Location of Intra- and Extracellular M. tuberculosis Populations in Lungs of Mice and Guinea Pigs during Disease Progression and after Drug Treatment

Abstract

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.

Evaluation of a Mouse Model of Necrotic Granuloma Formation Using C3HeB/FeJ Mice for Testing of Drugs against Mycobacterium tuberculosis

“…The hypoxic nature of necrotic TB granulomas has been reported in other animal models for tuberculosis infection, such as guinea pigs, rabbits, and nonhuman primates (23,62), but is generally not observed in the mouse model of infection (45). We evaluated oxygen tension in the late stage of the Kramnik model using immunohistochemical detection of pimonidazole hydrochloride (Hypoxyprobe) adducts throughout the progression of disease.…”

“…In addition, encapsulation of lesions by fibrous connective tissue may represent a physical barrier to drug penetration, which was recently indicated in a study using the rabbit model (49). We have shown in animal models that develop necrotic lesions that the majority of bacilli are extracellular and admixed with debris from necrotic host cells (23). Because these lesions more closely mimic what occurs in humans with naturally occurring TB, it is desirable to evaluate any promising new TB drug regimen under similar, clinically relevant in vivo conditions.…”

“…At each sacrifice, the right caudal lung lobe was infused with 10% neutral buffered formalin and preserved until processed for histopathological assessment. Five-micrometer-thick paraffin sections were stained with TB auramine-rhodamine T as per the manufacturer's instructions (Becton, Dickinson, Sparks, MD), with modifications in order to visualize bacteria as well as the surrounding lung tissue (23). A combination fluorescent staining method was used by combining auramine O and rhodamine B to stain the bacteria (AR; Becton, Dickinson), hematoxylin QS for staining tissue (HQS; Vector Laboratories, Inc., Burlingame, CA), and 4=,6-diamidino-2-phenylindole dihydrochloride for staining nuclei (DAPI; Sigma Chemical Co.).…”

The Pathology of Mycobacterium tuberculosis Infection

“…It was previously believed that Mtb were sparse in the caseum, based on Ziehl-Neelsen acid-fast staining, with most of the detectable bacilli persisting at the periphery. 126 A recently developed acid-fast technique using Auramine O and Rhodamine B revealed that the majority of bacilli are actually in the necrotic core in guinea pig caseous granulomas, 102 but the viability of these bacilli remains unclear. Mtb adapt to this environment by preferentially using fatty acids in their metabolism, slowing down active replication, and increasing cell wall thickness, entering a so-called dormant state.…”

Limited Activity of Clofazimine as a Single Drug in a Mouse Model of Tuberculosis Exhibiting Caseous Necrotic Granulomas

“…In addition, the bacilli within human pulmonary granulomas, especially those involved in cavitary disease and transmission, are believed to be primarily extracellular (19). However, the bacilli within the lungs of traditional mouse models of infection are predominantly intracellular (20). Therefore, the efficacy of compounds that preferentially accumulate within macrophages could be overestimated by testing in an animal model that lacks necrotic lesions containing abundant extracellular bacilli.…”