1. CHOLERA Vibrio cholerae
Dr.Salma

2. Gram Negative Curved Bacilli
There are certain microorganisms that share this type
of morphology which is sometimes is described as
comma-shaped. The following m.o. are curved gram negative bacilli:
1. Vibrio.
2. Aeromonas.
3. Plesiomonas.
4. Campylobacter.
5. Helicobacter

3. Characteristics of Gram Negative Curved Bacilli:
These m.o. are:
1. Gram negative curved bacilli.
2. Motile by a single polar flagellum.
3. Many live in fresh water, and some in sea water (called marine m.o.).
4. Some of these m.o. can cause diarrhoea in humans.

4. Vibrio species (cont’d)
Gram stain
Curved design

7. Family: Vibrionacea Genus : Vibrio
Genus Vibrio is classified practically into 10 important species:
1. Vibrio cholerae : Causes epidemic and pandemic cholera.
2. Non-cholera Vibrios: Cause ear, wound, soft tissue, and extra-intestinal infections. The important species of this group of m.o. are:
V. mimicus, V.vulnificus, V. hollisae*, V. fluvialis*,
V. damsela*, V. furnissi*, V. alginolyticus, V. metschnikovii.
= Group F Vibrio.
3. Vibrio parahaemolyticus: Causes gastroenteritis, and
possibly extra-intestinal infections.

8. Species of Vibrio

9. Vibrio cholerae:
Pacini in 1854 obseved this microorganism, and Robet Koch in 1883 described it as a comma-shaped m.o.
Vibrios have 2 important antigens; H (shared), and O (specific) which is lipopolysaccharides. There are at least 139 O - antigen groups.
Vibrio cholerae of O group 1 (o1) and 139 (O139) cause the classical disease cholera, while the non-O1/ non-O139
V. cholerae cause cholera-like disease.
Both O1 and O 139 V.cholerae can be of two biotypes:
1. Classical
2. El-Tor
Then both biotypes can be subgrouped into serotypes
according to 3 antigenic factors (A, B, and C) :
Ogawa (A,B), Inaba (A,C), and Hikojima (A,B.C).

10. Cholera: Transmitted to humans via contaminated food and water
Part of normal flora of brackish water and estuaries
associated with algal blooms (plankton), which are influenced by the temperature of the water. 8
- lots of pictures
Human beings are also one of the reservoirs of the pathogenic form of Vibrio cholerae.”8

11. Historical Perspective:
First Pandemic
spread out of India to
Europe
spread from India -> Middle
East (reaching Turkey &
Persia) -> Far East
(Singapore, Japan, Philippines)
Second pandemic
Entered US through NY and New Orleans -> proceeded through entire country
PICTURE: Title: 'Hoping for the Spread of Cholera', c Caption: 'Hoping for the Spread of Cholera', c Lithograph featuring a caricature of an undertaker in hat and tailcoat, gleefully rubbing his hands in anticipation of increased business from a cholera epidemic. He is seen looking down at a paper reporting recent deaths. The first major cholera epidemic struck England in 1831, and was initially attributed to 'miasma' (foul vapours in the atmosphere). However, observations made by John Snow ( ) during the epidemics of 1848 and 1854 established the role of polluted water in the spread of the disease.
had unusual virulence1
disease was new to the area -> thought it was a new disease

12. Recent Cholera Pandemics
7th pandemic:
V. cholerae O1 biotype El Tor
Began in Asia in 1961
8th pandemic (??)
V. cholerae O139 Bengal
Began in India in 1992 and spread to Asia, Europe and U.S.

13. Cholera affected countries

14. Classification: O1 Antigen

15. Vibrio cholerae (O-1 and O-139):
These m.o. are highly aerobic, very actively motile (described as shooting stars or darting motility), and Pacini and Koch were able to detect these m.o. with the help of this type of motility. The best way to detect motility is by dark-field or contrast microscopy. After a prolonged culture, the m.o. become straight (similar to enteric bacilli). These m.o. can tolerate temprature of range

16. Biochemical characteristics of V. cholerae:
They ferment glucose, maltose, mannite, and sucrose with acid production only. These m.o. are respiratory (oxidase positive) and fermentative (catalase positive). They give positive cholera-red reaction on nitrate peptone medium by reducing nitrate to nitrite, and produce indol (+ve) on tryptophan medium. When the latter 2 tests are combined, red colour is also produced due to nitroso-indol when H2SO4 is added. The TSI shows A/A profile (yellow colour). The optimum pH for the growth of these m.o. is 7.0, but they can tolerate up to They are very sensitive to acidic pH (less than 6), therefore, they are quite susceptible to gastric juice (a major barrier against
V. cholerae). V. cholerae is similar to El-Tor, but it differs in being capsulated.

