Diversity of Multi-Drug Resistant Avian Pathogenic Escherichia coli (APEC) Causing Outbreaks of Colibacillosis in Broilers during 2012 in Spain

RESEARCH ARTICLE Diversity of Multi-Drug Resistant Avian Pathogenic Escherichia coli (APEC) Causing Outbreaks of Colibacillosis in Broilers during 2012 in Spain Marc Solà-Ginés 1 , Karla Cameron-Veas 1 , Ignacio Badiola 1 , Roser Dolz 1 , Natalia Majó 1,2 , Ghizlane Dahbi 3 , Susana Viso 3 , Azucena Mora 3 , Jorge Blanco 3 , Nuria Piedra-Carrasco 4 , Juan José González-López 4 , Lourdes Migura-Garcia 1 * 1Centre de Recerca en Sanitat Animal (CReSA)—Institut de Recerca i Tecnologia Agroalimentàries (IRTA), Campus UAB, Barcelona, Spain, 2Departament de Sanitat i Anatomia Animals, Universitat Autònoma de Barcelona, Bellaterra (Cerdanyola del Vallés), Spain, 3Laboratorio de Referencia de E.coli, Departamento de Microbioloxía e Parasitoloxía, Facultade de Veterinaria, Universidade de Santiago de Compostela, Lugo, Spain, 4Servei de Microbiologia, Hospital Vall d’Hebron, Universitat Autònoma de Barcelona, Barcelona, Spain *[email protected] Abstract Avian pathogenic Escherichia coli (APEC) are the major cause of colibacillosis in poultry production. In this study, a total of 22 E.coli isolated from colibacillosis field cases and 10 avian faecal E.coli (AFEC) were analysed. All strains were characterised phenotypically by susceptibility testing and molecular typing methods such as pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). The presence of 29 virulence genes associated to APEC and human extraintestinal pathogenic E.coli (ExPEC) was also evaluated. For cephalosporin resistant isolates, cephalosporin resistance genes, plasmid location and replicon typing was assessed. Avian isolates belonged to 26 O:H serotypes and 24 sequence types. Out of 22 APEC isolates, 91% contained the virulence genes predictors of APEC; iutA,hlyF,iss,iroN and ompT. Of all strains, 34% were considered ExPEC. PFGE analysis demonstrated a high degree of genetic polymorphism. All strains were multi-resistant, including those isolated from healthy animals. Eleven strains were resistant to cephalosporins; six contained bla CTX-M-14 , two bla SHV-12 , two bla CMY-2 and one bla SHV-2 . Two strains harboured qnrA, and two qnrA together with aac(6’)-Ib-cr. Additionally, the emergent clone O25b:H4-B2-ST131 was isolated from a healthy animal which harboured bla CMY-2 and qnrS genes. Cephalosporin resistant genes were mainly associated to the presence of IncK replicons. This study demonstrates a very diverse population of multi-drug resistant E.coli containing a high number of virulent genes. The E.coli population among broilers is a reservoir of resistance and virulence-associated genes that could be transmitted into the community through the food chain. More epidemiological studies are necessary to identify clonal groups and resistance mechanisms with potential relevance to public health. PLOS ONE | DOI:10.1371/journal.pone.0143191 November 23, 2015 1/14 OPEN ACCESS Citation: Solà-Ginés M, Cameron-Veas K, Badiola I, Dolz R, Majó N, Dahbi G, et al. (2015) Diversity of Multi-Drug Resistant Avian Pathogenic Escherichia coli (APEC) Causing Outbreaks of Colibacillosis in Broilers during 2012 in Spain. PLoS ONE 10(11): e0143191. doi:10.1371/journal.pone.0143191 Editor: W.C. Yam, The University of Hong Kong, CHINA Received: June 3, 2015 Accepted: November 1, 2015 Published: November 23, 2015 Copyright: © 2015 Solà-Ginés et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper. Funding: This work was partially supported by the grants AGL201128836 and AGL2013-47852-R from the Ministerio de Economía