Contents lists available at ScienceDirect Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid Research paper Identification and characterization of plasmid-mediated quinolone resistance determinants in Enterobacteriaceae isolated from healthy poultry in Brazil Joseane Cristina Ferreiraa, Rafael Antonio Casarin Penha Filhob, Ana Paula Yorika Kuayea, Leonardo Neves Andradea, Angelo Berchieri Juniorb, Ana Lúcia da Costa Darinia,⁎ a School of Pharmaceutical Sciences of Ribeirão Preto, São Paulo University (USP), Ribeirão Preto, SP 14040-903, Brazil b School of Agricultural and Veterinary Sciences, São Paulo State University (UNESP), Jaboticabal, SP 14884-900, Brazil A R T I C L E I N F O Keywords: qnr PMQR Food-producing animal Chicken ColE-like plasmid A B S T R A C T The expression of plasmid-mediated quinolone resistance (PMQR) genes confers low-level quinolone and fluoroquinolones resistance alone. However, the association to chromosomal resistance mechanisms determines an expressively higher resistance in Enterobacteriaceae. These mechanisms are horizontally disseminated within plasmids and have contributed to the emergence of bacteria with reduced susceptibility or resistant to therapies worldwide. The epidemiological characterization of PMQR dissemination is highly relevant in the scientific and medical context, to investigate the dissemination within enterobacteria, from different populations, including humans and food-producing animals. In the present study, 200 Enterobacteriaceae isolates were harvested from poultry with cloacal swabs and identified as Escherichia coli (90.5%), Escherichia fergusonii (5.5%), Klebsiella oxytoca (2.5%) and Klebsiella pneumoniae (1.5%). Among isolates evaluated, 46 (23%) harboured PMQR genes including qnrB (43/200), qnrS (2/200) and aac(6′)-Ib-cr (1/200). All isolates carrying PMQR genes showed multidrug-resistance phenotype. The 36 E. coli isolates showed 18 different PFGE types. All E. fergusonii isolates showed the same PFGE type. The two Klebsiella oxytoca belonged to two different PFGE types. The phylogenetic groups A, B1, and D were found among the E. coli harboring PMQR genes. Based on the phylogenetic analysis and PFGE, the population structure of E. coli isolates was diverse, even within the same farm. All isolates car- rying qnrB and qnrS genes also harboured ColE-like plasmids. The Southern blot hybridization using the S1-PFGE revealed that the qnrB genes were located on low molecular weight plasmids, smaller than 10Kb. Resistance plasmids were sequenced and showed 100% identity with plasmid pPAB19-3. The association of PMQR genes with mobile genetic elements, such as transferable plasmids, favours the selection and dissemination of (fluoro) quinolones resistant bacteria among food-producing animals, and may play an important role in the current increased prevalence of resistant bacteria in different environments reported worldwide. 1. Introduction The introduction of fluoroquinolones in clinical therapies occurred in the 1980s and the advantage of oral administration associated with the bactericidal activity against Gram-negative microorganisms con- tributed to the frequent election of this class of antimicrobials to treat infections caused by Escherichia coli, Salmonella and other Enterobacteriaceae in humans and animals, to date (Dalhoff, 2012; Vanni et al., 2014; Zawack et al., 2016). Among different fluoroquinolone resistance mechanisms, chromo- somal