Culturability of Mycobacterium tuberculosis cells isolated from murine macrophages: a bacterial growth factor promotes recovery

Very little is known about the culturability and viability of mycobacteria following their phagocytosis by macrophages. We therefore studied populatio ns of the avirulent 'Academia' strain of Mycobacterium tuberculosis isolate d from murine peritoneal macrophage lysates several days post-infection in vivo. The resulting bacterial suspensions contained a range of morphologica l types including rods, ovoid forms and coccoid forms. Bacterial viability measured using the MPN method (dilution to extinction in liquid medium) was often much higher than that measured by CFU (plating on solid medium). Via bility in the MPN assay was further enhanced when the Micrococcus luteus pr otein, Rpf, was incorporated into the liquid culture medium at picomolar co ncentrations. Rpf is an example of a family of autocrine growth factors fou nd throughout the high G+C cohort of Grant-positive bacteria including M. t uberculosis. M. tuberculosis cells obtained from macrophages had altered su rface properties, as compared with bacteria grown in vitro. This was indica ted by loss of the ability to adsorb bacteriophage DS6A, a reduced tendency to form clumps, acquisition of ethidium bromide stainability following hea t treatment, and loss of Rpf-mediated resuscitation following freezing and thawing. These results indicate that a proportion of 'unculturable' M. tube rculosis cells obtained from macrophages is either injured or dormant and t hat these cells may be recovered or resuscitated using Rpf in liquid medium . (C) 2000 Federation of European Microbiological Societies. Published by E lsevier Science B.V. All rights reserved.