Abstract
Recently, we introduced a distinct Mycobacterium intracellulare INT-5 genotype, distantly related to other genotypes of M. intracellulare (INT-1 to -4). The aim of this study is to determine the exact taxonomic status of the M. intracellulare INT-5 genotype via genome-based phylogenetic analysis. To this end, genome sequences of the two INT-5 strains, MOTT-H4Y and MOTT-36Y were compared with M. intracellulare ATCC 13950T and Mycobacterium yongonense DSM 45126T. Our phylogenetic analysis based on complete genome sequences, multi-locus sequence typing (MLST) of 35 target genes, and single nucleotide polymorphism (SNP) analysis indicated that the two INT-5 strains were more closely related to M. yongonense DSM 45126T than the M. intracellulare strains. These results suggest their taxonomic transfer from M. intracellulare into M. yongonense. Finally, we selected 5 target genes (argH, dnaA, deaD, hsp65, and recF) and used SNPs for the identification of M. yongonese strains from other M. avium complex (MAC) strains. The application of the SNP analysis to 14 MAC clinical isolates enabled the selective identification of 4 M. yongonense clinical isolates from the other MACs. In conclusion, our genome-based phylogenetic analysis showed that the taxonomic status of two INT-5 strains, MOTT-H4Y and MOTT-36Y should be revised into M. yongonense. Our results also suggest that M. yongonense could be divided into 2 distinct genotypes (the Type I genotype with the M. parascrofulaceum rpoB gene and the Type II genotype with the M. intracellulare rpoB gene) depending on the presence of the lateral gene transfer of rpoB from M. parascrofulaceum.
Introduction
Members of the Mycobacterium avium complex (MAC) are the most important nontuberculous mycobacteria (NTM) in terms of clinical and epidemiological aspects [1]. Traditionally, MAC includes two species: M. avium and M. intracellulare [2–4]. In addition to these 2 species, recent advances in molecular taxonomy have fueled the identification of novel species within the MAC [5–10]. Our group introduced a novel species Mycobacterium yongonense, which was closely related to M. intracellulare, from a Korean patient with pulmonary symptoms [11]. Notably, M. yongonense possessed a distinct RNA polymerase gene (rpoB) sequence that was identical to M. parascrofulaceum, which is a distantly related scotochromogen, suggesting the acquisition of the rpoB gene via a potential lateral gene transfer (LGT) event [12, 13]. Recently, M. yongonense strains causing pulmonary disease were also isolated from patients in Italy [14]. However, it should be noted that these strains harbored rpoB sequences that were almost identical to M. intracellulare and not M. parascrofulaceum, suggesting the possibility of the existence of another group of M. yongonense strains.
Our group reported that M. intracellulare-related strains from Korean patients showed genetic diversity. This diversity could be used to divide the strains into a total of five distinct groups (INT-1 to -5) via the molecular taxonomic approach using three independent chronometer molecules: hsp65, the internal transcribed spacer 1 (ITS-1) region and the 16S rRNA gene [15]. Of these genotypes, the INT-5 strains were distantly related to other genotypes of M. intracellulare (INT-1 to -4). We also introduced the complete genome sequences of two INT-5 strains, MOTT-H4Y and MOTT-36Y [16, 17], showing that they were more closely related to the genome of M. yongonense DSM 45126T than M. intracellulare ATCC 13950T, despite they have rpoB sequences identical to M. intracellulare, but not M. parascrofulaceum. Furthermore, our recent study indicated that they harbored a novel insertion element (IS) sequence (ISMyo2) specific to M. yongonense [18]. Collectively, it suggests that MOTT-H4Y and MOTT-36Y might be variants of M. yongonense that were not subject to the rpoB LGT event from M. parascrofulaceum. Recently, it has been also reported that M. yongonense may be misidentified as one of the M. intracellulare strains [14, 19]. Therefore, the establishment of consensus guidelines is needed for the exact species delineation between M. intracellulare and M. yongonense.