mutations in gyrA and/or parC genes in quinolone-resistance- determining region (QRDR), may change the antibiotic target site, consequently reducing the therapeutic efficacy. This mechanism asso- ciated to the expression of plasmid-mediated quinolone resistance (PMQR) genes increases the resistance level of the bacteria, frequently reaching impracticable minimal inhibitory concentration (MIC) (Harada and Asai, 2010; Gagliotti et al., 2008). The main PMQR de- terminants reported are the qnr genes (qnrA, qnrB, qnrC, qnrD and qnrS) that mediate resistance to quinolones by protecting the antimicrobial target protein, type II DNA topoisomerases, from the action of these antimicrobials (Rodriguez-Martinez et al., 2011). Moreover, there are other PMQR determinants, for example, aac(6′)-Ib–cr genes, which https://doi.org/10.1016/j.meegid.2018.02.003 Received 23 October 2017; Received in revised form 9 January 2018; Accepted 3 February 2018 ⁎ Corresponding author at: Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo (USP), Ribeirão Preto, SP 14040-903, Brazil. E-mail address: aldarini@fcfrp.usp.br (A.L.d.C. Darini). Infection, Genetics and Evolution 60 (2018) 66–70 Available online 07 February 2018 1567-1348/ © 2018 Published by Elsevier B.V. T http://www.sciencedirect.com/science/journal/15671348 https://www.elsevier.com/locate/meegid https://doi.org/10.1016/j.meegid.2018.02.003 https://doi.org/10.1016/j.meegid.2018.02.003 mailto:aldarini@fcfrp.usp.br https://doi.org/10.1016/j.meegid.2018.02.003 http://crossmark.crossref.org/dialog/?doi=10.1016/j.meegid.2018.02.003&domain=pdf codify acetyltransferase, an aminoglycoside modifying enzyme able to acetylate fluoroquinolones due to the “–cr variant” and the QepA and OqxAB efflux pump proteins, that decrease susceptibility to hydrophilic fluoroquinolones. The expression of these genes associated with other resistance mechanisms, such as alteration of the target site, porin al- terations, and overexpression of chromosomal efflux systems have been contributing to the increased level of quinolone resistance as well as the selection of resistant clones in enterobacteria (Strahilevitz et al., 2009; Tamang et al., 2011). The prevalence of qnrB genes is higher than other qnr genes in Enterobacteriaceae isolates from human, animal, and environment (Poirel et al., 2012) with about 80 different alleles assigned in the Lahey website (http://www.lahey.org/qnrStudies/), currently. The qnrB19 gene has been reported worldwide in Enterobacteriaceae isolated from healthy humans, clinical infections and food-producing animals (Dionisi et al., 2009; Karczmarczyk et al., 2010; Pallecchi et al., 2011). This gene has been reported in plasmids IncN, IncX and small rolling- circle-replication (RCR) plasmids (Carattoli, 2013). The Xer-mediated site-specific recombination is often identified in RCR plasmids, espe- cially ColE1, and has been suggested as the source of acquisition of the resistance gene (Tran et al., 2012). Most of the qnr-carrying plasmids share high similarity with ColE1 replicon (Carattoli, 2013). This study was conducted to evaluate the prevalence of PMQR determinants and characterize plasmids harboring these genes in Enterobacteriaceae iso- lated from poultry in Brazil. 