鸡肉生产链中空肠弯曲杆菌PFGE分型及耐喹诺酮类药物gyrA基因突变分析

发布时间：2018-10-13 18:38

【摘要】：空肠弯曲杆菌(Campylobacter ieiuni, C.jejuni)是一种重要的食源性病原菌,食用受到污染的食品可引起腹泻或食物中毒,严重的可导致败血症、反应性关节炎、格林巴利综合症等,这些疾病都离不开抗生素的治疗,然而随着各种抗生素的广泛使用,空肠弯曲杆菌对抗生素的耐药性已引起世界的广泛关注。因此,本文从雅安养鸡场、屠宰场和市场中分离收集空肠弯曲杆菌,对鸡肉生产链(养殖场-屠宰场-市场)中空肠弯曲杆菌的污染及耐药情况、脉冲场凝胶电泳(PFGE)分型和耐喹诺酮类药物gyrA基因突变检测进行了分析。参考GB/T4789.9-2008、SN0175-2010、ISO10272-1:2006、FDA/BAM:2001、 USDA/FSIS:1998等方法,从鸡肉生产链中采集样品519份,分离鉴定出空肠弯曲杆菌180株,分离率为34.87%。其中,养鸡场和屠宰场鸡粪样空肠弯曲杆菌分离率分别为74.29%和66.38%,屠宰场和市场的鸡肉样中空肠弯曲杆菌的分离率分别为17.33%和13.66%。采用肉汤微量稀释法,空肠弯曲杆菌对8种抗生素(组合)的药敏实验结果表明,180株空肠弯曲杆菌对环丙沙星、左氟沙星、四环素和克林霉素的耐药率较高,分别为94.44%、93.89%、91.67%和78.33%；对红霉素(20.00%)、链霉素(20.56%)、庆大霉素(17.78%)、氟苯尼考(12.22%)呈现出不同的耐药率；多重耐药率为100%。从养殖场到市场,鸡肉生产链中空肠弯曲杆菌对上述8种抗生素的耐药率呈持平或上升趋势。鸡肉生产链中空肠弯曲杆菌的污染严重,对喹诺酮类药物、四环素和克林霉素的耐药率较高。采用Sma I对83株空肠弯曲杆菌进行PFGE分型,通过Info Quest(?) FP (version5.0)指纹图谱分析软件的聚类分析,结果产生了45种不同的谱型。基因分型结果表明空肠弯曲杆菌在鸡肉生产链中存在水平传播现象,部分耐药表型相同的菌株其基因型相似度较高,说明空肠弯曲杆菌的耐药表型与基因型之间有一定的相关性。采用错配扩增突变分析PCR (MAMA PCR)技术,检测137株耐环丙沙星的空肠弯曲杆菌的gyrA基因257位突变情况,结果有131株空肠弯曲杆菌在gyrA基因257位发生点突变,检测突变率为95.62%。10株对环丙沙星敏感的菌株均没有检测出gyrA基因的突变。gyrA基因耐药决定区的测序结果表明对环丙沙星耐药的菌株MJF31和MJF53都在第257位碱基发生了C→T(苏氨酸到异亮氨酸)的突变,而对环丙沙星敏感的菌株JF9和JF136均未发生C→T(苏氨酸到异亮氨酸)突变。本研究分析了鸡肉生产链中空肠弯曲杆菌的污染和耐药情况,为监测空肠弯曲杆菌的污染情况和耐药性提供了基础数据；对鸡肉生产链中的空肠弯曲杆菌进行PFGE分型,为研究空肠弯曲杆菌在食品链中的传播机制奠定了基础,同时采用MAMAPCR技术对耐喹诺酮类药物的空肠弯曲杆菌的gyrA基因进行了检测,为研究细菌耐药性的产生机制提供了参考。论文研究结果具有一定的理论意义和实际应用价值。
[Abstract]:Campylobacter jejuni (Campylobacter ieiuni, C.jejuni) is an important foodborne pathogen. Eating contaminated food can lead to diarrhea or food poisoning, severe sepsis, reactive arthritis, Guillain-Barre syndrome, etc. These diseases can not be separated from the treatment of antibiotics. However, with the wide use of various antibiotics, the antibiotic resistance of Campylobacter jejuni has attracted worldwide attention. Therefore, campylobacter jejuni was isolated and collected from Ya'an chicken farm, slaughterhouse and market, and the contamination and drug resistance of Campylobacter jejuni in chicken production chain (farm slaughterhouse market) were studied. Pulsed field gel electrophoresis (PGE) (PFGE) typing and quinolone-resistant gyrA gene mutation were analyzed. Referring to GB/T4789.9-2008,SN0175-2010,ISO10272-1:2006,FDA/BAM:2001, USDA/FSIS:1998 and other methods, 519 samples were collected from chicken production chain, 180 strains of Campylobacter jejuni were isolated and identified, the isolation rate was 34.87%. The isolation rates of Campylobacter jejuni were 74.29% and 66.38% in chicken manure, and 17.33% and 13.66% in chicken samples from abattoir and market, respectively. The susceptibility of Campylobacter jejuni to 8 kinds of antibiotics (combinations) was studied by broth microdilution method. The results showed that 180 strains of Campylobacter jejuni were resistant to ciprofloxacin, levofloxacin, tetracycline and clindamycin, which were 94.4449.8991.67% and 78.33%, respectively. The resistance rates of erythromycin (20.00%), streptomycin (20.56%), gentamicin (17.78%) and florfenicol (12.22%) were different, and the multidrug resistance rate was 100% for erythromycin, streptomycin (20.56%), gentamicin (17.78%) and florfenicol (12.22%). From farm to market, the resistance rate of Campylobacter jejuni to the above eight antibiotics in chicken production chain was flat or rising. The contamination of Campylobacter jejuni in chicken production chain was serious and the resistance rate of tetracycline and clindamycin to quinolones was higher. 83 strains of Campylobacter jejuni were genotyped by PFGE with Sma I. Info Quest (? The clustering analysis of FP (version5.0) fingerprint analysis software resulted in 45 different spectral types. The genotyping results showed that Campylobacter jejuni spread horizontally in the chicken production chain, and the genotypic similarity of some strains with the same drug resistance phenotype was higher. The results showed that there was a certain correlation between drug resistance phenotype and genotype of Campylobacter jejuni. A mismatch amplified mutation analysis (PCR (MAMA PCR) technique was used to detect the gyrA gene 257 mutation in 137 ciprofloxacin resistant Campylobacter jejuni strains. The results showed that 131 Campylobacter jejuni strains had a point mutation in gyrA gene 257 position. The mutation rate was 95.62.10 strains sensitive to ciprofloxacin did not detect the mutation of gyrA gene. The results of sequencing of the determining region of gyrA gene showed that both MJF31 and MJF53 of ciprofloxacin resistant strains had C + at the 257th base of ciprofloxacin. T (threonine to isoleucine) mutation, The C T (threonine to isoleucine) mutation was not found in the ciprofloxacin sensitive strains JF9 and JF136. In this study, the contamination and drug resistance of Campylobacter jejuni in chicken production chain were analyzed, which provided basic data for monitoring the contamination and drug resistance of Campylobacter jejuni, and the PFGE typing of Campylobacter jejuni in chicken production chain was carried out. In order to study the transmission mechanism of Campylobacter jejuni in food chain, MAMAPCR technique was used to detect the gyrA gene of Campylobacter jejuni resistant to quinolones. The results of this paper have certain theoretical significance and practical application value.
【学位授予单位】：四川农业大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R155.31

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