Defining the bacterial factors that modulate sensitivity to B-lactam in CA-MRSA

描述（由申请方提供）：最近健康个体中社区获得性耐甲氧西林金黄色葡萄球菌（CA-MRSA）菌株引起的感染病例在全球范围内引起了关注。CA-MRSA菌株与医院获得性MRSA（HA-MRSA）的不同之处在于其基因组背景和动物模型中的毒力增加。沙门氏菌对甲氧西林耐药。金黄色葡萄球菌是由mecA介导的，mecA嵌入在称为SCCmec的移动的遗传元件（21-67 kb）内。MRSA中有五种类型的SCCmec，它们在mec复合体（mecI-mecR 1-mecA）、整合质粒、转座子、IS元件和其他辅助基因方面不同。USA 300和MW 2（USA 400）是两种最常见的CA-MRSA分离株，携带SCCmec IV型，而USA 100和USA 200是常见的HA-MRSA，含有II型。我们实验室最近的研究表明，青霉素结合蛋白4（PBP 4）的缺失足以导致CA-MRSA中苯唑西林和萘夫西林耐药性降低16倍，但在HA-MRSA中则不然。这一发现得到了头孢西丁的证实，头孢西丁是一种不可逆结合PBP 4的半合成β-内酰胺，其与苯唑西林对CA-MRSA菌株表现出协同杀伤作用，但对HA-MRSA菌株则没有。由于所有MRSA菌株都含有mecA，这些数据表明编码PBP 2A的mecA不是CA-MRSA菌株中甲氧西林耐药性的唯一决定因素。基于SCCmec类型的差异，我们假设CA-MRSA菌株USA 300和USA 400中SCCmec IV型内的辅助基因在不存在pbp 4或存在头孢西丁的情况下赋予对苯唑西林（也是萘夫西林）的敏感性。为了定义细菌决定簇，我们开发了两个目的：I）在不存在pbp 4或存在PBP 4结合β-内酰胺如头孢西丁的情况下，定位赋予苯唑西林/萘夫西林敏感性的细菌决定簇; II）在CA-MRSA菌株USA 300和USA 400中，表征在头孢西丁存在的情况下赋予苯唑西林敏感性的遗传决定簇。通过这些研究，我们将确定IV型SCCmec中负责该表型的基因（目的1）。将构建USA 300和USA 400中这些基因的突变体，并在抗生素敏感性、细胞壁代谢和细胞壁形态方面进行表征（Aim II）。在完成这些研究后，我们试图确定影响CA-MRSA苯唑西林耐药性的遗传决定因素。这些遗传决定因素可能是开发新型抗菌治疗的目标。 公共卫生相关性：社区获得性耐甲氧西林金黄色葡萄球菌（CA-MRSA）已成为主要的健康问题。与医院获得性耐甲氧西林沙门氏菌相反。金黄色葡萄球菌、CA-MRSA的毒性更强，可影响健康个体。我们发现CA-MRSA中的“阿喀琉斯之踵”在于青霉素结合蛋白4（PBP 4）是CA-MRSA中的甲氧西林耐药性所必需的。在CA-MRSA中，在pbp 4基因缺失后，存在参与苯唑西林敏感性的基因。我们的目标是确定和表征这些基因，赋予苯唑西林敏感性。

DESCRIPTION (provided by applicant): Recent cases of infections caused by community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) strains in healthy individuals have raised concerns worldwide. CA-MRSA strains differ from hospital-acquired MRSAs (HA-MRSA) by virtue of their genomic background and increased virulence in animal models. Resistance to methicillin in S. aureus is mediated by mecA which is embedded within a mobile genetic element (21-67 kb) called SCCmec. There are five types of SCCmec in MRSA, which differ with respect to the mec complex (mecI-mecR1-mecA), integrated plasmids, transposons, IS elements and other accessory genes. USA300 and MW2 (USA400), the two most common CA-MRSA isolates, carry the SCCmec type IV while USA100 and USA200, which are common HA-MRSAs, contain type II. Recent studies from our lab have shown that a loss of penicillin-binding protein 4 (PBP4) is sufficient to cause a 16-fold reduction in oxacillin and nafcillin resistance in CA-MRSA, but not in HA- MRSA. This finding was confirmed with cefoxitin, a semi-synthetic beta-lactam that binds PBP4 irreversibly, which exhibits synergistic killing with oxacillin against CA-MRSA strains but not against HA-MRSA strains. As all MRSA strains contain mecA, these data indicate that that mecA encoding PBP2A is not the sole determinant of methicillin resistance in CA-MRSA strains. Based on the differences in SCCmec types, we hypothesize that the accessory genes within SCCmec type IV in CA-MRSA strains USA300 and USA400 confer sensitivity to oxacillin (also nafcillin) in the absence of pbp4 or in the presence of cefoxitin. To define the bacterial determinants, we have developed two aims: I) mapping the bacterial determinants that confer sensitivity to oxacillin/nafcillin in the absence of pbp4 or in the presence of PBP4-binding beta-lactam such as cefoxitin; II) characterization of the genetic determinants that confer oxacillin sensitivity in the presence of cefoxitin in CA-MRSA strains USA300 and USA400. With these studies, we will identity the genes in type IV SCCmec responsible for this phenotype (Aim 1). Mutants of these genes in USA300 and USA400 will be constructed and characterized with respect to antibiotic sensitivity, cell wall metabolism and cell wall morphology (Aim II). Upon completion of these studies, we seek to identify genetic determinants that impact oxacillin resistance in CA-MRSA. These genetic determinants may be targets for the development of novel antimicrobial therapy. PUBLIC HEALTH RELEVANCE: Community-acquired methicillin resistant Staphylococcus aureus (CA-MRSA) has become a major health problem. Contrary to hospital-acquired methicillin resistant S. aureus, CA-MRSAs are more virulent and can affect healthy individuals. We have found an "Achilles Heel" in CA-MRSA in that penicillin binding protein 4 (PBP4) is required for methicillin resistance in CA-MRSA. There are genes in CA-MRSA involved in this oxacillin sensitivity upon deletion of the pbp4 gene. Our goal is to identify and characterize these genes that confer oxacillin sensitivity.