So, the aim of the current study is to determine the exact taxonomic status of the two INT-5 strains, MOTT-H4Y and MOTT-36Y with the M. intracellulare rpoB sequences but with genomic sequences closely related to M. yongonense via genome-based phylogenetic analysis.
Materials and Methods
Mycobacterial Strains
A total of 16 clinical isolates were used in this study. These clinical isolates were collected from the Asan Medical Center (Seoul, Republic of Korea) and Seoul National University Hospital (Division of Pulmonary & Critical Care Medicine, Seoul, Republic of Korea). These strains were identified into genotypes by phylogenetic analysis based on hsp65, ITS1 and 16S rRNA gene sequences [15] and rpoB sequence analysis [6, 20] (S1 Table). These strains were grouped [15] as follows: five INT-1 strains (Asan 29591, 29778, 36309, 37128, and 37721), five INT-2 strains (Asan 36638, 37016, 38392, 38402, and 38585), and six INT-5 strains (Asan 36527 and 36912, MOTT-68Y, MOTT-H4Y, MOTT-36Y and Rhu). For genomic DNA extraction, the clinical isolates were cultured on Middlebrook 7H10 agar plates supplemented with OADC (BD GmbH, Heidelberg, Germany) for 7–10 days in a 5% CO2 incubator at 37°C. Genomic DNA was prepared by the bead beater-phenol extraction method as previously described [21].
Complete Genome Sequence-Based Phylogenetic Analysis
For the phylogenetic analysis of the two INT-5 strains (MOTT-H4Y and MOTT-36Y), their genome sequences [MOTT-H4Y (Genbank accession No. AKIG00000000) and MOTT-36Y (Genbank accession No. NC_017904)] [16, 17] were compared with those of M. yongonense DSM 45126T (Genbank accession No. NC_021715) [13], M. parascrofulaceum ATCC BAA-614T (Genbank accession No. ADNV00000000), and 3 strains belonging to M. intracellulare [one INT-1 strain: M. intracellulare MOTT-64 (GenBank accession No. NC_016948) and two INT-2 strains: M. intracellulare ATCC 13950T (GenBank accession No. NC_016946) and M. intracellulare MOTT-02 (GenBank accession No. NC_016947)] [22–24]. These genome sequences were subjected to whole-genome multiple sequence alignments using the neighbor-joining method [25] by the Mauve Genome Alignments software (http://darlinglab.org/mauve/mauve.html). A phylogenetic tree was generated using the aligned genome sequences and visualized by the TreeViewX program (http://darwin.zoology.gla.ac.uk/~rpage/treeviewx/); additionally, a Venn diagram was constructed by the BLASTCLUST program. The minimum length coverage and identity threshold in BLASTCLUST were 0.9 and 95%, respectively.
