Abstract
Mycobacterium abscessus, as a species, has been increasingly implicated in respiratory infections, notably in cystic fibrosis patients. The species comprises 3 subspecies, which can be difficult to identify. Since they differ in antibiotic susceptibility and clinical relevance, developing a routine diagnostic tool discriminating Mycobacterium abscessus at the subspecies level is a real challenge. Forty-three Mycobacterium abscessus species isolates, previously identified by multilocus sequence typing, were analyzed by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). A subspecies identification algorithm, based on five discriminating peaks, was drawn up and validated by blind identification of a further 49 strains, 94% of which (n = 46) were correctly identified. Two M. abscessus subsp. massiliense strains were misidentified as M. abscessus subsp. abscessus, and for 1 other strain identification failed. Inter- and intralaboratory reproducibility tests were conclusive. This study presents, for the first time, a classification algorithm for MALDI-TOF MS identification of the 3 M. abscessus subspecies. MALDI-TOF MS proved effective in discriminating within the M. abscessus species and might be easily integrated into the workflow of microbiology labs.
INTRODUCTION
Nontuberculous mycobacteria (NTM) are now well established as significant pathogens in cystic fibrosis (CF) patients (1). The prevalence of respiratory tract NTM in CF varies considerably both worldwide and within a given country: 4% (2) to 13% (3) reported in the United States, 2.7% in Sweden (4), 6.6% in France (5), and 22.6% in Israel (6). Mycobacterium abscessus species is the most prevalent NTM in CF, followed by Mycobacterium avium complex species, together accounting for 70% of NTM (5). M. abscessus species is an emergent pathogen, notably for CF patients, who are at high risk of colonization and pulmonary infection (5, 7). Three subspecies, M. abscessus subsp. abscessus, M. abscessus subsp. massiliense, and M. abscessus subsp. bolletii (8–10), have been distinguished, although this classification is still being debated (11). The subspecies are not equally resistant to chemotherapeutic agents (7), notably clarithromycin (12). Bastian et al. demonstrated that M. abscessus subsp. massiliense was usually susceptible due to two nucleotide deletions in the erm(41) gene, the genetic support of clarithromycin resistance, whereas M. abscessus subsp. bolletii was always resistant (12). Furthermore, the response is generally better in M. abscessus subsp. massiliense than in subsp. abscessus or subsp. bolletii (7, 13). Moreover, chronic macrolide therapy was recently shown to favor the emergence of M. abscessus species (14), making it crucial to differentiate subspecies to optimize treatment. Noneradication may impair health status, preventing lung transplantation (15).
Subspecies typing is mainly molecular. Single-gene sequencing is not sufficiently discriminating, due to frequent horizontal gene transfers between subspecies (16); the optimal method sequences several housekeeping genes (17). However, multilocus sequence typing (MLST) is cumbersome and expensive, whereas matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) is cheap (if we do not include the cost of the machine), rapid, and powerful (18, 19), showing 84 to 98.8% correct species-level identification (20–23), including bacteria only identifiable on 16S rRNA gene sequencing (24). Studies showed that MS identification required only $0.50 worth of reagents and 6 to 8.5 min per isolate (20) and constituted a promising alternative for bacterial identification in developing countries (21). Several MALDI-TOF MS mycobacterial identification studies (25–30) reported systematic difficulty in distinguishing very close species, notably the three M. abscessus subspecies. Recent progress using spectrum cluster analysis (31) considered only two M. abscessus species. The present study describes, for the first time, typing of all three subspecies using MALDI-TOF MS, with a validated portable classification algorithm based on a data set of discriminating peaks.
MATERIALS AND METHODS
Mycobacterial collections.
A working collection of 40 clinical M. abscessus species isolates was first analyzed with both multilocus sequence analysis (MLSA) and MALDI-TOF MS. Isolates were provided by 8 French hospitals (Brest, n = 18; Bordeaux, n = 9; Nantes, n = 4; Tours, n = 3; Cholet, n = 1; Limoges, n = 1; Montpellier, n = 1; and Paris, n = 3) and were epidemiologically unrelated. There were 37 sputum samples, including 33 from CF patients, and 2 blood culture samples and 1 bronchial aspiration sample, all identified as M. abscessus species by the Hain GenoType Mycobacterium CM assay. Three reference strains (M. abscessus subsp. abscessus CIP 104536, M. abscessus subsp. massiliense CIP 108297, and M. abscessus subsp. bolletii CIP 108541) were added to this collection.
