Department of Infection Control Science, Juntendo University, 2-1-1 Hongo, Bunkyo-Ku, Tokyo 113-8421, Japan.
Antimicrob Agents Chemother. 2011 Aug;55(8):3870-81. doi: 10.1128/AAC.01563-10. Epub 2011 May 31.
Vancomycin-intermediate Staphylococcus aureus (VISA) is generated from vancomycin-susceptible Staphylococcus aureus by multiple spontaneous mutations. We previously reported that sequential acquisition of mutations in the two-component regulatory systems vraSR and graRS was responsible for the VISA phenotype of strain Mu50. Here we report on the identification of a novel set of regulator mutations, a deletion mutation in two-component regulatory system walRK (synonyms, vicRK and yycFG), and a truncating mutation in a proteolytic regulatory gene, clpP, responsible for the raised vancomycin resistance in a laboratory-derived VISA strain, LR5P1-V3. The contributory effect of the two mutations to vancomycin resistance was confirmed by introducing the walK and clpP mutations into the vancomycin-susceptible parent strain N315LR5P1 by a gene replacement procedure. The vancomycin MIC of N315LR5P1 was raised from 1 to 2 mg/liter by the introduction of the walK or clpP mutation, but it was raised to 4 mg/liter by the introduction of both the walK and clpP mutations. The vancomycin MIC value of the double mutant was equivalent to that of strain LR5P1-V3. Like VISA clinical strains, LR5P1-V3 and the double mutant strain LR5P1walKclpP exhibited a thickened cell wall, slow growth, and decreased autolytic activity. Transcriptional profiles of the mutants with gene replacements demonstrated that introduction of both the walK and clpP mutations could alter expression of dozens or hundreds of genes, including those involved in cell envelope and cellular processes, intermediary metabolism, and information pathway. A mutation prevalence study performed on 39 worldwide clinical VISA strains showed that 61.5, 7.7, 10.3, and 20.5% of VISA strains harbored mutations in walRK, clpP, graRS, and vraSR, respectively. The mutation of walRK was most frequently carried by VISA strains. Together, these results suggested that the mutations of walK and clpP identified in LR5P1-V3 constitute a new combination of genetic events causing vancomycin resistance in Staphylococcus aureus.
耐万古霉素中间金黄色葡萄球菌（VISA）是由万古霉素敏感金黄色葡萄球菌通过多次自发突变产生的。我们之前报道过，两组分调节系统 vraSR 和 graRS 的连续获得突变导致了 Mu50 菌株的 VISA 表型。在这里，我们报告了一组新的调节基因突变，两组分调节系统 walRK（同义词，vicRK 和 yycFG）的缺失突变，以及一个蛋白水解调节基因 clpP 的截断突变，这些突变导致实验室衍生的 VISA 菌株 LR5P1-V3 的万古霉素耐药性升高。通过基因替换程序将 walK 和 clpP 突变引入万古霉素敏感亲本菌株 N315LR5P1，证实了这两个突变对万古霉素耐药性的贡献作用。N315LR5P1 的万古霉素 MIC 值从 1 升高到 2mg/L，通过引入 walK 或 clpP 突变，但通过引入 walK 和 clpP 突变，MIC 值升高到 4mg/L。双突变体的万古霉素 MIC 值与 LR5P1-V3 菌株相当。与 VISA 临床株一样，LR5P1-V3 和双突变体菌株 LR5P1walKclpP表现出细胞壁增厚、生长缓慢和自溶活性降低。基因替换突变体的转录谱表明，同时引入 walK 和 clpP 突变可以改变数十或数百个基因的表达，包括参与细胞包膜和细胞过程、中间代谢和信息途径的基因。对 39 株全球临床 VISA 株进行的突变流行研究表明，61.5%、7.7%、10.3%和 20.5%的 VISA 株分别携带 walRK、clpP、graRS 和 vraSR 突变。walRK 突变在 VISA 株中最常见。总之，这些结果表明，在 LR5P1-V3 中发现的 walK 和 clpP 突变构成了金黄色葡萄球菌万古霉素耐药性的新的遗传事件组合。
