Characterization of Emerging Pathogens Carrying blaKPC-2 Gene in IncP-6 Plasmids Isolated From Urban Sewage in Argentina

Abstract

Untreated wastewater is a reservoir for multidrug-resistant bacteria, but its role in the spread of antibiotic resistance in the human population remains poorly investigated. In this study, we isolated a KPC-2-producing ST2787 Klebsiella quasipneumoniae subsp. quasipneumoniae (WW14A), recovered from raw sewage at a wastewater treatment plant in Argentina in 2018 and determined its complete genome sequence. Strain WW14A was resistant to all β-lactams, ciprofloxacin and amikacin. A core genome phylogenetic analysis indicated that WW14A was closely related to a GES-5-producing Taiwanese strain isolated from hospital wastewater in 2015 and it was clearly distinct from strains isolated recently in Argentina and Brazil. Interestingly, blaKPC-2 was harbored by a recently described IncP-6 broad-spectrum plasmid which was sporadically reported worldwide and had never been reported before in Argentina. We investigated the presence of the IncP-6 replicon in isolates obtained from the same sampling and found a novel non-typable/IncP-6 hybrid plasmid in a newly assigned ST1407 Enterobacter asburiae (WW19C) also harboring blaKPC-2. Nanopore sequencing and hybrid assembly of strains WW14A and WW19C revealed that both IncP-6 plasmids shared 72% of coverage (~20 kb), with 99.99% of sequence similarity and each one also presented 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 (~11 kb) in both plasmids contained a Tn3 transposon disrupted by a Tn3-ISApu-flanked element and the core sequence was composed by ΔISKpn6/blaKPC-2/ΔblaTEM-1/ISKpn27. Both strains also carried genes conferring resistance to heavy metals (e.g., arsenic, mercury, lead, cadmium, copper), pesticides (e.g., glyphosate), disinfectants, and several virulence-related genes, posing a potential pathogenic risk in the case of infections. This is the first study documenting blaKPC-2 associated with IncP-6 plasmids in K. quasipneumoniae and Enterobacter cloacae complex from wastewater in Argentina and highlights the circulation of IncP-6 plasmids as potential reservoirs of blaKPC-2 in the environment.

Novel insights related to the rise of KPC-producing Enterobacter cloacae complex strains within the nosocomial niche

“…(2009) and was later called variant 1. Also, a similar genetic platform was described in Argentina in 2011 ( Gomez et al., 2011 ), in 2018 ( De Belder et al., 2018 ), and more recently in 2021 ( Ghiglione et al., 2021 ), whereas an identical platform was found in our institution ( Knecht et al, 2022 ). In the genetic platform of both K. pneumoniae HA7pKpn and E. hormaechei HA2pEho, Δ bla TEM-1 was missing, and nucleotides between IS Kpn27 and bla KPC-2 were only 254 bp.…”

“…These multiple events of loss and/or gain of mobile genetic elements that also include insertions, deletions, or homologous recombination coincide with what was identified for the dissemination of bla KPC-2 in previous works. Similar plasmids show traces of multiple events, with partial mobile genetic elements scattered throughout its genetic platform ( Botts et al., 2017 ; Brandt et al., 2019 ; Ghiglione et al., 2021 ).…”

“…A very closely related genetic platform carrying bla KPC-2 was found contemporary to strains isolated from inpatient M71 in environmental strains Enterobacter absuriae WW14A and Klebsiella quasipneumoniae subsp. quasipneumoniae WW19C, which were recovered from the same sewage network of our institution in 2018 ( Ghiglione et al., 2021 ). Therefore, our study also reinforces the need for studying the spread of critical acquired ARG within the One Health perspective.…”

“…The most common variants of the gene that codifies for KPC are bla KPC-2 and bla KPC-3 ( Brandt et al., 2019 ), which have become endemic in several countries ( Frost et al., 2019 ). Argentina is among these countries, and apart from clinical isolates, bla KPC-2 has also been recently detected in sewage ( Ghiglione et al., 2021 ). The bla KPC genes have been found in more than 257 different representative KPC plasmids ( Brandt et al., 2019 ) belonging to diverse Inc groups and sizes, and with several features that account for their success.…”

Molecular insights into novel environmental strains of Klebsiella quasipneumoniae harboring different antimicrobial-resistance genes

“…Emerging antimicrobial resistance genes (ARGs) conferring resistance to last-resort antibiotics including cephalosporins, carbapenems, and tigecycline, have been reported in clinical and environmental isolates. Such genes include bla CTX−M−2 , bla DHA−1 , bla NDM , bla IMP , bla KHM−1 , bla OXA−48 , bla OXA−162 , bla OXA−10 , bla KPC , tet(A), and tet(K) genes (9)(10)(11)(12). Environmental settings, especially water surfaces, soil, animal waste, and sewage treatment plants, are implicated in promoting the distribution of ARGs and as a result act as a reservoir of multidrug-resistant (MDR) K. quasipneumoniae (13).…”