JAC Advance Access published online on February 29, 2008
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkn070
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Original research |
Thioridazine and chlorpromazine inhibition of ethidium bromide efflux in Mycobacterium avium and Mycobacterium smegmatis
1 Unit of Mycobacteriology, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Rua da Junqueira 96, 1349-008 Lisboa, Portugal 2 UPMM, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Rua da Junqueira 96, 1349-008 Lisboa, Portugal 3 Center for Infectious Diseases and Travel Medicine, University Hospital, Hugstetter Strasse 55, D-79106 Freiburg, Germany 4 Centro de Recursos Microbiológicos (CREM), Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal
Received 24 October 2007; returned 21 December 2007; revised 13 December 2007; accepted 30 January 2008
* Corresponding author. Tel: +351-21-365-2653; Fax: +351-21-363-2105; E-mail: lamaral{at}ihmt.unl.pt
Objectives: Therapy of AIDS patients infected with Mycobacterium avium is problematic due to its intrinsic resistance to antibiotics. We have characterized the efflux pump activity of M. avium wild-type strain through an automated fluorometric method and correlated it with intrinsic resistance to antibiotics.
Methods: M. avium ATCC 25291T and Mycobacterium smegmatis mc2155 were evaluated for accumulation and efflux of ethidium bromide in the presence or absence of the efflux pump inhibitors (EPIs) thioridazine, chlorpromazine, verapamil and the proton uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP). For this purpose, a new automated fluorometric method was used that separately assesses accumulation and extrusion of ethidium bromide.
Results: The automated fluorometric method described in this paper allowed the detection and quantification of ethidium bromide transport across M. avium and M. smegmatis cell walls. Accumulation of ethidium bromide was found to be temperature-dependent and significantly increased by EPIs thioridazine, chlorpromazine, verapamil and CCCP in a concentration-dependent manner. Efflux of ethidium bromide under optimum conditions of temperature and glucose is inhibited by the above agents. At half their intrinsic MICs, both thioridazine and chlorpromazine, similarly to verapamil and CCCP, significantly increased the susceptibility of M. avium to erythromycin, suggesting an effect upon an efflux pump with ethidium bromide and erythromycin as substrates. A similar effect was observed for M. smegmatis with verapamil only.
Conclusions: M. avium and M. smegmatis intrinsic resistance is affected by EPIs such as thioridazine or chlorpromazine, an effect that might be important in research and development of new, more effective antimycobacterial therapies.
Key Words: mycobacteria , phenothiazines , efflux pumps , efflux pump inhibitors , automated fluorometric method
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