Diversity and antimicrobial resistance of pathogenic and non pathogenic Escherichia coli isolated in the broiler production chain

Avian pathogenic Escherichia coli (APEC) causes extraintestinal diseases in broilers and share common virulence factors with extraintestinal pathogenic E. coli (ExPEC) isolated in humans suggesting the zoonotic potential of APEC [1]. APEC are characterised by a high intra-population diversity in terms of serogroup, MLST and virulence genes [2]. The aim of the present study was to investigate the diversity and antimicrobial resistance of E. coli isolated from lung, spleen, pericardium and brain of broilers affected by colisepticemia and treated with enrofloxacin. In three different farms with ongoing infections, at day 0 (before starting the treatment), day 5 (last day of treatment), day 10 and day 20 (after treatment), 10 birds were humanly euthanized and their organs investigated for E. coli isolation by standard microbiological procedures. A total of 179 E. coli were isolated and submitted to pulsed field gel electrophoresis (PFGE) and minimum inhibitory concentration (MIC) to gain insights on their genetic diversity and antimicrobial susceptibility. Based on metadata as well as MIC and PFGE data, 31 isolates were whole genome sequenced on MiSeq platform (Illumina, 250 bp reads, paired ends). Genomic diversity by SNP calling and in silico MLST as well as antimicrobial resistance and virulence-associated genes were analysed with publicly available bioinformatics pipelines. According to PFGE results, in all three farms the genetic diversity was overall low before the treatment and increases along and after. At day 0, E. coli isolates can be gathered in 3 - 6 clusters at 95% similarity. Whereas at day 5, 10 and 20 the number of clusters rises up to 10-16 with the majority of single-isolate clusters. Similar results were confirmed by MLST and SNP calling analyses on the sequenced genomes. MIC data showed a significant increase of resistance to enrofloxacin and flumequine in E. coli isolates collected at day 5, 10 and 20 in comparison to day 0. Along with this drug, a significant increase of resistance to trimethoprim/sulfametoxazole (SXT) was observed in one of the three farms. These results suggest that the antibiotic was effective in eradicating the initial pathogen strains responsible for the colibacillosis infections. In all farms, fluoroquinolone resistant E. coli were collected after enrofloxacin treatment. In one farm, a high number of SXT resistant strains arose, although their SXT resistance genes were genetically unrelated to enrofloxacin resistance genes/mutations. Finally, the gross pathological lesions encountered before the treatment on diseased birds disappeared after the treatment in all organs except for lungs, suggesting the lower virulence of E. coli isolates collected after the treatment from spleen, pericardium and brain.