Phylogenetic Analysis Based on rpoB and 35 Selected Target Gene Sequences or Single Nucleotide Polymorphisms (SNPs) of the 35 Selected Target Gene Sequences
To analyze the sequence differences among the 3 M. intracellulare strains (M. intracellulare ATCC 13950T, M. intracellulare MOTT-02, and M. intracellulare MOTT-64), 2 INT-5 strains (MOTT-H4Y and MOTT-36Y) and M. yongonense DSM 45126T, the rpoB gene and 35 additional gene sequences were selected from the genome sequences. In the selected 35 genes, 10 genes (argH, cya, glpK, hsp65, murC, pta, recA, secA1 and sodA) [26–29] were included for mycobacterial MLST analysis, and other 25 genes were were randomly selected in the housekeeping genes without any standards. The list of chosen genes is as follows: adenylate kinase (adk), argininosuccinate lyase (argH), chorismate synthase (aroC), shikimate 5-dehydrogenase (aroE), F0F1 ATP synthase subunit beta (atpD), adenylate cyclase (cya), cytochrome b6 (cytB), ATP-dependent RNA helicase, dead/death box family protein (deaD), chromosomal replication initiation protein (dnaA), DNA primase (dnaG), molecular chaperone DnaK (dnaK), chaperone protein (dnaJ), 3-oxoacyl-(acyl-carrier-protein) reductase (fabG), cell division protein FtsZ (ftsZ), fumarate hydratase (fumC), malate synthase G (glcB), glutamine synthetase type I (glnA), glycerol kinase (glpK), fructose 1,6-bisphosphatase II (glpX), 6-phosphogluconate dehydrogenase (gnd), DNA gyrase subunit B (gyrB), heat-shock protein 65 kD (hsp65), myo-inositol-1-phosphate synthase (ino1), NAD-dependent DNA ligase LigA (ligA), ATP-dependent DNA ligase (ligB and ligC), UDP-N-acetylmuramate-L-alanine ligase (murC), endonuclease III (nth), glucose-6-phosphate isomerase (pgi), phosphoglycerate kinase (pgk), phosphate acetyltransferase (pta), recombinase A (recA), recombination protein F (recF), preprotein translocase subunit SecA (secA1), and [Mn]-superoxide dismutase (sodA) (Table 1). The retrieved rpoB gene or the concatenated 35 selected gene sequences were multiply aligned using the ClustalW method in the MEGA 4.0 software [30]. Using the multiple alignment matrix, phylogenetic trees were constructed using the neighbor-joining method [25] in the MEGA 4.0 software [30]. The bootstrap values were calculated from 1,000 replications.
Table 1. Total and M. yongonense-group related-SNPs from targeted 35 genes of Mycobacterium intracellulare strains.
| No. | Genes | Compared nucleotide size (bp) | Total SNPs (n) | M. yongonense-group related-SNPs (n) |
|---|---|---|---|---|
| 1 | adk | 534 | 49 | 0 |
| 2 | argH | 1,431 | 199 | 10 |
| 3 | aroC | 1,206 | 134 | 0 |
| 4 | aroE | 888 | 134 | 0 |
| 5 | atpD | 1,461 | 164 | 0 |
| 6 | cya | 1,554 | 207 | 5 |
| 7 | cytB | 1,704 | 185 | 0 |
| 8 | deaD | 1,704 | 228 | 14 |
| 9 | dnaA | 1,503 | 193 | 11 |
| 10 | dnaG | 1,953 | 250 | 5 |
| 11 | dnaJ | 1,149 | 124 | 2 |
| 12 | dnaK | 1,860 | 118 | 2 |
| 13 | fabG | 768 | 80 | 0 |
| 14 | ftsZ | 1,161 | 108 | 0 |
| 15 | fumC | 1,407 | 152 | 0 |
| 16 | glcB | 2,169 | 183 | 0 |
| 17 | glnA | 1,437 | 106 | 1 |
| 18 | glpK | 1,527 | 223 | 2 |
| 19 | glpX | 987 | 101 | 0 |
| 20 | gnd | 1,521 | 147 | 0 |
| 21 | gyrB | 2,034 | 245 | 4 |
| 22 | hsp65 | 1,626 | 103 | 6 |
| 23 | ino1 | 1,095 | 81 | 2 |
| 24 | ligA | 2,082 | 257 | 3 |
| 25 | ligB | 1,530 | 167 | 0 |
| 26 | ligC | 1,056 | 173 | 3 |
| 27 | murC | 1,479 | 207 | 0 |
| 28 | nth | 792 | 84 | 0 |
| 29 | pgi | 1,665 | 203 | 0 |
| 30 | pgk | 1,236 | 187 | 0 |
| 31 | pta | 2,160 | 328 | 3 |
| 32 | recA | 1,053 | 49 | 0 |
| 33 | recF | 1,158 | 235 | 18 |
| 34 | rpoB | 3,390 | 185 | 0 |
| 35 | secA1 | 2,829 | 268 | 3 |
| 36 | sodA | 624 | 118 | 0 |
SNPs were extracted from the multiple alignments of rpoB gene sequences and the 35 selected gene sequences from the 3 M. intracellulare strains (M. intracellulare ATCC 13950T, M. intracellulare MOTT-02, and M. intracellulare MOTT-64), 2 M. intracellulare INT-5 strains (MOTT-H4Y and MOTT-36Y), M. yongonense DSM 45126T and M. parascrofulaceum ATCC BAA-614T. Then, the patterns were compared. Additionally, the extracted SNP sequences were concatenated and used to construct a phylogenetic tree as described above.
