Use of the INNO-LiPA-MYCOBACTERIA Assay (Version 2) for Identification of Mycobacterium avium-Mycobacterium intracellulare-Mycobacterium scr
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微生物临床杂志 2005年第6期
Service de Microbiologie-Immunologie Biologique, Hpital Antoine Beclere, Clamart
Service de Microbiologie, Hpital Saint Louis
Centre National de Reference des Mycobacteries, Institut Pasteur, Paris, France
ABSTRACT
Using INNO-LiPA-MYCOBACTERIA (Lipav1; Innogenetics) and the AccuProbe (Gen-Probe Inc./bioMerieux) techniques, 35 Mycobacterium avium-Mycobacterium intracellulare-Mycobacterium scrofulaceum (MAC/MAIS) complex strains were identified between January 2000 and December 2002. Thirty-four of 35 isolates were positive only for the MAIS complex probe by Lipav1 and were further analyzed by INNO-LiPA-MYCOBACTERIA version 2 (Lipav2), hsp65 PCR restriction pattern analysis (PRA), and ribosomal internal transcribed spacer (ITS), hsp65, and 16S rRNA sequences. Lipav2 identified 14 of 34 strains at the species level, including 11 isolates positive for the newly specific MAC sequevar Mac-A probe (MIN-2 probe). Ten of these 11 isolates corresponded to sequevar Mac-A, which was recently defined as Mycobacterium chimerae sp. nov. Among the last 20 of the 34 MAIS isolates, 17 (by hsp65 PRA) and 18 (by hsp65 sequence) were characterized as M. avium. Ten of the 20 were identified as Mac-U sequevar. All these 20 isolates were identified as M. intracellulare by 16S rRNA sequence except one isolate identified as Mycobacterium paraffinicum by 16S rRNA and ITS sequencing. One isolate out of 35 isolates that was positive for M. avium by AccuProbe and that was Mycobacterium genus probe positive and MAIS probe negative by Lipav1 and Lipav2 might be considered a new species. In conclusion, the new INNO-LiPA-MYCOBACTERIA allowed the identification of 40% of the previously unidentified MAIS isolates at the species level. The results of the Lipav2 assay on the MAIS isolates confirm the great heterogeneity of this group and suggest the use of hsp65 or ITS sequencing for precise identification of such isolates.
INTRODUCTION
Molecular biology has made a great impact on the diagnosis of mycobacterial infection, considerably reducing the delay of reporting results to clinicians (14). AccuProbe (Gen-Probe Inc./bioMerieux) was the first molecular test developed and commercialized for the rapid identification of mycobacteria from culture-positive specimens (21). Detection of 16S rRNA by a chemiluminescent species-specific probe allowed the identification of mycobacterial isolates belonging to the Mycobacterium tuberculosis complex, the Mycobacterium avium complex (MAC/MAIS) (including species-specific probes for M. avium and Mycobacterium intracellulare), Mycobacterium kansasii (29), and Mycobacterium gordonae. However, the need for rapid identification of more mycobacterial species in parallel has resulted in the development of several in-house PCR-based assays, consisting of the amplification of several conserved genes followed by the characterization of the amplicons ensured by restriction enzyme analysis or sequencing (4-6, 9, 15-17, 22-24, 28). The 16S rRNA sequence is often considered as a gold standard for bacterial identification but has demonstrated lower variability with several examples of species difficult to separate within the genus Mycobacterium (31). The 65-kDa heat shock protein gene is highly conserved in mycobacteria, with several polymorphic regions allowing its use for identification purposes. PCR restriction pattern analysis (PRA) of an hsp65 441-bp sequence has been developed, leading to catalogues of hsp65 PRA patterns (28). The digestion product separated by agarose gel electrophoresis appears as bands whose patterns are usually species specific. In addition to the hsp65 gene, the 16S-23S rRNA internal transcribed spacer (ITS) region has been shown to be more discriminative than the 16S rRNA itself (6, 9, 10). A detailed understanding of ITS data was developed for the heterogeneous MAIS group, allowing the separation and classification of species belonging to this group as sequevars, and for other mycobacteria, including the M. tuberculosis complex (11), leading to the development of a line probe assay (INNO-LiPA-MYCOBACTERIA). This assay has been the first commercial DNA probe test able to identify the most frequently isolated mycobacterial species in parallel (18, 19, 27, 30). After amplification of the ITS, the biotinylated amplified DNA product is hybridized with 14 specific oligonucleotide probes immobilized as parallel lines on membrane strips. These include probes for the identification of strains belonging to the M. tuberculosis complex, M. kansasii (groups I, II, and III), Mycobacterium xenopi, M. gordonae, Mycobacterium chelonae (groups I, II, and III), and MAC (named MAIS complex to include M. scrofulaceum) with species-specific probes for M. avium, M. intracellulare, and M. scrofulaceum. Our laboratories have used this method for several years (reference 18 and J. L. Herrmann et al., unpublished results) and have encountered few difficulties in the identification of nontuberculous mycobacteria, with the exception of several isolates belonging to the MAIS group that were positive only with the complex-specific MAIS oligonucleotide probe. The aim of the study was (i) to clarify why strains were positive for the MAIS probe only and not with any specific probes for the complex members, (ii) to determine the contribution of several molecular markers (hsp65, ITS, and 16S rRNA) to identify these peculiar isolates at the species level, and (iii) to assess the diagnostic value of the INNO-LiPA-MYCOBACTERIA version 2.
