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Journal of Clinical Microbiology logoLink to Journal of Clinical Microbiology
. 2011 Jun;49(6):2296–2303. doi: 10.1128/JCM.02602-10

Web-Accessible Database of hsp65 Sequences from Mycobacterium Reference Strains,

Jianli Dai 1,4,*, Yuansha Chen 1,2, Michael Lauzardo 3,4
PMCID: PMC3122750  PMID: 21450960

Abstract

Mycobacteria include a large number of pathogens. Identification to species level is important for diagnoses and treatments. Here, we report the development of a Web-accessible database of the hsp65 locus sequences (http://msis.mycobacteria.info) from 149 out of 150 Mycobacterium species/subspecies. This database can serve as a reference for identifying Mycobacterium species.

TEXT

Included among the mycobacteria are a large number of clinically important pathogens, both obligate (e.g., M. tuberculosis and M. leprae) and opportunistic (e.g., M. avium, M. kansasii, etc.). The impact of mycobacteria on human morbidity and mortality is hard to overstate. Although tuberculosis (TB) is arguably one of the most important infectious diseases in the world, the incidence of disease due to nontuberculous mycobacteria (NTM) has been steadily increasing worldwide (6, 27, 42, 48, 59) and has likely far surpassed TB in the United States (7). Thus, the American Thoracic Society and the Infectious Diseases Society of America have recommended identifying the clinically significant NTM to the species level upon the diagnosis of nontuberculous mycobacterial diseases (17).

The Mycobacterium genus currently includes 150 species/subspecies (http://www.bacterio.cict.fr/m/mycobacterium.html), and the number has been increasing exponentially (Fig. 1), making identification difficult and challenging. Current identification based on biochemical tests of culture is slow and inadequate to differentiate among closely related mycobacteria, especially for those mycobacteria that are biochemically inert and slowly growing. In contrast, molecular identification methods based on PCR and nucleotide sequencing dramatically shorten the detection time and improve the accuracy of identification. The most common genomic loci used in molecular identification are the 16S rRNA gene (25), 16S-23S rRNA internal transcribed spacer (15), hsp65 (40, 47), and rpoB (22). Almost all recent publications on new mycobacterial species compared sequences from multiple loci to those of established species, and hsp65 is always included. A previous study has shown a 99.1% agreement between identification using hsp65 sequencing and a conventional method combining Accuprobes, biochemical test panels, or 16S rRNA gene sequencing (33), suggesting that hsp65 sequencing is an effective method for identifying mycobacterial species. It was also suggested that completeness of the sequence database was critical for this identification method (33). To our knowledge, there is no publicly accessible hsp65 sequence database that covers all currently validated mycobacterial species. Laboratorians and researchers have to rely on the sequences deposited in public databases, such as GenBank, EMBL, and DDBJ. The vast majority of these mycobacterial entries are from uncharacterized strains or undetermined species, and problematic sequence entries, such as base errors, incomplete sequences, invalid or misidentified species, and even species-strain mismatches, are frequently present. This makes identification by searching public databases onerous and error prone. To facilitate the taxonomic identification of mycobacterial isolates, we have developed a Web-accessible database of mycobacterial hsp65 sequences from 149 species/subspecies, excluding the problematic GenBank entries mentioned above.

Fig. 1.

Fig. 1.

Numbers of approved Mycobacterium species/subspecies from 1896 to 2010.