y Competitividad (España) and grants CN2012/303 and EM2014/001 (Consellería de Cultura, Educación e Ordenación Universitaria, Xunta de Galicia and the European Regional Development Fund, ERDF). Work from LMG is supported by the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA) and the European Social Fund. The funders Introduction Escherichia coli is a bacterium widespread in the intestine of animals and humans, and a pathogen that can induce enteric and extraintestinal infections. In particular, avian pathogenic E.coli (APEC) is the main cause of colibacillosis in poultry farms; a syndrome associated to airsacculitis, perihepatitis, pericarditis, and sometimes fatal septicemia. APEC strains are responsible for the mortality of 3–4% of the animals in a farm, and for the reduction of 2–3% of egg production [1], resulting in an economic burden to the poultry industry [2]. In many cases, the fundamental cause of the disease remains unclear, since the infection with E.coli is associated to the presence of Mycoplasma gallisepticum or respiratory viruses, such as Newcastle virus or Infectious Bronchitis virus [3]. Several virulence genes are implicated in avian colibacillosis such as adhesins, toxins, antihost defence factors, iron acquisition systems, autotransporters and the IbeA protein [4]. Subtractive hybridization studies have demonstrated sequence homology between specific DNA regions of APEC and human extraintestinal pathogenic E.coli (ExPEC) [5]. Additionally, the presence of similar virulence genes found in both, APEC and ExPEC strains, suggested that APEC strains may act as zoonotic pathogens and reservoir of virulence causing human infections [6–8]. According to Johnson et al. (2003), a strain could be considered ExPEC if exhibits two or more of the following virulence genes; pap (P fimbriae), sfa/foc (S/F1C fimbriae), afa/ dra (Dr binding adhesins), iutA (aerobactin receptor), and kpsM II (group 2 capsule synthesis) [9]. ExPEC strains are more often derived from virulence-associated B2 and D phylogroups [10]. The successful treatment of avian colibacillosis caused by APEC strains mainly depends on the use of antimicrobials. However, increasing resistance to critically important antimicrobials, such as third-generation cephalosporins and fluoroquinolones, is nowadays common in E.coli from poultry origin [11]. These resistances can be transmitted to humans via the food supply [12,13]. In particular, E.coli producing extended-spectrum beta-lactamases (ESBLs) and plasmid mediated AmpC beta-lactamases have increased considerably in the last years [14]. Normally, these genes are located on plasmids, and can be transferred by conjugation to other bacterial species [11]. Some of the virulence factors for APEC and ExPEC can also be harboured on plasmids. Particularly, ColV plasmids yield some virulence genes such as hlyF, ompT,iss and cvaC surrounding the replicon RepFIB [15]. Several studies have described APEC strains in the literature [16]. However, not many studies have combined extensive characterization at the serotype level, virulence-associated genes, molecular typing techniques, molecular determination of resistance mechanisms and mobile genetic elements involved in transfer of resistance. For this reason, the objective of this study was to discriminate and to perform such characterization of highly pathogenic E.coli causing outbreaks of colibacillosis in 13 different broiler farms throughout Spain, and compare them to avian faecal E.coli (AFEC) obtained from healthy animals. Additionally, the identification of clones more prone to cause disease has been assessed. Materials and Methods