Comprehensive Phenotypic and Genotypic Analysis of Multidrug-Resistant and Virulent Enterococcus spp. from Untreated Hospital Wastewater and River Water in Baghdad, Iraq

Hospital wastewater is a critical reservoir for antimicrobial-resistant bacteria, which poses considerable threats to human well-being and aquatic environments, particularly in regions lacking effective wastewater treatment. This work investigated the prevalence, antimicrobial resistance (AMR) patterns, and antibiotic resistance genes (ARGs) of Enterococcus spp. isolated from untreated hospital effluent and downstream river water in Baghdad, Iraq. Species identification was performed using molecular confirmation of the tuf gene, followed by antimicrobial susceptibility testing against nine antibiotics. Multidrug resistance (MDR) and multiple antibiotic resistance (MAR) indices were calculated. PCR was used to detect seven ARGs. Of the 65 isolates, 92.3% were identified and verified as belonging to the Enterococcus genus. E. faecalis (35%), E. faecium (27%), and E. hirae (18%) were the dominant species. High rates of resistance (61% at Site A and 55% at Site B), with MDR detected in 48% of isolates, significantly higher in hospital effluent (65%) than in river water. The mean MAR index was 0.46, ranging from 0.58 in Site A to 0.30 in Site B. E. faecium exhibited the highest resistance levels to most antibiotics, followed by E. faecalis , while E. hirae showed comparatively lower resistance. Molecular screening identified seven major ARGs, with tetM being most prevalent (55%), followed by ermB (45%) and vanA (28.3%). E. faecium carried the highest ARG burden, including vanA (75%). These findings demonstrate that untreated hospital discharges are a major source of MDR Enterococcus and ARGs dissemination, underscoring the urgent need for effective wastewater treatment under a One Health framework.