SummaryMycobacterium species, including Mycobacterium tuberculosis and Mycobacterium leprae, are among the most potent human bacterial pathogens. The discovery of cytosolic mycobacteria challenged the paradigm that these pathogens exclusively localize within the phagosome of host cells. As yet the biological relevance of mycobacterial translocation to the cytosol remained unclear. In this current study we used electron microscopy techniques to establish a clear link between translocation and mycobacterial virulence. Pathogenic, patientderived mycobacteria species were found to translocate to the cytosol, while non-pathogenic species did not. We were further able to link cytosolic translocation with pathogenicity by introducing the ESX-1 (type VII) secretion system into the nonvirulent, exclusively phagolysosomal Mycobacterium bovis BCG. Furthermore, we show that translocation is dependent on the C-terminus of the early-secreted antigen ESAT-6. The C-terminal truncation of ESAT-6 was shown to result in attenuation in mice, again linking translocation to virulence. Together, these data demonstrate the molecular mechanism facilitating translocation of mycobacteria. The ability to translocate from the phagolysosome to the cytosol is with this study proven to be biologically significant as it determines mycobacterial virulence.

Despite the widespread use of bacillus Calmette-Gué rin vaccination, Mycobacterium tuberculosis infection remains globally the leading cause of death from a single infectious disease. The complicated and often protracted dynamics of infection and disease make clinical trials to test new tuberculosis vaccines extremely complex. Preclinical selection of only the most promising candidates is therefore essential. Because macaque monkeys develop a disease very similar to humans, they have potential to provide important information in addition to small animal models. To assess the relative merits of rhesus and cynomolgus monkeys as screens for tuberculosis vaccines, we compared the efficacy of bacillus Calmette-Gué rin vaccination and the course of infection in both species. Unvaccinated rhesus and cynomolgus monkeys both developed progressive disease with high levels of C-reactive protein, M. tuberculosis-specific IgG, and extensive pathology including cavitation and caseous necrosis. Bacillus Calmette-Gué rin vaccination protected cynomolgus almost completely toward the development of pathology, reflected in a striking 2-log reduction in viable bacteria in the lungs compared with nonvaccinated animals. Rhesus, on the other hand, were not protected efficiently by the bacillus Calmette-Gué rin. The vaccinated animals developed substantial pathology and had negligible reductions of colony-forming units in the lungs. Comparative studies in these closely related species are likely to provide insight into mechanisms involved in protection against tuberculosis.T uberculosis (TB) is the leading global cause of death from a single infectious disease (1, 2), accelerated by the HIV epidemic and appearance of multidrug-resistant Mycobacterium tuberculosis (3). The efficiency of bacillus Calmette-Guérin, the only TB vaccine available for humans, ranges between 0 and 85% (4). Improved vaccines are needed urgently, and a large number of new TB vaccine candidates and delivery systems is being tested in small animal models (5-7). Human trials to evaluate new TB vaccines will be very complex, will often occur in bacillus Calmette-Guérin-immunized populations, will be of long duration, and require large cohorts (8). Effective screens to select only the most promising candidates, delivery systems, and formulations for clinical testing will be extremely valuable. Up to now, almost all efficacy testing of TB vaccine candidates has been in the mouse and͞or guinea pig (9). A more human-like response to TB has been described in the closely related rhesus (Macaca mulatta) and cynomolgus (Macaca fascicularis) macaques (reviewed in ref. 10). This similarity in response may be because several host molecules implicated in TB infections are present in man and nonhuman primates but are not found, or differ fundamentally, in mice and guinea pigs. For example, many primate species, including human, share the presence of functional Mhc-DR, -DQ, and -DP regions (11). The relevance of this structural similarity is reflected in the observation that spec...

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In this study, 2275 clinical isolates of 49 species of non-tuberculous mycobacteria isolated in The Netherlands were subjected to standardised drug susceptibility testing using the Middlebrook 7H10 agar dilution method. Clarithromycin and rifabutin were most active, with 87% and 83% of all isolates, respectively, being susceptible. Susceptibility to ciprofloxacin (44%) and amikacin (32%) was limited and was mostly restricted to Mycobacterium kansasii, Mycobacterium xenopi, Mycobacterium fortuitum and phylogenetically related species. Susceptibility to isoniazid (0.5%), rifampicin (37%), ethambutol (35%) and streptomycin (33%) was rare; susceptibility to cycloserine, clofazimine and prothionamide was generally restricted to slow growers, although prothionamide also had activity against M. fortuitum and related species. Significant discrepancies between in vitro and in vivo activity exist. To improve the utility of drug susceptibility testing, the selection of drugs should be changed to more drugs with proven clinical efficacy correlating with in vitro susceptibility.