Visualization of M. tuberculosis in Animal Models the lungs of an M. tuberculosis infected guinea pig 4 weeks post-aerosol infection. Secondary lesions are distinguished from primary granulomas by the lack of central necrosis in the former (H&E staining, 1006 magnification). (D, E) Low (D) and high (E) magnifications of a primary granuloma from the lungs of an M. tuberculosis infected guinea pig 4 weeks after aerosol infection. The majority of AR+ bacilli are extracellular within the necrotic core (c) (AR, 1006 and 2006 magnifications). (F) High magnification of area demarcated in figure 5E (square). Extracellular AR+ bacilli in the necrotic core of primary granulomas exist as single cells or are situated in clusters (AR, 10006 magnification). (G, H) Confocal micrographs of a necrotic granuloma showing an acellular necrotic core (c) with extracellular AR+ bacilli (red) (AR and DAPI, 2006and 6306 magnifications). (I) A primary granuloma from the lungs of an M. tuberculosis infected guinea pig 10 weeks after aerosol infection showing advanced calcification and calcification of the necrotic core (c) and the acellular rim (R) surrounding the core (H&E staining, 1006magnification). (J) Fluorescent image of a primary lung granuloma from an M. tuberculosis infected guinea pig 10 weeks after aerosol infection. Extracellular AR+ bacilli are present within the necrotic core (c) and the acellular rim (R) surrounding the necrotic regions (AR, 2006magnification). (K) Cropped image from figure 5I (square) showing extracellular AR+ stained bacilli in the acellular rim. (L) Fluorescent image of a necrotic primary lung granuloma from an M. tuberculosis infected guinea pig treated for 6 weeks with INH. Extra-cellular AR+ bacilli are primarily within the core (c) of the partially calcified lytic necrosis, and to a lesser extent within the acellular, uncalcified rim (R) (AR, 2006 and 4006 magnifications). (M) A low magnification of the remnant of a primary lung granuloma in an M. tuberculosis infected guinea pig treated for 6 weeks with TMC207. The primary granuloma shows a caseous necrotic core (c) surrounded by inflammatory cells (H&E staining, 1006 magnification). (N) Fluorescent image of the caseous necrotic core (c) in the primary lung granuloma shown in figure 5M taken from a serial tissue section. The few extra-cellular AR+ bacilli remaining after TMC207 treatment are primarily located within the central core of caseous necrosis (AR, 4006 magnification). doi:10.1371/journal.pone.0017550.g005 determined after plating of the organ homogenates on solid agar with little knowledge relating to pathology, location of the bacilli in the various animal models, or the potential differential drug activity in various locations in the lung. In this manuscript, we provide a comprehensive microscopic and histopathologic analysis of the disease progression of the animal models used in-house for TB drug development with a main focus on the location of the bacilli in the lung. M. tuberculosis is generally thought of as being an ‘intracellular organism’ living in (and perhaps needing) its host cell, the macrophage. We show that this is not necessarily the case. M. tuberculosis bacilli were visualized via a new rapid staining method combining fluorescent acid-fast AR for staining bacteria, hema- toxylin QS for staining tissue and DAPI for staining nuclei. This fluorescent staining method enabled us to easily visualize the bacteria in the tissue across a whole lung granuloma, whereas in past studies using the Ziehl-Neelsen method by brightfield microscopy, only limited information per microscopic field could be collected using high magnification. Confocal microscopy then gave us a three dimensional view of M. tuberculosis within a granuloma. Our earlier work using standard Ziehl-Neelsen staining suggested that the remaining bacilli after drug treatment in the guinea pig model were primarily present just inside the zone of incomplete necrosis within the acellular, fibrotic rim of necrotic lung lesions [23]. However, with this improved staining and detection method, the rim proved to be merely one of the locations. In this comprehensive study, we demonstrate that the majority of bacteria in the guinea pig model are more central within the core area of the necrotic lesion. In the mouse models studied here, we found that the majority of bacilli were indeed intracellular within macrophages through most of the infection. In the immunocompetent C57BL6 mice, AR+ bacilli were clearly intracellular, and this was especially evident in the foamy macrophages located outside of the lymphocyte region as described earlier [65,66,67]. In the immunocompromised GKO model, considerably more bacilli per macrophage were visualized by the AR staining method. Interestingly, in the late stages of infection in M. tuberculosis infected GKO mice, the granulomatous lesions begin to exhibit significant necrosis with the bacilli progressively becoming extracellular (with about half of the bacilli eventually being extracellular). The GKO mice form massive granulomas that contain multiple focal-points of necrotic cellular debris accumulating in alveolar air spaces. These necrotic areas increase in number and size and in a late stage can collectively coalesce into a single core (which is also observed in guinea pig, as well as in human lesions). As a confirmation of necrosis in the late stage GKO mouse model, immunofluorescence for tubulin was used that showed a clear loss of the cytoskeleton structure of the eucaryotic cells. The presence of macrophages and debris in the airways in this late stage of the GKO model shows pathological similarity to lung inflammation in cavitary TB patients. Since an earlier study using the guinea pig model showed these necrotic lesions to be hypoxic using pimonidazole [23], this led us to investigate the oxygen tension in lungs of M. tuberculosis infected GKO mice. Hypoxia has been postulated as one of the environmental conditions which transitions mycobacteria in vitro into a non-replicating phase, thereby affecting their responsiveness to drugs [7,68]. In the late stage of the GKO model, a clear hypoxic zone was found at the periphery of the necrotic regions. The inability of pimonidazole to stain the necrotic tissue itself was due to the lack of viable cells which are required to enzymatically create detectable adducts, however this necrotic tissue is also likely to be hypoxic. The guinea pig model was studied, which shows more complex pathology with a heterogeneity of lesion types and stages within one animal at any one time. The location of bacilli in guinea pig lung lesions was also studied by fluorescent AR staining. Intracellular bacilli were present in inflammatory lesions only in the first weeks after aerosol infection showing equal numbers of intra- and extracellular bacilli visualized throughout the granulo- ma. From one month after low dose aerosol infection, the bacilli were mainly extracellular within the necrotic core and surround- ing acellular rim of the primary granulomas. The ‘core of the lesion’ is here referred to as the centre of necrotic guinea pig lesion that calcifies and mineralizes, whereas the ‘rim of the lesion’ is defined here as the edge of incomplete necrosis which has not yet calcifieded. Confocal microscopy clearly showed that the core of the granuloma is indeed acellular and devoid of intact nuclei and is the location where most bacteria are. The few intracellular bacilli found were either located in the outer lymphocyte and epithelioid macrophage cell layer of primary granulomas or in secondary lesions, although this latter location rarely contained any detectable AR+ M. tuberculosis bacilli at all. The reason for finding only very few acid-fast bacilli in the secondary granulomas is not entirely clear. The secondary lesions in the guinea pig model are thought to originate mainly from bacilli after hematogenous dissemination from the primary lesions, which occurs only after the immune response is already activated. Therefore, bacillary replication in the secondary lesions is likely curtailed due to the now activated immune response [51]. The difference in cytokine profiles in primary and secondary lesions has been described earlier by others with secondary lesions showing a strong anti- inflammatory response [69]. For TB drug development, it is important to realize that in case of progressive disease most PLoS ONE | www.plosone.org 9 March 2011 | Volume 6 | Issue 3 | e17550