Groups of 5 mice were euthanized after 0, 2, 5 and 7 days of drug treatment. For the long term treatment experiment, drug treatment was started 3 weeks post-infection and continued for 12 weeks. Five infected mice were euthanized at the start of treatment as pretreatment controls. INH was administered at 25 mg/kg and RIF at 20 mg/kg, 5 days per week via oral gavage. Mice were euthanized at 2, 6, and 12 weeks after the start of treatment. Guinea pig TB infection model Four to five month-old, female Hartley guinea pigs (Charles River, Wilmington, MA) weighing approximately 500 g each were exposed to a low-dose aerosol of M. tuberculosis in a Madison aerosol chamber device. Our standard infection and treatment protocol for guinea pigs was followed as previously described [50,86]. Guinea pigs were delivered a low inoculum resulting in approximately 20–30 lesions in the lungs. At 30 days post- infection, 5 guinea pigs were euthanized to determine the bacterial load at the start of treatment. Each guinea pig was drug treated by administering each dose in the back of the mouth (5 guinea pigs per group). Control groups received daily oral administration of 1 ml of 40% (wt/vol) sucrose. Drug treated groups were administered INH at 30 mg/kg for 5 days per week, or TMC207 at 15 mg/kg. Earlier pharmacokinetic data using validated HPLC assays yielded an AUC 0–24 of 18.22 hr*mg/ ml for INH at 30 mg/kg [45]. Our earlier in vivo efficacy data on TMC207 in the guinea pig model showed the 15 mg/kg dose as a safe and highly efficacious dose [23]. Bacterial enumeration on agar plates Mice were euthanized by CO2 inhalation, and left lung lobes were aseptically removed and disrupted in a tissue homogenizer as previously described [44]. The number of viable organisms was determined by plating serial dilutions on nutrient Middlebrook 7H11 agar plates (GIBCO BRL, Gaithersburg, MD) containing cycloheximide at 10 mg/mL and carbenicillin at 50 mg/mL. Guinea pigs were euthanized by sodium barbital injection (Sleepaway; Fort Dodge Laboratories), and organs were aseptically removed and plated as previously described [23]. Statistical analysis to assess the significance of treatment efficacy results on bacterial numbers remaining after drug treatment was performed for mouse models [44] and guinea pig models [23], as described before. Combined staining procedure to visualize bacteria with AR and surrounding tissue The caudal right lung lobe for mice and guinea pigs was infused in situ with 10% neutral-buffered formalin. Four micron thick paraffin sections were stained with TB Auramine-Rhodamine T as recommended by Becton-Dickinson (Sparks, MD), with modifica- tions in order to visualize bacteria in the surrounding lung tissue. A combination staining method was developed by using both auramine O and rhodamine B to detect acid fast bacteria (AR; Becton Dickinson), hematoxylin QS for staining tissue (HQS; Vector Laboratories, Inc., Burlingame, CA) and 496-diamidino-2- phenylindole dihydrochloride for staining nuclei (DAPI; Sigma Chemical Co., St. Louis, MO). Tissue sections were dewaxed in xylene and rehydrated through a graded alcohol series, then stained with TB Auramine-Rhodamine T for 30 min. After washing excess stain with ddH2O, slides were decolorized with TB Decolorizer TM (BD) until the stain appeared dissolved. Counterstaining was performed with hematoxylin QS for about 5 sec. After washing excess hematoxylin with ddH2O, slides were Visualization of M. tuberculosis in Animal Models stained for 15 min. with DAPI (200 ng/ml final concentration) and washed in ddH2O. Entire lung tissue sections were scored based on: 1) estimating the number of auramine-rhodamine positive (AR+) bacilli per tissue section in mice and accurately counting the AR+ bacilli in guinea pig tissues (numbers are presented as percentages), 2) bacillary appearance (individual or clustered), and 3) intra or extra-lesional presence. For mouse lung tissues, bacterial numbers were generated from 2 non-sequential lung lobe sections for every timepoint for five mice per treatment group. Scoring sheets were generated for every lung section with the number of lesions showing bacilli for every section and the approximate number of the bacilli per lesion for the C57BL/6 mice (tables not shown). For the GKO mouse model, the percentages of bacteria present per lesion versus the whole lesion was estimated due to the high bacillary burden. For guinea pig tissues, the actual bacterial numbers were counted from 2 non-sequential lung lobe sections from three guinea pigs from every treatment group (tables not shown), for the primary (necrotic) and secondary (inflammatory) lesions. The improvement of the pathology after drug treatment in the guinea pig lung was evaluated blindly by a veterinary pathologist by scoring the lung involvement of the inflammation, as described earlier [24,45]. Immunofluorescence Five micron sections of lung tissue were dewaxed in xylene and rehydrated through graded alcohols. Antigen retrieval was performed using the RetrieverTM 2100 which pressure cooks at 121uC for 15 minutes using Target Retrieval Buffer solution S3307 (DAKO, Carpinteria, California). Blocking was performed with 1% goat serum in PBS (Biomeda, Foster City, California) for 30 minutes. The slides were incubated at 4uC overnight with a monoclonal mouse anti-tubulin antibody (Cell Signaling Technol- ogy, Danvers, Massachusetts) and a rabbit polyclonal anti-TB whole cell lysate minus LAM (Antibody E293, CSU TB Vaccine Testing and Research Materials Contract, Colorado). Subsequent- ly, the slides were washed using PBS and the antibody was detected with an Alexafluor 488 labeled goat anti-mouse IgG and also an Alexafluor 568 labeled goat anti-rabbit IgG (Invitrogen, Carlsbad, California). The slides were washed in PBS, mounted with ProLongH Gold antifade reagent with DAPI (Invitrogen, Carlsbad, California) and photographed under a fluorescent microscope. Hypoxia staining with pimonidazole Pimonidazole staining was used as described before [19,23] to detect hypoxic regions in the lungs of GKO mice. Pimonidazole is a 2-nitroimidazole that is able to identify regions of hypoxia (,4 mM O2 saturation or O2 tensions of 10 mm Hg) in animal organs after injection. Pimonidazole forms protein adducts with thiol groups in cells adjacent to hypoxic regions and the adducts are detected with a monoclonal antibody. M. tuberculosis-infected GKO mice were injected intraperitoneally (i.p.) with pimonidazole hydrochloride (Chemicon, Hampshire, United Kingdom) at a dose of 60 mg/kg mouse body weight dissolved in 16 PBS at 1.5 h prior to sacrifice at days 15, 17, 20, 22, 25, and 29 after low dose aerosol infection. Five micron thick sections of paraformaldehyde- fixed, paraffin-embedded tissue were cut and mounted on slides for processing. Tissue sections were deparaffinized with xylene before performing antigen retrieval with pronase (Fisher Scientific, Schwerte, Germany) for 40 min. at 40uC. Endogenous peroxidase activity was reduced with 1% hydrogen peroxide in TBS for 20 min. at room temperature (RT) in the dark. The blocking reagents avidin D and biotin were then added to each slide for PLoS ONE | www.plosone.org 12 March 2011 | Volume 6 | Issue 3 | e17550