Factors influencing the in vitro growth of Mycobacterium leprae: effect of inoculum

Factors responsible for the in vitro growth of Mycobacterium leprae in Dhople-Hanks (DH) medium, and also to improve the technique devised earlier, and the source of the M. leprae used as inoculum, were investigated. M. leprae were obtained from armadillos and nude mice, both inoculated earlier with human- or armadillo-derived M. leprae. The growth of M. leprae in DH medium was monitored using two biochemical indicators. Normal growth was obtained when inocula were from livers and spleens of M. leprae-infected armadillos. The M. leprae harvested from the footpads of nude mice failed to multiply in the same medium. Using inocula from livers and spleens of infected armadillos, a gradual decrease in inoculum size resulted in a proportionately slower multiplication of M. leprae. When the DH medium was supplemented with whole M. leprae, or cell-free extracts of M. leprae, from irradiated livers and spleens of infected armadillos, nude mouse-derived M. leprae exhibited growth in the DH medium in accord with that obtained using armadillo-derived M. leprae. Similar results were obtained with cell-free extracts of M. leprae harvested from non-irradiated livers and spleens of infected armadillos, but no growth was obtained when the medium was supplemented with extracts from livers or spleens of normal armadillos. These results indicate the possible existence of a growth factor in armadillo-derived M. leprae.     

Drug effects on intracellular mycobacteria determined by mass spectrometric analysis of the Na(+)-to-K+ ratios of individual bacterial organisms

The successful establishment of a drug screening system for intracellular cultivable and noncultivable mycobacteria based on the mass spectrometric determination of bacterial viability is described. To compare drug efficacies on intra- and extracellular mycobacteria, the mycobacteria were subjected to drug treatment either after phagocytosis by the mouse macrophage cell line RAW 264.7 or in cell-free medium. After reisolation, their viability was monitored by analyzing the intrabacterial sodium-to-potassium ratios for a limited number of individual organisms. This approach offers a reliable and quick tool for monitoring the influence of intracellular growth and of additional permeation barriers on intracellular drug efficacy and will thus provide useful information for the rational development and testing of optimized antimycobacterial drugs. In particular, the methodology is applicable to the noncultivable species Mycobacterium leprae, because the mass spectrometric analysis of the intrabacterial sodium-to-potassium ratio allows the determination of bacterial viability independent from their ability to multiply in vitro. Because of the improved metabolic activity of intracellularly growing M. leprae compared with that of extracellularly growing M. leprae, the spectrum of antileprosy drugs that can be tested in vitro could even be extended to those interfering with DNA replication and cell division.     

Gas embolism in obstetrics and gynecology. A review

Recent data suggest that the clinical course of reactional states in leprosy is closely related to the cytokine profile released locally or systemically by the patients. In the present study, patients with erythema nodosum leprosum (ENL) were grouped according to the intensity of their clinical symptoms. Clinical and immunological aspects of ENL and the impact of these parameters on bacterial load were assessed in conjunction with patients' in vitro immune response to mycobacterial antigens. In 10 out of the 17 patients tested, BI (bacterial index) was reduced by at least 1 log from leprosy diagnosis to the onset of their first reactional episode (ENL), as compared to an expected 0.3 log reduction in the unreactional group for the same MDT (multidrug therapy) period. However, no difference in the rate of BI reduction was noted at the end of MDT among ENL and unreactional lepromatous patients. Accordingly, although TNF-alpha (tumor necrosis factor) levels were enhanced in the sera of 70.6% of the ENL patients tested, no relationship was noted between circulating TNF-alpha levels and the decrease in BI detected at the onset of the reactional episode. Evaluation of bacterial viability of M. leprae isolated from the reactional lesions showed no growth in the mouse footpads. Only 20% of the patients demonstrated specific immune response to M. leprae during ENL. Moreover, high levels of soluble IL-2R (interleukin-2 receptor) were present in 78% of the patients. Circulating anti-neural (anti-ceramide and anti-galactocerebroside antibodies) and anti-mycobacterial antibodies were detected in ENL patients' sera as well, which were not related to the clinical course of disease. Our data suggest that bacterial killing is enhanced during reactions. Emergence of specific immune response to M. leprae and the effective role of TNF-alpha in mediating fragmentation of bacteria still need to be clarified.     