A test collection of 49 M. abscessus species (CNR-MyRMA [French National Reference Center for Mycobacteria and Resistance of Mycobacteria to Antituberculosis) was blind-tested by MALDI-TOF MS and compared with the CNR-MyRMA's previous erm(41) and hsp65 gene MLST. All isolates were frozen at −80°C before subculture on 5% sheep-blood agar (bioMérieux, Marcy l'Etoile, France) in 5% CO2 at 37°C for 2 days.
Multilocus sequence typing.
The 40 working collection isolates and 3 reference strains were analyzed by MLST. DNA extraction was performed in 1% Tris-EDTA (TE)/Triton X-100 buffer by a 30-min 86°C thermal lysis. DNA was collected after 5 min of 10,400 × g centrifugation and frozen at −35°C before analysis. MLST was used to sequence 7 housekeeping genes (argH, cya, glpK, gnd, murC, pta, and purH), following the method of Macheras et al. (17). A phylogenetic tree based on the nucleotide sequences of the supergene obtained by concatenation of the 7 M. abscessus species loci was generated using MEGA v5 software (www.megasoftware.net/) with the unweighted-pair group method with arithmetic mean (UPGMA). Statistical support per node was evaluated by a bootstrap test on 100 replicates.
MALDI-TOF MS. (i) Sample preparation.
A 10-μl loop of mycobacterial biomass was obtained from colonies grown on 5% sheep blood agar (bioMérieux, Marcy l'Etoile, France) in 5% CO2 at 37°C for 2 days. Extraction followed the manufacturer's instructions for inactivated mycobacteria bead preparation (Bruker Daltonics, Bremen, Germany).
(ii) MALDI-TOF MS acquisition.
One microliter supernatant from each extract and 1 μl standard (IVD mass calibration standard; Bruker Daltonics) were spotted onto a polished steel MSP 96 target plate (Bruker Daltonics). After drying, 1 μl matrix solution (saturated α-cyano-4-hydroxycinnamic acid in 50% acetonitrile and 2.5% trifluoroacetic acid; Bruker Daltonics) was overlaid onto each spot. Mass spectra were acquired on a microflex LT MALDI-TOF mass spectrometer (Bruker Daltonics) configured with Bruker flexControl software using the default settings. Spectra were analyzed by MALDI Biotyper software (version 3.0) with the Mycobacterium 2.0 database (20).
MS peak-based algorithm for subspecies identification. (i) Defining the algorithm.
Twelve clinical isolates that were well defined by MLST (M. abscessus subsp. abscessus, n = 4; M. abscessus subsp. massiliense, n = 4; and M. abscessus subsp. bolletii, n = 4) and 3 reference strains were analyzed by MALDI-TOF MS. For each, 24 mass spectra were checked visually for characteristic subspecies-discriminating peaks, using ClinProTools 2.0 software (Bruker Daltonics). To strengthen possible discriminating peaks, 28 additional multilocus sequence-typed isolates from the working collection were also analyzed. One spectrum for each was checked for characteristic peaks, using flexAnalysis 2.4 software (Bruker Daltonics), following MLST identification. An algorithm was determined, based on discriminating peaks.
(ii) Validation.
The algorithm was evaluated blindly on the 49 test collection strains.
(iii) Robustness and reproducibility.
To assess the intralaboratory reproducibility and impact of subculturing, the mass spectra of the isolates cultured in 5 replicates on 5 repeated subcultures of 5% blood agar (bioMérieux) were compared for each subspecies. To assess the interlaboratory portability and impact of the culture medium, 10 isolates (M. abscessus subsp. abscessus, n = 4; M. abscessus subsp. massiliense, n = 4; and M. abscessus subsp. bolletii, n = 2) were sent to Bruker Daltonics (Bremen, Germany). After 2 days of culture on 5% blood agar and 4 days on Löwenstein-Jensen (LJ) medium, the MALDI-TOF MS protocol and peak analysis were implemented as above.