2. Materials and methods 2.1. Isolation of bacteria From 2011 to 2012, a total of 200 isolates were screened from 200 healthy chickens from two different poultry Farms in Sao Paulo State, Brazil. The procedure for sampling was previously approved by the Institutional Animal Use and Ethics Committee (Proc. n. 12.1.248.53.7). The cloacal swab samples were seeded onto MacConkey agar plates containing ciprofloxacin (2 μg/mL) and incubated for 24 h/ 37 °C. Colonies were identified by biochemical standard methods, confirmed by API 20E test (BioMérieux, France) and, in some cases, the identification was performed by VITEK®2 (BioMérieux, France). 2.2. Screening of PMQR genes by multiplex PCR The total genomic DNA of the isolates was extracted as described previously (Bolano et al., 2001), and used in the PCR for the identifi- cation of the PMQR genes in all isolates. The genes qnrA, qnrB, qnrS (Cattoir et al., 2007), qnrC, qnrD (Wang et al., 2009), acc(6′)-Ib-cr, qepA and oqxAB (Minarini et al., 2008) were searched as previously de- scribed. Both strands of the amplicons were sequenced using the ABI 3730 sequencer (Applied Biosystems, USA) with the same primers used for PCR. 2.3. Antimicrobial susceptibility The antimicrobial susceptibility of E. coli isolates with PMQR genes were assessed by agar disk diffusion method following the re- commendations by Clinical Laboratory Standards Institute (CLSI, 2002), using breakpoints recommended by CLSI (2013). Fifteen anti- microbial drugs (Oxoid, UK) were tested: amoxillin/clavulanic acid (20/10 μg), piperacillin/tazobactam (100/10 μg), cefotaxime (30 μg), ceftazidime (30 μg), cefepime (30 μg), cefoxitin (30 μg), aztreonam (30 μg), ertapenem (10 μg), nalidixic acid (30 μg), ciprofloxacin (5 μg), levofloxacin (5 μg), enrofloxacin (5 μg), tetracycline (30 μg), genta- micin (10 μg), trimethoprim-sulfamethoxazole (1.25/23.75 μg) and chloramphenicol (30 μg). Multidrug-resistance (MDR) phenotype was characterized by non-susceptibility or resistance to at least one agent in three or more antimicrobial classes (Canton and Ruiz-Garbajosa, 2011; Magiorakos et al., 2012). 2.4. Pulsed-field gel electrophoresis (PFGE) Genomic DNA of qnr positive E. coli isolates were digested with restriction enzyme XbaI (Fermentas, USA) and the macrorestriction separation was carried out using pulsed-field gel electrophoresis (PFGE) in the CHEF-DRIII System (Bio-Rad Laboratories, USA). The assay was performed in 1,2% agarose PFGE gels at 6.0 V/cm with an initial/final switch time of 10/40 s and an angle of 120° at 14 °C for 24 h. Macrorestriction patterns were analysed using the BioNumerics 5.0 software (Applied Maths, USA). Relationship among PFGE-types was analysed by the Dice similarity index to obtain a dendrogram with clusters. The dendrogram was constructed using the unweighted-pair group method using average linkage algorithm (UPGMA). The minimum threshold value for similar PFGE patterns was defined at 85%. 2.5. Phylogenetic analysis The phylogenetic groups were determined according to the pre- viously described method (Clermont et al., 2000), classifying isolates to one of the four phylogenetic groups (A, B1, B2, or D) based on the presence of chuA, yjaA genes and TspE4C2 DNA fragment. 2.6. Plasmid replicon typing Plasmids were investigated by PCR-based Replicon Typing (PBRT) method using primers for 18 major incompatibility (Inc) groups de- scribed (FIA, FIB, FIC, HI2, I1-I, L/M, N, P, W, T, A/C, K, B/O, X, Y, F, FIIA, HI1) among Enterobacteriaceae as described previously (Carattoli et al., 2005). ColE plasmids were also searched by previously described method (Garcia-Fernandez et al., 2009). The Inc groups were de- termined in parental strains to characterize all plasmids, including the non-conjugative. 