Contribution of efflux pump activity of macrolide resistance in M. avium complex.

Mycobacterium avium complex (MAC), comprising M. avium and M. intracellulare, is clinically important since it can cause severe infections in AIDS patients and other immunocompromised individuals. Therapy of MACinfections is problematic due to the intrinsic resistance of these bacteria to many of the available antimicrobial drugs. The use of the macrolides clarithromycin and azithromycin has improved the outcome of MAC infections, but therapeutic failure is still a major problem. We have recently shown that efflux pumps of MAC play an important role on this resistance phenotype. In fact, increased activity of efflux pumps is known to contribute to a multidrug resistance phenotype by extruding a wide variety of chemically and structurally unrelated compounds from the cell, preventing them from reaching their cellular targets. Thus, the characterization of such efflux pumps is crucial for the design of new antimycobacterial therapeutic strategies.In this work, we have studied the efflux pump activity in MAC clinical strains by a fluorometric method that detects efflux activity on a real-time basis, and evaluated the contribution of active efflux to the resistance to macrolides.The results to be presented show that resistance to clarithromycin was significantly reduced in the presence of efflux pump inhibitors (EPIs) such as the calcium-channel inhibitors thioridazine or chlorpromazine and the calcium ion influx inhibitor verapamil. The same EPIs were effective in decreasing the efflux of ethidium bromide (a common efflux pump substrate) from MAC cells, as shown by fluorometric analysis. Moreover, the retention of [14C]-Erythromycin by the same inhibitors demonstrated that active efflux contributes to MAC resistance to macrolides.In conclusion, this study demonstrates that efflux pumps play an important role in MAC resistance to antibiotics, par-ticularly to macrolides, and opens the possibility to explore the usefulness of these EPIs, already used in clinical practice for other purposes, such as thioridazine, as adjuvants to enhance the effectiveness of the therapeutic regimens against MAC infections