Vibrio cholerae

 

Vibrio cholerae

Vibrio cholerae is the most important species that causes cholera, a severe epidemic diarrheal disease. Dehydration and death can occur rapidly within hours of infection. Vibrio cholerae belongs to the family Vibrionaceae. Members of this family are aerobes and facultative anaerobes, nonsporing, and positive for catalase and oxidase. They reduce nitrates to nitrites and can grow on ordinary media. Vibrio species are oxidase-positive, Gram-negative curved bacilli that are motile by the presence of a polar flagellum. The name "vibrio" comes from their characteristic vibratory motility. They are asporogenous and noncapsulated.

Properties of the Bacteria

• Morphology:
• V. cholerae are Gram-negative bacilli with rounded or slightly pointed ends, measuring 1–3 µm in length and 0.5–0.8 µm in diameter.
• They are typically comma-shaped. S-shaped or spiral forms may be seen.
• They can be frequently pleomorphic in old cultures.
• In stained films of mucous flakes from cholera cases, they are typically arranged in parallel rows, described as "fish in stream" appearance.
• They are actively motile by a single polar flagellum, showing a darting type of motility, appearing as a "swarm of gnats" under the microscope.
• They are nonsporing, noncapsulated, and nonacid-fast.
• Culture:
• V. cholerae is strongly aerobic and grows best under aerobic conditions, with scanty and slow growth under anaerobic conditions.
• It grows within a temperature range of 16–40°C (optimum 37°C).
• It grows better in an alkaline medium (pH 7.4–9.6, optimum 8.2).
• Sodium chloride (0.5–1%) is required for optimal growth, although it can grow in the absence of salt, unlike other halophilic bacteria. High concentrations (5% and above) are inhibitory.
• It grows well on a wide variety of media, including nonselective (nutrient agar, MacConkey agar, blood agar, gelatin agar, peptone water) and special media (transport, enrichment, and selective media).
• Nonselective media: On nutrient agar, they produce moist, translucent colonies with a bluish or greenish tinge after overnight incubation. Colonies are about 1–2 mm in diameter and emit a distinctive odor. On MacConkey agar, colonies are initially colorless but become reddish on prolonged incubation due to late lactose fermentation. V. cholerae biotype Eltor produces hemolytic colonies on blood agar, while biotype Classical strains show greenish discoloration that later clears. In gelatin stab culture, they produce infundibuliform or napiform liquefaction. In peptone water, growth forms a fine surface pellicle in 6–9 hours.
• Transport media: Cary–Blair medium (solid, buffered alkaline pH 8) is suitable for Vibrio spp., as well as Salmonella and Shigella spp.. Alkaline peptone water (APW) and Monsur’s taurocholate tellurite peptone water are liquid enrichment media also used as transport media due to their high pH.
• Enrichment media: Liquid media with a high pH (Alkaline peptone water, Monsur’s taurocholate tellurite peptone water) suppress other intestinal bacteria and favor V. cholerae growth.
• Selective media: Useful for isolation from feces, including thiosulfate citrate bile salt sucrose (TCBS) medium (pH 8.6), Monsur’s gelatin taurocholate trypticase tellurite agar (GTTA) medium, and alkaline bile salt agar (BSA). TCBS medium contains thiosulfate, citrate, bile salts, sucrose, and bromothymol blue. V. cholerae produces large, yellow convex colonies due to sucrose fermentation and acid production. V. parahaemolyticus produces blue-green colonies on TCBS. Alkaline BSA (pH 8.2) colonies are similar to those on nutrient agar.
• Biochemical reactions:
• It is catalase positive and oxidase positive.
• It ferments sugars with acid only (no gas). It ferments glucose, sucrose, maltose, mannitol, and mannose. It is a late lactose fermenter.
• It does not ferment arabinose, inositol, and dulcitol.
• It forms indole and reduces nitrates to nitrites.
• It shows a positive cholera red reaction (reddish-pink color in peptone water) due to nitroso indole formation (from indole and nitrite) after adding concentrated sulfuric acid to a 24-hour peptone water culture.
• It is methyl red positive and urease test negative.
• It liquefies gelatin and decarboxylates lysine and ornithine, but not arginine.
• Biotypes Classical and Eltor show variable Voges–Proskauer reaction, hemolysis of sheep RBCs, and hemagglutination of chick RBCs.
• Other properties:
• It is most susceptible to heat, drying, acids, and common disinfectants. Killed at 56°C for 30 minutes and rapidly in gastric juice of normal acidity.
• It is resistant to high alkalinity.
• It remains viable for 1–2 weeks in fresh seawater and up to 30 days in pure tap water. Survival on fruits is 1–5 days at room temperature and a week in the refrigerator. Survival is shorter in grossly contaminated water like river Ganga water, possibly due to bacteriophages. It survives for several days in untreated human excreta.

