Iron-cofactored superoxide dismutase inhibits host responses to Mycobacterium tuberculosis.

Superoxide dismutase (SOD) is a ubiquitous metalloenzyme in aerobic organisms that catalyzes the conversion of superoxide anion to hydrogen peroxide. Mycobacterium tuberculosis is unusual in that it secretes large quantities of iron-cofactored SOD. To determine the role of SOD in pathogenesis, we constructed mutants of M. tuberculosis H37Rv with reduced SOD production. Compared with controls, SOD-diminished isolates were more susceptible to killing by hydrogen peroxide. The isolates were markedly attenuated, exhibiting nearly 100,000-fold fewer bacilli than virulent control strains in the lungs and spleens of C57BL/6 mice 4 wk after intravenous inoculation. In the lung, SOD-attenuated M. tuberculosis induced robust interstitial mononuclear cell infiltration within 24 h and many cells were apoptotic by TUNEL staining, whereas virulent H37Rv exhibited minimal early inflammatory response and only rare interstitial mononuclear cell apoptosis. During prolonged infections, C57BL/6 mice tolerated SOD-attenuated M. tuberculosis better than BCG, exhibiting 68% greater weight gain, quicker eradication of bacilli from the spleen, and less alveolar lung infiltration. These results establish the importance of SOD in the pathogenesis of tuberculosis. Its effect appears to be mediated in part by inhibiting innate host immune responses, including early mononuclear cell infiltration of infected tissues and apoptosis.

Durable cure for tuberculosis: rifalazil in combination with isoniazid in a murine model of Mycobacterium tuberculosis infection.

Rifalazil (formerly known as KRM-1648) in combination with isoniazid has been found to be more active than rifampin/isoniazid. Administration of rifalazil/isoniazid for 12 weeks resulted in continued apparent sterilization of organs 6 months after cessation of therapy. In this study we evaluated the durability of rifalazil/isoniazid treatment. Female CD-1 mice were infected with Mycobacterium tuberculosis ATCC 35801 (strain Erdman). Rifalazil and isoniazid were given in combination for 6 and 12 weeks; no mycobacteria could be cultured from spleens and lungs at both the 6-week and 12-week time points. After completing treatment, groups of mice treated with rifalazil/isoniazid for 6 or 12 weeks were observed without any additional treatment. These observation groups were compared to groups of rifalazil/isoniazid-treated mice (6 and 12 weeks) given dexamethasone for 7 and 8 weeks, respectively. Modest regrowth was noted in the spleens and lungs of the group treated with rifalazil/isoniazid for 6 weeks. Regrowth in the 6-weeks group was enhanced slightly by treatment with dexamethasone. In contrast, no regrowth was noted in the 12-weeks rifalazil/isoniazid group, and treatment with dexamethasone did not result in any regrowth.

Evaluation of rifalazil in long-term treatment regimens for tuberculosis in mice.

Previous experiments with rifalazil (RLZ) (also known as KRM-1648) in combination with isoniazid (INH) demonstrated its potential for short-course treatment of Mycobacterium tuberculosis infection. In this study we investigated the minimum RLZ-INH treatment time required to eradicate M. tuberculosis in a murine model. RLZ-INH treatment for 6 weeks or longer led to a nonculturable state. Groups of mice treated in parallel were killed following an observation period to evaluate regrowth. RLZ-INH treatment for a minimum of 10 weeks was necessary to maintain a nonculturable state through the observation period. Pyrazinamide (PZA) was added to this regimen to determine whether the treatment duration could be further reduced. In this model, the addition of PZA did not shorten the duration of RLZ-INH treatment required to eradicate M. tuberculosis from mice. The addition of PZA reduced the number of mice in which regrowth occurred, although the reduction was not statistically significant.

High-dose isoniazid therapy for isoniazid-resistant murine Mycobacterium tuberculosis infection.

