摘要
目的观察Walk基因突变所致的异质性万古霉素中介耐药金黄色葡萄球菌(h-VISA)的生物学特性变化,进一步揭示h-VISA对万古霉素敏感性下降的机制。方法收集山东省11家三甲医院耐甲氧西林金黄色葡萄球菌(MRSA)菌株200株,用含有16 g/L的胰酶消化酪蛋白和4μg/ml的万古霉素脑心浸液琼脂筛选法进行初筛h-VISA,菌群分析曲线法(PAP/AUC)对初筛阳性的菌株进行确认。Illumina Hiseq 2500测序技术对筛选出的h-VISA进行全基因组测序,观察有无Walk基因的突变。透射电镜观察其细胞壁的厚度,利用Trinton-X 100对细胞的破坏性原理,检测Walk突变h-VISA菌株的自行活性,RT-PCR技术测定Walk基因调控的细胞壁代谢相关基因的表达情况。结果200株MRSA菌株中初筛阳性的h-VISA菌株有14例,PAP-AUC方法最终确认h-VISA菌株有6株,6株h-VISA菌株中,5株检测到Walk基因的突变,突变方式均为点突变;Walk基因突变的h-VISA菌株细胞壁较其他菌株明显增厚(P<0.05);Trinton-X 100诱导的Walk突变阳性的h-VISA菌株自溶能力较其他菌株明显减弱(P<0.05);walk基因调控的细胞壁代谢相关基因如atlA,ssaA,isaA,lytM表达明显下调(P<0.05)。结论在h-VISA菌株中,存在较高比例的walk基因突变,突变的h-VISA菌株通过下调细胞壁代谢相关基因的表达,导致其细胞壁明显增厚,自溶活性明显减弱,使其对万古霉素敏感性减弱。
OBJECTIVE To observe the changes of biological characteristics of heterogeneous vancomycin-intermediate Staphylococcus aureus(h-VISA)caused by mutation of Walk gene so as to reveal the resistance mechanism of h-VISA to vancomycin.METHODS A total of 200 strains of methicillin-resistant Staphylococcus aureus(MRSA)were collected from 11 three-A hospitals in Shandong Province.The h-VISA were preliminarily screened by brain heart infusion agar containing 4μg/ml vancomycin and 16 g/L casein then confirmed by flora analysis curve method(PAP/AUC).The whole genome sequencing of h-VISA strains was performed by the Illumina Hiseq 2500 system so as to observe the mutation of walk gene,and the thickness of the cell wall was observed by transmission electron microscopy,the self-dissolution of the walk mutant h-VISA strains was detected by using the destructive principle of Trinton-X100 on cells,and the expression of cell wall metabolism-related genes that were regulated by walk gene were detected by RT-PCR.RESULTS There were 14 cases of h-VISA strains that were preliminarily screened positive in 200 strains of MRSA.Totally 6 strains of h-VISA were finally confirmed by PAP-AUC method,5 of which were detected with mutations in the walk gene,and the all of the mutations were point mutations.The cell wall of the h-VISA strains with the mutation of the walk gene was significantly thicker than that of other strains(P<0.05);the self-dissolution ability of the h-VISA strains with positive walk mutation induced by Trinton-X100 was significantly weaker than other strains(P<0.05).The expression of cell wall metabolism-related genes such as atlA,ssaA,isaA and lytM regulated by walk gene were down-regulated obviously in the h-VISA strains with walk mutation(P<0.05).CONCLUSION There are large proportions of strains with the walk gene mutation among the h-VISA strains.The mutant h-VISA strains can down-regulate the expression of cell wall metabolism-related genes,resulting in a significant thickening of the cell wall,and the self-dissolutio
作者
刘明涛
李慧
燕小辉
朱建波
董春忠
常刚
李超
李凯述
欧阳修河
LIU Ming-tao;LI Hui;YAN Xiao-hui;ZHU Jian-bo;DONG Chun-zhong;CHANG Gang;LI Chao;LI Kai-shu;OUYANG Xiu-he(Binzhou People's Hospital,Binzhou,Shandong 256610,China)
出处
《中华医院感染学杂志》
CAS
CSCD
北大核心
2020年第13期1964-1969,共6页
Chinese Journal of Nosocomiology
基金
山东省重点研发基金资助项目(2018GSF118222)。
