# Evaluation of the Ribosomal Protein S1 Gene (rpsA) as a Novel Biomarker for Mycobacterium Species Identification

Authors: Hongfei Duan; Guan Liu; Xiaobo Wang; Yuhong Fu; Qian Liang; Yuanyuan Shang; Naihui Chu; Hairong Huang
Journal: BioMed Research International
 (2015)

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## Abstract

Objectives. To evaluate the resolution and reliability of the rpsA gene, encoding ribosomal protein S1, as a novel biomarker for mycobacteria species identification. Methods. A segment of the rpsA gene (565 bp) was amplified by PCR from 42 mycobacterial reference strains, 172 nontuberculosis mycobacteria clinical isolates, and 16 M. tuberculosis complex clinical isolates. The PCR products were sequenced and aligned by using the multiple alignment algorithm in the MegAlign package (DNASTAR) and the MEGA program. A phylogenetic tree was constructed by the neighbor-joining method. Results. Comparative sequence analysis of the rpsA gene provided the basis for species differentiation within the genus Mycobacterium. Slow- and rapid-growing groups of mycobacteria were clearly separated, and each mycobacterial species was differentiated as a distinct entity in the phylogenetic tree. The sequences discrepancy was obvious between M. kansasii and M. gastri, M. chelonae and M. abscessus, M. avium and M. intracellulare, and M. szulgai and M. malmoense, which cannot be achieved by 16S ribosomal DNA (rDNA) homologue genes comparison. 183 of the 188 (97.3%) clinical isolates, consisting of 8 mycobacterial species, were identified correctly by rpsA gene blast. Conclusions. Our study indicates that rpsA sequencing can be used effectively for mycobacteria species identification as a supplement to 16S rDNA sequence analysis.


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## Body

## 1. Introduction

Members of the genusMycobacterium are widespread in nature and range from harmless saprophytic species to strict pathogens that cause serious human and animal diseases. Both slow-growing mycobacteria and rapid-growing mycobacteria can cause human infections. Traditionally, taxonomy based on biochemical characteristics has been used for species determination of mycobacteria, but this approach is limited due to the overlapping biochemical and phenotypic patterns among the different mycobacterial species. Another approach using analysis of cell-wall fatty acid and mycolic acid composition is also limited by profile similarity among some emerging nontuberculosis mycobacteria (NTM) [1]. 16S rDNA homologue gene sequence comparison has been used as an important method for the mycobacterial species identification; however, ambiguous results have been obtained either due to the presence of more than one copy of the 16S rDNA gene within the genome, for example, inM. celatum andM. terraecomplex [2, 3], or due to sequence homology between species [4]. Therefore, alternative phylogenetic markers which are capable of complementing 16S rRNA gene would be useful for the phylogenetic study and species identification of the genusMycobacterium.Ribosomal protein S1 (rpsA), which is in the 30S ribosome subunit, contains the S1 domain that has been found in a large number of RNA-associated proteins.RpsA is a vital protein involved in protein translation and the ribosome-sparing process of translation. In addition, it has been reported thatRpsA is the target of pyrazinoic acid, the active form of the antituberculosis drug pyrazinamide [5].Mycobacterium tuberculosis has a single copy of therpsA gene in the genome [6], while the sequence homology among different mycobacteria species in which the gene has already been sequenced is between 86.7 and 100%. This suggests thatrpsA may be suitable for phylogenetic study of the genusMycobacterium. In this paper, we report an evaluation of therpsA gene as a novel biomarker for mycobacteria species identification.


