Abstract
We 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.
TEXT
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, high-level clarithromycin resistance was defined as an MIC of ≥8 μg/ml on day 5, and inducible resistance was defined as an increase of the clarithromycin MIC from ≤2 μg/ml on day 5 to ≥8 μg/ml on day 14. Aminoglycoside resistance was defined as an amikacin MIC of ≥64 μg/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 according to the manufacturer's recommendations.
Of the 50 M. abscessus isolates studied, 46 (92%) exhibited subspecies identification results concordant with the results obtained by DNA sequencing (Table 1). The banding patterns of three isolates were not attributed to a subspecies, namely, of one M. abscessus subsp. abscessus and two M. abscessus subsp. massiliense isolates. DNA sequencing of the unidentified M. abscessus subsp. massiliense isolates did not reveal deletion at position 64/65 within erm(41); such a deletion is a typical feature of M. abscessus subsp. massiliense (5, 6). The unidentified M. abscessus subsp. abscessus isolate did not exhibit sequence abnormalities within the 16S rRNA, erm(41), or hsp65 gene amplicon. One other isolate, identified as M. abscessus subsp. abscessus by gene sequencing, was identified as M. abscessus subsp. bolletii by the GenoType NTM-DR.
TABLE 1.
Sequencing and Phenotypic Resistance Results of 50 Mycobacterium abscessus Isolatesa
| No. | Mycobacterium abscessus subspecies (sequencing) | Position 28 in erm(41) | Clarithromycin MIC (μg/ml) on day 5 | Clarithromycin MIC (μg/ml) on day 14 | rrl positions 2058/2059 | Amikacin MIC (μg/ml) on day 5 | rrs position 1408 |
|---|---|---|---|---|---|---|---|
| 1 | Abscessus | T (R) | 0.5 (S) | >16 (R) | AA | 4 (S) | A |
| 2 | Abscessus | T (R) | 2 (S) | >16 (R) | AA | 4 (S) | A |
| 3 | Abscessus | C (S) | 0.5 (S) | 2 (S) | AA | 16 (S) | A |
| 4 | Abscessus | C (S) | 0.25 (S) | 0.25 (S) | AA | 8 (S) | A |
| 5 | Abscessus | C (S) | >16 (R) | >16 (R) | CA (R) | >64 (R) | G (R) |
| 6 | Massiliense | T (S) | 0.06 (S) | 0.5 (S) | AA | 8 (S) | A |
| 7 | Massiliense | T (S) | >16 (R) | >16 (R) | AG (R) | >64 (R) | G (R) |
| 8 | Massiliense | T (S) | 0.25 (S) | 0.5 (S) | AA | 4 (S) | A |
| 9 | Massiliense | T (S) | 0.25 (S) | 1 (S) | AA | >64 (R) | G (R) |
| 10 | Abscessus | T (R) | 0.5 (S) | >16 (R) | AA | 4 (S) | A |
| 11 | Abscessus | T (R) | 0.25 (S) | >16 (R) | AA | 4 (S) | A |
| 12 | Massiliense | T (S) | >16 (R) | >16 (R) | CA (R) | >64 (R) | G (R) |
| 13 | Massiliense | T (S) | >16 (R) | >16 (R) | CA (R) | >64 (R) | G (R) |
| 14 | Massiliense | T (S) | 0.5 (S) | 1 (S) | AA | 16 (S) | A |
| 15 | Massiliense | T (S) | 0.5 (S) | 1 (S) | AA | 16 (S) | A |