MATERIALS AND METHODS
Between January 2000 and December 2002, 495 nontuberculous mycobacteria isolates were cultured, analyzed, and identified by AccuProbe (Gen-Probe Inc./bioMerieux, Marcy l'Etoile, France), and/or by INNO-LiPA-MYCOBACTERIA v1 (Lipav1; Innogenetics, Ghent, Belgium) and/or by classical methods. All strains were single clinical isolates (one isolate per patient), grown in Middlebrook medium supplemented with oleic acid-albumin-dextrose-catalase and PANTA (consisting of polymyxin B, amphotericin B, nalidixic acid, trimethoprim, and azlocillin) (960-MGIT; Becton-Dickinson, Le Pont de Claix, France, and BacTAlert, bioMerieux, Marcy l'Etoile, France). The majority of identified mycobacterial species were M. xenopi (182), M. kansasii (24), M. gordonae (72), M. chelonae (56), Mycobacterium abscessus (9), Mycobacterium fortuitum-Mycobacterium peregrinum complex (55), and Mycobacterium marinum (8). Eighty-nine isolates (18%) were identified as belonging to the MAIS complex. Within this group, 36 (40.5%) were identified as M. avium, 17 (19.1%) as M. intracellulare, and 1 (1.1%) as M. scrofulaceum. Of the remaining 35 isolates (39.3%), 34 isolates (38.2%) were positive only for the MAIS-Lipav1 probe, and 1 isolate (1.1%) identified as M. avium by AccuProbe was Mycobacterium genus probe positive but MAIS probe negative by Lipav1 (Table 1). These 35 isolates were further analyzed by hsp65 PRA sequencing, ITS sequencing, 16S rRNA sequencing, and the newer INNO-LiPA-MYCOBACTERIA v2 (Lipav2; Innogenetics, Ghent, Belgium).
For each sample, all amplifications and sequences were performed on the same DNA extract. Identifications made by Lipav1 and Lipav2 and by AccuProbe were performed according to the manufacturer's recommendations and using Auto-LiPA automated instrumentation (Innogenetics, Ghent, Belgium) for the LiPA tests. The procedure is similar to procedures described previously (18, 30). In comparison with Lipav1, the updated Lipav2 had new probes specific for Mycobacterium celatum, Mycobacterium genavense, Mycobacterium simiae, M. marinum-Mycobacterium ulcerans, Mycobacterium malmoense, Mycobacterium haemophilum, and Mycobacterium smegmatis and a second probe (MIN-2) specific for MAC sequevar Mac-A, for M. intracellulare. The hsp65 PRA was performed as described by Telenti et al. (28). Amplicons were further sequenced using the forward primer Tb11 (5'-ACCAACGATGGTGTGTCCAT-3') as described previously (28) after purification on a QIAGEN column as described by the supplier (QIAGEN, France). The 16S rRNA sequencing was performed as previously described (16), and the ITS sequencing was performed on purified amplicons made for Lipav1 and Lipav2 by Innogenetics (Ghent, Belgium) (6, 20). Comparison of the sequence data was performed using the Basic Local Alignment Search Tool (BLAST) (2, 3) for hsp65 (>375 bp) and the RIDOM Mycobacteria project (http://www.ridom.de/mycobacteria/) for 16S rRNA sequences (13). Alignments were given based on a single nucleotide difference, and final identification was chosen to correspond to the highest percentage of similarity given by the software (Table 1). Sequences were put in clusters according to the unweighted-pair group method using arithmetic averages, and dendrograms were drawn using the Kodon v2.04 (Applied Maths, St. Martens Latem, Belgium) software package.
Nucleotide sequence accession numbers. ITS, hsp65, and 16S rRNA nucleotide sequences for isolates falling into new clusters (isolates 4 to 13 and 18 to 27) and for isolates considered new sequevars (isolates 3, 14, 28, 29, 31, 34, and 35) were deposited in the GenBank database under accession numbers AY858989 to AY859052.
RESULTS
Thirty-five clinical isolates belonging to the MAIS complex were selected because they were not identifiable to the species level or because of discrepant results between AccuProbe and Lipav1 (Table 1, and Fig. 1 to 3). Thirty-four isolates presented only a MAIS-Lipav1-positive probe, without an identification at the species level (Table 1, isolates 1 to 34), whereas 1 isolate identified as M. avium by AccuProbe did not react with the MAIS-Lipav1 probe (Table 1, isolate 35).
Lipav2 allowed identification of 14 out of the 35 (40%) isolates to the species level (Table 1, isolates 1 to 14): isolate 1 was identified as M. haemophilum and isolates 2 to 14 were identified as M. intracellulare (Table 1). Isolates 2 and 3 were positive for the MIN-1-Lipav2 species-specific probe (Table 1). Isolates 4 to 14 were positive for the MIN-2-Lipav2 species-specific probe (Table 1). Nine isolates out of 11 MIN-2-positive isolates were identified as M. intracellulare by AccuProbe (Table 1, isolates 4 to 12). The 14 Lipav2 identifications were confirmed by hsp65 PRA and by hsp65 sequencing (Table 1). With the exception of the M. haemophilum isolate for which 16S rRNA was not sequenced, all 13 sequenced isolates (isolates 2 to 14) were also identified by 16S rRNA as M. intracellulare (Table 1). ITS sequencing, which defines sequevars, confirmed the identification of M. haemophilum (Table 1) and identified 10 MIN-2-Lipav2-positive isolates out of 11 as sequevar Mac-A (Table 1, isolates 4 to 13). This sequence has 100% homology to the newly defined species M. chimerae sp. nov. (38). Isolate 14 (MIN-2-Lipav2 positive) was considered an unknown sequevar, with the closest homology being obtained for sequevar Mac-C (Table 1). Isolates 2 and 3, the two MIN-1-Lipav2-positive isolates, were identified as MIN-C sequevar (Table 1).