Isolation A total of 22 tissue swabs of culled-animals affected with colibacillosis arrived to the laboratory between January and March 2012. The samples were obtained as part of routine care. Samples were taken from chickens already sacrificed for diagnostic purposes following the procedures according to the requirements of the Ethics Committee of Animal and Human Experimentation of the Universitat Autònoma de Barcelona (Permit Number DMAH-4239 that specifically APEC in Broilers in Spain PLOS ONE | DOI:10.1371/journal.pone.0143191 November 23, 2015 2/14 had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. permits euthanasia of chickens). Animals were euthanized using intravenous sodium pentobarbital (100 mg/kg, Dolethal, Vétoquinol, Cedex, France) in the wing vein. The method to sacrifice the animals follows the welfare rules stated in the European Directive 86/609/CEE. None of the authors of this manuscript were involved in manipulating or sacrificing the chickens. All the samples were collected from clinical cases submitted to the Diagnostic Service of the Veterinary School of the Universitat Autònoma de Barcelona. Swabs were taken from 13 broiler farms located in nine different regions of Spain (Fig 1). Ten E.coli strains isolated from faeces of healthy animals collected in nine farms were also included in the study. The samples were plated onto MacConkey agar and incubated overnight at 37°C. Three lactose-positive colonies for each plate were selected and confirmed to be E.coli by PCR [17]. Subsequently, one representative was selected for further studies. Serotyping Determination of O and H antigens was carried out using the method previously described by Guinée et al. with all available O (O1 to O181) and H (H1 to H56) antisera [18]. Non-typeable isolates were denoted as ONT or HNT and non-motile isolates were denoted as HNM. All antisera were obtained and absorbed with the corresponding cross-reacting antigens to remove the nonspecific agglutinins. The O and H antisera were produced in the Laboratorio de Referencia de E.coli (LREC, Lugo, Spain). O25a and O25b subtypes were determined by PCR [19]. Fig 1. PFGE dendogram illustrating the phenotypic and genotypic relationship of the strains and the cephalosporin resistance genes. PFGE, pulsed-field gel electrophoresis; CR., cephalosporin resistance genes; FQ., flouoroquinolone resistance genes; Am: Ampicillin (WT8mg/L); Ctx: Cefotaxime (WT0.25mg/L); Caz: Ceftazidime (WT0.5mg/L); Fox: Cefoxitin (WT8mg/L); Fep: Cefepime (WT0.125mg/L); Nal: Nalidixic acid (WT16mg/L); Ci: Ciprofloxacin (WT0.064mg/L); Gm: Gentamicin (WT2mg/L); Sm: Streptomycin (WT16mg/L); Km: Kanamycin (WT8mg/L);. Cm: Chloramphenicol (WT16mg/L); Ff: Florfenicol (WT16mg/L); Tm: Trimethoprim (WT2mg/L); Su: Sulphamethoxazole (WT64mg/L); Tc: Tetracycline (WT8mg/L); Cs: Colistin (WT2mg/L). a Isolates are divided in APEC (N) and AFEC (GN) strains. b Location of the strains is named in order to the different regions of Spain where farms were localized. doi:10.1371/journal.pone.0143191.g001 APEC in Broilers in Spain PLOS ONE | DOI:10.1371/journal.pone.0143191 November 23, 2015 3/14 Phylogeny, pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) Isolates were separated in phylogroups (A, B1, B2, C, D, E or F) according to a method previously described [20,21]. PFGE was performed as described elsewhere [22]. The results were analysed by Fingerprinting II Informatix software (Applied Maths, Sint-Martens-Latem, Belgium). PFGE-types were separated based on differences of at least one band in the restriction profiles. The analysis of the bands generated was carried out using the Dice coefficient