RESULTS
MLST.
The MLST dendrogram revealed 3 clusters, perfectly congruent with the 3 subspecies (see the reference strain positions in Fig. 1), that accurately identified the 40 clinical isolates. The working collection thus comprised 24 M. abscessus subsp. abscessus, 10 M. abscessus subsp. massiliense, and 6 M. abscessus subsp. bolletii isolates and 3 reference strains.
FIG 1.
Relatedness of the 43 isolates of Mycobacterium abscessus species analyzed by UPGMA, using MEGA v5.2 software and the Kimura two-parameter mutation model of genetic distance. The nucleotide differences between supergene sequences obtained by the multilocus sequence analysis method of Macheras et al. (17) were used for subspecies identification. Bootstrap values are shown at the major nodes. Isolates can be divided into three clusters, corresponding to the three M. abscessus subspecies: M. abscessus subsp. abscessus (the reference strain of which, CIP 104536, is here referred to as B29), M. abscessus subsp. massiliense (reference strain CIP 108297, here B30), and M. abscessus subsp. bolletii (reference strain CIP 108541, here B31). The reference strains are indicated by asterisks.
MS peak-based algorithm.
All 92 isolates were perfectly identified as M. abscessus species (score range, 1.818 to 2.380; median, 2.235; interquartile range [IQR], 2.118 to 2.352) and correctly differentiated from Mycobacterium. chelonae. M. abscessus subspecies mass spectra were too similar for conclusive identification at the subspecies level using the MALDI Biotyper (Bruker Daltonics) standard algorithm.
The first 12 working collection isolates and 3 reference strains generated 360 spectra (24/isolate). Accurate analysis with ClinProTool software highlighted 9 subspecies-discriminating peaks: m/z 2,081, 3,108, 3,123, 3,378, 3,463, 4,641, 4,871, 6,385, and 6,728. These 9 peaks were then sought in the mass spectra of the other 28 working collection isolates. Four were not systematically found (m/z 4,641, 4,871, 6,385, and 6,728); 5 discriminating peaks (Fig. 2) were therefore used for the subspecies classification algorithm (Fig. 3). The algorithm first screens 2 characteristic peaks (m/z 2,081 and 3,123); their simultaneous presence indicates M. abscessus subsp. abscessus or bolletii and the absence M. abscessus subsp. massiliense. Second, subspecies-characteristic peaks are sought to discriminate M. abscessus subsp. abscessus (m/z 3,378) from M. abscessus subsp. bolletii (m/z 3,463) or to confirm M. abscessus subsp. massiliense (m/z 3,108 and 3,378). In the 40 clinical isolates tested by MALDI-TOF MS, there was only 1 failure, for a subsp. abscessus isolate (no. B18) (Table 1), which was not identified because its peaks (m/z 2,081, 3,123, 3,463, and 3,378) matched no subspecies combination; all M. abscessus subsp. massiliense and M. abscessus subsp. bolletii isolates were unequivocally identified.
FIG 2.
MALDI-TOF spectral profiles and characteristic peaks for each of the three Mycobacterium abscessus subspecies. Accurate analysis with ClinProTool software highlighted five discriminating peaks (m/z 2,081, 3,123, 3,378, 3,463, and 3,378).
FIG 3.
Classification algorithm. The three Mycobacterium abscessus subspecies are identified according to the presence (+) or absence (−) of the five characteristic peaks. The numbers of isolates in the working collection (n = 40) are indicated, with the exception of one isolate that could not be identified.
TABLE 1.
