Multiple β-Lactam Resistance Gene-Carrying Plasmid Harbored by Klebsiella quasipneumoniae Isolated from Urban Sewage in Japan.

The continuous emergence of carbapenemase-producing Enterobacteriaceae (CPE) presents a great public health challenge. Mitigation of CPE spread in the environment is crucial, particularly from a One Health perspective. Here we describe the isolation of CPE strain SNI47 from influent water of a sewage treatment plant in Japan. SNI47 was identified as Klebsiella quasipneumoniae subsp. quasipneumoniae by phylogenetic analysis and was resistant to beta-lactams, including carbapenems. Of four plasmids detected from SNI47, the 185,311-bp IncA/C2 plasmid (pTMSNI47-1), which carried 10 drug resistance genes, including genes for four beta-lactamases (bla(CTX-M-2), bla(DHA-1), bla(KHM-1), and bla(OXA-10)), was transferred to Escherichia coli J53 via conjugation. The MICs of all tested beta-lactams for the transconjugant were higher than for the recipient. We constructed recombinant plasmids, into which each beta-lactamase gene was inserted, and used them to transform E. coli DH5 alpha cells, demonstrating that KHM-1 enhanced carbapenem resistance. In addition, these beta-lactamases were responsible for a wide-spectrum beta-lactam resistance acquisition with mutual compensation. KHM-1, recognized as a rare type of metallo-beta-lactamase, was detected in a transferable plasmid, from a sewage treatment plant, involved in horizontal gene transfer. The detection of such plasmids raises a health risk alarm for CPE dissemination. IMPORTANCE In our investigation of urban wastewater in Japan, carbapenem-resistant Klebsiella quasipneumoniae subsp. quasipneumoniae was isolated that carried the pTMSNI47-1 plasmid, which carries four beta-lactamase genes and has transferability among Enterobacteriaceae. pTMSNI47-1 was found to encode a rarely reported carbapenemase, KHM-1. Cooperative effects of beta-lactamases encoded by pTMSNI47-1 appeared to have broad-spectrum resistance to beta-lactams. The detection of the KHM-1 gene in urban wastewater suggests that such a rare antimicrobial resistance (AMR) gene can be pooled in the environment, potentially emerging as an AMR determinant in a pathogen. When the number of beta-lactamase resistance genes is increased in one plasmid, the transfer of this plasmid can confer broad-spectrum resistance to beta-lactams, even if the individual gene confers narrow-spectrum resistance. The present study adds important information about the potential risk of sewage treatment plants as reservoirs and environmental suppliers of AMR genes, contributing to the public health from a One Health perspective.