Isolation, Identification, Antibiotic Resistance And Pulsed-field Gel Electrophoresis Analysis Of Campylobacter Spp. From Chicken Slaughter And Processing Segments

Campylobacter is one of the major bacterial food-borne pathogens causing acute bacterial gastroenteritis in humans worldwide and the main cause of acute gastroenteritis, guillain-barre syndrome (Guillain-wilkes-barre), acute neuromuscular paralysis reactive arthritis and other diseases. With the widespread use of various antibiotics, antibiotic resistance of Campylobacter spp. had increased and its resistance can be spread through the food chain to the crowd. Therefore, to analyze the prevalence and antibiotic resistance of Campylobacter spp. from three different processing steps in chicken production chain (before killing-after evisceration-after segmentation) is significance for food safety. This study aimed to analysis the infection of Campylobacter spp. isolated from three different processing steps in Sichuan Province, and to analysis the antimicrobial resistance and pulsed-field gel electrophoresis (PFGE) typing in chicken production.1. Isolation and identification of Campylobacter spp. GB4789.9-2008. enrichment culture, selective plate separation, biochemical identification methods were used for isolation of Campylobacter from 651 samples which were collected from chicken slaughter and processing segments in 2012 to 2013. And then three genes were selected for identification of the genus Campylobacter. There were the 16S rRNA (Campylobacter spp), the hipO(C.jejuni) and ceuE (C.coli). Multiplex PCR assay (m-PCR) with three sets of primers was developed for identification of C.jejuni and C.coli. According this method,301 Campylobacter isolates were obtained from 651 samples, including 160 C.jejuni,130 C.coli, and 11 unidentified Campylobacter isolates. The total separation rate of Campylobacter was 46.24%. 96.35% of the Campylobacter isolates were C.jejuni and C.coli. The occurrences of Campylobacter contamination before slaughtering, after evisceration and in chicken products were 56.10%,31.00% and 17.00%.2. Antimicrobial susceptibility testing The isolated Campylobacter spp were tested for the antimicrobial susceptibility against 8 kinds of antibiotics by using agar dilution method. The susceptibility results were determined by the standard of Clinical and Laboratory Standards Institute (CLSI,2010). The results indicated that the resistant rates for Campylobacter isolates were 93.02% against ciprofloxacin. The Campylobacter isolates also exhibited a high rate of resistance to levofloxacin(87.71%), tetracycline (87.71%) and clindamycin(80.07%), showing different antimicrobial resistance to gentamicin(67.11%), erythromycin(49.50%), streptomycin(31.89%) and florfenicol(13.29%).93.69% of the isolates showed multidrug (ttere or more antibiotics) resistance and have a total of 52 kinds of resistance patterns.3. Antimicrobial resistance mechanism testing3.1. Prevalence of class 1 integrons and the gene cassettes Class 1 integrons were detected in 148 Campylobacter spp. isolates (98.67%), and the integrons positive isolates in C.jejuni and C.coli was 98.72% (77/78) and 98.53% (67/68). The occurrences of Campylobacter integrons positive isolates before slaughtering, after evisceration and Chicken products was 98.04%,100.00% and 100.00%.78 of these were positive for the resistance gene cassette and all produced a 1000 bp amplicon which were identical to the nucleotide sequence of aadA2 encoding an aminoglycoside adenyl transferase which confers aminoglycoside resistance.3.2. Mutations within the QRDRs of gyrA genes 145 campylobacter spp. isolates from the 150 selected strains resistant to ciprofloxacin were examined for mutations in the QRDRs of gyrA by using MAMA-PCR to detect the mutations of the C257T (Thr-86-Ile) mutation in the quinolone resistance determining region (QRDR) associated with quinolone resistance. The results shows that 93.10%(135/145) of the strains were positive to the mutations in the QRDRs of gyrA and the mutations rate for C.jejuni andC.coli was 97.26%(71/73) and 89.71%(61/68). No mutations were observed in the fluoroquinolone-sensitive strain. By DNA sequence analysis, the replacemen of C-257-T leading to a Thr-86-Ile substitution in gyrA was happened in strain 12-JF156 and 12-JF181 which resistance to fluoroquinolone. No mutations were observed in strain 12-JF19 and 13-JF60 which sensitive to fluoroquinolone.3.3. Occurrence of tetO 130 Campylobacter spp. isolates were tetracycline-resistant from the 150 strains.94.62%(123/130) of the tetracycline-resistant strains were positive for the resistance of tetO gene. The detection rate of tetO gene for C.jejuni, C.coli and other Campylobacter was 96.61% (57/59),94.12%(64/68) and 66.67%(2/3).3.4. Mutations within 23S rRNA 83 Campylobacter spp. isolates were erythromycin-resistant from the 150 strains. By PCR and DNA sequence analysis, 96.39%(80/83) of the erythromycin-resistant strains have a mutation of A-2075-G in 23S rRNA sequences. The mutations rate for C.jejuni, C.coli and other Campylobacter was 95.00%(19/20),96.72 (59/61) and 100.00%(2/2).4. The PFGE typing of Campylobacter spp. isolates Using restriction enzyme sma I to research the classification of the 150 multi-drug resistant isolates and through the clustering analysis software. The result show that 78 Cjejuni strain s PFGE stripe similarity between 42.10%-42.10% and have 33 different genotype,68 C.coli strain`s PFGE stripe similarity between 54.01%-54.01%, and have 15 genotypes,4 of other campylobacter spp. strain s PFGE stripe between similarity between 43.24%-43.24%, and have 4 different spectral genotype. The study indicated that the phenomenon of Campylobacter transmission along the chicken production chain does exist.This study analysed the pollution, resistance, resistance mechanism and PFGE typing of Campylobacter spp. in chicken production chain, it provided the basic data for the monitoring of Campylobacter spp. contamination and resistance, and also laid the foundation for the study of the mechanisms for the dissemination of Campylobacter spp. in the chicken production chain. The findings of this paper have certain theory significance and the pratical value.