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

描述（由申请人提供）：最近在健康个体中由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引起了全世界的关注。CA-MRSA菌株不同于医院获得性mrsa (HA-MRSA)，因为它们的基因组背景和在动物模型中增加的毒力。金黄色葡萄球菌对甲氧西林的耐药性是由嵌入在一个名为SCCmec的移动遗传元件（21-67 kb）中的mecA介导的。MRSA的scmec有五种类型，它们在mec复合体（mecI-mecR1-mecA）、整合质粒、转座子、IS元件和其他辅助基因方面有所不同。USA300和MW2 （USA400）是两种最常见的CA-MRSA分离株，携带SCCmec IV型，而USA100和USA200是常见的ha - mrsa，含有II型。我们实验室最近的研究表明，在CA-MRSA中，青霉素结合蛋白4 （PBP4）的缺失足以使oxacillin和nafcillin耐药性降低16倍，而在HA- MRSA中则没有。头孢西丁证实了这一发现，头孢西丁是一种半合成的β -内酰胺，可与PBP4不可逆结合，对CA-MRSA菌株具有协同杀伤作用，但对HA-MRSA菌株没有协同杀伤作用。由于所有MRSA菌株都含有mecA，这些数据表明编码PBP2A的mecA并不是CA-MRSA菌株耐甲氧西林的唯一决定因素。基于SCCmec类型的差异，我们假设CA-MRSA菌株USA300和USA400中SCCmec IV型的附属基因在缺乏pbp4或存在头孢西丁的情况下对oxacillin（也包括nafcillin）敏感。为了确定细菌决定因素，我们制定了两个目标：1)绘制在缺乏pbp4或存在pbp4结合的β -内酰胺（如头孢西丁）时赋予oxacillin/nafcillin敏感性的细菌决定因素；II) CA-MRSA菌株USA300和USA400在头孢西丁存在时赋予oxacillin敏感性的遗传决定因素的表征。通过这些研究，我们将确定IV型SCCmec中负责这种表型的基因（目的1）。这些基因在USA300和USA400中的突变体将被构建，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耐氧西林的遗传决定因素。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）已成为一个主要的健康问题。与医院获得性耐甲氧西林金黄色葡萄球菌相反，ca - mrsa毒性更强，可影响健康个体。我们发现CA-MRSA的“致命弱点”是青霉素结合蛋白4 （PBP4）是CA-MRSA耐甲氧西林所必需的。CA-MRSA中有一些基因参与了pbp4基因缺失后对邻苯西林的敏感性。我们的目标是鉴定和表征这些赋予oxacillin敏感性的基因。

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.