Application of SNP Analysis to MAC Clinical Isolates
To confirm the different SNP patterns between the INT-5 strains and other M. intracellulare strains (INT-1 or INT-2 strains), five genes of other M. intracellulare clinical isolates, which proved to have more M. yongonense-group related signature SNPs than others, were amplified and sequenced for further analysis. The five selected genes were argH, dnaA, deaD, hsp65 and recF. Genomic DNA from each of the five INT-1 strains (Asan 29591, 29778, 36309, 37128, and 37721), five INT-2 strains (Asan 36638, 37016, 38392, 38402, and 38585), and four INT-5 strains (Asan 36527 and 36912, MOTT-68Y and Rhu) was used to amplify the five selected genes. As a positive and a negative control of PCR of 5 genes, genomic DNA of M. intracellulare ATCC 13950T and distilled water were also used. The five primer sets were as follows: argH, argH_F 5’–TGA GCA AGT CCA CCC ATT TC– 3’ and argH_R 5’–TGG CGT CGA TGG AGT TGT C– 3’; dnaA, dnaA_F 5’–ACG AGC CTC AAC CGC C– 3’ and dnaA_R 5’–CTC ACG GCA CAG GTA CAT CG–R’; deaD, DEAD_F 5’–GGA ATA CAA GCA GGT GGC ACT– 3’ and DEAD_R 5’–GCG TTC GTA GTC CTG GAC CA– 3’; hsp65, hspF3 5’–ATC GCC AAG GAG ATC GAG CT– 3’ and hspR4 5’–AAG GTG CCG CGG ATC TTG TT– 3’ and recF, recF_F 5’–GAA ATC CCT GTC TGG CGC– 3’ and recF_R 5’–TCA TGC GCG CAT CTC C– 3’. Template DNA and each primer pair (20 pmol) were added to the PCR premix (AccuPower PCR PreMix, Bioneer), and PCR was conducted by subjecting the samples to 5 min at 95°C, followed by 30 cycles of 95°C for 30 s, 58–60°C for 30 s, and 72°C for 1 min, and a final extension at 72°C for 5 min. The PCR reaction was performed in a MyCycler (Bio-RAD). The PCR products were detected and purified using the MEGAquick-Spin Total Fragment DNA Purification kit (iNtRON) for direct sequencing. Sequencing reactions were performed using an MJ Research PTC-225 Peltier Thermal Cycler and ABI PRISM BigDye Terminator Cycle Sequencing kits with the AmpliTaq DNA polymerase (FS enzyme, Applied Biosystems) following the manufacturer’s protocols. The obtained sequences were aligned using the MegAlign software package (DNASTAR), and then the SNPs in the sequences were analyzed.
Results
Genome Sequence-Based Phylogenetic Analysis of Two INT-5 Strains, MOTT-H4Y and MOTT-36Y
The phylogenetic relationships between 3 M. intracellulare strains (ATCC 13950T, MOTT-02, and MOTT-64), 2 INT-5 strains (MOTT-H4Y and MOTT-36Y) and one M. yongonense strain (DSM 45126T) were analyzed using the genome sequence information (Fig 1). All 6 strains were clustered together in a branch. The strains were separated into two different branches: one including 3 M. intracellulare strains (ATCC 13950T, MOTT-02, and MOTT-64) and the other including M. yongonense DSM 45126T and the two INT-5 strains, MOTT-H4Y and MOTT-36Y. This result indicated that the two INT-5 strains were more closely related to M. yongonense DSM 45126T than the 3 M. intracellulare strains (ATCC 13950T, MOTT-02, and MOTT-64) (Fig 1).
