Discovery of novel inhibitors of the NorA multidrug transporter of Staphylococcus aureus.

Four novel inhibitors of the NorA efflux pump of Staphylococcus aureus, discovered through a virtual screening process, are reported. The four compounds belong to different chemical classes and were tested for their in vitro ability to block the efflux of a well-known NorA substrate, as well as for their ability to potentiate the effect of ciprofloxacin (CPX) on several strains of S. aureus, including a NorA overexpressing strain. Additionally, the MIC values of each of the compounds individually are reported. A structure-activity relationship study was also performed on these novel chemotypes, revealing three new compounds that are also potent NorA inhibitors. The virtual screening procedure employed FLAP, a new methodology based on GRID force field descriptors.

Inability of a reserpine-based screen to identify strains overexpressing efflux pump genes in clinical isolates of Staphylococcus aureus.

Overexpression of efflux pump genes conferring multidrug resistance (MDR) in Staphylococcus aureus results in reduced susceptibility to select biocides, dyes and fluoroquinolones. Reserpine is commonly used as an inhibitor of MDR efflux pumps and previous work from our laboratory using a reserpine-based screen to identify clinical isolates with an efflux phenotype revealed that nearly one-half overexpressed norA-B-C, mepA or mdeA. The accuracy of reserpine in predicting efflux pump gene overexpression in clinical strains was examined in detail. Bloodstream isolates of S. aureus previously classified as non-effluxing strains by the reserpine screen underwent gene expression analysis using quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). The reserpine screen failed to identify many strains shown by qRT-PCR to overexpress one or more MDR efflux pump genes. Microdilution susceptibility testing with and without reserpine also failed to predict efflux pump activity. Although gene expression does not always correlate with protein translation, our results indicate that in clinical S. aureus isolates the use of reserpine to predict the contribution of efflux to reduced susceptibility is not dependable. All strains used in studies designed to assess MDR efflux pump gene expression in clinical isolates should be evaluated by a method independent of in vitro susceptibility testing.

Methicillin-Resistant Staphylococcus aureus: A Growing Risk in the Hospital and in the Community.

Methicillin-resistant Staphylococcus aureus is a common and continuously growing cause of nosocomial and community-acquired infections. The type, disease severity, and clinical outcomes of these infections, as well as the genotypic and susceptibility patterns of the bacteria differ according to the setting in which the infection occurs-a healthcare facility or the community setting. The incidence of these infections in the community setting has been growing consistently in the past decade or so. In addition, resistance to the many current antibiotics used to treat these infections is also growing, further complicating management. Rapid-diagnosis tests and new therapeutic agents are constantly under investigation. The authors review the current understanding of the epidemiology, clinical manifestations, and management of methicillin-resistant Staphylococcus aureus infection, including the growing problem of resistance. In addition, they discuss promising diagnostic and therapeutic alternatives, as well as new control strategies to prevent its transmission or the development of infection among carriers.

Multidrug efflux pump overexpression in Staphylococcus aureus after single and multiple in vitro exposures to biocides and dyes.

Biocides and dyes are commonly employed in hospital and laboratory settings. Many of these agents are substrates for multiple-drug resistance (MDR)-conferring efflux pumps of both Gram-positive and Gram-negative organisms. Several such pumps have been identified in Staphylococcus aureus, and mutants overexpressing the NorA and MepA MDR pumps following exposure to fluoroquinolones have been identified. The effect of exposure to low concentrations of biocides and dyes on the expression of specific pump genes has not been evaluated. Using quantitative reverse-transcription PCR we found that exposure of clinical isolates to low concentrations of a variety of biocides and dyes in a single step, or to gradually increasing concentrations over several days, resulted in the appearance of mutants overexpressing mepA, mdeA, norA and norC, with mepA overexpression predominating. Overexpression was frequently associated with promoter-region or regulatory protein mutations. Mutants having significant increases in MICs of common pump substrates but no changes in expression of studied pump genes were also observed; in these cases changes in expression of as-yet-unidentified MDR pump genes may have occurred. Strains of S. aureus that exist in relatively protected environments and are repeatedly exposed to sublethal concentrations of biocides can develop efflux-related resistance to those agents, and acquisition of such strains poses a threat to patients treated with antimicrobial agents that are also substrates for those pumps, such as ciprofloxacin and moxifloxacin.