Resistance to critically important antibiotics in hospital wastewater from the largest Croatian city

Abstract

The emergence of extended-spectrum β-lactamase (ESBL)- and especially carbapenemases in Enterobacterales has led to limited therapeutic options. Therefore, it is critical to fully understand all potential routes of transmission, especially in high-risk sources such as hospital wastewater. This study aimed to quantify four enteric opportunistic pathogens (EOPs), total, ESBL- and carbapenem-resistant coliforms and their corresponding resistance genes (two ESBL and five carbapenemase genes) and to characterize enterobacterial isolates from hospital wastewater from two large hospitals in Zagreb over two seasons. Culturing revealed similar average levels of total and carbapenem-resistant coliforms (3.4 × 10⁴ CFU/mL), and 10-fold lower levels of presumptive ESBL coliforms (3 × 10³ CFU/mL). Real-time PCR revealed the highest E. coli levels among EOPs (10⁵ cell equivalents/mL) and the highest levels of the bla_KPC gene (up to 10^-1 gene copies/16S copies) among all resistance genes examined. Of the 69 ESBL- and 90 carbapenemase-producing Enterobacterales (CPE) isolates from hospital wastewater, all were multidrug-resistant and most were identified as Escherichia coli, Citrobacter, Enterobacter, and Klebsiella. Among ESBL isolates, bla_CTX-M-15 was the most prevalent ESBL gene, whereas in CPE isolates, bla_KPC-2 and bla_NDM-1 were the most frequently detected CP genes, followed by bla_OXA-48. Molecular epidemiology using PFGE, MLST and whole-genome sequencing (WGS) revealed that clinically relevant variants such as E. coli ST131 (bla_CTX-M-15/bla_TEM-116) and ST541 (bla_KPC-2), K. pneumoniae ST101 (bla_OXA-48/bla_NDM-1), and Enterobacter cloacae complex ST277 (bla_KPC-2/bla_NDM-1) were among the most frequently detected clone types. WGS also revealed a diverse range of resistance genes and plasmids in these and other isolates, as well as transposons and insertion sequences in the flanking regions of the bla_CTX-M, bla_OXA-48, and bla_KPC-2 genes, suggesting the potential for mobilization. We conclude that hospital wastewater is a potential secondary reservoir of clinically important pathogens and resistance genes and therefore requires effective pretreatment before discharge to the municipal sewer system.

Keywords: Antibiotic resistance; Carbapenemase; ESBL; Enterobacterales; Hospital wastewater; Multidrug.