Staphylococcus aureus is a pathogen responsible for a wide variety of life-threatening diseases such as bacteremia, endocarditis, and pneumoniae. S. aureus has been a major concern in recent years due to the rampant spread of antibiotic resistance. The ability of S. aureus to form biofilms aids in the spread of antibiotic resistance as biofilms are a known hotspot for horizontal gene transfer. Biofilms also protect cells from host immune responses and antibiotics, making these infections very difficult to treat. The matrix of S. aureus biofilms can be made of polysaccharides, protein, and DNA. In these studies, we sought to elucidate how biofilm composition correlates with source of isolation in S. aureus strains, the role of biofilm-related genes in biofilm composition, and the potential role of biofilm eDNA in horizontal gene transfer. The composition and strength of biofilms made by a variety of hospital and meat-associated strains of S. aureus was measured using crystal violet (CV) staining and DNase or proteinase K treatment. Biofilm polysaccharide concentration was also measured using the phenol sulfuric-acid assay. We found that biofilms of hospital-associated isolates tend to have more protein and polysaccharides while those of meat isolates contain significantly more DNA. We also investigated the effects that biofilm-related genes have on biofilm formation and composition by analyzing specific transposon mutants of genes suggested by previous studies to play a role in biofilm development. Transposon insertions in agrA, atl, clfA, fnbA, purH, and sarA significantly weakened biofilms as compared to a wild-type control, whereas the acnA insertion mutant produced a significantly stronger biofilm. Biofilms formed from these mutant strains were treated (or mock-treated) with DNase or proteinase K and tested with phenol and sulfuric acid to determine what role these genes play in biofilm composition. We found that the atl and sarA insertion mutants produced biofilms with greater polysaccharide concentrations than the wild-type. Since many of the isolates produced biofilms composed of DNA, we investigated the potential role of this extracellular DNA in horizontal gene transfer. Strains with complementary antibiotic resistances and susceptibilities were paired together and co-cultured together in a biofilm and plated onto double antibiotic plates to select for possible gene transfer. Putative gene transfer was found to be largely biofilm dependent and enhanced with the addition of subinhibitory concentrations of antibiotics added to the biofilm. Potential transformation was also shown to naturally occur in many strains when naked DNA was added to a single strain biofilm and was also aided with the addition of subinhibitory antibiotics.



College and Department

Life Sciences; Microbiology and Molecular Biology



Date Submitted


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Staphylococcus aureus, antibiotic resistance, biofilm, horizontal gene transfer, MRSA



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Life Sciences Commons