Surveillance of ESKAPE pathogens in wastewater environments

Abstract

Wastewater constitutes a considerable source of environmental contamination, notably as a medium for the proliferation and dissemination of antibiotic-resistant bacteria (ARB) and antibiotic-resistance genes (ARGs). The ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonasaeruginosa, and Enterobacter species) that form part of ARBs are notable for their elevated antibiotic resistance and their common link to severe, frequently hospital-acquired infections, representing a substantial global health risk. The presence of ESKAPE pathogens in the environment constitutes a potential threat to public health, as exposure to these bacteria may result in infections that are challenging to treat due to their intrinsic antibiotic resistance. The One Health Approach necessitates the development of efficient surveillance strategies to prevent and manage the dissemination of ESKAPE pathogens and antibiotic resistance in human, veterinary, and environmental settings. These surveillance strategies have been widely implemented in clinical settings, but few have been implemented in the wastewater environment. In addition, ESKAPE pathogen surveillance in the wastewater environment, especially at wastewater treatment plants (WWTPs) in South Africa, remains unexplored. To contribute towards the One Health Approach, this study aimed to evaluate the presence and characteristics of ESKAPE pathogens in the wastewater environment and to use the data obtained to propose a surveillance programme for the North West and Gauteng provinces of South Africa. The first objective of the study was to compare agar-based enumeration with real-time PCR (RT-PCR) approaches to detecting ESKAPE pathogens in WWTP influent, effluent, and downstream sites. Agar-based enumeration detected all species, while RT-PCR identified only four (E. faecium, S. aureus, K. pneumoniae, and A. baumannii). Enterobacter sp. was most prevalent in agar-based enumeration, while K. pneumoniae was predominant in RT-PCR. Both methods corresponded with high ESKAPE pathogen levels in WWTP influent, gradually decreasing in WWTP effluent. RT-PCR indicated higher downstream ESKAPE pathogen levels than agar-based enumeration. The highest removal efficiency (100%) was observed at some WWTPs, but traces of pathogens remained in downstream sites, indicated by removal efficiency below 99% at WWTPs. The second objective of the study quantified ARGs at WWTP influent, effluent, and downstream sites. Among the six plasmid-mediated AmpC (pAmpC) ARGs, blaMOX had the highest levels, while blaDHA had the lowest. blaMOX was detected in 80% of WWTP effluent and downstream sites. The int1 gene, representing environmental ARGs, was more prevalent than sul1, with 80% detection across all WWTPs. While many WWTPs significantly reduced ARG levels, traces were still discharged into the environment. These findings reinforced concerns that conventional WWTPs cannot fully eliminate antibiotics, ARBs, and ARGs, posing potential risks to receiving water bodies.

The third objective of the study was to examine the antibiotic resistance, virulence factors and genomic characteristics of K. pneumoniae from WWTP influent and effluent using culture-based and genomic approaches. β-lactam resistance detected in culture-based tests was confirmed by PCR. Whole genome sequencing (WGS) identified diverse antibiotic resistance genes (ARGs), including ESBLs, and plasmid analysis revealed horizontal gene transfer mechanisms. Virulence genes related to adhesion, biofilm formation, and iron acquisition were also detected. Genomic comparisons showed a shared "core" genome but unique environmental clusters, indicating niche-specific adaptations. These findings emphasized the need for improved surveillance and management to curb antibiotic resistance spread in wastewater ecosystems. The fourth objective of the study was to monitor K. pneumoniae and pAmpC genes at a functional WWTP in the North West province for 12 months. Findings showed positive correlations between K. pneumoniae, pAmpC genes, and physicochemical parameters, indicating their influence on pathogen and ARG presence and dissemination. K. pneumoniae was detected throughout the year, peaking in WWTP influent (June 2021) and WWTP effluent (February 2021), aligning with pAmpC gene trends where blaFOX and blaMOX were most prevalent. While notable reductions were observed (pAmpC genes removal efficiency: 80%–99%, K. pneumoniae removal efficiency: 6%–98%), no reduction occurred in February 2021. This objective confirmed that even well-functioning WWTPs cannot completely eliminate priority pathogens and ARGs, leading to their release into water bodies. In conclusion, the overall findings of this study could be used in policymaking strategies aimed at wastewater-based epidemiology and ESKAPE pathogens surveillance.