immunocompetent mice due to the immune response and show a changed metabolism in the chronic disease state in mice [11,36,82]. The results show an advantage of the GKO model as a first in vivo screen to assess potential in vivo activity. The high numbers of actively replicating bacilli in GKO mice make for a large dynamic window when testing novel experimental com- pounds for in vivo activity. The findings presented in this paper have at least three important implications for TB drug development. First, it is important in TB drug development to realize that the majority of the bacilli in an advanced disease state are extracellular organisms located in necrotic lesions, as is also seen in human cavitary disease [83]. Most mouse models are modeling activity of single compounds or drug regimens against intracellular bacteria, and because of this limitation other animal models with a more progressive pathology are required to evaluate drug efficacy against potentially persistent, extracellular bacilli in hypoxic, necrotic lesions. A second implication is that a decrease in inflammation might not be a good measure of antibactericidal activity. Recently, different imaging methods have been intro- duced as methods to follow the effect of TB drug therapy in real time such as PETscan (which requires the uptake of radio-labeled glucose by actively metabolizing cells) and CATscan based imaging modalities [21,63]. Our results show that rapid resolution of non-necrotic inflammatory lesions (such as the secondary lesion in the guinea pig model) might not adequately reflect the bactericidal activity of a drug as these lesions only contain very few bacilli. And thirdly, the immune response as well as the rapid structural organization of the tuberculous granuloma is usually seen as a favorable host response as it contains bacilli locally, thus preventing the progression of the disease. However, the immune response and its effect on lung pathology may decrease the drug responsiveness of the bacilli. In the context of drug therapy, the necrotic granuloma may in addition also present a physical barrier to effective treatment and to the host immune response. Therapies aiming at preventing and minimizing necrosis may be beneficial in eliminating bacilli that persist in necrotic lesions in the face of standard TB therapy. In summary, the differences in host immune response among the different animal models infected with M. tuberculosis result in a wide variety of granulomatous lesion types which will eventually determine efficacy of a TB drug regimen. Increasing our understanding on lesion morphologies as well as the location of bacilli among the different animal models is vital to designing different levels of stringency for testing new drugs. Materials and Methods Ethics Statement All experimental protocols were approved with written consent by the Animal Care Use Committee of Colorado State University (approval numbers ACUC # 04-302A-06 and ACUC # 06- 221A-03) which abides by the USDA Animal Welfare Act and the Public Health Service Policy on Humane Care and Use of Laboratory Animals. Bacterial Isolates The virulent M. tuberculosis Erdman strain (TMC 107; ATCC 35801) has been used as a standard for drug evaluations in mice in our laboratory, and the strain was propagated as previously described [14,44]. Briefly, M. tuberculosis Erdman was grown to mid-log phase in Proskauer-Beck Medium containing 0.05% Tween 80 (Sigma Chemical Co., St. Louis, MO) and stored in vials frozen at 270uC until use. Visualization of M. tuberculosis in Animal Models The strain M. tuberculosis H37Rv (Trudeau Institute, Saranac Lake, NY) has been used as a standard in our laboratory for guinea pig infection studies [23,84]. M. tuberculosis H37Rv was grown from low passage seed lots in Proskauer-Beck liquid medium containing 0.05% Tween 80 to early mid-log phase and frozen in aliquots at 270uC until needed. Chemicals and drugs Isoniazid (INH), rifampin (RIF) and ethambutol (EMB) were obtained from Sigma Chemical Co. (St. Louis, MO). Moxifloxacin (MXF) and gatifloxacin (GAT) were kindly provided by Southern Research Institute (SRI) (Birmingham, AL). All drugs, except for RIF, were dissolved in water. RIF was dissolved in 100% dimethyl sulfoxide (DMSO) prior to dilution in distilled water (5% final DMSO concentration). Drug formulations prepared in distilled water were prepared weekly and stored at 4uC. For the guinea pig studies, INH was dissolved in 40% (wt/vol) sucrose and administered in 1 ml per guinea pig. INH was prepared weekly in distilled sucrose water and stored at 4uC. TMC207 was kindly provided by Dr. K. Andries (Tibotec, Belgium) and was prepared in a hydroxypropyl-ß-cyclodextrin solution (CD) as described before [23]. Immunocompromised mouse TB infection model Several experiments using different lengths of treatment were performed,. Briefly, eight- to ten-week-old female specific pathogen- free, C57BL/6-Ifngtm1ts (GKO) mice were purchased (Jackson Laboratories, Bar Harbor, Maine). These mice have a disrupted interferon-c gene, which renders them highly susceptible to tuberculosis [47]. The standard infection and treatment protocol for this model was performed as extensively validated and previously described [43]. Mice were exposed to a low-dose aerosol infection with the M. tuberculosis strain Erdman ( TMC 107; ATCC 35801) in a Glas-Col inhalation exposure system (Glas-Col Inc., Terre Haute, IN) [14]. One day after low dose aerosol infection, three mice were euthanized to verify bacterial uptake of 50 to 100 CFU per mouse. Each treatment group consisted of 4–5 mice for every subsequent time point. Treatment was initiated 18 days after low dose aerosol infection (standard protocol) and lasted up to 28 days after aerosol infection. Untreated mice cannot succumb to disease 28–30 days after aerosol infection. INH was administered at 25 mg/kg, GAT and MXF at 100 mg/kg, and all drugs were administered via oral gavage for 7 days/week. One control group of untreated, infected mice was euthanized by CO2 inhalation at the start of treatment, and a second control group after the cessation of treatment. Mice were euthanized after 2, 5, 7, and 10, days after the start of treatment. Immunocompetent mouse TB infection model Several experiments using different lengths of treatment were performed and the protocols are described below. Six- to 8-week- old female specific pathogen-free immunocompetent C57BL/6 mice (Charles River, Wilmington, MA) were infected via a low dose aerosol exposure to M. tuberculosis Erdman. The standard infection and treatment protocol for this model were performed as previously described [14,44,85]. Three mice were euthanized one day post low dose aerosol to verify bacterial uptake of 50 to 100 CFU per mouse. For the short term treatment experiment, drug treatment started three weeks after low dose aerosol infection (standard protocol) and lasted for 7 days. INH was administered at 25 mg/ kg, EMB at 150 mg/kg, MXF at 100 mg/kg and RIF at 10 mg/ kg, and drugs were administered via oral gavage for 7 days. PLoS ONE | www.plosone.org 11 March 2011 | Volume 6 | Issue 3 | e17550