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J Clin Microbiol. 2004 Jul; 42(7): 3000–3011.
PMCID: PMC446301

Assessment of Partial Sequencing of the 65-Kilodalton Heat Shock Protein Gene (hsp65) for Routine Identification of Mycobacterium Species Isolated from Clinical Sources


We assessed the ability of an in-house database, consisting of 111 hsp65 sequences from putative and valid Mycobacterium species or described groups, to identify 689 mycobacterial clinical isolates from 35 species or groups. A preliminary assessment indicated that hsp65 sequencing confirmed the identification of 79.4% of the isolates from the 32 species examined, including all Mycobacterium tuberculosis complex isolates, all isolates from 13 other species, and 95.6% of all M. avium-M. intracellulare complex isolates. Identification discrepancies were most frequently encountered with isolates submitted as M. chelonae, M. fortuitum, M. gordonae, M. scrofulaceum, and M. terrae. Reexamination of isolates with discrepant identifications confirmed that hsp65 identifications were correct in a further 40 isolates. This brought the overall agreement between hsp65 sequencing and the other identification methods to 85.2%. The remaining 102 isolates had sequence matches below our acceptance criterion, had nondifferential sequence matches between two or more species, were identified by 16S rRNA sequencing as a putative taxonomic group not contained in our database, or were identified by hsp65 and 16S rRNA gene sequencing as a species not in our biochemical test database or had conflicting identifications. Therefore, to incorporate the unconfirmed isolates it was necessary to create 29 additional entries in our hsp65 identification database: 18 associated with valid species, 7 indicating unique sequences not associated with valid or putative species or groups, and 4 associated with unique, but currently described taxonomic groups. Confidence in the hsp65 sequence identification of a clinical isolate is best when sequence matches of 100% occur, but our data indicate that correct identifications can be confidently made when unambiguous matches exceeding 97% occur, but are dependent on the completeness of the database. Our study indicates that for hsp65 sequencing to be an effective means for identifying mycobacteria a comprehensive database must be constructed. hsp65 sequencing has the advantage of being more rapid and less expensive than biochemical test panels, uses a single set of reagents to identify both rapid- and slow-growing mycobacteria, and can provide a more definitive identification.

In 1989, Hance et al. (18) reported amplifying a fragment of the 65-kDa heat shock protein gene (hsp65) to detect and, coupled with species-specific probes, identify mycobacteria from clinical samples. After this, Plikaytis et al. (37) and Telenti et al. (56) described, using separate gene regions, the successful identification of mycobacteria by using restriction digest analysis of amplified hsp65 fragments (hsp65 PRA). The technique, as described by Telenti et al., has been frequently investigated as a means of identifying mycobacteria and, although hsp65 PRA is an accepted means of identifying mycobacteria, it does have functional limitations, some of which have been addressed (3, 6, 10, 11, 13, 15, 19, 39, 40, 43, 49, 55, 69, 71).

Kapur et al. (23), recognizing the limitations of hsp65 PRA and the potential advantage of generating direct unambiguous data, were the first to sequence the hsp65 amplicon generated by the Telenti primers as a means for identifying mycobacteria. This technique has since been used by many investigators to identify species, as well as characterize and define groups within a number of mycobacteria, but, like hsp65 PRA, the method has limitations (31, 40, 48, 52, 53, 61, 62, 67). In spite of a large number of publications describing the use of hsp65 sequencing to identify mycobacteria, we are not aware of any publication describing its integration into a diagnostic laboratory as the primary means of identification (6, 14, 23, 31, 40, 52, 53). Therefore, we assessed the viability of using hsp65 sequencing to identify all mycobacteria routinely isolated by a clinical mycobacteriology laboratory, and we report here on the results of the present study.


Mycobacterial isolates.