Classification and Typing

• The genus Vibrio has at least 33 species, with 12 implicated in human infections. V. cholerae, V. parahaemolyticus, and V. vulnificus are the most prominent. Other species can cause gastroenteritis and wound infections.
• V. cholerae O1 has two biotypes: Classical and Eltor. They differ in hemolysis of sheep RBCs, agglutination of chick erythrocytes, Voges–Proskauer test, polymyxin B sensitivity, and susceptibility to specific phages. Biotype Eltor is generally associated with less severe illness but higher carrier rates and has largely replaced the Classical biotype in many regions, although Classical strains have resurged in some areas like Bangladesh.
• V. cholerae O1 is further divided into serotypes based on O antigens: Ogawa (AB), Inaba (AC), and Hikojima (ABC).
• Phage typing can further subdivide V. cholerae O1 biotypes Classical (5 types based on 4 phages) and Eltor (6 types based on 5 phages).
• Heiberg (1934) classified Vibrios into six groups based on fermentation of mannose, sucrose, and arabinose; V. cholerae belongs to group I.
• Serotypes from O2 to O139 are known as noncholera vibrios (nonagglutinable vibrios or V. cholerae non-O1). V. cholerae O139 is a notable noncholera vibrio that can cause epidemic cholera and can infect individuals previously infected with O1 strains. Other noncholera vibrios can cause mild to severe diarrheal disease and extraintestinal infections.

Pathogenesis and Immunity

• Cholera is a toxin-mediated disease, with cholera toxin (CTx) being the key virulence factor.
• Virulence factors of V. cholerae include:
• Cholera toxin (CTx): Inhibits absorption of sodium and chloride and causes hypersecretion of water and electrolytes by activating adenylate cyclase and overproducing cAMP. It is structurally and functionally similar to the heat-labile enterotoxin of Escherichia coli.
• Toxin coregulated pilus (TCP): Helps in adherence to mucosal cells of the intestine.
• Accessory colonization factor (ACF): Helps in adhesion to the intestinal mucosa.
• Hemagglutination-protease (hap; mucinase): Induces intestinal inflammation and helps release vibrios from the mucosa to the intestinal lumen.
• Neuraminidase: Increases toxin receptors for V. cholerae.
• Siderophores: Cause sequestration of iron.
• Upon reaching the small intestine, V. cholerae uses motility, chemokines, and enzymes (hemagglutinin and protease) to reach the mucous layer. Hemagglutinin and protease break down mucin and fibronectin. Bacteria then adhere to the intestinal wall, facilitated by TCP. The production of CTx, TCP, and other virulence factors is regulated by Tox R gene products.
• CTx binds to receptors on intestinal epithelial cells, leading to disruption of sodium transport and activation of chloride transport, causing sodium chloride accumulation in the intestinal lumen. This results in a large secretion of water, overcoming absorptive capacity and causing diarrhea.
• V. cholerae O139 has a similar pathogenic mechanism but produces a unique O139 LPS and an immunologically related O antigen capsule, enhancing virulence and resistance to human serum.
• Natural infection with V. cholerae O1 biotype Classical usually protects against subsequent infection with both Classical and Eltor strains. However, infection with biotype Eltor does not always protect against biotype Classical.
• Live oral vaccines induce local immunity by producing IgA antibodies in the gut, but this immunity is short-lived.

Epidemiology

• V. cholerae is found naturally in estuary and marine environments worldwide, with marine ecosystems associated with plankton being the primary habitat.
• The number of bacteria in contaminated water increases during warmer months.
• It is never found in normal humans; in infected individuals, it inhabits the small intestine.
• Reservoirs include the aquatic environment and infected humans. Transmission occurs through ingestion of contaminated water and food, especially seafood. Achlorhydric individuals are more vulnerable to infection, and even small doses can be infective.
• Cholera remains a major health problem in many parts of the world, including the Indian subcontinent and sub-Saharan Africa, occurring as endemic, epidemic, or pandemic disease.
• The seventh pandemic was caused by V. cholerae biotype Eltor.
• In 1992, V. cholerae O139 emerged in India as a cause of epidemic cholera.

Laboratory Diagnosis

• Specimens: Stool is the primary specimen.
• Microscopy: Dark-field microscopy can demonstrate characteristic motility and its inhibition by antisera, a rapid method for examining stool or enriched cultures. Direct immunofluorescence is another rapid method for demonstrating vibrios in stool. Gram-stained stool smears are not recommended for diagnosis.
• Culture: Specimens in holding media are inoculated into enrichment media (alkaline peptone water) for 6-8 hours, then onto selective (TCBS, GTTA) and nonselective media (BSA, MacConkey agar, blood agar). Direct plating onto these media can also be done. Plates are incubated at 37°C overnight. V. cholerae produces characteristic yellow colonies on TCBS and nonlactose-fermenting colonies on MacConkey agar.
• Identification: Colonies are identified by a series of biochemical tests including oxidase test, utilization of amino acids (lysine, ornithine, arginine), fermentation of sugars, sheep cell hemolysis, chick cell agglutination, VP test, polymyxin B sensitivity, cholera red reaction, catalase test, and serotyping using specific polyvalent antisera.
• Serotyping: Suspected V. cholerae are tested by slide agglutination with specific O1 antisera. Positive results are followed by testing with Inaba and Ogawa sera for serotyping. Hikojima strains agglutinate with both. Colonies not agglutinating with O1 antisera are tested with H antisera and can be identified as non-O1 cholera vibrios. Non-O1 isolates are tested for O139 using specific antisera.
• Biotyping: V. cholerae O1 isolates are tested to differentiate between Classical and Eltor biotypes based on hemolysis of sheep RBCs, agglutination of chick RBCs, sensitivity to polymyxin B, VP test, and sensitivity to bacteriophages.
• Isolated strains for phage typing can be sent to reference centers.

Prevention and Control

• Preventive measures include general sanitation and hygiene, ensuring safe drinking water and proper food handling.
• Cholera vaccines are available.

Treatment

• Treatment involves prompt and adequate replacement of lost fluid and electrolytes. Antibiotics can also be used to reduce the duration and severity of the illness