Detection of Vibrio cholerae and Vibrio parahaemolyticus by molecular and culture based methods from source water to household container-stored water at the point-of-use in South African rural communities

Abstract

Detection methods for Vibrio cholerae and Vibrio parahaemolyticus which included the culture based approach with polymerase chain reaction (PCR) confirmation, PCR detection without enrichment and PCR with a pre-enrichment were developed and their performance evaluated. PCR assays targeted the SodB (V. cholerae species), Flae (V. parahaemolyticus species), 16S rRNA (Vibrio and Enterobacteriacea species) genes (Multiplex 1) and V. cholerae O1 and V. cholerae O139 rfb genes, ctxA (cholera toxin) gene and 16S rRNA gene (Multiplex 2). These methods were used to determine the occurrence of selected Vibrios in source water as well as in household container-stored water. The combination of filtration, enrichment and PCR method provided a sensitive and specific method for the detection of selected Vibrios in water samples. The PCR with a pre-enrichment method detected as few as 4-10 cfu/100 mL of selected Vibrios and PCR detection without the enrichment method detected as few as 40-100 cfu/100 mL of selected Vibrios. The inclusion of an enrichment period allows detection of culturable bacteria. As an application of the developed methods, V. cholerae and V. parahaemolyticus were detected in the source water used by the population and in the water-storage containers. The results indicate that Vibrio species in the containers could have originated from the source water and survive in biofilms inside the containers.