17. Culture of V. cholerae:
This m.o. can be cultivated on different media:
1. Alkaline Peptone Water (APW; Sea Water):
This medium is of pH 8.5 – 9.0 and used for the primary (first) isolation from clinical specimens. The m.o. grow forming a surface pellicle within 8 hours. Then, from this medium the m.o. can be sub- cultured on solid media (see below). Many m.o. can not tolerate such pH, but
Pseudomonas does.
2. Thiosulfate Citrate Bile Salt Sucrose Agar (TCBS):
The non-inoculated medium is olive green in colour, and contains an indicator called bromo-thymol blue.
V.Cholerae is sucrose- fermenter, and due to acid production, the pH drops and colour of the medium and colonies become yellow. Aeromonas can produce similar chages in this medium.

19. 3. Tellurite Taurocholate Gelatin Agar (TTGA):
Cont/…culture V. cholerae
3. Tellurite Taurocholate Gelatin Agar (TTGA):
On this medium the colonies show iridescence and are surrounded by cloudy zone of gelatin hydrolysis.
4. Meat Extract Agar:
On this medium the colonies appear translucent green to red bronzy in colour and in old cultures the colonies become opaque and rough.
5. Taurocholate Pepton Broth:
This medium can be used primary cultivation of the m.o., then sub-cultured on other media.
6. MacConkey’s Agar:
V.Cholerae colonies are of non-lactose fermenters.
7. Blood Agar:
El-Tor growth shows beta-haemolysis ( vesus the classical).
In general, the colonies of V. cholerae are convex, smooth, granular.

20. Practical procedure of cultivation of V. cholerae:.
1. Direct stool examination:
a. Motility: Dark field or contrast microscopy.
B. Microscopical exam.: mucous, epith. cells, and large numbers of m.o., but no pus cells are seen.
c. Fluorescent antibody staining.
2. 2 ml of faeces + 20 ml APW pH 8.0 – 8.5 are incubated for 5 hours.
At the same time, TCBS is heavily inoculated, and
left for overnight.
3. From the APW, TCBS and another APW are
inoculated and left either for 5 hours or overnight.
4. The colonies are subjected to further identification (see below):

21. Further Identification of V. cholerae: 1. String test:
A drop of 0.5% Na-deoxycholate is mixed with a colony (?); after 60 seconds, with a loop there will a thread of tenacious nature. If positive, it means V. cholerae.
2. Polyvalent anti-sera (anti-O1, anti- O139):
To confirm that the V. cholerae belongs to O-1 or
O-139, agglutination test with specific polyvalent sera and emulsified colonies is carried out.
3. Biochemical tests including cholera red reaction.
4. Differentiation between classical & El-Tor Biotypes:
See the following Table.
5. Serotyping: by specific antisera against A, B and A antigenic factors to determine Ogawa, Inaba & Hikojima.

22. Table: Differentiation between Classical and El-Tor biotypes of V
Table: Differentiation between Classical and El-Tor biotypes of V. cholerae.
tests
classical
El-Tor
Voges-Proskauer - +
Chicken RBC Agglutination
Polymyxin-B sensitivity (50 U)
Group IV cholera- phage sensitivity
Bet – Haemolysis
(On Blood Agar)

23. Factors of Pathogenicity of V. cholerae:
1. Adherance and Motility:
The actively motile m.o. can adhere to the intestinal wall (pathogenic), and vice versa for the non-motile (non-pathogenic). V.cholerae does not usually reach to the blood stream.
2. Enterotoxin:
It is an important factor in the pathogenicity of the m.o. and has a M.W. of Daltons. It is composed of of 6 light chains of 8000 Daltons each, and one heavy chain of Daltons which has 2 portions:
a. Toxic portion (A1): 98% protein, 1% lipid, and 1% CHO .
b. Stabilizer portion (A2).
The A1 portion increases the intracellular cAMP of the intestinal epithelium. The cAMP prevents reabsorption of Na (1 X), excretion of NaHCO3 (2 X),chloride and K (3-5 X). This leads to accumulation of water in large quantities (> 20 liters/day), acidosis, hypokalaemia, and death.