Fig 1. Phylogenetic tree based on whole-genome sequences of 3 Mycobacterium intracellulare group, 2 INT-5 group, M. yongonense and other mycobacterial strains.
The tree was calculated using the neighbor-joining method by the Mauve Genome Alignment software and visualized by the TreeViewX program. The bar indicates the number of substitutions per nucleotide position.
To assess the number of genes shared between each genome, we performed a BLASTCLUST analysis on the four genomes (M. intracellulare ATCC 13950T, MOTT-H4Y, and M. yongonense DSM 45126T or M. intracellulare ATCC 13950T, MOTT-36Y, and M. yongonense DSM 45126T). At the level of 95% identity, M. intracellulare MOTT-36Y or MOTT-H4Y shared more orthologous coding sequences (CDSs) with M. yongonense DSM 45126T (4,271/5,128 CDSs, 83.3% or 4,287/5,020 CDSs, 85.4%, respectively) than M. intracellulare ATCC 13950T (4,101/5,128 CDSs, 80.0% or 4,052/5,020 CDSs, 80.7%, respectively) (Fig 2). This finding supported the results of our phylogenetic study that the two INT-5 strains might belong to M. yongonense rather than to M. intracellulare (Fig 1).
Fig 2. Venn diagrams based on genome information of INT-5 strains.
Venn diagrams showing orthologous ORFs among four mycobacterial species as determined by BLASTCLUST analysis. Numbers in parenthesis include paralogous ORFs.
Phylogenetic Analysis of Two INT-5 Strains, MOTT-H4Y and MOTT-36Y Based on the rpoB Gene Sequences and the Sequences of 35 Selected Target Genes
The taxonomic signature of M. yongonense was previously reported to be based on the rpoB gene sequence. The sequence of this gene is identical to the distantly related species M. parascrofulaceum, which enables the separation of the 2 closely related species M. intracellulare and M. yongonense [11, 12]. Therefore, to obtain the exact taxonomic delineation of the two INT-5 strains we compared their taxonomic location by phylogenetic analysis based on the sequences of rpoB and 35 selected target genes.
The entire sequences of rpoB and the 35 selected genes were retrieved from the genome sequences of 6 mycobacterial strains [3 M. intracellulare strains (M. intracellulare ATCC 13950T, MOTT-02, and MOTT-64), 2 INT-5 strains (MOTT-36Y and MOTT-H4Y) and M. yongonense DSM 45126T] (Table 1) and subjected to phylogenetic analysis. In the rpoB gene (3,375 to 3,462 bp)-based phylogenetic analysis, the two INT-5 strains MOTT-H4Y and MOTT-36Y were clustered into the group including the M. intracellulare strains (M. intracellulare ATCC 13950T, MOTT-02, and MOTT-64) and were separated from M. yongonense DSM 45126Tand M. parascrofulaceum ATCC BAA-614 (Fig 3A). However, in the phylogenetic analyses based on the sequences of the 35 selected genes, the two INT-5 strains MOTT-H4Y and MOTT-36Y were clustered into M. yongonense DSM 45126T and separated from the other 3 M. intracellulare strains with a high bootstrap value (> 99%), as shown in the genome sequence-based phylogenetic analysis (Figs 1 and 3B). These results suggest that there may be a distinct M. yongonense genotype having an rpoB gene sequence that is almost identical to M. intracellulare.
Fig 3. Neighbor-joining phylogenetic tree based on the rpoB or 35 concatenated genes from 6 Mycobacterium intracellulare strains.
