Multidrug resistant Acinetobacter baumannii,

描述(由申请人提供)：多重耐药革兰氏阴性菌(MDR-GNB)对成功的抗菌化疗构成重大威胁。在MDR-GNB中，对头孢菌素类、碳青霉烯类和喹诺酮类耐药的鲍曼不动杆菌正在成为一个全球性的问题。我们对从治疗伊拉克/科威特(伊拉克自由行动)和阿富汗(持久自由行动)受伤的平民和军事人员的军事医疗设施中分离到的多药耐药鲍曼不动杆菌的分析表明，不动杆菌衍生头孢菌素酶(ADC)、OXA-23和OXA-58类似碳青霉烯酶是头孢他啶和亚胺培南耐药的主要原因。我们了解到：1)ADC是C类酶中唯一的头孢菌素酶；2)外排泵与喹诺酮类耐药密切相关；3)AdeABC外排泵的调节是鲍曼不动杆菌多药耐药表型表达的核心。迫切的挑战是了解β-内酰胺耐药的遗传和氨基酸序列要求，ADC和OXA碳青霉烯酶的动力学性质和原子结构，以及MDR鲍曼不动杆菌外排泵的贡献。该建议的目的是：1)确定ADC-7β-内酰胺酶的原子结构并测试新型的硼酸和磺酸过渡态抑制剂，以揭示指导新抑制剂设计的重要构效关系；2)探索OXA碳青霉烯酶对亚胺培南水解所需的氨基酸并确定OXA-23的结构；3)阐明多药耐药鲍曼不动杆菌群体感应和生物膜形成的作用；以及4)通过DNA测序确定多药耐药分离株外排泵(AdeABC)调节区(Ader-ade)的遗传变异性。我们将把我们的ADER-ADES序列分析与一种基于快速自动聚合酶链式反应的电喷雾电离质谱仪方法相关联，该方法与碱基组成分析相结合，可以检测突变。实现这些目标将实现对蛋白质和底物相互作用以及导致MDR表型的遗传属性的前所未有的了解。这项工作还将作为鲍曼不动杆菌新耐药表型的快速诊断和预测的范例，形成评估新的β-内酰胺类药物的基础，并展示酶药物靶标如何影响底物亲和力和催化作用。

DESCRIPTION (provided by applicant): Multi-drug resistant gram-negative bacteria (MDR-GNB) present a significant threat to successful antimicrobial chemotherapy. Among MDR-GNB, Acinetobacter baumannii that are resistant to cephalosporins, carbapenems, and quinolones are becoming a global problem. Our analysis of MDR A. baumannii isolates recovered from military medical facilities treating civilian and military personnel injured in Iraq/Kuwait (Operation Iraqi Freedom) and Afghanistan (Operation Enduring Freedom) showed that the Acinetobacter derived cephalosporinase (ADC), OXA-23 and OXA-58 like carbapenemases are the major reasons for ceftazidime and imipenem resistance. We learned that: 1) ADC is a unique cephalosporinase among the class C enzymes; 2) efflux pumps contribute significantly to quinolone resistance; and 3) the regulation of the AdeABC efflux pump is central to the expression of the MDR phenotype in A. baumannii. The pressing challenge is to understand the genetic and amino acid sequence requirements for beta-lactam resistance, kinetic properties and atomic structures of the ADC and OXA carbapenemases, and the contribution of the efflux pump in MDR A. baumannii. The aims of this proposal are: 1) to determine the atomic structure of the ADC-7 beta-lactamase and to test novel boronic acid and sulfone transition state inhibitors in order to reveal important structure activity relationships that will guide new inhibitor design; 2) to explore the amino acid requirements for imipenem hydrolysis by OXA carbapenemases and to determine the structure of OXA-23; 3) to elucidate the role of quorum sensing and biofilm formation in MDR A. baumannii; and 4) to determine the genetic variability of the regulatory region (adeR-adeS) of the efflux pump (AdeABC) of MDR isolates by DNA sequencing. We will correlate our sequence analysis of adeR-adeS with a rapid automated PCR based electrospray ionization mass spectrometry method linked with base composition analysis that can detect mutations. Accomplishing these goals will achieve an unprecedented understanding of the protein and substrate interactions and genetic properties responsible for the MDR phenotype. This work will also serve as a paradigm for the rapid diagnosis and prediction of novel MDR phenotypes in A. baumannii, form a basis for the evaluation of novel beta-lactams, and show how enzyme drug targets influence substrate affinity and catalysis.