The emergence of extended-spectrum β-lactamases (ESBLs)- and especially carbapenemases in Enterobacterales has led to limited therapeutic options; therefore, it is critical to fully understand all potential pathways of their transmission, especially in high-risk sources such as hospital wastewater. The aim of this study was to isolate and characterize ESBL- and carbapenemase-producing Enterobacterales (CRE) from untreated wastewater of two major hospitals in Zagreb, Croatia.
Phenotypic testing was performed to identify ESBL or carbapenemase producers. Antibiotic susceptibility was tested by agar disk diffusion and broth microdilution methods. Genotypic antibiotic resistance profiles of isolates were analyzed by PCR and whole genome sequencing (WGS), while molecular epidemiology was studied by PFGE and MLST.
Sixty-nine ESBL and 90 CRE isolates were isolated from hospital wastewater; all were multidrug resistant and were mostly identified as Escherichia coli, Klebsiella pneumoniae, Citrobacter, and Enterobacter cloacae complex. Among ESBL isolates, blaCTX-M-15 was the most prevalent ESBL gene, while among CRE isolates, blaKPC-2 and blaNDM-1 were the most frequently detected carbapenemases, especially in Citrobacter spp. followed by blaOXA-48, especially in K. pneumoniae. The most frequently detected sequence types included clinically relevant variants E. coli ST131 (blaCTX-M-15/blaTEM-116) and ST541 (blaKPC-2), K. pneumoniae ST101 (blaOXA-48/blaNDM-1) and E. cloacae ST277 (blaKPC-2/ blaNDM-1). WGS also showed that these isolates contained resistance genes to multiple antibiotic classes and a diverse plasmidome previously associated with clinically relevant ESBL or carbapenemases. Finally, analysis of the genomic context of blaCTX-M, blaOXA-48, and blaKPC-2 showed that these genes were associated with mobile genetic elements, particularly transposons and insertion sequences, indicating the potential for mobilisation.
Our results suggest that hospital wastewater is a reservoir for critical priority pathogens that exhibit resistance mechanisms to antibiotics critical to human health and provide a pathway for their dissemination in water systems. Effective treatment of this wastewater with advanced treatment methods such as UV and ozone treatment must therefore be ensured to reduce or stop the spread of resistant bacteria and ARGs in the natural environment.