Newly Identified mechanisms of Ceftaroline Resistance in MRSA Clinical Strains

 描述（由申请方提供）：耐甲氧西林金黄色葡萄球菌（MRSA）是一种重要的传染性人类病原体，可引起皮肤和软组织感染至危及生命的疾病。新兴的研究表明，MRSA感染对患有慢性基础疾病（如囊性纤维化（CF），糖尿病和骨髓炎）的个体具有显著的临床影响，其中抗生素压力和代谢适应可能有利于金黄色葡萄球菌的适应性，以建立长期持久性。头孢洛林（CPT）是FDA批准的唯一一种靶向PBP 2a的头孢菌素，具有很强的抗菌活性，包括MRSA。我们检查了在美国首次分离的CPT高（CPT-HR）和CPT中（CPT-IR）耐药MRSA菌株的分子基础，这些菌株对CPT的MIC分别为2-8 μg/ml和≥32 μg/ml。尽管mecA基因突变与CPT耐药相关，但其分子机制尚未完全阐明。我们的数据表明，CPT-HR菌株具有三个SNP，其中两个在PBP 2a的青霉素结合口袋中，导致直接作为转肽酶活性位点的看门人的氨基酸替换，而第三个突变定位于非青霉素结合或二聚化结构域。与mecA的变化相关，在pbp 2中也发现了非同义突变。此外，其他相关的非同义SNP存在于thyA（编码胸苷酸合成酶的基因）和mutS和mutL（DNA错配修复）基因中。此外，我们确定CPT-IR菌株显示出CPT诱导的mecA表达的非常显著的增加，伴随着PBP 2中的突变，一起可能干扰CPT完全抑制PBP 2a和PBP 2a/PBP 2相互作用的能力。CPT-IR和CPT-HR的一个共同特点是对亚胺培南（IPM）高度耐药，本文旨在阐明CPT-HR和CPT-IR在MRSA临床菌株中的分子基础，为临床治疗耐CPT的MRSA提供新的抗菌药物组合。本提案的具体目的是：具体目的#1研究CPT耐药中PBP 2扰动的机制意义具体目的#2定义S。金黄色葡萄球菌代谢适应性与CPT耐药性发展过程中的thyA突变有关。具体目标#3确定遗传决定因素并分析碳青霉烯类在临床MRSA菌株获得CPT耐药性中的作用。

 DESCRIPTION (provided by applicant): Methicillin-resistant Staphylococcus aureus (MRSA) is an important infectious human pathogen responsible for diseases ranging from skin and soft tissue infections to life threatening diseases. Emerging research has demonstrated that MRSA infections have a significant clinical impact on individuals with chronic underlying diseases such as cystic fibrosis (CF), diabetes and osteomyelitis in where antibiotic pressure and metabolic adaptions may favor the adaptability of S.aureus to establish long persistence. Ceftaroline (CPT) is the only FDA-approved cephalosporin targeting PBP2a with strong antibacterial activity including MRSA. We have examined the molecular basis of both CPT-high(CPT-HR) and -intermediate (CPT-IR) resistant MRSA strains isolated for the first time in the USA displaying MICs to CPT of 2-8 μg/ml and ≥32 μg/ml, respectively. Although mutations in the mecA gene have been associated to CPT resistance, the molecular mechanisms have not been fully elucidated. Our data suggest that the CPT-HR strain harbored three SNPs, two of them in the penicillin-binding pocket of PBP2a that result in replacement of amino acids directly acting as gatekeeper to the transpeptidase active site, while the third mutation localizes at the non-penicillin binding or dimerization domain. Related tochanges in mecA, non-synonymous mutations were also identified in pbp2. In addition, other relevant nonsynonymous SNPs were present in thyA (gene coding for the thymidylate synthase) and mutS and mutL (DNA mismatch repair) genes. Moreover, we determined that CPT-IR strains displayed a very significant increase in CPT-induced mecA expression that, accompanied with mutations in PBP2, together may interfere with CPT's ability to repress completely both PBP2a and PBP2a/PBP2 interactions. A common feature consistently found in CPT-IR and -HR strains was their high level of resistance to imipenem (IPM).This proposal intends to elucidate the molecular bases of CPT-HR and CPT-IR in MRSA clinical strains, and to provide new therapeutic antimicrobial combinations for the treatment of CPT-resistant MRSA infections. The Specific Aims of this proposal are: Specific Aim#1 To investigate the mechanistic significance of PBP2 perturbations in CPT-resistance Specific Aim #2 To define S. aureus metabolic adaptations linked to thyA mutations during development of CPT-resistance. Specific Aim #3 To identify the genetic determinates and analyze the role of carbapenems in the acquisition of CPT-resistance in clinical MRSA strains.