Impact of urban wastewater on antibiotic resistance gene prevalence in peri-urban soils

Thesis Abstract

Urban wastewater, often containing diverse microbial assemblages and antibiotic residues, is increasingly recognized as a key driver of antibiotic resistance gene (ARG) dissemination in peri-urban environments. This study addresses the rising concern that municipal effluents applied to peri-urban soils as irrigation or waste stabilization inputs contribute to the prevalence and distribution of ARGs, potentially altering soil microbiomes and posing risks to environmental and public health. The aim is to quantify ARG prevalence in soils influenced by urban wastewater and to identify environmental, anthropogenic, and microbial drivers of ARG abundance and diversity. Specific objectives are (i) to quantify the abundance of targeted ARGs (including blaTEM, blaCTX-M, tetA, sul1, and intI1) in soils receiving urban wastewater compared with control soils without recent wastewater exposure; (ii) to determine associations between ARG prevalence and physicochemical soil properties (pH, organic matter, moisture, electrical conductivity) and wastewater-derived contaminants (ciprofloxacin, tetracycline, and sulfamethoxazole residues); (iii) to characterize microbial community structure and potential pathogens using 16S rRNA gene sequencing and shotgun metagenomics; (iv) to evaluate spatial and temporal variation in ARGs across five peri-urban sites over two seasons; and (v) to examine the mediated effects of horizontal gene transfer indicators, including intI1 abundance, on ARG dissemination. The study adopts a cross-sectional and seasonal design, integrating quantitative molecular assays with ecological analyses. The population comprises soils from peri-urban zones influenced by treated and untreated urban wastewater in a metropolitan region. A stratified random sampling scheme yields 150 soil samples (75 wastewater-exposed and 75 control) collected across five sites in two seasons (dry and wet), with triplicate subsamples per plot to ensure replication. Data collection employs quantitative PCR (qPCR) for ARG and integrase gene copy numbers, digital droplet PCR (ddPCR) for low-abundance targets, high-throughput 16S rRNA sequencing for microbial community profiling, and metagenomic sequencing on a subset (n=30) to resolve ARG contexts and mobile genetic elements. Physicochemical analyses include soil pH, organic matter, texture, moisture content, and extractable antibiotic residues quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Statistical analyses proceed in a hierarchical framework descriptive statistics to summarize ARG distributions, multivariate generalized linear models (GLMs) to test associations between ARG abundance and environmental predictors, and redundancy analysis (RDA) to relate microbial community composition to ARG and environmental variables. Structural equation modeling (SEM) will assess direct and indirect pathways linking wastewater exposure, antibiotic residues, mobile genetic elements, and ARG prevalence. A theoretical basis is anchored in the One Health concept and the ecological resistome framework, with theoretical underpinnings from the Environmental Selection Theory and the Human-Environment-Microbiome continuum. Anticipated findings include higher normalized ARG abundances (gene copies per gram soil) and greater intI1 prevalence in wastewater-exposed soils, correlated with residual antibiotic concentrations and shifts in microbial taxa associated with Gram-negative pathogens and opportunistic opportunists. Temporal analyses are expected to reveal seasonal amplification of ARGs in wet seasons due to increased moisture and mobilization. The study contributes to knowledge by linking urban wastewater management practices to soil resistome dynamics, clarifying mechanisms of ARG enrichment, and identifying environmental reservoirs and mobile genetic contexts that facilitate ARG dissemination in peri-urban landscapes. The results will inform risk assessment frameworks and wastewater governance policies, suggesting optimization of treatment levels, targeted soil management interventions, and monitoring strategies. The main conclusion is that urban wastewater input significantly elevates ARG prevalence and mobilization potential in peri-urban soils, with implications for groundwater quality and downstream food safety. Recommendations include adopting advanced treatment steps to reduce antibiotic residues, implementing buffer zones and controlled land application practices, developing routine soil resistome surveillance using qPCR/ddPCR and metagenomics, and integrating resistome considerations into urban water management policies to mitigate environmental AMR spread.