牛源金黄色葡萄球菌的分离鉴定、耐药性分析及木糖醇对生物被膜形成的干预

Isolation,identification and bacterial resistance of dairy cows’ Staphylococcus aureus and intervention of biofilm formation by xylitol

在金黄色葡萄球菌临床分离株耐药表型分析基础上,探索不同质量浓度木糖醇对该菌生物被膜形成的干预,旨在为耐药性研究及奶牛乳房炎治疗提供新思路。通过金黄色葡萄球菌专用显色培养基、PCR等方法分离鉴定金黄色葡萄球菌;按照K-B法测定临床分离株对17种常用抗菌药物的敏感性;采用刚果红平板试验、96孔板和置片法分析分离菌生物被膜形成能力;应用置片法、超声波洗脱结合平板计数考察不同质量浓度木糖醇对生物被膜形成黏附阶段的影响。结果显示:金黄色葡萄球菌的检出率较高达60.7%(236/389);分离株对青霉素、阿莫西林以及氨苄西林耐药率高达98.31%～99.58%,且均为多重耐药;刚果红试验结果表明,74.6%(176/236)的分离株能形成生物被膜;96孔板法发现分离株形成生物被膜能力按强、中、弱依次占34.7%(82/236)、51.6%(122/236)和13.6%(32/236);置片法提示培养4 d可得到成熟的金黄色葡萄球菌生物被膜;随机选取标准菌株ATCC29213和9株分离株(生物被膜形成能力强、中、弱各3株)发现,与不用木糖醇(2.28×10~5)相比,经0.125,0.250,0.500 g/mL的木糖醇处理后黏附到盖玻片上的菌落数(分别为1.51×10~5,1.37×10~5,1.10×10~5)分别降低了33.8%(P<0.01),39.9%(P<0.01)和51.8%(P<0.01)。由此推断:金黄色葡萄球菌临床分离菌株多重耐药严重,生物被膜形成普遍,0.125～0.500 g/mL的木糖醇能有效抵抗金黄色葡萄球菌黏附,从而干扰生物被膜的形成。

Based on phonotype analysis of the bacterial resistance of Staphylococcus aureus in clinical isolates from dairy farms,different concentrations of xylitol were applied to investigate the intervention of biofilm formation,aiming to provide new insights for anti-bacterial resistance study and clinical control of cow mastitis.Staphylococcus aureus were isolated and identified via selective medium culture and PCR techniques,susceptibility testing to 17 commonly used antimicrobials was subsequently determined by K-B method;in addition,the capitibility of biofilm formation of the isolates was characterized by Congo red plate test,followed by 96-well plate and film placement approach;the effects of different concentrations of xylitol against bacterial adhesion were scrutinized by plate placement,ultrasonic elution and plate counting.Results showed that the detection rate of Staphylococcus aureus from milk was as high as 60.7%(236/389);the isolates had severe resistance to penicillin,amoxicillin and ampicillin(98.31%-99.58%),and all were multi-resistant;Congo red test showed 74.6%(176/236)of the isolates could form biofilm,and the 96-well plate method exhibited that the ability of the isolates to form biofilms according to strong,medium and weak was 34.7%(82/236),51.6%(122/236)and 13.6%(32/236).Additionally,it took 4 days for the establishment of the mature of the biofilm.Xylitol at 0.125,0.250 and 0.500 g/mL can reduce the colonies adhered to cover glass by 33.8%(P<0.01),39.9%(P<0.01)and 51.8%(P<0.01),respectively.The result can drew the conclusion that the multi-drug resistance of Staphylococcus aureus isolates is severe and the biofilm formation is prevalent.Xylitol can effectively resist the adhesion of Staphylococcus aureus.Its effective concentration of anti-adhesion is 0.125-0.500 g/mL.