## 2. Materials and Methods

### 2.1. Mycobacterial Reference Strains and Clinical Isolates

42 type and reference strains of the genusMycobacterium (Table 1), 172 clinical NTM isolates, and 16M. tuberculosis complex (MTC) isolates were investigated. All the type and reference strains were purchased from the American Type Culture Collection (ATCC) and all the clinical isolates used in this study were obtained from the Clinical Database and Sample Bank of tuberculosis of Beijing, National Clinical Lab on Tuberculosis, Beijing Chest Hospital. All the clinical NTM isolates were identified to the species level by sequence alignment of at least two of the following: 16S rDNA, 16-23S rRNA gene internal transcribed spacer (ITS), and rpoB and hsp65 genes as described before [2, 7–9]. Following sequencing, the 172 NTM clinical strains were found to include 10 strains ofM. avium, 75 strains ofM. intracellulare, 23 strains ofM. kansasii, 39 strains ofM. abscessus, 17 strains ofM. fortuitum complex, 7 strains ofM. gordonae, and 1 strain ofM. neoaurum.Table 1
PercentagerpsA sequence match among type strain.
Species
Type strain
First match (%)a
Second match (%)a
M.  thermoresistibile
ATCC19527
M.  senegalense (100%)
M.  porcinum (98.7%)
M.  senegalense
ATCC35796
M.  thermoresistibile (100%)
M.  porcinum (98.7%)
M.  porcinum
ATCC33776
M.  senegalense/M.  thermoresistibile (98.7%)
M.  fortuitum (98.3%)
M.  fortuitum
ATCC6481
M.  porcinum (98.3%)
M.  senegalense/M.  thermoresistibile (98.1%)
M.  phlei
ATCC11758
M.  porcinum (96.5%)
M.  fortuitum (96.1%)
M.  gadium
ATCC27726
M.  porcinum/M.  fortuitum (95.7%)
M.  smegmatis (95.3%)
M.  flavescens
ATCC14474
M.  smegmatis (96.6%)
M.  vaccae (96.0%)
M.  smegmatis
ATCC19420
M.  vaccae/M.  parafortuitum/M.  triviale (96.6%)
M.  senegalense/M.  thermoresistibile (96.3%)
M.  vaccae
ATCC15483
M.  diernhoferi/M.  duvalii (96.8%)
M.  parafortuitum/M.  triviale (96.6%)
M.  parafortuitum
ATCC19686
M.  triviale (100%)
M.  terrae/M.  austroafricanum (96.6%)
M.  triviale
ATCC23292
M.  parafortuitum (100%)
M.  terrae/M.  austroafricanum (96.6%)
M.  neoaurum
ATCC25795
M.  diernhoferi/M.  duvalii (96.4%)
M.  porcinum (95.5%)
M.  diernhoferi
ATCC19340
M.  duvalii (100%)
M.  vaccae (96.8%)
M.  duvalii
ATCC43910
M.  diernhoferi (100%)
M.  vaccae (96.8%)
M.  aurum
ATCC23366
M.  gilvum (93.9%)
M.  chubuense/M.  vanbaalenii (93.0%)
M.  chubuense
ATCC27278
M.  gilvum (96.6%)
M.  vanbaalenii (96.0%)
M.  gilvum
ATCC43909
M.  vanbaalenii (97.2%)
M.  terrae/M.  austroafricanum (97.0%)
M.  vanbaalenii
PYR-1
M.  terrae/M.  austroafricanum (99.8%)
M.  gilvum (97.2%)
M.  terrae
ATCC15755
M.  austroafricanum (100%)
M.  vanbaalenii (99.8%)
M.  austroafricanum
ATCC33464
M.  terrae (100%)
M.  vanbaalenii (99.8%)
M.  rhodesiae
ATCC27024
M.  terrae/M.  austroafricanum (93.9%)
M.  obuense (93.7%)
M.  obuense
ATCC27023
M.  porcinum /M.  fortuitum (96.1%)
M.  senegalense/M.  thermoresistibile (96.5%)
M.  aichiense
ATCC27280
M.  sphagni (95.8%)
M.  vaccae (94.7%)
M.  sphagni
ATCC33027
M.  aichiense (95.8%)
M.  porcinum (95.5%)
M.  tokaiense
ATCC27282
M.  porcinum (95.3%)
M.  smegmatis (93.7%)
M.  chelonae
ATCC35752
M.  obuense (93.9%)
M.  phlei (93.5%)
M.  abscessussubspeciesmassiliense
str. GO 06
M.  abscessus (98.7%)
M.  sphagni (91.4%)
M.  abscessus
ATCC19977
M.  abscessus  subspecies massiliense (98.7%)
M.  chubuense (92.0%)
M.  tuberculosisH37Rv
ATCC27294
M.  tuberculosisH37Ra/M.  africanum/M.  bovisBCG/M.  bovis (100%)
M.  canettii (99.8%)
M.  tuberculosisH37Ra
ATCC25177
M.  tuberculosis H37Rv/M.  africanum/M.  bovisBCG/M.  bovis (100%)
M.  canettii (99.8%)
M.  africanum
ATCC25420
M.  tuberculosis H37Ra /M.  tuberculosis Rv/M.  bovisBCG/M.  bovis (100%)
M.  canettii (99.8%)
M.  bovisBCG
ATCC35735
M.  tuberculosis H37Ra /M.  tuberculosis H37Rv /M.  africanum/M.  bovis (100%)
M.  canettii (99.8%)
M.  bovis
ATCC19210
M.  tuberculosis H37Ra/M.  tuberculosisH37Rv/M.  africanum/M.  bovisBCG (100%)
M.  canettii (99.8%)
M.  canettii
CIPT140010059
M.  tuberculosis/M.  africanum/M.  bovisBCG/M.  bovis (99.8%)
M.  asiaticum (93.2%)
M.  xenopi
ATCC19250
M.  asiaticum (93.0%)
M.  aviumsubspeciesparatuberculosis (92.4%)
M.  avium
ATCC25291
M.  avium subspecies paratuberculosis  (99.6%)
M.  asiaticum (95.8%)
M.  aviumsubspeciesparatuberculosis
K-10
M.  avium (99.4%)
M.  asiaticum (96%)
M.  malmoense
ATCC29571
M.  intracellulare(94.3%)
M.  szulgai (93.9%)
M.  intracellulare
ATCC13950
M.  avium (95.6%)
M.  aviumsubspeciesparatuberculosis  (95.4%)
M.  scrofulaceum
ATCC19981
M.  asiaticum (95.4%)
M.  szulgai (95.1%)
M.  marinum
ATCC927
M.  ulcerans (99.6%)
M.  szulgai (96.0%)
M.   ulcerans
Agy99
M.  marinum (99.6%)
M.  szulgai (96.0%)
M.  szulgai
ATCC25799
M.  marinum/M.  ulcerans (96.0%)
M.  asiaticum (95.6%)
M.  gordonae
ATCC14470
M.  asiaticum (95.3%)
M.  kansasii (95.1%)
M.  asiaticum
ATCC25276
M.  simiae (97.7%)
M.  kansasii (96.4%)
M.  kansasii
ATCC12478
M.  asiaticum (96.3%)
M.  gastri (95.8%)
M.  gastri
ATCC15754
M.  kansasii (95.8%)
M.  asiaticum (94.5%)
Rhodococcus equi
103S
M.  smegmatis (90.3%)
M.  intracellulare (90.1%)
aBeside the queried type strain itself, the first matched or the second matched type strain.