Type and reference strains were purchased from the American Type Culture Collection (ATCC) or the Deutsche Sammlung von Mikroorganismen und Zelkulturen GmbH (DSM), Braunschweig, Germany (Table (Table1).1). The strains were reconstituted and cultured according to the manufacturer's instructions at the appropriate temperature. To ascertain purity, reference and clinical strains received from other researchers, and biochemically typical strains from our laboratory were cultured on Lowenstein-Jensen medium at the appropriate temperature. Mycobacterium lepraemurium and M. visibile were received as paraffin-mounted tissue samples, which were deparaffinized; the tissue was minced; and the DNA was extracted by using a tissue extraction kit (Qiagen, Inc., Valencia, Calif.) according to the manufacturer's instructions.

List of valid and putative type strains of Mycobacterium species sequences

Conventional identification.

Isolates from clinical sources or our culture collection had been identified by using AccuProbe (Gen-Probe), which is specific for M. avium-M. intracellulare complex, M. tuberculosis complex, M. gordonae, or M. kansasii with confirmatory biochemical testing. All negative AccuProbe isolates were identified by using 16S rRNA gene sequencing or phenotypic identification, such as standard biochemical tests, pigment production, and growth rate and colony characteristics. Isolates were cultured to Lowenstein-Jensen medium to determine growth rate and colony morphology and, based upon these characteristics, subjected to a panel of biochemical tests. Phenotypic testing used at British Columbia Centre for Disease Control may include the following: growth at 25, 32, 37, and 45°C; arylsulfatase (3 days); semiquantitative catalase (>45 mm); 68°C catalase; Tween hydrolysis (5 or 10 days); tellurite reduction; niacin production and nitrate reduction; pyrazinamidase (4 days); and iron uptake and growth on 5% sodium chloride and MacConkey agar without crystal violet. Isolates generously donated from other researchers were accepted as identified unless hsp65 identification did not match the submitted nomenclature.

DNA extraction.

Bacteria were harvested by removing approximately 1 mm3 of biomass with sterile wooden sticks and were placed into a Mycobacterium lysis tube (AccuProbe; GenProbe, Inc., San Diego, Calif.) containing glass beads and 500 μl of 0.5× TBE buffer (Gibco/Invitrogen Corp., Carlsbad, Calif.). Bacteria were killed by heating them to 95 ± 5°C for 10 min and then lysed by sonication at 35 kHz for 15 min. Extracts were centrifuged for 2 min at 12,000 × g to pellet the cellular debris, and 200 μl of supernatant was transferred to a clean 0.6-ml microcentrifuge tube. All mycobacterial isolates received in paraffin-embedded tissue blocks were processed by heating them to 95°C in a BioOven (Baxter) to melt the paraffin, and a sterile disposable scalpel blade was used to excise ca. 25 mg of tissue, which was placed in a sterile 1.5-ml microcentrifuge tube. The excised tissue was then washed with 1 ml of xylene to dissolve the remaining paraffin in the tissue and centrifuged at 15,000 × g for 5 min at room temperature. The supernatant was discarded, and the process was repeated. The tissue was washed in 1 ml of 80% ethanol to remove any remaining xylene and centrifuged at 15,000 × g for 5 min at room temperature. The supernatant was discarded, and the process was repeated. The cleaned tissue was allowed to air dry in a biological safety cabinet for 30 min and then lysed by using a QIAamp DNA minikit according to the manufacturer's protocol.

DNA amplification.

