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-Ginés1, Karla Cameron-Veas1, Ignacio Badiola1, Roser Dolz1, Natalia Majó1,2, Ghizlane Dahbi3, Susana Viso3, Azucena Mora3, Jorge Blanco3, Nuria Piedra-Carrasco4, Juan José González-López4, Lourdes Migura-Garcia1* 1 Centre de Recerca en Sanitat Animal (CReSA)—Institut de Recerca i Tecnologia Agroalimentàries (IRTA), Campus UAB, Barcelona, Spain, 2 Departament de Sanitat i Anatomia Animals, Universitat Autònoma de Barcelona, Bellaterra (Cerdanyola del Vallés), Spain, 3 Laboratorio de Referencia de E. coli, Departamento de Microbioloxía e Parasitoloxía, Facultade de Veterinaria, Universidade de Santiago de Compostela, Lugo, Spain, 4 Servei de Microbiologia, Hospital Vall d’Hebron, Universitat Autònoma de Barcelona, Barcelona, Spain 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 AGL2011- 28836 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 * 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 blaCTX-M-14, two blaSHV-12, two blaCMY-2 and one blaSHV-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 blaCMY-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 APEC in Broilers in Spain 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. 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 str (...truncated)