Effect of Transport at Ambient Temperature on Detection and Isolation of Vibrio cholerae from Environmental Samples

Abstract

It has long been assumed that prolonged holding of environmental samples at the ambient air temperature prior to bacteriological analysis is detrimental to isolation and detection of Vibrio cholerae, the causative agent of pandemic cholera. The present study was aimed at understanding the effect of transporting environmental samples at the ambient air temperature on isolation and enumeration of V. cholerae. For water and plankton samples held at ambient temperatures ranging from 31°C to 35°C for 20 h, the total counts did not increase significantly but the number of culturable V. cholerae increased significantly compared to samples processed within 1 h of collection, as measured by culture, acridine orange direct count, direct fluorescent-antibodydirect viable count (DFA-DVC), and multiplex PCR analyses. For total coliform counts, total bacterial counts, and DFA-DVC counts, the numbers did not increase significantly, but the culturable plate counts for V. cholerae increased significantly after samples were held at the ambient temperature during transport to the laboratory for analysis. An increase in the recovery of V. cholerae O1 and improved detection of V. cholerae O1 rfb and ctxA also occurred when samples were enriched after they were kept for 20 h at the ambient temperature during transport. Improved detection and isolation of toxigenic V. cholerae from freshwater ecosystems can be achieved by holding samples at the ambient temperature, an observation that has significant implications for tracking this pathogen in diverse aquatic environments.Cholera is an highly epidemic diarrheal disease which continues to devastate populations of many developing countries in which socio-economic conditions are poor, sanitary systems and public hygiene are rudimentary, and safe drinking water is limited. The conventional microbiological methods for enumerating and isolating Vibrio cholerae involve cultural, biochemical, and immunological assays (10). Several investigators have developed PCR and DNA probe-based techniques for direct detection of pathogenic Vibrio species (2,3,8). A variety of other techniques for direct detection of V. cholerae in clinical and environmental samples, including chromatographic assays (28), direct fluorescent-monoclonal-antibody methods (1, 11), and monoclonal antibody-based kits (4), have also been described. The laboratory protocols for isolation of V. cholerae from clinical specimens from cholera patients include both direct plating and broth enrichment methods (20). In the case of environmental samples, despite the many methods that have been devised, overnight enrichment in alkaline peptone broth with isolation on thiosulfate-citrate-bile salts-sucrose (TCBS) medium immediately after collection is most frequently employed (5, 23).In Bangladesh, cholera epidemics are recurrent. However, the lack of laboratory facilities in remote areas of the country require samples to be transported from the site of collection to a central laboratory before processing, and road links are poor. In most c...

Occurrence of Vibrio cholerae in fish and water from a reservoir and a neighboring channel in Ouagadougou, Burkina Faso

“…The absence of V. cholerae O1 in our samples may be due to the high prevalence of non-O1/non-O139 strains, which are believed to be indigenous members of the aquatic ecosystem and which can prevent isolation of the truly pathogenic strains [14]. Holding samples from freshwater ecosystems at an ambient temperature ranging from 31°C to 35°C for 20 hours before processing has been found to selectively increase the number of culturable vibrios [15].…”

Environmental Surveillance for Toxigenic Vibrio cholerae in Surface Waters of Haiti

“…A 24-hour holding time has been shown to be beneficial for V. cholerae culture. 15 Using multiple sample collection and analytical methods, toxigenic V. cholerae O1 was detected in Haitian water sources more than 2 years after the start of the cholera epidemic. The association of V. cholerae ctxA gene detections with higher E. coli concentrations and turbidity indicate that V. cholerae contamination may have been associated with inadequate sanitation and fecal runoff into water bodies.…”

“…Water samples for V. cholerae testing were held in a cooler at ambient temperature for 24 hours to improve detection by culture. 15 Some samples were held for 48 hours before processing as a result of logistical constraints.…”

Role of Zooplankton Diversity in Vibrio cholerae Population Dynamics and in the Incidence of Cholera in the Bangladesh Sundarbans

“…The two areas under study were reported previously by Alam et al (1,2). Briefly, Bakerganj is located in the southern district of Barisal, about 70 km north of the coast of the Bay of Bengal and approximately 300 km southwest of Dhaka, the capital city of Bangladesh (Fig.…”

“…Also, during the process of filtration, 500 ml of filtrate from the 20-m net was collected and analyzed for planktonic (unattached, free-living) bacteria. Both water and plankton samples were transported at ambient temperature to the International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR, B), Dhaka, and processed within 12 to 16 h of collection (2).…”