Cellular responses of Mycobacterium tuberculosis to antimycobacterial agents
The effects of clinically important antimycobacterial drugs on Mycobacterium tuberculosis at the single cell level using cytochemical indicators of cellular activity were studied. Procedures based on rhodamine 123 (R 123) uptake as an indicator of cytoplasmic membrane energisation, propidium iodide (PI) exclusion and iodonitrophenyltetrazolium chloride (INT) reduction were established. Some cells in every preparation were found to resist labelling by all of the procedures applied. This proportion was highest in broth culture (up to 70%) and lowest in cell suspensions prepared from agar spread plates. R123 uptake in growing cells was reversibly sensitive to carbonylcyanide m-chlorophenylhydrazone (CCCP), an uncoupler of oxidative phosphorylation. Several tuberculocidal treatments (70°C/30 min, 70% ethanol and 4% formaldehyde) lead to development of uncoupler insensitive R 123 labelling of dead cells, demonstrating the requirement for a physiologically validated procedure where labelling was unambiguously attributed to membrane energisation. All antimycobacterial drug treatments (isoniazid, rifampicin, ethambutol, streptomycin and capreomycin) produced an excess of between 1 and 4 orders of magnitude of uncoupler sensitive R 123 labelling cells over culturable units. Thus, large populations of active but nonculturable (ABNC) cells were produced by antimycobacterial drugs commonly used in the treatment of tuberculosis. Non-labelling and ABNC cell populations were further investigated using a GFP reporter strain and by exposure to the lytic mycobacteriophage D29. In addition to demonstrating many of the potential pitfalls that may be encountered when the results of cellular activity/integrity assays are equated with viability/nonviability, these studies illustrate the heterogeneous nature of M tuberculosis cultures and the extent to which bulk analysis may give a misleading picture of cellular composition and physiology. Although the significance of the non-labelling and ABNC cells observed remains to be established, we speculate that these populations may have implications for the chemotherapy of tuberculosis.