The increased prevalence of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae in the environment is a serious global health issue. In this regard, anthropogenic activities related to the overuse of antimicrobial agents in medicine (human and veterinary) and agriculture have contributed to the environmental reservoirs of resistant bacteria, which can be directly or indirectly transmitted to humans and other animals (Amos et al. 2014; Reinthaler et al. 2013). Previous studies have shown that intestinal carriage of ESBL-producing Enterobacteriaceae is frequent, in both hospital and community settings (Valverde et al. 2008; Lim et al. 2014). ESBL-producing bacteria can be released into the wastewater network and public wastewater treatment plants (WWTPs) (Bréchet et al. 2014). WWTPs can process sewage from several sources, providing a hotspot for horizontal transfer of ESBL-encoding genes between bacteria from many origins. Thus, ESBL variants found in bacterial populations in wastewaters represent the ESBL dominating in environments in contact with broad-spectrum cephalosporins and other beta-lactams used in human and veterinary medicine. Among all ESBL families, CTX-M enzymes have been the most frequent ESBLs reported worldwide. In Brazil, although the production of CTX-M enzymes has become the most prevalent mechanism of acquired resistance to broad-spectrum cephalosporins in gram-negative bacteria from clinical samples (Fernandes et al. 2009; Tollentino et al. 2011; Carvalho-Assef et al. 2014), poultry (Fernandes et al. 2009; Silva et al. 2013; Ferreira et al. 2014), food-producing and companion animals (Aizawa et al. 2014; Leigue et al. 2015), retail chicken meat (Casella et al. 2015), hospital wastewater (Chagas et al. 2011), and urban rivers (Oliveira et al. 2014), there is no data available regarding the detection of CTX-M-producing bacteria in public WWTPs. So, the aim of this study was to investigate the presence of ESBL-producing bacteria and the genetic background of genes encoding ESBLs in public WWTPs.

From June to December of 2009, ten sludge and ten sewage samples from five different public WWTPs, from São Paulo, Brazil, were screened for the presence of ampicillin-resistant Enterobacteriaceae, using LB broth and MacConkey agar containing 100 μg/mL of ampicillin. Two hundred strains were recovered, from which seven (3.5 %) were ESBL-producing Enterobacteriaceae, detected through the double-disk synergy method (CLSI 2012), using LB agar amended with 250 μg/mL of cloxacillin for natural AmpC-producing bacteria (Picão et al. 2009). No carbapenemase-producing bacteria were detected, and MIC measures for nine beta-lactam compounds were determined using Etest® strips (Biomérieux, France). ESBL-encoding genes, class 1 integrons, and insertion sequences (IS26, ISCR1, and ISEcp1) were screened by PCR and direct DNA sequencing (Table 1). Mapping of ESBL genetic environment was assessed through PCR reactions combining bla ESBL forward and reverse primers with forward and reverse primers targeting the mobile elements. Amplification products were purified and directly sequenced. Transformation experiments, using One Shot® TOP 10 Electrocomp™ Escherichia coli (Invitrogen, CA, USA) as recipients, were carried out, and transformed cells were selected in Luria Bertani agar containing cefotaxime 4 μg/mL. PCR and sequencing (Table 1) were carried out to confirm the presence of bla ESBL genes and mobile elements in E. coli transformants. PCR-based replicon typing (PBRT) was carried out (Carattoli et al. 2005, 2015), and plasmids were extracted and characterized by S1-PFGE as previously described (Dropa et al. 2015). Finally, multilocus sequence typing (MLST) was carried out to characterize E. coli, Klebsiella pneumoniae, and Enterobacter cloacae sequence types (STs) (http://mlst.warwick.ac.uk/mlst/dbs/Ecoli; http://www.pasteur.fr/recherche/genopole/PF8/mlst/Kpneumoniae.html; http://pubmlst.org/ecloacae/).

Table 1 Primers used in this study for PCR and sequencing
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In Table 2 are the quoted MIC values for β-lactams and ESBL genotypes. According to sequencing results, three E. coli, one K. pneumoniae, and one Enterobacter cloacae, each one from a different WWTP, carried the bla CTX-M-8 gene while one E. coli and one K. pneumoniae, from the same WWTP, harbored the bla CTX-M-15 gene. ESBL-encoding bla SHV-28 gene was also identified in the K. pneumoniae strain harboring the bla CTX-M-15 ESBL gene. All strains carried class 1 integrase genes and insertion sequences IS26 and ISEcp1. Mapping of genetic environment revealed that ESBL genes were not associated with class 1 integrons, and that bla CTX-M-8 and bla CTX-M-15 genes were actually associated with IS26. For the IS26-bla CTX-M-8 array, the 5′-3′ bla CTX-M-8 was flanked upstream by a 3′-5′ IS26 truncating a 3′-5′ IS10, whereas for the IS26-bla CTX-M-15, the 5′-3′ bla CTX-M-15 was flanked upstream by a 3′-5′ IS26 interrupting a 5′-3′ ISEcp1 (Fig. 1). bla SHV-28 was not associated with neither class 1 integrons nor any of the studied insertion sequences.

