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

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.

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.