Date of Award


Degree Name

Master of Science


Molecular Biology Microbiology and Biochemistry

First Advisor

Olson, Michael


The glycopeptide vancomycin is commonly used to treat a variety of bacterial infections, especially multi-drug resistant species of bacteria such as Staphylococcus aureus. While vancomycin remains an effective treatment for Staphylococcal infections, strains of vancomycin-intermediate Staphylococcus aureus (VISA) and vancomycin-resistant Staphylococcus aureus (VRSA) strains have emerged. One mechanism for the increased antibiotic (vancomycin-intermediate) resistance is due to acquisition of various mutations within different genes that alter the cell wall physiology making vancomycin ineffective. Biofilm development is a bacterial mode of growth that can lead to mutations within the bacterial genome and allow for advantageous traits such as increased antibiotic resistance. The biofilm environment can be harsh, having niches that are often nutrient and oxygen deficient, leading to damaged DNA. This DNA damage induces the SOS response to repair double-stranded breaks in DNA, and enables bacterial survival. However, DNA repair via the SOS response is an error-prone system that often results in mutations within the genome. We hypothesize that the acquisition of vancomycin intermediate resistance is an unintended consequence within the S. aureus biofilm environment. To assess the hypothesis, both wildtype and RecA/LexA deficient biofilms were grown in microtiter assays with and without the addition of sub-inhibitory concentrations of vancomycin. Efficiency of plating techniques were used to quantify the subpopulation of biofilm-derived S. aureus cells that developed vancomycin intermediate resistance. Microtiter assays and efficiency of plating techniques were repeated using multiple strains of S. aureus. Experimentation was repeated by comparing the subpopulation of biofilm-derived and planktonic culture cells that grew in intermediate-concentrations of vancomycin with three additional strains of S. aureus. Mutagenesis that occurs within the biofilm environment was further assessed by plating both biofilm-derived and planktonic culture cells on sheep blood agar and tryptic soy agar supplemented with streptomycin, novobiocin, or rifampicin, and quantifying the non-hemolytic variants that grew on blood agar, or the number of colonies that grew in the presence of an antibiotic, respectively. The biofilm results were then compared to the results from wildtype and RecA/LexA deficient planktonic cultures and used to determine the impact of the S. aureus biofilm environment in the acquisition of vancomycin intermediate resistance. The results indicate that a larger subpopulation of cells derived from wildtype biofilms grew in increased concentrations of vancomycin (4 µg/ml) as compared to the planktonic counterpart. The subpopulation of cells derived from wildtype biofilms was also higher than all subpopulations of RecA/LexA deficient biofilm and planktonic cultures. Further experimentation indicates that this phenomenon may not be specific to all strain backgrounds of S. aureus. Additionally, growth with sub-inhibitory concentrations of vancomycin did not exhibit an exaggerated subpopulation of cells in biofilm environments or planktonic cultures that could grow in intermediate-concentrations of vancomycin, however standard antibiotic testing suggests that the mechanism by which point mutations occur in planktonic conditions may be mediated by the RecA and SOS response system. Bacteria that live in a biofilm community are often subjected to harsh environments. In order to survive, the SOS response system will be activated to repair damaged DNA. This error prone process will result in mutational changes and increased genetic diversity. The VISA phenotype may be a result of the diversity that occurs within the biofilm environment. While the VISA phenotype would be an unintended consequence of genetic diversity and gene transfer in the biofilm setting, it demonstrates that mutations that occur within the biofilm environment allow for S. aureus to better adapt to new environments, including the presence of widely used antibiotics such as vancomycin.




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