Water scarcity is one of the main challenges in sustainable development particularly in developing countries. The use of wastewater effluent for irrigation of crops is common in most water-stressed countries as this alleviates pressure on fresh water supply. Wastewater treatment plants (WWTPs) are regarded as hotspots for antibiotic resistance determinants; antibiotic resistant bacteria (ARB), antibiotic resistance genes (ARGs) and mobile genetic elements (e.g. plasmids). These antibiotic resistance determinants are consequently introduced into the soil and fresh produce through irrigation with effluent wastewater. Microcosm and field surveillance studies in Palapye and Gaborone respectively, were carried out to evaluate the impact of wastewater effluent irrigation in soil and vegetables in agricultural settings. Culturebased, molecular (PCR), 16S rRNA gene metagenomics and shotgun metagenomics methods were used to determine the occurrence, abundance, diversity and overall dynamics of ARB and ARGs in effluent irrigated soil and vegetables. Clinically relevant bacteria (Campylobacter, Listeria, Pseudomonas, E.coli, Enterobacter, Staphylococcus and Shigella species) were targeted and isolated from wastewater effluent, effluent irrigated soils and selected vegetables. The results revealed a significant reduction in total viable bacterial quantities in the storage tank containing effluent used for microcosm irrigation. A shift in bacterial community profile was observed as notable reduction in proteobacteria and increase in firmicutes phyla from the microcosm soil following wastewater irrigation. Antibiotic resistance genes; beta-lactamase resistance gene (blaTEM), tetracycline resistance gene (tetA), aminoglycoside resistance gene (aadA), sulfonamide resistance gene (sul1), trimethoprim resistant dihydrofolate (dfrA) were all identified by PCR in Gaborone wastewater treatment plant (GWWTP) effluent but only blaTEM, aadA and dfrA were detected in the soil from an agricultural field irrigated using effluent from GWWTP. Shotgun metagenomics revealed diverse ARGs belonging to different classes of antibiotics; aminoglycoside, beta-lactam, trimethoprim, macrolide, glycopeptide, tetracycline, sulfonamide, quinolone and oxazolidinone in Palapye wastewater treatment plant (PWWTP) effluent used in the irrigation of the microcosm experiment. However only blaTEM and aadA were identified in the microcosm soil, and only beta-lactamase gene blaTEM was detected on vegetable surfaces following irrigation with PWWTP effluent wastewater. The results from this study demonstrated the short and long-term impact of wastewater irrigation which results in persistence and possible dissemination of wastewater-associated ARB and ARGs into agricultural soils and vegetables. Moreover, this study enhances our understanding of antibiotic resistance dynamics and highlights the importance of monitoring antibiotic resistance in agro-systems, which is critical for informing policies aimed at sustainable use of wastewater effluent in water-stressed countries.

Thesis (MSc Biological Sciences) --Botswana International University of Science and Technology, 2021