The type strain hsp65 sequences of 147 mycobacterial species/subspecies were downloaded from GenBank. M. lepraemurium and M. leprae do not have type strains due to difficult cultivation. We chose M. lepraemurium TS130 and M. leprae TN as their reference strains and obtained their hsp65 sequences with the GenBank sequence accession numbers AY550232 (34) and NC_002677 (11). Multiple identical GenBank sequences from the type strain of the same species were combined into a single entry in our database (Table 1). We trimmed the sequences to 401 bp, which corresponds to nucleotide positions 165 to 565 of the M. tuberculosis H37Rv hsp65 gene and can be amplified and sequenced using primers Tb11 and Tb12 (47). Entries that did not completely cover this 401-bp hsp65 locus were excluded from our database. Using previously described methods (13), we determined and verified 47 hsp65 sequences and submitted them to GenBank (bold accession numbers in Table 1). As a result of our verification, the incorrect sequences from M. asiaticum, M. flavescens, M. intracellulare, M. porcinum, M. senegalense, M. septicum, and M. szulgai in GenBank were excluded from our database, and only verified sequences of these species were adopted. Sequence similarities were determined by MEGA 5.02 (http://www.megasoftware.net/). Currently, there are a total of 143 unique sequences from 149 species/subspecies in this database. Identical sequences are found among the three M. avium subspecies, between two M. fortuitum subspecies, and among four M. tuberculosis complex members (i.e., M. bovis, M. caprae, M. microti, and M. tuberculosis). A BLAST server based on this database has been developed (http://msis.mycobacteria.info). Query sequences are accepted in FASTA format by copying and pasting or file uploading and then searched against the database using NCBI's BLASTN program. The output results will show 20 best hits to suggest their taxonomic categories at species level. The pairwise alignments and the percentages of identities are shown in the results as well. PhyML 3.0 (18) was used to generated a maximum-likelihood phylogeny of these 149 Mycobacterium species/subspecies (Fig. 2) that is the most complete so far, covering 99.3% of the Mycobacterium genus (the only missing species is M. pinnipedii, an M. tuberculosis complex member). Slowly growing mycobacteria (SGM) and rapidly growing mycobacteria (RGM) are clearly separated, except that the slowly growing M. tusciae, M. hiberniae, M. nonchromogenicum, and M. triviale are grouped with the RGM.

Table 1.

List of the Mycobacterium species/subspecies, the reference strains (all are type strains except M. leprae TN and M. lepraemurium TS130), and the GenBank sequence accession numbers for their hsp65 sequences from published studies used to generate the database

