title: 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 São Paulo, Brazil.
abstract: The release of extended-spectrum β-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 São 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 São 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.
authors.affiliation: Public Health Laboratory, School of Public Health, University of São Paulo, Avenida Dr. Arnaldo 715, Cerqueira César, 01146-904, São Paulo, SP, Brazil. milenadropa@gmail.com.
authors.forename: Milena
authors.identifier: http://orcid.org/0000-0003-1459-915X
authors.initials: M
authors.lastname: Dropa
authors.affiliation: Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, Avenida Professor Lineu Prestes, 1374, Butantã, 05508-900, São Paulo, SP, Brazil.|Department of Clinical Analysis, School of Pharmacy, University of São Paulo, Avenida Professor Lineu Prestes, 580, Butantã, 05434-070, São Paulo, SP, Brazil.
authors.forename: Nilton
authors.initials: N
authors.lastname: Lincopan
authors.affiliation: Public Health Laboratory, School of Public Health, University of São Paulo, Avenida Dr. Arnaldo 715, Cerqueira César, 01146-904, São Paulo, SP, Brazil.
authors.forename: Livia C
authors.initials: LC
authors.lastname: Balsalobre
authors.affiliation: Public Health Laboratory, School of Public Health, University of São Paulo, Avenida Dr. Arnaldo 715, Cerqueira César, 01146-904, São Paulo, SP, Brazil.
authors.forename: Danielle E
authors.initials: DE
authors.lastname: Oliveira
authors.affiliation: Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, Avenida Professor Lineu Prestes, 1374, Butantã, 05508-900, São Paulo, SP, Brazil.
authors.forename: Rodrigo A
authors.initials: RA
authors.lastname: Moura
authors.affiliation: Department of Clinical Analysis, School of Pharmacy, University of São Paulo, Avenida Professor Lineu Prestes, 580, Butantã, 05434-070, São Paulo, SP, Brazil.
authors.forename: Miriam Rodriguez
authors.initials: MR
authors.lastname: Fernandes
authors.affiliation: Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, Avenida Professor Lineu Prestes, 1374, Butantã, 05508-900, São Paulo, SP, Brazil.
authors.forename: Quézia Moura
authors.initials: QM
authors.lastname: da Silva
authors.affiliation: Public Health Laboratory, School of Public Health, University of São Paulo, Avenida Dr. Arnaldo 715, Cerqueira César, 01146-904, São Paulo, SP, Brazil.
authors.forename: Glavur R
authors.initials: GR
authors.lastname: Matté
authors.affiliation: Environmental Company of São Paulo State (CETESB), Avenida Professor Frederico Hermann Jr, 345, Pinheiros, 05489-900, São Paulo, SP, Brazil.
authors.forename: Maria I Z
authors.initials: MI
authors.lastname: Sato
authors.affiliation: Public Health Laboratory, School of Public Health, University of São Paulo, Avenida Dr. Arnaldo 715, Cerqueira César, 01146-904, São Paulo, SP, Brazil.
authors.forename: Maria H
authors.initials: MH
authors.lastname: Matté
mesh_terms: D015373:Bacterial Typing Techniques; D001938:Brazil; D004755:Enterobacteriaceae / Q000201:enzymology / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000096:biosynthesis*; D000068617:Genetic Background; D007711:Klebsiella pneumoniae / Q000201:enzymology / Q000235:genetics*; D058885:Multilocus Sequence Typing; D010957:Plasmids; D016133:Polymerase Chain Reaction; D062065:Wastewater / Q000382:microbiology*; D018508:Water Purification; D001618:beta-Lactamases / Q000096:biosynthesis*
chemical_list: D029968:Escherichia coli Proteins; D062065:Wastewater; D001618:beta-Lactamases; C473109:beta-lactamase CTX-M-15; C000599759:blaCTX-M-8 protein, E coli
keywords: Brazil; CTX-M-15; CTX-M-8; ESBL; Resistance; SHV-28; Sewage