This left us with 21 MAIS isolates not identified at the species level (by Lipav1 and Lipav2) (Table 1, isolates 15 to 34), including the M. avium isolate previously identified by AccuProbe that was negative for the MAIS-Lipav1 and MAIS-Lipav2 probes (Table 1, isolate 35). Additional DNA molecular techniques were necessary for a complete identification of these 21 isolates. By hsp65 PRA, isolates 16 to 29 and 31 to 33 were identified as M. avium; isolates 15, 30, and 34 were identified as M. intracellulare; and isolate 35 was identified as M. simiae (Table 1). Results of hsp65 sequencing confirmed the PRA results except for isolate 35, for which hsp65 sequencing identified no close match but a weak homology to M. avium complex, and isolates 30 and 34, identified by hsp65 PRA as M. intracellulare but as M. avium (isolate 30) and M. avium complex (isolate 34) by hsp65 sequencing (Table 1). Inside the group of 18 M. avium isolates identified by hsp65 sequence, ITS sequencing identified 9 isolates as Mac-U sequevars (Table 1, isolates 18 to 25 and isolate 27), 1 as a Mac-Q sequevar (isolate 30), 2 as Mac-E sequevars (isolates 16 and 17), 1 as a Mac-R sequevar (isolate 32), and 4 as unknown sequevars (isolates 26, 28, 29, and 31) (Table 1). Isolate 33 did not give an ITS sequence result that could be interpreted. Isolate 15, the unique M. intracellulare isolate by hsp65 sequence, was identified as a Mac-C sequevar (Table 1). The ITS sequence identified isolate 34 as M. paraffinicum and isolate 35 as M. intracellulare with weak homology (Table 1). It is of importance to note that the hsp65 sequence comparison for isolate 34 did not pick up M. paraffinicum, potentially due to the absence of this sequence in the database. The 16S rRNA sequence results identified isolates 15 to 33 as M. intracellulare as a first choice and M. avium as a second choice. Isolate 15, the unique Mac-C sequevar in this group, was identified as M. intracellulare by hsp65 PRA, hsp65 sequence, and 16S rRNA sequence (Table 1). Isolate 34 was confirmed by 16S rRNA as M. paraffinicum, and isolate 35 was identified as being most related to Mycobacterium interjectum and M. simiae by 16S rRNA sequencing (Table 1).
To fully summarize the results of the molecular markers, specific dendrograms were constructed using the unweighted-pair group method using arithmetic averages algorithm. This approach allows a more precise definition and identification of the close relationships between bacterial isolates using sequence data for specific molecular marker (ITS, hsp65, and 16S rRNA in our study). Two main clusters of clinical isolates were clearly individualized by ITS marker; one cluster was related to the Mac-A (or M. chimerae) sequevar, and one cluster was related to the Mac-U sequevar (Fig. 1). The Mac-A cluster was separated into two groups of isolates by the hsp65 molecular marker, one comprising isolates 4 to 6 and a second comprising isolates 7 to 13 (Fig. 2). Similarly, the Mac-U cluster was more heterogeneous according to the hsp65 molecular marker. Three additional isolates were included in the cluster: isolates 28 and 29, identified as Mac-Q-like sequevars, and isolate 33, for which no ITS sequence could be obtained (Fig. 2). By comparison, the 16S rRNA sequence encompassing region A individualized three clusters (Fig. 3): the Mac-A cluster with the addition of isolate 2, the Mac-U cluster with five more isolates (isolates 14, 15, 28, 31, and 33), and the last one comprising four isolates (isolates 16, 17, 29, and 32) also shown to be closely related by ITS and hsp65 molecular markers.
DISCUSSION
The ability to differentiate three species, three subspecies, and 28 serovars within the MAIS complex remains difficult despite existing methods of serotyping, DNA hybridization, or DNA sequencing (5, 9, 36). A number of clinical isolates and reference strains have been negative by both AccuProbes and by ITS species-specific probes (references 6 and 7 and the present study). Therefore, clinical identification is often limited to assigning an isolate merely to the MAIS complex, frequently considered by the clinician as an M. avium isolate. The use of molecular biology for such heterogeneous isolates, in addition to shorter delays for identification (26), has provided easy access to the precise identification of mycobacteria for any clinical microbiologist. Our study demonstrates that Lipav2 represents a valuable improvement because it is simple to perform and allows the identification of an additional 14 of the 89 MAIS isolates (15.7%, or 68 identified isolates out of 89) compared to Lipav1 (54 identified isolates out of 89). Its precision is confirmed by all molecular markers tested including 16S rRNA. This improvement will enable any mycobacteriologist to identify 21 mycobacterial species directly from a positive culture in less than a working day (37; L. Lebrun and J. L. Herrmann, unpublished results).
Second, our study highlights the difficulty of choosing specific genetic markers in a very heterogeneous group of clinical isolates, as for several strains of the MAIS complex, hsp65 and 16S rRNA sequencing did not classify the isolates into the same groups (Fig. 2 and 3). Examples of such discrepancies are found exclusively among isolates that are positive for the Lipav2-MAIS probe, where heterogeneity is high. For the isolates positive for the Lipav2-MIN-1 or MIN-2 probe, identification is clear and similar by all molecular markers.
Compared to other molecular markers, 16S rRNA data are available for the majority of bacteria, and the 16S rRNA sequencing method, therefore, is considered by many as the gold standard for identification and taxonomic purposes. However, although its routine use to distinguish well-resolved species might be sufficient, very recently divergent species may not be recognizable (8, 37). Examples of the limited power of 16S rRNA sequencing in the mycobacteria genus exist for strains related to M. intracellulare species, with some intraspecific allelic diversity, and was demonstrated in the Lipav2-MAIS probe-positive isolates (reference 22 and our study). The hsp65 or ITS molecular markers have been shown to be more discriminating first in the very heterogeneous MAIS complex (Fig. 1 to 3) (6, 9, 20, 25; our study) and then for other atypical mycobacteria (M. kansasii groups, for example) (1). Despite the advantage of being available in any laboratory performing PCR techniques and DNA restriction, hsp65 PRA has several well-known drawbacks (lack of standardization and slight variability in fragment length, for example) (22). Species with overlapping patterns and/or multiple patterns within a single species do exist (22, 31). An identification was given for each isolate by hsp65 PRA in our study. All were confirmed by hsp65 sequencing except isolates 30, 34, and 35, which means that an M. avium or an M. intracellulare hsp65 PRA result does not always correlate with the gene sequence result.
