Screening for carbapenemase-producing Escherichia coli and Klebsiella pneumoniae in freshwater, bathing water and hospital continuum in Belgium

• Neither CP E. coli nor CP K. pneumoniae could be isolated from bathing water • Surface water and hospital continuums contain CP E. coli and CP K. pneumoniae • Hospital effluent plays an important role in the dissemination of CP bacteria • A diversity of CP genes was found in the sequenced bacterial genomes This study focused on carbapenemase-producing (CP) Escherichia coli and Klebsiella pneumoniae in the aquatic environment in Belgium, based on the screening of three hospital continuums (hospital-WWTP-receiving river), 20 surface waters and 29 bathing waters in 2022. The strains were enumerated and isolated from CHROMagar SuperCARBA medium. Phenotypic tests including disk combination tests to identify the No carbapenemase-producing (CP) E. coli nor CP K. pneumoniae was isolated from bathing waters, while 24 CP E. coli and 22 CP K. pneumoniae were isolated from 6 of the 20 surface water samples and from hospital continuums. Of the 24 CP E. coli strains, 20 had the bla KPC gene (83.3%) and 4 the bla OXA gene (16.6%), while of the 22 CP K. pneumoniae strains, the number of strains with carbapenemases genes was: 11 bla KPC (50.0%), 9 bla OXA (40.9%), one bla VIM (4.5%), one bla NDM (4.5%). The genomes of 11 CP E. coli and 14 CP K. pneumoniae were sequenced, and all possessed carbapenemase genes - bla KPC-3 (n=12), bla OXA-48 (n=10), bla OXA-244 (n=1), bla VIM-1 (n=1) and bla NDM-5 (n=1) - mostly coupled to ESBL-encoding genes. This study demonstrates that CP E. coli and CP K. pneumoniae are disseminated in the Belgian aquatic environment and suggest the role of hospital effluents in this dissemination.