Vibrio

Vibrios are Gram-negative, rigid, curved rods that are

actively motile by means of a polar flagellum.
The name ‘Vibrio’ is derived from the characteristic
vibratory motility (from vibrare, meaning to vibrate). They
are asporogenous and noncapsulated.
The most important member of the genus is Vibrio
cholerae, the causative agent of cholera.
It was first isolated by Koch from cholera patients in
Egypt, though it has been observed earlier by Pacini.
Other pathogenic vibrios, are Vibrio parahaemolyticus
and Vibrio vulnificus.
 VIBRIO CHOLERAE
Morphology :
Short, curved, cylindrical rod with rounded or slightly
pointed ends.
The cell is typically comma shaped (hence the old name V
comma) but the curvature is often lost on subculture.
 S-shaped or spiral form may be seen due to two or more
cells lying end to end. Pleomorphism is frequent in old
cultures.
In stained films of mucus flakes from acute cholera cases,
the vibrios are seen arranged in parallel rows, described
by Koch as the ‘fish in stream’ appearance.
It is actively motile, with a single polar flagellum.
 The motility is of the darting type, and when acute
cholera stool or a young culture is examined under the
microscope, the actively motile vibrios have a ‘swarm of
gnats’ appearence.
The vibrios stain readily with aniline dyes and are Gram
negative and non-acid fast.
Culture Characteristics
The cholera vibrio is strongly aerobic, growth being
scanty and slow anaerobically. It grows within a
temperature range of 16-40⁰C (optimum 37⁰C).
 Growth is better in an alkaline medium, the range of pH
being 6.4-9.6 (optimum 8.2).
NaCl (0.5-1%) is required for optimal growth though high
concentration (6% and above) are inhibitory.
It grows well on ordinary media.
On nutrient agar, colonies are moist , translucent, round
discs with a bluish tinge in transmitted light with a
distinctive odour.
 On MacConkey’s agar, the colonies are colourless at first
but become reddish on prolonged incubation due to the
late fermentation of lactose.
On blood agar, colonies are initially surrounded by a zone of
greening, which later become clear due to hemodigestion.
In peptone water, growth occurs in about six hours as a fine
surface pellicle, which on shaking breaks up into
membranous pieces.
In gelatin stab culture, infundubliform (funnel shaped) or
napiform(turnip shaped) liquefaction occurs in three days
at 22ᵒ C.
Special Media
A number of special media have been employed for the
cultivation of Vibrio cholera. They may be classified as
follows :
Holding or transport media
1. Venkatraman – Ramakrishnan (VR) medium
2. Cary-Blair medium
3. Autoclaved sea water.
Enrichment media
1. Alkaline peptone water at a pH of 8.6;
2. Monsur’s taurocholate tellurite peptone water
Plating media
1. Alkaline bile salt agar (BSA)
2. Monsur’s gelatin taurocholate trypticase tellurite
agar(GTTA)
3. TCBS medium : This medium, containing thiosulfate,
citrate, bile salts and sucrose. Available commercially and
is very widely used at present. Cholera vibrios produce
large yellow convex colonies which may become green on
continued incubation.
Biochemical reactions :
Carbohydrate metabolism is fermentative, producing acide,
but no gas. Ferment glucose, mannitol, maltose, mannose
and sucrose but not inositol, arabinose or lactose, though
lactose may be split very slowly.
Indole is formed and nitrates are reduced to nitrites. These
two properties contribute to the ‘cholera red reaction’
which is tested by adding a few drops of concentrated
sulphuric acid to a 24-hour peptone water culture. With
cholera vibrios, a reddish pink colour develops due to the
formation of nitroso-indole.
String test: A loopfull of growth is mixed with a drop of 0.5%
sodium deoxycholate in saline on a slide. If test positive
suspension loses its turbidity , becomes mucoid and forms a
string when loop is withdrawn slowly from suspension.
PATHOGENECITY
Cholera
Cholera is an acute diarrheal disease caused by
V. cholerae.
 In most severe form, cholera is a dramatic and terrifying
illness in which profuse painless watery diarrhea and
copious effortless vomiting may lead to hypovolemic
shock and death in less than 24 hours.
In treated cases, the disease may last 4-6 days, during
which period the patient may pass a total volume a liquid
stool equal to twice his body weight.
Clinical Feature of Cholera
 All the clinical features of severe cholera result from this
massive loss of fluid and electrolytes.
 The cholera stool is typically a colourless watery fluid with
flecks of mucus, said to resemble water in which rice has
been washed (hence called ‘rice water stools’). It has a
characteristic inoffensive sweetish odour. Its a bicarbonate
rich isotonic electrolyte solution, with little protein.