and unweighted pair group method with arithmetic averages (optimization of 1.25% and position tolerance 1.25%). MLST was carried out as previously described according to the protocol and primers specified on the E.coli MLST web site (http://mlst.ucc.ie/mlst/dbs/Ecoli)[23]. Sequences were analysed with Vector NTI advance 11 software (InforMax, Inc., Bethesda, MD). Detection of virulence-associated genes All strains were tested by PCR for 29 ExPEC and APEC virulence-associated genes (Tables 1 and 2)[13,24]. The genes described previously by Johnson et al. (2008) as the minimal predictors of APEC virulence; iroN,ompT,hlyF,iutA and iss were detected by a multiplex PCR [24]. Virulence scores were calculated for each isolate as the sum of all virulence-associated genes detected; pap,sfa-foc and kpsM II were counted only once. Statistical analysis Differences in the prevalence between different groups were determined by Fisher’s exact test as described before [25]. Virulence scores were compared by the use of Mann-Whitney U test. Statistical analyses were performed using GraphPad Prsim, version 3.1 software (GraphPad Software, Inc., San Diego, CA). Antimicrobial susceptibility testing Disc diffusion was performed according to CLSI guidelines using the following discs (Oxoid, Basingstoke, UK): cefoxitin, 30 mg; cefepime, 30 mg; ceftazidime, 30 mg; cefotaxime, 30 mg; cefotaxime+clavulanic acid, 30+10 mg; and ceftazidime+clavulanic acid, 30+10 mg. The disc combinations of cefotaxime and cefotaxime/clavulanic acid, ceftazidime and ceftazidime/clavulanic acid were used for the identification of ESBLs; cefoxitin was used for the detection of AmpC-type beta-lactamase [26]. All isolates were susceptibility tested using a minimum inhibitory concentration (MIC)-based broth microdilution (VetMIC GN-mo, National Veterinary Institute, Uppsala, Sweden) as described before [25]. Isolates were considered to be wild type (WT) or non-WT based on epidemiological cut-off values according to EUCAST (http://www. eucast.org/). Resistance genes All strains exhibiting resistance to third-generation cephalosporins (cefotaxime and ceftazidime) were tested by PCR methods for the presence of the bla CTX-M ,bla SHV ,bla TEM ,bla CMY-1 and bla CMY-2 genes as described by Hasman et al.[27]. Detection of plasmid-mediated AmpC beta-lactamase genes was assessed by multiplex PCR [28]. Sequencing of both strands of amplicons was performed. The presence of the fluoroquinolone resistance genes aac(6’)-Ib-cr,qnrA, qnrB,qnrS,qepA and oqxAB was also assessed [29,30]. APEC in Broilers in Spain PLOS ONE | DOI:10.1371/journal.pone.0143191 November 23, 2015 4/14 Plasmid DNA analysis Isolates exhibiting resistance to cephalosporins were selected for plasmid characterization. Plasmid replicons tested were elected according to the presence of determinant resistance genes (HI1, HI2, I1, X, L/M, N, FIA, FIB, W, Y, P, FIC, A/C, T, FIIA and K) [31] and were identified using the PCR-based replicon typing method previously described [32,33]. Plasmids detection and sizing was performed on all the isolates by S1-nuclease PFGE of total DNA [34]. Restriction fragments from S1-PFGE gels were transferred onto a positively charged nylon membrane and hybridised with specific probes for bla CTX-M-14 ,bla TEM ,bla SHV ,bla CMY and for each replicon that was previously identified. Results A total of 22 E.coli were recovered from 13 different farms distributed throughout Spain during 2012. Additionally 10 isolates from healthy animals collected in nine different farms were also included in the study to make a total of 32 E.coli isolates. Serotypes A total of 16 different