### 2.2.rpsA Gene Amplification and Sequencing

DNA was released from cultured mycobacteria by boiling the cultured mycobacterial suspension in TE buffer for 10 min. After centrifugation, the supernatant was used for PCR amplification [10]. The primers used were forward primer, 5′-CCCTACATCGGCAAGGAG-3′, position 487–504 in therpsA gene ofMycobacterium tuberculosis, GenBank accession number NC_000962.2, and reverse primer, 5′-TGTCGATGACCTTGACCATC-3′, position 1032–1051 in therpsA gene ofMycobacterium tuberculosis, GenBank accession number NC_000962.2. The amplified product was 565 bp. PCR products were purified and sequenced by a commercial company (YINGJUN Biotech Company, Beijing, China) using ABI 3730 DNA Analyzer (Applied Biosystems, California, USA).


### 2.3. Sequence Analysis and the Phylogenetic Tree Constructions

In addition to the 42 reference strains, therpsA sequences of 5 other mycobacterial species were obtained directly from GenBank, includingM. avium subspeciesparatuberculosis (GenBank accession number NC_002944.2),M. ulcerans (GenBank accession number NC_008611.1),M. vanbaalenii (GenBank accession number NC_008726.1),M. abscessus subspeciesmassiliense (GenBank accession number NC_018150.1), andM. canettii (GenBank accession number NC_015848.1). All sequences were aligned and homology was calculated by using the multiple alignment algorithm in the MegAlign package (Windows version 7.1.0; DNASTAR, Madison, Wis). 527 bp of sequence (excluding 38 nucleotides at each end of the amplicon, corresponding to the primer binding sites) was analyzed for the phylogenetic tree construction by the neighbor-joining method using the MEGA 5.05 package (http://www.megasoftware.net/mega.php). A bootstrap analysis (1000 repeats) usingRhodococcus equi (GenBank accession number NC_006361.1) as the outgroup was performed to evaluate the topology of the phylogenetic tree.


### 2.4. Species Identification of the Clinical Isolates

The 188 clinical isolates were analyzed blindly. Sequences, minus the known PCR primer sequences, were assembled and edited by using SeqMan software (version 7.1.0; DNASTAR, Madison, WI). Isolates were identified by comparing sequences by using a FASTA BLASTn search with MegAlign (version 7.1.0; DNASTAR, Madison, WI) to an in-house database of sequences consisting of type and reference strains from external culture collections.