PCR amplification of a 441-bp fragment of the hsp65 gene was performed for all type and reference strains, as well as for the isolates. This was accomplished by adding 21.25 μl of UltraPURE Distilled DNase, RNase-Free Water (Gibco/Invitrogen) and 0.625 μl of 20 μM Tb11 5′-ACCAACGATGGTGTGTCCAT-3 and Tb12 5′-CTTGTCGAACCGCATACCCT-3 primers, as described by Telenti et al., and 2.5 μl of bacterial lysates to puRe Taq Ready-to-Go PCR beads (Amersham Biosciences, Piscataway, N.J.) (56). The amplification was performed by using a Perkin-Elmer 9600 GeneAmp PCR System (Perkin-Elmer-Cetus, Foster City, Calif.) with a program of 45 cycles at 96°C for 40 s, 60°C for 50 s, and 72°C for 1 min, followed by a final extension at 72°C for 7 min. Then, 2.5 μl of each post-PCR mixture was subjected to electrophoresis in a 2% agarose gel containing 1% ethidium bromide and an appropriate size standard to confirm that a 441-bp fragment had been amplified. The amplified DNA was cleaned by using a QIAquick PCR purification kit (Qiagen) according to the manufacturer's instructions, and the DNA was quantified in sterile, disposable UVettes (Eppendorf, Hamburg, Germany) with a UV absorbance at 260 nm as determined by using a BioChrom Ultrospec 3100 Pro spectrophotometer (BioChrom, Ltd., Cambridge, United Kingdom) and then diluted with sterile distilled water to a final concentration of 10 ng/μl.

hsp65 sequencing.

Sequences for Mycobacterium canariense (AY255477), Mycobacterium nebraskiae (AY368457), Mycobacterium sherrisii (AY365190), and M. leprae (NC_002677) were obtained from GenBank and edited as required. All other sequences were determined by using an ABI Prism 310 or 3100 genetic analyzer (Applied Biosystems, Foster City, Calif.) and BigDye terminator cycle sequencing ready reaction kit (version 3.0 or 3.1; Applied Biosystems). Sequencing reactions consisted of 2 μl of BigDye terminator cycle sequencing reagents, 6 μl of BigDye terminator cycle sequencing buffer, 1.6 μl of 2 μM concentrations of primers Tb11or Tb12, and 3 or 1 μl of standardized amplicon eluate added to the Tb11 and Tb12 reactions, respectively, as well as sufficient UltraPURE Distilled DNase, RNase-Free Water (Gibco/Invitrogen) to make a 20-μl reaction. It was determined that sequencing reactions were optimal at different template concentrations (unpublished data) and that the best-quality sequence with regard to read length, minimal baseline activity, least amount of spurious base insertions, and base call quality occurred when 10 ng of DNA was used for the Tb12 primer reaction and 30 ng of DNA was used for the Tb11 primer reaction. Cycle sequencing was performed by using a Perkin-Elmer 9600 GeneAmp PCR system programmed for 25 cycles at 96°C for 10 s, 50°C for 5 s, and 60°C for 4 min. Sequencing products were cleaned according to the manufacturer's instructions (Applied Biosystems), suspended in 25 μl of ABI template suppression reagent or Hi-Di formamide as appropriate (Applied Biosystems), heat denatured at 95°C for 2 min, placed on wet ice for 2 min, and mixed by using a vortex mixer prior to being analyzed with an ABI Prism 310 or 3100 genetic analyzer according to the manufacturer's protocol.

16S rRNA gene sequencing.

Isolates were sequenced, as required, at the National Reference Centre for Mycobacteria, National Microbiology Laboratory, Winnipeg, Manitoba, Canada, as previously described or in our laboratory by the method described by Lane et al. (28, 63). Identifications were assigned to isolates by comparing their sequences to RIDOM (http://www.ridom-rdna.de) and using interpretations as previously described (63).

PCR and sequencing controls.

Negative PCR amplification controls, which consisted of sterile distilled water being added to the primary PCR mixture instead of DNA template, were processed with each batch of isolates tested. The presence of any bands in these controls indicated contamination had occurred, and in such cases the entire batch was discarded and the process was repeated from biomass harvesting. Also, an isolate of M. asiaticum ATCC 25276T of known sequence was included from the biomass-harvesting stage to act as a positive control in all PCR and sequencing reactions, as well as to determine the polymerase fidelity and base call accuracy. Any deviation from the known sequence resulted in the entire batch being discarded, and the analysis was repeated from biomass harvesting.

Analysis of sequence data.

