A High-Throughput Microfluidic qPCR Platform for the Simultaneous Quantification of Fecal Indicator Bacteria, Microbial Source Tracking Markers, and Pathogens in Surface Waters and Municipal Wastewater

Abstract

The contamination of water with enteric pathogens is known to result in adverse human health outcomes. Untreated recreational waters may carry an increased risk of gastrointestinal illnesses for water recreators, especially if waters are contaminated with sewage. Current recreational water quality standards are based on outdated methods that cannot pinpoint fecal sources of pollution or accurately predict the risk of pathogen infection. There is a critical need to elucidate the correlations between fecal pollution sources and pathogens in recreational waters. This study expanded upon previous work to develop a novel Microfluidic qPCR (MFQPCR) platform for the simultaneous detection of microbial source tracking (MST) markers, fecal indicator bacteria (FIB), and pathogens in a single water sample. Eighty previously validated TaqMan probe-based assays were applied for use in 96.96 and 192.24 GE chips from Fluidigm. Results showed that 71 of these assays were capable of quantifying genes within acceptable qPCR quality criteria with uniform concentrations and cycling conditions. Multiple host-specific MST markers, FIB, and pathogens were quantified in wastewater influent and effluent, surface waters, and fecal samples. While virulence factor genes and pathogenic species were infrequently detected, a few significant relationships were observed between pathogens and human MST markers. By these associations, linear regression models were generated to estimate the occurrence of Mycobacteria spp. from MST marker quantities and additional physicochemical parameters. This MFQPCR technology is a promising tool for amassing comprehensive pathogen datasets. Downstream applications of these data, including Quantitative Microbial Risk Assessment, may be highly impactful in the development of strategies for more accurate and timely water quality monitoring.