H37rv atcc 27294

Virulent Mtb strain H37Rv ATCC 27294 and the avirulent strain H37Ra ATCC 25177 were purchased from American Type Culture Collection (ATCC, Manassas, VA). All mycobacteria were provided from the International Tuberculosis Research Center (ITRC, Changwon, Gyeongsangnam-do, South Korea). These strains were cultured in Middlebrook 7H9 broth (Difco Laboratories, Detroit, MI) supplemented with 0.02% glycerol and 10% (vol/vol) oleic acid-albumin-dextrose-catalase (OADC, Becton Dickinson, Sparks, MD) for 25–28 days at 37°C. Age- and sex-matched C57BL/6 mice were infected with Mtb H37Ra, and the mycobacteria preparation protocol was performed as previously described [29 (link)]. Briefly, 6-week-old mice per group were intravenous initial infectious dose with 10⁷ CFU Mtb H37Ra. The infected mice were euthanized at 6 weeks after infection to analyze immune responses. CD4⁺ T-cells were isolated from the spleens of H37Ra-infected mice using a MACS column.

M. tuberculosis H37Rv (ATCC 27294) (American Type Culture Collection, Manassas, VA, USA) and clinical strains provided and characterized by the TB National Reference Laboratory from the Portuguese National Institute of Health Dr. Ricardo Jorge (INSA) were grown in Middlebrook’s 7H9 medium supplemented with 10% OADC enrichment (BD Difco, Franklin Lakes, NJ, USA), 0.02% glycerol, and 0.05% tyloxapol (Merck, KGaA, Darmstadt, Germany) at 37 °C. The clinical strain (INSA code 33427) is susceptible to streptomycin, isoniazid, rifampicin, and pyrazinamide; the MDR strain (INSA code 34192) is resistant to all those antibiotics in addition to ethionamide. The XDR strain (INSA code 163761) is resistant to all of the previous named antibiotics, and, additionally, to amikacin, kanamycin, capreomycin, moxifloxacin, and ofloxacin. All experimental procedures using live Mtb strains were performed in the biosafety level 3 laboratory at the Faculty of Pharmacy of the University of Lisbon, maintaining the national and European containment level 3 laboratory management and biosecurity standards based on applicable EU directives. The faculty’s biological safety committee has also approved all those procedures.

H37Ra (ATCC 35835) and H37Rv (ATCC 27294) strains of M. tuberculosis were purchased from the American Type Culture Collection (ATCC, Manassas, Virginia, United States of America). Clinically isolated MDR (KMRC 00116–00023, KMRC 00116–00072, KMRC 00116–00082, KMRC 00116–00087, KMRC 00116–00107, KMRC 00116–00111, KMRC 00116–00122, KMRC 00116–00150, KMRC 00116–00181, KMRC 00116–00185, KMRC 00116–00232), XDR (KMRC 00203–00197, KMRC 00203–00052, KMRC 00203–00060, KMRC 00203–00063, KMRC 00203–00085, KMRC 00203–00092, KMRC 00203–00117, KMRC 00203–00128, KMRC 00203–00169), and TDR (KMRC 00203–00005, KMRC 00203–00051, KMRC 00203–00151) strains were purchased from the Korean Microorganism Resource Center (KMRC) (Chungbuk, Korea). Clinically isolated TDR strains were found to be resistant to kanamycin (30 μg/ml), capreomycin (40 μg/ml), prothionamide (40 μg/ml), cycloserine (30 μg/ml), para-aminosalicylic acid (1 μg/ml), ofloxacin (4 μg/ml), rifabutin (20 μg/ml), moxifloxacin (2 μg/ml), amikacin (30 μg/ml), and levofloxacin (2 μg/ml) by the absolute concentration method using Lowenstein–Jensen medium.