Vibrio cholerae

Agent of disease
Vibrio cholerae is a comma shaped gram-negative flagellated bacterium which was first isolated, cultured and characterized by Robert Koch in Germany in 1883 (Figure 1). Bacteria invades the small intestine and produce enterotoxin which disrupts ion transport by intestinal epithelial cells. This interference results in the disease known as Cholera which remains one of the biggest killers in the world, especially in countries with poor sanitation and lack of clean water. From 150 serogroups, two groups O1 and O139 have been associated with epidemics and pandemics of cholera, both sharing identical biochemical and cultural characteristics.

Stela Ardelean Topics in Public Health Microbiology

Inoculation and Isolation

V.cholerae will grow on a number of commonly used agar media, but isolation from faecal specimens is readily achieved with specialized media: Alkaline peptone water (APW) may be used as an enrichment broth, useful when only a few bacteria are present, as is the case with asymptomatic carriers or convalescent patients. Thiosulfate citrate bile salts sucrose agar (TCBS) may be used as the selective agar medium but it is not used for direct testing with V.cholerae antisera. APW can be inoculated with liquid stool, faecal suspension or a rectal swab followed by 6 to 8 hours incubation at 35 to 37C. Following incubation TCBS subcultures should be prepared with one to two loopfuls of APW from the surface of the broth, since V.cholerae favour growing in this area. Isolation from TCBS selective agar TCBS plates are inoculated and incubated at 35 to 37C for 18 to 24 hours. Colonies suspicious for V.cholerae will have a diameter of 2 to 4 mm, shiny appearance and a yellow colour as a result of sucrose fermentation in the medium (Figure 3). Each type of sucrose-fermenting colony from the TCBS plate is selected and a heart infusion agar (HIA) slant is inoculated. The HIA slants are incubated at 35 to 37C for up to 24 hours although after 6 hours adequate growth may be observed to allow serologic tests to be conducted. Slide serology with O1 and O139 antisera is sufficient for a presumptive identification.

Screening Tests
A number of Screening tests are conducted for suspected V.cholerae, the main one being the Oxidase test, although further tests may be carried out. The Oxidase test is conducted with fresh growth from an HIA slant and a positive reaction will result immediately in dark purple bacteria (Figure 4). String test is performed by suspending 18 to 24 hours bacteria growth from HIA in an aqueous solution of 0.5% sodium deoxycholate, a positive test resulting in DNA being lysed resulting in a viscous solution (Figure 5). Kligler iron agar (KIA) and triple sugar iron agar (TSI) can also detect V.cholerae, the former resulting in red slant, the latter yellow acid (Figure 6).

Figure 1 Vibrio cholerae

Figure 2 Contaminated water

Figure 5 Positive string test with V.cholerae

Figure 6 Reactions of V.cholerae in KIA (left) and TSI (right)

Infection and Symptoms

The bacteria V.cholerae can be found in brackish waters and they thrive and proliferate in the sheets of blue-green algae but they are also found in faeces from severely infected individuals. Infection with bacteria is predominantly through the consumption of untreated water but also contaminated food. The incubation period varies from a few hours up to five days. Children are at high risk of the disease as well as immunocompromised individuals (Figure 2). Symptoms associated with V. cholerae infection are: watery diarrhoea which leads to dehydration, vomiting and exhaustion.

Treatment
Loss of fluid and minerals caused by severe diarrhoea requires prompt oral rehydration with salts and intravenous administration of fluids in severe cases.

References
1.http://www.cdc.gov/ncidod/dbmd/diseaseinfo/cholera/ch6.pdf 2.Faruque, S.M., Albert, M.J. and Mekalanos,J.J.(1998) Epidemiology, Genetics, and Ecology of ToxigenicVibrio cholerae. Microbiol. Mol. Biol. Rev. 62(4):1301-1314 Figure 3 Growth of V.cholerae on TCBS Figure 4 Positive test oxidase of V. cholerae