High-throughput diagnosis of human pathogens and fecal contamination in marine recreational water.

Waterborne pathogens and their associated diseases are major threats to public health, and surveillance of pathogens and identification of the sources of pollution are imperative for preventing infections. However, simultaneously quantitative detection of multiple pathogens and pollution sources in water environments is the major challenge. In this study, we developed and validated a highly sensitive (mostly >80%) and highly specific (>99%) high-throughput quantitative PCR (HT-qPCR) approach, which could simultaneously quantify 68 marker genes of 33 human pathogens and 23 fecal markers of 10 hosts. The HT-qPCR approach was then successfully used to investigate pathogens and fecal pollution in marine recreational water samples of Xiamen, China. Totally, seven pathogenic marker genes were found in 13 beach bathing waters, which targeted Acanthamoeba spp., Clostridium perfringens, enteropathogenic Escherichia coli, Klebsiella pneumoniae, Vibrio cholera/V. parahaemolyticus and Legionella spp.. Fecal markers from human and dog were the most frequently detected, indicating human and dog feces were the main contamination in the recreational waters. Nanopore sequencing of full-length 16S rRNA gene revealed that 28 potential human pathogens were detected and electrical conductivity, salinity, oxidation-reduction potential and dissolved oxygen were significantly correlated with the variation in bacterial community. Our results demonstrated that HT-qPCR approach had the potential rapid quantification of microbial contamination, providing useful data for assessment of microbial pathogen associated health risk and development of management practices to protect human health.