The use of isoniazid (INH) for the treatment of INH-resistant Mycobacterium tuberculosis infection has been controversial. The purpose of the present studies was to determine if there is a dose response with INH for INH-susceptible M. tuberculosis Erdman (ATCC 35801), and whether high-dose INH (100 mg/kg of body weight) was more effective than standard-dose INH (25 mg/kg) for therapy of tuberculosis infections caused by INH-resistant mutants of M. tuberculosis Erdman. Six-week-old CD-1 mice were infected with approximately 10(7) viable mycobacteria. Early control groups of infected but untreated mice were euthanized by CO(2) inhalation 1 week later when treatment was initiated. INH (25, 50, 75, and 100 mg/kg) was given by gavage 5 days/week for 4 weeks. Late control groups of untreated mice and treated mice were sacrificed 2 days after the last dose of drug. Spleens and right lungs were removed aseptically and homogenized, and viable cell counts were determined by titration on 7H10 agar plates. In the next study, INH at 100 mg/kg was compared to INH at 25 mg/kg against an isogenic mutant of M. tuberculosis Erdman (INH MIC, 2 microg/ml) and the parent strain. This mutant was found to have a mutation in the KatG protein (Phe to Leu at position 183). In the first study, there was no dose response with increasing doses of INH. In the second study, there was no significant difference between the reduction of viable cell counts for mice treated with INH at 100 mg/kg and that for mice treated with INH at 25 mg/kg (parent or INH-resistant mutant). These preliminary results suggest that INH may be useful in combination therapy of M. tuberculosis infections caused by low-level INH-resistant organisms (INH MICs, 0.2 to 5 microg/ml) and that higher doses of INH are unlikely to be more efficacious than the standard 300-mg/day dose.

Enhanced in vitro activity of WR99210 in combination with dapsone against Mycobacterium avium complex.

WR99210, a dihydrofolate reductase inhibitor, has promising in vitro activity against Mycobacterium avium complex (MAC). The in vitro activities of WR99210 alone and in combination with a fixed concentration of dapsone (0.5 microgram/ml) were evaluated against 35 clinical MAC isolates by a broth dilution method. The MIC at which 50% of isolates were inhibited (MIC50) and MIC90 of WR99210 alone were 2 and 8 micrograms/ml, respectively. The MIC50 and MIC90 of WR99210 in combination with dapsone were 0.25 and 4 micrograms/ml, respectively. Overall, 75% of the MAC isolates displayed enhanced susceptibility to the combination.

Therapy of multidrug-resistant tuberculosis: lessons from studies with mice.

The activities of antituberculosis agents were evaluated in a murine tuberculosis model using a drug-resistant isolate. A multidrug-resistant clinical isolate from a recent outbreak of tuberculosis in the New York State correctional system was used for infection. Approximately 10(7) viable Mycobacterium tuberculosis ATCC 49967 (strain CNL) organisms were given intravenously to 4-week-old female outbred mice. Treatment was started 1 day after infection and given for 4 weeks. Spleens and lungs were homogenized, and viable cell counts were determined. Statistical analysis indicated that ethionamide, sparfloxacin, ofloxacin, capreomycin, clarithromycin, and clofazimine are active in the murine test system with this multidrug-resistant tuberculosis isolate. Sparfloxacin is the most active quinolone. Despite in vitro resistance, isoniazid has moderate activity. In vitro susceptibility data coupled with evaluation of agents against drug-resistant isolates in the murine system should provide information necessary to design clinical trials for treatment of infections with these organisms.

Activity of clarithromycin against Mycobacterium avium complex infection in beige mice.

The activity of clarithromycin alone and in combination with other antimycobacterial agents was evaluated in the beige (C57BL/6J bgj/bgj) mouse model of disseminated Mycobacterium avium complex (MAC) infection. A dose-response experiment was performed with clarithromycin at 50, 100, 200, or 300 mg/kg of body weight administered daily by gavage to mice infected with approximately 10(7) viable MAC. A dose-related reduction in spleen and liver cell counts was noted with treatment at 50, 100, and 200 mg/kg. The difference in cell counts between treatment at 200 and 300 mg/kg was not significant. Clarithromycin at 200 mg/kg of body weight was found to have activity against three additional MAC isolates (MICs for the isolates ranged from 1 to 4 micrograms/ml by broth dilution). Clarithromycin at 200 mg/kg in combination with amikacin, ethambutol, temafloxacin, or rifampin did not result in increased activity beyond that seen with clarithromycin alone. Clarithromycin in combination with clofazimine or rifabutin resulted in an increase in activity beyond that seen with clarithromycin alone. The combination of clarithromycin with clofazimine or rifabutin should be considered for evaluation in the treatment of human MAC infections.