Identifications obtained using MALDI-TOF MS and gene sequencing
| Strain | Results of MALDI-TOF MS identification |
M. abscessus subsp. identified by molecular typingb | ||||||
|---|---|---|---|---|---|---|---|---|
| At a characteristic peak ofa: |
Reliability score | M. abscessus subsp. | ||||||
| 2,081 | 3,108 | 3,123 | 3,378 | 3,463 | ||||
| B1 | + | − | + | − | − | 1.818 | bolletii | bolletii |
| B2 | + | − | + | − | − | 2.218 | bolletii | bolletii |
| B3 | + | − | + | − | + | 2.257 | bolletii | bolletii |
| B4 | + | − | + | − | + | 2.040 | bolletii | bolletii |
| B5 | − | + | − | + | − | 2.223 | massiliense | massiliense |
| B6 | + | − | + | + | − | 2.336 | abscessus | abscessus |
| B7 | + | − | + | + | − | 2.380 | abscessus | abscessus |
| B8 | − | + | − | + | − | 2.107 | massiliense | massiliense |
| B9 | + | − | + | + | − | 2.300 | abscessus | abscessus |
| B10 | + | − | + | + | − | 2.285 | abscessus | abscessus |
| B11 | − | + | − | + | − | 2.227 | massiliense | massiliense |
| B12 | + | − | + | − | − | 2.086 | bolletii | bolletii |
| B13 | + | − | + | + | − | 2.112 | abscessus | abscessus |
| B14 | − | + | − | + | − | 2.176 | massiliense | massiliense |
| B15 | + | − | + | + | − | 2.207 | abscessus | abscessus |
| B16 | + | − | + | + | − | 2.174 | abscessus | abscessus |
| B17 | + | − | + | + | − | 2.214 | abscessus | abscessus |
| B18c | + | − | + | + | + | 1.987 | Not identified | abscessus |
| B19 | + | − | + | − | + | 2.321 | bolletii | bolletii |
| B20 | + | − | + | + | − | 2.279 | abscessus | abscessus |
| B21 | − | + | − | + | − | 2.246 | massiliense | massiliense |
| B22 | + | − | + | + | − | 2.252 | abscessus | abscessus |
| B23 | + | − | + | + | − | 2.203 | abscessus | abscessus |
| B24 | − | − | + | + | − | 2.180 | abscessus | abscessus |
| B25 | − | + | − | + | − | 2.097 | massiliense | massiliense |
| B26 | + | − | + | + | − | 2.350 | abscessus | abscessus |
| B27 | − | + | − | + | − | 2.173 | massiliense | massiliense |
| B28 | + | − | + | + | − | 2.270 | abscessus | abscessus |
| B29d | + | − | + | + | − | 2.296 | abscessus | abscessus |
| B30d | − | + | − | + | − | 1.926 | massiliense | massiliense |
| B31d | + | − | + | − | + | 1.999 | bolletii | bolletii |
| B32 | + | − | + | + | − | 2.294 | abscessus | abscessus |
| B33 | + | − | + | + | − | 2.275 | abscessus | abscessus |
| B34 | + | − | + | + | − | 2.290 | abscessus | abscessus |
| B35 | + | − | + | + | − | 2.261 | abscessus | abscessus |
| B36 | + | − | + | + | − | 2.333 | abscessus | abscessus |
| B37 | + | − | + | + | − | 2.304 | abscessus | abscessus |
| B38 | + | − | + | + | − | 2.307 | abscessus | abscessus |
| B39 | + | − | + | + | − | 2.157 | abscessus | abscessus |
| B40 | − | + | − | + | − | 2.356 | massiliense | massiliense |
| B41 | − | + | − | + | − | 2.301 | massiliense | massiliense |
| B42 | + | − | + | + | − | 2.021 | abscessus | abscessus |
| B43 | − | + | − | + | − | 2.256 | massiliense | massiliense |
| P1 | + | − | + | + | − | 2.234 | abscessus | abscessus |
| P2 | − | + | − | + | − | 2.144 | massiliense | massiliense |
| P3 | + | − | + | + | − | 2.175 | abscessus | abscessus |
| P4 | + | − | + | − | + | 2.253 | bolletii | bolletii |
| P5 | − | + | − | + | − | 2.015 | massiliense | massiliense |
| P6 | − | + | − | + | − | 2.155 | massiliense | massiliense |
| P7 | + | − | + | + | − | 2.133 | abscessus | abscessus |
| P8 | − | + | − | + | − | 1.983 | massiliense | massiliense |
| P9 | + | − | + | − | + | 2.077 | bolletii | bolletii |
