Development of Multiplex RT qPCR Assays for Simultaneous Detection and Quantification of Faecal Indicator Bacteria in Bathing Recreational Waters

Abstract

In this study, we designed and validated in silico and experimentally a rapid, sensitive, and specific multiplex RT qPCR for the detection and quantification of faecal indicator bacteria (FIB) used as microbiological references in marine bathing water regulations (Escherichia coli and intestinal enterococci). The 16S rRNA gene was used to quantify group-specific enterococci and Escherichia/Shigella and species-specific such as Enterococcus faecalis and E. faecium. Additionally, a ybbW gene encoding allantoin transporter protein was used to detect E. coli. An assessment of marine coastal systems (i.e., marine water and sediment) revealed that intestinal enterococci were the predominant group compared to Escherichia/Shigella. The low contribution of E. faecalis to the intestinal enterococci group was reported. As E. faecalis and E. faecium were reported at low concentrations, it is assumed that other enterococci of faecal origin are contributing to the high gene copy number of this group-specific enterococci. Moreover, low 16S rRNA gene copy numbers with respect to E. faecalis and E. faecium were reported in seawater compared to marine sediment. We conclude that marine sediments can affect the quantification of FIBs included in bathing water regulations. Valuing the quality of the marine coastal system through sediment monitoring is recommended.