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.
