Date of Award
Doctor of Philosophy
Molecular Biology, Microbiology and Biochemistry
Staphylococcus aureus is a gram-positive bacterial pathogen that causes diseases such as skin and soft tissue infections, osteomyelitis, and endocarditis, and it is listed by the World Health Organization as a global priority pathogen owing to prevalence of infections and antibiotic resistant strains. S. aureus one of six ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) pathogens that pose a threat to current antibiotic treatments due to their propensity for acquiring antibiotic resistance to an array of different antibiotics. We sought to determine mechanisms underlying how S. aureus develops antibiotic resistance and how the bacterium obtains carbon from the host. Vancomycin is commonly used to treat multi-drug resistant species of bacteria such as S. aureus Strains of vancomycin-intermediate Staphylococcus aureus (VISA) and vancomycin-resistant Staphylococcus aureus (VRSA) strains have emerged and threaten the viability of vancomycin as an effective treatment. The harsh environment within biofilm formations gives way to genetic diversity mediated by the RecA/SOS response, and we hypothesized that development of a VISA subpopulation within biofilm growth may be a result of mutations that occur during this error prone DNA repair mechanism. Both wildtype and RecA/LexA deficient biofilms and planktonic cultures were grown with and without sub-inhibitory concentrations of ciprofloxacin (0.25 µg/ml) and vancomycin (0.5 µg/ml). The efficiency of plating technique was used to quantify the subpopulation of S. aureus cells with the VISA phenotype (MIC: 4 – 8 µg/ml). RecA expression was measured via RT-PCR and fluorescence using a gfp reporter strain. Antibiotic testing was done as an alternate means of quantifying mutagenesis. A larger subpopulation of cells derived from wildtype biofilms (4.16 x 102 CFUs) grew in increased concentrations of vancomycin (4 µg/ml) as compared to the planktonic counterpart (1.53 x 101 CFUs). Wildtype biofilms (4.16 x 102 CFUs) also exhibited increased growth compared to strains with inactive recA (8.15 x 101 CFUs) or lexA genes (3.00 x 101 CFUs). When measuring recA expression using a gfp reporter strain, we found that recA is upregulated in biofilms treated with ciprofloxacin (258.2 RFUs) and vancomycin (197 RFUs), compared to biofilms grown without sub-inhibitory antibiotics (158.2 RFUs). The VISA phenotype observed in our study is an unintended consequence of the natural stress within biofilms, and our results further suggest that the biofilm environment allows for bacteria to better adapt to the presence of widely used antibiotics. The S. aureus genome contains the nan locus, a set of genes that enables S. aureus to catabolize sialic acid (N-acetylneuraminic acid [Neu5Ac]) as part of its central metabolism. Bacterial species utilize sialic acid for a variety of functions, including metabolic processes and evasion of the host immune system. We hypothesize S. aureus’ ability to catabolize Neu5Ac may confer a growth or competitive advantage to S. aureus during its colonization of the host. Specifically, our study analyzes biofilm growth using Neu5Ac as a primary source of carbon and nitrogen. To assess S. aureus’ ability to catabolize Neu5Ac in a biofilm environment, knock-out mutants within the nan locus were created in the S. aureus laboratory strain HG001. Biofilm growth was analyzed using crystal violet assays, and biofilm cultures derived from microtiter assays were plated on TSA, after which colony forming units (CFUs) were calculated. These experiments were repeated in co-culture with Streptococcus pneumoniae to assess growth in the presence of a sialidase. The results of the crystal violet and microtiter assays suggest that while glucose is the primary carbon source for S. aureus in both planktonic and biofilm conditions, Neu5Ac supplementation may provide S. aureus with a nominal growth advantage compared to growth without an additional carbon source. Pilot co-culture experiments demonstrate sufficient growth of S. aureus and S. pneumoniae together in co-culture, and S. aureus may acquire Neu5Ac by utilizing other bacterial species’ sialidase activity. However, further experimentation is required to understand the importance of the Neu5Ac catabolic pathway in S. aureus under various growth conditions and in a polymicrobial environment.
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