Repression of beta-actin synthesis and persistence of ribosomal protein synthesis after infection of HeLa cells by herpes simplex virus type 1 infection are under translational control

Multibacillary (MB) leprosy patients treated with multidrug therapy (MDT) or MDT + immunotherapy (IMT) with BCG + heat-killed Mycobacterium leprae were tested annually for their ability to proliferate in vitro to the mycobacterial antigens BCG, M. leprae soluble extract, and intact M. leprae. IgM antibody responses to phenolic glycolipid I (PGL-I) were measured, as well as serum nitrite levels in patients' sera, before, during and after treatment. Patients who received only MDT did not present cellular reactivity to intact M. leprae antigens, in contrast to the results obtained with BCG, which elicited reactivity at time zero, that increased after treatment. Regarding PGL-I antibody variations in relation to the initial value, we observed a statistically significant marked decrease at the end of 2 years which continued to fall in successive evaluations. MB patients showed high initial serum nitrite concentrations which dropped drastically with treatment. This decay was apparently associated with the bacillary load present in these patients. The group submitted to IMT + MDT showed high and long-lasting T-cell responses to mycobacterial antigens in a significant number of initially unresponsive MB patients. There was a marked increase to M. leprae soluble extract and BCG, as well as a more variable response to whole bacilli. The antibody levels in this group of patients are sustained for a somewhat longer period and decreased more slowly during the 5-year follow up.     

Down-regulatory effect of Mycobacterium leprae cell wall lipids on phagocytosis, oxidative respiratory burst and tumour cell killing by mouse bone marrow derived macrophages

The authors have previously demonstrated that lipids from Mycobacterium leprae cell walls inhibit macrophage functions and are endowed with anti-inflammatory properties in vivo. To investigate these observations further, the authors describe here the influence of dead M. leprae or of the lipids extracted from the cell wall of the mycobacterium, enclosed in liposomes, on the phagocytic, oxidative respiratory burst and tumouricidal ability of bone marrow derived macrophages in vitro. Dead M. leprae or its cell wall lipids abrogated the oxidative respiratory burst and phagocytic ability of mouse bone marrow derived macrophages. A dose-dependent inhibitory effect of the bacterial lipid extract on tumour cell killing by lipopolysaccharide (LPS)-activated bone marrow derived macrophages was demonstrated. However, when delipidated M. leprae was added to cultures of bone marrow derived macrophages, immune phagocytosis and superoxide production was up-regulated. Mycobacterium leprae or its lipids did not appear to be toxic to those cells assayed by the MTT (methyl thiazol tetrazolium) test. These data, added to our preceding observations, support the hypothesis that the down-regulatory activity of M. leprae wall lipids on macrophage function might be one of the evasive mechanisms of the bacterium to enable it to perpetuate itself in the host tissues.     

Analysis of cytokine production by Mycobacterium-reactive T cells. Failure to explain Mycobacterium leprae-specific nonresponsiveness of peripheral blood T cells from lepromatous leprosy patients

Recent analyses of antimycobacterial T cells clones from a small number of individuals indicate that mycobacteria preferentially induce Th cells that produce high levels of IFN-gamma and no or little IL-4 in Mycobacterium leprae-resistant tuberculoid leprosy (TT) patients and healthy subjects, whereas in one study M. leprae-induced Ts clones from polar lepromatous leprosy (LL) patients showed a reciprocal cytokine secretion profile and mediated their suppressive activity via the release of high levels of IL-4. We have evaluated these findings in peripheral blood T cells from a larger panel of TT and LL patients as well as healthy individuals. Mycobacterium-reactive T cell lines generated from the PBMC of these individuals were tested for cytokine secretion and proliferative capacity in response to M. leprae, Mycobacterium tuberculosis, and various individual mycobacterial Ag. The lepromatous pole of the leprosy spectrum was additionally investigated by analyzing the cytokine-secretion profile of M. leprae-induced (suppressor) T cell clones as well as primary ex vivo PBMC. All T cell lines from healthy individuals and TT patients responding to M. leprae, M. tuberculosis, or individual Ag, produced high levels of IFN-gamma and TNF-alpha but little or no IL-4 and IL-6. At the lepromatous pole, T cell lines failed to proliferate upon stimulation with M. leprae but in some cases produced significant levels of IFN-gamma. No IL-4 or IL-6 secretion was observed in response to M. leprae. These lines displayed strong proliferation and Th1-like cytokine production upon stimulation with M. tuberculosis. Similarly, stimulation of primary PBMC from LL patients with M. leprae or M. tuberculosis resulted in the release of IFN-gamma but no detectable IL-4 production. Control tetanus toxoid-reactive T cell lines from the same individuals instead produced large amounts of IL-4 and low levels of IFN-gamma. The analysis of M. leprae-induced T cell clones, including those with known suppressive activity, revealed that all lepromatous T cell clones produced large amounts of IFN-gamma. Most of these clones released no or little IL-4, but some clones produced higher levels of IL-4 in addition to IFN-gamma. Most clones tested produced IL-10 as well. The suppressor activity of suppressor T cell clones could not be inhibited by a neutralizing anti-IL-4 antibody and only in one case by neutralizing anti-IL-10 antibody. Anti-IL-4 and anti-IL-10 could not overcome the M. leprae-specific unresponsiveness observed in primary PBMC from LL patients.(ABSTRACT TRUNCATED AT 400 WORDS)     