| P10 | + | − | + | + | − | 2.092 | abscessus | abscessus |
| P11 | + | − | + | + | − | 2.116 | abscessus | abscessus |
| P12 | − | + | − | + | − | 1.940 | massiliense | massiliense |
| P13 | − | + | − | − | − | 1.920 | massiliense | massiliense |
| P14 | + | − | + | + | − | 1.876 | abscessus | abscessus |
| P15 | + | − | + | + | − | 2.188 | abscessus | abscessus |
| P16 | + | − | + | + | − | 2.172 | abscessus | abscessus |
| P17 | − | + | − | + | − | 2.015 | massiliense | massiliense |
| P18c | − | − | + | + | − | 2.035 | Not identified | massiliense |
| P19 | + | − | + | + | − | 2.148 | abscessus | abscessus |
| P20 | + | − | + | + | − | 2.302 | abscessus | abscessus |
| P21 | + | − | + | + | − | 2.050 | abscessus | abscessus |
| P22c | + | − | + | + | − | 2.108 | abscessus | massiliense |
| P23 | + | − | + | + | − | 1.858 | abscessus | abscessus |
| P24 | + | − | + | − | + | 1.956 | bolletii | bolletii |
| P25 | − | + | − | + | − | 2.090 | massiliense | massiliense |
| P26 | + | − | + | + | − | 2.161 | abscessus | abscessus |
| P27 | + | − | + | + | − | 2.246 | abscessus | abscessus |
| P28 | + | − | + | + | − | 2.111 | abscessus | abscessus |
| P29 | + | − | + | − | + | 2.202 | bolletii | bolletii |
| P30 | + | − | + | + | − | 2.242 | abscessus | abscessus |
| P31 | + | − | + | + | − | 1.973 | abscessus | abscessus |
| P32 | − | + | − | + | − | 2.039 | massiliense | massiliense |
| P33 | − | + | − | + | − | 1.977 | massiliense | massiliense |
| P34 | + | − | + | − | − | 2.302 | bolletii | bolletii |
| P35 | − | + | − | + | − | 1.961 | massiliense | massiliense |
| P36 | − | + | − | + | − | 2.048 | massiliense | massiliense |
| P37 | − | + | − | + | − | 1.921 | massiliense | massiliense |
| P38 | + | − | + | − | + | 2.027 | bolletii | bolletii |
| P39 | + | − | + | + | − | 1.858 | abscessus | abscessus |
| P40 | + | − | + | + | + | 2.120 | bolletii | bolletii |
| P41 | + | − | + | + | − | 2.100 | abscessus | abscessus |
| P42 | + | − | + | − | + | 2.064 | bolletii | bolletii |
| P43 | + | − | + | − | + | 2.184 | bolletii | bolletii |
| P44 | + | − | + | − | + | 2.177 | bolletii | bolletii |
| P45c | + | − | + | + | − | 2.194 | abscessus | massiliense |
| P46 | + | − | + | − | + | 2.297 | bolletii | bolletii |
| P47 | + | − | + | − | + | 1.916 | bolletii | bolletii |
| P48 | + | − | + | − | + | 1.945 | bolletii | bolletii |
| P49 | + | − | + | − | + | 2.235 | bolletii | bolletii |
+, present; −, absent.
MLSA identification for the working collection (B) and hsp65 and erm(41) gene sequencing for the testing collection (P).
Isolates showing discrepant results.
Reference strain.
Validation.
Forty-six of the 49 blindly tested CNR-MyRMA strains were correctly identified using MALDI-TOF MS and the 5-peak algorithm (Table 1). For 1 subsp. massiliense strain (no. P18), the mass spectra harbored only 1 peak of the set (m/z 3,378), precluding species identification. Two M. abscessus subsp. massiliense strains (no. P22 and P45) were misidentified as M. abscessus subsp. abscessus, having all 3 characteristic peaks (m/z 2,081, 3,123, and 3,378) and not that (m/z 3,108) of M. abscessus subsp. massiliense (Fig. 3). These discrepant identifications were checked by MLST: the MS scores (all >2.00) appeared reliable; and the second extraction and MS analysis retrieved similar spectra. Finally, 94% of strains (n = 46/49) were correctly identified by MALDI-TOF MS and the peak-based algorithm, with 4% (n = 2) misidentification and 2% (n = 1) nonidentification.