## 2.1. Mycobacterial Reference Strains and Clinical Isolates

42 type and reference strains of the genusMycobacterium (Table 1), 172 clinical NTM isolates, and 16M. tuberculosis complex (MTC) isolates were investigated. All the type and reference strains were purchased from the American Type Culture Collection (ATCC) and all the clinical isolates used in this study were obtained from the Clinical Database and Sample Bank of tuberculosis of Beijing, National Clinical Lab on Tuberculosis, Beijing Chest Hospital. All the clinical NTM isolates were identified to the species level by sequence alignment of at least two of the following: 16S rDNA, 16-23S rRNA gene internal transcribed spacer (ITS), and rpoB and hsp65 genes as described before [2, 7–9]. Following sequencing, the 172 NTM clinical strains were found to include 10 strains ofM. avium, 75 strains ofM. intracellulare, 23 strains ofM. kansasii, 39 strains ofM. abscessus, 17 strains ofM. fortuitum complex, 7 strains ofM. gordonae, and 1 strain ofM. neoaurum.Table 1
PercentagerpsA sequence match among type strain.
Species
Type strain
First match (%)a
Second match (%)a
M.  thermoresistibile
ATCC19527
M.  senegalense (100%)
M.  porcinum (98.7%)
M.  senegalense
ATCC35796
M.  thermoresistibile (100%)
M.  porcinum (98.7%)
M.  porcinum
ATCC33776
M.  senegalense/M.  thermoresistibile (98.7%)
M.  fortuitum (98.3%)
M.  fortuitum
ATCC6481
M.  porcinum (98.3%)
M.  senegalense/M.  thermoresistibile (98.1%)
M.  phlei
ATCC11758
M.  porcinum (96.5%)
M.  fortuitum (96.1%)
M.  gadium
ATCC27726
M.  porcinum/M.  fortuitum (95.7%)
M.  smegmatis (95.3%)
M.  flavescens
ATCC14474
M.  smegmatis (96.6%)
M.  vaccae (96.0%)
M.  smegmatis
ATCC19420
M.  vaccae/M.  parafortuitum/M.  triviale (96.6%)
M.  senegalense/M.  thermoresistibile (96.3%)
M.  vaccae
ATCC15483
M.  diernhoferi/M.  duvalii (96.8%)
M.  parafortuitum/M.  triviale (96.6%)
M.  parafortuitum
ATCC19686
M.  triviale (100%)
M.  terrae/M.  austroafricanum (96.6%)
M.  triviale
ATCC23292
M.  parafortuitum (100%)
M.  terrae/M.  austroafricanum (96.6%)
M.  neoaurum
ATCC25795
M.  diernhoferi/M.  duvalii (96.4%)
M.  porcinum (95.5%)
M.  diernhoferi
ATCC19340
M.  duvalii (100%)
M.  vaccae (96.8%)
M.  duvalii
ATCC43910
M.  diernhoferi (100%)
M.  vaccae (96.8%)
M.  aurum
ATCC23366
M.  gilvum (93.9%)
M.  chubuense/M.  vanbaalenii (93.0%)
M.  chubuense
ATCC27278
M.  gilvum (96.6%)
M.  vanbaalenii (96.0%)
M.  gilvum
ATCC43909
M.  vanbaalenii (97.2%)
M.  terrae/M.  austroafricanum (97.0%)
M.  vanbaalenii
PYR-1
M.  terrae/M.  austroafricanum (99.8%)
M.  gilvum (97.2%)
M.  terrae
ATCC15755
M.  austroafricanum (100%)
M.  vanbaalenii (99.8%)
M.  austroafricanum
ATCC33464
M.  terrae (100%)
M.  vanbaalenii (99.8%)
M.  rhodesiae
ATCC27024
M.  terrae/M.  austroafricanum (93.9%)
M.  obuense (93.7%)
M.  obuense
ATCC27023
M.  porcinum /M.  fortuitum (96.1%)
M.  senegalense/M.  thermoresistibile (96.5%)
M.  aichiense
ATCC27280
M.  sphagni (95.8%)
M.  vaccae (94.7%)
M.  sphagni
ATCC33027
M.  aichiense (95.8%)
M.  porcinum (95.5%)
M.  tokaiense
ATCC27282
M.  porcinum (95.3%)
M.  smegmatis (93.7%)
M.  chelonae
ATCC35752
M.  obuense (93.9%)
M.  phlei (93.5%)
M.  abscessussubspeciesmassiliense
str. GO 06
M.  abscessus (98.7%)
M.  sphagni (91.4%)
M.  abscessus
ATCC19977
M.  abscessus  subspecies massiliense (98.7%)
M.  chubuense (92.0%)
M.  tuberculosisH37Rv
ATCC27294
M.  tuberculosisH37Ra/M.  africanum/M.  bovisBCG/M.  bovis (100%)
M.  canettii (99.8%)
M.  tuberculosisH37Ra
ATCC25177
M.  tuberculosis H37Rv/M.  africanum/M.  bovisBCG/M.  bovis (100%)
M.  canettii (99.8%)
M.  africanum
ATCC25420
M.  tuberculosis H37Ra /M.  tuberculosis Rv/M.  bovisBCG/M.  bovis (100%)
M.  canettii (99.8%)
M.  bovisBCG
ATCC35735
M.  tuberculosis H37Ra /M.  tuberculosis H37Rv /M.  africanum/M.  bovis (100%)
M.  canettii (99.8%)
M.  bovis
ATCC19210
M.  tuberculosis H37Ra/M.  tuberculosisH37Rv/M.  africanum/M.  bovisBCG (100%)
M.  canettii (99.8%)
M.  canettii
CIPT140010059
M.  tuberculosis/M.  africanum/M.  bovisBCG/M.  bovis (99.8%)
M.  asiaticum (93.2%)
M.  xenopi
ATCC19250
M.  asiaticum (93.0%)
M.  aviumsubspeciesparatuberculosis (92.4%)
M.  avium
ATCC25291
M.  avium subspecies paratuberculosis  (99.6%)
M.  asiaticum (95.8%)
M.  aviumsubspeciesparatuberculosis
K-10
M.  avium (99.4%)
M.  asiaticum (96%)
M.  malmoense
ATCC29571
M.  intracellulare(94.3%)
M.  szulgai (93.9%)
M.  intracellulare
ATCC13950
M.  avium (95.6%)
M.  aviumsubspeciesparatuberculosis  (95.4%)
M.  scrofulaceum
ATCC19981
M.  asiaticum (95.4%)
M.  szulgai (95.1%)
M.  marinum
ATCC927
M.  ulcerans (99.6%)
M.  szulgai (96.0%)
M.   ulcerans
Agy99
M.  marinum (99.6%)
M.  szulgai (96.0%)
M.  szulgai
ATCC25799
M.  marinum/M.  ulcerans (96.0%)
M.  asiaticum (95.6%)
M.  gordonae
ATCC14470
M.  asiaticum (95.3%)
M.  kansasii (95.1%)
M.  asiaticum
ATCC25276
M.  simiae (97.7%)
M.  kansasii (96.4%)
M.  kansasii
ATCC12478
M.  asiaticum (96.3%)
M.  gastri (95.8%)
M.  gastri
ATCC15754
M.  kansasii (95.8%)
M.  asiaticum (94.5%)
Rhodococcus equi
103S
M.  smegmatis (90.3%)
M.  intracellulare (90.1%)
aBeside the queried type strain itself, the first matched or the second matched type strain.


