Anti-Infective Research Laboratory, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, 259 Mack Ave, Detroit, MI 48201, USA.
J Infect Dis. 2013 Jul;208(1):67-74. doi: 10.1093/infdis/jit127. Epub 2013 Mar 28.
We used 2 in vitro experimental systems to compare phenotypic and genotypic changes that accompany selection of mutants of methicillin-resistant Staphylococcus aureus (MRSA) strain JH1 with low-level vancomycin resistance similar to the type found in vancomycin-intermediate S. aureus (VISA).
The previously described MRSA strain JH1 and its vancomycin-intermediate mutant derivative JH2, both of which were recovered from a patient undergoing vancomycin chemotherapy, were used in this study. Mutants of JH1 were selected in vitro by means of a pharmacokinetic/pharmacodynamic (PK/PD) model of simulated endocardial vegetations (SEVs) and by exposure to vancomycin in laboratory growth medium. Phenotypic abnormalities of JH1 mutants generated by each in vitro experimental system were compared to those of JH2, and whole genomes of 2 in vitro JH1 mutants were sequenced to identify mutations that may be associated with an increased vancomycin minimum inhibitory concentration.
JH1R1 was selected from the PK/PD model, and JH1R2 was selected in laboratory growth medium. Both mutants displayed reduced vancomycin and daptomycin susceptibility and phenotypic alterations (eg, thicker cell walls and abnormal autolysis) that are typical of in vivo VISA mutants. Genome sequencing of JH1R1 identified point mutations in 4 genes, all of which were different from the mutations described in JH2, including 1 mutation in yycG, a component of the WalKR sensory regulatory system. Sequencing of the JH1R2 genome identified mutations in 7 genes, including 2 in rpoB.
Our findings indicate that JH1 is able to develop VISA-type resistance through several alternative genetic pathways.
我们使用了 2 种体外实验系统来比较耐甲氧西林金黄色葡萄球菌（MRSA）菌株 JH1 的低水平万古霉素耐药突变体的表型和基因型变化，这些变化类似于发现的万古霉素中介金黄色葡萄球菌（VISA）的类型。
本研究使用了先前描述的 MRSA 菌株 JH1 及其万古霉素中介突变体衍生物 JH2，这两种菌株均从接受万古霉素化疗的患者中分离出来。通过模拟心内膜赘生物（SEV）的药代动力学/药效学（PK/PD）模型和在实验室生长培养基中暴露于万古霉素，体外选择 JH1 的突变体。通过每种体外实验系统生成的 JH1 突变体的表型异常与 JH2 进行比较，并对 2 种体外 JH1 突变体的全基因组进行测序，以鉴定可能与万古霉素最小抑菌浓度增加相关的突变。
JH1R1 是从 PK/PD 模型中选择的，JH1R2 是在实验室生长培养基中选择的。这两种突变体均表现出对万古霉素和达托霉素的敏感性降低以及表型改变（例如，细胞壁增厚和异常自溶），这些改变是体内 VISA 突变体的典型特征。JH1R1 的基因组测序确定了 4 个基因中的点突变，这些突变都与 JH2 中的突变不同，包括 WalKR 感觉调节系统的一个组成部分 yycG 中的 1 个突变。JH1R2 基因组的测序确定了 7 个基因中的突变，包括 rpoB 中的 2 个突变。
我们的研究结果表明，JH1 能够通过几种替代遗传途径发展出 VISA 型耐药性。
