Identification of the resistant mechanisms, particularly a novel mechanism, is
important for the development of surrogate markers that can be combined with other known resistance determinants to improve the rapid detection of drug-resistant M. tuberculosis strains. Methods Mycobacterial strains and culture conditions Mycobacterium tuberculosis clinical strains (one strain per Belinostat mouse patient) were obtained from the Drug-Resistant Tuberculosis Research Laboratory, Drug-Resistant Tuberculosis Research Fund, Siriraj Foundation, Faculty of Medicine Siriraj Hospital, Mahidol University. They were isolated between 2004 and 2011 from new and previously treated patients with both known and unknown HIV status. This study was approved by the Siriraj Ethics Committee, Mahidol University, Bangkok, Thailand (Certificate of Approval No. Si 208/2005). The mycobacteria were cultured on Löwenstein-Jensen (LJ) medium (BBL, Epigenetics Compound Library USA) and incubated
buy Poziotinib at 37°C for 3-4 weeks. Species identification and antimycobacterial susceptibility testing were performed using in-house one-tube multiplex PCR  and the standard proportion method [40, 41], respectively. Isolation of genomic DNA One loop of mycobacterial cells grown on solid medium was scraped and suspended in 500 μl of TE buffer (10 mM Tris-HCl (pH8.0), 1 mM EDTA). The cells were inactivated by heating at 80°C for 20 min and subsequently harvested by centrifugation at 6,000xg at 4°C for 10 min. The cells were resuspended in 400 μl of Tris-EDTA-Tween-lysozyme solution (10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.5% (v/v) Tween 80, 2 mg/ml lysozyme (Amresco, USA)), and the mixtures were then incubated at 37°C for 3 h. SDS and proteinase K were added to the cell suspension to generate final concentrations of 1% (w/v) and 1 mg/ml, respectively, prior to incubation at 37°C for 1 h. Then, 80 μl of 5 M NaCl and 80 μl of 10% (w/v) cetyl trimethyl ammonium bromide (CTAB) (Sigma, USA) were added to the suspension, and the suspension was immediately heated at 65°C for 15 min. An equal volume of chloroform-isoamyl
L-NAME HCl alcohol (24:1) (v/v) was added to the suspension. The aqueous DNA phase was separated by centrifugation at 12,000xg for 5 min and mixed again with an equal volume of chloroform-isoamyl alcohol (24:1) (v/v). DNA was precipitated by adding 0.1 volume of 3 M sodium acetate (pH 5.3) and 2.5 volumes of ice-chilled absolute ethanol, followed by incubation at -70°C for 30 min. DNA was separated by centrifugation at 12,000xg at 4°C for 15 min. Total nucleic acid was washed once with 500 μl of ice-chilled 70% ethanol, dried, and resuspended in 20 μl of TE buffer. RNaseA (Qiagen, Germany) was added to the total nucleic acid solution to generate a final concentration of 0.5 μg/μl, and the tube was subsequently incubated at 37°C for 1 h.