## 2.2.rpsA Gene Amplification and Sequencing

DNA was released from cultured mycobacteria by boiling the cultured mycobacterial suspension in TE buffer for 10 min. After centrifugation, the supernatant was used for PCR amplification [10]. The primers used were forward primer, 5′-CCCTACATCGGCAAGGAG-3′, position 487–504 in therpsA gene ofMycobacterium tuberculosis, GenBank accession number NC_000962.2, and reverse primer, 5′-TGTCGATGACCTTGACCATC-3′, position 1032–1051 in therpsA gene ofMycobacterium tuberculosis, GenBank accession number NC_000962.2. The amplified product was 565 bp. PCR products were purified and sequenced by a commercial company (YINGJUN Biotech Company, Beijing, China) using ABI 3730 DNA Analyzer (Applied Biosystems, California, USA).


## 2.3. Sequence Analysis and the Phylogenetic Tree Constructions

In addition to the 42 reference strains, therpsA sequences of 5 other mycobacterial species were obtained directly from GenBank, includingM. avium subspeciesparatuberculosis (GenBank accession number NC_002944.2),M. ulcerans (GenBank accession number NC_008611.1),M. vanbaalenii (GenBank accession number NC_008726.1),M. abscessus subspeciesmassiliense (GenBank accession number NC_018150.1), andM. canettii (GenBank accession number NC_015848.1). All sequences were aligned and homology was calculated by using the multiple alignment algorithm in the MegAlign package (Windows version 7.1.0; DNASTAR, Madison, Wis). 527 bp of sequence (excluding 38 nucleotides at each end of the amplicon, corresponding to the primer binding sites) was analyzed for the phylogenetic tree construction by the neighbor-joining method using the MEGA 5.05 package (http://www.megasoftware.net/mega.php). A bootstrap analysis (1000 repeats) usingRhodococcus equi (GenBank accession number NC_006361.1) as the outgroup was performed to evaluate the topology of the phylogenetic tree.


## 2.4. Species Identification of the Clinical Isolates

The 188 clinical isolates were analyzed blindly. Sequences, minus the known PCR primer sequences, were assembled and edited by using SeqMan software (version 7.1.0; DNASTAR, Madison, WI). Isolates were identified by comparing sequences by using a FASTA BLASTn search with MegAlign (version 7.1.0; DNASTAR, Madison, WI) to an in-house database of sequences consisting of type and reference strains from external culture collections.


