肉鸡源弯曲菌的分离、多重PCR鉴定及其耐药性分析

Isolation,multiple PCR identification and antimicrobial resistance analysis of Campylobacter spp.from chicken

目的了解肉鸡屠宰加工过程中弯曲菌的污染及其耐药情况。方法根据GB 4789.9-2008,采用增菌培养、选择性平板分离、生化鉴定从肉鸡屠宰过程中分离疑似弯曲菌,并以弯曲菌属的16SrRNA、空肠弯曲菌的Hip O和结肠弯曲菌的Ceu E为靶基因,采用三重PCR方法对疑似菌株进行鉴定。采用琼脂稀释法对弯曲菌阳性菌株进行8种常用抗生素的药敏试验,参考CLSI标准(2010)判定药敏结果。结果根据形态特征、生化指标及PCR鉴定结果,从651份样品中,鉴定出空肠弯曲菌160株、结肠弯曲菌130株,其他弯曲菌11株,弯曲菌总分离率为46.24%;其中屠宰前、屠宰过程中及屠宰后鸡肉中弯曲菌的污染率分别为56.10%、31.00%、17.00%,可能存在由屠宰前向屠宰后传播趋势。药敏试验表明,弯曲菌对环丙沙星、左氧氟沙星、四环素和林可霉素的耐药率较高,分别为93.02%、87.71%、87.71%、80.07%,对庆大霉素、红霉素、链霉素、氟苯尼考的耐药率分别为67.11%、49.50%、31.89%、13.29%。结论肉鸡屠宰过程中弯曲菌的污染及其耐药情况较为严重,应该加强卫生和抗生素使用监督。

Enrichment culture, selective plate separation, and biochemical identification methods were used for isolation of Campylobacter from 651 samples collected from chicken slaughter and processed for segments. And then three genes were selected for identification of the genus Campylobacter, which were the 16S rRNA （Campylobacter spp. ）, the Hip 0 （C. je- juni） and Ceu E （C. coli）. Multiplex PCR assay （m PCR） with three sets of primers was developed for identification of C. je- juni and C. coll. Finally, the isolated Campylobacter spp. were tested for the antimicrobial susceptibility against 8 kinds of an tibiotics by using agar dilution method. The susceptibility results were determined by the standard of Clinical and Laboratory Standards Institute （CLSI, 2010）. According to this method, 301 Campylobacter isolates were obtained from 651 samples, in- cluding 160 C. jejuni, 130 C. coli, and 11 unidentified Campylobacter isolates. The total separation rate of Campylobacter was 46.24~. The occurrences of Campylobacter contamination before slaughtering, after evisceration and chicken products were 56.10%, 31.00% and 17. 00% respectively. 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%）. It＇s indicated that Campylobacter spp. isolates from different processing steps were widespread and according to the antimicrobial susceptibility testing, and the resistant rates for Campylobacter spp. isolates were serious.