Vibrio Cholerae: Characteristics, Pathogenesis, and Lab Diagnosis

Cholera is an acute gut infection caused by ingesting food or water contaminated with the bacterium Vibrio cholerae O1 or O139.  Other serogroups of Vibrio cholerae may cause diarrheal disease and other infections.

• Vibrio parahaemolyticus is an important cause of enteritis.
• Characterized by copious (voluminous watery stools), painless, watery diarrhea often accompanied by vomiting
• Death occurs by dehydration (hypovolemic shock or acidosis) if left untreated.
• Cholera is preventable; up to 80% of cholera cases can be successfully managed with oral rehydration salts (ORS).

Characteristics of Vibrio cholerae

• Gram-negative curved bacilli (comma-shaped)
• Highly motile (darting type of motility) with a single polar flagellum (monotrichous)
• Oxidase-positive, catalase-positive
• Not a normal human flora
  • Primary habitat – brackish or marine water
  • Transmitted by the ingestion of seafood, contaminated  food, and water
• Ability to cause epidemics and pandemics
• Vibrios are sensitive to low pH and die rapidly in solution below pH 6 and tolerate alkaline media (pH 8.5)
• Antigen
  • Somatic O (>200 serotypes, agents of cholera O1, O139)
  • Flagellar H

Vibrio cholerae strains

V. cholerae is divided into serogroups according to the nature of its O cell wall antigen. Two serogroups of V. cholerae O1 and O139  cause outbreaks. V. cholerae O1 causes most outbreaks, while O139 is confined to South-East Asia. Non-O1 and non-O139 V. cholerae can cause mild diarrhea but do not cause epidemics.

The O1 organisms have two biotypes, called classic and El Tor, and three serotypes called Ogawa, Inaba, and Hikojima. Biotypes are based on differences in biochemical reactions, whereas serotypes are based on antigenic differences.

People at risk

The risk of cholera is highest in areas where basic infrastructure is unavailable, such as peri-urban slums and refugees with limited access to safe drinking water and proper waste disposal. People with low immunity – such as malnourished children or people living with HIV – are at a greater risk of death if infected.

Mechanism of action of cholera toxin

1. V cholerae toxin consists of two subunits, A (A1: the toxic moiety and A2) and B.
2. B subunit binds to the receptor GM1 ganglioside in the intestinal cell membrane and internalizes subunit A.
3. ADP-ribosylation of the G protein of Adenylate cyclase occurs; it inhibits the action of GTPase and activates the adenylate cyclase.
4. Activated adenylate cyclase catalyzes the transformation of ATP to cAMP.
5. Increased levels of cAMP stimulate the cells to secrete ions into the intestinal lumen actively.
6. To maintain osmotic stabilization, the cells then secrete fluid into the lumen.
7. Patients become hydrated and hypotensive rapidly as the fluid is drawn from an intravascular fluid store of the body.

Signs and Symptoms of cholera

• According to WHO, up to 75% of people infected with Vibrio cholerae O1 or O139 don’t develop any symptoms.
  • Bacteria may present in their feces for 7–14 days after infection and are shed back into the environment.
• Among people who develop symptoms,
  • 80% have mild or moderate symptoms,
  • while around 20% develop acute watery diarrhea (rice watery stools).
• The incubation period of 6-48 hours
• Begins with the abrupt onset of watery diarrhea which is followed by several diarrheal episodes which may be accompanied by vomiting.
• Muscle cramps may occur as water and electrolytes are lost from body tissues.
• Disease outcome depends upon the
  • extent of water and electrolyte loss and
  • adequacy of water and electrolyte repletion therapy.
• If untreated, death can occur from hypovolemic shock, metabolic acidosis, and uremia.

Laboratory Diagnosis

• Sample: Fresh diarrheal stool or stool collected in Cary Blair transport medium.
• Macroscopic examination: Rice watery stool with mucus flecks 
• The laboratory diagnosis of cholera is based on colony morphology, culture characteristics, biochemical reactions, and serological identification by slide agglutination using specific antisera. However, a presumptive diagnosis of cholera can be made by an immobilization test.

I) Immobilization test: A rapid presumptive diagnosis of cholera can be made by observing the wet smear for the distinctive rapid to and fro movement (darting movement) of V. cholerae O1 and O139 due to their single polar flagellum. The movement can be stopped by adding one drop of V. cholerae O1 and O139 antiserum respectively.

Hanging drop method for Vibrio cholerae is one of the easy but most popular tests used for the presumptive diagnosis.

II)  Oxidase test: on performing an oxidase test from a pure sub-culture on nutrient agar, a positive reaction is observed. Oxidase test should not be performed directly from TCBS or MacConkey agar as acidification of these media may result in false-negative oxidase tests.
(Note: However, Aeromonas spp also gives a positive oxidase test result so further confirmation is necessary by culture and serotyping)

Cultural characteristics

• Fresh stool can be directly plated on a non-selective medium like MacConkey agar and a selective medium such as Thiosulphate Citrate Bile Salt Sucrose (TCBS) agar.
• However, in the case of a rectal swab, the swab stick should be dipped in 10 ml of alkaline peptone water (APW) and incubate for 6-8 hours. After incubation, inoculation on solid media should be done only from the pellicle formed at the upper layer of the broth. (Note: The broth should not be shaken before plating)
• After 18-24 hrs of incubation at 37°C observe the colony morphology.
  • MacConkey agar:  Appearance of pale, non-lactose fermenting,1-2 mm in diameter, flat with a serrated margin.
  • TCBS: Button-shaped yellow colonies of 1-2 mm diameter.

Biochemical Reactions

Serological Reactions

• Pick up the colonies resembling V. cholerae and perform a slide agglutination test using polyvalent and serotype-specific antisera for V. cholerae O1; if negative, perform an agglutination test with O139 antiserum.
• Place two drops of normal saline on a slide side by side and emulsify colonies resembling V. cholerae from a nonselective medium on both.
• Add a drop of polyvalent O1 antisera to one of the suspensions and tilt the slide to and fro. Observe for agglutination within a minute.
• If positive, continue the same procedure with monospecific Ogawa and Inaba antisera. If a positive report is seen with Ogawa antisera, report it as Ogawa. Report it as ‘Inaba’ if a positive result is seen with Inaba antisera, but if positive with both antisera,  report it as Hikojima.
• If none of the above shows a positive reaction, perform an agglutination test with O139 antiserum.

Biotyping

• V. cholerae O1 can be further differentiated into two biotypes by the following tests.
• Differentiation of the Classical and El Tor biotypes of V. cholerae serogroup O1.

Key: S = Sensitive, R = Resistant, + = Positive, – =  Negative

References

1. Ojeda Rodriguez, J. A., & Kahwaji, C. I. (2022). Vibrio cholerae Infection. In StatPearls. StatPearls Publishing.
2. Faruque, S. M., Albert, M. J., & Mekalanos, J. J. (1998). Epidemiology, genetics, and ecology of toxigenic Vibrio cholerae. Microbiology and molecular biology reviews : MMBR, 62(4), 1301–1314. https://doi.org/10.1128/MMBR.62.4.1301-1314.1998
3. Yoon, S. H., & Waters, C. M. (2019). Vibrio cholerae. Trends in microbiology, 27(9), 806–807. https://doi.org/10.1016/j.tim.2019.03.005