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1 Department of Microbiology, College of Medicine, Seoul National University, 28 Yongon-dong, Chongno-gu, Seoul 110-799, Korea
2 Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Ulsan College of Medicine, Asan Medical Center, Seoul 138-600, Korea
3 Department of Laboratory Medicine, University of Ulsan College of Medicine, Asan Medical Center, Seoul 138-600, Korea
4 The Korean Institute of Tuberculosis, The Korean National Tuberculosis Association, Seoul 137-140, Korea
5 Department of Microbiology, College of Medicine, Konkuk University, Chungju 380-230, Korea
6 Institute of Hansen's Disease, The Catholic University Medical College, Seoul 137-7014, Korea
Correspondence
Bum-Joon Kim
kbumjoon{at}snu.ac.kr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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The GenBank/EMBL/DDBJ accession numbers for the partial hsp65 sequences determined in this study are AF057449–AF057493.
Details of reference strains, including GenBank accession numbers, trees comparing the hsp65 and 16S rRNA genes of the same sample of reference strains and details of a protocol to distinguish M. tuberculosis complex and NTM isolates are available as supplementary material in IJSEM Online.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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; Wayne & Kubica, 1986). Moreover, this culture-based taxonomic approach is laborious, complex and usually impeded by the slow growth of Mycobacterium species in clinical laboratories.
Several macromolecular sequence comparisons have been conducted to delimit more clearly species boundaries within the genus Mycobacterium and to determine phylogenetic relationships between species within this genus (Kim et al., 1999; Pitulle et al., 1992; Rogall et al., 1990; Roth et al., 1998; Stahl et al., 1990; Stone et al., 1995; Takewaki et al., 1994). In particular, phylogenetic approaches targeting the 16S rRNA gene have been widely used and have demonstrated natural relationships among species within this genus (Pitulle et al., 1992; Rogall et al., 1990; Stahl & Urbance, 1990). However, this phylogenetic approach also has its limitations, for example, due to the existence, albeit rare, of two-copy genes with different sequences in a single organism (Ninet et al., 1996; Reischl et al., 1998), or with respect to the sequence identities of Mycobacterium kansasii and Mycobacterium gastri, which are obviously different species (Goodfellow & Magee, 1998; Rogall et al., 1990; Stahl & Urbance, 1990). Another example of 16S rRNA sequence identity between two species is between M. avium and Mycobacterium paratuberculosis (Goodfellow & Magee, 1998). Other examples of the limitations of relying on a single target molecule to determine phylogenetic relationships and for species identification among species in a genus have also been reported (Clayton et al., 1995; Fox et al., 1992; Palys et al., 1997; Turenne et al., 2001). Therefore, alternative phylogenetic and identification methods capable of complementing 16S rRNA gene analysis should be developed for the phylogenetic study and species identification of the genus Mycobacterium.
In recent years, the 60 kDa heat-shock protein family (HSP60 or HSP65), because of their highly conserved primary structures and ubiquity, have been considered as useful phylogenetic markers in several eubacterial genera (Chang et al., 2003; Kwok et al., 1999; Lee et al., 2003; Ringuet et al., 1999; Viale et al., 1994). It is also known that almost all bacterial species have a single copy of the hsp60 or hsp65 gene in their genome (Segal & Ron, 1996), which means that this gene is not easily transferred from one bacterium to another and that a phylogenetic approach, targeting this gene, is probably suitable for phylogenetic studies of closely related species or strains (Kwok et al., 1999). The hsp65 gene has also been used successfully for species identification of cultured clinical isolates of the genus Mycobacterium, although there are some drawbacks in the application of methods targeting the hsp65 gene, a single-copy gene, for the direct detection of mycobacterial organisms in clinical specimens due to limited sensitivity. However, identifications based on this gene are usually based on PCR-restriction fragment length polymorphism analysis (PRA) targeting the 441 bp Telenti fragment of the hsp65 gene (Devallois et al., 1997; Hafner et al., 2004; Telenti et al., 1993). Although phylogenetic and molecular epidemiological studies based on the hsp65 sequence have also been performed, the phylogenetic relationships of only a limited number of reference strains, i.e. ‘fast growers’, were analysed (Ringuet et al., 1999). Molecular epidemic studies were also limited to the sequence databases of only some specific species, like the M. avium complex (Smole et al., 2002) and Mycobacterium scrofulaceum (Swanson et al., 1996). Therefore, we undertook this study to perform a phylogenetic analysis and to develop species identification methods of mycobacteria, based on 604 bp hsp65 sequences of 56 reference and 101 clinical strains, in order to evaluate the usefulness of this gene as a alternative target molecule for identification and phylogenetic analysis of mycobacteria.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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) and 70 strains for blinded hsp65 direct sequencing analysis (Table 2) were used in this study. The 53 Mycobacterium reference strains and 89 clinical isolates that had already been identified by conventional biochemical testing and rpoB PRA analysis (Kim et al., 2001) were provided by the Korean Institute of Tuberculosis (KIT). The single strain of Mycobacterium leprae (Thai 53 strain) and 16 punch-biopsy specimens obtained from active lesions of patients diagnosed on the basis of histological findings, acid-fast bacterium staining and the amplification of DNA encoding an 18-kDa protein (Williams et al., 1990) were provided or performed by the Institute of Hansen's Disease, Catholic University Medical College, Seoul, Korea. Two heat-killed reference strains of M. kansasii (subspecies II and III) were kindly provided by Veronique Vincent (TB and Mycobacteria Lab, Institute of Pasteur, Paris, France). The type strain of T. paurometabola (KCTC 9821T) was provided by the Korean Collection for Type Cultures (KCTC). The 70 non-tuberculous mycobacterium (NTM) clinical isolates for the blinded hsp65 direct sequencing analysis were identified by conventional biochemical testing and provided by the Department of Internal Medicine at the Asan Medical Center.