## 3. Results

### 3.1.rpsA Sequence Alignment of the Reference Strains

Between 85.4% and 100% sequence homology (interspecies divergence, 0% to 14.6%) was observed among the 42 tested reference strains and the 5 additional mycobacterial species whose sequences were obtained from the GenBank database (Table2). All therpsA gene sequences of the analyzed mycobacteria strains were distinct from the outgroup strainR. equi. Among the 19 reference strains of the slow-growingMycobacterium genus, 11 strains were greater than 97% homology, including 5M. tuberculosis complex strains. Among the 28 reference strains of the rapid-growingMycobacterium genus, 14 strains were greater than 97% homology (Table 1). The pathogenicM. kansasii was easily differentiated from the nonpathogenicM. gastri (95.8% homology), while those two species were not distinguishable by the 16S rDNA sequence alignment. The sequence homologies between various species were 91.8% betweenM. chelonae andM. abscessus, 95.6% betweenM. avium andM. intracellulare, and 93.9% betweenM. szulgai andM. malmoense. However, the sequence homology between other species was higher; for example, it was 99.6% betweenM. ulcerans andM. marinum and 98.7% betweenM. abscessus subspeciesmassiliense andM. abscessus. All members of theM. tuberculosis complex had identical sequences as didM. senegalense andM. thermoresistibile,M. parafortuitum andM. trivial,M. diernhoferi andM. duvalii, andM. austroafricanum andM. terrae.Table 2
Sequence pair distances of 47 type strains of mycobacteria and 1 out-group strain determined byusing the multiple alignment algorithm in the MegAlign package (version 7.1.0; DNASTAR, Madison, WI).
Percent identity
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
1
—
100.0
98.7
98.1
96.0
95.3
95.4
96.4
96.2
94.5
94.5
95.1
94.5
94.5
92.0
93.9
94.1
94.3
94.5
94.5
92.8
95.6
93.7
94.7
93.2
92.4
88.8
89.2
91.1
91.1
91.1
91.1
91.1
91.3
91.5
93.9
94.1
91.3
93.0
92.8
92.6
92.6
92.4
93.4
93.4
92.4
92.8
89.2
1
M.  thermoresistibile
2
0.0
—
98.7
98.1
96.0
95.3
95.4
96.4
96.2
94.5
94.5
95.1
94.5
94.5
92.0
93.9
94.1
94.3
94.5
94.5
92.8
95.6
93.7
94.7
93.2
92.4
88.8
89.2
91.1
91.1
91.1
91.1
91.1
91.3
91.5
93.9
94.1
91.3
93.0
92.8
92.6
92.6
92.4
93.4
93.4
92.4
92.8
89.2
2
M.  senegalense
3
1.3
1.3
—
98.3
96.6
95.8
95.8
96.8
96.8
94.7
94.7
95.6
95.4
95.4
92.0
94.1
95.4
94.9
95.1
95.1
93.0
96.2
94.7
95.6
94.5
93.0
89.4
89.8
91.1
91.1
91.1
91.1
91.1
91.3
92.0
94.5
94.7
92.0
93.4
93.5
93.2
93.2
92.8
93.7
93.7
93.0
92.8
89.4
3
M.  porcinum
4
1.9
1.9
1.7
—
96.2
95.8
95.8
96.2
96.2
94.1
94.1
95.4
94.5
94.5
92.0
94.3
94.5
94.3
94.5
94.5
93.9
96.2
94.9
94.9
93.7
93.7
90.1
90.5
90.9
90.9
90.9
90.9
90.9
91.1
92.0
94.7
94.9
91.3
93.2
93.2
93.0
93.4
92.8
93.5
93.7
92.8
93.0
89.2
4
M.  fortuitum
5
4.1
4.1
3.5
3.9
—
94.3
95.4
96.4
96.0
93.9
93.9
93.4
94.3
94.3
91.5
94.3
94.3
94.5
94.7
94.7
91.5
94.3
94.1
94.9
92.8
91.1
89.8
90.3
90.1
90.1
90.1
90.1
90.1
90.3
91.1
93.5
93.7
91.3
93.4
93.0
93.5
93.5
93.0
93.7
93.4
93.0
92.0
89.6
5
M.  phlei
6
4.9
4.9
4.3
