GenoType NTM-DR for Identifying Mycobacterium abscessus Subspecies and Determining Molecular Resistance

Abstract

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...

Evaluation of the GenoType® NTM DR for Subspecies Identification and Determination of Drug Resistance in Clinical M. abscessus Isolates

“…GenoType® NTM DR showed concordance with pDST in 94% for Clarithromycin resistance and in 94% for Amikacin resistance, improving to 98% and 96%, respectively, after repeat pDST. Similar results have recently been published by Kehrmann et al [18] and Mougari et al [19], however discordance of subspecies identification or resistance results did not take into account WGS data.…”

Whole-Genome Sequencing for Predicting Clarithromycin Resistance in Mycobacterium abscessus

“…All isolates which had any mutation of position 2269, 2270, or 2271 (Escherichia coli numbering, positions 2057, 2058, and 2059, respectively) in rrl were resistant to clarithromycin ( We did not observe any isolates with an rrl mutation which also harbored a T28C mutation in erm (41). Where this occurred in isolates reported in the literature, they were always resistant (15,16). Of 37 isolates with a T28C mutation in erm (41) and no other relevant mutations, 84% (31/37) were susceptible to clarithromycin, 11% (4/31) had intermediate susceptibility, and 5% (2/31) were resistant.…”

Whole genome sequencing for predicting clarithromycin resistance in Mycobacterium abscessus

“…http://dx.doi.org/10.1101/251918 doi: bioRxiv preprint first posted online Jan. 24, 2018; C1409T and G1491T were reported as causing resistance. [18][19][20] One article described rpsL as being important for aminoglycoside resistance in M. abscessus , but no specific SNPs were identified by the authors. 21 For ciprofloxacin, mutations in gyrA amino acid positions 92 and 96 and gyrB position 492 ( M. abscessus numbering) were associated with resistance.…”

“…Interestingly a recent study showed the Hain GenoType NTM-DR line probe assay incorrectly predicted subspecies in 8% of samples, presumably because it lacks the whole genome resolution provided by sequencing and is vulnerable to between species recombination. 18 Despite analysing all mutations occurring in erm(41) and rrl for the full collection of genomes, we were unable to predict all clarithromycin resistance. This may be because there are other genes implicated or due to unreliable DST results.…”