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). The aqueous phase was transferred to another clean tube and the DNA pellet was precipitated by adding isopropanol and then solubilized with 60 µl TE buffer (10 mM Tris/HCl, 1 mM EDTA, pH 8·0). Two microlitres of purified DNA was used as a template in the PCRs.
PCR amplification of a 644 bp fragment of the hsp65 gene.
A set of primers [forward primer HSPF3 (5'-ATCGCCAAGGAGATCGAGCT-3'; positions 163–182 in the hsp65 gene of Mycobacterium tuberculosis, GenBank accession no. M15467), reverse primer HSPR4 (5'-AAGGTGCCGCGGATCTTGTT-3'; positions 806–787)] were used for the amplification of 644 bp PCR products from Mycobacterium strains (Fig. 1). Template DNA (50 ng) and 20 pmol of each primer were added to a PCR mixture tube (AccuPower PCR PreMix; Bioneer) containing 1 U Taq DNA polymerase, 250 µM of each dNTP, 50 mM Tris/HCl (pH 8·3), 40 mM KCl, 1·5 mM MgCl2 and gel-loading dye and the volume was then adjusted with distilled water to 20 µl. The reaction mixture was subjected to 30 cycles of amplification (60 s at 95 °C, 45 s at 62 °C and 90 s at 72 °C) followed by a 5 min extension at 72 °C (model 9600 Thermocycler; Perkin-Elmer Cetus). The PCR products were electrophoresed in a 3 % agarose gel and visualized with ethidium bromide under UV light.
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Analysis of the sequence data and phylogenetic tree construction.
The partial hsp65 DNA sequences (604 bp), excluding both primers, from 56 mycobacterial reference strains, T. paurometabola KCTC 9821T and 105 clinical strains were aligned and their similarities were calculated using the multiple-alignment algorithm in the MEGALIGN software package (DNASTAR, Windows version 3.12e) to analyse the similarity levels among different species and within clinical isolates of the same species of the genus Mycobacterium and to select signature nucleotides specific to M. tuberculosis complex strains.
Two hsp65 phylogenetic trees were constructed. One used 41 mycobacterial reference strains for comparison with the 16S rRNA gene tree conducted from the same strains and the other used all 56 reference strains for analysis of phylogenetic relationships of overall strains. The hsp65 tree was inferred by using the neighbour-joining (Saitou & Nei, 1987), maximum-likelihood (Felsenstein, 1993) and maximum-parsimony (Fitch, 1972) methods using T. paurometabola KCTC 9821T as an outgroup. Evolutionary-distance matrices were generated according to the model of Jukes & Cantor (1969). The neighbour-joining and maximum-parsimony methods were carried out using MEGA version 2.1 (Kumar et al., 2001) and the maximum-likelihood method (DNAML) was carried out using PHYLIP version 3.5 (Felsenstein, 1993). The resulting trees and topology were evaluated by bootstrap analyses (Felsenstein, 1985) based on 1000 resamplings. The 16S rRNA gene sequences (1396–1427 bp, positions 30–1443 in the 16S rRNA gene sequence numbering of M. tuberculosis) of the same 41 species were aligned and compared. The 16S rRNA gene tree was inferred by using the neighbour-joining method and compared with the hsp65 tree.
hsp65 direct sequencing analysis.