Sequences were assembled and edited by using SeqMan software (DNAStar, Madison, Wis.), and the primer sequence was deleted to decrease fragment homology. Isolates were identified by comparing unknown sequences by using a FASTA BLASTn search to an in-house database of sequences consisting of type and reference strains from external culture collections (2, 68). CLUSTAL analysis was performed by using MegAlign (DNAStar) when isolates did not match >97% against a species type strain. When this occurred, isolates were assigned a temporary nomenclature of “Mycobacterium species” with our assigned accession number. These isolates were then incorporated into future versions of our database so that it became heuristic or self-learning.

GenBank sequence accession numbers.

The unique hsp65 sequences determined in the present study were deposited in GenBank under accession numbers AY550207 to AY550239.


No incidences of hsp65 gene amplification or sequencing failure, interprimer sequence variation from 401 nucleotides (nt), or control failures occurred during the analysis of the 111 Mycobacterium type and reference strains or the 689 mycobacterial isolates. Amplification occurred in 23 nonmycobacterial isolates; three were Nocardia spp. and 20 were Tsukamurella spp., which were easily differentiated from mycobacteria. This was expected, since the use of hsp65 PRA to identify Nocardia and related genera had been described earlier (69). Phylogenetic analysis of putative species M. lepraemurium and M. visibile indicated that they were related to M. leprae and, for analysis purposes, they were included with the slow-growing mycobacteria. Among the 50 slow-growing Mycobacterium species examined, 15 incidences occurred where type strains were >97% related (Fig. (Fig.11 and Table Table2).2). Of the 61 rapid-growing Mycobacterium species examined, 25 incidences occurred in which type strains were >97% related (Fig. (Fig.22 and Table Table2).2). With the exception of species within the M. tuberculosis complex and subspecies of M. avium and M. fortuitum, no rapid or slow-growing species had identical matches.

FIG. 1.
Phylogenetic tree for single representatives of both putative and valid species and groups of slow-growing mycobacteria and anchored by Nocardia asteroides as determined by the CLUSTAL W algorithm of MegAlign (version 5.01; DNAStar). An asterisk indicates ...
FIG. 2.
Phylogenetic tree for single representatives of both putative and valid species and groups of fast-growing mycobacteria and anchored by N. asteroides as determined by the CLUSTAL W algorithm of MegAlign (version 5.01; DNAStar). An asterisk indicates no ...
Percentage sequence match for putative and valid Mycobacterium spp.

We found that a criterion of first distinct hsp65 sequence match ≥97% confirmed the identification of 547 of 689 (79.4%) isolates (Table (Table3.).3.). Identification discrepancies were found in 20 of 34 species or groups (Table (Table3).3). No discrepancies were found in the species submitted as M. asiaticum, M. branderi, M. goodii, M. holsaticum, M. lentiflavum, M. mucogenicum, M. neoaurum, M. nonchromogenicum, M. obuense, M. peregrinum, M. phlei, M. szulgai, M. thermoresistible, or M. tuberculosis. Identification confirmation may have occurred within these species since they are reliably identified by biochemical tests or commercial probes or, in the case of the more recent species, were identified by 16S rRNA gene sequencing. Although identifications for most isolates of the M. avium-M. intracellulare complex were confirmed, identification discrepancies did occur (Table (Table3).3). The type strains for these two species vary by 2% (Table (Table2)2) and are quite easily differentiated, a situation reflected by most of the clinical isolates, and we were able to confirm the identification of 218 of 228 isolates, 174 of 228 (76.3%) as M. avium and 44 of 228 (19.3%) as M. intracellulare. Of the 10 discrepant isolates, 5 were attributed to a putative M. intracellulare variant and 5 isolates failed to meet our identification criteria (30).