Species/subspeciesa Reference strain GenBank accession no. (reference[s])b
M. abscessus CIP 104536 AY458075 (2), AF547802 (19)
ATCC 19977 EF486338 (24), AY498743 (43), NC_010397 (41), JF491290
M. africanum CIP 105147 AF547803 (14)
ATCC 25420 FJ617583 (20), JF491313
M. agri CIP 105391 AY438080
M. aichiense ATCC 27280 AY299147 (23)
DSM 44147 AF547804 (14)
M. alvei CIP 103464 AF547805 (14)
M. aromaticivorans JS19b1 DQ841182 (19)
M. arosiense DSM 45069 JF491321
T1921 EU370531 (4)
ATCC BAA-1401 GQ153297 (49)
M. arupense DSM 44942 EU191917, GQ214503 (29), JF491325
AR30097 DQ168662 (9)
M. asiaticum ATCC 25276 AY299133 (23), GU362517 (13)
M. aubagnense CIP 108543 AY859677 (1)
CCUG 50186 DQ987727
M. aurum ATCC 23366 AF350414 (64), FJ172326 (44)
CIP 104465 AY438081
M. austroafricanum CIP 105395 AF547807 (14)
M. avium subsp. avium ATCC 25291 AF126030 (28), EU239779 (5), GQ153289 (49), JF491291
CIP 104244 AF547808 (14)
M. avium subsp. paratuberculosis CIP 103963 AF547809 (14)
ATCC 19698 AY299137 (23)
M. avium subsp. silvaticum ATCC 49884 EU239781 (5)
CIP 103317 AF547810 (14)
M. boenickei CIP 107829 AY943195
M. bohemicum CIP 105811 AF547811 (14)
M. bolletii CCUG 50184 DQ987724
CIP 108541 EU266576, AY859675 (1), FJ607778 (26)
M. botniense DSM 44537 AF547812 (14)
M. bouchedurhonense CIP 109827 HM602039
M. bovis CIP 105234 AF547813 (14)
ATCC 19210 JF491332
M. branderi CIP 104592 AF547815 (14)
M. brisbanense DSM 44680 AB456564, JF491333
CIP 107830 AY943196
M. brumae CIP 103465 AF547816 (14)
M. canariasense 502329 AY255477 (21)
DSM 44828 JF491316
M. caprae CIP 105776 AF547884 (14)
M. celatum ATCC 51131 AY299180 (23), JF491292
CIP 106109 AF547817 (14)
M. chelonae CIP 104535 AF547818 (14), AY458074 (2)
ATCC 35752 JF491293
M. chimaera CIP 107892 GQ153296 (49), AY943198
DSM 44623 EU239783 (5)
M. chitae CIP 105383 AF547819 (14)
ATCC 19627 AY299149 (23)
M. chlorophenolicum CIP 104189 AF547820 (14)
M. chubuense CIP 106810 AF547821 (14)
M. colombiense CIP 108962 EU239785 (5), GQ153298 (49)
M. conceptionense CIP 108544 AM902957 (39), EU191920, AY859678 (1)
M. confluentis CIP 105510 AF547822 (14)
M. conspicuum CIP 105165 AF547823 (14)
M. cookie CIP 105396 AF547824 (14)
M. cosmeticum LTA-388 AY449730 (12)
DSM 44829 DQ124111
M. crocinum czh-42 DQ533998 (19)
M. diernhoferi CIP 105384 AF547825 (14)
M. doricum DSM 44339 AF547826 (14)
M. duvalii CIP 104539 AF547827 (14)
M. elephantis CIP 106831 AF547828 (14)
M. fallax CIP 81.39 AF547829 (14)
ATCC 35219 JF491294
M. farcinogenes ATCC 35753 AY299150 (23)
DSM 43637 AF547830 (14)
NCTC 10955 AY458073 (2)
M. flavescens ATCC 14474 AF350413 (64), GU362519 (13)
M. florentinum DSM 44852 DQ350162, JF491317
M. fluoranthenivorans DSM 44556 DQ350157, JF491318
M. fortuitum subsp. acetamidolyticum CIP 105423 AF547832 (14)
ATCC 35931 JF491314
M. fortuitum subsp. fortuitum CIP 104534 AF547833 (14)
ATCC 6841 JF491295
M. frederiksbergense DSM 44346 AF547834 (14)
M. gadium CIP 105388 AF547835 (14)
M. gastri CIP 104530 AF547836 (14)
ATCC 15754 JF491315
M. genavense DSM 44424 AF547837 (14)
M. gilvum DSM 44503 AF547838 (14)
M. goodii CIP 106349 AF547839 (14)
ATCC 700504 AY458071 (2)
M. gordonae CIP 104529 AF547840 (14)
ATCC 14470 AF434734
M. haemophilum CIP 105049 AF547841 (14)
ATCC 29548 AY299185, GQ245967, JF491296
M. hassiacum CIP 105218 AF547842 (14)
M. heckeshornense DSM 44428 AF547843 (14)
M. heidelbergense CIP 105424 AF547844 (14)
M. hiberniae DSM 44241 AY438083
ATCC 49874 JF491297
M. hodleri CIP 104909 AF547845 (14)
M. holsaticum DSM 44478 AY438084
M. houstonense ATCC 49403 AY458077 (2)
DSM 44676 DQ987725
M. immunogenum CIP 106684 AY458081 (2), EU266577
M. insubricum DSM 45132 JF491319
FI-06250 EF584487 (51)
M. interjectum DSM 44064 AF547846 (14)
ATCC 51457 JF491298
M. intermedium CIP 104542 AF547847 (14)
ATCC 51848 AY299187
M. intracellulare ATCC 13950 AF126035 (28), DQ284774 (53), GQ153290 (49), JF491299
TMC 1406 U85633 (46)
M. kansasii CIP 104589 AF547849 (14)
ATCC 12478 AF434739, AY299189, JF491300
M. komossense CIP 105293 AY438649
M. kubicae CIP 106428 AF547850 (14)
ATCC 700732 AY373458 (23)
M. kumamotonense CST 7247 AB239920 (32)
CCUG 51961 EU191915
DSM 45093 JF491323
M. kyorinense KUM 060204 AB370171 (37)
DSM 45166 HM602040
