Abstract Description

Antimicrobial resistance (AMR) is recognised as one of the major global public health threats, posing significant challenges to the advancement of modern medicine. Extended-spectrum beta-lactamases (ESBLs) are particularly concerning because they confer resistance to third-generation cephalosporins. Wastewater is a crucial factor in the spread of antimicrobial resistance, especially regarding ESBL Escherichia coli. In South Africa, where antibiotic resistance is an increasing concern, wastewater serves as a significant reservoir for antibiotic resistance genes in both urban and rural areas. This study aims to investigate the prevalence of ESBL E. coli in human wastewater in the Makana municipality, located in the Eastern Cape, using the adapted Tricycle protocol to phenotypically and genotypically confirm the presence of key ESBL genes. Water samples were collected from June to September 2025. Membrane filtration and culturing methods were utilised to isolate E. coli, and confirmed using PCR. Molecular identification of diarrheagenic E. coli (DEC) pathotypes was performed using multiplex PCR. The antibiotic susceptibility test was used to assess resistance against commonly used antibiotics and ESBL resistance. Phenotypically confirmed ESBL E. coli were subjected to PCR for phylogenetic analysis. Analyses revealed the occurrence of DEC pathotypes and increased resistance to the antibiotics, including ESBL strains. The findings from this research will provide essential data to inform public health responses and contribute to global surveillance efforts to combat AMR. This aligns with key Sustainable Development Goals (SDGs), particularly those related to improving health and well-being (SDG 3) and ensuring access to clean water and sanitation (SDG 6).