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

Marija Kvesić; Ivica Šamanić; Anita Novak; Željana Fredotović; Mia Dželalija; Juraj Kamenjarin; Ivana Goić Barišić; Marija Tonkić; Ana Maravić

doi:10.3389/fmicb.2022.858821

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

Front Microbiol. 2022 May 6:13:858821. doi: 10.3389/fmicb.2022.858821. eCollection 2022.

Authors

Marija Kvesić^{1

2}, Ivica Šamanić³, Anita Novak^{4

5}, Željana Fredotović³, Mia Dželalija³, Juraj Kamenjarin³, Ivana Goić Barišić^{4

5}, Marija Tonkić^{4

5}, Ana Maravić³

Affiliations

¹ Center of Excellence for Science and Technology, Integration of Mediterranean Region, University of Split, Split, Croatia.
² Doctoral Study of Biophysics, Faculty of Science, University of Split, Split, Croatia.
³ Department of Biology, Faculty of Science, University of Split, Split, Croatia.
⁴ School of Medicine, University of Split, Split, Croatia.
⁵ University Hospital Split, Split, Croatia.

Abstract

The rapid and ongoing spread of carbapenemase-producing Enterobacteriaceae 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 Enterobacteriaceae (nine Escherichia coli, four Klebsiella pneumoniae and two Citrobacter freundii) 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 E. coli isolates belonged to serotype O21:H27 and sequence type (ST) 2795, while K. pneumoniae 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 bla _KPC-2 and bla _OXA-48. The bla _OXA-48 was associated with IncL-type plasmids in E. coli and K. pneumoniae. Importantly, the bla _KPC-2 in four E. coli isolates was located on ~40 kb IncP6 broad-host-range plasmids which recently emerged as bla _KPC-2 vesicles, providing first report of these bla _KPC-2-bearing resistance plasmids circulating in E. coli in Europe. This study also represents the first evidence of XDR and potentially virulent strains of KPC-producing E. coli in coastal waters and the co-occurrence of bla _KPC-2 and bla _OXA-48 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.

Keywords: Croatia; KPC; OXA-48; carbapenemase-producing Enterobacteriaceae; coastal waters; marine environment.