Dissemination of Carbapenemase-producing Enterobacterales through outlet water and gulls at a wastewater treatment plant in Sweden

The emergence of carbapenemase-producing Enterobacterales (CPE) is a threat to human and animal health since carbapenems are often used to treat severe infections. In the environment, water and wastewater treatment plants (WWTP) has been suggested having an important part in the dissemination of antimicrobial resistant bacteria (ARB). The objective of this study was to investigate presence of CPE among aquatic birds, Black headed gulls (Croicocephalus ridibundus) in Sweden, interacting with environments impacted by WWTPs. Sampling of composite WWTP inlet and outlet, surface seawater from key aquatic bird habitats and freshly deposited gull faeces was done on six separate occasions May to September 2021. Inlet, outlet and water samples were filtrated through Sterivex-GP PES 0.22 µM until clogged. Samples were enriched in brain heart infusion (BHI) broth with vancomycin (8 mg/L) followed by selective screening of putative CPE on mSuperCarbaTM (CHROMagar). Identification was done with MALDI TOF. Antimicrobial susceptibility testing was performed according to EUCAST. Meropenem resistant isolates were selected for whole genome short and long read sequencing. Hybrid assemblies were analyzed for resistance genes, mobile genetic elements and phylogenetic relatedness via Centers for Genomic Epidemiology, CLC genomic workbench, RAST, BRIG and Clinker. Twenty putative CPE were collected and subjected to sequencing, resulting in 17 identified Enterobacterales isolates carrying carbapenemase genes. blaGES-5 was carried on an IncP plasmid in Escherichia coli, Raoultella ornithinolytica and in Klebsiella spp.. blaIMI-3 was carried on an IncFII(Yp) plasmid in Enterobacter ludwigii. Phylogentic analysis of R. ornithinolytica and E. ludwigii indicate that the gulls carry the same strains that are found in the WWTP samples. In addition, blaOXA-181 located on a COLKP3 plasmid was found in an E. coli and blaOXA-244 was chromosomally located in an E. coli. We report the first finding of CPE in animals in Sweden. In addition, WWTP can be considered hotspots for AMR dissemination as our data suggest both horizontal gene transfer and clonal propagation of ARB, which are further spread by aquatic birds interacting with anthropogenic influenced environments.