Reproducibility.
Intralaboratory reproducibility was assessed by comparing the mass spectra of a given strain cultured simultaneously on 5 different plates and 5 repeated subculture plates. Spectra were identical, and the 5 discriminating peaks were consistent.
Comparisons with the results of Bruker Daltonics assays demonstrated interlaboratory reproducibility. All 10 strains showed mass spectra identical to those found in our laboratory; the 5 discriminating peaks were found on both LJ medium and blood agar, allowing unequivocal identification using the peak-based algorithm.
DISCUSSION
In this study, we sought to assess the performance of MALDI-TOF MS for subspecies-level typing of M. abscessus species because the present methods fail to enable accurate subspecies identification for the optimal clinical management of M. abscessus species infection (14).
MS-based identification of conventional bacteria is 95% reliable (20); for mycobacteria, however, the protocols remain to be validated. In 2006, the first reports of NTM identification by MALDI-TOF MS demonstrated its value and also demonstrated the difficulty of distinguishing closely related species (27, 28). Subsequent studies described MALDI-TOF MS mycobacteria identification (18, 25–27, 29–31), but only one focused on M. abscessus species (31). The present study of the largest M. abscessus species collection to date confirmed the results of the initial report by Saleeb et al. (25). After optimized protein extraction, all 92 isolates were perfectly identified as M. abscessus complex species, with a median score of 2.235.
The real challenge is to distinguish the 3 M. abscessus subspecies. Precise identification within a given complex (e.g., M. abscessus species, M. avium complex, or M. tuberculosis complex) is a recurring problem, whatever the software (MALDI Biotyper or Andromas) (18, 25, 26). Even so, many members of bacterial complexes, such as the Burkholderia complex, Streptococcus bovis/equinus, or Bacteroides, have been accurately typed by MALDI-TOF MS (32–34). Recently, Teng et al. provided proof of concept for accurate discrimination between two species by MALDI-TOF MS (31). The present study extends the concept with (i) a classification algorithm based on analysis of 40 MLSA-typed isolates, (ii) validation by a blind test on 49 genotyped strains, and (iii) reproducibility testing. The algorithm yielded 94% reliability, 2% uncertainty, and 4% error. The accuracy reported was poorer than the 100% accuracy reported by Teng et al. (31), but the present study covered not 2 but all 3 M. abscessus subspecies. The failures may be explained by the degree of horizontal gene transfer in the evolution of M. abscessus species (17), some strains displaying a hybrid proteome, with biomarker signatures of both M. abscessus subsp. massiliense and M. abscessus subsp. abscessus, just as some strains display allelic signatures of both M. abscessus subsp. massiliense and M. abscessus subsp. abscessus (17, 35). Both errors were misidentification of M. abscessus subsp. massiliense as M. abscessus subsp. abscessus.
Because M. abscessus species is an important respiratory pathogen worldwide, algorithm robustness and interlaboratory reproducibility are crucial. The present reliable subspecies identification was independent of the cultivation method (sheep blood agar or LJ medium and culture time) and of the experimenter (French or German laboratory). However, all the isolates were from France, which may introduce bias. None of the specific peaks of the Taiwanese collection of Teng et al. were found, despite an identical extraction protocol (31). The algorithm therefore requires testing in a larger, worldwide collection to check the hypothesis of different biogeographic MS profiles for a given species.
MALDI-TOF MS seemed to discriminate effectively within M. abscessus species, with 100% accuracy for M. abscessus subsp. bolletii and M. abscessus subsp. abscessus and a very low error rate for M. abscessus subsp. massiliense. It thus provides considerable advantages in speed and costs and might be easily integrated into the workflow of Mycobacterium laboratories, as we experienced in our own laboratory.
ACKNOWLEDGMENTS
We thank Markus Timke (Bruker Daltonics) for wise advice and Peio Mogabure (Bruker Daltonics) for significant support as a go-between. We also thank Pascale Bemer, Jean-Louis Gaillard, Sylvain Godreuil, Philippe Lanotte, Cécile Le Brun, Christian Martin, and Olivia Peuchant for providing some of the isolates used in this study.
Footnotes
Published ahead of print 9 July 2014
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