, 2007), and this activity appears to contribute to survival DAPT ic50 under starvation at the dormant stage of growth (Jackson et al., 1989; Deb et al., 2009). Here, we analysed the biochemical characteristics and their relationship to susceptibility to environmental stress, such as oxidative stress, nitrosative stresses and pH changes, among BCG substrains. Mycobacterium bovis BCG strains Australia (ATCC 35739), Birkhaug (ATCC 35731), Connaught (ATCC 35745), Danish (ATCC 35733), Glaxo (ATCC 35741), Mexico (ATCC 35738), Montreal (ATCC 35735), Pasteur (ATCC 35734), Phipps (ATCC 35744), Tice (ATCC 35743), Russia (ATCC 35740) and M. tuberculosis strain

H37Rv (ATCC 25618) were purchased from American Type Culture Collection (ATCC, Manassas, MK2206 VA). BCG-Moreau, M. bovis (JATA) and Mycobacterium smegmatis were provided by Dr M. Takahashi (The Research Institute of Tuberculosis Japan Anti-tuberculosis Association, Kiyose, Tokyo, Japan). BCG-Japan (Tokyo 172) was purchased from Japan BCG Laboratory (Kiyose, Tokyo, Japan). BCG-Sweden (vaccine

seed) was provided by Dr S. Yamamoto (Japan BCG Laboratory). Mycobacterium avium strains 724S and 2151SmO were kindly provided by Drs J. Inamine and E. Torsten (Colorado State University, Fort Collins, CO). Bacterial culture and freeze stocking were performed as reported by Hayashi et al. (2009). Tests for nitrate reduction, catalase, Tween 80 hydrolysis, urease, pyrazinamidase and resistance to thiophene 2-carboxylic acid hydrazide (TCH) were performed by standard procedures except as described below (Gangadharam & Jenkins, 1998). Nitrate

reduction was performed by the classical procedure with liquid reagent. Pyrazinamidase activity was tested Coproporphyrinogen III oxidase on Middlebrook 7H11 broth (BD, Franklin Lakes, NJ) instead of Dubos broth. Resistance to TCH was determined on solid Ogawa medium containing 1 or 10 μg mL−1 TCH. Niacin accumulation was detected using the Kyokuto Niacin Test (Kyokuto Pharmaceutical Industries, Tokyo, Japan) in accordance with the manufacturer’s instruction. Degradation of p-amino salicylate (PAS) was determined according to Tsukamura (1961). Mycobacterium tuberculosis, M. bovis, M. avium and M. smegmatis were used as controls. In the urease test, urease-deficient recombinant BCG (Mukai et al., 2008) was used as a negative control. The human monocytic cell line THP-1 (ATCC TIB202) was purchased from ATCC and maintained in RPMI 1640 medium containing 100 U mL−1 penicillin G and 5% heat-inactivated fetal bovine serum (FBS). THP-1 cells were stimulated with 10 nM phorbol 12-myristate 13-acetate (PMA; Wako Pure Chemical Industries, Osaka, Japan) for 24 h to be differentiated to macrophages. Cells were washed three times with culture medium and used for the assays. Bone marrow was isolated from the tibias and femurs of C57BL/6J female mice at 4–8 weeks of age. Bone marrow cells haemolysed in 0.