O serogroups, 15 flagellar H antigens and 26 different O:H serotypes were identified (Table 1). The most prevalent serotypes were: O3:H26, O5:H10, O5:H51 and O78:H9. Additionally, the emergent clone O25b:H4 was detected in a commensal isolate. Phylogeny, PFGE and MLST Within the APEC strains (n = 22) the phylotyping identified six strains belonging to group A (27%), six to group B1 (27%), four to group D (18%), three to group E (14%), two to group C (9%) and one to group B2 (5%). For the commensal strains (n = 10), 60% belonged to phylogroup A, and the remaining 40% to B1, B2, D and F phylogroups (10% each) (Table 1). XbaI-PFGE analysis showed a high degree of genetic polymorphism. A total of 31 different PFGE restriction profiles were identified among the 32 E.coli isolates (Fig 1). Only two isolates were epidemiologically related and belonged to the same farm. Among the APEC subgroup (n = 22), MLST analyses identified 18 STs, while six STs were found among AFEC isolates (n = 10) (Table 1). Within the APEC strains, four belonged to the ST168 clonal complex (Cplx), three to the ST350 Cplx, two to ST156 Cplx, and two to the highly pathogenic ST23 Cplx (Table 1). Within the AFEC isolates, the most common Cplx was ST10 (n = 3), followed by ST165 Cplx (n = 3) (Table 1). The emergent pandemic multirresistant clone O25b:H4-B2-ST131 was found among the AFEC isolates. Detection of virulence-associated genes The prevalence of 29 virulence-associated genes is shown in Tables 1and 2. Regarding the five virulence genes associated to APEC (Fig 1); 81%, 78%, 75%, 72% and 72% of the 32 E.coli strains yielded amplicons for iss,ompT,iutA,iroN and hlyF, respectively (Table 1). The prevalence of these genes was higher in APEC isolates (91% of the APEC strains harboured all of the mentioned genes), when compared to the AFEC isolates. In general, the presence of virulenceassociated genes in AFEC strains was low, with 80% of the strains having from zero to three of the previously mentioned virulence genes (Table 1). According to the number of virulenceassociated genes, 34% of the isolates were considered ExPEC. APEC in Broilers in Spain PLOS ONE | DOI:10.1371/journal.pone.0143191 November 23, 2015 5/14 Table 1. Distribution of virulence-associated gene profiles, phylogeny, serotyping and MLST results among all 32 strains. Isolate Phylo. Serotype ST Cplx Virulence gene profiles a AFEC GN2215 D ONT:H4 ST117 iss,fimH,fyuA,irp-2,cdtB,traT,malX,tsh GN2216 A O53:H18 ST10 ST10 Cplx iss,fimH,traT GN2221 B2 O25b:H4 ST131 iroN,ompT,iutA,iss,fimH,cvaC,irp-2,kpsM II-K1,traT,ibeA,malX,usp,tsh GN2222 F O83:HNT ST648 iroN,ompT,iutA,iss,fimH,cvaC,astA,kspM II-K5,traT,malX,tsh GN2223 A ONT:HNT ST10 ST10 Cplx fimH,fyuA,irp-2,traT GN2233 A O3:H26 ST165 ST165 Cplx fimH,fimAvMT78,traT GN2234 A O2:H40 ST10 ST10 Cplx iroN,ompT,iss,fimH,fyuA,traT GN2235 A O3:HNM ST165 ST165 Cplx fimH,fimAvMT78,traT GN2236 B1 O127: H37 ST297 ompT,iutA,iss,fimH,cvaC,traT,malX,tsh GN2237 A O3:H26 ST189 ST165 Cplx fimH,fimAvMT78,traT APEC N107 B1 ONT:H16 ST295 iroN,ompT,hlyF,iutA,iss,fimH,cvaC,traT,tsh N108 C O78:H9 ST23 ST23 Cplx iroN,ompT,hlyF,iutA,iss,fimH,fyuA,astA,traT,tsh N109 B1 ONT:H28 ST156 ST156 Cplx iroN,ompT,hlyF,iutA,iss,fimH,astA,irp-2,traT N116 D O25a: [H4] ST624 iroN,ompT,hlyF,iutA,iss,fimH,cvaC,kpsM III,traT,malX N113 A O5:H10 ST93 ST168 Cplx iroN,ompT,hlyF,iutA,iss,astA,kpsM II-K2 N114 A O6:H16 ST48 ST10 Cplx iroN,ompT,hlyF,iutA,iss,fimH,fimAvMT78,kpsM III,traT N125 A O88:HNM ST1137 iroN,ompT,hlyF,iutA,iss,fimH,cvaC,astA,fimAvMT78,traT N126 B1 ONT:H51 ST889 iroN,ompT,hlyF,iutA,iss,fimH,cvaC,traT,tsh N127b D ONT:H51 ST156 ST156 Cplx