Common complications are muscular cramps, renal
failure , pulmonary edema, cardiac arrhythmias and
paralytic ileus.
 The clinical severity of cholera varies widely, from the
rapidly fatal disease to a transient asymptomatic
colonization of the intestine by the vibrios.
The incidence of mild and asymptomatic infections is
more with EI Tor vibrios than with the classical cholera
vibrios.
 The incubation period varies from less than 24 hours to
about five days. The clinical illness may begin slowly with
mild diarrhea and vomiting in 1-3 days or abruptly with
sudden massive diarrhea.
Pathogenesis
Vibrio Cholerae enters through orally contaminated food
& water.
Vibrios are highly susceptible to acids and gastric acidity
provides an effective barrier against small doses of
vibrios.
In small intestine vibrios cross the protective layer of mucus
and reach epithelial cells by chemotaxis, motility, mucinase
and other proteolytic enzymes. Adhesion to epithelial cell &
colonisation may be facilitated by toxin coregulated pilus (TCP).
Enterotoxin
Vibrios multiplying on the intestinal epithelium and produce
an enterotoxin called Cholera Toxin/ CT.
 Cholera toxin binds to GM1 ganglioside on surface of the
jejunal epithelial cells and activates cellular adenly cyclase
which converts ATP to CAMP and causes accumulation of cyclic
AMP.
CAMP increases the outflow of electrolytes & water to gut
lumen & causes diarrhea.
All clinical manifestations and complications of cholera result
from massive loss of water and electrolytes.
Laboratory Diagnosis:
1. Specimen:
Stool – collected in the acute stage of the disease, before
administration of antibiotics. Specimen best collected by
inserting a lubricated catheter into rectum and letting the
liquid to collect directly into a screw capped bottle.
Rectal swab- made with good quality of cotton wool,
absorbing about 0.1-0.2 ml of fluid. Useful in collecting
specimens from convalescents as there is no watery
diarrhea. Swabs must be moistened with transport
medium before sampling.
Collection from bed pans is not good. Vomitus is not
useful.
2. Transport: As cholera vibrios may die in a few hours it
is preserved at 4⁰C or in holding or transport medium. VR
medium and Cary-Blair medium used for this.
3. Microscopy: Diagnosis by direct microscopy is not
recommended. For rapid diagnosis darting motility of
vibrio and its inhibition by specific antiserum can be
demonstrated under dark field or phase contrast
microscope.
4. Culture: On reaching lab specimen sent in enrichment
media is incubated for 6 -8 hrs including transit time.
Specimens transmitted in transport media is inoculated
into enrichment media and incubated 6-8 hrs before
being plated on selective and a nonselective media.
Plating media- BSA and MA used as nonselective and
TCBS as selective media. Plates should not be older than
3-5 days and plates should be completely dried. Plates
kept for overnight incubation at 37⁰C.
5. Identification:
Slide agglutination : colonies suspected to be of vibrios
are picked by a sterile straight wire and tested for slide
agglutination with cholera O subgroup I serum. If positive
agglutination done by ogawa, inaba sera. If agglutination
negative with one colony repeat the test with atleast 5
more colonies. If it is positive – isolates further tested for
biochemical reactions to differentiate between E1 Tor or
classical cholera vibrios.
Biochemical reactions : sugar fermentation, string test,
cholera red reaction. The strain may be sent to National
institute of cholera and Enteric disease at Kolkata.
6. Serology: Not used in diagnosis.
7. Testing of water sample: Enrichment or filtration
methods are used.
Enrichment- 900 ml of water is added to 100 ml tenfold
concentrated peptone water at pH9.2. incubated at 37⁰C
for 6-8 hrs. a second enrichment done before plating in
selective media.
Filtration: Membrane filter technique performed and
filter paper is placed on a selective medium.
Prophylaxis:
General measures such as provision of protected water
supply and improvement of environmental sanitation.
Vaccines:
Parentral – killed suspensions containing 8000 millions
V.cholerae per ml. Given by subcutaneous or intramuscular
injections. Duration of protection is only 3-6 months.
Injectable vaccines doesnot produce local immunity in the
intestinal mucosa.
Oral vaccine: 2 types used
- Killed oral whole cell vaccines with or without B fragment
of CT toxin.
Live oral vaccines- Classical or E1Tor with their toxin genes
deleted.
Treatment
Prompt and adequate replacement of lost fluid and
electrolytes.
Oral administration of fluid containing glucose and
electrolytes.
Antibacterial therapy is of secondary importance.Oral
tetracyclin is used.
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Microbiology/Lesson-27.pdf