4.3
5.9
—
95.1
95.3
94.1
93.7
93.7
94.7
94.1
94.1
91.3
93.7
93.5
93.4
93.5
93.5
93.2
93.7
93.7
93.7
93.0
92.4
88.6
89.0
90.7
90.7
90.7
90.7
90.7
90.9
91.5
94.3
94.1
90.7
93.2
92.0
91.8
92.2
91.5
93.0
93.2
91.8
91.5
88.0
6
M.  gadium
7
4.7
4.7
4.3
4.3
4.7
5.1
—
96.6
96.0
95.4
95.4
94.7
94.7
94.7
93.0
95.1
95.3
95.6
95.8
95.8
93.2
94.1
94.9
94.7
92.4
92.0
90.1
90.5
92.4
92.4
92.4
92.4
92.4
92.6
92.8
94.9
94.7
93.0
94.1
94.9
93.7
93.7
93.5
93.9
94.3
92.6
93.4
90.3
7
M.  flavescens
8
3.7
3.7
3.3
3.9
3.7
4.9
3.5
—
96.6
96.6
96.6
95.3
96.2
96.2
92.0
96.2
96.2
96.4
96.2
96.2
93.5
94.7
94.5
94.5
93.9
91.5
90.5
90.9
91.1
91.1
91.1
91.1
91.1
91.3
92.2
93.7
93.5
92.0
94.3
93.5
93.2
93.2
93.0
93.4
93.5
92.4
92.2
90.9
8
M.  smegmatis
9
3.9
3.9
3.3
3.9
4.1
6.1
4.1
3.5
—
96.6
96.6
95.1
96.8
96.8
92.4
95.8
95.6
96.2
96.4
96.4
93.0
95.6
94.9
94.7
92.4
92.0
91.5
91.8
90.9
90.9
90.9
90.9
90.9
91.1
92.2
94.3
94.5
92.4
93.9
92.8
93.4
93.4
93.2
93.9
93.5
92.8
92.8
89.2
9
M.  vaccae
10
5.7
5.7
5.5
6.1
6.3
6.5
4.7
3.5
3.5
—
100.0
94.9
96.2
96.2
93.0
94.9
95.8
96.4
96.6
96.6
93.0
94.3
92.8
93.0
92.4
91.7
89.8
90.1
89.9
89.9
89.9
89.9
89.9
90.1
91.5
92.6
92.4
91.3
93.2
92.0
91.1
91.1
91.3
91.8
91.5
90.3
91.7
89.0
10
M.  parafortuitum
11
5.7
5.7
5.5
6.1
6.3
6.5
4.7
3.5
3.5
0.0
—
94.9
96.2
96.2
93.0
94.9
95.8
96.4
96.6
96.6
93.0
94.3
92.8
93.0
92.4
91.7
89.8
90.1
89.9
89.9
89.9
89.9
89.9
90.1
91.5
92.6
92.4
91.3
93.2
92.0
91.1
91.1
91.3
91.8
91.5
90.3
91.7
89.0
11
M.  triviale
12
5.1
5.1
4.5
4.7
7.0
5.5
5.5
4.9
5.1
5.3
5.3
—
96.4
96.4
91.8
94.5
95.3
95.1
95.3
95.3
94.1
95.1
92.8
92.2
92.6
93.0
90.3
90.7
89.9
89.9
89.9
89.9
89.9
90.1
91.3
93.2
92.8
89.9
92.0
92.0
91.7
91.7
91.1
91.8
92.4
90.9
89.9
88.2
12
M.  neoaurum
13
5.7
5.7
4.7
5.7
5.9
6.1
5.5
3.9
3.3
3.9
3.9
3.7
—
100.0
92.6
95.3
95.8
95.6
95.8
95.8
94.1
94.9
94.1
93.7
92.8
92.2
91.3
91.7
89.4
89.4
89.4
89.4
89.4
89.6
90.7
92.0
91.8
91.1
93.0
91.8
91.5
91.5
91.3
91.7
91.8
91.3
90.5
89.8
13
M.  diernhoferi
14
5.7
5.7
4.7
5.7
5.9
6.1
5.5
3.9
3.3
3.9
3.9
3.7
0.0
—
92.6
95.3
95.8
95.6
95.8
95.8
94.1
94.9
94.1
93.7
92.8
92.2
91.3
91.7
89.4
89.4
89.4
89.4
89.4
89.6
90.7
92.0
91.8
91.1
93.0
91.8
91.5
91.5
91.3
91.7
91.8
91.3
90.5
89.8
14
M.  duvalii
15
8.4
8.4
8.4
8.4
9.1
9.3
7.4
8.5
8.0
7.4
7.4
8.6
7.8
7.8
—
93.0
93.9
93.0
93.2
93.2
91.7
91.8
91.1
91.1
89.4
91.1
89.8
90.1
89.2
89.2
89.2
89.2
89.2
89.4
88.8
90.1
90.5
91.5
90.5
90.5
89.6
89.8
89.8
89.2
90.1
89.6
89.0
87.7
15
M.  aurum
16
6.3
6.3
6.1
5.9
5.9
6.5
5.1
3.9
4.3
5.3
5.3
5.7
4.9
4.9
7.4
—
96.6
96.0
95.8
95.8
93.9
94.7
94.7
93.5
92.8
92.4
91.8
92.4
90.5
90.5
90.5
90.5
90.5
90.7
91.7
93.2
93.0
91.1
92.6
92.4
92.6
93.0
91.3
92.4
93.5
91.5
90.9
88.4
16
M.  chubuense
17
6.1
6.1
4.7
5.7
5.9
6.8
4.9
3.9
4.5
4.3
4.3
4.9
4.3
4.3
6.3
3.5
—
97.2
97.0
97.0
94.1
95.6
94.5
94.1
93.5
93.4
91.3
91.7
90.3
90.3
90.3
90.3
90.3
90.5
91.3
93.0
93.2
92.0
92.8
93.2
92.6
93.0
91.7
92.8
92.8
91.5
91.1
90.1
17
M.  gilvum
18
5.9
5.9
5.3
5.9
5.7
7.0
4.5
3.7
3.9
3.7
3.7
5.1
4.5
4.5
7.4
4.1
2.9
—
99.8
99.8
94.1
94.9
92.8
93.4
92.6
93.0