iroN,ompT,hlyF,iutA,iss,fimH,traT,tsh N128 E ONT:H27 ST350 ST350 Cplx iroN,ompT,hlyF,iutA,iss,fimH,cvaC,astA,irp-2,fimAvMT78,papEF,papG,traT, tsh N129 B1 O15:H10 ST101 ST101 Cplx iroN,ompT,hlyF,iutA,iss,cvaC,astA,sfa/focDE,traT,tsh N130 A O5:H51 ST93 ST168 Cplx iroN,ompT,hlyF,iutA,iss,astA,kspM II-K2 N131 A O5:H51 ST93 ST168 Cplx iroN,ompT,hlyF,iutA,iss,astA,kspM II-K2 N132 E O102: H25 ST57 ST350 Cplx iroN,ompT,hlyF,iutA,iss,fimH,traT,tsh N133 A O5:H10 ST93 ST168 Cplx iroN,ompT,hlyF,iutA,iss,kpsM II-K2 N134 B1 O159: H28 ST539 ompT,hlyF,iutA,iss,fimH,irp-2,traT N135 B2 O2:H1 ST429 iroN,ompT,hlyF,iutA,iss,fimH,fyuA,cvaC,irp-2,kpsM II-K1,traT,ibeA,malX,usp, tsh N136 D ONT:H4 ST830 iroN,ompT,hlyF,iutA,iss,fimH,fyuA,traT,malX (Continued) APEC in Broilers in Spain PLOS ONE | DOI:10.1371/journal.pone.0143191 November 23, 2015 6/14 Statistical analysis Significant differences (P= 0.035) were observed in the number of virulence-associated genes (hlyF,astA,iroN,iutA and ompT) found between APEC (mean, 9.2; range 5 to 16) and AFEC (mean, 6.7; range 3 to 13) isolates (Table 2). Statistical difference was found also comparing the virulence-associated genes of the isolates of phylogroup A for APEC and AFEC (P=0.013;virulence score 7.2 vs 4.2). The strains belonging to phylogroup B2 exhibited the highest virulence score (mean, 14.5; range, 13 to 16) whereas the phylogroup A exhibited the lowest (mean, 5.8; range, 3 to 11). Additionally, significant differences were observed comparing B2 and D phylogroups (mean, 11.1; range 8 to 16) with A and B1 phylogroups (mean 7.4; range 3 to 13) (P= 0.008). Antimicrobial susceptibility testing and resistance genes All the analyzed strains were multi-resistant (resistant to more than 3 antimicrobial families), including those isolated from healthy animals. Furthermore, 50% were resistant to more than eight antimicrobials. Susceptibility testing detected 11 strains resistant to cephalosporins (34%); six bla CTX-M-14 , two bla SHV-12 , two bla CMY-2 and one bla SHV-2 . Two of these strains belonged to AFEC isolates. Two isolates were resistant to cefoxitin, and the resistance mechanism involved could not be determined. In addition, 88% of the isolates were resistant to nalidixic acid and 91% to ciprofloxacin. Additionally, 91% of the strains were resistant to tetracycline, 78% to ampicillin, 69% to streptomycin, 63% to sulfamethoxazole, 59% to trimethoprim 34% to cefotaxime, 31% to ceftazidime, 19% to kanamycin, 16% to gentamicin, 13% to cefoxitin, 13% to chloramphenicol and 6% to florfenicol. No resistance to cefepime and colistin was observed among the isolates (Fig 1). The presence of the qnrS gene was only confirmed in the isolate belonging to O25b: H4-B2-ST131. Finally, two of the APEC strains exhibited qnrA and two qnrA together with aac (6’)-Ib-cr. The genes qnrB,qepA and oqxAB were not found in this strain collection. Plasmid localisation of cephalosporin resistance genes PCR-based replicon typing among the 11 cephalosporin resistant E.coli isolates showed that IncFIB replicon was present in all analysed isolates (Table 3). The replicons IncI1, IncN, IncK, IncY, IncP, IncFIA, IncHI1 and IncHI2 were also detected (Table 3). Table 1. (Continued) Isolate Phylo. Serotype ST Cplx Virulence gene profiles a N137 D O11:H15 ST3161 iroN,ompT,hlyF,iutA,iss,fimH,cvaC,traT N138 C O78:H9 ST650 ST23 Cplx iroN,ompT,hlyF,iutA,iss,fimH,traT N139 B1 O45:H8 ST533 iroN,ompT,hlyF,fimH,astA,traT,malX N140 E O119: H27 ST350 ST350 Cplx iutA,fimH,astA,fimAvMT78 Phylo, phylogroup; ST, sequence type; Cplx, clonal complex. Adhesins fimH (D-mannose-specific adhesin of type I fimbriae), fimAvMT78 (FimA variant MT78 of