Alterations in the transcriptome and antibiotic susceptibility of Staphylococcus aureus grown in the presence of diclofenac.

BACKGROUND: Diclofenac is a non-steroidal anti-inflammatory drug (NSAID) which has been shown to increase the susceptibility of various bacteria to antimicrobials and demonstrated to have broad antimicrobial activity. This study describes transcriptome alterations in S. aureus strain COL grown with diclofenac and characterizes the effects of this NSAID on antibiotic susceptibility in laboratory, clinical and diclofenac reduced-susceptibility (DcRS) S. aureus strains. METHODS: Transcriptional alterations in response to growth with diclofenac were measured using S. aureus gene expression microarrays and quantitative real-time PCR. Antimicrobial susceptibility was determined by agar diffusion MICs and gradient plate analysis. Ciprofloxacin accumulation was measured by fluorescence spectrophotometry. RESULTS: Growth of S. aureus strain COL with 80 µg/ml (0.2 × MIC) of diclofenac resulted in the significant alteration by ≥2-fold of 458 genes. These represented genes encoding proteins for transport and binding, protein and DNA synthesis, and the cell envelope. Notable alterations included the strong down-regulation of antimicrobial efflux pumps including mepRAB and a putative emrAB/qacA-family pump. Diclofenac up-regulated sigB (σB), encoding an alternative sigma factor which has been shown to be important for antimicrobial resistance. Staphylococcus aureus microarray metadatabase (SAMMD) analysis further revealed that 46% of genes differentially-expressed with diclofenac are also σB-regulated. Diclofenac altered S. aureus susceptibility to multiple antibiotics in a strain-dependent manner. Susceptibility increased for ciprofloxacin, ofloxacin and norfloxacin, decreased for oxacillin and vancomycin, and did not change for tetracycline or chloramphenicol. Mutation to DcRS did not affect susceptibility to the above antibiotics. Reduced ciprofloxacin MICs with diclofenac in strain BB255, were not associated with increased drug accumulation. CONCLUSIONS: The results of this study suggest that diclofenac influences antibiotic susceptibility in S. aureus, in part, by altering the expression of regulatory and structural genes associated with cell wall biosynthesis/turnover and transport.

sarA inactivation reduces vancomycin-intermediate and ciprofloxacin resistance expression by Staphylococcus aureus.

It is known that multiple genome-wide transcriptional changes often accompany the development of antimicrobial resistance and occur in response to challenge with antimicrobial agents. We now show that inactivation of the staphylococcal accessory gene regulator sarA, which controls at least tens of genes in Staphylococcus aureus, leads to dramatic reductions in vancomycin and ciprofloxacin resistance in vancomycin-intermediate and ciprofloxacin-resistant strains of S. aureus. This is particularly evident when judged by antimicrobial-gradient plate analysis or population analysis profiles. Whilst the intact sarA cistron is required for full vancomycin resistance expression by vancomycin-intermediate S. aureus (VISA), sarA expression as determined by quantitative real-time polymerase chain reaction was found to be VISA strain-dependent. Reductions in vancomycin resistance expression levels following sarA inactivation do not necessarily include an alteration in autolysis. Expression of sarR, the negative regulator of sarA, was downregulated in two VISA mutants, and transcription of the alternative sigma factor sigB was downregulated in one VISA strain. This study contributes to a growing body of evidence demonstrating the importance of loci previously identified to control virulence in the regulation of clinically relevant antibiotic resistance mechanisms.

The mouse A/HeJ Y chromosome: another good Y gone bad.

In both humans and mice there are numerous reports of Y chromosome abnormalities that interfere with sex determination. Recent studies in the mouse of one such mutation have identified Y chromosome nondisjunction during preimplantation development as the cause of abnormal testis determination that results in a high frequency of true hermaphroditism. We report here that the mouse Y chromosome from the A/HeJ inbred strain induces similar aberrations in sex determination. Our analyses provide evidence, however, that the mechanism underlying these aberrations is not Y chromosome nondisjunction. On the basis of our findings, we postulate that a mutation at or near the centromere affects both the segregation and sex-determining properties of the A/HeJ Y chromosome. This Y chromosome adds to the growing list of Y chromosome aberrations in humans and mice. In both species, the centromere of the Y is structurally and morphologically distinct from the centromeres of all other chromosomes. We conclude that these centromeric features make the human and mouse Y chromosomes extremely sensitive to minor structural alterations, and that our studies provide yet another example of a good Y chromosome gone 'bad.'