The susceptibility was determined according to the breakpoints recommended by the Clinical and Laboratory Standards

Institute (CLSI) (23). Two differently sized products were amplified by PCR using the ermF-ermR1 primer set. Specifically, the PCR products amplified using the template DNA from M. abscessus and M. bolletii had a length of 673 bp. However, the erm(41) DNAs amplified from M. massiliense isolates were much smaller (397 bp) than those of the other two species (Fig. 1), from which deletion was assumed by PCR only FK228 in vitro without any sequence analysis of the single M. massiliense isolate (16). These findings were consistently observed in all of the clinical isolates and type strains evaluated in this study. This enabled us to use the erm(41) PCR for the simple differentiation method of M. massiliense from M. abscessus and M. bolletii. All of the M. massiliense strains were clearly

distinguished from M. abscessus and M. bolletii. Interestingly, two clinical isolates were further confirmed to be M. massiliense simply by erm(41) PCR, when they were originally identified by additional sequence analysis of sodA and 16S-23S ITS after see more the discordant results from sequence analysis of rpoB and hsp65. They had the typical erm(41) sequence of M. massiliense. In addition, no amplicon was produced when PCR was conducted using a template DNA from M. chelonae. When the nucleotide sequences of M. massiliense, M. bolletii and M. abscessus were compared, the erm(41) sequences (522 bp) of M. abscessus and M. bolletii showed higher than 98.3%

similarity. However, even though M. massiliense is closely related to these two species, the sequence of its erm(41) contained only 246 nucleotides due to two deletions (Fig. 2a). Because of polymorphic nucleotides Amylase in the M. abscessus (11 of 522 nucleotides) and M. massiliense (two of 246 nucleotides) erm(41) sequences (Fig. 2b, c), intra-species similarities of these two species were 98.7–100% and 99.2–100%, respectively. Furthermore, a variation of either A (61.2%) or G (38.8%) was found in the first nucleotide of the 64th codon (466th nucleotide of 156th codon in M. abscessus numbering) in the M. massiliense isolates. Specifically, the type strain of M. massiliense had A, whereas all M. abscessus and M. bolletii had G at this site. When compared to M. abscessus and M. bolletii, M. massiliense isolates contained two deletion sites on the basis of aligned sequences. These two deletions of M. massiliense were equivalent to those of the erm(41) deletion mutant of M. abscessus (GenBank accession no. EU590128). In addition, the T28C transition of erm(41), referred by Nash et al. (16), was detected in erm(41) of M. abscessus and M. bolletii isolates (7/48, 14.6%). However, none of the M. massiliense isolates had the T28C transition of erm(41) (0/49, 0%). On the basis of erm(41) sequences, 49 clinical isolates of M. massiliense were separated into two possible clonal groups.