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 degrees C to 35 degrees 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-antibody-direct 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.

FIG. 1.

Direct detection of V. cholerae O1 rfb and ctxA after immediate processing (IP) or delayed processing (DP) of samples of water (WA), the 64-μm plankton fraction (ZP), and the 20-μm plankton fraction (PP) by multiplex PCR. The first two gels from the left show the PCR results for sampling round 1, in which amplification of V. cholerae O1 rfb and ctxA occurred after immediate processing in the water sample and remained unchanged after delayed processing even after 20 h of preincubation at the ambient temperature. The third and fourth gels from the left show the PCR results for sampling round 2 after immediate processing and delayed processing, respectively. The amplification of the V. cholerae O1 rfb and ctxA genes that occurred in water and zooplankton samples but not in phytoplankton (20-μm fraction) samples after immediate processing, however, occurred in samples subjected to delayed processing (i.e., after 20 h of incubation at the ambient temperature). +Ve Con, positive control for V. cholerae O1 rfb and ctxA.