The mechanism(s) by which Mycobacterium tuberculosis crosses the alveolar wall to establish infection in the lung is not well known. In an attempt to better understand the mechanism of translocation and create a model to study the different stages of bacterial crossing through the alveolar wall, we established a two-layer transwell system. M. tuberculosis H37Rv was evaluated regarding the ability to cross and disrupt the membrane. M. tuberculosis invaded A549 type II alveolar cells with an efficiency of 2 to 3% of the initial inoculum, although it was not efficient in invading endothelial cells. However, bacteria that invaded A549 cells were subsequently able to be taken up by endothelial cells with an efficiency of 5 to 6% of the inoculum. When incubated with a bicellular transwell monolayer (epithelial and endothelial cells), M. tuberculosis translocated into the lower chamber with efficiency (3 to 4%). M. tuberculosis was also able to efficiently translocate across the bicellular layer when inside monocytes. Infected monocytes crossed the barrier with greater efficiency when A549 alveolar cells were infected with M. tuberculosis than when A549 cells were not infected. We identified two potential mechanisms by which M. tuberculosis gains access to deeper tissues, by translocating across epithelial cells and by traveling into the blood vessels within monocytes.Infection caused by M. tuberculosis represents one of the great tragedies in world history. Approximately 3 million people die annually of the disease (7), despite the availability of cheap, efficacious, and curative therapy for tuberculosis.Once more, it seems clear that the improvement of the knowledge about the mechanisms employed by M. tuberculosis to infect the host will certainly offer new opportunities for the development of both effective therapy and vaccine.M. tuberculosis is inhaled into the respiratory tract, eventually reaching the alveolar space. It has been assumed that the bacterium is ingested by alveolar macrophages and subsequently gains access to the bloodstream by being transported by the alveolar macrophages and blood monocytes across the alveolar wall (10). Recently, however, it was demonstrated by several groups that M. tuberculosis invades and survives within human type II alveolar epithelial cells in vitro (3, 14, 17), and a possible role for alveolar epithelial cells in vivo has been postulated. In fact, the chance that M. tuberculosis would encounter an alveolar epithelial cell (the average human male has 1,500 type II and 28,000 type I alveolar epithelial cells [22]) is significantly greater than encountering an alveolar macrophage (50 macrophages per alveolus [8]). Therefore, the participation of type II alveolar epithelial cells, alveolar macrophages, and blood monocytes in the translocation of M. tuberculosis across the alveolar wall is currently poorly understood. Previous work has established the use of an in vitro model with a bilayer with alveolar epithelial cells and human lung endothelial cells (6). Using this model, i...

Mycobacterium avium complex infections occur in 30%-80% of patients with AIDS. Recent evidence supports the gastrointestinal tract as the source of M. avium. Although a reproducible animal model exists, a model more closely resembling the infection in AIDS patients is needed to answer pertinent questions regarding response to therapy and prophylaxis. Beige mice were infected orally (1 x 10(8) or 1 x 10(4) cfu, five doses), and consistent, reproducible disseminated infections after 4 and 8 weeks, respectively, were obtained. Bacteremia was observed in none to 70% of the animals depending on the strain used, and mortality ranged from none to 33%, also depending on the strain used. Concomitant ingestion of ethanol (4% of daily dietary calories) was associated with a significant increase in the number of viable bacteria recovered from liver, spleen, and appendix compared with animals not receiving ethanol. The orally infected animal model closely resembles M. avium infection in humans and may be important in investigating prophylaxis and therapy of this infection.

Moxifloxacin activity against Mycobacterium avium complex (MAC) was evaluated in vitro against 25 strains. The MIC was determined to range from 0.125 to 2.0 g/ml. In addition, U937 macrophage monolayers infected with MAC strain 101 (serovar 1) were treated with moxifloxacin (0.25 to 8 g/ml) daily, and the number of intracellular bacteria was quantitated after 4 days. Moxifloxacin showed inhibitory activity at 0.5 g/ml and higher. To assess the activity of moxifloxacin containing regimens in vivo, we infected C57BL bg ؉ /bg ؉ mice with 3 ؋ 10 7 MAC strain 101 bacteria intravenously. One week later treatment was begun with the following: (i) moxifloxacin (50 mg/kg/day or 100 mg/kg/day), ethambutol (100 mg/kg/day), or a combination of moxifloxacin and ethambutol; or (ii) moxifloxacin (100 mg/kg/day), azithromycin (200 mg/kg/day), or rifabutin (40 mg/kg/ day) as oral monotherapy; or (iii) all permutations of two-drug therapy or all three drugs in combination. All groups contained at least 14 animals, and the control group received the drug vehicle. After 4 weeks, quantitative blood cultures were obtained and the number of bacteria in liver and spleen was quantitated. Moxifloxacin, ethambutol, and azithromycin were active as single agents in liver, spleen, and blood. Rifabutin showed inhibitory activity only in the blood. Two-drug combinations containing azithromycin were no more active than azithromycin alone. Similarly, the three-drug combination was not more active than azithromycin alone in the spleen. Rifabutin did not add to the activity of any other single agent or two-drug combination. Moxifloxacin at both concentrations in combination with ethambutol was significantly more active than each drug alone.

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Despite the development of several agents, new classes of antimicrobials with activity against the Mycobacterium avium complex (MAC) are needed. Based on a broad screening of compounds, we found that mefloquine has MICs of 8 to 16 μg/ml by the BACTEC system and 16 μg/ml by broth microdilution for five MAC strains tested. An expansion of the screening with broth microdilution to 24 macrolide-susceptible strains and 6 macrolide-resistant strains determined that the MIC for all strains was 16 μg/ml. To determine the intracellular activity of mefloquine, U937 macrophage monolayers infected with MAC strain 101, 100, or 109 (serovars 1, 8, and 4) were treated with mefloquine daily, and the number of intracellular bacteria was quantitated after 4 days. Significant growth inhibition against the three MAC strains at concentrations greater than or equal to 10 μg/ml (P < 0.05) was obtained. Due to the encouraging anti-MAC activity, in vivo efficacy in beige mice infected with MAC 101 was evaluated. Animals were treated with 5, 10, 20, or 40 mg/kg of body weight daily, three times a week, twice a week, or once a week for 4 weeks, and bacteria were quantitated in blood, liver, and spleen. No toxicity was observed with any of the treatment regimens. Mefloquine had borderline bactericidal activity at a dosage of 40 mg/kg daily (100% inhibition compared with a 1-week control), and significant inhibition was obtained at dosages of 40 mg/kg three times a week, as well as 20 mg/kg daily. Mefloquine had no significant effect on bacteremia. A combination of mefloquine and ethambutol showed significantly more activity than did either drug alone in liver, spleen, and blood; the combination was also bactericidal againstM. avium. Although safety is a potential concern, mefloquine and related compounds deserve further investigation as anti-MAC therapies.