Comparison of mycobacteria identified by biochemical test panels, AccuProbe tests, and 16S rRNA gene sequencinga

Many of the discrepancies were attributed to isolates submitted as M. chelonae (33), M. fortuitum (17), M. gordonae (42), M. avium-M. intracellulare complex (10), M. scrofulaceum (8), and M. terrae (9). Reexamination of data and further testing of isolates indicated that 23 isolates previously identified as M. chelonae were probably M. abscessus, and another 4 were M. mucogenicum. Sequence distance analysis of the type strains indicated that M. chelonae is 92.8 and 91.5% related, respectively, and is easily differentiated from the latter species. A further six isolates were identified as the putative species M. fuerthensis, but examination of biochemical test characteristics did not support the hsp65 identification (U. Reischl, unpublished data) and the identifications were left as discrepant. When the biochemical data from 13 isolates of M. fortuitum were reexamined it was determined that they were probably M. peregrinum, corroborating the hsp65 identification. Three isolates were identified as M. mucogenicum (2) and M. senegalense (1), species that may be difficult to distinguish from M. fortuitum by biochemical tests (8, 66). Sequence distance analysis indicated that M. fortuitum was 93.5% related to M. mucogenicum, 97% related to M. peregrinum, and 97.8% related to M. senegalense and that the species should be easily distinguished from one another, and we included these identifications as being correct. This brought the agreement to 587 of 689 (85.2%) isolates. The inability of our type strain database to identify 42 of 120 isolates of M. gordonae is a major factor for the poor corroboration between methods. We used MegAlign to group the isolates that had not been identified as M. gordonae and selected centrostrains to create three further database entries. This allowed us to include the 42 outlying M. gordonae isolates. We applied this process to all other isolates not identified by sequences from valid and putative species.


This study assessed the feasibility of sequencing the 65-kDa heat shock protein gene hsp65 as the primary means for the routine identification of mycobacteria from clinical sources. This technique, although widely published in the literature, has not, to the best of our knowledge, been examined as the primary means for identifying all mycobacteria isolated in clinical mycobacteriology laboratories (3, 14, 23, 31, 40, 52, 53). This may be due to the cost of purchasing a DNA sequencer and the perceived cost of reagents or to the difficulty in sequencing DNA (9). Recent improvements in DNA sequencers, base calling software, and the cost of associated reagents are such that most mycobacteriology laboratories could now perform this technique (9, 62). Analyses of reagents and labor determined that hsp65 sequencing costs were similar those for 16S rRNA gene sequencing, which have been recently calculated at approximately $30 (U.S. dollars) per sample. This has been determined to be less expensive than the identification of isolates by biochemical testing (9). On primary examination, hsp65 sequencing is a more expensive means of identifying the limited species for which a commercial species-specific probe exists (unpublished data) (9). Primary identification of 405 of 689 (58.8%) of the isolates we analyzed was accomplished by using commercial probes. However, erroneous probe testing, coupled with rudimentary confirmatory biochemical testing as suggested by the probe manufacturer, can result in the use of probes being equal to or more expensive than identification by hsp65 sequencing. A review of a small percentage of the isolates identified by commercial probes or biochemical testing showed many cases where isolates were tested by probe, found to be negative and identified by biochemical tests, an isolate was tested with same probe more than once or was tested with multiple species-specific probes, with the second or third probe yielding an identification (unpublished data). These incurred costs would have been avoided; since we are now able to identify all isolates our laboratory encounters with a single sequencing reaction. hsp65 sequencing also has the additional capability of determining species within the M. avium-M. intracellular complex and the ability to correctly identify species that are known to have false-positive probe reactions (12, 13, 44).

In our opinion, the main deterrents for the primary use of hsp65 sequencing as a routine means of identifying mycobacteria reside with the need for a comprehensive database and the ability to determine the differential percentage match between clinical isolates and valid species (40). Therefore, the purpose of the present study was twofold: first, to produce a scrutinized database that contained hsp65 sequences for all valid and putative mycobacteria species or groups isolated by our laboratory and, second, to attempt to determine what percentage sequence match constituted a correct identification for these isolates. We constructed our own hsp65 sequence database since previously deposited hsp65 sequences contained in internet sequence repositories have not been examined for correctness or completeness, and no repository contains entries for all valid and putative species of mycobacteria isolated from clinical sources (62; unpublished observations). Also, although guidelines exist for the use of sequencing the 16S rRNA gene as a means of identifying bacteria, including mycobacteria, there are limited or no guidelines for identifying mycobacteria using other gene sequences, including the hsp65 gene (22, 23, 31, 48).

