ISOLATION METHODS FOR MOLECULAR DETECTION AND ANTIBIOTIC RESISTANCE PATTERN OF CAMPYLOBACTER SPP IN LAYER CHICKENS

Abstract

This study was conducted to compare two culture methods for the isolation of Campylobacter spp from commercial layer chickens and subsequently confirmed by Polymerase Chain Reaction assays (PCR). Furthermore, the antimicrobial resistance profiles of PCR positive Campylobacter isolates were determined.Cloacal swab samples (550) from chickens randomly selected from five poultry farms in the four geographical zones in Ogun State were cultured for Campylobacter using modified charcoal Cefoperazone deoxycholate agar (MCCDA) and an improved culture method involving Preston broth pre-enrichment and subsequent subculture on Mueller Hinton agar with Campylobacter growth supplements. Putative isolates were later confirmed by PCR assay and sequencing analysis.Other isolates that grew on MCCDA and confirmed by sequencing analysis are Enterococcus faecalis, Escherichis coli, Comamonas kerstli and Pseudomonas aeroginusa . The antibiotic resistant profile of all the isolates were evaluated genotypically for resistance genes to tetracyclines (tetO), multiclasses (cmeB), aminoglycosides (aphA-3-1) and β-lactams (Blaoxa-61) using multiplex PCR (mPCR), and phenotypically for chlortetracycline, tylosin, streptomycin, ciprofloxacin and erythromycin resistance by microbroth dilution method which correspond to the antibiotic resistance genes. The apparent prevalence of Campylobacter was 16.8% by MCCDA while none of the isolates was positive to PCR. Meanwhile, prevalence rate of 26% was obtained using Preston broth pre-enrichment and Mueller Hinton agar with Campylobacter growth supplements, of which 11/50 (22%) of the isolates was confirmed positive by PCR. Genotypic characterization of PCR positive isolates showed 10/11(90%) were C. coli, 1/11(10%) other Campylobacter species and 0% C. jejuni. All the isolates carried both tetO and cmeB resistant genes. The results of minimum inhibitory concentration presented all PCR positive isolates had resistance of 10/10(100%), 9/10(90%), 6/10(60%), 9/10(90%), and 8/10(80%) to tetracycline, ciprofloxacin, erythromycin, spectinomycin and tylosin respectively. In addition, all isolates carried multiple resistance to most antibiotics tested which are commonly used in poultry practice in Nigeria. Campylobacter spp in the study areas showed diverse genotypic characteristics, and gene mediated multidrug resistance.