Genetic background of novel sequence types of CTX-M-8-and CTX-M-15-producing Escherichia coli and Klebsiella pneumoniae from public wastewater treatment plants in Sao Paulo, Brazil

The release of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae to the environment is a public health issue worldwide. The aim of this study was to investigate the genetic background of genes encoding ESBLs in wastewater treatment plants (WWTPs) in So Paulo, southeastern Brazil. In 2009, during a local surveillance study, seven ESBL-producing Enterobacteriaceae strains were recovered from five WWTPs and screened for ESBL genes and mobile genetic elements. Multilocus sequence typing (MLST) was carried out, and wild plasmids were transformed into electrocompetent Escherichia coli. S1-PFGE technique was used to verify the presence of high molecular weight plasmids in wild-type strains and in bla (ESBL)-containing E. coli transformants. Strains harbored bla (CTX-M-8), bla (CTX-M-15), and/or bla (SHV-28). Sequencing results showed that bla (CTX-M-8) and bla (CTX-M-15) genes were associated with IS26. MLST revealed new sequence types for E. coli (ST4401, ST4402, ST4403, and ST4445) and Klebsiella pneumoniae (ST1574), except for one K. pneumoniae from ST307 and Enterobacter cloacae from ST131. PCR and S1-PFGE results showed CTX-M-producing E. coli transformants carried heavy plasmids sizing 48.5-209 kb, which belonged to IncI1, IncF, and IncM1 incompatibility groups. This is the first report of CTX-M-8 and SHV-28 enzymes in environmental samples, and the present results demonstrate the plasmid-mediated spread of CTX-M-encoding genes through five WWTPs in So Paulo, Brazil, suggesting WWTPs are hotspots for the transfer of ESBL genes and confirming the urgent need to improve the management of sewage in order to minimize the dissemination of resistance genes to the environment.