bWe studied the performance of a new line probe assay for identifying the subspecies and determining the macrolide and aminoglycoside resistance levels of 50 Mycobacterium abscessus isolates. Agreement of GenoType NTM-DR results with sequencing and phenotypic resistance results was 92% for subspecies identification and 98% for determining molecular and phenotypic resistance. The Mycobacterium abscessus complex is divided into three subspecies: M. abscessus subsp. abscessus, M. abscessus subsp. massiliense, and M. abscessus subsp. bolletii. Their differentiation is of clinical interest, because subspecies differ in antibiotic resistance and treatment response in M. abscessus lung disease (1, 2). Identification of the members of M. abscessus relies on sequencing of multiple genes (3, 4). Inducible macrolide resistance in M. abscessus is conferred by the presence of the inducible methylase Erm(41) (5, 6), whereas high-level clarithromycin resistance is attributed to mutations at position 2058 or 2059 in the peptidyltransferase-binding region of the 23S rRNA gene (rrl) (7). A single point mutation at position 1408 in rrs of the 16S rRNA gene is responsible for the high level of resistance against aminoglycosides, another category of first-line antibiotics (8).GenoType NTM-DR (Hain Lifescience, Nehren, Germany) is a new line probe assay that enables M. abscessus subspecies identification and the simultaneous determination of antibiotic resistance to macrolides and aminoglycosides of mutations at position 28 in erm(41) (5, 6), position 2058/2059 in rrl, and position 1408 in rrs. We studied the ability of this assay to characterize 50 M. abscessus isolates (28 M. abscessus subsp. abscessus isolates, 19 M. abscessus subsp. massiliense isolates, and 3 M. abscessus subsp. bolletii isolates). Furthermore, 4 Mycobacterium chelonae isolates were analyzed. M. chelonae is closely related to M. abscessus. The two species share the same biochemical features, have highly similar 16S rRNA sequences, and are frequently summarized as the M. chelonae/abscessus complex (9). Results of the GenoType NTM-DR assay were compared with the results of sequencing the hsp65, erm(41), rrl, rrs, and 16S rRNA genes, which were obtained previously (10) or sequenced within this study as described in reference 10. Additionally, molecular resistance results of all M. abscessus isolates were compared with results of phenotypic resistance to clarithromycin and amikacin published by Rueger et al. (10), who used the broth microdilution method in RAPMYCO Sensititre 96-well plates. According to the CLSI breakpoints, highlevel clarithromycin resistance was defined as an MIC of Ն8 g/ml on day 5, and inducible resistance was defined as an increase of the clarithromycin MIC from Յ2 g/ml on day 5 to Ն8 g/ml on day 14. Aminoglycoside resistance was defined as an amikacin MIC of Ն64 g/ml.Isolates were grown in mycobacterial growth indicator tube (MGIT) liquid medium (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) at 37°C. GenoType NTM-DR was performed...