A cis-regulatory element within the 5'' flanking region of arylsulfatase gene of sea urchin, Hemicentrotus pulcherrimus

Mycobacteria generally have thick cell walls and contain large amounts of lipid, making them resistant to DNA extraction. Five methods, namely, extensive enzymic digestion method (M1), 2-min mechanical glass-bead disruption method (M2), thermal shock method (M3), modified conventional enzymic digestion method (M4), and manual disruption with modified conventional enzymic digestion method (M5), were used to compare their effectiveness and simplicity in extracting DNA from slowly growing mycobacteria (Mycobacterium leprae, M. lepraemurium and M. bovis BCG), and a rapidly growing mycobacterium (M. phlei). The highest DNA yield was obtained by M2 from M. lepraemurium which produced 2.82 micrograms DNA/mg wet weight of cells, representing a theoretical yield of 78%. M3 gave the lowest DNA yield; 0.01 microgram DNA/mg wet weight of cells of M. lepraemurium was obtained. M4, in which proteinase K was used, is more effective than M1, in which subtilisin and pronase were used. M5 yielded a higher amount of DNA, but it required more manipulations to extract DNA as compared to M4. Extraction of DNA of M. leprae from nude mice is more difficult than that of M. leprae from armadillos by all of the methods used. These results suggest that the biosynthetic capabilities of these two forms of M. leprae may vary, depending on their cultural conditions and/or strain differences. Our results have shown that both M2 and M4 are the simplest, most effective and time-saving methods which are suitable for every routine laboratory to extract DNA from slowly and rapidly growing mycobacteria.     

Role of macrophages in defective cell mediated immunity in lepromatous leprosy. I. Factor(s) from macrophages affecting protein synthesis and lymphocyte transformation

Macrophages from lepromatous leprosy patients specifically show reduced protein synthesis in the presence of M. leprae. They also produce, as a result of interaction with M. leprae, factor(s) that reduce protein synthesis in normal macrophages as well as block lymphocyte transformation in normal leukocyte cultures in the presence of M. leprae as the antigen. These observations implicate a defective macrophage system in lepromatous leprosy patients.     

A light and electron microscopic study of cytoplasmic phospholipase A2 and cyclooxygenase-2 in the hippocampus after kainate lesions

Tamoxifen, the major adjuvant drug treatment for estrogen-dependent breast cancer, has been shown previously to affect both estrogen-dependent and calcium/calmodulin-dependent pathways. In the current study, we developed an in vitro slice system to study the effects of tamoxifen on ATP levels in hypothalamic (HTH) and preoptic areas (POA) of the rat brain. Baseline data showed that, following a 2-h incubation, HTH and POA slices had comparable ATP levels to hippocampal slices, a system used extensively by researchers examining the metabolic responsiveness of the hippocampal region (HPC) of the brain. HTH-POA slice ATP levels remained steady for 2, 4 and 6 h, but fell to 11% of initial levels by 12 h. Neurons from HTH-POA slices incubated for 4 h appeared healthy and demonstrated robust protein synthesis as measured autoradiographically by incorporation of [3H]leucine. We explored the effects of tamoxifen (TAM), fluphenazine (FLU) and estradiol (E2) on ATP levels in HTH and POA slices. The effects of TAM were complex: a 4-h incubation with 10-6 M TAM led to decreased ATP levels in HTH (but not POA), and a 4-h incubation with 10-8 M led to increased ATP levels in POA (but not HTH); a 15-min exposure to 10-6 M TAM decreased ATP levels in POA (but not HTH) slices, while the exposure of slices to the lower concentration of TAM was without effect in either area. As with higher concentrations of TAM, 4-h incubation with 10-6 M FLU decreased ATP levels in HTH (but not POA), while incubation with E2 did not affect slice ATP levels. These data are consistent with the hypothesis that both TAM and FLU alter ATP levels in HTH slices via calmodulin- or calcium-mediated processes.     

