bacteria are primarily extracellular organisms within a matrix of cellular debris and calcified cellular components. TB drug treatment aims to sterilize lesions from bacteria and should therefore target to eradicate these initial necrotic lesions from the extracellular bacteria. Interestingly, a diffuse pink staining effect was frequently seen after AR staining in primary necrotic granulomas of the guinea pigs after day 30 (figure 5L). This pink haze was evident throughout the necrotic granuloma and was also seen in the GKO mouse in areas of advanced necrosis at day 28 post infection (figure 2H) but not earlier, and was never observed in the standard C57BL/6 mouse. It is not entirely clear what this diffuse staining represents, however, it is possible that AR staining detects free mycolic acids either from degraded dead bacilli or from bacilli that shed mycolic acids, a feature reported for in vitro pellicle-grown M. tuberculosis [70]. We have shown free mycolic acids to stain readily with rhodamine but not auramine in our laboratory (data not shown) in an ongoing study to elucidate the potential target of the AR stain. Once the vast differences in bacillary locations in the different animal models were recorded, we studied the effect of drug treatment on the clearance of bacilli in these different locations in the lungs using the new fluorescent acid-fast AR stain combined with hematoxylin QS and DAPI. Results showed that for both mouse models the reduction of the AR+ bacilli after drug treatment was largely homogenous across the lesion. Necrosis was never seen in the lungs of the drug-treated GKO mice as the onset of necrosis only occurs late in infection after treatment has initiated. In the unorganized lesion structure of the GKO mice, a very rapid clearance of AR+ bacilli was observed, presumably by the large number of phagocytic cells and macrophages present. Therefore, the AR staining method can in fact be used for the GKO model as a readout for drug efficacy. In immunocompetent mice, clearance of the AR+ bacilli occurs slower . After drug treatment, the clearance of bacilli is homogenous across the lesions; the remaining bacilli are intracellular, either in epithelioid mFs within the LC cuff or in foamy mFs located outside the LC cuff. Although difficult to quantify, a slight trend was observed of relatively more bacilli remaining in the foamy mFs versus the epithelioid mFs after drug treatment. Whether this is due to slower clearance of dead bacilli by the various macrophage celltypes, or to the bacilli in the foamy mFs being less responsive to drugs has to be further investigated. Interestingly, the foamy mFs have more recently been described as being the key participants in both sustaining persistent bacteria and contributing to tissue pathology that leads to cavitation and the release of infectious bacilli [65– 67,71,72]. Cardona et al. hypothesize this foamy macrophage population to be dynamic and drain from the alveolar spaces resulting in dissemination and generating intragranulomatous necrosis [72]. In the guinea pig model of M. tuberculosis infection, drug treatment reduced the bacterial load in the lungs heterogeneously over the different lung lesion types. While drug treatment completely reversed lung inflammation associated with secondary lesions relatively quickly, the resolution of the primary granulomas required more intensive treatment regimens [45,53]. These results are in keeping with classical studies by Smith et al., who showed that INH, RIF and PZA chemotherapy sterilized secondary lesions but had far less effect on the primary lesions [73]. None of the earlier studies, however, described the location of the bacilli in the lung. We observed that six weeks of INH treatment of M. tuberculosis guinea pigs only had a moderate effect on the AR+ bacilli visualized mainly present in the primary granulomas, whereas in the guinea pigs treated with TMC207 for 6 weeks an Visualization of M. tuberculosis in Animal Models almost complete eradication of AR+ bacteria in both lesion types was observed. The clearance of the bacteria in the necrotic lesions is generally very slow, and therefore AR staining may not be a good alternative to CFU enumeration for evaluating drug efficacy in guinea pigs, unless the bacteria are lysed after drug treatment (which is in fact observed after TMC207 treatment in vitro, personal communication, K. Andries). The few remaining AR+ bacilli after TMC207 treatment were found extracellular, in the core of the primary lesion, a microenvironment of residual primary lesion necrosis. These observations further highlight the importance of a TB drug to eradicate the persisting, extracellular bacteria remaining within the necrotic lesions. Although acid-fast stains have been used for diagnostic purposes for decades, the exact cellular component of M. tuberculosis recognized by the dyes is still being debated. Fuchsin, the main component of Ziehl-Neelsen and Kinyoun acid-fast stain, has been shown to stain the vastly complex lipid portion of the mycobacterial cell wall. However, less is known about the target of the combined auramine O-rhodamine B stain. Auramine O is believed to bind to mycolic acids and nucleic acids [74–76]. Although rhodamine B has been used in numerous M. tuberculosis studies, the exact staining target of M. tuberculosis has yet to be elucidated [74]. Preliminary studies in our laboratory show that rhodamine B alone stains M. tuberculosis quite readily in culture and tissue sections and also stains purified cell wall components (data not shown). To understand what bacterial population is visualized with the AR staining method in this study, we need to keep two characteristics of the stain in mind. First, the AR stain might not visualize all bacterial populations present in the lesion, we might not see the earlier described acid-fast negative bacterial subpop- ulation(s) [77,78]. Recently, we also reported on an acid-fast- negative but immunofluorescence-positive M. tuberculosis popula- tion in vitro as well as in vivo [12]. Secondly, the stain cannot differentiate between live and recently killed, intact [and not yet cleared] tubercle bacilli. Despite these limitations, the purpose of the combined staining method was to illustrate the differences in locations of the AR+ bacilli and their clearance (or lysis) caused by drug treatment, throughout entire lung sections across our animal models. To understand the interaction between immunology, pathology and the location and state of the M. tuberculosis bacillus, the role of the immune response was studied by comparing single drug treatment effects in the immunocompromised GKO mice versus the immunocompetent C57BL/6 mice. The immune response is generally believed to be favorable to the host by containing and aiding in the eradication of bacilli due to the induction of protective cytokines and other immune molecules [6,79]. From the perspective of TB therapeutics however, the immune response appears to be an unfavorable response in that it renders the bacilli less responsive to drugs, as is shown by the results described here. Drug activity was found to be far more pronounced for all compounds evaluated in the GKO mice when compared to the immunocompetent C57BL/6 mice. Drugs administered were INH, which is mainly active against replicating bacilli [68], as well as RIF and/or quinolones (MXF, GATI), which are known to be effective against replicating bacteria as well as sterilizing compounds [80,81]. This result is not entirely surprising because the bacilli are actively replicating in the GKO mice without the interference of adaptive immunity of the host. In fact, M. tuberculosis bacteria in GKO mice replicate about once a day, which is the same replication rate as in vitro cultures. However, drugs showed far less activity in the C57BL/6 mice with any given compound when compared to the GKO mice. Earlier papers have elegantly shown that bacteria are slowing their replication rate in PLoS ONE | www.plosone.org 10 March 2011 | Volume 6 | Issue 3 | e17550