Virulence Determinants in Staphylococcus Bacteremia

描述（由申请人提供）：由金黄色葡萄球菌引起的感染跨越广泛的临床范围，从无症状的鼻运输到心内膜炎、骨和关节感染以及致命性休克。金黄色葡萄球菌感染率的上升和社区获得性菌株的出现，促使人们需要更多地了解这种新兴病原体的毒力决定因素，并评估它们在金黄色葡萄球菌菌血症患者预后中的作用。评估这些毒力决定因素在人类中的作用受到限制，因为缺乏大量、特征明确的血液金黄色葡萄球菌分离株。这种临床资源是由Vance Fowler（合作研究者）开发的，当时他创建了世界上最大的具有前瞻性特征的金黄色葡萄球菌菌血症（SAB）患者分离物集合之一。严格的临床分组定义应用于该收集（金黄色葡萄球菌菌血症组或SABG），最大限度地减少了患者异质性，这是评估临床严重程度和细菌毒力决定因素之间潜在关联的关键组成部分。在最初的R01应用程序中，使用多位点序列分型（MLST）和阵列比较基因组杂交（aCGH）来确定379个SABG分离株的系统发育关系，并确定基因组差异（例如，存在或不存在毒力因子，移动遗传元件等）。在这个亚群中，两个克隆复合物（CC5和CC30）显示出更复杂感染的显著趋势。对SAGA的阵列CGH分析表明，CC5和CC30内的分离株携带毒力因子的移动遗传元件（MGE）频率增加，并且不同CC内分离株的MGE内部序列差异显著。此外，在整个金黄色葡萄球菌基因组中发现核苷酸多态性，但在MGE和细菌表面蛋白中富集。基于我们的研究结果和其他实验数据表明基因组多态性和重排在金黄色葡萄球菌毒力中发挥重要作用，我们建议：1)利用小鼠脓毒症模型验证与CC5和CC30以及候选毒力基因毒力相关的假设；2)完成高通量基因组测序和比较分析，鉴定金黄色葡萄球菌分离亚群（复杂感染组：CIG）的基因组多态性和新基因；3)小鼠脓毒症模型中CIG分离株全基因组全表达分析；4)利用全基因组关联研究确定与毒力和差异基因表达相关的基因组多态性；4)选择毒力候选基因的完全等位基因置换，并在小鼠模型中进行测试。公共卫生相关性：金黄色葡萄球菌的遗传因素在引起和决定感染和疾病的严重程度方面起着关键作用。本研究提出通过检测已知参与的基因和鉴定可能参与感染的新基因，在大量临床分离的致病葡萄球菌中鉴定这些变异。从这项研究中获得的知识可能潜在地用于开发新的和可能的预防性治疗来根除复杂的葡萄球菌感染。

DESCRIPTION (provided by applicant): Infections caused by Staphylococcus aureus span a wide clinical spectrum, ranging from asymptomatic nasal carriage to endocarditis, bone and joint infections and lethal shock. Increasing rates of S. aureus infection and the emergence of community-acquired strains drive the need for an increased understanding of the virulence determinants of this emerging pathogen and evaluating the role they play in outcome of patients with S. aureus bacteremia. Evaluating the role of these virulence determinants in humans was limited by the absence of a large, well-characterized collection of bloodstream S. aureus isolates. Such a clinical resource was developed by Vance Fowler (Co-Investigator), when he created one of the world<s largest collections of prospectively characterized isolates from patients with S. aureus bacteremia (SAB). Stringent clinical grouping definitions were applied to this collection (the S. aureus bacteremia group or SABG), which minimized patient heterogeneity, a critical component in evaluating potential associations between clinical severity and bacterial virulence determinants. In the original R01 application, multilocus sequence typing (MLST) and array comparative genome hybridization (aCGH) were used to determine phylogenetic relationships and to identify genomic differences (e.g., presence or absence of virulence factors, mobile genetic elements, etc.) among a subset of 379 SABG isolates. Within this subset, two clonal complexes (CC5 and CC30) showed a significant trend toward more complicated infection. Array CGH analysis of SAGA indicates that isolates within CC5 and CC30 are associated with an increased frequency of mobile genetic elements (MGE) carrying virulence factors and that MGE contain a remarkable level of internal sequence divergence between isolates in different CC. Furthermore, nucleotide polymorphisms are found throughout the S. aureus genomes, but are enriched in MGE and bacterial surface proteins. Based on our results and other experimental data suggesting that genomic polymorphisms and rearrangements have significant roles in S. aureus virulence, we now propose to: 1) use the murine sepsis model to test hypotheses related to virulence of CC5 and CC30 and candidate virulence genes; 2) complete high-throughput genome sequencing and comparative analysis to identify genomic polymorphisms and novel genes in a subset of S. aureus isolates (complicated infection group: CIG); 3) complete genome wide expression analysis of CIG isolates in the murine sepsis model; 4) use genome wide association studies to identify genomic polymorphisms associated with virulence and differential gene expression and 4) complete allelic-replacement of selected virulence candidates and test in murine model. PUBLIC HEALTH RELEVANCE: Genetic factors in Staphylococcus aureus play a critical role in causing and determining the severity of infection and disease. This study proposes to identify these variations in a large collection of clinically isolated, disease-causing Staphylococci by examining genes known to be involved and identifying new genes that may be involved in infection. Knowledge acquired from this study could potentially be used to develop novel and possibly preventive therapies to eradicate complicated staphylococcal infections.