The 280-bp rRNA region or ITS, which is transcribed but does not encode a final product, allows more sequence variation than 16S rRNA. It is characterized by a higher rate of polymorphisms, which allows a more precise species definition. However, in some cases the presence of intraspecies variability may result in confusion (8, 12). Inside the MAIS complex, isolates typed as being M. avium and M. intracellulare by other techniques belonged to sequevars Mac-A to Mac-G and Min-A to Min-D, respectively (6, 7, 11). Several Lipav1 isolates positive only for the MAIS probe but identified as M. intracellulare by AccuProbe are now identified as such by the new Lipav2 due to the addition of the Min-2- or Mac-A-specific probe (32; our study). These isolates belonged to sequevar Mac-A. As described recently, the ITS sequence of the Mac-A sequevar is 100% homologous with the newly defined species M. chimerae sp. nov. (33). Ten isolates of 35 in our study, with 100% homology to sequevar Mac-A, could be named M. chimerae sp. nov. The MAC or MAIS intermediates belonged to sequevars Mac-B to Mac-O and Mac-Q to Mac-U. Within the group of isolates positive only for the Lipav2-MAIS probe, hsp65 sequencing gave the highest homology to M. avium compared to 16S rRNA, which favored identification of M. intracellulare-like isolates. Overlapping of the mismatches in the comparison of 16S rRNA sequences made it difficult to unambiguously identify the best match, although one M. intracellulare reference strain was always picked up as the first choice. However, ITS and hsp65 dendrograms (Fig. 1 and 2) allow a clearer distinction between two clusters of clinical isolates, including the Mac-A cluster, compared to the 16S rRNA dendrogram. The second observed cluster (10 of 35 isolates) belonged to Mac-U (Table 1, isolates 18 to 27, and Fig. 1).
Several isolates (Table 1, isolates 3, 14, 26, 27, 28, 29, and 31) were not 100% homologous to a defined sequevar. For all these isolates, the definition of new sequevars can be achieved if we apply the definitions given by Frothingam and Wilson (9) and De Smet et al. (6) for the identification of a new sequevar.
Isolate 34 was identified as M. paraffinicum by ITS and 16S rRNA sequencing. M. paraffinicum is synonymous with M. scrofulaceum, distinguished mainly by a negative urease reaction (35). This sequence homology explained the positive hybridization with the Lipav1 and Lipav2 MAIS complex probe for this clinical isolate, as this probe includes the recognition of M. scrofulaceum. However, the slight ITS sequence difference explained the negative reaction with the M. scrofulaceum species-specific probe. For isolate 35, different results were found by each molecular marker (with very low homology), indicating that this isolate should be considered a new mycobacterial species not yet genetically and phenotypically defined.
Overrepresentation of two sequevars (Mac-A and Mac-U) can be correlated with a higher frequency of colonization or disease in humans. All studied strains were isolated from patients with pulmonary disorders (bronchiectasias and lung cancer) (data not shown). None of the Mac-U isolates had characteristics that fulfill the American Thoracic Society criteria (34) and, therefore, have to be considered responsible for colonization of the patient's lungs. Interestingly, all Mac-U isolates were isolated from native Africans. Two patients had three and four smear-negative, culture-positive specimens with Mac-A isolates. One of them died rapidly with major changes shown on the chest X ray. Mac-A has also previously been observed in pulmonary disease (33). All the other Mac-A isolates were obtained from unique smear-negative, culture-positive specimens (L. Lebrun and J. L. Herrmann, unpublished results). With more precision in the identification of mycobacteria, microbiologists in collaboration with clinicians will be able to follow up such new isolates to gain a more complete understanding of the behavior of mycobacteria either as commensal or opportunistic pathogens.
In conclusion, the availability of molecular tests such as the Lipav2 in the mycobacteriology laboratory will allow increased efficiency in identification. Precise identification can then be correlated with clinical disease and will allow the clinician to consider such isolates as potential or opportunistic pathogens. The question of which molecular tool to use is difficult to answer. Lipav2 is simple to use but fails to identify a few isolates. Sequencing of the 16S rRNA and the hsp65 genes will always provide a result but in some cases may not identify the tested isolate. In addition, the use of this technique may not be feasible in all mycobacteriology laboratories because of the requirement for extensive equipment. When isolates belong to well-defined species such as those in the Lipav2-MIN-1 or Lipav2-MIN-2 probe-positive isolates, 16S rRNA sequencing is sufficiently discriminating. However, when heterogeneity in the mycobacterial population is increased, a more precise result will require either ITS or hsp65 sequencing, as is the case for the isolates that were probe positive only for Lipav2-MAIS.
ACKNOWLEDGMENTS
We are grateful to Ben Marshall for critical reading of the manuscript, to Rebecca Millecamps and Wouter Mijs for their valuable help in sequence comparisons and dendrograms, and to Koenrad De Smet, Roland Brosch, and Laura Vernoux for their helpful comments and support.
