Graduation Year

2012

Document Type

Thesis

Degree

M.S.

Degree Granting Department

Biology (Cell Biology, Microbiology, Molecular Biology)

Major Professor

James T. Riordan

Keywords

Bacteriology, Fluoroquinolones, Gram-positive, Microbiology, NSAIDs

Abstract

Growth of Staphylococcus aureus with the NSAID salicylate increases phenotypic resistance (SAPAR), and the frequency at which heritable resistance occurs to various antibiotics (SAGAR). This study describes the effect of salicylate on heritable and phenotypic resistance to a set of antibiotics for laboratory and multi-drug resistant strains of S. aureus and investigates the link between resistance and SAGAR. Drug gradient plates were used to determine phenotypic resistance to antibiotics targeting DNA replication, transcription, translation and the cell wall in the presence or absence of salicylate. To measure heritable resistance, mutation frequencies were determined for each antibiotic in the presence and absence of salicylate. Salicylate significantly increased mutation frequency of SH1000 to ciprofloxacin 27- fold from 4.9 x 10-8 to 8.5 x 10-7. A significant 8.5- fold increase was observed for LAC from 5.2 x 10-7 to 2.1 x 10-6. Conversely, salicylate significantly decreased mutation frequency for SH1000 to lincomycin 0.035-fold from 3.4 x 10-7 to 1.3 x 10-7. Deletion of the general stress sigma factor sigB encoding σB in SH1000 resulted in decreased heritable and phenotypic resistance, signifying the importance of σB in the full expression of both phenotypes. Metabolite profiling revealed downregulation of glycolysis, TCA, pentose phosphate pathway, and amino acid metabolism. The downregulation of the TCA cycle was confirmed as observed through an increase in NAD+ at growth toxic concentrations of salicylate. Salicylate has been shown to result in ROS accumulation and disruption of proton motive force in mitochondria. SAGAR was only detected for fluoroquinolones, which have been shown to impair TCA cycle and result in ROS accumulation. Examination of ROS under growth-toxic concentrations of salicylate did not reveal a significant increase in ROS levels. Also, the combination of ciprofloxacin and salicylate did not result in an increase in ROS levels. Despite this, addition of the antioxidant glutathione abrogated SAGAR for ciprofloxacin in SH1000 but not for SAPAR. Analysis of SAGAR with NSAIDs benzoate and acetyl salicylic acid revealed a necessity for the ortho hydroxyl group on salicylate to fully express SAGAR. These results suggest that salicylate has pleiotropic effects on S. aureus that include antimicrobial resistance, altered metabolic flux and accumulation of ROS as well as unidentified regulatory genes.

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