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

• Published in: The Science of the total environment Vol. 870; p. 161805
• Main Authors: Puljko, Ana, Rozman, Svjetlana Dekić, Barišić, Ivan, Maravić, Ana, Jelić, Marko, Babić, Ivana, Milaković, Milena, Petrić, Ines, Udiković-Kolić, Nikolina
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 20.04.2023
• Subjects: Anti-Bacterial Agents - pharmacology; Antibiotic resistance; Bacterial Proteins - genetics; beta-Lactamases - genetics; Carbapenemase; Carbapenems - pharmacology; Croatia; Enterobacter - genetics; Enterobacterales; ESBL; Escherichia coli; Hospital wastewater; Hospitals; Klebsiella - genetics; Klebsiella - metabolism; Klebsiella pneumoniae; Microbial Sensitivity Tests; Multidrug; Multilocus Sequence Typing; Wastewater; Croatia; Carbapenemase; ESBL; Multidrug; Enterobacterales; Hospital wastewater; Antibiotic resistance
• ISSN: 0048-9697; 1879-1026
• DOI: 10.1016/j.scitotenv.2023.161805

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 blaKPC 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, blaCTX-M-15 was the most prevalent ESBL gene, whereas in CPE isolates, blaKPC-2 and blaNDM-1 were the most frequently detected CP genes, followed by blaOXA-48. Molecular epidemiology using PFGE, MLST and whole-genome sequencing (WGS) revealed that clinically relevant variants such as E. coli ST131 (blaCTX-M-15/blaTEM-116) and ST541 (blaKPC-2), K. pneumoniae ST101 (blaOXA-48/blaNDM-1), and Enterobacter cloacae complex ST277 (blaKPC-2/blaNDM-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 blaCTX-M, blaOXA-48, and blaKPC-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. [Display omitted] •Hospital wastewater contained high concentration of blaKPC and Escherichia coli.•The blaCTX-M-15 gene dominated among ESBL isolates.•The blaKPC-2 and blaNDM-1 genes dominated among carbapenemase-producing isolates.•Various high-risk enterobacterial clones were detected.•These clones harboured a diverse plasmidome and exhibited multidrug resistance.