Three type strains [M. abscessus (ATCC 19977T), M. massiliense (KCTC 19086T= CIP 108297T) and M. bolletii (KCTC 19281T= CIP 108541T)], and 101 M. abscessus-M. chelonae group clinical isolates (M. abscessus, 46; M. massiliense, 49; M. bolletii, two; and M. chelonae, four strains) were used in the present study. In addition to the 85 strains that were used in a previous report (7), 16 strains (Inje collection) were newly included. Mycobacteria were cultivated on Ogawa media or blood agar plates at 37°C under 5% CO2

for 4 days, after which they were subjected to clarithromycin susceptibility testing and sequence analysis. Total DNAs were extracted from cultured colonies using the bead beater-phenol extraction method (17) and used as templates for PCR. The following primer pairs were used: ermF (5′-GAC CGG GGC CTT CTT CGT GAT-3′) and ermR1 (5′-GAC TTC CCC GCA CCG selleck chemicals llc ATT CC-3′) for the whole erm(41) (GenBank accession No. CU458896) and primers 19 (5′-GTA GCG AAA TTC CTT GTC GG-3′) and 21 (5′-TTC CCG CTT AGA TGC TTT CAG-3′) for 23S rRNA gene (18). Template DNA (approximately 50 ng) and 20 pmol of each primer were added to a PCR mixture tube (AccuPower PCR PreMix; Bioneer, Daejeon, Korea) that contained 1 unit of Taq DNA polymerase, 250 μM deoxynucleotide triphosphate, 10 mM Tris-HCl (pH 8.3), 10 mM KCl, 1.5 mM MgCl2, and gel loading dye. The final volume was

then adjusted to 20 μl with distilled water, after which the reaction mixture DNA Damage inhibitor was amplified using a model 9700 Thermocycler (Perkin-Elmer Cetus, Norwalk, NJ, USA). The PCR products were purified using QIAEX II gel extraction Inositol monophosphatase 1 kits (Qiagen, Hilden, Germany), and were then sequenced directly using forward and reverse primers on an Applied Biosystems automated sequencer (model 377) using BigDye Terminator Cycle Sequencing kits (Applied Biosystems, Warrington, UK). Both strands were sequenced as a cross-check. The resultant 23S rRNA gene and erm(41) sequences were aligned using ClustalW in the MEGA 4.0 (19) and the sequence similarities were analyzed using MegAlign software (DNAStar, Madison, WI, USA) (20). Mycobacterium tuberculosis erm(37) and M. abscessus erm(41) were retrieved from the

GenBank and used to compare with newly determined sequences. The newly determined erm(41) sequences of M. massiliense (accession no. FJ358487 to FJ358490), M. bolletii (accession no. FJ358491), and M. abscessus (accession no. FJ358483 to FJ358486) were deposited in GenBank. M. abscessus (ATCC 19977T), M. massiliense (KCTC 19086T= CIP 108297T), and M. bolletii (KCTC 19281T= CIP 108541T), which are known for their susceptibility to clarithromycin, were used as controls. The MIC of clarithromycin were determined in microtiter plates (21) using the broth dilution method with slight modification as described previously (7). To prepare a stock solution, clarithromycin (Boryung, Seoul, Korea) was solubilized in distilled water with glacial acetic acid (2 μl/ml) (22).