Comparison of ESKAPE Pathogen Levels in Wastewater and Receiving Water Bodies Using Agar-Based Enumeration and Real-Time PCR

The ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) are key multidrug-resistant organisms increasingly recognized outside clinical settings. Their persistence in wastewater raises concerns regarding the efficacy of conventional treatment processes and the dissemination of antimicrobial resistance. This study aimed to detect and quantify ESKAPE pathogens in wastewater treatment plants (WWTPs) and downstream environments using agar-based enumeration and real-time PCR (RT-PCR). Culture methods detected all targeted species, with Enterobacter spp. being most abundant and A. baumannii least prevalent. RT-PCR quantified four species (E. faecium, S. aureus, K. pneumoniae, and A. baumannii), identifying K. pneumoniae as dominant. Both approaches revealed higher concentrations in influents that declined after treatment, although RT-PCR indicated elevated downstream levels, suggesting incomplete removal. The highest removal efficiency (100%) was observed for S. aureus and A. baumannii in WWTPs B and J, and the lowest (54.1%) for S. aureus in WWTP K. Integrating culture and molecular methods improved detection sensitivity and provided complementary insights. These results demonstrate that conventional treatment may not fully eliminate ESKAPE pathogens and underscore the need to include them in wastewater-based surveillance for antimicrobial resistance monitoring.