PFGE Typing And MAMS-PCR Assay For The Detection Of Point Mutations In Gyra Of Campylobacter Jejuni Isolated From Chicken Production Chain

Campylobacter jejuni is a kind of important foodborne pathogens, which may cause diarrhea or food poisoning, even septicemia, reactive arthritis, Guillain-Barre syndrome. All these diseases are inseparable from the treatment of antibiotics. However, with the widespread use of various antibiotics, antibiotic resistance of Campylobacter jejuni had attracted worldwide attention. To analyze the prevalence and antibiotic resistance of Campylobacter jejuni in chicken production chain (farms-slaughterhouse-market), samples of chicken feces and chicken meat were collected in Sichuan Province, pulsed-field gel electrophoresis (PFGE) typing and gyrA gene of quinolone resistance mutations detection were analuzedAccording to the standards of GB/T4789.9-2008, SN0175-2010, ISO10272-1:2006, FDA/BMA:2001and USDA/FSIS:1998, an effective isolation method was developed for suspected Campylobacter jejuni of chicken origin. The isolates were acurately identified by the duplex PCR based on the sequence of16S rDNA and hipO gene. A total of519samples of the retail raw meat and feces were examined for the presence of Campylobacter jejuni. From the chicken production chain,180Campylobacter jejuni strains were isolated, and the isolation rate was34.87%. The higher contamination rates were observed in feces from farms and slaughterhouse (74.29%and66.38%, respectively). Whereas, the contamination rates in chicken from slaughterhouse and market were lower (17.33%and13.66%, respectively). The antibiotic resistant patterns of the180Campylobacter jejuni isolates were examined by the broth micro-dilution method. Resistance to ciprofloxacin was the most common (94.44%), followed by levofloxacin (93.89%), tetracycline (91.67%), clindamycin (78.33%), erythromycin (20.00%), streptomycin(20.56%),gentamicin (17.78%) and florfenicol (12.22%). All isolates showed multidrug (two or more antibiotics) resistance. From the farms to the market, the tendency of the resistance rates of Campylobacter jejuni to all eight antibiotics was faired or rising. This demonstrated that the Campylobacter jejuni were widespread in chicken production chain, and the resistance rate to the quinolones, tetracycline and clindamycin was higher. Genotypic characterization of all Campylobacter jejuni isolates (n=83) was performed by pulsed-field gel electrophoresis (PFGE). The DNA of all the isolates was digested by Sma I. The computer software program Info Quest(?) FP (version5.0) was employed for numerical analysis of Sma I macrorestriction patterns. The PFGE genotyping of83Campylobacter jejuni isolates yielded45SmaI PFGE types. The genotyping results showed that the phenomenon of horizontal transmission of Campylobacter jejuni existed in chicken production chain. Some strains with the same part of the resistance phenotype had higher genotype similarity. It demonstrated that the resistance phenotypes were related to genotypes of Campylobacter jejurMismatch amplification mutation assay PCR (MAMA PCR) was used to detect the mutation of gyr A. The results indicated that137Campylobacter jejuni which were showed resistance to ciprofloxacin mutated at257in the gyr A gene, the mutation rate was95.62%, while other10sensitive ones had no mutation. Sequencing results of the resistance determining region in gyr A gene showed Ciprofloxacin resistant strains MJF31and MJF53have Câ†'T mutations at257, however, Câ†'T mutation did not occur in ciprofloxacin sensitive strains JF9and JF136.This study analysed the pollution and the resistance of Campylobacter jejuni in chicken meat production chain for the first time, it provided the basic data for the monitoring of Campylobacter contamination and resistance. PFGE typing of Campylobacter jejuni in chicken production chain laid the foundation for the study of the mechanisms for the dissemination of Campylobacter jejuni in the food chain. At the same time, Campylobacter jejuni gyr A gene of quinolone resistance was detected by the MAMA PCR. it may be useful for the study of the mechanism of bacterial resistance to antibiotics. The thesis results have some theoretical significance and practical application value.