Metagenomics-Based Environmental Monitoring of Antibiotic Resistance: Towards Standardization

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Virginia Tech


Antibiotic resistance (AR) is a critical and looming threat to human health that requires action across the One Health continuum (humans, animals, environment). Coordinated surveillance within the environmental sector is largely underdeveloped in current National Action Plans to combat the spread of AR, and a lack of effective study approaches and standard analytical methods have led to a dearth of impactful environmental monitoring data on the prevalence and risk of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in aquatic environments. In this dissertation, integrated surveillance approaches of surface water and wastewater systems are demonstrated, and efforts are made towards standardizing both metagenomic- and culture-based techniques for globally comparable environmental monitoring. A field study of differentially-impacted watersheds on the island of Puerto Rico post-Hurricane Maria demonstrated the effectiveness of metagenomics in defining direct impact of anthropogenic stress and human fecal contamination on the proliferation of ARGs in riverine systems. The contribution of treated wastewater effluents to the dissemination of highly mobile and clinically-relevant ARGs and their connection to local clinical settings was also revealed. At the international scale, a transect of conventional activated sludge wastewater treatment plants (WWTPs), representing both US/European and Asian regions, were found to significantly attenuate ARG abundance through the removal of total bacterial load and human fecal indicators, regardless of influent ARG compositions. Strong structural symmetry between microbiome and ARG compositions through successional treatment stages suggested that horizontal gene transfer plays a relatively minor role in actively shaping resistomes during treatment. Risk assessment models, however, indicated high-priority plasmid-borne ARGs in final treated effluents discharged around the world, indicating potentially increased transmission risks in downstream environments. Advancements were also made toward standardizing methods for the generation of globally representative and comparable metagenomic- and culture-based AR monitoring data via two comprehensive and critical literature reviews. The first review provides guidance in next-generation sequencing (NGS) studies of environmental AR, proposing a framework for experimental controls, adequate sequencing depths, appropriate use of public databases, and the derivation of datatypes that are conducive for risk assessment. The second review focuses on antibiotic-resistant Enterococcus spp. as robust monitoring targets and an attractive alternative to more widely adopted Gram-negative organisms, while proposing workflows that generate universally equivalent datatypes. Finally, quantitative metagenomic (qMeta) techniques were benchmarked using internal reference standards for high-throughput quantification of ARGs with statistical reproducibility.



antibiotic resistance, surface water, wastewater, metagenomics, next-generation sequencing, enterococcus, standardization, quantitative metagenomics