2.7. Determination of the PMQR To determine the plasmids harboring the PMQR genes, PFGE was performed, after digestion with S1 nuclease (S1-PFGE). The Lambda Ladder PFG Marker and Low Range PFG Marker (Biolabs, USA) were used as size standards. The Southern blot and hybridization were per- formed using DNA probes specific for quinolone resistance genes pre- pared with AlkPhos Direct Labeling and Detection System with CDP- Star (GE Healthcare Life Sciences, USA) (Sambrook et al., 1989). 2.8. Characterization of ColE-like plasmids The plasmids were extracted using PureYield™ Plasmid Miniprep System (Promega, USA), according to manufacturer instructions. The ColE-like plasmids were characterized using the PCR-based method described elsewhere (Pallecchi et al., 2010). The complete sequence of these plasmids was obtained by Sanger sequencing method (ABI 3730 sequencer, Applied Biosystems, USA) and analysed using Chromas Pro (Technelysium, Australia) and BLAST (http://www.ncbi.nlm.nih.gov/ BLAST/) softwares. 3. Results and discussion Antimicrobial resistance has been exponentially noticed in Enterobacteriaceae isolated from infected (Dalhoff, 2012) and colonized humans (Woerther et al., 2013) and also from food and food-producing animals (Bardon et al., 2013; Clemente et al., 2015; Liebana et al., 2013; Szmolka et al., 2011; Tamang et al., 2011). Two hundred ci- profloxacin resistant Enterobacteriaceae isolates were isolated from 200 cloacal swabs streaked on MacConkey agar plates, selecting one colony J.C. Ferreira et al. Infection, Genetics and Evolution 60 (2018) 66–70 67 http://www.lahey.org/qnrStudies/ http://www.ncbi.nlm.nih.gov/BLAST/ http://www.ncbi.nlm.nih.gov/BLAST/ per plate after incubation. Among the identified isolates, 90.5% were E. coli (181/200), 5.5% Escherichia fergusonii (11/200), 2.5% Klebsiella oxytoca (5/200) and 1.5% Klebsiella pneumoniae (3/200). Out of 200 Enterobacteriaceae isolates examined in this study, 23% (46/200) carried PMQR genes including qnrB (43/200), qnrS (2/200) and aac(6′)-Ib-cr (1/200). Among Qnr-producing isolates carrying qnrB, 33 were E. coli isolates (qnrB19, n= 29; qnrB5, n=4), 7 E. fergusonii (qnrB19), 2 K. oxytoca (qnrB19) and 1 K. pneumoniae (qnrB19). The qnrS1 was found in two E. coli isolates and aac(6′)-Ib-cr gene was found in one E. coli isolate (Table 1). None isolate was positive for qnrA, qnrC, qnrD, qepA and oqxAB genes. In our work, the most prevalent PMQR determinant was qnrB (n= 43), found in different bacterial species. Other research groups have also reported this gene as the most frequent PMQR determinant, followed by qnrS (Cattoir and Nordmann, 2009; Rodriguez-Martinez et al., 2011; Strahilevitz et al., 2009). The qnrB19 (n=39) was the most prevalent gene detected in our study, followed by qnrB5 (n= 4). The gene qnrB19 was reported in other countries, in E. coli from chickens, however, in most cases, in lower prevalence (Ben Sallem et al., 2014; Fortini et al., 2011; Literak et al., 2013; Oh et al., 2016) than described in the present study. Our results show a high prevalence of qnrB19 in poultry produced for meat consumption. There is few data about PMQR determinants either in humans or in animals in Brazil, therefore the prevalence of these genes is poorly understood. Another study in Brazil, also reported the gene qnrB19 