Applied Microbiology cases

Vibrio cholerae: Cholera
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Several students of a primary school in a village fell ill. All of them were admitted to local hospital following vomiting and diarrhea. Purging was effortless and the feces was non-odorant and rice-watery.

What is your diagnosis?
It is a case of gastoenteritis, probably cholera.

Which are the pathogens that can cause gastroenteritis?
Common bacteria that can cause gastroenteritis include vibrio cholerae and Enterotoxigenic E.coli (ETEC).

What is the pathogenesis of cholera?
Cholera is basically an enteric disease and the transmission involves feco-oral route. Vibio cholerae is transmitted following ingestion of contaminated food or water. Ingestion of large numbers of bacilli (approximately 106) can initiate infection in a susceptible person. Once they survive the gastric juices, they reach small intestine and colonize the epithelial lining and produce cholera toxin. They adhere to the intestinal wall mediated by toxin-coregulated pilus (TCP). Hemagglutinin/protease is a zinc-dependent protease, which cleaves the mucin
and fibronectin and may serve to facilitate the spread and excretion of vibrios within the intestine in stools by detaching them from the intestinal walls. Incubation period can be short (1-3 days). Cholera toxin is holotoxin consisting of A subunit and B subunit. The B subunit binds to specific ganglioside receptors (GM1) on the epithelial cells of small intestine and facilitates the entry of A subunit where it activates adenylate cyclase. Stimulation of adenylate cyclase causes an increased production of cAMP, which leads to hypersecretion of water and electrolytes into the lumen and inhibition of sodium reasborption.

What is the specimen collected and how is the condition diagnosed with the aid of laboratory?
Freshly passed feces is collected in a sterile wide mouthed container, if no feces is available rectal swabs may also be taken. In case of delay, the feces may be transported in transport medium such as Cary Blair medium or VR medium. The enrichment medium alkaline peptone water may also be used as transport medium. Direct gram stained smear and hanging drop preparation may be useful. Feces is inoculated on to MacConkey's agar, and on to selective media such as TCBS Agar and incubated at 37oC overnight. The sample may also be inoculated into enrichment broth such as alkaline peptone water and subcultured after 6 hours of incubation on to MacConkey's agar for subsequent overnight incubation.

What is your observation?
Hanging drop preparation or darkground microscopy shows actively motile (darting-type) bacilli. Vibrio can be detected by immobilization test, wherein addition of vibrio antisera renders the bacilli in feces non-motile. Gram stain of mucus flakes in feces shows several curved, gram negative bacilli. Pale (non-lactose fermenting), smooth, low convex, circular colonies is seen on MacConkey's agar. On further incubation, colonies turned pink due to late lactose fermentation. TCBS agar shows yellow coloured colonies. Surface pellicle is noticed in alkaline peptone water. Gram stained smear of the colony shows curved gram negative bacilli, hanging drop shows actively motile bacilli, catalase test and oxidase test are positive. Results of biochemical reactions are: positive indole test, negative urea hydrolysis, positive citrate utilization, positive MR test and negative VP test. TSI agar shows acid slant/acid butt with no gas or H2S. The isolate ferments mannose and sucrose with acid production. Other tests that help in identifying Vibrio are string test and nitrosoindole test. The isolate is identified as Vibrio cholerae.

How do you identify the species?
Slide agglutination with antiserum against H antigen confirms that it is a Vibrio sps. Further slide agglutination using O1 antiserum is used to identify V. cholerae O1 from NAG vibrios. Serotyping by slide agglutination is performed to identify Ogawa, Inaba or Hikojima serotypes. Biotyping is undertaken by performing chick RBC agglutination, Hemolysis, VP test, susceptibility to Polymyxin B and Phage IV to differentiate ElTor from Classical biotype.

Is it necessary to identify the serotype and biotype?
Identity of the serotype and biotype is not important for treatment.  Serotyping and biotyping is only epidemiologically significant.

Is antibiotic treatment necessary?
The mainstay of treatment is replacement of water and electrolytic, restoration of acid-base balance; role of antibiotics is secondary and serves to eradicate bacteria.

What are the complications of cholera?
After dehydration, hypoglycemia is the most common lethal complication of cholera in children. Acidosis in cholera is a result of bicarbonate loss in stools and accumulation of lactate. Acidemia occurs when respiratory compensation is unable to sustain a normal blood pH. Hypokalemia results from potassium loss in the stool.

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