Application of metagenomic and molecular microbiology techniques to elucidate sources of fecal pollution and anthropogenic antibiotic resistance genes to surface water: a step towards a “One Health” approach to watershed management

The use of fecal indicator bacteria, such as Escherichia coli, is a widely established regulatory and monitoring practice to detect surface water contamination associated with fecal pollution. However, the detection or quantification of fecal indicator bacteria alone does not accurately inform the sources of fecal pollution. The development of molecular and metagenomic methods that target the DNA of microorganisms has resulted in a host of new tools for monitoring fecal pollution and its sources, as well as for understanding emerging microbial threats, such as antimicrobial resistance. Antimicrobial resistance is a critical “One Health” challenge presenting a high risk to humans, animals, and environment health, causing over 2.8 million illnesses and 35 thousand deaths annually in the U.S. Anthropogenic fecal pollution is a major source of contamination of high health risk antibiotic resistance genes (ARGs) to water sources, including recreational waters and drinking water sources. The overall goal of this research was to develop and demonstrate a watershed-focused approach for monitoring anthropogenic fecal pollution and associated antibiotic resistance. The following specific objectives were addressed, using the West Run Watershed (Morgantown, WV) as a model study watershed: 1) differentiate sources of fecal contamination and their impact on water quality using microbial source tracking (MST) approaches, 2) identify ARG monitoring targets that are strong indicators of overall antibiotic resistance and associated human health risk, and 3) assess the loading of ARG monitoring targets and identify sources of ARG-containing fecal pollution in surface water. In a study of the mixed land-use West Run Watershed, coupled monitoring of the FIB E. coli and host-specific MST markers was applied to effectively differentiate sources and quantify fecal contamination. Human-associated MST markers were primarily found at developed sites both with and without access to centralized sewage collection facilities, indicating septic or sewer failure as the major source of fecal contamination in the surface water. Further, environmental factors were found to significantly impact fecal marker loading. For example, low pH conditions associated with acid mine drainage sources in the watershed may have contributed to inactivation or loss of culturability in E. coli, while human fecal markers remained detectable. To establish a framework for environmental monitoring of antimicrobial resistance in wastewater and impacted environments, metagenomic sequencing data was compiled from wastewater collected in 64 different countries. Sequencing data was analyzed based on ARG clinical relevance, geographic ubiquity, and associations with mobile genetic elements to identify four indicators for monitoring antimicrobial resistance: oqxA, ermB, sul1, and mexE, from high-health risk concern groups. Monitoring of these identified indicator ARGs in the surface water revealed that sewer or septic failure or illicit discharge in urban residential areas and agriculture were likely sources of fecal ARGs, as informed by MST. Finally, identifying the sources of fecal contamination containing ARGs and better understanding the impact of environmental factors through this research will help to develop sustainable and effective watershed management plans to mitigate downstream risks and promote a One Health approach to managing water quality