Investigations of the effects of some antimicrobials and anti-inflammatory substances on the transcriptomes and proteins of the clinical staphylococci isolates

The aim of our study was to investigate the in vitro effects of antimicrobials, alone or in combination with anti-inflammatory drugs on Staphylococci strains. For this purpose, before gene expression studies, investigations of the susceptibilities of three clinical methicillin resistant Staphylococcus aureus (MRSA) isolates and standard S. aureus ATCC strain with antimicrobials (moxifloxacin, vancomycin, ciprofloxacin, clindamycin and gentamicin) and anti-inflammatory agents (diclofenac, naproxen, ibuprofen, acetylsalicylic acid ve acetaminophen) by disc diffusion test and microdilution assay, synergy studies (checkerboard method) and alteration of minimum inhibitory concentration (MIC) studies were performed with antimicrobials and other anti-inflammatory agents. After exposure to the agents, gene espression ratios of virulence factor coding genes and regulator genes (spa, sarA, RNAIII, sigB, mepA, mgrA ve srtA) in Staphylococcus aureus strains were investigated at RNA level by quantitative real time reverse transcriptase polymerase chain reaction (RT-qPCR) method. Then by evaluating the detected inhibitory/inducing concentrations of agents at RNA expression level, isolation of bacterial protein and quantification of protein assay were performed, and staphylococcal protein A (spa) expression levels were investigated by Western blot analysis. According to disc diffusion results, among the five anti-inflammatory drugs, diclofenac was found to be the highest inhibition zone diameters. Ibuprofen inhibition zone diameters were detected lower than diclofenac. According to the microdilution method results; diclofenac at 195.31 µg/ml and 390.63 µg/ml concentrations showed inhibitory activity against two strains. The MIC value of acetylsalicylic acid against ATCC strain was determined as 390.63 µg/ml. According to the checkerboard assay results; combination of gentamicin+acetaminophen against three strains, combinations of gentamicin+diclofenac, gentamicin+ibuprofen, vancomycin+ acetylsalicylic acid, ciprofloxacin+diclofenac and vancomycin+naproxen against two strains, were determined synergistic, respectively. According to alteration of MIC studies, the most effective combinations were evaluated as gentamicin with diclofenac, ibuprofen and naproxen and clindamycin with diclofenac. In addition, ciprofloxacin+diclofenac combination significantly reduced the MIC levels of ciprofloxacin against two clinical isolates and ATCC strain. MIC levels of all anti-inflammatory drugs inhibited the growth of clinical isolates and ATCC strain with 4 and 16 hours incubation. The effects of antibiotics and anti-inflammatory drugs with different concentrations on expression of virulence factors and regulator genes depended on the drugs and the virulence factor/regulator gene. According to the transciptional analyses, despite the increase in the gene expression of mepA, the MICs of ciprofloxacin and gentamicin were decreased, and we believed that this alteration was mepA-independent. The results of immunoblotting study has showed that; spa protein expression has been shown to be reduced by MICs of anti-inflammatory drugs and combinations, and induced by sub-MIC levels of gentamicin, vancomycin, acetylsalicylic acid, diclofenac and naproxen. Therefore, anti-inflammatory agents may be the therapeutic agents against staphylococcal infections by modulating the virulence factors and regulator genes. Further studies with phenotyphic assays, transcriptional and protein analyses are necessary to research relations between the resistance and the drugs and to understand the mechanisms of the anti-inflammatory and other drugs against bacterial strains.