【摘要】：金黃色葡萄球菌是引起奶牛乳房炎的主要病原菌之一。近年來(lái)由于抗菌藥物的廣泛使用,其耐藥性及多重耐藥逐年增加,尤其是金黃色葡萄球菌形成生物被膜以后,不僅耐藥能力極大提高,還能逃避機(jī)體免疫作用,長(zhǎng)期存活并引起慢性感染,因此奶牛乳房炎反復(fù)感染、治療困難。研究金黃色葡萄球菌生物被膜的形成能力和機(jī)制有助于為奶牛乳房炎臨床預(yù)防和治療提供新的思路。本研究主要采集寧夏地區(qū)奶牛臨床型和隱性型乳房炎乳樣,采用革蘭氏染色鏡檢、科瑪嘉金黃色葡萄球菌顯色培養(yǎng)基初步篩選以及PCR方法擴(kuò)增16SrDNA、nuc基因進(jìn)行金黃色葡萄球菌的分離鑒定;再根據(jù)CLSI標(biāo)準(zhǔn)委員會(huì)推薦的肉湯微量稀釋法,對(duì)鑒定出的金黃色葡萄球菌進(jìn)行14種常用抗菌藥物的MIC值測(cè)定。結(jié)果顯示,共分離鑒定出265株金黃色葡萄球菌,其對(duì)萬(wàn)古霉素、多西環(huán)素的敏感率達(dá)到了 90%以上;對(duì)頭孢唑啉、慶大霉素、四環(huán)素、環(huán)丙沙星、諾氟沙星的敏感率在80%以上;其中耐藥率較高的是氨芐西林、青霉素,耐藥率在60%左右。分離的菌株主要以3、4耐的居多,所占比例分別為13.6%、15.5%,分離的菌株最多耐受的抗菌藥物達(dá)到10種。利用剛果紅法培養(yǎng)金黃色葡萄球菌生物被膜,再經(jīng)結(jié)晶紫染色法檢測(cè)生物被膜的形成能力。結(jié)果顯示,265株牛源金黃色葡萄球菌中有181株可以形成生物被膜,陽(yáng)性率達(dá)到68.3%;在181株生物被膜陽(yáng)性菌中,弱粘附、中等粘附、強(qiáng)粘附菌株所占比例分別為47.5%、30.4%、22.1%。采用PCR擴(kuò)增技術(shù)對(duì)265株金黃色葡萄球菌進(jìn)行生物被膜相關(guān)基因檢測(cè)。結(jié)果顯示,基因sigB、sar、agr、icaA、icaD 以及 icaR 的檢出率分別為 91.7%、77.4%、57.4%、72.5%、87.2%及90.6%。本研究通過(guò)對(duì)寧夏地區(qū)奶牛乳房炎金黃色葡萄球菌耐藥性分析以及生物被膜的檢測(cè),初步揭示了本地區(qū)牛源金黃色葡萄球菌的耐藥情況以及生物被膜的流行情況,為指導(dǎo)獸醫(yī)臨床合理使用抗菌藥物提供了理論依據(jù),也為進(jìn)一步進(jìn)行生物被膜的研究奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：寧夏大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S852.611

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 張倩;屈勇剛;于會(huì)舉;劉功成;劉鴿;張培生;;石河子地區(qū)奶牛乳房炎源金黃色葡萄球菌的分離鑒定及耐藥性分析[J];黑龍江畜牧獸醫(yī);2017年02期

2 朱玉龍;嚴(yán)燕燕;顧蓓青;俞惠宇;祝慧華;;653株金黃色葡萄球菌臨床分布及耐藥性分析[J];現(xiàn)代預(yù)防醫(yī)學(xué);2014年21期

3 胡曉豐;史云;戚麗華;王勇;宋宏彬;張傳福;劉雪林;;金黃色葡萄球菌生物膜形成機(jī)制研究進(jìn)展[J];生物技術(shù)通訊;2014年05期

4 桂中玉;李琳;李冰;徐振波;;金黃色葡萄球菌耐藥性與生物被膜能力的鑒定[J];現(xiàn)代食品科技;2014年03期

5 聶鑫;高原;;細(xì)菌生物被膜檢測(cè)方法研究進(jìn)展[J];黑龍江畜牧獸醫(yī);2012年21期

6 李紀(jì)兵;陸春;;體內(nèi)生物膜研究進(jìn)展[J];微生物學(xué)雜志;2012年05期

7 譚翰清;林鳳;譚海芳;陳華;程潔萍;區(qū)意;高向雄;葉堅(jiān);;醫(yī)院分離金黃色葡萄球菌的毒力基因和耐藥基因的研究[J];中國(guó)消毒學(xué)雜志;2011年06期

8 陳鐵柱;李曉聲;曾文魁;杜堯;譚財(cái)福;;細(xì)菌生物膜耐藥機(jī)制的研究與進(jìn)展[J];中國(guó)組織工程研究與臨床康復(fù);2010年12期

9 孫鳳軍;枉前;夏培元;;臨床分離金黃色葡萄球菌生物膜相關(guān)基因的PCR分析[J];第三軍醫(yī)大學(xué)學(xué)報(bào);2009年15期

10 胡金樹;王彥榮;趙建宏;;細(xì)菌生物被膜的防治進(jìn)展[J];中國(guó)誤診學(xué)雜志;2009年10期

相關(guān)博士學(xué)位論文 前1條

1 周田華;頭孢他啶陽(yáng)離子脂質(zhì)體復(fù)合n-HA/β-TCP陶瓷支架的制備、體外溶出機(jī)理及其體外抗金黃色葡萄球菌生物膜的研究[D];第三軍醫(yī)大學(xué);2012年

相關(guān)碩士學(xué)位論文 前1條

1 譚磊;基于PCR的牛源金黃色葡萄球菌基因分型研究[D];黑龍江八一農(nóng)墾大學(xué);2008年

，

本文編號(hào)：2195006