carried by ColE- like plasmids in four ExPEC isolates from poultry (Cunha et al., 2017). In this study, all isolates carrying PMQR genes (46/46) showed MDR phenotype. Among these isolates, the resistance to antibiotics tested was: amoxicillin-clavulanic acid (100%), cefotaxime (100%), ceftazidime (56%), cefepime (15%), cefoxitin (63%), aztreonam (41%), nalidixic acid (NAL 100%), ciprofloxacin (CIP 91%), levofloxacin (LEV 82%), enrofloxacin (ENR 100%), tetracycline (89%), gentamicin (24%), trimethoprim-sulfamethoxazole (63%), and chloramphenicol (46%). None isolate showed resistance to piperacillin/tazobactam and erta- penem (Tables 1 and 2). NAL, CIP and LEV are mainly used to treat urinary tract infections and respiratory infections in human patients. However, ENR is a fluoroquinolone exclusively developed for use in veterinary medicine (Lopez-Cadenas et al., 2013) and has been ex- tensively used as a prophylactic measure in poultry farms, to reduce intestinal infections by Salmonella and other pathogens, which may have contributed for the selection of fluoroquinolone resistance among these animals. Furthermore, the oral use of ENR also contributes for selection of resistance to non-quinolone antimicrobials in commensal E. coli isolated from chickens (Jurado et al., 2015). The use of other an- tibiotic classes, such as beta-lactam antibiotics, could advantage co- selection of fluoroquinolones non-susceptible E. coli from retail poultry (Ingram et al., 2013), when mobile genetic elements (MGE), carrying multiple antimicrobial determinants are involved, as explained else- where (Canton and Ruiz-Garbajosa, 2011). As shown in Table 1, the 36 E. coli isolates carrying PMQR genes exhibited 18 different PFGE types. The qnrB resistance genes (qnrB19 and qnrB5) were present in different E. coli isolates classified in showed 15 PFGE types (A to O), demonstrating the dissemination of the re- sistance gene within a diverse bacterial population. Among the different PFGE types the major cluster “E” included 12 isolates producing qnrB19. Two different PFGE types were found in the two E. coli isolates carrying qnrS1 (Q and R). The E. coli carrying aac(6′)-Ib-cr belonged to Table 1 Phenotypic and genotypic characteristics of PMQR-harboring E. coli isolates from poultry. Isolates Species Farm Resistance PMQR gene Plasmid family Phylogenetic group PFGE-type 157a E. coli 2 AMC, CTX, CAZ, FOX, NAL, CIP, LEV, GEN, TET, SXT qnrB19 ColE, HI1, I1, F A A 167 E. coli 2 AMC, CTX, FOX, NAL, CIP, LEV, GEN, TET, SXT qnrB19 ColE, FIB, F A A 166a E. coli 2 AMC, CTX, CAZ, FOX, NAL, CIP, LEV, GEN, TET, SXT qnrB19 ColE, FIB, F A B 109 E. coli 2 AMC, CTX, CAZ, FOX, NAL, CIP, LEV, TET, SXT qnrB19 ColE, K A C 107 E. coli 2 AMC, CTX, CAZ, FOX, NAL, CIP, LEV, TET, SXT, CHL qnrB19 ColE, FIB, F D D 110 E. coli 2 AMC, CTX, CAZ, FOX, NAL, CIP, LEV, TET, SXT, CHL qnrB19 ColE, F D E 112 E. coli 2 AMC, CTX, CAZ, FOX, NAL, CIP, LEV, TET, CHL qnrB19 ColE, FIB, F D E 115 E. coli 2 AMC, CTX, CAZ, ATM, FOX, NAL, CIP, LEV, TET, CHL qnrB19 ColE, FIB, F D E 125a E. coli 2 AMC, CTX, NAL, CIP, LEV, TET, CHL qnrB19 ColE, FIB, F D E 127 E. coli 2 AMC, CTX, CAZ, ATM, FOX, NAL, CIP, LEV, TET, CHL qnrB19 ColE, FIB, F D E 135 E. coli 2 AMC, CTX, CAZ, ATM, FOX, NAL, CIP, LEV, TET, CHL qnrB19 ColE, FIB, F D E 138 E. coli 2 AMC, CTX, CAZ, ATM, FOX, NAL, CIP, LEV, TET, CHL qnrB19 ColE, FIB, F D E 141b E. coli 2 AMC, CTX, FOX, NAL, CIP, LEV, TET, CHL qnrB19 ColE, FIB, F D E 