异质性万古霉素中介金黄色葡萄球菌的流行性及mgrA基因对万古霉素耐药性影响的研究

The epidemiology of heterogeneous vancomycin-intermediated Staphylococcus aureus and study on impact of mgrA gene in Staphylococcus aureus on vancomycin resistance

目的 监测郑州大学第一附属医院近5年来异质性万古霉素中介金黄色葡萄球菌(heterogeneous vancomycinintermediated Staphylococcus aureus, hVISA)的流行性,并进一步研究金黄色葡萄球菌mgrA基因对万古霉素耐药性的影响。方法采用琼脂稀释法和E-test法检测万古霉素对金黄色葡萄球菌的最小抑菌浓度(minimum inhibitory concentration, MIC);MHA5T和菌群曲线分析法(PAP/AUC)检测hVISA,Real-time RT-PCR法测定hVISA和non-hVISA菌株中mgrA基因的mRNA表达水平,通过SOE-PCR+同源重组的方法敲除mgrA基因。结果 823株MRSA中共检出hVISA 147株,检出率为17.9%,hVISA菌株mgrA基因的mRNA表达水平是non-hVISA菌株的2.65倍,明显高于non-hVISA菌株(t=7.326, P<0.001),成功构建mgrA基因缺失株,并发现mgrA基因缺失株万古霉素耐药性有所下降。结论 本地区hVISA的流行率为17.9%,并且5年来检出率呈逐年上升的趋势,mgrA基因可能与万古霉素耐药性有关,本研究推测mgrA基因下调可使万古霉素耐药性下降,研究为进一步明确金黄色葡萄球菌对万古霉素的耐药机制提供了新的理论基础。

Objective To investigate the prevalence of heterogeneous vancomycin-intermediate Stapylococcus aureus(hVISA)from the First Affiliated Hospital of Zhengzhou University over the past five years and study the impact of mgrA gene in Staphylococcus aureus on vancomycin resistance.Methods The minimum inhibitory concentration(MIC)of vancomycin to S.aureus were determined by the agar dilution method and the E-test method.hVISA strains were detected by MHA5T and the population analysis profile/area under the curve method(PAP/AUC).The mRNA expression levels of mgrA gene in hVISA and non-hVISA isolates were detected using real-time RT-PCR assay.The mgrA gene knockout was implemented by the SOE-PCR+homologous recombination method.Results 147 hVISA strains were detected among 823 MRSA strains,the prevalence of hVISA was 17.9%.The mRNA levels of mgrA in hVISA isolates was 2.65-fold higher than those in non-hVISA isolates,being significantly greater among the hVISA isolates than the non-hVISA isolates(t=7.326,P<0.001).The mgrA gene deletion strains was successfully constructed.Furthermore,we found that the deletion of mgrA gene could decrease vancomycin resistance.Conclusion The prevalenc of hVISA was 17.9%in the region,and the detection rate of hVISA showed an increasing trend over the past five years.The mgrA gene might be associated with vancomycin resistance,and we hypothesized that the downregulated mgrA gene may decrease vancomycin resistance.Our result provides a new theoretical basis for further clarification of the vancomycin resistance mechanisms to Staphylococcus aureus.