Submarine Outfalls of Treated Wastewater Effluents are Sources of Extensively- and Multidrug-Resistant KPC- and OXA-48-Producing Enterobacteriaceae in Coastal Marine Environment

The rapid and ongoing spread of carbapenemase-producing has led to a global health threat. However, a limited number of studies have addressed this problem in the marine environment. We investigated their emergence in the coastal waters of the central Adriatic Sea (Croatia), which are recipients of submarine effluents from two wastewater treatment plants. Fifteen KPC-producing (nine , four and two ) were recovered, and susceptibility testing to 14 antimicrobials from 10 classes showed that four isolates were extensively drug resistant (XDR) and two were resistant to colistin. After ERIC and BOX-PCR typing, eight isolates were selected for whole genome sequencing. The isolates belonged to serotype O21:H27 and sequence type (ST) 2795, while isolates were assigned to STs 37 and 534. Large-scale genome analysis revealed an arsenal of 137 genes conferring resistance to 19 antimicrobial drug classes, 35 genes associated with virulence, and 20 plasmid replicons. The isolates simultaneously carried 43-90 genes encoding for antibiotic resistance, while four isolates co-harbored carbapenemase genes and . The was associated with IncL-type plasmids in and . Importantly, the in four isolates was located on ~40 kb IncP6 broad-host-range plasmids which recently emerged as vesicles, providing first report of these -bearing resistance plasmids circulating in in Europe. This study also represents the first evidence of XDR and potentially virulent strains of KPC-producing in coastal waters and the co-occurrence of and carbapenemase genes in this species. The leakage of these strains through submarine effluents into coastal waters is of concern, indicating a reservoir of this infectious threat in the marine environment.