1. Gram negative rods VibrionaceaeVibrio

2. General charcters of Vibrionaceae Gram negative, curved, comma shaped bacilli Motile by single polar flagella Non spore forming Non capsulated Most vibrios have relatively simple growth factor requirements and grow well in alkaline pH Facultative anaerobes Vibrios are capable of both respiratory & fermentative metabolism i.e. O+/F+. Oxidase and catalase positive Natural inhabitants of aquatic environment

3. Species of Vibrio Vibrios Vibrio cholerae Cause Cholera Classical type V. cholerae El-Tor-type V. El-Tor V. parahaemolyticus cause Gastroenteritis Allied vibrios Saprophytic

4. Species of Vibrio V. cholerae is the causative agent of cholera –V. cholerae divided serologically into 6 groups based on somatic O-antigens –V. cholerae O1 and O139 are the most important agents that cause cholera –V. El-Tor is O1 serotype that cause disease similar to cholera but milder Vibrio parahaemolyticus is the cause of acute gastroenteritis following ingestion of contaminated sea-food such as raw fish V. cholerae & V. parahaemolyticus, are pathogens of human, produce diarrhea, but in ways those are entirely different. –V. parahaemolyticus is an invasive organism affecting the colon –V. cholerae is noninvasive, affecting the small intestine through secretion of an enterotoxin. Allied Vibrios are a large group of organisms; some of them are saprophytic while others cause disease in animals

5. Difference between O1 V. choleae Classical & El-Tor type Vibrio El-Tor V. cholerae Hemolytic Non hemolytic Hemolysis PositiveNegative Voges- Prosakauer ResistantSensitive Polymyxin B resistance

6. Cholera Cholera is toxin mediated, a severe diarrheal disease caused by V. cholerae O1 & 139 serotype and others. It is endemic in southern Asia (India, Pakistan, and Bangladesh). Transmission is by contaminated water or food through oral-fecal routs. Incubation period of the disease is 1-4 days. Sudden onset of intense vomiting and rice water diarrhea with rapid dehydration. The disease progresses from the first liquid stool to shock in 4-12 hours, with death following in 18 hours to several days.

7. Identification of V. cholerae Specimen and microscopical examination: Specimen and microscopical examination: –Rice watery stool or rectal swap collected in acute stage of disease –Dark-field microscopy of stool specimen from patients with cholera reveal large numbers of Vibrio (short, curved rods) with a characteristic motility that gives the appearance of shooting stars Culture: Culture: –Inoculation of rice water stool in enrichment media (alkaline peptone water, pH8.5), in which the organisms multiply rapidly and tend to form pellicle at the surface of the medium after 6-8 h at 37 C. –Subculture is made into Thiosulphate Citrate Bile Sucrose (TCBS) agar.

8.  TCBS medium is selective because  Also, contains bile salts  High conc. of thiosulfate and citrate & strong alkalinity of this medium (pH9)  TCBS medium is differential because  It contains sucrose  It contains bromothymol blue  Some species ferment sucrose & others not ferment  Sucrose fermenting Vibrio spp (V. cholerae) appears as yellow colonies  Sucrose non fermenting Vibrio spp (V. parahemolyticus) appears as blue to green colonies Alkaline pH: blue Neutral pH: green Acidic pH: yellow  Sucrose fermentation on TCBS is the gold standard in its identification kills most intestinal commensals Identification of V. cholerae Growth on TCBS  Principle

9. 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

10. Reaction on TCBS Yellow colonies of V. cholorae due to sucrose fermentation

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

12. Practical work Gram stain Culture on TCBS