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...

Diarrhea continues to be one of the most common causes of morbidity and mortality among infants and children in the developing world (45). Escherichia coli, a gram-negative bacillus, is among the normal microbial flora that exists in the healthy human intestine (11). However, some of the E. coli serotypes cause diarrhea or food poisoning, mostly in infants and children. Following the recognition of certain pathotypes of E. coli as human diarrheagenic pathogens and the establishment of a serotyping system, serovars associated with diarrheal diseases have gained increasing interest (47). At coli strains revealed 47%, 30%, and 6% to belong to EPEC, ETEC, and EHEC pathotypes, respectively. The majority of the 160 strains tested were resistant to commonly used antimicrobial agents. Plasmid profiling showed a total of 17 different bands ranging from 1.3 to 40 kb. However, 35% of the strains did not contain any detectable plasmid, implying no correlation between plasmid and drug resistance. Although virulence gene profiling revealed 97 (61%) of the strains to harbor the gene encoding heat-stable enterotoxin (ST), 2 for the gene encoding Shiga toxin (Stx), and none for the gene for heat-labile enterotoxin (LT), serotype-based pathotyping of E. coli was not fully supported by this gene profiling. A dendrogram derived from the PFGE patterns of 22 strains of three predominant serogroups indicated two major clusters, one containing mainly serogroup O55 and the other O8. Three strains of identical PFGE profiles belonging to serogroup O55 were isolated from three distinct areas, which may be of epidemiological significance. Finally, it may be concluded that serotype-based pathotyping may be useful for E. coli strains of clinical origin; however, it is not precise enough for reliably identifying environmental strains as diarrheagenic. Phenotypic and Molecular Characteristics of