Cytokine patterns at the site of mycobacterial infection

Distinct patterns of T cell cytokine production have been shown to influence the outcome of infection in mouse models and humans. Th1 or Type 1 cytokines, interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) are generally associated with resistance to infection, whereas Th2 or Type 2 cytokines, IL-4 and IL-10 are associated with progressive disease. Leprosy is a useful model for studying the role of cytokines in modulating T cell responses in human infectious disease. Infection by Mycobacterium leprae results in disease manifestations that encompass an immunological spectrum. Tuberculoid patients are able to restrict the growth of the pathogen and mount strong T cell responses to M. leprae. In contrast, lepromatous patients manifest disseminated infection and their T cells weakly respond to M. leprae. We have found that tuberculoid leprosy lesions have a predominance of CD4+ T cells producing the Type 1 cytokine pattern. Secondly, IL-12 mRNA was expressed at 10-fold higher levels in tuberculoid lesions as compared to lepromatous lesions and that IL-12 promotes the selective expansion of the Type 1 cytokine producing cells. In contrast, lepromatous lesions contain CD8+ IL-4-producing cells that suppress antigen-specific T cell responses and promote the outgrowth of additional suppressor T cells. IL-10, also expressed at higher levels in lepromatous as compared to tuberculoid lesions, was found to be produced by macrophages, effectively inhibiting cytokine production and macrophage activity.     

Absence of complete homologous immunity in hepatitis B infection after massive exposure

Ascorbic acid was fed to mice in concentrations of 0.05%, 0.15%, and 0.45% w/w in the diet. Six months after inoculation of M. leprae into the foot pads, there were significantly fewer acid-fast bacilli harvested from animals receiving 0.15% and 0.45% w/w ascorbic acid than from control mice. On the other hand, M. leprae did multiply in mice fed ascorbic acid while no multiplication at all was observed in animals fed dapsone, clofazimine or rifampin. No toxic effects of ascorbic acid were noted in these mice.     

Activity of rifabutin, clarithromycin, ethambutol, sparfloxacin and amikacin, alone and in combination, against Mycobacterium avium complex in human macrophages

Disseminated infection with Microbacterium avium complex (MAC) in patients with AIDS is currently treated with a combination of antimycobacterial agents in order to prevent the selection of resistant mutant strains. Although clinical and microbiological responses can generally be achieved within a few weeks, relapses are common and require modification of the combination regimen or identification of effective alternate therapies. In this study we investigated the activities of rifabutin 0.5 mg/L, sparfloxacin 1 mg/L, clarithromycin 4 mg/L, amikacin 16 mg/L and ethambutol 2 mg/L, alone and in combination, against nine strains of M. avium isolated from the blood of patients with AIDS in order to identify regimens with the greatest therapeutic potential. Macrophages derived from human monocytes were infected with M. avium and inoculated with a single drug or a combination of drugs; cfu counts were performed at 0, 4 and 7 days after infection. At day 4 and at day 7, the combination of rifabutin, clarithromycin, amikacin and sparfloxacin displayed the highest degree of activity. However, the activity did not differ significantly from that of the combination of rifabutin, clarithromycin and ethambutol. The results of this study confirm the activity of combinations including rifabutin and clarithromycin (+/- ethambutol) in human monocyte-derived macrophages and suggest potentially useful associations in incorporating sparfloxacin and amikacin.     

