Assessing the Efciency of Detection of Vibrio cholerae Genetic Determinants Through Waterbody Vibrioflora Monitoring System

Abstract

Objective of the study was to assess the effectiveness of PCR screening of Vibrio cholerae genetic determinants in samples from surface water reservoirs for optimization of the cholera microbiological monitoring system.Materials and methods. The study was carried out as a part of the vibrioflora monitoring in surface water bodies in Irkutsk city. The study design included: 1) PCR screening of V. cholerae genetic determinants in nutrient-enriched (1 % peptone water) samples from surface water reservoirs during the monitoring period (824 samples); 2) studying of the V. cholerae DNA accumulation dynamics applying PCR assay of the samples from surface water reservoirs during cultivation on the enriched media (16 samples in dynamics); 3) experimental study of the detected V. cholerae concentrations in samples from surface water reservoirs. Species-specifc (hlyA, toxR) and serogroup-specifc (wbeT, wbfR) V. cholerae determinants were indicated in PCR with hybridization-fluorescent and electrophoretic detection.Results and discussion. At the frst stage it was found that the proportion of the positive samples through PCR screening (33.9 %) exceeded the percentage of the positive samples in bacteriological examination (19.3 %) (t=6.6; p<0,01). In the assessment of DNA accumulation dynamics, a decrease in the threshold cycle (Ct) by 1.2–5.2 times was recorded, indicating an increase in the V. cholerae concentration and proving the detection of genetic determinants of viable forms during PCR screening. An extended study of PCR-positive but bacteriologically negative samples made it possible to additionally isolate 4 V. cholerae cultures. However, there were no differences in the sensitivity of PCR screening and bacteriological analysis in the experiment with water samples artifcially contaminated with V. cholerae unlike the analysis of the enriched native samples. It can be determined by the metabolism and adaptation peculiarities of the microorganism in different environmental conditions. The results of the integrated study indicate the epidemiological effectiveness of PCR screening which gives grounds to recommend its application in monitoring studies of vibrioflora from environment after preliminary enrichment on liquid nutrient media in the work of federal, territorial, and regional laboratories.

Keywords

• vibrio cholerae
• epidemiological surveillance
• monitoring
• surface water bodies
• molecular-genetic analysis
• pcr screening