Sequence divergence analysis of our database indicated that of the 50 slow-growing and 61 rapid-growing Mycobacterium species included, 15 incidences and 25 incidences occurred, respectively, where species were >97% related, with a few species differentiated by only a single nucleotide (Table (Table2).2). This allowed us to determine, prior to examining clinical isolates, where identification difficulties might occur and provided an impression of the sequence match required to accurately identify a clinical isolate. Of note is the fact that hsp65 sequencing was not able to distinguish between members of the M. tuberculosis complex, with the exception of the recently described species M. canettii, which varied by a single nucleotide from the other species (36, 65). Nor was it able to differentiate the subspecies of M. fortuitum or M. avium (unpublished data). Therefore, all M. tuberculosis isolates had equal matches with M. tuberculosis and M. bovis but were identified as M. tuberculosis complex, and all isolates of M. fortuitum or M. avium were reported to the species level only.

A review of the literature, as well as the currently valid species list (www.bacterio.cict.fr), indicated that the utilization of molecular or other techniques are detecting an ever-increasing number of new species, subgroups within current species, or “taxonomic orphans” isolated from clinical specimens or other sources (1, 3-5, 7, 16, 17, 20, 22, 24, 25-27, 30, 32-35, 38, 41, 42, 44-47, 50-54, 57-61, 63, 64, 67). The description of new species and groups from clinical sources can overwhelm the identification capability of current biochemical testing protocols, with some species or groups unlikely or not able to be differentiated by these tests (8, 66; A. Roth, unpublished data). In the present study, with the exceptions listed previously, all defined species and named groups had unique sequences, although some were closely related. Therefore, our hsp65 sequence database was more comprehensive than our biochemical test database and allowed us to reliably identify or exclude isolates as M. abscessus, M. avium, M. branderi, M. chelonae, M. fortuitum, M. goodii, M. heckeshornense, M. holsaticum, M. interjectum, M. intracellulare, M. lentiflavum, M. mucogenicum, M. novocastrense, M. obuense, M. peregrinum, M. tuberculosis, and M. szulgai, as well as more recently an isolate of M. hiberniae that was not included in our study. Many of these species are only recently described, are not included in our biochemical test database, may be difficult to identify by biochemical testing, or may not be recognized because they are infrequent causes of infection in humans (5, 8, 16, 17, 21, 24, 27, 29, 34, 39, 41, 42, 45, 47, 70, 71, 72). Also, the current quality of hsp65 sequencing performed allows for the integration of hsp65 sequences deposited in GenBank directly into our database, with sequences for Mycobacterium canariense (AY255477), M. nebraskiae (AY368457), and M. sherrisii (AY365190) being examples. This provides us with the ability to constantly upgrade our database and the capability to identify the most recently described species.

It became apparent during the course of the present study that the sequences from some isolates did not meet our identification criterion and were not able to be confirmed or identified. Fortunately, more-comprehensive 16S rRNA databases exist for the identification of mycobacteria, and we were able to assign to putative species or groups nine clusters of isolates on the basis of 16S rRNA gene sequencing. However, the remaining isolates had sequences that were not sufficiently related to any deposited 16S rRNA sequences to assign them to any species or group. Therefore, we created 20 database entries based on these unique hsp65 sequences and described them as a variant of the species they had been identified as by probe or biochemical testing or, when not identified by any of the other methods, we described them as a unique species. Without these additional 29 hsp65 sequences we would have considered hsp65 sequencing unsuitable as a primary means of identifying mycobacteria from clinical sources.

