Characterization of Avian Pathogenic Escherichia Coli and Extended Spectrum Beta-Lactamase Producing Escherichia Coli Isolated from Chickens in Enugu State, Nigeria.

ABSTRACT The aim of this study was to characterize avian pathogenic Escherichia coli (APEC) and extended spectrum beta-lactamase (ESBL)-producing Escherichia coli strains isolated from chicken in Enugu State, Nigeria. Dead and moribund chickens collected from twenty four farms in 6 LGAs of the state and organ samples from chicken submitted to our departmental necropsy unit were used for the study. A total of 182 samples comprising of 100 broiler and 82 layers diagnosed of colibacillosis were collected and processed for isolation and identification of APEC strains, following standard bacteriological procedures. Eight stocked cultures of ESBLproducing E. coli strains isolated from 210 healthy broilers at Ikpa market Nsukka were also used in the study. The isolated APEC strains were cultured on blood agar and congo red agar to test for haemolysis and congo red uptake, respectively. Antibacterial resistance profile of the APEC strains was determined using the disc diffusion technique. Ten antibacterial agents were used. A multiplex polymerase chain reaction (PCR) was performed to ascertain the presence of 12 virulence genes (FimH, pap, felA, sfa, fac, csgA, tsh, cvaC, kpsII, iss, iutA, and cnf) among the APEC strains. The ESBL enzyme type and phylogenetic groups of the ESBL-producing E. coli were determined by the conventional PCR. The sequence type (ST) of the ESBL strains was identified by multi-locus sequence typing. Data generated were analyzed by Chi square and correlation coefficient tests using Graph Pad Prism 5. Significance was accepted at 5% probability level. Avian pathogenic E. coli strains were isolated from 57 (31.3%) of the 182 chickens examined. Eleven (19.2%) of 57 APEC strains were haemolytic while 41(71.9%) were positive for congo red uptake. The APEC strains recorded high resistance of 95%, 86%, 76% and 74% xvi against tetracycline, streptomycin, ciprofloxacin and ampicillin, respectively, and moderate resistance (47%) to gentamicin. None of the strains was resistant to imipenem. The APEC strains demonstrated 30 resistance patterns with TE-S-CN-CIP-AMP being the most predominant; occurring in 7 strains. Out of the 12 virulence gene investigated, 9 (FimH, pap, felA, csgA, tsh, cvaC, kpsII, iss and iutA) were present in the APEC strains, with FimH gene being the most prevalent (96%). The virulence genes occurred in various combinations with fimH-csgA-iss being the most predominant (17.6%). There was a positive but non- significant relationship (r = 0.03; P = 0.83) between the number of virulence genes possessed and the number of antibacterial agents to which the strains were resistant. There was no significant association (P>0.05) between the virulence gene and the chicken type and source of isolation. Extended spectrum β-lactamase gene types among the ESBL-producing E. coli strains were CTX-M-1, CTX-M-15 and TEM; with CTX-M-15 occurring in all the strains. Five of the 8 ESBL-producing strains belonged to phylogroup A while 3 to group B1. Three STs viz: ST10 (2 strains), ST226 (2 strains) and ST3625 (3 strains) were identified while 1 strain was untypeable. ST3625 was identified as a new ST with a new icd allele. In conclusion, this study has established the presence of APEC strains from chickens in Enugu State. These APEC strains harboured virulence associated genes and were multi-resistant with cefoxitin and imipenem being the drugs of choice for treatment. It is recommended that more research be carried out involving other APEC virulence associated genes and other poultry species. Proper sanitary measures should be employed in poultry farms and during meat handling and preparation. The public should be made to understand the dangers inherent in eating improperly boiled chicken meat.