Mechanism Of Inhibition Of Macrolides On Staphylococcus Aureus Virulence Factor

Staphylococcus aureus (S. aureus) is one of the most common pathogens ofcommunity-acquired and nosocomial diseases with a significant morbidity andmortality. Infections due to this organism have a variety of clinical manifestations,ranging from localized skin infections to severe sepsis, including toxic-shocksyndrome. The pathogenicity of S. aureus, similar to that of other Gram-positivebacteria, to a great extent, depends on the production of numerous extracellularvirulence factors. S. aureus produces a wide array of toxins that determine, at least inpart, the pathogenesis of infection. Consequently, the clinical efficacy of antibiotics isnot only determined by their respective bactericidal or bacteriostatic activity andpharmacokinetics but also by their action on bacterial virulence factor release,especially at subinhibitory concentrations. Therefore, seeking to released antibioticalters S. aureus virulence factor production for the treatment of S. aureus diseases thathas great great scientific value and clinical significance.To obtain a comprehensive picture of the antibacterial effects of macrolides, theMinimum inhibitory concentrations (MICs) and minimum bactericidal concentrations(MBCs) of azithromycin, clarithromycin and erythromycin against Staphylococcalaureus ATCC19213, clinical isolates SA2985, Staphylococcal aureus8325-4and S.aureus hla mutant DU1090grown in suspension were detemined, microbrothdilution assays were performed in line with Clinical and Laboratoy Standard Institute(CLSI) guidelines. For microorganisms grown in biofilm, minimum biofilm inhibitoryconcentrations (MBICs) and minimum biofilm bactericidal concentrations (MBBCs)were determined using plating of swabbing method. The effect of macrolides on S.aureus biofilm formation was further validation by biofilm formation and detachmentassays. The results showed that azithromycin having good antimicrobial activityagainst S. aureus were grown in suspension or biofilm. Among them, clinical isolatesSA2985resistant to erythromycin. Furthermore, the three drugs showed lower bactericidal capacity on biofilm bacteria. Biofilm formation assay showed thatsubinhibitory macrolides can be effective inhibition of biofilm formation, and provedthat biofilm structure of the main components of the proteinaceous material.The effects of macrolides on virulence factor expression by S. aureus weredetermined via western blot, hemolysin assay and fibrinogen adhesion experiment.The results showed that macrolides suppressed the secretion of virulence factors in adose dependent manner. Among them, azithromycin significantly inhibited theexpression of α-haemolysin, staphylococcal enterotoxin A (SEA), SEB. Whileclarithromycin and erythromycin inhibitted α-haemolysin and SEB secretion only.Hemolysin assay and fibrinogen adhesion experiment showed that subinhibitoryazithromycin remarkably repressed S. aureus haemolysin and fibrinogen adhesion.In addition, the suppression of azithromycin on expression levels of hla, sea, seb,and agrA in S. aureus ATCC29213were also evaluated by real-time reversetranscription (RT)-PCR analysis. The results showed that subinhibitory azithromycininhibited the expression of hla、sea、seb and agrA at different levels.The results of the present study suggest that subinhibitory macrolides inhibitsvirulence factor release by S. aureus, which might be especially helpful for thetreatment of S. aureus infections, where both bactericidal as well as anti-toxin activitymay be advantageous.