M. lacus DSM 44577 AY438090
M. leprae TN NC_002677 (11)
M. lepraemurium TS130 AY550232 (34)
M. lentiflavum CIP 105465 AF547851 (14)
M. llatzerense MG13 AM421341 (16)
DSM 45343 JF491330
M. madagascariense CIP 104538 AF547852 (14)
M. mageritense CIP 104973 AY458070 (2), AF547853 (14)
M. malmoense CIP 105775 AF547854 (14)
ATCC 29571 GQ153293 (49), JF491301
M. mantenii NLA000401474 FJ232523 (60)
CIP 109863 HM602041
M. marinum ATCC 927 AY299134 (23), AF456470 (55), AB548715
NCTC 2275 AF271346 (45)
CIP 104528 AF547855 (14)
M. marseillense 5356591 EU239787 (5)
CIP 109828 HM602037
M. massiliense CIP 108297 EU191919, EF486339 (24), EU266578
CCUG 48898 AY596465 (3)
M. microti CIP 104256 AF547856 (14)
ATCC 19422 AY299135 (23)
M. monacense DSM 44395 EU191918, JF491320
M. montefiorense DSM 44602 AY943204
ATCC BAA-256 AY027785 (31)
M. moriokaense CIP 105393 AF547857 (14), AY859680 (1)
M. mucogenicum ATCC 49650 AY299155, AY458079 (2)
M. murale CIP 105980 AF547859 (14)
M. nebraskense DSM 44803 DQ124110
ATCC BAA-837 GQ153294 (49)
UNMC-MY1349 AY368457 (35)
M. neoaurum ATCC 25795 AY299156, FJ172320 (44), JF491302
CIP 105387 AF547860 (14)
M. neworleansense CIP 107827 AY943199
ATCC 49404 AY458076 (2), AY496143 (62)
M. nonchromogenicum DSM 44164 AF547861 (14)
ATCC 19530 AY299136 (23), AF434732, JF491303
M. noviomagense NLA000500338 EU600390 (57)
M. novocastrense CIP 105546 AF547862 (14)
M. obuense CIP 106803 AF547863 (14)
M. pallens czh-8 DQ533997 (19)
M. palustre DSM 44572 AY943200
M. paraffinicum ATCC 12670 GQ153287 (49)
M. parafortuitum CIP 106802 AF547864 (14)
M. parascrofulaceum ATCC BAA-614 AY337274 (52), GQ153295 (49)
CIP 108112 AY943201
M. paraseoulense DSM 45000 HM602042, JF491324
31118 DQ536402
M. parmense CIP 107385 HM022199
M. peregrinum NCTC 10264 AM902953 (39)
CIP 105382 AY458069 (2), AF547865 (14)
ATCC 14467 AY299159 (23)
M. phlei ATCC 11758 AY299158 (23)
CIP 105389 AF547866 (14)
M. phocaicum CCUG 50185 DQ987726
CIP 108542 AY859676 (1), EU266579
M. porcinum ATCC 33776 AY496137 (62), JF491326
M. poriferae CIP 105394 AF547868 (14)
M. pseudoshottsii NCTC 13318 AM902956 (39), DQ987722
ATCC BAA-883 AY571788
M. psychrotolerans DSM 44697 HM602035
M. pulveris CIP 106804 AF547869 (14)
M. pyrenivorans DSM 44605 JF510463
M. rhodesiae CIP 106806 AF547870 (14)
M. riyadhense NLA000201958 EU921671 (56)
M. rufum JS14 DQ841181 (19)
M. rutilum czh-117 DQ841180 (19)
M. salmoniphilum ATCC 13758 DQ866777 (63)
M. saskatchewanense NRCM 00-250; ATCC BAA-544 AY208858 (54)
CIP 108114 AY943203
DSM 44616 JF491331
M. scrofulaceum ATCC 19981 GQ153288 (49), AF434733, AY299138 (23), JF491304
CIP 105416 AF547871 (14)
M. senegalense NCTC 10956 AM902954 (39)
ATCC 35796 AY684045 (61), JF491327
M. senuense DSM 44999 FJ268582, JF491328
05-832 DQ536409 (36)
M. seoulense DSM 44998 EU191916, JF491322
M. septicum ATCC 700731 AY373457 (23), AY496142 (62)
DSM 44393 JF491329
M. setense CIP 109395 EU371505 (50)
M. shimoidei DSM 44152 AF547874 (14)
ATCC 27962 JF491305
M. shottsii NCTC 13215T AM902955 (39), DQ987723
ATCC 700981 AY550225 (34), EU619895
M. simiae CIP 104531 AF547875 (14)
ATCC 25275 GQ153292 (49), AF434730, JF491306
M. smegmatis ATCC 19420 AY458065 (2), JF491307
CIP 104444 AF547876 (14)
M. sphagni DSM 44076 AF547877 (14)
M. stomatepiae DSM 45059T AM902968 (38, 39)
M. szulgai ATCC 35799 AF350412 (64), AY299141 (23), JF491308
CIP 104532 AF547878 (14)
M. terrae ATCC 15755 AF257468, AF434736, AY299142 (23)
CIP 104321 AF547879 (14)
M. thermoresistibile CIP 105390 AF547880 (14)
M. timonense CIP 109830 HM602038
M. tokaiense CIP 106807 AF547881 (14)
ATCC 27282 JF491309
M. triplex ATCC 700071 AY027786 (31), GQ153291 (49)
CIP 106108 AF547882 (14)
M. triviale DSM 44153 AF547883 (14)
ATCC 23292 AF434737, AY299143 (23), JF491310
M. tuberculosis ATCC 27294 AY299144 (23), JF491311
H37Rv NC_000962 (8, 10)
M. tusciae CIP 106367 AF547887 (14)
M. ulcerans ATCC 19423 AY299145 (23), AB548723, AF271096 (45)
M. vaccae CIP 105934 AF547889 (14)
ATCC 15483 JF491312
M. vanbaalenii DSM 7251 AY438091
PYR-1 NC_008726
M. vulneris NLA000700772 EU834054 (58)
M. wolinskyi CIP 106348 AF547890 (14)
ATCC 700010 AY299164 (23), AY458064 (2)
M. xenopi CIP 104035 AF547891 (14)
ATCC 19250 AF434738, AY373454 (23)
a