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Wayne, L. G., R. C. Good, E. C. Bttger, R. Butler, M. Dorsch, T. Ezaki, W. Gross, V. Jonas, J. Kilburn, P. Kirschner, M. I. Krichevsky, M. Ridell, T. M. Shinnick, B. Springer, E. Stackebrandt, I. Tarnok, Z. Tarnok, H. Tasaka, V. Vincent, N. G. Warren, C. A. Knott, and R. Johnson. 1996. Semantide- and chemotaxonomy-based analyses of some problematic phenotypic clusters of slowly growing mycobacteria, a cooperative study of the International Working Group on Mycobacterial Taxonomy. Int. J. Syst. Bacteriol. 46:280-297.(Lea Lebrun, Franois-Xavie)
Service de Microbiologie, Hpital Saint Louis
Centre National de Reference des Mycobacteries, Institut Pasteur, Paris, France
ABSTRACT
Using INNO-LiPA-MYCOBACTERIA (Lipav1; Innogenetics) and the AccuProbe (Gen-Probe Inc./bioMerieux) techniques, 35 Mycobacterium avium-Mycobacterium intracellulare-Mycobacterium scrofulaceum (MAC/MAIS) complex strains were identified between January 2000 and December 2002. Thirty-four of 35 isolates were positive only for the MAIS complex probe by Lipav1 and were further analyzed by INNO-LiPA-MYCOBACTERIA version 2 (Lipav2), hsp65 PCR restriction pattern analysis (PRA), and ribosomal internal transcribed spacer (ITS), hsp65, and 16S rRNA sequences. Lipav2 identified 14 of 34 strains at the species level, including 11 isolates positive for the newly specific MAC sequevar Mac-A probe (MIN-2 probe). Ten of these 11 isolates corresponded to sequevar Mac-A, which was recently defined as Mycobacterium chimerae sp. nov. Among the last 20 of the 34 MAIS isolates, 17 (by hsp65 PRA) and 18 (by hsp65 sequence) were characterized as M. avium. Ten of the 20 were identified as Mac-U sequevar. All these 20 isolates were identified as M. intracellulare by 16S rRNA sequence except one isolate identified as Mycobacterium paraffinicum by 16S rRNA and ITS sequencing. One isolate out of 35 isolates that was positive for M. avium by AccuProbe and that was Mycobacterium genus probe positive and MAIS probe negative by Lipav1 and Lipav2 might be considered a new species. In conclusion, the new INNO-LiPA-MYCOBACTERIA allowed the identification of 40% of the previously unidentified MAIS isolates at the species level. The results of the Lipav2 assay on the MAIS isolates confirm the great heterogeneity of this group and suggest the use of hsp65 or ITS sequencing for precise identification of such isolates.
INTRODUCTION
Molecular biology has made a great impact on the diagnosis of mycobacterial infection, considerably reducing the delay of reporting results to clinicians (14). AccuProbe (Gen-Probe Inc./bioMerieux) was the first molecular test developed and commercialized for the rapid identification of mycobacteria from culture-positive specimens (21). Detection of 16S rRNA by a chemiluminescent species-specific probe allowed the identification of mycobacterial isolates belonging to the Mycobacterium tuberculosis complex, the Mycobacterium avium complex (MAC/MAIS) (including species-specific probes for M. avium and Mycobacterium intracellulare), Mycobacterium kansasii (29), and Mycobacterium gordonae. However, the need for rapid identification of more mycobacterial species in parallel has resulted in the development of several in-house PCR-based assays, consisting of the amplification of several conserved genes followed by the characterization of the amplicons ensured by restriction enzyme analysis or sequencing (4-6, 9, 15-17, 22-24, 28). The 16S rRNA sequence is often considered as a gold standard for bacterial identification but has demonstrated lower variability with several examples of species difficult to separate within the genus Mycobacterium (31). The 65-kDa heat shock protein gene is highly conserved in mycobacteria, with several polymorphic regions allowing its use for identification purposes. PCR restriction pattern analysis (PRA) of an hsp65 441-bp sequence has been developed, leading to catalogues of hsp65 PRA patterns (28). The digestion product separated by agarose gel electrophoresis appears as bands whose patterns are usually species specific. In addition to the hsp65 gene, the 16S-23S rRNA internal transcribed spacer (ITS) region has been shown to be more discriminative than the 16S rRNA itself (6, 9, 10). A detailed understanding of ITS data was developed for the heterogeneous MAIS group, allowing the separation and classification of species belonging to this group as sequevars, and for other mycobacteria, including the M. tuberculosis complex (11), leading to the development of a line probe assay (INNO-LiPA-MYCOBACTERIA). This assay has been the first commercial DNA probe test able to identify the most frequently isolated mycobacterial species in parallel (18, 19, 27, 30). After amplification of the ITS, the biotinylated amplified DNA product is hybridized with 14 specific oligonucleotide probes immobilized as parallel lines on membrane strips. These include probes for the identification of strains belonging to the M. tuberculosis complex, M. kansasii (groups I, II, and III), Mycobacterium xenopi, M. gordonae, Mycobacterium chelonae (groups I, II, and III), and MAC (named MAIS complex to include M. scrofulaceum) with species-specific probes for M. avium, M. intracellulare, and M. scrofulaceum. Our laboratories have used this method for several years (reference 18 and J. L. Herrmann et al., unpublished results) and have encountered few difficulties in the identification of nontuberculous mycobacteria, with the exception of several isolates belonging to the MAIS group that were positive only with the complex-specific MAIS oligonucleotide probe. The aim of the study was (i) to clarify why strains were positive for the MAIS probe only and not with any specific probes for the complex members, (ii) to determine the contribution of several molecular markers (hsp65, ITS, and 16S rRNA) to identify these peculiar isolates at the species level, and (iii) to assess the diagnostic value of the INNO-LiPA-MYCOBACTERIA version 2.