Table 2 Characteristics and genetic background of CTX-M-8- and CTX-M-15-producing Enterobacteriaceae in public wastewater treatment plants in southeastern Brazil
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Fig. 1
figure 1

Genetic context of bla CTX-M-15 and bla CTX-M-8 genes in Enterobacteriaceae isolated from public wastewater treatment plants in Brazil. Top, the association of bla CTX-M-15 with IS26 and ISEcp1. Bottom, the association of bla CTX-M-8 with IS26 and IS10. (t) truncated sequence

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MLST results revealed that E. coli strains belonged to new STs. Regarding K. pneumoniae, while the strain co-producing CTX-M-15 and SHV-28 belonged to the ST307, the CTX-M-8-producing strain belonged to the new ST1574 (Table 2). MLST also revealed that E. cloacae FSP1471/09 belonged to ST131.

S1-PFGE results showed that all wild-type strains possessed one or two high molecular weight plasmids, ranging from 48.5 to 209 kb (Table 2), which were successfully electroporated into E. coli Top 10 recipient strains. Each electrocompetent E. coli received only one of the high molecular weight plasmids, harboring bla CTX-M genes and their respective genetic platforms. In K. pneumoniae, the bla SHV-28 gene was not co-transferred with bla CTX-M-15, suggesting chromosomal location. Otherwise, E. coli strains harbored IncI1 plasmids carrying bla CTX-M-8, whereas the IncM1 backbone was detected in CTX-M-8-producing K. pneumoniae and E. cloacae. On the other hand, CTX-M-15-producing E. coli carried IncFIA and IncFIB, and the CTX-M-15-producing K. pneumoniae strain was not typeable by PBRT.

This study shows the presence of CTX-M-type extended-spectrum β-lactamases among members of the Enterobacteriaceae family recovered from public WWTPs in Brazil. The genetic background of bla CTX-M genes was associated with IS26 insertion sequences, harbored by high molecular weight plasmids. MLST analysis revealed new STs for CTX-M-15-producing E. coli, different from STs usually identified in clinical settings. So, the spread of this gene through sewage could be mediated by transferable IncF-type plasmids, which could be part of CTX-M-15 route from hospitals to the environment. An elegant comparison of resistance patterns in clinical- and sewage sludge-originated E. coli (Reinthaler et al. 2013) showed that resistance patterns found in medical environment are reflected in sludge samples, and also that the spread of cephalosporin resistance to the environment could be more effective than other kinds of resistance, due to strains’ fitness and/or changes in their genetic characteristics, like the presence of mobile elements.

Regarding the CTX-M-8 enzyme, this ESBL variant was first described in Brazil and originated a new cluster of CTX-M enzymes (Bonnet et al. 2000). Curiously, CTX-M-8-producing Enterobacteriaceae have been sporadically identified in clinical settings (Minarini et al. 2009; García-Fulgueiras et al. 2011; Sennati et al. 2012), whereas it has been prevalent among isolates from food-producing animals and chicken meat (Jouini et al. 2007; Dhanji et al. 2010; Aizawa et al. 2014; Eller et al. 2014; Kawamura et al. 2014; Casella et al. 2015), suggesting that the studied WWTPs include waste from animal sources. Indeed, the presence of CTX-M-8-producing bacteria was confirmed in the collected samples from all five WWTPs, showing new STs for E. coli and K. pneumoniae.

Besides CTX-M-type ESBLs, this study describes for the first time the detection of an environmental SHV-28-producing K. pneumoniae. This ESBL has already been reported in clinical samples from Brazilian studies (Cergole-Novella et al. 2010; Tollentino et al. 2011; Veras et al. 2011), supporting their dissemination from clinical to water environment.

In summary, we hereby report the spread of plasmidial-located IS26-associated bla CTX-M genes through five public WWTPs in Brazil, along with the first description of CTX-M-8 and SHV-28 in environmental samples. Identification of novel STs of CTX-M-type-producing E. coli and K. pneumoniae suggests that WWTPs provide an environmental hotspot for the transfer of bla CTX-M genes.

Nucleotide sequence accession numbers. KT001471 to KT001477.