type 1 fimbriae), papEF and papG (P fimbria subunits), and sfa/focDE (S fimbrial adhesin/putative F1C fimbrial adhesin); toxins cdtB (cytolethal distending toxin), hlyF (hemolysin F), and astA (EAST1, enteroaggregative E. coli heat-stable toxin); siderophores fyuA (yersiniabactin), iutA (aerobactin), iroN (novel catecholate siderophore receptor), and irp-2 (iron repressible associated with yersiniabactin synthesis); protectins kpsM (groups II and III, specifically targeting the K1, K2 and K5 genes of group II capsules), cvaC (ColV, colicin V from serum resistance-associated plasmids), iss (surface exclusion serum survival protein), and traT (serum resistance); miscellaneous virulence genes ompT (protease), ibeA (invasion of brain endothelium), malX (PAI, pathogenicity island marker), and usp (uropathogenic-specific protein, bacteriocin). a Virulence-associated genes shown in boldface are the five genes characteristics of APEC strains. doi:10.1371/journal.pone.0143191.t001 APEC in Broilers in Spain PLOS ONE | DOI:10.1371/journal.pone.0143191 November 23, 2015 7/14 Table 2. Distribution and characterization of virulence-associated genes and phylogroups of the 32 isolates. Virulence gene (s) a Total isolates (%) (n = 32) APEC isolates (%) (n = 22) AFEC isolates (%) (n = 10) A (%) (n = 12) B1 (%) (n = 7) B2 (%) (n = 2) D (%) (n = 5) C, E and F (%) (n = 6) B2/D (%) (n = 7) A/B1 (%) (n = 19) A/ APEC (%) (n = 6) A/ AFEC (%) (n = 6) P value b APEC vs AFEC P value b A/ APEC vs A/ AFEC P value b B2/D vs A/ B1 Adhesins fimH 28 (88%) 18 (82%) 10 (100%) 8 (67%) 7 (100%) 2 (100%) 5 (100%) 5 (83%) 7 (100%) 15 (79%) 2 (33%) 6 (100%) fimAvMT78 7 (22%) 4 (18%) 3 (30%) 5 (42%) 0 2 (100%) 02 (33%) 2 (29%) 5 (26%) 2 (33%) 3 (50%) papEF 1 (3%) 1 (5%) 0 0 0001 (17%) 0000 papG 1 (3%) 1 (5%) 0 0 0001 (17%) 0000 sfa/focDE 1 (3%) 1 (5%) 0 0 1 (14%) 00001(5%) 0 0 afa/draBC 0 0 0 0 00000 0 00 Toxins cnf1 0 0 0 0 00000 0 00 cdtB 1 (3%) 0 1 (10%) 0 0 0 1 (20%) 01 (15%) 000 sat 0 0 0 0 00000 0 00 hlyA 0 0 0 0 00000 0 00 hlyF*22 (72%) 22 (100%) 0 6 (50%) 6 (86%) 1 (50%) 4 (80%) 4 (67%) 5 (71%) 12 (63%) 6 (100%) 0<0.0001 0.0022 astA 12 (38%) 11 (50%) 1 (10%) 5 (42%) 3 (43%) 01 (20%) 3 (50%) 0 8 (42%) 5 (83%) 0 0.0496 0.0152 tsh 12 (38%) 8 (36%) 4 (40%) 0 4 (57%) 2 (100%) 2 (40%) 4 (67%) 4 (57%) 4 (21%) 0 0 Siderophores fyuA 9 (28%) 5 (23%) 4 (40%) 2 (17%) 1 (14%) 2 (100%) 3 (60%) 1 (17%) 5 (71%) 3 (16%) 0 2 (33%) 0.0138 iutA*24 (75%) 21 (95%) 2 (20%) 6 (50%) 6 (86%) 2 (100%) 4 (80%) 6 (100%) 6 (86%) 12 (63%) 6 (100%) 0<0.0001 0.0022 iroN*24 (72%) 21 (95%) 3 (30%) 7 (58%) 6 (86%) 2 (100%) 4 (80%) 5 (83%) 6 (86%) 13 (68%) 6 (100%) 1 (17%) 0.0003 0.0152 irp-2 8 (25%) 5 (23%) 3 (30%) 1 (8%) 3 (43%) 2 (100%) 1 (20%) 1 (17%) 3 (43%) 4 (21%) 0 1 (17%) Protectins kpsM II 7 (22%) 5 (23%) 2 (20%) 4 (33%) 0 2 (100%) 01 (17%) 2 (29%) 4 (21%) 4 (67%) 0 kpsM II-K1 2 (6%) 1 (5%) 1 (10%) 0 0 2 (100%) 002 (29%) 000 kpsM II-K2 4 (13%) 4 (18%) 0 4 (33%) 000004(2%) 4 (67%) 0 kpsM II-K5 1 (3%) 0 1 (10%) 0 0001 (17%) 0000 kpsM III 2 (6%) 2 (9%) 0 1 (8%) 0 0 1 (20%) 01 (15%) 1 (5%) 1 (17%) 0 cvaC 11 (34%) 9 (41%) 3 (30%) 1 (8%) 4 (57%) 2 (100%) 2 (40%) 2 (33%) 4 (57%) 5 (26%) 1 (17%) 0 iss*26 (81%) 20 (91%) 6 (60%) 8 (67%) 7 (100%) 2 (100%) 5 (100%) 5 (83%) 7 (100%) 15 (79%) 6 (100%) 2 (33%) (Continued) APEC in Broilers in Spain PLOS ONE | DOI:10.1371/journal.pone.0143191 November 23, 2015 8/14 Plasmid-sizes varied between 90 and 120-kb, with the exception of two plasmids; pST131-2 and pST350-1 with 250 and 180-kb, respectively (Table 3). The most common incompatibility group detected by Southern blot was IncK (n = 5). Specific probe hybridization of S1-digested