Kuroda Makoto, Sekizuka Tsuyoshi, Matsui Hidehito, Ohsuga Jun, Ohshima Toshio, Hanaki Hideaki
Pathogen Genomics Center, National Institute of Infectious Diseases, Shinjuku, Japan.
Infection Control Research Center, Kitasato University, Minato-ku, Japan.
Front Microbiol. 2019 Aug 14;10:1882. doi: 10.3389/fmicb.2019.01882. eCollection 2019.
Vancomycin (VAN)-intermediate-resistant s (VISA) is continually isolated globally, with a systematic review suggesting a prevalence of 2% in all blood culture samples. Most VISA strains exhibit common characteristics, such as a thickened cell wall, reduced autolysis, and attenuated virulence. Here, based on multi-omics approaches, we have characterized clinical VISA isolates obtained through prolonged antimicrobial treatment in a single patient. All VISA isolates were isogenic, based on multi-locus sequence typing (MLST) ST5, SCC type II (2A), and type t17639. Core-genome single nucleotide variations (SNVs) found among thirteen isolates during the patient's hospitalization, indicated clonality, but not notable genetic features of the VISA phenotype. We determined the complete genome sequence of VAN-susceptible strain KG-03 (minimum inhibitory concentration [MIC] 0.5 μg/mL) and two VISA strains, KG-18 and KG-22 (MIC 8.0 and 4.0 μg/mL, respectively). Comparative genome analysis showed remarkable strain-specific IS insertions. RNA-Seq transcriptome analysis revealed IS-mediated overexpression of the two-component system in VISA KG-18, possibly leading to modulation of cell wall integrity ( and ) and surface charge ( and ). In addition, secretome analysis indicated that cell wall-anchored proteins (Protein A, SasG, and SdrD) were significantly decreased. KG-18 and KG-22 exhibit thickened cell wall, and are relatively resistant to lysostaphin, which cleaves a staphylococcus-unique pentaglycine chain in the peptidoglycan. We conclude that KG-18 achieved reduced susceptibility to VAN by IS-mediated WalKR overexpression, leading to a markedly thickened cell wall for trapping free VAN molecules with redundant D-Ala-D-Ala targets. In addition, a positively charged surface with lysyl-phosphatidylglycerol and depolarization of wall teichoic acid could contribute to inhibiting cationic daptomycin and VAN antimicrobial activity. Comparative omics approaches in this study strongly suggest that fully complete and annotated genome sequences will be indispensable for characterizing overall VISA phenotype.
万古霉素（VAN）中介耐药金黄色葡萄球菌（VISA）在全球范围内不断被分离出来，一项系统评价表明，在所有血培养样本中其流行率为2%。大多数VISA菌株表现出共同特征，如细胞壁增厚、自溶减少和毒力减弱。在此，基于多组学方法，我们对通过在一名患者中进行长期抗菌治疗获得的临床VISA分离株进行了特征分析。基于多位点序列分型（MLST）ST5、SCC II型（2A）和t17639型，所有VISA分离株均为同基因。在患者住院期间，在13株分离株中发现的核心基因组单核苷酸变异（SNV）表明存在克隆性，但没有VISA表型的显著遗传特征。我们测定了万古霉素敏感菌株KG-03（最低抑菌浓度[MIC]为0.5μg/mL）以及两株VISA菌株KG-18和KG-22（MIC分别为8.0和4.0μg/mL）的全基因组序列。比较基因组分析显示出显著的菌株特异性插入序列（IS）插入。RNA测序转录组分析显示，在VISA KG-18中，IS介导双组分系统的过表达，这可能导致细胞壁完整性（和）以及表面电荷（和）的调节。此外，分泌蛋白组分析表明，细胞壁锚定蛋白（蛋白A、SasG和SdrD）显著减少。KG-18和KG-22表现出细胞壁增厚，并且对溶葡萄球菌素相对耐药，溶葡萄球菌素可切割肽聚糖中葡萄球菌特有的五肽甘氨酸链。我们得出结论，KG-18通过IS介导的WalKR过表达实现了对万古霉素敏感性降低，导致细胞壁明显增厚，以捕获带有多余D-Ala-D-Ala靶点的游离万古霉素分子。此外，带正电荷的赖氨酰磷脂酰甘油表面以及壁磷壁酸的去极化可能有助于抑制阳离子达托霉素和万古霉素的抗菌活性。本研究中的比较组学方法强烈表明，完整且注释完整的基因组序列对于全面表征VISA表型将是不可或缺的。