| 16 | Massiliense | T (S) | 0.25 (S) | 0.5 (S) | AA | 16 (S) | A |
| 17 | Massiliense | T (S) | 0.25 (S) | 0.5 (S) | AA | 16 (S) | A |
| 18 | Abscessus | C (S) | 0.06 (S) | 0.12 (S) | AA | 8 (S) | A |
| 19 | Massiliense | T (S) | 0.5 (S) | 0.5 (S) | AA | 8 (S) | A |
| 20 | Abscessus | T (R) | 2 (S) | >16 (R) | AA | 16 (S) | A |
| 21 | Abscessus | T (R) | 0.5 (S) | >16 (R) | AA | 16 (S) | A |
| 22 | Abscessus | T (R) | >16 (R) | >16 (R) | TA (R) | 32 (I) | A |
| 23 | Abscessus | T (R) | 4 (I) | >16 (R) | AA | 16 (S) | A |
| 24 | Massiliense | T (S) | 0.25 (S) | 1 (S) | AA | 8 (S) | A |
| 25 | Massiliense | T (S) | 0.06 (S) | 0.5 (S) | AA | 8 (S) | A |
| 26 | Massiliense | T (S) | 0.25 (S) | 2 (S) | AA | 8 (S) | A |
| 27 | Massiliense | T (S) | 0.25 (S) | 0.25 (S) | AA | 8 (S) | A |
| 28 | Massiliense | T (S) | 1 (S) | 2 (S) | AA | 8 (S) | A |
| 29 | Abscessus | T (S) | 1 (S) | >16 (R) | AA | 32 (I) | A |
| 30 | Abscessus | T (R) | 0.5 (S) | >16 (R) | AA | 16 (S) | A |
| 31 | Abscessus | T (R) | 0.25 (S) | >16 (R) | AA | 8 (S) | A |
| 32 | Abscessus | T (R) | 1 (S) | >16 (R) | AA | >64 (R) | A |
| 33 | Abscessus | T (R) | 0.25 (S) | 16 (R) | AA | 4 (S) | A |
| 34 | Abscessus | T (R) | 1 (S) | >16 (R) | AA | 4 (S) | A |
| 35 | Abscessus | T (R) | 0.25 (S) | 16 (R) | AA | 8 (S) | A |
| 36 | Massiliense | T (S) | 0.25 (S) | 1 (S) | AA | 8 (S) | A |
| 37 | Abscessus | T (R) | 1 (S) | >16 (R) | AA | 32 (I) | A |
| 38 | Abscessus | C (S) | 0.12 (S) | 0.25 (S) | AA | 16 (S) | A |
| 39 | Abscessus | C (S) | 0.25 (S) | 1 (S) | AA | 8 (S) | A |
| 40 | Massiliense | T (S) | 0.12 (S) | 0.25 (S) | AA | 16 (S) | A |
| 41 | Abscessus | T (R) | 0.5 (S) | >16 (R) | AA | 8 (S) | A |
| 42 | Massiliense | T (S) | >16 (R) | >16 (R) | CA (R) | 8 (S) | A |
| 43 | Abscessus | T (R) | 0.5 (S) | >16 (R) | AA | 32 (I) | A |
| 44 | Abscessus | T (R) | 2 (S) | >16 (R) | AA | 32 (I) | A |
| 45 | Bolletii | T (R) | 8 (R) | >16 (R) | AA | 16 (S) | A |
| 46 | Bolletii | T (R) | 4 (I) | >16 (R) | AA | 16 (S) | A |
| 47 | Abscessus | C (S) | >16 (R) | >16 (R) | AG (R) | >64 (R) | G (R) |
| 48 | Abscessus | T (R) | >16 (R) | >16 (R) | GA (R) | 16 (S) | A |
| 49 | Bolletii | T (R) | 16 (R) | >16 (R) | AA | 16 (S) | A |
| 50 | Abscessus | C (S) | >16 (R) | >16 (R) | AG (R) | >64 (R) | G (R) |
Disagreements with GenoType NTM-DR results are marked in bold. One M. abscessus subsp. abscessus isolate was identified as M. abscessus subsp. bolletii by the GenoType NTM-DR. The three other disagreeing isolates did not yield species-specific band patterns in the GenoType NTM-DR. R, resistant; S, susceptible; I, intermediate; C, cytosine; T, thymine; A, adenine; G, guanine.
