Characteristics of Carbapenemase-Producing Enterobacteriaceae in Wastewater Revealed by Genomic Analysis

Wastewater is considered a major source of antibiotic-resistant bacteria released into the environment. Here, we characterized carbapenemase-producing Enterobacteriaceae (CPE) in wastewater by whole-genome analysis. Wastewater samples (n = 40) were collected from municipal wastewater treatment plants and hospital wastewater in Japan and Taiwan. Samples were screened for CPE using selective media, and the obtained isolates were sequenced using an Illumina MiSeq. The isolates (n = 45) included the following microorganisms: Klebsiella quasipneumoniae (n = 12), Escherichia coli (n = 10), Enterobacter cloacae complex (n = 10), Klebsiella pneumoniae (n = 8), Klebsiella variicola (n = 2), Raoultella ornithinolytica (n = 1), Citrobacter freundii (n = 1), and Citrobacter amalonaticus (n = 1). Among the 45 isolates, 38 harbored at least one carbapenemase-encoding gene. Of these, the bla_GES (bla_GES-5, bla_GES-6, and bla_GES-24) genes were found in 29 isolates. The genes were situated in novel class 1 integrons, but the integron structures were different between the Japanese (In1439 with bla_GES-24 and In1440 with bla_GES-5) and Taiwanese (In1441 with bla_GES-5 and In1442 with bla_GES-6) isolates. Other carbapenemase-encoding genes (bla_VIM-1, bla_NDM-5, bla_IMP-8, bla_IMP-19, and bla_KPC-2) were found in one to three isolates. Notably, class 1 integrons previously reported among clinical isolates obtained in the same regions as the present study, namely, In477 with bla_IMP-19 and In73 with bla_IMP-8, were found among the Japanese and Taiwanese isolates, respectively. The results indicate that CPE with various carbapenemase-encoding genes in different genetic contexts were present in biologically treated wastewater, highlighting the need to monitor for antibiotic resistance in wastewater.