Identification of ESKAPEE pathogens from wastewater treatment plants in Vhembe District, Limpopo, South Africa

Abstract

Background: The ESKAPEE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter species, and Escherichia coli) are a WHO priority pathogen group of highly antibiotic-resistant bacteria that pose a significant threat to public health. Wastewater treatment plants (WWTPs) play a crucial role in removing harmful pathogens and other pollutants from domestic and industrial effluents. In South Africa, many WWTPs, especially in rural districts, are overloaded or poorly maintained, resulting in the discharge of untreated or partially treated wastewater into the environment. Objective: This study investigated the presence and prevalence of ESKAPEE pathogens in wastewater and associated rivers in the Vhembe District, Limpopo, South Africa. Methodology: A total of 48 samples were collected from two WWTPs and their adjacent rivers. Bacteria were isolated from WWTP influent, effluent, upstream and downstream river samples using standard microbiological culture techniques and were identified using the VITEK® 2 Compact system. The antibiotic susceptibility profiles of the isolates were determined, and molecular confirmation was performed using PCR and Sanger sequencing. Results: All seven ESKAPEE pathogens were detected in influent samples, with E. coli, E. faecium, and K. pneumoniae being the most prevalent. Effluent samples showed reductions in pathogen detection, with rates ranging from 20% to 55%, reflecting partial removal by conventional treatment processes. River and effluent samples contained multiple multidrug-resistant (MDR) isolates, indicating incomplete pathogen removal. The highest resistance levels were observed against β-lactams, cephalosporins, and vancomycin, whereas carbapenems remained largely effective. Sanger sequencing confirmed that the amplified resistance genes shared 98–100% sequence similarity with globally circulating clinical MDR strains, suggesting gene flow between environmental and clinical reservoirs. Conclusion: WWTPs significantly reduced total bacterial counts but failed to eliminate viable MDR pathogens. These results highlight the role of WWTPs as potential reservoirs and dissemination routes for antimicrobial-resistant bacteria and emphasize the need for improved wastewater management to mitigate environmental and public health risks in South Africa.