血培养中异质性万古霉素中介金黄色葡萄球菌的分布及特征分析

Distribution and characteristics of heterogeneous vancomycin-intermediate Staphylococcus aureus in blood culture

目的研究血培养中异质性万古霉素中介金黄色葡萄球菌(heterogeneous vancomycin-intermediate Staphylococcus aureus,hVISA)的流行性及分子生物学特点。方法采用MHA5T(含有5μg/ml替考拉宁的MH琼脂)和菌群曲线分析法(populats profiles/area under the curve,PAP/AUC)检测hVISA,PCR方法对hVISA菌株进行SCCmec(staphylococcal cassette chromosome mec)﹑多位点序列分型(multilocus-sequence typing,MLST)﹑金黄色葡萄球菌蛋白A(Staphylococcus aureus protein A,spa)和附属基因调节子(accessory gene regulator,agr)分型检测,TritonX-100诱导自溶性检测hVISA菌株和万古霉素敏感金黄色葡萄球菌(vancomycin-sensitive Staphylococcus aureus,VSSA)菌株的自溶性差异,real-time PCR方法检测hVISA和VSSA菌株中vraR、mgrA、icaA、icaR、pbp4和agr基因的表达差异。结果血培养中甲氧西林耐药金黄色葡萄球菌(methicillin-resistant Staphylococcus aureus,MRSA)的检出率为39.5%,136株MRSA中共检出hVISA 31株,hVISA的阳性率为22.8%,hVISA菌株的万古霉素最小抑菌浓度(MIC)主要集中在1.5μg/ml(占54.8%)和2μg/ml(占25.8%),而VSSA菌株万古霉素MIC主要分布在0.5μg/ml(占46.7%)和0.75μg/ml(占39.0%)。hVISA的主要流行克隆为ST239-SCCmecⅢ-t030-agrⅠ型,有22株,占71.0%,与VSSA相比,hVISA菌株自溶性有所下降(χ^2=13.583,P=0.032)。RT-PCR结果显示与VSSA菌株相比,hVISA菌株中vraR、mgrA和icaA表达水平分别升高了1.58倍、1.53倍和1.06倍(P<0.01),而icaR、agr和pbp4基因的表达水平分别下降了0.85倍、0.61倍和1.03倍(P<0.05)。结论hVISA的流行率高达22.8%,主要流行克隆为ST239-SCCmecⅢ-t030-agrⅠ型,应引起临床高度重视,注意抗生素的合理使用,加强院感防控,避免hVISA菌株流行克隆的传播以及VISA和万古霉素耐药金黄色葡萄球菌(VRSA)的产生。

Objective To investigate the epidemiological and molecular biological characteristics of heterogeneous vancomycin-intermediate Staphylococcus aureus(hVISA)in blood culture.Methods hVISA was detected using Mueller-Hinton agar containing 5μg/ml of teicoplanin(MHA5T)and Populats profiles/area under the curve(PAP/AUC).Staphylococcal cassette chromosome mec(SCCmec),Staphylococcus aureus protein A(spa)and accessory gene regulator(agr)typing and multilocus-sequence typing(MLST)were analyzed using PCR.Difference in autolysis between hVISA and vancomycin-sensitive Staphylococcus aureus(VSSA)isolates were evaluated with Triton X-100-inducd autolysis.Expression of vraR,mgrA,icaA,icaR,pbp4 and agr genes in hVISA and VSSA strains were detected by real-time PCR.Results The positive detection rate of methicillin-resistant Staphylococcus aureus(MRSA)in blood culture was 39.5%(136/344)in our hospital.Among the MRSA strains,there were 31 strains of hVISA(22.8%).The minimum inhibitory concentrations(MIC)of vancomycin were mainly 1.5μg/ml(54.8%)and 2μg/ml(25.8%)against hVISA isolates,and 0.5μg/ml(46.7%)and 0.75μg/ml(39.0%)against VSSA isolates.The predominant clone of hVISA was ST239-SCCmecⅢ-t030-agrⅠaccounting for 71.0%(22/31).The autolysis of hVISA isolates decreased significantly as compared with that of VSSA isolates(χ^2=13.583,P=0.032).Compared with VSSA strains,the expression of vraR,mgrA and icaA genes in hVISA strains increased by 1.58,1.53 and 1.06 times(P<0.01),while the expression of icaR,agr and pbp4 genes decreased by 0.85,0.61 and 1.03 times(P<0.05).Conclusions The prevalence rate of hVISA in our hospital reached 22.8%and the main epidemic clone was ST239-SCCmecⅢ-t030-agrⅠ,which should be paid great attention to clinically.Rational use of antibiotics,strengthening the prevention and control of nosocomial infection,and avoiding the spread of hVISA strains and the emergence of VISA and VRSA(vancomycin-resistance Staphylococcus aureus)were also necessary.