Decoding multidrug-resistant pathogen dynamics for clinically-relevant wastewater surveillance - Multidrug-resistant bacteria (MDRB) contribute increasingly to morbidity, mortality, and healthcare costs. Extended-spectrum beta-lactam- and carbapenem-resistant Enterobacterales (ESBL-E and CRE) are MDRB of particular concern due to their demonstrated ability to evolve into highly transmissible clones and acquire and spread antibiotic resistance determinants. Traditional epidemiological surveillance typically focuses on outbreaks of MDRB causing clinical infections, underestimating the burden of these pathogens within hospital systems. Broader efforts that account for asymptomatic carriage and environmental and community reservoirs would be ideal to track and mitigate the spread of MDRB. Wastewater surveillance has proven an effective tool for public health pathogen monitoring, as shown with SARS-CoV-2, but has not been established in clinical settings. This proposal will develop new systems to leverage wastewater for clinically applicable, proactive, and readily deployable MDRB monitoring. In Aim 1, we will establish standardized longitudinal surveillance strategies to inform infection control responses. We will use long-read metagenomics and novel bioinformatic approaches to rapidly identify significant changes in relative or absolute abundance of ESBL-E or CRE compared to site-specific baselines. We will also establish methods to translate wastewater testing data into interpretable “action thresholds” for use by hospital and clinical teams. In Aim 2, we will identify factors enabling the emergence of novel ESBL-E and CRE genotypes in the wastewater environment. Wastewater sampling can identify novel resistant genotypes before detection of clinical infections. We will further develop our novel Metapore-C technique to link bacterial hosts with resistance gene-harboring mobile elements and will use this approach to identify environmental factors such as wastewater antibiotic levels and plumbing design associated with acquisition of resistance. Lastly, in Aim 3 we will devise wastewater testing methodologies suited to resource-limited clinical settings. Given the costs and infrastructure needed for comprehensive clinical surveillance, wastewater testing is better poised to aid in mitigation of MDRB under resource constraints. Yet, current wastewater surveillance approaches are often impractical in such settings. Our strategies for reducing per-sample costs and the analytical burden of wastewater data interpretation, as piloted at a pediatric hospital in Gaborone, Botswana, will serve as a proof-of-concept for wastewater MDRB testing in diverse contexts. Overall, this project will significantly broaden the ability of wastewater surveillance to inform hospital and clinical care efforts, while establishing best practices for global surveillance of antimicrobial resistance in hospital wastewater.