MATERIALS AND METHODS
Between January 2000 and December 2002, 495 nontuberculous mycobacteria isolates were cultured, analyzed, and identified by AccuProbe (Gen-Probe Inc./bioMerieux, Marcy l'Etoile, France), and/or by INNO-LiPA-MYCOBACTERIA v1 (Lipav1; Innogenetics, Ghent, Belgium) and/or by classical methods. All strains were single clinical isolates (one isolate per patient), grown in Middlebrook medium supplemented with oleic acid-albumin-dextrose-catalase and PANTA (consisting of polymyxin B, amphotericin B, nalidixic acid, trimethoprim, and azlocillin) (960-MGIT; Becton-Dickinson, Le Pont de Claix, France, and BacTAlert, bioMerieux, Marcy l'Etoile, France). The majority of identified mycobacterial species were M. xenopi (182), M. kansasii (24), M. gordonae (72), M. chelonae (56), Mycobacterium abscessus (9), Mycobacterium fortuitum-Mycobacterium peregrinum complex (55), and Mycobacterium marinum (8). Eighty-nine isolates (18%) were identified as belonging to the MAIS complex. Within this group, 36 (40.5%) were identified as M. avium, 17 (19.1%) as M. intracellulare, and 1 (1.1%) as M. scrofulaceum. Of the remaining 35 isolates (39.3%), 34 isolates (38.2%) were positive only for the MAIS-Lipav1 probe, and 1 isolate (1.1%) identified as M. avium by AccuProbe was Mycobacterium genus probe positive but MAIS probe negative by Lipav1 (Table 1). These 35 isolates were further analyzed by hsp65 PRA sequencing, ITS sequencing, 16S rRNA sequencing, and the newer INNO-LiPA-MYCOBACTERIA v2 (Lipav2; Innogenetics, Ghent, Belgium).
For each sample, all amplifications and sequences were performed on the same DNA extract. Identifications made by Lipav1 and Lipav2 and by AccuProbe were performed according to the manufacturer's recommendations and using Auto-LiPA automated instrumentation (Innogenetics, Ghent, Belgium) for the LiPA tests. The procedure is similar to procedures described previously (18, 30). In comparison with Lipav1, the updated Lipav2 had new probes specific for Mycobacterium celatum, Mycobacterium genavense, Mycobacterium simiae, M. marinum-Mycobacterium ulcerans, Mycobacterium malmoense, Mycobacterium haemophilum, and Mycobacterium smegmatis and a second probe (MIN-2) specific for MAC sequevar Mac-A, for M. intracellulare. The hsp65 PRA was performed as described by Telenti et al. (28). Amplicons were further sequenced using the forward primer Tb11 (5'-ACCAACGATGGTGTGTCCAT-3') as described previously (28) after purification on a QIAGEN column as described by the supplier (QIAGEN, France). The 16S rRNA sequencing was performed as previously described (16), and the ITS sequencing was performed on purified amplicons made for Lipav1 and Lipav2 by Innogenetics (Ghent, Belgium) (6, 20). Comparison of the sequence data was performed using the Basic Local Alignment Search Tool (BLAST) (2, 3) for hsp65 (>375 bp) and the RIDOM Mycobacteria project (http://www.ridom.de/mycobacteria/) for 16S rRNA sequences (13). Alignments were given based on a single nucleotide difference, and final identification was chosen to correspond to the highest percentage of similarity given by the software (Table 1). Sequences were put in clusters according to the unweighted-pair group method using arithmetic averages, and dendrograms were drawn using the Kodon v2.04 (Applied Maths, St. Martens Latem, Belgium) software package.
Nucleotide sequence accession numbers. ITS, hsp65, and 16S rRNA nucleotide sequences for isolates falling into new clusters (isolates 4 to 13 and 18 to 27) and for isolates considered new sequevars (isolates 3, 14, 28, 29, 31, 34, and 35) were deposited in the GenBank database under accession numbers AY858989 to AY859052.
RESULTS
Thirty-five clinical isolates belonging to the MAIS complex were selected because they were not identifiable to the species level or because of discrepant results between AccuProbe and Lipav1 (Table 1, and Fig. 1 to 3). Thirty-four isolates presented only a MAIS-Lipav1-positive probe, without an identification at the species level (Table 1, isolates 1 to 34), whereas 1 isolate identified as M. avium by AccuProbe did not react with the MAIS-Lipav1 probe (Table 1, isolate 35).
Lipav2 allowed identification of 14 out of the 35 (40%) isolates to the species level (Table 1, isolates 1 to 14): isolate 1 was identified as M. haemophilum and isolates 2 to 14 were identified as M. intracellulare (Table 1). Isolates 2 and 3 were positive for the MIN-1-Lipav2 species-specific probe (Table 1). Isolates 4 to 14 were positive for the MIN-2-Lipav2 species-specific probe (Table 1). Nine isolates out of 11 MIN-2-positive isolates were identified as M. intracellulare by AccuProbe (Table 1, isolates 4 to 12). The 14 Lipav2 identifications were confirmed by hsp65 PRA and by hsp65 sequencing (Table 1). With the exception of the M. haemophilum isolate for which 16S rRNA was not sequenced, all 13 sequenced isolates (isolates 2 to 14) were also identified by 16S rRNA as M. intracellulare (Table 1). ITS sequencing, which defines sequevars, confirmed the identification of M. haemophilum (Table 1) and identified 10 MIN-2-Lipav2-positive isolates out of 11 as sequevar Mac-A (Table 1, isolates 4 to 13). This sequence has 100% homology to the newly defined species M. chimerae sp. nov. (38). Isolate 14 (MIN-2-Lipav2 positive) was considered an unknown sequevar, with the closest homology being obtained for sequevar Mac-C (Table 1). Isolates 2 and 3, the two MIN-1-Lipav2-positive isolates, were identified as MIN-C sequevar (Table 1).
