Characterizing Antibiotic-Resistant Bacteria in Urban Wastewater Systems

Thesis Abstract

Urban wastewater systems represent a critical nexus for the amplification and dissemination of antibiotic-resistant bacteria (ARB) and resistance genes, posing risks to public health through environmental exposure and horizontal gene transfer. This study investigates the prevalence, diversity, and drivers of ARB in municipal wastewater streams to inform mitigation strategies and policy interventions. The aim is to characterize (i) the spatial and temporal patterns of ARB distribution across influent, primary, secondary effluent, and reclaimed water within a large metropolitan wastewater network; (ii) the resistome and associated mobile genetic elements by metagenomic profiling; and (iii) the environmental and operational factors that correlate with ARB abundance and resistance gene loads. Specific objectives include quantify culturable ARB using selective culture-based methods; determine the prevalence of extended-spectrum beta-lactamase (ESBL) and carbapenemase producers via targeted PCR assays; profile the resistome with shotgun metagenomics and annotate resistance determinants against the Comprehensive Antibiotic Resistance Database (CARD); assess the influence of wastewater parameters (chemical oxygen demand, ammonia, nutrient load, temperature, pH) and treatment stage on ARB and resistance gene abundance using multivariate modeling; identify co-occurrence networks between ARB taxa and resistance genes; and compare ARB and resistome signatures with downstream receiving waters to evaluate potential environmental dissemination. A mixed-methods, longitudinal field study will be conducted over 12 months in three sentinel wastewater treatment facilities serving a combined population of about 2.5 million residents. The population of interest comprises bacteria within municipal influent and effluent streams, framed as a dynamic environmental reservoir of antimicrobial resistance. A stratified sampling approach will collect quarterly composite samples from influent, primary effluent, secondary effluent, and tertiary/disinfected discharges, totaling 48 samples per site (144 samples overall). Culture-based assays will enumerate aerobic and facultative anaerobic ARB on selective media (e.g., MacConkey agar with cefotaxime, ceftazidime, and meropenem), followed by species identification via MALDI-TOF MS and confirmation of resistance phenotypes through disk diffusion according to CLSI guidelines. PCR screening will detect key resistance determinants (blaCTX-M, blaKPC, blaNDM, blaOXA-48-like) and integron-associated genes (class 1 and 2). Shotgun metagenomic sequencing will be performed on a subset of samples (n=24 per site) to characterize the resistome and mobilome, with bioinformatic analysis conducted against CARD, ResFinder, and mobile genetic element databases. Physicochemical parameters (COD, BOD, ammonia, nitrate, phosphate, turbidity, temperature, pH) and operational data ( Hydraulic Retention Time, sludge age, disinfection efficacy) will be collected in parallel. Data analysis will employ descriptive statistics for ARB abundance, multivariate generalized linear models to identify predictors of ARB counts and resistance gene abundance, and redundancy analysis to relate microbial community structure to environmental variables. Network analysis will elucidate co-occurrence patterns between taxa and resistance determinants. A theoretical framework incorporating the One Health concept and the Resistance Selection Pressure model will anchor interpretation, complemented by the ecological risk assessment approach to quantify potential human and environmental exposure. Expected findings include higher ARB concentrations and resistome complexity in influent and variable reductions across treatment stages, with residual ARB and genes detected in secondary and tertiary effluents, and detectable signatures in downstream waters. The study anticipates identifying critical factors driving ARB persistence, such as high organic load, suboptimal disinfection, and horizontal gene transfer potential via integrons. The contribution to knowledge lies in providing a comprehensive, lifecycle-based assessment of ARB and resistome dynamics within urban wastewater systems, evidencing links between treatment performance and antimicrobial resistance dissemination, and offering data-driven recommendations for process optimization, monitoring frameworks, and policy strategies to mitigate environmental and public health risks. The main conclusion will emphasize the necessity of integrated wastewater management to curtail resistance spread, with recommendations including enhanced disinfection protocols, routine resistome monitoring, and targeted interventions at the influent stage to reduce ARB load entering treatment plants.