宁夏地区牛源金黄色葡萄球菌生物被膜相关基因检测及耐药性研究

发布时间：2018-08-21 07:34

【摘要】：金黄色葡萄球菌是引起奶牛乳房炎的主要病原菌之一。近年来由于抗菌药物的广泛使用,其耐药性及多重耐药逐年增加,尤其是金黄色葡萄球菌形成生物被膜以后,不仅耐药能力极大提高,还能逃避机体免疫作用,长期存活并引起慢性感染,因此奶牛乳房炎反复感染、治疗困难。研究金黄色葡萄球菌生物被膜的形成能力和机制有助于为奶牛乳房炎临床预防和治疗提供新的思路。本研究主要采集宁夏地区奶牛临床型和隐性型乳房炎乳样,采用革兰氏染色镜检、科玛嘉金黄色葡萄球菌显色培养基初步筛选以及PCR方法扩增16SrDNA、nuc基因进行金黄色葡萄球菌的分离鉴定;再根据CLSI标准委员会推荐的肉汤微量稀释法,对鉴定出的金黄色葡萄球菌进行14种常用抗菌药物的MIC值测定。结果显示,共分离鉴定出265株金黄色葡萄球菌,其对万古霉素、多西环素的敏感率达到了 90%以上;对头孢唑啉、庆大霉素、四环素、环丙沙星、诺氟沙星的敏感率在80%以上;其中耐药率较高的是氨苄西林、青霉素,耐药率在60%左右。分离的菌株主要以3、4耐的居多,所占比例分别为13.6%、15.5%,分离的菌株最多耐受的抗菌药物达到10种。利用刚果红法培养金黄色葡萄球菌生物被膜,再经结晶紫染色法检测生物被膜的形成能力。结果显示,265株牛源金黄色葡萄球菌中有181株可以形成生物被膜,阳性率达到68.3%;在181株生物被膜阳性菌中,弱粘附、中等粘附、强粘附菌株所占比例分别为47.5%、30.4%、22.1%。采用PCR扩增技术对265株金黄色葡萄球菌进行生物被膜相关基因检测。结果显示,基因sigB、sar、agr、icaA、icaD 以及 icaR 的检出率分别为 91.7%、77.4%、57.4%、72.5%、87.2%及90.6%。本研究通过对宁夏地区奶牛乳房炎金黄色葡萄球菌耐药性分析以及生物被膜的检测,初步揭示了本地区牛源金黄色葡萄球菌的耐药情况以及生物被膜的流行情况,为指导兽医临床合理使用抗菌药物提供了理论依据,也为进一步进行生物被膜的研究奠定了基础。
[Abstract]:Staphylococcus aureus is one of the main pathogens of cow mastitis. In recent years, due to the widespread use of antimicrobial agents, their drug resistance and multidrug resistance have increased year by year, especially after the formation of biofilm of Staphylococcus aureus, not only the drug resistance ability has been greatly improved, but also the immune function of the body can be avoided. Long-term survival and chronic infection, so cow mastitis repeated infection, treatment is difficult. To study the ability and mechanism of biofilm formation of Staphylococcus aureus is helpful to provide new ideas for clinical prevention and treatment of mastitis in dairy cattle. In this study, clinical and recessive mastitis milk samples of dairy cows in Ningxia were collected and examined by Gram staining. Primary screening of Colour medium of Colour Staphylococcus aureus and amplification of 16s rDNA nuc gene by PCR were used to isolate and identify Staphylococcus aureus, and then according to the broth dilution method recommended by CLSI Standard Committee, The MIC values of 14 common antimicrobial agents were determined for the identified Staphylococcus aureus. The results showed that 265 strains of Staphylococcus aureus were isolated and identified, the sensitivity to vancomycin and doxycycline was over 90%, and the sensitivity to cefazolin, gentamycin, tetracycline, ciprofloxacin and norfloxacin was over 80%. Among them, ampicillin and penicillin were the most resistant, and the resistance rate was about 60%. Most of the isolates were resistant to 3D4, which accounted for 13.615.5%, and the most resistant strains were 10 kinds of antimicrobial agents. The biofilm of Staphylococcus aureus was cultured by Congo red method and the ability of biofilm formation was examined by crystal violet staining. The results showed that 181 strains of Staphylococcus aureus from cattle could form biofilm, the positive rate was 68.3%, and among 181 strains of biofilm positive bacteria, the proportion of weak adhesion, moderate adhesion and strong adhesion was 47.5% and 30.40.21%, respectively. The biofilm related genes of 265 strains of Staphylococcus aureus were detected by PCR amplification. The results showed that the positive rates of icaR and sigma were 91.7%, 77.4% and 72.5%, 87.2% and 90.6%, respectively. In this study, the resistance of staphylococcus aureus in dairy cattle and the detection of biofilm were analyzed, and the resistance of Staphylococcus aureus and the prevalence of biofilm in this area were preliminarily revealed. It provides a theoretical basis for the rational use of antimicrobial drugs in veterinary clinic and lays a foundation for the further study of biofilm.
【学位授予单位】：宁夏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.611

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