KPC-157 and KPC-181 carbapenemases produced by Citrobacter freundii ST522 and Klebsiella pneumoniae ST258 isolated from wastewater

Abstract

Background While novel KPC variants continue to emerge among clinically relevant Enterobacterales from hospital settings, their occurrence in impacted aquatic environments has been poorly investigated. We hereby report KPC-157 and KPC-181, allelic variants of KPC-2, produced by Citrobacter freundii and Klebsiella pneumoniae isolated from wastewater in Brazil.Methods Antimicrobial susceptibility was determined by disk diffusion and broth microdilution, whereas carbapenemase production was evaluated by inhibitor-based methods. Genome sequencing was performed combining short-read (Illumina HiSeq) and long-read (Oxford Nanopore) technologies, with further bioinformatic analyses. In silico KPC modeling was carried out using Yasara/PyMOL. Horizontal transfer and plasmid stability were assessed by conjugation and serial passage experiments, respectively. Epidemiological tracking of KPC-2 allelic variants and KPC-bearing plasmids was performed using publicly available genomes.Results Carbapenem-resistant C. freundii strain M21 [sequence type (ST) 522] and K. pneumoniae strains M16 and M18 (ST258), harboring blaKPC−157 and blaKPC−181 genes, respectively, were recovered from a sewage treatment plant. KPC-157 and KPC-181 differed from KPC-2 by single amino acid substitutions (Asn132Ser and Glu275Asp, respectively) that do not affect the main kinetic behavior, preserving the classical KPC-2 resistance phenotype (i.e., carbapenem resistance and ceftazidime-avibactam susceptibility). KPC-2 allelic variants were embedded in Tn4401 transposons. KPC-157 was carried on an IncN2 plasmid, while KPC-181 was associated with an IncFIB(pQil)/IncFII(K) plasmid.Conclusion The identification of KPC-157 and KPC-181 in wastewater highlights the role of polluted environments in harboring novel KPC variants from high-risk Enterobacterales clones and reinforces the importance of continued antimicrobial resistance surveillance beyond hospital settings.