Tuberculosis remains a leading cause of death worldwide, despite the availability of effective chemotherapy and a vaccine. Bacillus Calmette-Gué rin (BCG), the tuberculosis vaccine, is an attenuated mutant of Mycobacterium bovis that was isolated after serial subcultures, yet the functional basis for this attenuation has never been elucidated. A single region (RD1), which is absent in all BCG substrains, was deleted from virulent M. bovis and Mycobacterium tuberculosis strains, and the resulting ⌬RD1 mutants were significantly attenuated for virulence in both immunocompromised and immunocompetent mice. The M. tuberculosis ⌬RD1 mutants were also shown to protect mice against aerosol challenge, in a similar manner to BCG. Interestingly, the ⌬RD1 mutants failed to cause cytolysis of pneumocytes, a phenotype that had been previously used to distinguish virulent M. tuberculosis from BCG. A specific transposon mutation, which disrupts the Rv3874 Rv3875 (cfp-10 esat-6) operon of RD1, also caused loss of the cytolytic phenotype in both pneumocytes and macrophages. This mutation resulted in the attenuation of virulence in mice, as the result of reduced tissue invasiveness. Moreover, specific deletion of each transcriptional unit of RD1 revealed that three independent transcriptional units are required for virulence, two of which are involved in the secretion of ESAT-6 (6-kDa early secretory antigenic target). We conclude that the primary attenuating mechanism of bacillus Calmette-Gué rin is the loss of cytolytic activity mediated by secreted ESAT-6, which results in reduced tissue invasiveness. B acillus Calmette-Guérin (BCG) was first isolated fromMycobacterium bovis after serial subculturing in ox bile medium (1, 2), when Drs. Calmette and Guérin set out to test the hypothesis that a bovine tubercle bacillus could transmit pulmonary tuberculosis after oral administration (1, 3, 4). However, unexpectedly after the 39th passage, the strain was unable to kill experimental animals (1, 2), and showed no reversion to virulence even after the authors had performed over 200 passages (3), which is consistent with the attenuating mutation being a deletion mutation. In proceeding studies, BCG was determined to be able to protect animals receiving a lethal challenge of virulent tubercle bacilli (5), and in 1921 was first used as an anti-tuberculous vaccine (6). Presently, an estimated 3 billion doses have been used to vaccinate the human population against tuberculosis, yet the mechanism that causes the attenuation of BCG remains unknown.Mahairas et al. (6) first compared the genomic sequences of BCG and M. bovis, by using subtractive hybridization, and found that there were three regions of difference (designated RD1, RD2, and RD3) present in the genome of M. bovis, but missing in BCG. Behr et al. (7), and others (8), later identified 16 large deletions, including RD1-RD3, which were present in the Mycobacterium tuberculosis genome but absent in BCG. Eleven of these 16 deletions were unique to M. bovis whereas the remaining 5 del...

BUD is an environmentally acquired infection strongly associated with exposure to river areas. Exposed skin may facilitate transmission. Until transmission is better defined, control strategies in BUD-endemic areas could include covering exposed skin.

Because of the emergence of Buruli ulcer disease, the World Health Organization launched a Global Buruli Ulcer Initiative in 1998. This indolent skin infection is caused by Mycobacterium ulcerans. During a study of risk factors for the disease in Ghana, adequate excisional skin-biopsy specimens were obtained from 124 clinically suspicious lesions. Buruli ulcer disease was diagnosed in 78 lesions since acid-fast bacilli (AFB) were found by histopathologic examination. Lesions with other diagnoses included filariasis (3 cases), zygomycosis (2 cases), ulcerative squamous cell carcinomas (2 cases), keratin cyst (1 case), and lymph node (1 case). Thirty-seven specimens that did not show AFB were considered suspected Buruli ulcer disease cases. Necrosis of subcutaneous tissues and dermal collagen were found more frequently in AFB-positive specimens compared with specimens from suspected case-patients (p<0.001). Defining histologic criteria for a diagnosis of Buruli ulcer disease is of clinical and public health importance since it would allow earlier treatment, leading to less deforming sequelae.

Mycobacterium tuberculosis establishes infection, progresses towards disease, and is transmitted from the alveolus of the lung. However, the role of the alveolar epithelium in any of these pathogenic processes of tuberculosis is unclear. In this study, lung epithelial cells (

We recently evaluated several tissue culture model systems for the study of invasion and intracellular multiplication of Mycobacterium tuberculosis. These model systems include a human alveolar pneumocyte epithelial cell line, a murine macrophage cell line (J774), and fresh human peripheral blood-derived macrophages. Our data indicated that the initial level of association of M. tuberculosis with human alveolar pneumocyte cells (2%) was less than that observed with fresh human peripheral blood macrophages (9%) or J774 murine macrophages (13%) within 6 h of the addition of the bacteria. M. tuberculosis replicated in association with the pneumocyte cells by more than 55-fold by day 7 postinfection. In contrast, total bacterial growth in the J774 cells and human macrophages was considerably less, with increases of only fourfold and threefold, respectively, over the same 7-day period. Amikacin, an aminoglycoside antimicrobial agent, was added to inhibit the growth of extracellular bacteria after the initial 6-h infection period. Decreases in viable counts were observed in all three cell cultures within the first 3 days after infection. However, unlike the case with either macrophage culture, intracellular bacterial CFU obtained from the infected pneumocytes increased by fourfold by day 7 after the addition of amikacin. These data indicate that M. tuberculosis infects and multiplies intracellularly in human lung epithelial cells and that these cells may be an alternative in vitro model for the study of intracellular multiplication of M. tuberculosis in the human lung.

Although most diseases due to pathogenic mycobacteria are caused by Mycobacterium tuberculosis, several other mycobacterial diseases—caused by M. ulcerans (Buruli ulcer), M. marinum, and M. haemophilum—have begun to emerge. We review the emergence of diseases caused by these three pathogens in the United States and around the world in the last decade. We examine the pathophysiologic similarities of the diseases (all three cause necrotizing skin lesions) and common reservoirs of infection (stagnant or slow-flowing water). Examination of the histologic and pathogenic characteristics of these mycobacteria suggests differences in the modes of transmission and pathogenesis, though no singular mechanism for either characteristic has been definitively described for any of these mycobacteria.

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A tissue culture bilayer system that mimics some aspects of early alveolar infection by Mycobacterium tuberculosis was developed. This model incorporates human lung epithelial type II pneumocyte (A549) (upper chamber) and endothelial cell (lower chamber) layers separated by a microporous membrane. This construction makes it possible to observe and quantify the passage of bacteria through the two layers, to observe the interaction of the bacteria with the various cell types, and to examine the basic mechanisms of immune cell recruitment to the site of infection. After 107 organisms were added to the upper chamber we microscopically observed large numbers of bacteria attached to and within the pneumocytes and we determined by viable-cell counting that a small percentage of the inoculum (0.02 to 0.43%) passed through the bilayer into the lower chamber. When peripheral blood mononuclear cells were added to the lower chamber, microscopic examination indicated a migration of the mononuclear cells through the bilayer to the apical surface, where they were seen associated with the mycobacteria on the pneumocytes. The added complexity of the bilayer system offers an opportunity to define more precisely the roles of the various lung cell types in the pathogenesis of early tuberculosis.