ABSTRACTPersistence ofMycobacterium tuberculosisremains a significant challenge for the effective treatment of tuberculosis in humans. In animals that develop necrotic lung lesions following infection withM. tuberculosis, drug-tolerant bacilli are present and persist in an extracellular microenvironment within the necrotic cores. In this study, we examined the efficacy of drug treatment in C3HeB/FeJ (Kramnik) mice that develop lesions with liquefactive necrosis, in comparison to BALB/c mice that develop nonnecrotic lesions following aerosol challenge. To accomplish this, Kramnik and BALB/c mice were infected by aerosol withM. tuberculosisand treated for 7 to 8 weeks with monotherapy using drugs with different modes of action. The efficacy of drug therapy was quantified by enumeration of bacterial load. The progression of disease and location and distribution of bacilli within lesions were visualized using various staining techniques. In the late stages of infection, Kramnik mice developed fibrous encapsulated lung lesions with central liquefactive necrosis containing abundant extracellular bacilli, whereas BALB/c mice formed nonnecrotic lesions with primarily intracellular bacilli. Necrotic lesions in Kramnik mice showed evidence of hypoxia by pimonidazole staining. Kramnik mice were significantly more refractory to drug therapy, especially for pyrazinamide. Metronidazole showed no bactericidal activity in either model. There were significantly higher numbers of drug-resistant colonies isolated from the Kramnik mice compared to BALB/c mice. These results suggest that the Kramnik mouse model will be a valuable model to test antituberculosis drugs, especially against bacilli that persist within necrotic lesions.

The lengthy chemotherapy of tuberculosis reflects the ability of a small subpopulation of Mycobacterium tuberculosis bacteria to persist in infected individuals. To date, the exact location of these persisting bacteria is not known. Lung lesions in guinea pigs infected with M. tuberculosis have striking similarities, such as necrosis, mineralization, and hypoxia, to natural infections in humans. Guinea pigs develop necrotic primary lesions after aerosol infection that differ in their morphology compared to secondary lesions resulting from hematogenous dissemination. In infected guinea pigs conventional therapy for tuberculosis during 6 weeks reduced the bacterial load by 1.7 logs in the lungs and, although this completely reversed lung inflammation associated with secondary lesions, the primary granulomas remained largely unaffected. Treatment of animals with the experimental drug R207910 (TMC207) for 6 weeks was highly effective with almost complete eradication of the bacteria throughout both the primary and the secondary lesions. Most importantly, the few remnants of acid-fast bacilli remaining after R207910 treatment were to be found extracellular, in a microenvironment of residual primary lesion necrosis with incomplete dystrophic calcification. This zone of the primary granuloma is hypoxic and is morphologically similar to what has been described for human lung lesions. These results show that this acellular rim may, therefore, be a primary location of persisting bacilli withstanding drug treatment. Tuberculosis (TB) is treatable by drugs, and the World HealthOrganization has promoted the use of "directly observed therapy" to administer effective regimens to infected patients. However, despite this, no new drug classes have been introduced in the last two to three decades and new, effective compounds are badly needed. A central problem, however, even when compliance problems are dealt with, is the sheer length of time needed for current drug regimens to ensure clearance of the infection without relapse (44). As a result, conventional drug regimens are usually of 6 to 9 months in length.The length of treatment is believed to represent the need to eradicate a small population of bacteria that persist within the lung and extrapulmonary tissues. In addition, these "persisters" likely are the source of reactivation TB that can then occur at a later date, including as a result of human immunodeficiency virus infection. The different mechanisms underlying mycobacterial persistence are not known but appear to represent some form of refractory state rather than true drug resistance (7,26,31). In vitro a subpopulation of 5 to 10% of Mycobacterium tuberculosis appears far less responsive to killing by drugs. In vivo ca. 99% of bacteria in mice are killed within 2 weeks of drug treatment, but then it requires at least 3 more months of treatment to clear the remaining 1% (19-21, 28, 40). This apparent drug tolerance of M. tuberculosis can be readily demonstrated and modeled in mice.Treatment of persisting bacteria in h...

The lengthy treatment regimen for tuberculosis is necessary to eradicate a small sub-population of M. tuberculosis that persists in certain host locations under drug pressure. Limited information is available on persisting bacilli and their location within the lung during disease progression and after drug treatment. Here we provide a comprehensive histopathological and microscopic evaluation to elucidate the location of bacterial populations in animal models for TB drug development.To detect bacilli in tissues, a new combination staining method was optimized using auramine O and rhodamine B for staining acid-fast bacilli, hematoxylin QS for staining tissue and DAPI for staining nuclei. Bacillary location was studied in three animal models used in-house for TB drug evaluations: C57BL/6 mice, immunocompromised GKO mice and guinea pigs. In both mouse models, the bacilli were found primarily intracellularly in inflammatory lesions at most stages of disease, except for late stage GKO mice, which showed significant necrosis and extracellular bacilli after 25 days of infection. This is also the time when hypoxia was initially visualized in GKO mice by 2-piminidazole. In guinea pigs, the majority of bacteria in lungs are extracellular organisms in necrotic lesions and only few, if any, were ever visualized in inflammatory lesions. Following drug treatment in mice a homogenous bacillary reduction across lung granulomas was observed, whereas in guinea pigs the remaining extracellular bacilli persisted in lesions with residual necrosis.In summary, differences in pathogenesis between animal models infected with M. tuberculosis result in various granulomatous lesion types, which affect the location, environment and state of bacilli. The majority of M. tuberculosis bacilli in an advanced disease state were found to be extracellular in necrotic lesions with an acellular rim of residual necrosis. Drug development should be designed to target this bacillary population and should evaluate drug regimens in the appropriate animal models.