This left us with 21 MAIS isolates not identified at the species level (by Lipav1 and Lipav2) (Table 1, isolates 15 to 34), including the M. avium isolate previously identified by AccuProbe that was negative for the MAIS-Lipav1 and MAIS-Lipav2 probes (Table 1, isolate 35). Additional DNA molecular techniques were necessary for a complete identification of these 21 isolates. By hsp65 PRA, isolates 16 to 29 and 31 to 33 were identified as M. avium; isolates 15, 30, and 34 were identified as M. intracellulare; and isolate 35 was identified as M. simiae (Table 1). Results of hsp65 sequencing confirmed the PRA results except for isolate 35, for which hsp65 sequencing identified no close match but a weak homology to M. avium complex, and isolates 30 and 34, identified by hsp65 PRA as M. intracellulare but as M. avium (isolate 30) and M. avium complex (isolate 34) by hsp65 sequencing (Table 1). Inside the group of 18 M. avium isolates identified by hsp65 sequence, ITS sequencing identified 9 isolates as Mac-U sequevars (Table 1, isolates 18 to 25 and isolate 27), 1 as a Mac-Q sequevar (isolate 30), 2 as Mac-E sequevars (isolates 16 and 17), 1 as a Mac-R sequevar (isolate 32), and 4 as unknown sequevars (isolates 26, 28, 29, and 31) (Table 1). Isolate 33 did not give an ITS sequence result that could be interpreted. Isolate 15, the unique M. intracellulare isolate by hsp65 sequence, was identified as a Mac-C sequevar (Table 1). The ITS sequence identified isolate 34 as M. paraffinicum and isolate 35 as M. intracellulare with weak homology (Table 1). It is of importance to note that the hsp65 sequence comparison for isolate 34 did not pick up M. paraffinicum, potentially due to the absence of this sequence in the database. The 16S rRNA sequence results identified isolates 15 to 33 as M. intracellulare as a first choice and M. avium as a second choice. Isolate 15, the unique Mac-C sequevar in this group, was identified as M. intracellulare by hsp65 PRA, hsp65 sequence, and 16S rRNA sequence (Table 1). Isolate 34 was confirmed by 16S rRNA as M. paraffinicum, and isolate 35 was identified as being most related to Mycobacterium interjectum and M. simiae by 16S rRNA sequencing (Table 1).
To fully summarize the results of the molecular markers, specific dendrograms were constructed using the unweighted-pair group method using arithmetic averages algorithm. This approach allows a more precise definition and identification of the close relationships between bacterial isolates using sequence data for specific molecular marker (ITS, hsp65, and 16S rRNA in our study). Two main clusters of clinical isolates were clearly individualized by ITS marker; one cluster was related to the Mac-A (or M. chimerae) sequevar, and one cluster was related to the Mac-U sequevar (Fig. 1). The Mac-A cluster was separated into two groups of isolates by the hsp65 molecular marker, one comprising isolates 4 to 6 and a second comprising isolates 7 to 13 (Fig. 2). Similarly, the Mac-U cluster was more heterogeneous according to the hsp65 molecular marker. Three additional isolates were included in the cluster: isolates 28 and 29, identified as Mac-Q-like sequevars, and isolate 33, for which no ITS sequence could be obtained (Fig. 2). By comparison, the 16S rRNA sequence encompassing region A individualized three clusters (Fig. 3): the Mac-A cluster with the addition of isolate 2, the Mac-U cluster with five more isolates (isolates 14, 15, 28, 31, and 33), and the last one comprising four isolates (isolates 16, 17, 29, and 32) also shown to be closely related by ITS and hsp65 molecular markers.
DISCUSSION
The ability to differentiate three species, three subspecies, and 28 serovars within the MAIS complex remains difficult despite existing methods of serotyping, DNA hybridization, or DNA sequencing (5, 9, 36). A number of clinical isolates and reference strains have been negative by both AccuProbes and by ITS species-specific probes (references 6 and 7 and the present study). Therefore, clinical identification is often limited to assigning an isolate merely to the MAIS complex, frequently considered by the clinician as an M. avium isolate. The use of molecular biology for such heterogeneous isolates, in addition to shorter delays for identification (26), has provided easy access to the precise identification of mycobacteria for any clinical microbiologist. Our study demonstrates that Lipav2 represents a valuable improvement because it is simple to perform and allows the identification of an additional 14 of the 89 MAIS isolates (15.7%, or 68 identified isolates out of 89) compared to Lipav1 (54 identified isolates out of 89). Its precision is confirmed by all molecular markers tested including 16S rRNA. This improvement will enable any mycobacteriologist to identify 21 mycobacterial species directly from a positive culture in less than a working day (37; L. Lebrun and J. L. Herrmann, unpublished results).
Second, our study highlights the difficulty of choosing specific genetic markers in a very heterogeneous group of clinical isolates, as for several strains of the MAIS complex, hsp65 and 16S rRNA sequencing did not classify the isolates into the same groups (Fig. 2 and 3). Examples of such discrepancies are found exclusively among isolates that are positive for the Lipav2-MAIS probe, where heterogeneity is high. For the isolates positive for the Lipav2-MIN-1 or MIN-2 probe, identification is clear and similar by all molecular markers.
