Characterization of Multi Drug Resistant (MDR) Escherichia coli Isolated from Chicken using Molecular and Bioinformatics Tools

Abstract

Global propagation of multidrug-resistant (MDR) Escherichia coli in food chains is a threat not only to animal health but also to human health. Some advanced countries have devised policies for antimicrobial use not only for humans but also for animal health treatment purposes. The poultry industry is an important component of Pakistan's gross domestic product. However, the Pakistani poultry industry faces several environmental conditions that threaten continued economic output, livestock health, and human health. One of these emerging threats is MDR E. coli. Until 2016 there was no policy in Pakistan regarding antimicrobial use or contaminant of antimicrobial resistance. Besides, public awareness regarding the presence of MDR pathogens in the food chain is scant. In this study total of 1,219 liver samples were collected from the National Reference Laboratory for Poultry Diseases in Pakistan via federal and provincial sentinel surveillance laboratories under a national surveillance program from 2015 to 2017. In which 511, liver samples were taken from culled layer and broiler chickens having poor birth growth and reduced appetite but not otherwise symptomatic for colibacillosis. Of which 265 isolations were identified as E. coli which were tested initially for antimicrobial resistance (AMR) against 21 priority antibiotics used in poultry in Pakistan. Out of 265 isolates, 105 were further checked against the extended spectrum of 31 antibiotics and analyzed through Next Generation Sequencing. The pattern of antibiotics resistance explains that commercial isolated E. coli has shown a higher percentage of resistance to penicillin, amoxicillin, lincomycin, oxytetracycline, doxycycline, erythromycin, sulfa-methazole-trimethoprim, spectinomycin, and nitrofurantoin as compared to the backyard poultry (domestic chicken), isolated E. coli. Moreover, the isolates from both commercial and backyard poultry have shown similar resistance patterns to two major groups of antibiotics, aminoglycosides (neomycin, gentamycin, streptomycin) and fluoroquinolones (flumequine, enrofloxacin, and norfloxacin, ciprofloxacin). However, some very important antibiotics like ceftiofur (thirdgeneration cephalosporin), colistin, and carbapenem were the least resistant drugs in both commercial and backyard poultry. The presence of colistin-resistant bacteria in chickens indicates a danger to public health in Pakistan. An Illumina whole-genome sequencing was performed on 92 E. coli isolates. Our analysis indicates that the isolates were predominantly from B1 and A clade. They harbour a diverse number of antibiotic resistance and virulence genes with no connection between phylogeny and antibiotic gene presence. However, some connection was found between phylogeny virulence gene, and SNP (single nucleotide polymorphism). Importantly, most of our isolates can be XX classified as multidrug-resistant with one displaying extended drug resistance. These results provide the highest resolution analysis from poultry-associated E. coli isolates in an area with a high endemic burden of antibiotic resistance. The isolated mcr-1 bearing strains E. coli 79 was used to transform colistin-resistant gene to the donor E. coli J53, via conjugation. E. coli 79 isolate was used as donors. While E. coli J53 (resistant to sodium-azide) was used as the recipient strain. Plasmid (mcr1_Inc12) was confirmed in our several strains by NGS data using Plasmid-finder. Prominently, the findingsof the study depict that out of92 strains 82were found to be MDR. One strain was reported as extensively drug-resistant (XDR) and 13 were mcr-1 positive E. coli. In Multiple locus Sequence Typing (MLST) data analysis, nine strains of serotype ST 117 E. coli (09/92) reported were from phylogroup B2. More interestingly two of our isolated strains EC-83 and EC-35-B have blaCTX M-15 genes. However, in our study 12 of our isolates carried blaCTX-M-15 is the second most common after blaT-EM-1B in the Extended-spectrum β-lactamase (ESBL) family. Furthermore, 08 sequence types (ST117, ST1011, ST2847, ST533, ST1324, ST2973, ST155, ST4516) have mcr 1 genes. One of the very interesting results in the study wasthe EC-60 strain, ofST131 serotype is found in many infections worldwide. The strain ST131 is blaCTX-M-1 gene having IncFIB (AP001918), IncFIC (F11), and Incl 1 plasmid replicons were reported in this strain. Overall, it is the first study from Pakistan that provide detailed information of antibiotic resistance pattern and genetic attributes of avian MDR E. coli. However, the limitation of the study was that with short Illumina reads, we were unable to unequivocally implicate ARGs or virulence genes present on mobilizable plasmids. Additionally, we do not have access to a chicken model of infection to demonstrate links between comparative pathogenicity of the strains and their virulence gene mosaic.