DNA demonstrated that isolate GN2221 harboured bla CMY-2 gene in a plasmid containing replicon IncK (pST131-3). The isolate N135 harbouring also bla CMY-2 gene in a 90-kb plasmid contained IncK and IncP replicons. Isolate GN2233 yielded bla CTX-M-14 in a 90-kb plasmid with two replicons (IncK and IncY). The presence of bla SHV-12 was confirmed in two isolates having a 120 and 90-kb plasmids of the IncI1 and unknown replicon family, respectively. N109 harboured bla SHV-2 in an IncK plasmid (pST156-2). Finally, no plasmid location could be confirmed for the CTX-M-14 gene of isolate N127b. Discussion This study has demonstrated the presence of different clones of APEC causing outbreaks of colibacillosis during the same period of time in different broiler farms from different regions of Spain. All these isolates were multirresistant, therefore therapeutic success may have been compromised, causing a serious economic burden to the broiler industry. Moreover, the AFEC strains were also resistant to critically important antimicrobials such as cephalosporins and Table 2. (Continued) Virulence gene (s) a Total isolates (%) (n = 32) APEC isolates (%) (n = 22) AFEC isolates (%) (n = 10) A (%) (n = 12) B1 (%) (n = 7) B2 (%) (n = 2) D (%) (n = 5) C, E and F (%) (n = 6) B2/D (%) (n = 7) A/B1 (%) (n = 19) A/ APEC (%) (n = 6) A/ AFEC (%) (n = 6) P value b APEC vs AFEC P value b A/ APEC vs A/ AFEC P value b B2/D vs A/ B1 traT 27 (84%) 17 (77%) 10 (100%) 8 (67%) 7 (100%) 2 (100%) 5 (100%) 5 (83%) 7 (100%) 15 (79%) 2 (33%) 6 (100%) Miscellaneous ompT*26 (78%) 22 (100%) 4 (40%) 7 (58%) 7 (100%) 2 (100%) 4 (80%) 3 (50%) 6 (86%) 14 (74%) 6 (100%) 1 (17%) 0.0002 0.0152 ibeA 2 (6%) 1 (5%) 1 (10%) 0 0 2 (100%) 002 (29%) 000 malX 8 (25%) 4 (18%) 4 (40%) 0 1 (14%) 2 (100%) 3 (60%) 1 (17%) 5 (71%) 1 (5%) 0 0 0.0018 usp 2 (6%) 1 (5%) 1 (10%) 0 0 2 (100%) 3 (60%) 05 (71%) 0 0 0 0.0003 Mean (range) virulence score c 8.2 (3– 16) 9.2 (5– 16) 6.7 (3– 13) 5.8 (3– 11) 10 (8– 13) 14.5 (13– 16) 9.8 (8– 11) 9.2 (5– 14) 11.1 (8–16) 7.4 (3– 13) 7.2 (5– 11) 4.2 (3– 6) 0.035 0.013 0.008 Adhesins fimH (D-mannose-specific adhesin of type I fimbriae), fimAvMT78 (FimA variant MT78 of type 1 fimbriae), papEF and papG (P fimbria subunits), sfa/focDE (S fimbrial adhesin/putative F1C fimbrial adhesin), and afa/draBC (Dr antigen specific adhesin); toxins cnf1 (cytotoxic necrotizing factor 1), cdtB (cytolethal distending toxin), sat (secreted autotransporter toxin), hlyA (α-hemolysin), hlyF (hemolysin F), and astA (EAST1, enteroaggregative E. coli heatstable toxin); siderophores fyuA (yersiniabactin), iutA (aerobactin), iroN (novel catecholate siderophore receptor), and irp-2 (iron repressible associated with yersiniabactin synthesis); protectins kpsM (groups II and III, specifically targeting the K1, K2 and K5 genes of group II capsules), cvaC (ColV, colicin V from serum resistance-associated plasmids), iss (surface exclusion serum survival protein), and traT (serum resistance); miscellaneous virulence genes ompT (protease), ibeA (invasion of brain endothelium), malX (PAI, pathogenicity island marker), and usp (uropathogenic-specific protein, bacteriocin). a Virulence-associated genes shown with asterisk are the five genes characteristics of APEC strains. b Pvalues (by Fisher’s exact test) are shown where P<0.05. c The virulence score was the number of virulence genes detected, adjusted for multiple detection of the pap,sfa and foc, and kpsM II operons. Virulence scores were compared by use of the Mann-Whitney U test. doi:10.1371/journal.pone.0143191.t002 APEC in Broilers in Spain PLOS ONE | DOI:10.1371/journal.pone.0143191 November 23, 2015 9/14