Use of polymerase chain reaction to assess efficacy of leprosy chemotherapy

The assessment of chemotherapy efficacy in leprosy is difficult, since the only reliable method for determining whether the causative organism, Mycobacterium leprae, is viable depends on its growth in mouse foot pads. In an attempt to replace this expensive, time-consuming test, methods based on the polymerase chain reaction (PCR) have been developed. These methods depend on detection of DNA, which is more susceptible to degradation on cell death than are other cell components, so should be a more accurate indicator of viability. We have used a specific PCR assay to detect M leprae DNA in skin biopsy samples from leprosy patients. By use of limiting dilution PCR (LD-PCR), the concentration of M leprae DNA in the original sample could be measured. The DNA concentration was more closely correlated with the morphological index (derived from a staining technique that distinguishes morphologically intact and damaged bacteria) than with the number of bacteria visible (bacterial index, BI, which counts both alive and dead bacteria). In a longitudinal study of multibacillary patients on multi-drug therapy, skin biopsy samples were collected before treatment and 3, 6, 12, and 24 months after the start of therapy. While the BI showed little or no change during treatment, the number of genomes detected by PCR fell sharply, in parallel with the MI. We propose that PCR can be used as a rapid measure of M leprae viability and that this approach can be used for monitoring individual leprosy patients and for assessment of existing and new regimens. The method may be applicable to other infectious diseases in which culture of the causative organism is slow or impossible.     

Polysome-dependent in vitro translation system capable of peptide chain reinitiation

A sensitive in vitro translation system has been developed which makes use of cellular polysomes as the source of mRNA and ribosomes. The soluble factors are derived from the preincubated S-30 fraction by centrifugation through a discontinuous sucrose gradient. Of the four fractions tested, fraction 1 (topmost fraction in the gradient) and fraction 2 (fraction sedimenting in 0.5 M sucrose) were stimulatory. These two fractions together yield the highest activity, corresponding to about 125 times the background incorporation. The polysome-directed system exhibits optimal activity in the range 1.8-2 mM Mg2+ and 125-175 mM KCl. The polysome-directed in vitro products exhibit a complexity comparable to the in vivo products resolved on the two-dimensional polyacrylamide gels of O'Farrell [O'Farrell, P. (1975) J. Biol. Chem. 250, 4007-4021]. The system is capable of active chain reinitiation as indicated by partial inhibition by 7-methylguanosine 5'-monophosphate and pactomycin and N-terminal end analysis of in vitro products. This system can also translate polysomes from diverse tissues such as mouse liver, rat liver, and rat brain. The levels and also the authenticity of translation of rat liver albumin and mouse liver carbamoyl phosphate synthetase I were tested by immunoprecipitation with monospecific antibodies. The results show that the major as well as the minor translation products are synthesized in this system at levels comparable to the physiological levels.     

Activity of two doses of rifampin against Mycobacterium leprae

In the course of a clinical trial designed to re-examine the bactericidal efficiency of 600-mg doses of rifampin (RMP) against Mycobacterium leprae, two doses of RMP, either 600 mg or 1200 mg, were administered 28 days apart to 29 previously untreated patients with lepromatous or borderline leprosy. Seven, 28, and 35 days after the start of the trial, skin biopsies were performed and immunologically normal mice were inoculated with 5 x 10(3) or 10(4) M. leprae in each hind foot pad. The patients assigned to the two regimens did not differ significantly in terms of sex, age, disease classification, bacterial index, or the concentration of M. leprae in the skin lesion biopsied for the inoculation of mice. The concentrations of organisms in the skin-biopsy specimens did not change significantly over the course of the trial among the patients, whether they were being treated by the first or the second regimen. The M. leprae recovered from specimens obtained from 21 of the patients, before beginning treatment, multiplied in a majority of the mice inoculated. The results of mouse inoculation confirmed the rapid bactericidal effects of RMP against M. leprae: a single dose of RMP rendered the organisms obtained from all but two of the patients incapable of multiplying in mice. No significant difference was demonstrated between the two regimens, nor was an additional effect of the second dose of RMP observed.     

Papillary thyroid carcinoma: MR diagnosis of lymph node metastasis

We investigated the in vitro antifungal activity of amphotericin B, alone and in combination with rifabutin, an inhibitor of bacterial RNA polymerase, against 26 clinical isolates of Aspergillus and 25 clinical isolates of Fusarium. Synergy or additivism between these drugs was demonstrated against all isolates tested. Amphotericin B MICs were reduced upon combination with rifabutin from a mean of 0.65 microg/ml to a mean of 0.16 microg/ml against Aspergillus, and from a mean of 0.97 microg/ml to a mean of 0.39 microLg/ml against Fusarium (P < 0.000001 for both). Similarly, the MICs of rifabutin were reduced upon combination with amphotericin B from a mean of >32 microg/ml to a mean of 1.1 microg/ml against both fungi (P < 0.000001 for both). These positive interactions were corroborated by a colony count study with two Fusarium isolates, for which treatment with the combination of subinhibitory concentrations of amphotericin B (at concentrations 2- and 4-fold less than the MIC) and rifabutin (at concentrations ranging from 4- to 64-fold less than the MIC) resulted in 3.2-log reductions in colony counts compared to those after treatment with either drug alone. Inhibition of RNA synthesis was shown to be the mechanism of antifungal activity. These results suggest that inhibition of fungal RNA synthesis might be a potential target for antifungal therapy.     