GenoType NTM-DR results matched 100% (50/50) of the erm(41) and rrs sequencing results and 98% (49/50) of the rrl sequencing results (Table 1). Overall, 9 isolates exhibited mutations within rrl, and 7 isolates exhibited mutations within rrs. One M. abscessus subsp. abscessus isolate exhibited a mutation in rrl that revealed neither a mutation band nor a wild-type band, a finding indicating the existence of a resistance mechanism not detected by the line probe assay.
The four M. chelonae isolates were correctly identified by the GenoType NTM-DR, and resistance results correlated with the sequencing results of rrs and rrl, which all exhibited a wild-type sequence (data not shown). erm(41) is absent in M. chelonae (5).
Phenotypic high-level clarithromycin resistance testing of the 50 M. abscessus isolates showed congruence with the molecular methods (Table 1). The 9 isolates, which had been tested resistant by rrl sequencing, exhibited phenotypic high-level resistance to clarithromycin (MICs > 16 μg/ml) after 5 days.
Phenotypic inducible clarithromycin resistance was detected by increases in the clarithromycin MICs from day 5 to day 14 in 18 M. abscessus subsp. abscessus isolates. These results are in line with the results of molecular testing by the GenoType NTM-DR and sequencing for these isolates. Two more M. abscessus subsp. abscessus isolates exhibited inducible and high-level clarithromycin resistance. A clarithromycin MIC of >16 μg/ml on day 5, explained by high-level clarithromycin resistance, masked the inducible resistance in these isolates. No inducible phenotypic macrolide resistance was found in M. abscessus subsp. massiliense, which is explained by the lack of a functional erm(41) gene (11). A thymine at position 28 is not accompanied by inducible clarithromycin resistance in this subspecies. Three M. abscessus subsp. bolletii isolates exhibited induced clarithromycin resistance but not high-level macrolide resistance, as determined by sequencing and the GenoType NTM-DR. These isolates showed MICs of 4–16 μg/μl on day 5 and >16 μg/ml on day 14, indicating a faster induction of resistance. Of the 50 M. abscessus isolates, 8 isolates exhibited phenotypic aminoglycoside resistance (3 M. abscessus subsp. abscessus and 5 M. abscessus subsp. massiliense isolates) with an amikacin MIC of >64 μg/ml. Seven of these isolates had been identified to be aminoglycoside resistant by the GenoType NTM-DR and sequencing of rrs position 1408. One isolate with an amikacin MIC of >64 μg/ml exhibited mutation at neither position 1408 nor positions 1406, 1409, or 1491 of rrs, which have been reported to cause aminoglycoside resistance in M. abscessus species (12). To summarize, the GenoType NTM-DR showed 98% correlation with molecular and phenotypic results for determining clarithromycin and aminoglycoside resistance. Phenotypic amikacin resistance of one M. abscessus subsp. abscessus isolate was not identified by the GenoType NTM-DR.
To the best of our knowledge, this is the first study to have evaluated a line probe assay for the simultaneous determination of M. abscessus subspecies and resistance to clarithromycin and aminoglycosides. The GenoType NTM-DR is a valuable test for characterizing most clinical M. abscessus isolates.
ACKNOWLEDGMENTS
Hain Lifescience reduced the price of the kits used in this study.
We declare no conflicts of interest.