Confidence in the correctness of the identification of clinical isolates depends on the degree of sequence homology between the isolate and a type strain, putative species, or group. Naturally, the level of confidence in the identification increases as the percentage sequence match approaches 100%, but it is also dependent on no two species or groups having identical sequences. Only 328 of 689 (47.6%) of the isolates we examined had sequences identical to a defined species or group, with 24 isolates being assigned to a species other than the primary identification. A decrease in the confidence of an isolate's identification occurs when the percentage match is <100%, which occurred in the remaining isolates investigated. In our experience, a match ≥99% is almost always correct; exceptions do occur with some species, such as the M. marinum-M. shottsii-M. ulcerans group, requiring a 100% match, or other species, such as M. avium, where a confirmed identification was obtained with a 97.3% match (Tables (Tables22 and and3)3) (38).

Using the criterion of the highest unambiguous sequence match exceeding 97%, hsp65 sequences confirmed the identification of 587 of 689 (85.2%) isolates. The inability of hsp65 sequencing to confirm the identification of some isolates was compounded since the majority of the isolates were identified by using biochemical techniques or commercial probes, both of which incorrectly identify some clinical isolates (8, 12, 30, 39, 41, 42, 43, 44, 45, 46, 47, 59, 71). We estimated that 85 isolates had been misidentified by using the latter techniques (Table (Table33).

Of the 102 identifications that were not confirmed by hsp65, the majority had a sequence match of <97%, had equal sequence matches with two or more valid species, or were identified as another species or group in spite of having differential biochemical test characteristics or positive probe results. This occurred most commonly with isolates primarily identified as M. avium-M. intracellular complex, M. chelonae, M. fortuitum, M. gordonae, M. scrofulaceum, or M. terrae, which in our opinion suggests inadequate taxonomy within the currently described species. Therefore, based on our experience and with our current database we are confident in identifying any isolate with an unambiguous sequence match exceeding 97%.

Our investigation provides evidence that hsp65 sequencing has the potential of being an accurate, reliable, and effective means for identifying clinical Mycobacterium isolates. It has the advantages over biochemical test profiles of being rapid, less expensive, and using a single set of reagents for identifying both fast- and slow-growing mycobacteria. The method has advantages over 16S rRNA gene sequencing for the identification of mycobacteria since it is more differential and there is no need to sequence a fragment larger than 401 nt (23, 40). However, for the method to be an efficient and cost-effective means of identification a comprehensive database must be available to be queried (9, 62; unpublished observations). Although hsp65 sequences for type strains of valid species are available in internet repositories, few entries exist for putative species and groups or undefined or biochemically aberrant groups, nor are there multiple sequences from the same species. We were aware that the lack of these sequences would be a major handicap in using hsp65 sequencing as a routine means of identifying mycobacteria from clinical sources. We were surprised that ca. 15% (102 of 689) of our clinical isolates would fall into this category. However, ca. 50% of these isolates were of questionable clinical significance but did represent identification failures. By far the most intriguing fact was the degree of sequence heterogeneity in M. gordonae, since we were only able to identify 65% (78 of 120) of the clinical isolates based on the hsp65 sequence for type strain with the discrepant isolates being identified as M. asiaticum, a species easily differentiated by biochemical testing (66). To overcome this, as well as other misidentifications, an additional 29 sequence entries had to be created and added to our database. These database additions were sufficient to allow us to assign all isolates in the present study to either a valid or putative species or group. Although our assessment continues, it is apparent that hsp65 sequencing has a place in the routine mycobacteriology laboratory, but only once a comprehensive in-house database is constructed and evaluated.


We thank N. Chin for acquisition of type strains; G. Appleyard, F. Baggi, B. Carbonnelle, K. Chemlal, B. Elliot-Brown, M. J. Garcia, L. Hall, D. Kiska, M. Pickard, G. Pfyffer, M. Rhodes, S. Smole, M. Takahashi, E. Tortoli, C. Truffot, C. Turenne, U. Reischel, and R. Wallace III for providing mycobacterial isolates or DNA; and R. Wallace III, B. Elliot-Brown, C. Turenne, and R. Behme for reviewing the manuscript.


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