(A) A tree based on the whole rpoB gene sequences from 3 M. intracellulare, 2 INT-5 strains, M. yongonense, and M. parascrofulaceum. (B) A tree based on the 35 concatenated gene sequences from 3 M. intracellulare, 2 INT-5 strains, M. yongonense, and M. parascrofulaceum. The bootstrap values were calculated from 1,000 replications and values <50% were not shown. The bar indicates the number of substitutions per nucleotide position. M. tuberculosis H37Rv and M. avium 104 were used as outgroups in the rpoB gene- or concatenated genes-based phylogenetic trees, respectively.
Phylogenetic Analysis of Two INT-5 Strains (MOTT-H4Y and MOTT-36Y) Based on Single Nucleotide Polymorphisms (SNPs) of the rpoB and 35 Targeted Genes
Multiple alignments of the rpoB and 35 gene sequences from the 3 M. intracellualre (M. intracellulare, MOTT-02 and MOTT-64), 2 INT-5 (MOTT-H4Y and MOTT-36Y), M. yongonense and M. parascrofulaceum showed that there were M. yongonense group- related SNPs in 17 genes [hsp65 (6 M. yongonense group-related SNPs/103 total SNPs), argH (10/199), cya (5/207), dnaJ (2/124), glpK (2/223), pta (3/328), recF (18/235), secA1 (3/268), deaD (14/228), dnaA (11/193), dnaG (5/250), dnaK (2/118), glnA (1/106), gyrB (4/245), ino1 (2/81), ligA (3/257), and ligC (3/173)] (Table 1). Detailed M. yongonense group-related SNP signatures are listed in Table 2.
Table 2. Details of M. yongonense group-related SNP signatures.
| Genes | M. yongonense group-related SNP signatures |
|---|---|
| argH | C105Ta C132G C138G C244T G303A C306G C322T G339C T566C C603A |
| cya | C399G C414G G432C C483G C504T |
| deaD | G204C G210C G216T G276A A315T C376T C648T G840A A894G T993C G1062C C1068G G1191A C1383T |
| dnaA | T222C C441G G639A G651C G714C G759A C921T C1035G G1080A A1326G G1341C |
| dnaG | C897G G921T C1350G C1488T G1560T |
| dnaJ | C849T C1008T |
| dnaK | C1476T T1509C |
| glnA | T1434A |
| glpK | T30C C723G |
| gyrB | C297G G375C C660T G702C |
| hsp65 | G198A C555G G633C C726T G1191C G1539C |
| ino1 | G291C G396A |
| ligA | A146C C441G G1986A |
| ligC | C384T G813A C933T |
| pta | C1368T C1371T C1464T |
| recF | C171T C249A C264T C279T G336A T429C A467G T534G G570C C579T G586C T660C G771T T796C T937C G963A C1009T G1123A |
| secA1 | G645C T717G C1854G |
All the nucleotide positions were determined from Mycobacterium intracellulare ATCC 13950T strain.
a Bold characters represent M. yongonense group-related SNPs.
In the case of rpoB gene, there was no M. yongonense group-related SNPs, however, rpoB gene of M. yongonense shared identical 151 SNPs with that of M. parascrofulaceum. A concatenated phylogenetic tree was constructed using the extracted SNP sequences. The tree showed that the two INT-5 strains were clustered into M. yongonense DSM 45126T and separated from the other 3 M. intracellulare strains based on the phylogenetic analyses of the complete genome sequences and 35 concatenated gene sequences (Figs 1, 3B and 4A).
Fig 4. Neighbor-joining phylogenetic tree based on concatenated SNPs.
(A) A concatenated SNP-based tree from 35 target genes of 3 M. intracellulare, 2 INT-5 strains, M. yongonense, and M. parascrofulaceum. (B) A concatenated SNP-based tree from 5 selected genes (argH, dnaA, deaD, hsp65 and recF) from 3 M. intracellulare, 2 INT-5 strains, M. yongonense, and M. parascrofulaceum and 14 clinical isolate strains. The indicated INT-groupings were assigned in a previous report [15]. The bootstrap values were calculated from 1,000 replications and values <50% were not shown. The bar indicates the number of substitutions per nucleotide position.