24. Cholera:
Cholera is an acute infectious disease characterized by severe vomiting and watery diarrhoea (rice water stool) resulting in dehydration and collapse. The disease is endemic in the Indian subcontinent and used to occur in world wide epidemics.
Infection occurs by oral route through contaminated food or drink and 10 trillions m.o. are required to cause cholera. Source of infection is a case or a carrier who excretes the organism in the stool. The incubation period is of 1-4 days. The organisms attach to the microvilli of the brush border of epithelial cells, where they multiply and liberate cholera enterotoxin which exerts the above mentioned effects. Convalescent carriers occur, but chronic carriers are rare. The stool contains mucous, epith. cells, large numbers of m.o. and no pus cells. .

25. Vibrio cholerae: Clinical Infections
Acute diarrheal disease
Spread through contaminated water, but also improperly preserved foods, including fish and seafood, milk, ice cream, and unpreserved meat
“Rice Water” stools which contains ~ vibrios/ gm.
Caused by cholera toxin or choleragen
Loss of electrolytes & water
Dehydration is usual cause of death
Man is the only host

26. Diagnosis of cholera: A. First case in non-endemic area:
Any comma-shaped motile m.o. detected in a stool sample should be thoroughly identified before giving a report as positive for cholera (see identification).
B. Second case during an epidemic:
In an established epidemic, cases can be diagnosed by direct stool examination mainly for motility, and immobilization by specific anti-O antisera. Then, continue the identification procedure. PCR assays have been developed for detection of cholera toxin genes.
Finally, mortality from untreated cholera may reach up to 25% - 50%.

27. Immunity against cholera:
The immunity against cholera could be achieved by:
1. Gastric juice: It is a major barrier against infection by V. cholerae.
2. Secretory IgA: Intestinal secretory IgA prevents the attachment of m.o. to the intestinal wall.
3. Vibriocidal IgG: Anti-toxic Ab, presents in the serum.
4. Solid immunity can stay for 3 years after infection.
5. Immunoprophylaxis:
a. Heat killed vaccine: Given in 2 subcutaneous doses. It stimulate anti-bacterial and not anti-toxic antibodies. Therefore, its role in protection is questionable.
b. Live oral vaccine: Protects against cholera enterotoxin, and is prepared by recombinant DNA tech.
c. O-139 : New vaccine using the Bengal strain (O139)

28. Identification of Vibrio cholreae
Gram stain:
Gram negative short rods, comma shaped, motile
Electron Micrograph of V cholerae
Rods with single polar flagella
Gram stain of Vibrio cholorae
Serology:
Diagnosis can be confirmed as well as serotyping done by agglutination
with specific antisera (O1, O139 antisera)

29. Identification of Vibrio Differentiation between SF & NSF by Growth on TCBS
Method:
TCBS agar is inoculated with tested organism recovered from alkaline peptone water using streak plate technique
Incubate the plate in incubator at 37 C/24 hrs
Results:
SF organism appears as yellow colonies (V. cholerae)
NSF organism appears as blue to green colonies (V. parahaemolyticus)
Flame & Cool 1 2 3 4 5

30. Treatment and control of cholera:
1. Intravenous fluid and electrolytes replacement: This is the most important part of the therapy.
2. Oral anti-microbial drugs: Have a secondary role in the treatment. Tetracyclines are the most effective drugs.
3. Control: The main control measures are:
a. Community and personal hygiene.
b. Isolation of cases and proper disposal of sewage.
c. Chemoprophylaxis by using tetracyclines for
exposed persons.
d. Immunoprophylaxis; see above (immunity).

31. Vibrio parahaemolyticus
These are gram negative, motile, curved, and
non-sucrose bacilli. They are halophilic (salt –loving) in tolerating 2-11 % NaCl depending on their biotypes. These are marine m.o., and cause diarrhoea in some countries with sea water as Japan. There are reports that these m.o. are detected in south Iraq (Basrah).
The diarrhoea is usually a self limiting condition and may be associated with constitutional symptoms as nasea, vomiting and fever. However, some times broad spectrum anti-microbial may be required as
co-trimoxazole.

32. Vibrio vulnificus
Found in marine environments along all coasts in the U.S.
Two categories of infections
Primary septicemia following consumption of contaminated shellfish, especially raw oysters; patients with liver dysfunction that results in increased levels of iron are predisposed
Wound infections following traumatic aquatic wound

33. Thank you