A unique hallmark of tuberculosis is the granulomatous lesions formed in the lung. Granulomas can be heterogeneous in nature and can develop a necrotic, hypoxic core which is surrounded by an acellular, fibrotic rim. Studying bacilli in this in vivo microenvironment is problematic as Mycobacterium tuberculosis can change its phenotype and also become acid-fast negative. Under in vitro models of differing environments, M. tuberculosis alters its metabolism, transcriptional profile and rate of replication. In this study, we investigated whether these phenotypic adaptations of M. tuberculosis are unique for certain environmental conditions and if they could therefore be used as differential markers. Bacilli were studied using fluorescent acid-fast auramine-rhodamine targeting the mycolic acid containing cell wall, and immunofluorescence targeting bacterial proteins using an anti-M. tuberculosis whole cell lysate polyclonal antibody. These techniques were combined and simultaneously applied to M. tuberculosis in vitro culture samples and to lung sections of M. tuberculosis infected mice and guinea pigs. Two phenotypically different subpopulations of M. tuberculosis were found in stationary culture whilst three subpopulations were found in hypoxic culture and in lung sections. Bacilli were either exclusively acid-fast positive, exclusively immunofluorescent positive or acid-fast and immunofluorescent positive. These results suggest that M. tuberculosis exists as multiple populations in most conditions, even within seemingly a single microenvironment. This is relevant information for approaches that study bacillary characteristics in pooled samples (using lipidomics and proteomics) as well as in M. tuberculosis drug development.

Metronidazole, which is used for the treatment of infections caused by anaerobic organisms, was evaluated in Mycobacterium tuberculosis-infected guinea pigs. M. tuberculosis can adapt to hypoxia, which is present in the primary lesions of infected guinea pigs. Metronidazole treatment (for 6 weeks at 100 mg/kg of body weight) resulted in no reduction in the bacillary burden and significantly worsened lesion inflammation.The excellent distribution of metronidazole (MET) in all organs and its good bactericidal activity, including its activity against quiescent bacteria, make it the compound of choice for the treatment of many infections caused by anaerobic organisms. Mycobacterium tuberculosis also has the ability to adapt to gradual oxygen depletion or stationary growth. This phenomenon has been studied extensively by Wayne and Hayes and has been described as a sequential progression through two stages, defined as nonreplicating phase 1 (NRP-1) and NRP-2 (15). MET has no effect on exponentially growing bacilli or on NRP-1 bacilli, but it is bactericidal for bacilli in stationary phase and NRP-2 (6, 16).Nonprogressive M. tuberculosis lesions in the lungs often have limited vascularization (due to necrosis and dystrophic mineralization), causing a limited O 2 supply. Bacterial persistence in these hypoxic lesions has been thought to be accompanied by susceptibility to MET. In mouse models of tuberculosis (TB), MET has failed to show consistent activity (5, 6, 10). This is not surprising, as hypoxia was found to be completely absent in the M. tuberculosis lesions of infected mice (1, 11), and the progression of disease rarely reaches the stages of extensive necrosis (2,3,8,12). Lung lesions in guinea pigs infected with M. tuberculosis, on the other hand, show caseous necrosis, mineralization, and hypoxia, which are also seen in natural infections in humans (13,14). Guinea pigs develop necrotic primary lesions that differ in their morphology compared to those of the secondary lesions that result after the activation of adaptive immunity (8). In an earlier paper, we described that the persisting, acid-fast bacilli are primarily found extracellularly in a hypoxic microenvironment of primary lesion necrosis and that only few a bacilli are localized within the secondary granulomas (8). The same study also showed evidence of hypoxia in these primary lesions of guinea pigs when pimonidazole was used (8), and therefore, we chose to explore the potential bactericidal activity of MET in the guinea pig model.To establish appropriate drug doses in guinea pigs for the anti-M. tuberculosis drugs used in this study, pharmacokinetic analysis was performed with a single dose each of isoniazid (INH), rifampin (RIF), pyrazinamide (PZA), and MET. All drugs were obtained from Sigma Chemical Co. (St. Louis, MO). INH, PZA, and MET were dissolved in distilled water, while RIF was dissolved in dimethyl sulfoxide (final concentration, 0.5%) with sucrose (40%, wt/vol) to increase its palatability. The pharmacokinetic data obtained by validated...