Vibrio cholerae
Dr. Sudheer Kher
Key Words
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Vibrio cholerae
Late lactose fermenter
Oxidase positive
String test
Hemodigestion
Choleragen (cholera
toxin)
• Rice water stool
• Fish in Stream
appearance
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Darting motility
Cholera red reaction
TCBS
Transport media
Classical Vs El Tor
Endemic
Severe Dehydration
Halophilic vibrios
Vibrios
• Gram negative, rigid, curved Comma shaped rods,
motile by means of a single polar flagellum.
• Origin of the term vibrio –> vibrare=vibrate
• Nonsporing & noncapsulated.
• Present in marine environment and surface water all
over the world.
• Important member Vibrio cholerae - first observed by
Pacini (1854) and first isolated by Robert Koch in Egypt
in 1883.
• Stained smears from mucus flakes in stool show parallel
arrangement called “fish in stream” appearance.
• Darting motility by a single polar flagellum describes as
“swarm of gnats”.
Cultural characteristics
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Strongly aerobic but can grow anaerobically.
Grows at 16-40 C optimum 37 C
pH 6.4 – 9.6 optimum 8.2
NaCl is required for growth at 0.5-1%
Grows on ordinary culture media
Nutrient agar – Moist translucent disc like
colonies, 1-2 mm diameter with bluish tinge in
transmitted light. Distinct odour present.
• MacConkey’s agar – Initially colourless later
become pink due to lactose fermentation (late
lactose fermenter).
Cultural characteristics
• Blood agar – Initially surrounded by zone
of greening, later the zone is clear due to
hemodigestion.
• Gelatin stab culture – Infundibuliform
liquifaction in 3 days at 22 C.
• Peptone water – In six hours fine surface
pellicle is formed. Turbidity and powdery
deposit later.
Special Media for V. cholerae
• Holding or Transport media –
 Venkataraman –
Ramakrishnan (VR)
medium –
20 g crude sea salt + 5 g
peptone in 1 litre of DW.
Adjust pH to 8.6-8.8
Dispense in screw
capped bottle 10-15 ml
1-3 ml stool added to
each bottle.
Vibrios do not multiply but
remain viable for
several weeks.
 Cary-Blair medium –
Buffered solution of Sodium
chloride, Sodium
thioglycollate. Disodium
phosphate and Calcium
chloride at pH 8.4 The
medium is suitable for
Salmonella, Shigella as well
as V. cholerae.
Enrichment media
• Alkaline peptone water at pH 8.6
• Monsur’s taurocholate tellurite peptone
water at pH 9.2
• Both these are good transport as well as
enrichment media
Plating media
• Alkaline Bile Salt Agar (BSA) –
– pH 8.2, widely used. Colonies like on Nutrient agar.
• Monsur’s Gelatin Taurocholate Trypticase
Tellurite Agar (GTTA) –
– Small translucent colonies with greyish black centre
and turbid halo. The colonies become 3-4 mm in 48
hrs.
• Thiosulphate Citrate Bile Salt Sucrose (TCBS)
medium –
– Widely used. Yellow colonies turning green on further
incubation.
String test
• Vibrio colonies identified by string test.
Loopful of growth mixed with a drop of
Sod. Deoxycholate on a slide.
• Positive test indicated by
– Loss of turbidity
– Mucoid appearance
– Formation of ‘string’ as the loop is drawn
away from suspension
Biochemical reactions
• Catalase & Oxisase tests are
positive.
• Glucose, Mannitol, Maltose,
Sucrose fermented with acid
production.
• Lactose fermented late.
• Indole formed & nitrates
reduced to nitrites. These two
give cholera red reaction. Few
drops of Conc. H2SO4 added to
24 hr peptone water culture.
Development of reddish pink
colour due to production of
nitroso-indole indicates
presence of V. cholerae.
• Methyl Red & urease tests
negative.
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Catalase
Oxidase
G
M
M
S
L
I
MR
U
Cholera red
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A
A
A
A
Late A
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Resistance
• Susceptible to heat, drying & acids.
• Can survive for 2-3 days on linen in dry
condition.
• Survive in tap water for 30 days, cold
storage food, for 30 days.
• El Tor strains survive better than classical
strains
• Susceptible to disinfectants including
chlorine.
