苯唑西林敏感耐甲氧西林金黄色葡萄球菌临床株耐药性及生物膜活性分析

Analysis on Drug Resistance and Biofilm Activities of Clinical Strains of Oxacillin-Susceptible Methicillin-Resistant Staphylococcus aureus(OS-MRSA)

目的:本研究旨在探讨苯唑西林敏感耐甲氧西林金黄色葡萄球菌(OS-MRSA)临床分离株的耐药性及生物膜形成能力,为了解OS-MRSA菌株的生物学特征、临床治疗方案选择提供实验依据。方法:采用微量肉汤稀释法检测金黄色葡萄球菌对苯唑西林的最低抑菌浓度(MIC);采用PCR方法检测mec A基因和mec C基因携带情况;采用多位点序列分型(MLST)、葡萄球菌蛋白A分型(Spa)、葡萄球菌染色体盒mec分型(SCCmec)和脉冲场凝胶电泳分型(PFGE)方法进行基因分型;采用结晶紫法检测OS-MRSA菌株的生物膜形成能力,并采用PCR方法检测OS-MRSA株生物膜形成相关基因携带情况。结果:本研究共收集金黄色葡萄球菌1500株,检出OS-MRSA菌株21株,检出率为1.40%。苯唑西林对OS-MRSA菌株的MIC值在0.25~1μg/ml范围内,头孢西丁纸片扩散法筛选出3株MRSA菌株(14.29%)。21株OS-MRSA株均携带mec A基因、不携带mec C基因。21株OS-MRSA株均对万古霉素、利奈唑胺敏感,对青霉素、红霉素、四环素、克林霉素、庆大霉素、左氧氟沙星、复方磺胺甲唑和利福平的耐药率分别为100.00%、76.19%、52.38%、47.62%、23.81%、14.29%、9.51%和4.76%。分子分型结果显示,ST398型占比最多(4株,19.05%),而SCCmecIV型菌株为优势型(11株,52.38%)。21株OS-MRSA株均具有生物膜形成能力,其中3株为强产膜株(14.29%),13株为中产膜株(61.90%)。PCR结果显示,所有菌株均携带生物膜相关基因ica A和ica D基因,fnbp B和Bap基因阳性率较低,分别为23.81%、4.76%。结论:本研究发现人源OS-MRSA株分离率及对临床常用抗菌药物耐药率均较低,但常规耐药表型方法容易漏诊,应联合耐药表型技术和基因检测技术以提高OS-MRSA检测准确性。OS-MRSA具有生物膜形成能力,不同菌株间产膜能力及生物膜相关基因分布差异显著,不同菌株生物膜形成机制的异质性及调控机制需要进一步阐明。

Objective:The purpose of this study was investigate the drug resistance and biofilm formation ability of clinical isolates of oxacillin-susceptible methicillin-resistant Staphylococcus aureus(OS-MRSA),so as to provide experimental basis for understanding the biological characteristics of OS-MRSA strains and the selection of clinic treatment options.Methods:Minimum inhibitory concentration(MIC)of Staphylococcus aureus against oxacillin was determined by broth microdilution method.PCR method was used to detect mecA gene and mecC gene.Multi locus sequence typing(MLST),Spa,SCCmec and PFGE were used for genotyping.The biofilm forming ability of OS-MRSA strain was detected by crystal violet method.The genes related to biofilm formation of OS-MRSA strain were detected by PCR.Results:In this study,1500 strains of Staphylococcus aureus were collected and 21 strains of OS-MRSA were detected,the detection rate was 1.40%.The MIC values of oxacillin against OS-MRSA strains were 0.25~1μg/ml,3 MRSA strains(14.29%)were screened by cefoxitin disk diffusion method.All of the 21 OS-MRSA strains carried mec A gene but not mecC gene.All the 21 OS-MRSA strains were sensitive to vancomycin and linezolid.The resistance rates of the 21 OS-MRSA strains to penicillin,erythromycin,tetracycline,clindamycin,gentamicin,levofloxacin,cotrimoxazole and rifampicin were 100.00%,76.19%,52.38%,47.62%,23.81%,14.29%,9.51%and 4.76%,respectively.The results of molecular typing showed that ST398 accounted for the largest proportion(4 strains,19.05%),while SCCmec IV was the dominant type(11 strains,52.38%).All the 21 OS-MRSA strains showed biofilm forming ability,among which 3 strains were strong biofilm producers(14.29%),and 13 strains were middle biofilm producers(61.90%).PCR results showed that all strains carried biofilm related genes icaA and icaD.The positive rates of fnbpB and Bap genes were low,only 23.81%and 4.76%.Conclusion:In this study,it is found that the isolation rate of human OS-MRSA strains and the resistance rate to commonly used antibiotics in clinic are low,but OS-MRSA strains are easily missed by the conventional drug resistance phenotypic method.Therefore,to improve the detection accuracy of OS-MRSA,the resistance phenotype technology and gene detection technology we should combined.OS-MRSA showed biofilm formation ability,and biofilm production ability and the distribution of biofilm related genes varied greatly among different strains.The heterogeneity of biofilm formation mechanism and regulatory mechanism of different strains need to be further clarified.