REFERENCES
- 1.Jeon K, Kwon OJ, Lee NY, Kim BJ, Kook YH, Lee SH, Park YK, Kim CK, Koh WJ. 2009. Antibiotic treatment of Mycobacterium abscessus lung disease: a retrospective analysis of 65 patients. Am J Respir Crit Care Med 180:896–902. doi: 10.1164/rccm.200905-0704OC. [DOI] [PubMed] [Google Scholar]
- 2.Koh WJ, Jeon K, Lee NY, Kim BJ, Kook YH, Lee SH, Park YK, Kim CK, Shin SJ, Huitt GA, Daley CL, Kwon OJ. 2011. Clinical significance of differentiation of Mycobacterium massiliense from Mycobacterium abscessus. Am J Respir Crit Care Med 183:405–410. doi: 10.1164/rccm.201003-0395OC. [DOI] [PubMed] [Google Scholar]
- 3.Adekambi T, Berger P, Raoult D, Drancourt M. 2006. rpoB gene sequence-based characterization of emerging non-tuberculous mycobacteria with descriptions of Mycobacterium bolletii sp. nov., Mycobacterium phocaicum sp. nov. and Mycobacterium aubagnense sp. nov. Int J Syst Evol Microbiol 56:133–143. doi: 10.1099/ijs.0.63969-0. [DOI] [PubMed] [Google Scholar]
- 4.Adekambi T, Reynaud-Gaubert M, Greub G, Gevaudan MJ, La Scola B, Raoult D, Drancourt M. 2004. Amoebal coculture of “Mycobacterium massiliense” sp. nov. from the sputum of a patient with hemoptoic pneumonia. J Clin Microbiol 42:5493–5501. doi: 10.1128/JCM.42.12.5493-5501.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Nash KA, Brown-Elliott BA, Wallace RJ Jr. 2009. A novel gene, erm(41), confers inducible macrolide resistance to clinical isolates of Mycobacterium abscessus but is absent from Mycobacterium chelonae. Antimicrob Agents Chemother 53:1367–1376. doi: 10.1128/AAC.01275-08. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Bastian S, Veziris N, Roux AL, Brossier F, Gaillard JL, Jarlier V, Cambau E. 2011. Assessment of clarithromycin susceptibility in strains belonging to the Mycobacterium abscessus group by erm(41) and rrl sequencing. Antimicrob Agents Chemother 55:775–781. doi: 10.1128/AAC.00861-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Brown-Elliott BA, Nash KA, Wallace RJ Jr. 2012. Antimicrobial susceptibility testing, drug resistance mechanisms, and therapy of infections with nontuberculous mycobacteria. Clin Microbiol Rev 25:545–582. doi: 10.1128/CMR.05030-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Prammananan T, Sander P, Brown BA, Frischkorn K, Onyi GO, Zhang Y, Bottger EC, Wallace RJ Jr. 1998. A single 16S ribosomal RNA substitution is responsible for resistance to amikacin and other 2-deoxystreptamine aminoglycosides in Mycobacterium abscessus and Mycobacterium chelonae. J Infect Dis 177:1573–1581. doi: 10.1086/515328. [DOI] [PubMed] [Google Scholar]
- 9.Brown-Elliott BA, Wallace RJ Jr. 2002. Clinical and taxonomic status of pathogenic nonpigmented or late-pigmenting rapidly growing mycobacteria. Clin Microbiol Rev 15:716–746. doi: 10.1128/CMR.15.4.716-746.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Ruger K, Hampel A, Billig S, Rucker N, Suerbaum S, Bange FC. 2014. Characterization of rough and smooth morphotypes of Mycobacterium abscessus isolates from clinical specimens. J Clin Microbiol 52:244–250. doi: 10.1128/JCM.01249-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Nessar R, Cambau E, Reyrat JM, Murray A, Gicquel B. 2012. Mycobacterium abscessus: a new antibiotic nightmare. J Antimicrob Chemother 67:810–818. doi: 10.1093/jac/dkr578. [DOI] [PubMed] [Google Scholar]
- 12.Nessar R, Reyrat JM, Murray A, Gicquel B. 2011. Genetic analysis of new 16S rRNA mutations conferring aminoglycoside resistance in Mycobacterium abscessus. J Antimicrob Chemother 66:1719–1724. doi: 10.1093/jac/dkr209. [DOI] [PMC free article] [PubMed] [Google Scholar]