Enzymatic inactivation and reactivation of chloramphenicol by Mycobacterium tuberculosis and Mycobacterium bovis

Mycobacterium tuberculosis and Mycobacterium bovis are inhibited by chloramphenicol. Chloramphenicol acetyltransferase (CAT) converts chloramphenicol to inactive diacetyl chloramphenicol, but a mycobacterial carboxylesterase hydrolyzes the diacetyl product to active chloramphenicol. The esterase activity was eliminated by proteinase K and heat treatment. Protein extracts of M. tuberculosis and M. bovis hydrolyzed four other ester substrates. cat was inserted into the chromosome of both M. tuberculosis and M. bovis resulting in a level of chloramphenicol resistance that could be used to select for transformants. CAT assays in the resistant strain of M. tuberculosis showed interference due to esterase activity. This interference could be eliminated with the addition of a heating step. (C) 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.