Purified PCR amplicons from 70 NTM clinical isolates were directly sequenced. In direct sequencing analysis, we used 644 bp PCR products as templates and used HSPR4, the reverse PCR primer, as a direct sequencing primer. Although approximately 500 bp sequences were obtained by direct sequencing, for reasons of simplicity only 422 bp sequences (positions 233–654 in the hsp65 gene of M. tuberculosis) were used for species differentiation (Fig. 1
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PRA with XhoI digestion.
Based on the determined hsp65 sequences of the 56 mycobacterial reference strains used in this study (GenBank accession numbers AF057449–AF057493), a single endonuclease (XhoI) was selected using MapDraw (version 3.14; DNASTAR) for the differential identification of the M. tuberculosis complex from NTM strains, and for the grouping of NTM strains. The recognition sites and fragment sizes of XhoI were also calculated using the MapDraw program. XhoI recognizes and cuts at three sites (positions 553, 637 and 703) on the mycobacterial hsp65 gene (Fig. 1). Differential identification between NTM and M. tuberculosis strains is based on the presence of a signature nucleotide specific to M. tuberculosis complex strains (position 705) (Table 3) in the third XhoI recognition sequence (703C
TCGAC). PCR was performed using the set of primers described above (HSPF3 and HSPR4) and using the same PCR conditions. Restriction enzyme XhoI (Takara) was then added to the 644 bp PCR product of the 56 reference strains. Briefly, 10 µl of the PCR products, 2 U enzyme and restriction buffer were transferred to a fresh microcentrifuge tube and water was added to a final volume of 20 µl per reaction. Digestion was performed for 2 h at 37 °C. Following digestion, the mixtures were electrophoresed in a 3 % agarose gel.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Comparison of sequence similarity of hsp65 and the 16S rRNA gene
When the nucleotide sequences of hsp65 and the 16S rRNA gene from the same 41 mycobacterial reference strains were compared, the 16S rRNA gene sequence similarity between any given pair was always higher than the corresponding hsp65 DNA sequence similarity, except for M. genavense ATCC 51233T and M. simiae ATCC 25275T, which had identical hsp65 sequences.
hsp65 DNA sequences of clinical mycobacteria isolates
The sequence similarities of hsp65 among the clinical isolates of the same species were investigated. The level of sequence similarity among strains in each species was more than 98·2 % (intraspecies divergence <1·8 %). In general, fewer than 11 nucleotide variations were observed among the clinical isolates. For clinical isolates of M. tuberculosis, M. leprae, M. scrofulaceum and Mycobacterium peregrinum, a sequence similarity of 100 % was observed, and 17 of 18 clinical isolates of M. kansasii showed similarity in the range 99·2–100 % with the M. kansasii type I reference strain (ATCC 12478T), with the other strain showed a similarity of 99·7 % with M. kansasii type II (Table 1).
Phylogenetic analysis based on the hsp65 sequences
On comparing the 16S rRNA tree based on the sequences of the reference strains with the phylogenetic trees based on hsp65 sequences, they were generally found to be topologically similar, in spite of some differences (Fig. 2a, b). This hsp65 tree confirms that the traditional division between rapidly and slowly growing mycobacteria is a natural division. The branching point of the rapidly growing members is close to that of total Mycobacterium strains, which shows that these are phylogenetically older than their slowly growing relatives.
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) that all three species of the M. terrae complex have rapid-grower-specific leucine (L) instead of methionine (M) at codon 468 of RpoB. This phenomenon is also partially supported by the result of biochemical testing (Goodfellow & Magee, 1998; Wayne & Kubica, 1986) that Mycobacterium triviale is the only slow-grower known to grow in the presence of 5 % NaCl, a trait it shares with rapid-growers. Secondly, the grouping of the strict pathogens M. leprae Thai 53 strain and M. tuberculosis complex strains and Mycobacterium haemophilum ATCC 29548T, the so-called ‘blood-loving mycobacterium’ distinct from other mycobacteria in its requirement for ferric ions in the form of haemin or ferric ammonium citrate, was observed in the hsp65 tree. This grouping reflecting pathogenic potential seems to be reliable and it was also supported by relatively high bootstrap values and conservation of different trees. Thirdly, an unusual branching of M. leprae Thai 53 strain, reflecting the rapid evolution of the hsp65 gene of this species, was also observed in the hsp65 tree. It may be associated with the extraordinarily low G+C content of the hsp65 gene of this species. These phylogenetic results emphasize that molecular markers other than the 16S rRNA gene must be applied to obtain a precise phylogenetic and evolutionary scheme.
Signature nucleotides and amino acids specific to M. tuberculosis complex strains
Signature nucleotides specific to M. tuberculosis complex strains were determined by analysing hsp65 sequence alignments, and seven signature nucleotides were detected at five codons (Table 3). Since the signature nucleotides in four codons (positions 76, 181, 200 and 235) were located only at synonymous sites of each codon, they did not affect the protein sequence, but the signature nucleotides in codon 240 were located at all three nucleotides and thus cause amino acid sequence variations [NTM, 240Q(CAG); M. tuberculosis complex, 240G(GGA)] (Table 3).