Prevalence of artificial hip implants and use of health services by recipients

Intraneural injection of 10-20 x 10(6) viable Mycobacterium leprae into the sciatic nerve of normal, unsensitized, Swiss white mice gives rise to a tuberculoid type of granulomatous response in 2 weeks. The same dose of viable M. leprae when injected into the sciatic nerves of unsensitized immunosuppressed mice (T200 x 5R) elicited a macrophage response. When macrophages were systemically immobilized using an intraperitoneal injection of silica quartz dust in normal mice, the lesion produced was of the lepromatous type, suggesting a role for the macrophage in the induction of the tuberculoid type of granulomatous response. In all of these in situ experiments, M. leprae failed to enter the Schwann cells.     

Bactericidal activity of a single-dose combination of ofloxacin plus minocycline, with or without rifampin, against Mycobacterium leprae in mice and in lepromatous patients

To develop a fully supervisable, monthly administered regimen for treatment of leprosy, the bactericidal effect of a single-dose combination of ofloxacin (OFLO) and minocycline (MINO), with or without rifampin (RMP), against Mycobacterium leprae was studied in the mouse footpad system and in previously untreated lepromatous leprosy patients. Bactericidal activity was measured by the proportional bactericidal method. In mouse experiments, the activity of a single dose of the combination OFLO-MINO was dosage related; the higher dosage of the combination displayed bactericidal activity which was significantly inferior to that of a single dose of RMP, whereas the lower dosage did not exhibit a bactericidal effect. In the clinical trial, 20 patients with previously untreated lepromatous leprosy were treated with a single dose consisting of either 600 mg of RMP plus 400 mg of OFLO and 100 mg of MINO or 400 mg of OFLO plus 100 mg of MINO. The OFLO-MINO combination exhibited definite bactericidal activity in 7 of 10 patients but was less bactericidal than the RMP-OFLO-MINO combination. Both combinations were well tolerated. Because of these promising results, a test of the efficacy of multiple doses of ROM in a larger clinical trial appears justified.     

Effects of overexpression of the alkyl hydroperoxide reductase AhpC on the virulence and isoniazid resistance of Mycobacterium tuberculosis

Mutations to the regulatory region of the ahpC gene, resulting in overproduction of alkyl hydroperoxide reductase, were encountered frequently in a large collection of isoniazid (INH)-resistant clinical isolates of Mycobacterium tuberculosis but not in INH-susceptible strains. Overexpression of ahpC did not seem to be important for INH resistance, however, as most of these strains were already defective for catalase-peroxidase, KatG, the enzyme required for activation of INH. Transformation of the INH-susceptible reference strain, M. tuberculosis H37Rv, with plasmids bearing the ahpC genes of M. tuberculosis or M. leprae did not result in a significant increase in the MIC. Two highly INH-resistant mutants of H37Rv, BH3 and BH8, were isolated in vitro and shown to produce no or little KatG activity and, in the case of BH3, to overproduce alkyl hydroperoxide reductase as the result of an ahpC regulatory mutation that was also found in some clinical isolates. The virulence of H37Rv, BH3, and BH8 was studied intensively in three mouse models: fully immunocompetent BALB/c and Black 6 mice, BALB/c major histocompatibility complex class II-knockout mice with abnormally low levels of CD4 T cells and athymic mice producing no cellular immune response. The results indicated that M. tuberculosis strains producing catalase-peroxidase were considerably more virulent in immunocompetent mice than the isogenic KatG-deficient mutants but that loss of catalase-peroxidase was less important when immunodeficient mice, unable to produce activated macrophages, were infected. Restoration of virulence was not seen in an INH-resistant M. tuberculosis strain that overexpressed ahpC, and this finding was confirmed by experiments performed with appropriate M. bovis strains in guinea pigs. Thus, in contrast to catalase-peroxidase, alkyl hydroperoxide reductase does not appear to act as a virulence factor in rodent infections or to play a direct role in INH resistance, although it may be important in maintaining peroxide homeostasis of the organism when KatG activity is low or absent.