148 E. coli 2 AMC, CTX, CAZ, ATM, FOX, NAL, CIP, LEV, TET, CHL qnrB19 ColE, FIB, F D E 149 E. coli 2 AMC, CTX, CAZ, ATM, FOX, NAL, CIP, LEV, TET, CHL qnrB19 ColE, FIB, F D E 114b E. coli 2 AMC, CTX, CAZ, ATM, FOX, NAL, CIP, LEV, TET, CHL qnrB19 ColE, FIB, F D E 122 E. coli 2 AMC, CTX, CAZ, ATM, FOX, NAL, CIP, LEV, TET, CHL qnrB19 ColE FIB, F D E 176 E. coli 2 AMC, CTX, CAZ, FOX, NAL, CIP, LEV, GEN, TET, SXT qnrB19 ColE, FIA, FIB, F D F 39a E. coli 1 AMC, CTX, CAZ, FOX, NAL, CIP, LEV qnrB19 ColE, K, B/O A G 74 E. coli 1 AMC, CTX, CAZ, ATM, FOX, NAL, CIP, LEV, TET qnrB19 ColE, K A G 91 E. coli 1 AMC, CTX, CAZ, FOX, NAL, CIP, LEV qnrB5 ColE, K A G 123b E. coli 2 AMC, CTX, NAL, CIP, LEV, GEN, TET qnrB19 ColE, K A H 136 E. coli 2 AMC, CTX, FOX, NAL, CIP, LEV, GEN, TET qnrB19 ColE, K A H 146 E. coli 2 AMC, CTX, NAL, CIP, LEV, GEN, TET qnrB19 ColE, K A H 105 E. coli 2 AMC, CTX, FOX, NAL, CIP, LEV, GEN, TET qnrB19 ColE, FIA A H 147 E. coli 2 AMC, CTX, FOX, NAL, CIP, LEV, GEN, TET qnrB19 ColE, K A H 86 E. coli 1 AMC, CTX, ATM, NAL, CIP, LEV, GEN, TET qnrB5 ColE, FIB, FIC, F A N 106 E. coli 2 AMC, CTX, CAZ, FOX, NAL, CIP, LEV, GEN, TET, SXT qnrB19 ColE, FIA, F D I 143 E. coli 2 AMC, CTX, FOX, NAL, CIP, LEV, SXT qnrB19 ColE, K A J 12 E. coli 1 AMC, CTX, CAZ, ATM, FOX, NAL, CIP, LEV, CHL qnrB19 ColE, FIC, K, F A K 37 E. coli 1 AMC, CTX, CAZ, ATM, FOX, NAL, CIP, LEV, TET qnrB19 ColE, K, B/O A L 89 E. coli 1 AMC, CTX, CAZ, ATM, FEP, NAL, CIP, LEV, TET, CHL qnrB5 ColE, F B1 M 82 E. coli 1 AMC, CTX, FEP, NAL, CIP, TET qnrB5 ColE, I1, FIB, F A O 49a E. coli 1 AMC, CTX, CAZ, ATM, FEP, NAL, CIP, LEV, TET, SXT, CHL aac(6′)-Ib-cr FIB, F,Y B1 P 01 E. coli 1 AMC, CTX, CAZ, ATM, FEP, NAL, CIP, TET qnrS1 ColE, I1, FIB, F, A/C B1 Q 93 E. coli 1 AMC, CTX, ATM, FEP, NAL, CIP, LEV, TET, SXT, CHL qnrS1 ColE, HI1, I1, FIB, F A R AMC: amoxicillin-clavulanic acid, CTX: cefotaxime, CAZ: ceftazidime, FEP: cefepime, FOX: cefoxitin, ATM: aztreonam, NAL: nalidixic acid, CIP: ciprofloxacin, LEV: levofloxacin, TET: tetracycline, CHL: chloramphenicol, SXT: trimethoprim-sulfamethoxazole, GEN: gentamicin. The plasmids that carried PMQR genes are underlined. J.C. Ferreira et al. Infection, Genetics and Evolution 60 (2018) 66–70 68 a different PFGE type (P). Results in Table 2, show that all E. fergusonii carrying qnrB19 belonged to the same PFGE type (Ef A). Although, the two Klebsiella oxytoca isolates carrying qnrB19 were from different PFGE types (Table 2). The evaluation of the resistant E. coli population from farm 1 and farm 2 shows nine different PFGE types in each farm, carrying the resistance genes, without any common PFGE type shared between farms. These findings suggest that the dissemination of the resistance genes is most likely associated to MGE (e.g. plasmids), ex- changed among different isolates, rather than with a unique resistant isolate prevailing. The phylogenetic groups A, B1, and D were found among the E. coli harboring PMQR genes. Most isolates were identified in the phylogenetic group A (50%), other 42% of the isolates were classified in the phylogenetic group D and 8% of the isolates were identified phylogenetic group B1 (Table 1). None of the isolates be- longed phylogenetic group B2. In addition, 14 qnrB19-producing be- longed to phylogenetic group D. Based on the phylogenetic analysis and PFGE, the population structure of E. coli isolates was diverse, even within the same poultry farm, characterizing a non-clonal