Location of Intra- and Extracellular M. tuberculosis Populations in Lungs of Mice and Guinea Pigs during Disease Progression and after Drug Treatment Donald R. Hoff1, Gavin J. Ryan1, Emily R. Driver1, Cornelius C. Ssemakulu2, Mary A. De Groote1, Randall J. Basaraba1, Anne J. Lenaerts1* 1 Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, Colorado, United States of America, 2 Council for Scientific and Industrial Research (CSIR), CSIR Biosciences, Pretoria, South Africa 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. Citation: Hoff DR, Ryan GJ, Driver ER, Ssemakulu CC, De Groote MA, et al. (2011) Location of Intra- and Extracellular M. tuberculosis Populations in Lungs of Mice and Guinea Pigs during Disease Progression and after Drug Treatment. PLoS ONE 6(3): e17550. doi:10.1371/journal.pone.0017550 Editor: Ludovic Tailleux, Institut Pasteur, France Received October 11, 2010; Accepted February 8, 2011; Published March 21, 2011 Copyright: 2011 Hoff et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Support was provided by the National Institutes of Health (NIH) grant RO-1 AI061505-01A1 and the NIH Contracts NO1 AI-95385 at Colorado State University, Fort Collins, and N01-AI-95364 at the Southern Research Institute, Birmingham, AL. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: lenaerts@colostate.edu Introduction The standard regimen for tuberculosis (TB) requires 6–9 months of daily, multidrug therapy to achieve sterilization without relapse [1,2]. The lengthy treatment regimen is thought to be necessary to eradicate a small sub-population of M. tuberculosis bacilli that persist in the face of drug pressure [3]. The ability of M. tuberculosis to adapt to a changing environment and persist in certain locations of the host despite a vigorous adaptive immune response, likely contributes to the difficulty in curing TB with antimicrobial drugs [3–6]. Several in vitro studies to date have shown that the environment surrounding M. tuberculosis can alter the metabolic state and replication rate of the bacilli thereby rendering them refractory to drug treatment [7–11]. In contrast to the controlled conditions of culture environments in vitro, very little information is available on the far more complicated in vivo conditions of the bacteria such as in human lung lesions [5]. We previously described that M. tuberculosis exists as multiple populations in most in vitro and in vivo conditions, even within a seemingly single microenvironment [12]. More understanding of the bacterial location in the lung throughout disease progression and the bacterial in vivo microenvironment is needed to elucidate the host-pathogen interaction further. Human TB infected lungs generally show a heterogeneity of lung lesion types often with necrosis in the center and surrounded by a peripheral rim of fibrosis [13]. A better understanding of tuberculosis lesion pathogenesis is emerging from evaluating animal models that demonstrate diverse in vivo responses to experimental infections. Animal infection models are essential to vaccine and drug development by generating the data that are a prerequisite for clinical trials. The standard laboratory mouse models have provided critical information on the efficacy of a novel compound in TB drug development as well as on safety and pharmacokinetics [14–17]. The drawback of the standard mouse infection models for tuberculosis is their lack of advanced lesion types as the progression of disease rarely reaches the stages of extensive necrosis and calcification in the lungs [18]. Numerous studies have demonstrated that the standard laboratory mouse fails PLoS ONE | www.plosone.org 1 March 2011 | Volume 6 | Issue 3 | e17550