The seven M. tuberculosis signature nucleotides, particularly the three nucleotides (GGA) concentrated at codon 240, might be used effectively for the development of M. tuberculosis detection methods. Actually, a specific polymorphism that distinguished M. tuberculosis from NTM strains in the XhoI PRA method developed in this study was also based on the recognition of a signature nucleotide at position 703.
Identification of clinical isolates by direct sequencing analysis of hsp65
We applied a direct sequencing protocol targeting 422 bp of hsp65, which enabled species differentiation of mycobacteria in a single sequencing reaction without a cloning procedure, to 70 NTM clinical isolates. No ambiguous results due to two-copy genes with different sequences were obtained using this direct sequencing protocol (data not shown). By referring to the phylogenetic tree constructed by using 56 reference strains, we were able to identify all 70 NTM strains to the species level. The result was concordant with that obtained by conventional biochemical testing and rpoB sequencing analysis (Kim et al., 1999). Furthermore, one Mycobacterium celatum strain misidentified as M. avium complex by conventional tests, seven M. kansasii strains and three Mycobacterium fortuitum isolates could be differentiated to the strain level (Table 2).
Compared with the approach targeting the 16S rRNA gene, hsp65 sequence-based analysis has several advantages. Firstly, hsp65 analysis provided a resolving power higher than 16S rRNA gene analysis for differentiation among species. Furthermore, separation between closely related species such as between Mycobacterium abscessus and Mycobacterium chelonae, between Mycobacterium szulgai and Mycobacterium malmoense and between M. kansasii and M. gastri is poorly achieved by 16S rRNA gene analysis due to the small amount of sequence variation (Kirschner et al., 1993), but these species can be clearly separated by hsp65 analysis. Secondly, in the multiple alignment of hsp65 sequences of 56 mycobacterial reference strains and 105 clinical isolates, no gaps or additions were found. This means that all the sequence information can be considered for phylogenetic analysis without deletion of gap sequences. This is not the case with the 16S rRNA gene. Thirdly, although rare, some mycobacterial strains (M. celatum and M. terrae complex) have two-copy 1S rRNA genes with different sequences in an organism (Ninet et al., 1996; Reischl et al., 1998). In this case, the application of direct sequencing requires some care in the interpretation of the results due to sequence polymorphisms. Analysis targeting the hsp65 gene, known as a single-copy gene, can resolve this problem. In fact, when direct sequencing protocols targeting 422 bp hsp65 fragments were applied to 70 clinical isolates, no ambiguous results were observed (data not shown).
XhoI PRA and M. tuberculosis complex-specific PCR
The XhoI PRA algorithm which could differentiate M. tuberculosis strains from NTM strains and mycobacterial strains into five groups [A (391, 150, 103 bp), B (391, 253 bp), C (391, 169, 84 bp), D (644 bp) and E (475, 169 bp)] was developed on the basis of the determined hsp65 DNA sequences of 56 mycobacterial reference strains (see Supplementary Fig. S2 available in IJSEM Online). When this PRA method was applied to 105 clinical isolates, all 25 M. tuberculosis strains could be clearly differentiated from the other 90 NTM strains, showing a distinct polymorphism (A type) on the 3 % agarose gel (Table 1).
Compared with the previous PRA targeting a 441 bp fragment of hsp65 (Devallois et al., 1997; Telenti et al., 1993), which required two restriction enzymes, our XhoI PRA method using only one cheap enzyme offers an easy, rapid and inexpensive procedure for the differential identification of M. tuberculosis and NTM strains. However, since it has limitations in the differentiation of all mycobacterial strains, novel PRA algorithms using several restriction enzymes for the differentiation of all the mycobacteria strains should be developed in future.
In the present study, the usefulness of hsp65 sequencing analysis in species identification and phylogenetic study of mycobacteria was evaluated with only 56 reference strains, about 50 % of those officially recognized at present, and clinical isolates of frequently encountered species. Therefore, hsp65 sequencing analysis of the other type strains and application of this method to the identification of infrequently encountered strains remain to be studied.
In conclusion, this study shows that a 604 bp fragment of hsp65 is a good alternative molecular marker not only for the phylogenetic analysis of mycobacteria but also for species identification of mycobacterial clinical isolates. The sequence divergence shown by this 604 bp hsp65 fragment is sufficient for it to be applied to the development of molecular methods for the detection of mycobacteria strains with clinical significance, such as M. tuberculosis.
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ACKNOWLEDGEMENTS |
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