dissemina- tion and demonstrating the potential of these genes and MGEs to be maintained by different E. coli populations. Isolates within phylogenetic group B2 reported, as the most virulent, frequently causing infections were not detected. Nevertheless, among the phylogenetic groups iden- tified in the present work, group D has also been involved in extra- intestinal infections, causing concerns in clinical settings. The phylo- genetic groups A and B1 are considered less virulent, however, were also described in human extraintestinal infections (Pitout, 2012). As shown in Table 1, in all isolates, the qnrB and qnrS genes were harboured on ColE-like plasmids. Moreover, 43 E. coli isolates carrying qnrB in ColE-like plasmids, also carried other replicon types, including IncHI1 (n= 1), IncI1 (n=2), IncFIA (n= 3), IncFIB (n=17), IncFIC (n=2), IncF (n=22), IncK (n=12) and IncB/O (n= 2). E. coli iso- lates harboring qnrS1 showed the presence of IncHI1 (n=1), IncI1 (n=2), IncFIB (n= 2) IncF (n= 2) and IncA/C (n= 1). The E. coli isolate carrying aac(6′)-Ib-cr showed IncFIB, IncF, and IncY, however it was not possible to determine which plasmid harboured this gene. As shown in Table 2, E. fergusonii isolates carrying qnrB gene in ColE-like plasmids, also harboured IncHI1 (n= 4), IncI1 (n= 7) and IncF (n=8). K. oxytoca isolates harboring qnrB also showed the replicon types IncFIB (n= 2), IncI1 (n= 7) and IncF (n=8). The Southern blot hybridization using the S1-PFGE revealed that the qnrB19 and qnrB5 genes were located on low-molecular weight plas- mids, smaller than 10Kb.The plasmids sequences from isolates carrying qnrB19 gene were analysed using GenBank database tools, and 100% identity with plasmid pPAB19-3 was found (GenBank accession number JN985534). The sequenced plasmids were determined as ColE-like plasmids and the size was 2989 bp. The ColE-like backbone found in- cluded regions for plasmid replication and mobilization. The qnr region was located near the Xer specific recombination site. The ISEcp1 was found in these plasmids downstream from psp gene (activator of the stress-inducible) and from qnr gene. Moreover, the presence of this MGE in different poultry Farms, bacterial species and E. coli isolates of different PFGE-types and phylogenetic groups, suggests the evidence of lateral gene transfer. The ColE plasmids identified in all our isolates shared the same genetic sequence as those reported in E. coli from hospital, in Argentina, which has been circulating since 2007 in that country (Tran et al., 2012). However, now we report this replicon associated to PMQR de- terminants in isolates from food-producing poultry in Brazil, suggesting the capacity to disseminate. Moreover, it has been proposed that this RCR plasmid arrangement could play a role in the evolution of plasmids and present a model for DNA swapping between plasmid DNA mediated by site-specific recombination events at oriT and a Xer target site (Tran et al., 2012). The plasmids pPAB19-1, pPAB19-2, pPAB19-3 (pPAB19-3 also detected here), and pPAB19-4 share extensive homology among themselves and with other previously described small qnrB19-harboring plasmids (Tran et al., 2012). Overall, as summarized in Tables 1 and 2, a high prevalence of Enterobacteriaceae, including E. coli, E. fergusonii and K. oxytoca, were found carrying PMQR determinant genes harboured in ColE-like plas- mids. Moreover, these isolates also harboured other important