Microbial Diversity and Antibiotic Resistance in Urban Wastewater Treatment Plants

Thesis Abstract

Urban wastewater treatment plants (WWTPs) play a critical role in managing wastewater and protecting public health; however, they are increasingly recognized as hotspots for microbial diversity and the proliferation of antibiotic-resistant bacteria (ARB), posing significant challenges to environmental and human health. The presence of diverse microbial communities and their potential to harbor and disseminate antibiotic resistance genes (ARGs) within WWTPs require comprehensive investigation to inform effective mitigation strategies. This study aims to assess microbial diversity and the prevalence of antibiotic resistance in selected urban WWTPs, with specific objectives including characterizing the microbial community composition across different treatment stages; quantifying and identifying ARGs through molecular techniques; elucidating the factors influencing resistance gene dissemination; and evaluating the potential risks of resistant bacteria release into receiving environments. The research employs a descriptive cross-sectional design, incorporating both culture-dependent and culture-independent methods for a holistic understanding of microbial dynamics. The study population comprises influent, activated sludge, effluent, and sludge digestate samples collected from three major urban WWTPs servicing populations of approximately 1 million residents each. A total of 36 samples (12 from each plant, representing different treatment stages) will be collected quarterly over a one-year period to account for temporal variability. Bacterial isolates will be obtained using selective media, identified via 16S rRNA gene sequencing, and their antibiotic susceptibility profiles determined through disk diffusion and minimum inhibitory concentration (MIC) assays aligned with Clinical and Laboratory Standards Institute (CLSI) guidelines. Concurrently, quantitative PCR (qPCR) targeting prevalent ARGs (e.g., blaTEM, mecA, tetM, sul1) will be employed to quantify resistance gene abundance, complemented by metagenomic sequencing to characterize overall microbial diversity and resistome profiles. Data analysis will include multivariate statistical techniques, particularly principal component analysis (PCA) and redundancy analysis (RDA), to explore relationships between microbial community structure, ARG presence, operational parameters, and environmental factors. Regression models will assess predictors of ARB and ARG prevalence, while diversity indices (Shannon, Simpson) will evaluate microbial community richness and evenness. Theoretical frameworks, including the One Health approach and the resistome concept, underpin the interpretive lens of the investigation, emphasizing the interconnectedness of environmental, animal, and human health in resistance dissemination. Expected findings indicate a high diversity of bacteria across all treatment stages, with a substantial proportion exhibiting multidrug resistance, particularly in influent and primary sludge stages. Elevated levels of specific ARGs are anticipated in influent and sludge samples, with a reduction observed in effluent stages, although residual resistance genes may persist, highlighting potential environmental dissemination risks. The research will reveal key operational and environmental factors influencing ARG dynamics, such as hydraulic retention time, organic load, and microbial community interactions. This study contributes to the growing body of knowledge by elucidating the microbial ecology and resistome of urban WWTPs, providing empirical evidence on the efficiency of current treatment processes in reducing antibiotic resistance dissemination. The findings will inform policymakers and wastewater management practitioners about the necessity of integrating resistance mitigation into standard treatment protocols. It is recommended that WWTPs adopt advanced treatment technologies, such as ultraviolet (UV) disinfection and membrane bioreactors, to further reduce ARB and ARG loads. Future research should explore longitudinal monitoring and the effectiveness of bioaugmentation strategies for microbial and resistance management. The study underscores the importance of adopting a One Health paradigm in wastewater management to curb the environmental spread of antibiotic resistance and safeguard public health.