes diarrhea, septicemia, osteomyelills, and wound infection,
usually in immunocompromised patients
b. Isolated from patients with and without diarrheal disease
3. Therapy - usually sensitive to aminoglycosides, tetracycline, and
cephalosporins
B GENUS PlESIOMONAS
1 Genus characteristics
a. A gram-negative rod with polar flagella found in tropics
and sub-tropics
b. Oxidase-positive - important to differentiate from
Shigella as they share common antigens
c. Has arginine, ornithine, and lysine decarboxylase distinguishes from Aeromonas.
2. Clinical manifestations
a. P. shigelloides primarily causes gastroenteritis
b. Has been isolated from blood and spinal fluid
CAMPYLOBACTER
A CENUS CHARACTERISTICS
1 Isolation of comma-shaped motile gram-negative organisms
from rectal or stool specimens cultured selective media containing
various antibiotics that inhibit other fecal flora
a. Campy-BAP-vanco, polymixin, trimethoprim and cephalothin
b. Skirrow's medium-vancomycin, polymixin B and trimethoprim
c. Butzler's medium
2. Microaerophilic - grows best in 5 % O2 (compared to 20%
atmospherically) present.
3. Do not oxidize or ferment carbohydrates.
4. Oxidase and catalase positive
5. Reservoir: domestic animals such as dogs, cows, and chicken
6. Fecal-oral transmission
B C. JEJUNI
Species characteristics
a. Grows at 39 C and 42C with candle jar, 42 will
inhibit growth of other bacteria that are present (including
C. intestinalis)
b. Nalidixic acid sensitive (in contrast to C.intestimalis)
2. Clinical manifestations: Enterocolitis
a. Invasive enteritis with bloody diarrhea, crampy
abdominal pains, malaise and fever – usually limited to 1week period; more common in children
b. Inflammatory proctitis in homosexuals
c. Reactive arthritis may follow in individuals HLA-B27(+).
3. Thefapy - erythromycin
C. C. INTESTINALIS (C. FETUS SUBSP. FETUS)
1. Species characteristics
a. Grows best at 25 C and 37 C and poorly at 42 C (in
contrast to C.jejuni)
b. Nalidixic asid resistant
2. Clinical manifestation
a. Bacteremia
b. Opportunistic pathogen in debilitated patients
c. May localize to peritoneum, pleura, lung, pericardium,
j joints, or meninges.
d. May cause localized diarrheal illness.
3. Therapy - aminoglycoside
C. HELICOBACTER PYLORI (previously C. pylori)
1. Species characteristics
a. Spiral-shaped, motile rod
b. Produces urease
c. Present in gastric mucosa of fewer than 20% of people
less than 30 years old but increases to greater than 50% of
people over 60 years old, even in asymptomatic patients
d. Optimal growth at pH 6 0-7.0; organism is sheltered
from lumenal acidity by embedding into gastric mucosa.
2 Clinical manifestations
a. Associated with antral gastritis and peptic ulcer disease
b. No direct evidence that organism causes disease but
strong association exists
c. Eradication of H. pylori will help heal peptic ulcer and
improvement of gastritis.
3. Diagnosis
a. Best made histologically via biopsy with ulceration and
gastritis seen with Giemsa stain;
characteristic intra-epithelial curved organisms
b Urease test is used for presumptive presence of H. pylori
4. Therapy - long term (1 month) therapy with bismuth
(Pepto Bismol), ampicillin, and metronidazole