This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Growth of S. aureus with salicylate at concentrations that induce multiple antimicrobial resistance (2 mM) leads to growth inhibition. Since slow-growing bacteria express increased antimicrobial resistance, growth inhibition by salicylate may contribute to the salicylate-inducible antimicrobial resistance mechanism. In addition, with collaborator Wayne Van Vorheiss (NMSU) we demonstrated that O2 consumption by SH1000 cultures was reduced (p 0.01) during exposure to 2 mM salicylate by 31.2% (13.0 vs 9.0 ul) and CO2 production was reduced (p 0.01) by 35.4% after 50 min (11.3 vs 17.4 ul). This reduced metabolic activity correlate with the transcriptional analyses findings. In particular, the expression of gapA2 which encodes glyceraldehyde-3-phosphate dehydrogenase is downregulated by salicylate. GapA activity is also decreased ~ 2-fold and while the GapA inhibitor iodoacetate inhibits GapA activity in untreated cell lysates, 2 mM salicylate had no effect on activity. This demonstrates that salicylate inhibits GapA activity at the level of transcription. Growth in the presence of salicylate also downregulated the transcriptional activity of pgi, which encodes glucose-6-phosphate isomerase. Pgi catalyzes the interconversion of glucose-6-phosphate to fructose-6-phosphate and its activity is also required for glycolysis to occur. In contrast to the genes above, the genes found within the S. aureus gluconate operon (gntRKP) gntK and gntP were significantly upregulated following growth in the presence of salicylate.The addition of gluconate in excess to growth media containing salicylate ameliorated the growth inhibitory effect(s) of salicylate. Since there is no evidence of an Entner-Doudoroff pathway in S. aureus, gluconate is probably initially oxidized via the pentose phosphate cycle (PPC) in S. aureus. We hypothesized that if the metabolism of the salicylate-treated cell relied on a gluconate metabolic pathway, than the addition of glucose and the associated catabolite repression would contribute to salicylate toxicity. As expected, the addition of glucose exacerbated the growth inhibitory effects of salicylate. Another gene repressed by growth in the presence of salicylate is pckA, which encodes phosphoenolpyruvate carboxykinase. PckA converts phophoenolpyruvate from oxaloacetate using GTP and is required for gluconeogenesis Collectively, we speculate that salicylate derepresses catabolite repression, inhibiting the glycolytic pathway and gluconeogenesis stiffling overall glucose metabolism, and induces gluconate oxidation initiated with the pentose phosphate cycle that provides energy and metabolites allowing for continued growth during salicylate exposure. More experimentation is required to determine how this alteration in central metabolism contributes to the salicylate-inducible multiple antimicrobial resistance mechanism. Topoisomerase IV is the primary target of fluoroquinolones in S. aureus, and independent single-step fluoroquinolone-resistant mutants of S. aureus possess mutations in the genes encoding both topoisomerase IV subunits, parE and parC. Fusidic acid binds to the complex of elongation factor G (EF-G), GTP/GDP and the ribosome, inhibiting the release of EF-G-GDP after the translocation step of peptide synthesis, thereby inhibiting growth of susceptible bacteria. Mutations in fusA, the chromosomally located gene encoding EF-G in S. aureus leads to fusidic acid resistance by reducing the affinity of the drug for the protein synthesis machinery. Growth of S. aureus in the presence of salicylate leads to a reduction in susceptibility to the antibiotics ciprofloxacin and fusidic acid. We have captured the genes parE and fusA via SCOTS analysis following growth in the presence of salicylate and confirmed the salicylate inducibility of genes via real-time PCR analysis. fusA also appeared upregulated by salicyate on the array analysis (1.16-fold). Since salicylate exposure elevates parE and fusA transcription, it is possible that an increased production of antimicrobial target increases intrinsic resistance to ciprofloxacin and fusidic acid respectively. Growth in the presence of salicylate also downregulates mgrA which encodes a negative regulator of genes encoding the antimicrobial efflux pumps, NorA, NorB, NorC, and Tet38. We speculate that early on during salicylate induction one or more of these efflux pump transcriptional units may be upregulated transiently leading to increased production of a pump(s), but then returns to its pre-induced activity level. Since growth of S. aureus in the presence of salicylate reduces susceptibility to fusidic acid and common house cleaners, and reduces mgrA expression, we hypothesized that mgrA mutants should demonstrate reduced susceptibility to fusidic acid and a common house cleaner. mgrA::cat mutants demonstrated reduced susceptibility to a common house cleaner (), yet increased susceptibility to fusidic acid. Furthermore, the addition of salicylate to all drug gradients led to reduced susceptibility in all mgrA::cat mutants. More research is required to determine if the efflux pumps controlled by mgrA are also contributing to the response of the staphylococcal cell to common house cleaners. Overall, since mgrA inactivation does not affect GapA activity, nor inhibit salicylate-inducible multiple antimicrobial resistance, it appears that salicylate response functions in a mgrA-independent fashion. Interestingly, salicylate-induction did not modulate the expression of MgrA-regulated virulence genes (e.g. agr, hla, sarS, and spa). However, the major bifunctional autolysin atl negatively regulated by MgrA, was upregulated by growth with salicylate . Besides mgrA, we note that the gene encoding the negative regulator of sarA, sarR was also downregulated by growth with salicylate. Previously we have demonstrated that sarA is required for the expression of intrinsic multiple antimicrobial resistance and sarA regulation was induced by salicylate in S. aureus. However, mutational and transcriptional analysis of sarA, demonstrate that the salicylate effect on multiple antimicrobial resistance is sarA-independent.
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