Compared to other molecular markers, 16S rRNA data are available for the majority of bacteria, and the 16S rRNA sequencing method, therefore, is considered by many as the gold standard for identification and taxonomic purposes. However, although its routine use to distinguish well-resolved species might be sufficient, very recently divergent species may not be recognizable (8, 37). Examples of the limited power of 16S rRNA sequencing in the mycobacteria genus exist for strains related to M. intracellulare species, with some intraspecific allelic diversity, and was demonstrated in the Lipav2-MAIS probe-positive isolates (reference 22 and our study). The hsp65 or ITS molecular markers have been shown to be more discriminating first in the very heterogeneous MAIS complex (Fig. 1 to 3) (6, 9, 20, 25; our study) and then for other atypical mycobacteria (M. kansasii groups, for example) (1). Despite the advantage of being available in any laboratory performing PCR techniques and DNA restriction, hsp65 PRA has several well-known drawbacks (lack of standardization and slight variability in fragment length, for example) (22). Species with overlapping patterns and/or multiple patterns within a single species do exist (22, 31). An identification was given for each isolate by hsp65 PRA in our study. All were confirmed by hsp65 sequencing except isolates 30, 34, and 35, which means that an M. avium or an M. intracellulare hsp65 PRA result does not always correlate with the gene sequence result.
The 280-bp rRNA region or ITS, which is transcribed but does not encode a final product, allows more sequence variation than 16S rRNA. It is characterized by a higher rate of polymorphisms, which allows a more precise species definition. However, in some cases the presence of intraspecies variability may result in confusion (8, 12). Inside the MAIS complex, isolates typed as being M. avium and M. intracellulare by other techniques belonged to sequevars Mac-A to Mac-G and Min-A to Min-D, respectively (6, 7, 11). Several Lipav1 isolates positive only for the MAIS probe but identified as M. intracellulare by AccuProbe are now identified as such by the new Lipav2 due to the addition of the Min-2- or Mac-A-specific probe (32; our study). These isolates belonged to sequevar Mac-A. As described recently, the ITS sequence of the Mac-A sequevar is 100% homologous with the newly defined species M. chimerae sp. nov. (33). Ten isolates of 35 in our study, with 100% homology to sequevar Mac-A, could be named M. chimerae sp. nov. The MAC or MAIS intermediates belonged to sequevars Mac-B to Mac-O and Mac-Q to Mac-U. Within the group of isolates positive only for the Lipav2-MAIS probe, hsp65 sequencing gave the highest homology to M. avium compared to 16S rRNA, which favored identification of M. intracellulare-like isolates. Overlapping of the mismatches in the comparison of 16S rRNA sequences made it difficult to unambiguously identify the best match, although one M. intracellulare reference strain was always picked up as the first choice. However, ITS and hsp65 dendrograms (Fig. 1 and 2) allow a clearer distinction between two clusters of clinical isolates, including the Mac-A cluster, compared to the 16S rRNA dendrogram. The second observed cluster (10 of 35 isolates) belonged to Mac-U (Table 1, isolates 18 to 27, and Fig. 1).
Several isolates (Table 1, isolates 3, 14, 26, 27, 28, 29, and 31) were not 100% homologous to a defined sequevar. For all these isolates, the definition of new sequevars can be achieved if we apply the definitions given by Frothingam and Wilson (9) and De Smet et al. (6) for the identification of a new sequevar.
Isolate 34 was identified as M. paraffinicum by ITS and 16S rRNA sequencing. M. paraffinicum is synonymous with M. scrofulaceum, distinguished mainly by a negative urease reaction (35). This sequence homology explained the positive hybridization with the Lipav1 and Lipav2 MAIS complex probe for this clinical isolate, as this probe includes the recognition of M. scrofulaceum. However, the slight ITS sequence difference explained the negative reaction with the M. scrofulaceum species-specific probe. For isolate 35, different results were found by each molecular marker (with very low homology), indicating that this isolate should be considered a new mycobacterial species not yet genetically and phenotypically defined.
Overrepresentation of two sequevars (Mac-A and Mac-U) can be correlated with a higher frequency of colonization or disease in humans. All studied strains were isolated from patients with pulmonary disorders (bronchiectasias and lung cancer) (data not shown). None of the Mac-U isolates had characteristics that fulfill the American Thoracic Society criteria (34) and, therefore, have to be considered responsible for colonization of the patient's lungs. Interestingly, all Mac-U isolates were isolated from native Africans. Two patients had three and four smear-negative, culture-positive specimens with Mac-A isolates. One of them died rapidly with major changes shown on the chest X ray. Mac-A has also previously been observed in pulmonary disease (33). All the other Mac-A isolates were obtained from unique smear-negative, culture-positive specimens (L. Lebrun and J. L. Herrmann, unpublished results). With more precision in the identification of mycobacteria, microbiologists in collaboration with clinicians will be able to follow up such new isolates to gain a more complete understanding of the behavior of mycobacteria either as commensal or opportunistic pathogens.
In conclusion, the availability of molecular tests such as the Lipav2 in the mycobacteriology laboratory will allow increased efficiency in identification. Precise identification can then be correlated with clinical disease and will allow the clinician to consider such isolates as potential or opportunistic pathogens. The question of which molecular tool to use is difficult to answer. Lipav2 is simple to use but fails to identify a few isolates. Sequencing of the 16S rRNA and the hsp65 genes will always provide a result but in some cases may not identify the tested isolate. In addition, the use of this technique may not be feasible in all mycobacteriology laboratories because of the requirement for extensive equipment. When isolates belong to well-defined species such as those in the Lipav2-MIN-1 or Lipav2-MIN-2 probe-positive isolates, 16S rRNA sequencing is sufficiently discriminating. However, when heterogeneity in the mycobacterial population is increased, a more precise result will require either ITS or hsp65 sequencing, as is the case for the isolates that were probe positive only for Lipav2-MAIS.
ACKNOWLEDGMENTS
We are grateful to Ben Marshall for critical reading of the manuscript, to Rebecca Millecamps and Wouter Mijs for their valuable help in sequence comparisons and dendrograms, and to Koenrad De Smet, Roland Brosch, and Laura Vernoux for their helpful comments and support.
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