characterized by numerous, small foci of eosinophilia that were devoid of cellular debris. A distinct increase in AR+ bacilli number was observed at 9 weeks post aerosol infection, AR+ bacilli (80– 85%) were found intracellular within mFs distributed throughout the central region of inflammatory lesions within the LC cuff (Fig. 4E, 4F). Fewer AR+ bacilli (15–20%) were found within foamy mFs that were located outside of the lymphocyte cuff in the surrounding alveolar air spaces. AR+ bacilli were not found in the areas of necrosis. The location of M. tuberculosis in the lungs of C57BL/6 mice was studied after 6 weeks of INH, RIF or MXF treatment. Over the 6 weeks treatment, the bacterial burden determined by plating on agar plates decreased in the lungs from 6.0060.07 Logs in the untreated control groups to 2.0860.16, 3.0660.38 and 1.7560.07 Log10CFU for INH, RIF and MXF, respectively. The occurrence of drug resistant colonies after treatment is not an issue in this model due to the low bacterial load at the start of treatment. After two weeks of treatment, minimal effect was observed on lesion pathology and distribution of the intracellular AR+ bacilli across the pulmonary lesions. After six weeks of therapy the pulmonary inflammation was markedly reduced, although the same cell morphology, spatial distribution and degree of necrosis was still observed in remaining lesions. Despite limited effect on lung lesion pathology, a clear decrease in the number of AR+ bacilli in lungs was evident at this time and this reduction was uniform across the lesion. The bacilli (80–85%) were located intracellularly in epithelioid mFs within the lymphocyte cuff composing the lesion cores (Fig. 4G), while the minority (15–20%) were found outside of the lymphocyte cuff in foamy mFs (Fig. 4H). Remarkably, these AR+ bacilli could still be visualized in these foamy mFs after drug treatment. Although AR+ bacilli were often found in close proximity to necrotic regions, none were found within these necrotic sites. Extracellular M. tuberculosis in the primary lung lesions in guinea pigs The location of bacilli in guinea pig lung sections was studied by using the combination staining of AR, hematoxylin QS and DAPI starting one month after low dose aerosol infection (which is the start of drug treatment in our standard laboratory protocol for the guinea pig model) [23,45,50]. Lung sections from guinea pigs studied four weeks after aerosol infection contained multiple granulomatous lesions with differing morphologies. The classic necrotic granulomas (also primary granuloma) were differentiated from inflammatory lesions (also secondary granuloma) based on the presence of necrosis surrounded by lymphocytic cell populations [22,23,25,49,51–53] (Fig. 5A, C). The AR+ bacilli (,70%) were dispersed predominantly throughout the necrotic areas of the necrotic granulomas (Fig. 5D, E). The bacilli are present extracellularly either as small clusters of bacteria or as single cells (Fig. 5F). Confocal microscopy was then used to confirm the location of AR+ bacilli within the acellular necrotic core of these granulomas (Fig. 5G, H). Fewer AR+ bacilli (,20%) were observed intracellularly within mFs found in close proximity to necrosis within primary lesions, and very few AR+ bacilli (,10%) were found within mW cells from secondary lesions. By 10 weeks post aerosol infection, the necrotic core of the primary granulomas progressed to a state of complete, dystrophic calcification [22,52] (Fig. 5I). A modest decrease in AR+ bacilli was observed, but in contrast to earlier time points, the AR+ bacilli (95%) were now almost exclusively extracellular within the necrotic core and surrounding, acellular rim of residual lesion necrosis in the primary granulomas (Fig. 5J, K). Most secondary Visualization of M. tuberculosis in Animal Models lesions contained very few intracellular, if any, detectable AR+ bacilli (data not shown). Over the 6 weeks treatment, the bacterial burden determined by plating on agar plates decreased in the lungs from 5.1260.28 Log10 CFU in the untreated control group to 3.7660.0.1 and 1.7860.26 Log10CFU for INH and TMC207, respectively. The occurrence of drug resistant colonies is not an issue in this model due to the low bacterial load at the start of treatment. INH treatment had a differential effect on the lung pathology of the primary versus the secondary lesions. Secondary lesions dimin- ished in size and were largely healed after 6 weeks of INH treatment as previously reported [23]. Primary granulomas, on the other hand, required prolonged therapy in order to see visible resolution of the pathology. Healed secondary lesions within the pulmonary parenchyma reatain normal alveolar structure except for thickened alveolar walls and a mild to moderate increase in cellularity. Healed primary lesions are characteried by residual foci of dystrophic calcification and fibrosis that replace alvoli or are ebedded in the fibrous stroma that supports airways and pulmonary lymphatic and blood vascuature [53]. The AR+ bacterial population in the primary lesions steadily declined throughout INH treatment. However, the decrease in AR+ bacilli was far less pronounced in guinea pigs compared to the mouse infection models treated with the same drugs. Similar to the untreated controls, the remaining AR+ bacilli (,95%) were extracellular located in primary granulomas and concentrated within the necrotic core and acellular rim, with only few intracellular bacilli in the surrounding mF and lymphocyte region (Fig. 5L). Very few bacteria (,5%) were found within mW cell types composing secondary lesions. Besides INH treatment, we evaluated the highly potent TMC207 in the guinea pig model to see if more intensive treatment would ultimately result in visible resolution of the more advanced necrotic lesions. Drug efficacy data of TMC207 and INH in the guinea pig model have been reported before [23], and the drug treatments were repeated for this study also using intermittent timepoints of 2, 4 and 6 weeks of treatment. Similar to INH treatment, 6 weeks of TMC207 treatment lead to an almost complete clearance of secondary lesions in guinea pigs. However, compared to INH, TMC207 was more effective at resolving some of the primary granulomas in guinea pigs when administered for 6 weeks. While the numbers of AR+ bacilli were far lower after TMC207 treatment than after INH treatment, the remaining AR+ bacilli were predominantly present in the necrotic cores and surrounding acellular rims of the remaining primary granulomas (Fig. 5M, N). Discussion TB drug discovery and development use a sequence of in vitro assays and animal models of M. tuberculosis infection to identify and select active experimental compounds. Mouse models provide important information regarding pharmacokinetics and in vivo efficacy of novel compounds [15–17,54–57]. Recently, other animal models which reflect the progression of lung pathology as seen in humans (to include necrosis, calcification and fibrosis of lesions) are being considered in later stages of drug development: such as the guinea pig [21,26,53,58–61], the rabbit [21,28], the non-human primate infection model for tuberculosis [27], the Kramnik mouse [62,63], and more recently the minipig model [64]. Surprisingly, there is limited to no information on the location of M. tuberculosis in the most widely used animal models for TB drug development. Up to date, the in vivo efficacy of a new compound is primarily measured by the reduction in bacillary load PLoS ONE | www.plosone.org 7 March 2011 | Volume 6 | Issue 3 | e17550