Gardener & Venkatraman’s Classification
Vibrio
Gp A
Cholera vibrios
Gp B
Biochem & Ag
heterogenous
Biochem similarities; Common H Ag
V. cholerae
Serogroups
O1
Non – 01
Currently 0-139
Classical
Ogawa
Inaba
El Tor
Biotypes
Hikojima
Serotypes
Classical Cholera Vs El Tor Vibrio
Test
Classical cholera
El Tor vibrio
Hemolysis
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Voges Proskauer
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Chick erythrocyte
agglutination
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Polymixin B sensitivity@
+
-
Group IV phage
susceptibility
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El Tor phage V
susceptibility
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@ 50 iu disc
O serotypes of Cholera vibrios
SEROTYPE
O ANTIGENS
Ogawa
AB
Inaba
AC
Hikojima
ABC
Transmission - V. cholerae
feces
water
– fresh
– salt
food
Cholera toxin (CT) Choleragen
• B binds to gangliosides
• provides channel for A
• A catalyses ADP-ribosylation
–regulator complex
– activates adenylate cyclase
CT determined by chromosomal gene.
Each molecule has 1 A & 5 B subunits
A – Active
B - Binding
A1 – Active
A2 - Linking
B attaches to GM1 ganglioside receptor of eneterocyte
Allowing entry of A subunit in enterocyte
Splits into A1 & A2
allows only biologically active A1 to be
linked with B
Prolonged activation of cellular
adenylate cyclate and accumulation of
cAMP
Outpouring of large qty of water +
electrolytes + Carbonates + K
Diarrhea
Cholera disease
• Massive fluid and electrolyte loss. Upto 20
litres per day.
• Leads to extracellular volume loss,
hemoconcentration, hypokalemia, base
deficit acidosis, shock.
• Leads to Muscular cramps, renal failure,
pulmonary edema, cardiac arrhythmias
and paralytic ileus.
• No fever, no WBCs in stool
Brackish water (less commonly brack water) is
water that has more salinity than fresh water, but not
as much as seawater.
Laboratory Diagnosis
• Specimen collection –
– Stool collected in acute stage before
administration of antibiotic
– Best collected by No. 26 or 28 rectal catheter
– Rectal swabs may be used particularly in
convalescent patients.
– Grossly – “Rice water stool”
– Not suitable
• Specimen from pans
• Vomitus
Transportation
• Whenever possible, specimen plated at bedside and
inoculated plates sent to the lab.
• Preserve at 4 C or in holding medium whenever delay is
more than few hours
 Cary – Blair medium
 VR fluid
 If delay is only few hours, transport medium can be used
 Monsur’s medium
 Alkaline peptone water
 If transport media are not available, filter paper strips can
be soaked in stool sample, sealed in plastic envelopes
and sent to lab.
Processing of specimens
• Direct microscopy –
– Not too reliabile
• Rapid diagnosis –
– Demonstration of characteristic darting motility
coupled with immobilization by specific antiserum.
• Enrichment media specimens –
– Incubate for 6-8 hrs inclusive of transit time followed
by streaking in selective & non-selective media.
– Direct plating can also be done before enrichment.
– Plating media used
• BSA
• MacConkey’s
• TCBS
Processing of specimens
• After overnight incubation, suspicious
colonies should be tested for Ag detection
using antisera.
• International reference centre located at
National Institute of Cholera & Enteric
Diseases (NICED), Calcutta, India.
Testing water samples for Vibrios
• Enrichment method –
– 900 ml water added to 100 ml tenfold
concentrated peptone water at pH 9.2,
incubated at 37 C for 6-8 hrs. A second
enrichment is done before plating on solid
media.
• Filtration technique –
– Filter water through Millipore membrane filter,
place it directly over the surface of selective
medium
Cholera -therapy
• massive secretion of ions/water into
gut lumen
• dehydration and death
• therapy
– fluid replacement
– antibiotic therapy
• vaccination
– partially effective
– not generally used
– international travelers
Prophylaxis & Treatment
• Prophylaxis
– Injectable killed vaccine gives 50-60% protection.
– Live oral vaccine using Texas star strain under study.
• Treatment –
– Prompt & adequate replacement of water and
electrolytes
• Oral or Intravenously
• Oral tetracycline reduces period of excretion of vibrio and
also fluid requirement.
Halophilic vibrios
• Have high requirement of salt, natural
habitat is sea water, marine life.
• Disease producing halophilic vibrios
– V. parahemolyticus
– V. alginolyticus
– V. vulnificus
Vibrio parahemolyticus
• Raw sea-food
• Grows best in high salt
• Food poisoning due to sea fish
•Out breaks in Japan and other countries
Vibrio alginolyticus
Associated with eye, ear,
wound infection in humans
exposed to sea water
Vibrio vulnificus
1. Wound infections in humans exposed to sea water
2. Consumption of infected oysters
penetration of gut
Severe septicemias
High mortality
Key Words
•
•
•
•
•
•
Vibrio cholerae
Late lactose fermenter
Oxidase positive
String test
Hemodigestion
Choleragen (cholera
toxin)
• Rice water stool
• Fish in Stream
appearance
•
•
•
•
•
•
•
•
Darting motility
Cholera red reaction
TCBS
Transport media
Classical Vs El Tor
Endemic
Severe Dehydration
Halophilic vibrios