Application of the M. yongonense-Related SNP Analysis to MAC Clinical Isolates
To develop SNP analysis to enable the selective identification of M. yongonense strains from the MAC strains, five genes (argH, deaD, dnaA, hsp65 and recF) were selected that possessed a higher number of M. yongonense group (M. yongonense DSM 45126T and two INT-5 strains MOTT-H4Y and MOTT-36Y) -related SNPs compared to the other genes. To explore the usefulness of this assay, sequence analysis of the five genes was applied to a total of 14 MAC clinical isolates from different Korean patients [five M. intracellulare INT-1 strains (Asan 29591, 29778, 36309, 37128 and 37721), five M. intracellulare INT-2 strains (Asan 36638, 37016, 38392, 38402 and 38585), and four INT-5 strains (Asan 36527, 36912, Rhu and MOTT-68Y)] and 7 was subjected to phylogenetic analysis.
All four INT-5 strains had 10 M. yongonense group-related SNPs in the partial argH gene sequence out of 10 M. yongonense group-related SNPs (from 105 nt to 657 nt). However, two INT-1 (Asan 29778 and 37721) and two INT-2 group (Asan 37016 and 38392) strains also shared one M. yongonense group-related SNP (G339C). All four INT-5 strains had 7 M. yongonense group-related SNPs in the partial dnaA gene sequence out of 7 M. yongonense group-related SNPs (from 627 nt to 1257 nt). However, one INT-2 group strain (Asan 38392) also shared one M. yongonense group-related SNP (C921T). All four INT-5 strains had 7 M. yongonense group-related SNPs in the partial deaD gene sequence out of 7 M. yongonense group-related SNPs (from 588 nt to 1191 nt). However, three INT-2 strains also shared one or four M. yongonense group-related SNPs (Asan 36638: C1068G; Asan 38392: C1068G; and Asan 38585: C648T, C681T, G1062C, and C1068G). In the partial hsp65 gene sequence with 4 M. yongonense group-related SNPs (from 192 nt to 726 nt), two INT-5 group strains (Asan 36527 and Asan 36912) had only one M. yongonense group-related SNPs (G198A) and the three INT-1 or INT-2 SNPs (C555, G633 and C726), while the other two strains (Rhu and MOTT-68Y) had 4 M. yongonense group-related SNPs. All of the INT-5 group strains had 11 M. yongonense group-related SNPs in the partial recF gene sequence out of 11 M. yongonense group-related SNPs (from 520 nt and 1131 nt). However, one INT-1 (Asan 36309: T660C and G1123A) and one INT-2 strain (Asan 38392: T660C) shared one or two M. yongonense group-related SNPs.
The phylogenetic analysis based on the concatenated SNP sequences (395 bp) extracted from the five target genes showed that all four INT-5 strains of M. yongonense may share were clearly separated from the other M. intracellulare clinical isolates (Fig 4B). These results suggested the usefulness of SNP analysis for the taxonomic separation of M. yongonense from closely related M. intracellulare strains.
Discussion
In the present study, our phylogenetic analysis based on complete genome sequences, multi-locus sequence typing (MLST) of 35 target genes, and single nucleotide polymorphism (SNP) analysis indicated that the two INT-5 strains, MOTT-H4Y and MOTT-36Y were more closely related to M. yongonense DSM 45126T than the M. intracellulare strains. This finding suggests the presence of another distinct genotype in M. yongonense that may not have been subjected to the LGT event of rpoB from M. parascrofulaceum. Therefore, M. yongonense could be divided into 2 distinct genotypes: one with the M. parascrofulaceum rpoB gene and the other with the M. intracellulare rpoB gene, depending on the presence of the LGT event of rpoB from M. parascrofulaceum (Figs 1 and 3). Here, we proposed the former and the latter as the M. yongonense Type I and Type II genotypes, respectively.
