Genomic characterization of multidrug-resistant Enterobacteriaceae isolated from wastewater reveals the circulation of emerging pathogens as a source of KPC-2 dissemination in Argentina

GHIGLIONE BÁRBARA; HAIM MARÍA SOL; PENZOTTI PEDRO; BRUNETTI FLORENCIA; D´AMICO GONZÁLEZ, G; DI CONZA JOSÉ; FIGUEROA ESPINOSA ROQUE A; NUÑEZ LIDIA; TORNELLO CARINA; MONTAVANO JULIÁN; GUTKIND GABRIEL; LINCOPAN, NILTON; POWER PABLO; DROPA MILENA

Simposio; GW4 AMR symposium 2020; 2020

Untreated wastewater is a reservoir for multidrug-resistant bacteria, but its role in the spread of antibiotic resistance in human population remains poorly investigated. In this study, we evaluated the diversity of antimicrobial resistance genetic elements in a raw sewage effluent from Buenos Aires, Argentina and characterized two KPC-2 producing strains by whole genome sequencing. A 1000-mL raw sewage sample was collected from a WWTP, filtered through 0.45μm membranes, from which DNA was extracted for PCR screening of plasmid incompatibility (Inc) groups; integrases (intI); ISCR1, IS26, IS10 and ISEcp1 transposases; wide-spectrum beta-lactamases (bla); and phosphoethanolamine-transferases (mcr). Another membrane was cultivated in McConkey agar. Antibiotic susceptibility testing was performed by disk diffusion, followed by bacterial MALDI-TOF/MS identification. Suspected β-lactamase producers were assessed by PCR. Klebsiella pneumoniae 14A (Kpn14A) and Enterobacter sp. 19CONT (Ent19CONT) were sequenced by Illumina platform. Inc F group plasmids were detected, as well as intI1, intI3 integrases, ISCR1, IS26, IS10 and ISEcp1 transposases. Different bla genes detected in total membrane DNA included blaCTX-M, blaOXA-48, blaVIM, blaNDM, blaPER-2, blaGES, blaCMY and blaKPC; mcr-1 and mcr-3 were also detected. KPC-2 producing isolates included K. pneumoniae, K. variicola and E. cloacae Complex. Two E. coli isolates harbored blaCTX-M-2, and 1 K. pneumoniae isolate both blaCTX-M-15 and blaKPC-2; blaIMP was present in K. oxytoca and P. aeruginosa, and blaVIM in P. aeruginosa. Both Kpn14A and Ent19CONT harbored two different but related IncP-6 plasmids carrying blaKPC-2. They were composed of three uniquely combined regions that were derived from other plasmids recently reported in different countries of South America, Asia, and Europe. The region harboring the carbapenem resistance gene (14 kb) contained a Tn3 transposon disrupted by an ISApu-flanked element and the core sequence was composed by ISKpn6/blaKPC-2/blaTEM-1/ISKpn27. Occurrence of genes encoding for resistance to last-choice antimicrobials, as well as mobile genetic elements, in potentially relevant MDR pathogens in a raw sewage sample may contribute to the spread of bacterial resistance. We document here the presence of two promiscuous blaKPC-2-IncP-6 plasmids recently described that appear to have been circulating in environmental bacteria in wastewater and human populations in different continents.