Vibrio cholerae

Gram negative curved bacilli
• Presented by:
• Zeena makkie &
Zainab khalid.

Gram negative curved bacilli
• There are certain M.O that share this type of
morphology which is sometimes described as
coma-shaped
• The following M.O are gram negative curved
bacilli:
1. Vibrio.
2. Aeromonas.
3. Plesiomonas.
4. Campylobacter.
5. Helicobacter.

General charectarestics:
• Gram negative curved bacilli.
• Motile by a single polar flagellum.
• Live in fresh water &sea water.
• It can cause diarrhoea in human.

Family: vibrinaceae
genus :vibrio
Genus vibrio is classified 10 important
species:
1.Vibrio cholerae:it can cause epedic
&pandemic cholera.
2.Non cholera vibrios:it can cause
ear,wound,soft tissue infection&extra
intestinal infections

• The most important species are:
V.mimicus, V .vulnificus, V .hollisae, V
.fluvialis,V.damesla,V. frunissi,V.
alginolyticus,V. metschnikovii.
3. V.parahaemolyticus:Causes
gastroenteritis,&extrintestinal infections.

• John Snow (1813-
1858):
• Water borne
transmission of
Cholera (1855)
Causative Agent
Discovery

• Filippo Pacini (1812-
1883)
– 1854: Cholera reaches
Florence, Italy. Pacini
discovers causative agent
– Publishes “Microscopical
Observations and
Pathological Deductions on
Cholera”
– 1965: Bacterium named
Vibrio cholerae Pacini 1854
Discovery

• Robert Koch (1843-
1910)
• 1884: Rediscovers
Vibrio cholerae
Discovery

Vibrio cholerae
• Have 2 imp antigens;H(shared)&O spesific which
is LPS.
• There are at least 139 O-antigen groups.
• Only of O group 1(o1)&139(o139) cause classical
cholera disease.
• While non O1-O139 can cause cholera like
disease
• Both o1-o139 of 2 bio type:
1. Classical .
2. El-Tor.

Both bio types can be sub grouped into
3 biotypes:

Differentiation between classical
and EL-TOr biotypes of V.cholera
tests
Voges-proskauer
Chicken RBC
agglutination
Polymyxin-B
sensitivity (50 U)
Group IV choleraphage
sensitivity
Bet-haemolysis (on
blood agar)
classical
_
_
+
+
_
EL-Tor
+
+
_
_
+

Vibrio cholrae(O-1&O139)
• These m.o are highly aerobic.
• Very actevly motile (described as
darting(shooting) motility.
• The best way to detect motility is by dark
feiled microscopy or contrast microscopy.
• After prolonged culture these m.o become
straight .
• Can tolerate temp. of range 18-37.

Biochemical characteristics of
V. cholerae:
• They ferment glucose,maltose&succarose.
with acid production only.
• Oxidase +ve &catalase +ve.
• Give ve cholera red reaction on nitrate
pepton media by reducing nitrate to nitrite.
• Indol test +ve on tryptophan media when
the lator 2 test are combined red coulor
also appear due to nitroso-indol when
H2SO4 is added.

• TSI is A/A.
• The optemal ph of growth is 7 but they can
tolerate up to 8,5-9.
• They are sensitive to acidic PH less than 6
so they are quite suseptable to gastric
juice.
• V.cholerae o-139 is like El-Tor but it
differs by being capsulated.

Culture of V. cholerae
1. Alkaline peptone water media:
PH of this media is 8.5-9 .
Used for primary cultivation from clinical
specimens.
Growth on it can be detected as a surface
pellicle within 8 h.
Sub culture on solid media .

2. Thiosulfate Citerate Bile salt
Sucrose agar (TCBS)
• TCBS is the medium of choice for the isolation of V.
cholerae.
• easy to prepare, requires no autoclaving .
• it has a relatively short shelf life once prepared (3 to 5
days) unless plates are carefully protected against
drying.
• The non –inoculated m.o is olive green.
• bromo thymol blue is the indicator.
• V.cholerae is sucrose – fermenter.
• Asid production drop the ph & change colour to yellow.

Overnight colonies of V. cholerae on TCBS agar are
large (2-4 mm) and yellow because of the fermentation
of sucrose. They are characteristically round, smooth,
glistening, and slightly flattened.

3- Tullerite Taurochelate Gelatine
Agar(TTGA)
TTGA is a selective and differential agar specific for the
isolation of V. cholerae .
has a relatively long shelf life after preparation.
overnight colonies of V. cholerae on TTGA tend to be
smaller (1 to 2 mm) than those from the TCBS agar.
Potassium tellurite, which is added to the medium to
increase selectivity .
Overnight growth of V. cholerae on TTGA agar appears
as small opaque colonies with slightly dark centers
opaque zone around colonies which resembles a halo,
which is due to the production of the enzyme gelatinase,
can be intensified by brief (15- to 30-minute) refrigeration
of the plate

TTGA

4. Meat extract agar.
• The colonies appear translucent green to
red pronzy in colour.
• On old culture the colonies appearopaque
&rough

5. Taurocholate pepton broth
• This media can be used for primary
cultivation .
• Sub cultured on other medias can be
done.

6. MacConkey s agar.
• They are non – lactose fermenters.
• Overnight colonies of V. cholerae on MAC
tend to be small to moderately sized (1 to
3 mm) and usually appear as lactosenegative
.

MacConkey agar

Blood agar.
• El-tor growth show beta haemolysis

Practical procedure of cultevation
of V. cholerae
1. direct stool examenation:
• motility: dark field microscopy.
• Microscopical
examination:mucous,epith.,cells& large
numbers of m.o,but no pus cells are
seen.
• Fluresent antibody staining

• 2 ml of faeses + 20 ml APW ph 8-8.5 are
incubated for 5 h.
• At the same time the TCBS is heavily
inoculated & left overnight.
3. From the TCBS,APW,an another APW
are inoculated & left 5 h.

Furtheer identification of V.
1. string test:
cholerae:
• a drop of 0.5% Na-deoxycholate is mixed
with a colony after 60 seconds with aloop
there will be a thread of tenacious like
nature it is +ve test for V.cholerae.
2. Poly valent antisera(anti O1,anti O139):
agglutination test with specific
antisera&emulsified colonies is carried
out to .

String test

3. Biochemical test :
• including cholera red reaction.
4. Serotyping by specific antisera against
A,B antigenic factors to determine
Ogawa,Inaba&Hikojima.

FACTORS OF PATHOGENISITY
1. Adherance &motility:
the actively motile m.o can adher to the
the intestinal wall(pathogenic)& visaversa
for the non motile(non pathogenic).
V.cholerae dose not usually reach to the
blood stream.

• 2. Enterotoxine :
it has amolecular wt.of 84ooo daltons.
it is composed of 6 light chains of 8000
each,&one heavy chain of 28000 daltons
which has 2 portions:
A-toxic portion(A1):98% protein,1%lipid
1%CHO
B-stabilizer portion(A2)

Cholera toxine

• The A1portion increase the intracellular
cAMPof the intestinal epithelium.
• The cAMP prevents reabsorption of Na
ion, excretion of NaHCO3,Cl&K ions.
• This lead to accumulation of water in large
quantities(>20 liters/day),acidosis
,hypokalemia& death.

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