Date of Award
Master of Science (MS)
School of Biological Sciences
Brian J. Wilkinson
Staphylococcus aureus is a major human pathogen that is well known for its ability to develop resistance to antimicrobial therapies. Recent studies have identified a combination of Î²-lactam antibiotic and daptomycin (DAP) to be highly effective at killing single and multi-drug-resistant S. aureus. Of particular interest is this drug combinationâ??s efficiency with which it kills strains classified as DAP-resistant methicillin-resistant Staphylococcus aureus (DAP-R MRSA). The goals of the present study were two-fold: to provide a physiological profile of a DAP-R S. aureus strain undergoing beta-lactam treatment by observing changes in autolytic rate, susceptibility to lytic enzymes, levels of peptidoglycan O-acetylation, release of lipoteichoic acid, and localization of the major cellular autolysin Atl. Additionally, the manner by which Î²-lactam+DAP therapy was bactericidal was explored by examining cell viability and culture optical density in response to DAP+Nafcillin (NAF). The strains used in this study were a well-characterized DAP-R MRSA D712 and its isogenic DAP-Susceptible MRSA precursor (DAP-S MRSA) D592. Growth of DAP-S and DAP-R MRSA in the presence of the nafcillin increased whole cell and crude cell wall (CCW) autolysis. Interestingly, while D712 initially exhibited decreased whole cell autolysis both strains responded remarkably similarly to triton X-100 stimulated autolysis. CCWs of both D592 and D712 were almost entirely resistant to autolysis, lysing only ~10% over four hours. After NAF treatment a significant increase in autolysis was observed, however, the increase was more pronounced in D592. NAF exposure also increased purified cell wall (PCW) susceptibility lysoszyme and lysostaphin. Similar to CCW autolysis, nearly complete resistance to lysozyme was reversed upon growth in NAF, in contrast, lysostaphin was effective at lysing PCW, and NAF exposure produces a pronounced effect. Interestingly, D712 exhibited slight lysostaphin resistance, a phenotype that may be worth investigating. Despite the dramatic increase in lysoszyme susceptibility, growth in NAF did not result in attenuation in the level of O-acetyl groups, a factor that has been previously implicated in lysozyme resistance. In addition, NAF exposure appeared to alter Atl processing. Cell wall protein extracts in equal amounts displayed a highly similar ability to lyse suspended Micrococcus luteus cells, providing evidence that Atl may be delocalized upon NAF treatment. Together, these data suggest that changes in cell wall structure caused by Î²-lactams that may render cells more susceptible to daptomycin, and elucidation of these factors provided clues to the complicated nature of DAP resistance and will guide future research.
Krzyskowski, Michael, "Î?- Lactam-Induced Changes in Cell Wall Composition and Autolytic Properties of Daptomycin Resistant Staphylococcus Aureus" (2016). Theses and Dissertations. 515.