QT-AMP: Sequencing PCR amplicons from Quanti-Tray wells to analyze enterococci communities

Enterococcus is ubiquitous in human feces and has been adopted as a useful indicator of human fecal pollution in water. Although regular enterococci monitoring only examines their numbers, identifying human-specific Enterococcus species or genotypes could help discriminate human fecal contamination from other environmental sources. We documented a new approach to characterize enterococci using a high-throughput 16S rRNA gene amplicon sequencing platform from Quanti Trays after following the counting of the most probable numbers of enterococci. We named this method QT-AMP (Quanti-Tray-based amplicon sequencing). We tested surface water samples collected from three rivers in southwest Florida. We detected 11 Enterococcus species from 45 samples in 1.1 million sequence reads. The method detected three rare species and eight cosmopolitan species (Enterococcus faecalis, E. faecium, E. casseliflavus, E. hirae, E. mundtii, E. gallinarum, E. avium, and E. durans) which have been commonly documented in previous studies. The approximate detection level of QT-AMP was four orders of magnitude higher than regular 16S rRNA gene amplicon sequencing. The current Enterolert MPN method only provides quantitative information but now we can look into the relative abundance of Enterococci species composition by accompanying Illumina sequencing. This QT-AMP could be a useful tool to streamline the quantification and identification of enterococci and could be used in various water management projects and human health risk assessment.