Wastewater as a Probable Environmental Reservoir of Extended-Spectrum-β-Lactamase Genes: Detection of Chimeric β-Lactamases CTX-M-64 and CTX-M-123.

The presence of antimicrobial-resistant bacteria and resistance genes in aquatic environments is a serious public health concern. This study focused on Escherichia coli possessing bla CTX-M genes in wastewater inflows. Twelve crude inflow water samples from wastewater treatment plant (WWTP) A and two samples each from three other WWTPs were collected in 2017 and 2018. A total of 73 E. coli isolates with 31 different sequence types (STs) harboring distinctive bla CTX-M gene repertoires were detected. In WWTP A influents, bla CTX-M-14 (14 isolates) was dominant, followed by bla CTX-M-15 (12 isolates) and bla CTX-M-27 (10 isolates). The chimeric bla CTX-M-64 and bla CTX-M-123 genes were each identified in one of the E. coli isolates from the same WWTP A inflow port. The bla CTX-M-27 gene was associated with five of seven B2-ST131 isolates, including three isolates of the B2-O25b-ST131-H30R/non-Rx lineage. One of the remaining two isolates belonged to the B2-O25b-ST131-H30R/Rx lineage harboring the bla CTX-M-15 gene. As for the B2-O25b-ST131-H30R/non-Rx lineage, two isolates with bla CTX-M-27 were recovered from each of the WWTP B and D influents, and one isolate with bla CTX-M-174 was also recovered from WWTP B influent. Whole-genome sequencing of chimeric bla CTX-M-harboring E. coli isolates revealed that the bla CTX-M-64 gene was integrated into the chromosome of ST10 E. coli B22 via ISEcp1-mediated transposition of a 9,467-bp sequence. The bla CTX-M-123-carrying IncI1 plasmid pB64 was 109,169 bp in length with pST108. The overall findings suggest that wastewater may act as a probable reservoir of clinically significant clonal lineages mediating antimicrobial resistance genes and chimeric genes that have not yet been identified from human isolates of domestic origin in Japan.IMPORTANCE Global spread of CTX-M-type extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae is a critical concern in both clinical and community settings. This dominance of CTX-M-type ESBL producers may be largely due to the successful international spread of epidemic clones, as represented by the extraintestinal pathogenic Escherichia coli (ExPEC) ST131. Our findings highlight the worrisome presence of diverse E. coli clones associated with humans, including ExPEC lineages harboring the most common bla CTX-M variants in untreated wastewater samples. Moreover, the chimeric genes bla CTX-M-64 and bla CTX-M-123, which have not yet been identified from human isolates of domestic origin in Japan, were identified. Exposure to untreated wastewater through combined sewer overflow caused by heavy rains derived from abnormal weather change could pose a risk for human health due to ingesting those antimicrobial-resistant bacteria.