Newly Identified mechanisms of Ceftaroline Resistance in MRSA Clinical Strains

 描述(申请人提供)：耐甲氧西林金黄色葡萄球菌(MRSA)是一种重要的人类感染性病原体，可导致从皮肤和软组织感染到危及生命的各种疾病。新出现的研究表明，MRSA感染对患有囊性纤维化(CF)、糖尿病和骨髓炎等慢性基础疾病的个人具有重大的临床影响，在这些疾病中，抗生素压力和代谢适应可能有利于金黄色葡萄球菌建立长期持久性的适应。头孢他林(CPT)是FDA批准的唯一针对PBP2a的头孢菌素，具有很强的抗菌活性，包括MRSA。我们研究了美国首次分离的耐CPT高(CPT-HR)和中耐CPT(CPT-IR)的耐甲氧西林金黄色葡萄球菌的分子基础，其MIC值分别为2-8μg/ml和≥32μg/ml。虽然mecA基因突变与CPT耐药有关，但其分子机制尚未完全阐明。我们的数据表明，CPT-HR株含有三个SNP，其中两个位于PBP2a的青霉素结合口袋中，导致氨基酸直接作为转肽酶活性部位的守门人，而第三个突变位于非青霉素结合或二聚区域。与mecA的改变有关，在pbp2中也发现了非同义突变。此外，Thya(胸苷合成酶编码基因)和MutS和muL(DNA错配修复)基因中还存在其他相关的非同义SNP。此外，我们确定CPT-IR菌株显示出非常显著的CPT诱导的mecA表达增加，伴随着PBP2的突变，共同可能干扰CPT完全抑制PBP2a和PBP2a/PBP2相互作用的能力。CPT-IR和-HR菌株的一个共同特征是对亚胺培南(IPM)的高度耐药，本研究旨在阐明CPT-HR和CPT-IR在MRSA临床菌株中的分子基础，并为治疗CPT耐药的MRSA感染提供新的抗菌药物组合。这项建议的具体目的是：具体目标1调查PBP2干扰在CPT耐药中的机制意义；具体目标2确定金黄色葡萄球菌在CPT耐药发展过程中与Thya突变相关的代谢适应。具体目标#3确定基因决定因素并分析碳青霉烯类抗生素在临床MRSA菌株获得CPT耐药中的作用。

 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.