replicon types. The increased resistance to quinolones and fluoroquinolones, determined by PMQR genes, was noticed in isolates that also showed resistance to other important antibiotics such as AMC, CTX, CAZ, ATM, GEN, CHL, TET. These characteristics were present in a diverse popu- lation of E. coli, as shown by PFGE and phylogeny, demonstrating that this dissemination of resistance has a non-clonal nature. The concern about the dissemination of PMQR genes is that these genes may facil- itate the selection of higher levels of quinolone resistance (Jacoby et al., 2014). It is difficult to determine the incidence and prevalence of this plasmid carrying these genes, however, silent dissemination may be occurring in many ecological settings and in different countries in South America. 4. Conclusion A high prevalence of PMQR determinants was found in Enterobacteriaceae isolated from healthy chickens. Different Enterobacteriaceae harboured these genes and the genes were found in two poultry Farms, showing the dissemination of resistant bacteria in food-producing animals, which may play an important role in the current increased prevalence of MDR bacteria in different environments reported worldwide. Therefore, the surveillance of resistant genes in the food chain is considered a key point to assist in the control of an- timicrobial resistance. Conflict of interest statement None to declare. Table 2 Phenotypic and genotypic characteristics of PMQR-harboring K. oxytoca, K. pneumoniae and E. fergusonii isolates from poultry. Isolates Species identified Farms Resistance PMQR gene Plasmid family PFGE 121a K. oxytoca 2 AMC, CTX, CAZ, FOX, NAL, CIP, LEV, TET, CHL qnrB19 ColE, FIB, F Ko A 137a K. oxytoca 2 AMC, CTX, CAZ, ATM, FOX, FEP, NAL, CIP, LEV, TET, SXT, CHL qnrB19 ColE, I1 Ko B 108a K. pneumoniae 2 AMC, CTX, NAL, CIP, LEV, TET, SXT, CHL qnrB19 ColE Kp A 117c E. fergusonii 2 AMC, CTX, NAL, CIP, LEV, TET, SXT qnrB19 ColE, I1, F Ef A 123a E. fergusonii 2 AMC, CTX, NAL, CIP, LEV, TET, CHL qnrB19 ColE, HI1, I1, F Ef A 151c E. fergusonii 2 AMC, CTX, NAL, CIP, LEV, TET, SXT, CHL qnrB19 ColE, HI1, I1, F Ef A 156b E. fergusonii 2 AMC, CTX, NAL, CIP, LEV, TET, SXT qnrB19 ColE, HI1, I1, F Ef A 157b E. fergusonii 2 AMC, CTX, CAZ, NAL, CIP, LEV, TET qnrB19 ColE, HI1, I1, F Ef A 169b E. fergusonii 2 AMC, CTX, NAL, CIP, LEV, TET, SXT qnrB19 ColE, I1, F Ef A 171b E. fergusonii 2 AMC, CTX, NAL, CIP, LEV, TET, SXT qnrB19 ColE, I1, F Ef A AMC: amoxicillin-clavulanic acid, CTX: cefotaxime, CAZ: ceftazidime, FEP: cefepime, FOX: cefoxitin, ATM: aztreonam, NAL: nalidixic acid, CIP ciprofloxacin, LEV: levofloxacin, TET: tetracycline, CHL: chloramphenicol, SXT: trimethoprim-sulfamethoxazole, GEN: gentamicin. 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http://refhub.elsevier.com/S1567-1348(18)30047-9/rf0215 http://refhub.elsevier.com/S1567-1348(18)30047-9/rf0215 http://refhub.elsevier.com/S1567-1348(18)30047-9/rf0215 Identification and characterization of plasmid-mediated quinolone resistance determinants in Enterobacteriaceae isolated from healthy poultry in Brazil Introduction Materials and methods Isolation of bacteria Screening of PMQR genes by multiplex PCR Antimicrobial susceptibility Pulsed-field gel electrophoresis (PFGE) Phylogenetic analysis Plasmid replicon typing Determination of the PMQR Characterization of ColE-like plasmids Results and discussion Conclusion Conflict of interest statement Acknowledgements References