ROLE OF BETA-LACTAMASE IN WOUND INFECTION PATHOGENESIS

尽管在手术中常规使用预防性抗体，但仍有35000人 金黄色葡萄球菌伤口感染在美国每年都会发生。 而与感染相关的金黄色葡萄球菌菌株通常看起来 在常规药敏试验、专门化验中对抗生素敏感 证明它们通常能够有效地水解 体外抗生素。临床研究表明，葡萄球菌的存活率 接种到伤口后，部分是由β-内酰胺酶介导的- 介导的预防性抗生素在体内的降解。少校 这项拟议工作的目的是阐明β-内酰胺酶在 金黄色葡萄球菌创面败血症的发病机制。最新生物化学研究 表明金黄色葡萄球菌有八种运动学上可以区分的类型 β-内酰胺酶。根据初步数据，假设 尽管表现出明显的结构相似性，但 不同β-内酰胺酶侧翼部分的异质性 活性位裂隙产生了它们之间的动力学差异。这一假说将 通过确定初级蛋白质结构的差异来进行评估， 根据核苷酸序列推断，在之前未测序的六个 葡萄球菌β-内酰胺酶的种类。随后，结构-功能 包括氨基酸替换的鉴定在内的关系(S) 对动力学异质性的解释将通过现场定向来探索 诱变及其嵌合β-内酰胺酶的构建 与两个β-受体相关的部分结构基因的重组 内酰胺酶类型。最后，各种β-内酰胺酶在 介导金黄色葡萄球菌在伤口接种后的存活 将使用皮下脓肿的活体模型来探索感染 仅在类型上不同的金黄色葡萄球菌的形成和同基因菌株 产β-内酰胺酶的量。更好地理解了 细菌逃避给予抗生素的手段的分子水平 预防感染可能有助于制定策略以减少这种情况的发生 这些病态的和潜在的致命感染。

Despite the routine use of prophylactic antibodies in surgery, 35,000 Staphylococcus aureus wound infections occur annually in the United States. Whereas infection-associated strains of S. aureus usually appear to be antibiotic-susceptible in routine susceptibility tests, specialized assays demonstrate that they are often capable of efficiently hydrolyzing the antibiotic in vitro. Clinical studies suggest that staphylococcal survival after inoculation into the wound is mediated, in part, by beta-lactamase- mediated degradation of the prophylactic antibiotic in vivo. The major objective of the proposed work is to clarify the role of beta-lactamase in the pathogenesis of S. aureus wound sepsis. Recent biochemical studies suggest that there are eight kinetically-distinguishable types of S. aureus beta-lactamase. Based upon preliminary data, it is hypothesized that despite exhibiting marked structural similarity, small regions of heterogeneity in the portions of the various beta-lactamase that flank the active site cleft produce their kinetic differences. This hypothesis will be evaluated by identifying the differences in primary protein structure, as deduced from the nucleotide sequence, of the six previously-unsequenced types of staphylococcal beta-lactamase. Subsequently, structure-function relationships including identification of the amino acid substitutions(s) that account for kinetic heterogeneity will be explored by site-directed mutagenesis and the construction of chimeric beta-lactamases after recombination of portions of the structural gene associated with two beta- lactamase types. Finally, the role of the various beta-lactamases in mediating S. aureus survival following wound inoculation with subsequent infection will be explored using an in vivo model of subcutaneous abscess formation and isogenic strains of S. aureus that differ only in the type and amount of beta-lactamase produced. An improved understanding at the molecular level of the means by which bacteria evade antibiotics given to prevent infection may help in developing strategies to reduce the occurance of these morbid and potentially mortal infections.