The sequences of 47 species/subspecies (boldface) were validated by our laboratory. The sequences of 40 species/subspecies (underlined) are supported by multiple GenBank records deposited by other research groups.

b

Accession numbers in bold are for sequences determined in our laboratory.

Fig. 2.

Fig. 2.

Maximum-likelihood phylogeny of Mycobacterium genus. PhyML 3.0 with default settings (18) and iTOL (30) were used to generate the circular phylogenic tree rooted with Nocardia farcinica (strain IFM 10152). Rapidly growing mycobacteria are in blue, while slowly growing mycobacteria are in red. The scale bar is equivalent to 0.02 substitution/site.

The popularity of species identification using hsp65 sequences has resulted in a large number of mycobacterial hsp65 sequences being deposited in public repositories. McNabb et al. also developed an in-house database, including 111 Mycobacterium species (34). The accuracy and coverage are crucial for the database to become a viable solution for species identification. Here, we report a publicly accessible hsp65 database with 99.3% coverage of the entire Mycobacterium genus, of which 47 species/subspecies have been verified in our laboratory (boldface in Table 1) and 40 entries are supported by multiple GenBank sequences and thus are considered confirmed sequences (underlined in Table 1). A 97% identity was previously suggested as a criterion for identifying a species using hsp65 sequences (34). However, pairwise comparison of these 149 species identified 219, 121, and 45 instances of sequence similarity greater than 97%, 98%, and 99%, involving 94 (63.1%), 82 (55.0%), and 42 (28.2%) species/subspecies, respectively (see the table in the supplemental material). This makes the identification of species with less than 100% matches challenging. Because the interspecies similarities vary from group to group in the phylogeny and change as the number of species/subspecies increases, investigators need to be cautious when assigning species/subspecies for these isolates. Further research is needed to establish a more reliable criterion and validate our database with clinical and environmental isolates. Nevertheless, our database provides the most comprehensive phylogenetic information on the mycobacterial hsp65 locus that can facilitate species identification in this genus.

Supplementary Material

[Supplemental material]

Acknowledgments

We thank Jack Crawford for thoughtful suggestions and helpful comments.

Footnotes

Supplemental material for this article may be found at http://jcm.asm.org/.

Published ahead of print on 30 March 2011.

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