To date, a total of 3 strains (M. yongonense DSM 45126T, Asan 36912 and Asan 36527) belonging to the M. yongonense Type I genotype have been introduced via our 2 recent reports [11, 12]. The Rhu strain used in this study was also identified as the Type I genotype by rpoB gene analysis (data not shown). In addition to MOTT-H4Y and MOTT-36Y, one additional strain (MOTT-68Y) used in this study was identified as the M. yongonense Type II genotype. Although detailed taxonomic proof is needed, the M. yongonense strains recently isolated in Italy have the potential to be included in the M. yongonense Type II genotype.
LGT is the major mechanism by which bacteria can acquire genetic diversity, guaranteeing their survival under harsh environmental conditions [31, 32]. However, it is generally accepted that mycobacteria are more resistant to LGT compared to other bacteria, possibly due to the unusually mycolic acid-rich cell wall structure and the relative scarcity of genetic elements such as plasmids and transposable elements [33–35]. Notably, because the M. yongonense strains were demonstrated to possess an rpoB gene that might have been laterally transferred from the distantly-related scotochromogenic species M. parascrofulaceum, these strains have gained increasing importance in the mycobacterial taxonomic fields. One of the noteworthy findings in this study is the identification of a novel genotype of M. yongonense without the rpoB gene from the LGT event in its genome. A genome comparison study between 3 mycobacterial groups [the M. yongonense Type I (subject to the LGT event) and Type II genotypes (without the LGT event) and M. parascrofulaceum (gene donor for LGT)] may provide novel insights into our understandings regarding mycobacterial LGT mechanisms.
In the present study, we developed an SNP analysis targeting 5 genes (argH, deaD, dnaA, hsp65 and recF) for the separation of M. yongonense from the closely related M. intracellulare strains. The concatenated 395-bp SNP-based phylogenetic analysis clearly separated 7 M. yongonense strains from 12 closely related M. intracellulare strains belonging to the INT-I and INT-2 genotypes, which were the first and the second most prevalent genotypes in Korean patients infected with M. intracellulare, respectively, with 83% bootstrap values (Fig 4A). This result suggests the feasibility of this assay for the selective identification of M. yongonense strains in clinical settings. Interestingly, this assay could not differentiate 4 Type I (DSM 45126T, Asan36527, Asan 36912, and Rhu) and 3 Type II strains (MOTT-H4Y, MOTT-36Y and MOTT-68Y) (Fig 4B), suggesting the potential for gene exchanges by LGT events between the 2 genotypes. Notably, a total of 39 M. yongonense signature SNPs out of the 395 selected SNPs were found. These SNPs could be used for the development of M. yongonense-specific molecular diagnostic methods.
In conclusion, our genome-based phylogenetic analysis indicated that the taxonomic status of the two INT-5 strains, MOTT-H4Y and MOTT-36Y previously identified as M. intracellulare should be revised to M. yongonense. Taken together, M. yongonense could be divided into 2 distinct genotypes depending on the presence of the LGT event of rpoB from M. parascrofulaceum: the Type I genotype with the M. parascrofulaceum rpoB gene and the Type II genotype with the M. intracellulare rpoB gene. Additionally, we developed a novel SNP-based phylogenetic analysis to enable the taxonomic identification of M. yongonense clinical strains.
Supporting Information
(XLSX)
Data Availability
All relevant data are within the paper and its Supporting Information files.
Funding Statement
Funded by www.htdream.kr, Korean Healthcare Technology R&D project, Ministry of Health, Welfare & Family Affairs, Republic of Korea (Grant No., HI13C15620000) (Bum-Joon Kim) and ernd.nrf.re.kr, National Research Foundation of Korea (Grant No., NRF-2014R1A1A2004008) (YHK). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Supplementary Materials
(XLSX)
Data Availability Statement
All relevant data are within the paper and its Supporting Information files.