90.7
91.1
90.1
90.1
90.1
90.1
90.1
90.3
91.3
92.8
92.6
91.3
92.4
92.8
92.0
92.0
91.3
92.0
92.4
91.3
92.0
89.2
18
M.  vanbaalenii
19
5.7
5.7
5.1
5.7
5.5
6.8
4.3
3.9
3.7
3.5
3.5
4.9
4.3
4.3
7.2
4.3
3.1
0.2
—
100.0
93.9
95.1
93.0
93.5
92.8
93.2
90.9
91.3
90.3
90.3
90.3
90.3
90.3
90.5
91.5
93.0
92.8
91.5
92.6
93.0
92.2
92.2
91.5
92.2
92.6
91.5
92.2
89.0
19
M.  terrae
20
5.7
5.7
5.1
5.7
5.5
6.8
4.3
3.9
3.7
3.5
3.5
4.9
4.3
4.3
7.2
4.3
3.1
0.2
0.0
—
93.9
95.1
93.0
93.5
92.8
93.2
90.9
91.3
90.3
90.3
90.3
90.3
90.3
90.5
91.5
93.0
92.8
91.5
92.6
93.0
92.2
92.2
91.5
92.2
92.6
91.5
92.2
89.0
20
M.  austroafricanum
21
7.6
7.6
7.4
6.3
9.1
7.2
7.2
6.7
7.4
7.4
7.4
6.1
6.1
6.1
8.9
6.3
6.1
6.1
6.3
6.3
—
93.7
91.8
91.7
91.1
92.6
88.8
89.2
89.4
89.4
89.4
89.4
89.4
89.6
90.3
91.5
91.7
90.1
90.9
90.9
89.9
90.1
89.6
90.7
91.3
90.9
89.9
89.6
21
M.  rhodesiae
22
4.5
4.5
3.9
3.9
5.9
6.6
6.1
5.5
4.5
5.9
5.9
5.1
5.3
5.3
8.7
5.5
4.5
5.3
5.1
5.1
6.5
—
93.7
94.5
93.4
94.1
90.3
90.7
90.3
90.3
90.3
90.3
90.3
90.5
90.5
93.7
93.9
90.3
92.6
92.8
92.2
92.2
91.8
92.6
93.0
91.7
90.9
89.2
22
M.  obuense
23
6.5
6.5
5.5
5.3
6.1
6.5
5.3
5.7
5.3
7.6
7.6
7.6
6.1
6.1
9.5
5.5
5.7
7.6
7.4
7.4
8.6
6.5
—
95.8
93.9
93.0
91.7
92.0
90.9
90.9
90.9
90.9
90.9
91.1
92.2
93.7
93.9
91.7
93.2
92.6
93.4
93.7
92.2
93.7
93.9
92.4
91.7
89.0
23
M.  aichiense
24
5.5
5.5
4.5
5.3
5.3
6.5
5.5
5.7
5.5
7.4
7.4
8.2
6.5
6.5
9.5
6.7
6.1
7.0
6.7
6.7
8.9
5.7
4.3
—
93.7
93.4
91.3
91.8
91.5
91.5
91.5
91.5
91.5
91.7
91.5
93.7
94.1
92.2
93.0
93.5
92.8
93.0
93.0
93.4
94.1
93.2
92.4
89.2
24
M.  sphagni
25
7.2
7.2
5.7
6.5
7.6
7.4
8.0
6.3
8.0
8.0
8.0
7.8
7.6
7.6
11.5
7.6
6.7
7.8
7.6
7.6
9.5
7.0
6.3
6.5
—
92.0
88.8
89.4
89.6
89.6
89.6
89.6
89.6
89.8
90.5
94.5
94.5
90.7
93.0
92.8
91.8
92.2
92.0
92.4
93.4
92.2
91.5
88.2
25
M.  tokaiense
26
8.0
8.0
7.4
6.5
9.5
8.0
8.4
9.1
8.4
8.9
8.9
7.4
8.2
8.2
9.5
8.0
7.0
7.4
7.2
7.2
7.8
6.1
7.4
7.0
8.4
—
91.5
91.8
89.6
89.6
89.6
89.6
89.6
89.8
89.8
92.2
92.6
89.4
90.5
91.1
90.7
91.1
89.8
92.0
91.7
90.7
90.3
87.1
26
M.  chelonae
27
12.1
12.1
11.5
10.6
11.0
12.4
10.6
10.1
9.1
11.0
11.0
10.4
9.3
9.3
11.0
8.6
9.3
9.9
9.7
9.7
12.1
10.4
8.9
9.3
12.1
9.1
—
98.7
86.7
86.7
86.7
86.7
86.7
86.9
88.4
88.4
88.8
88.4
88.6
89.4
89.6
89.6
88.4
90.1
89.8
88.2
87.1
87.1
27
M.  abscessus
28
11.7
11.7
11.0
10.1
10.4
11.9
10.1
9.7
8.6
10.6
10.6
9.9
8.9
8.9
10.6
8.0
8.9
9.5
9.3
9.3
11.7
9.9
8.4
8.6
11.5
8.7
1.3
—
87.5
87.5
87.5
87.5
87.5
87.7
88.8
88.8
89.2
88.8
89.0
89.8
89.9
89.9
89.0
90.5
90.1
88.6
87.5
87.7
28
M.  abscessus
29
9.5
9.5
9.5
9.7
10.6
10.0
8.0
9.5
9.7
10.8
10.8
10.8
11.5
11.5
11.7
10.1
10.4
10.6
10.4
10.4
11.5
10.4
9.7
9.1
11.3
11.3
14.6
13.7
—
100.0
100.0
100.0
100.0
99.8
90.9
91.7
92.0
90.9
91.7
92.2
92.0
91.8
92.0
92.8
93.4
92.2
91.8
88.0
29
M.  tuberculosis
30
9.5
9.5
9.5
9.7
10.6
10.0
8.0
9.5
9.7
10.8
10.8
10.8
11.5
11.5
11.7
10.1
10.4
10.6
10.4
10.4
11.5
10.4
9.7
9.1
11.3
11.3
14.6
13.7
0.0
—
100.0
100.0
100.0
99.8
90.9
91.7
92.0
90.9
91.7
92.2
92.0
91.8
92.0
92.8
93.4
92.2
91.8
88.0
30
M.  tuberculosis
31
9.5
9.5
9.5
9.7
10.6
10.0
8.0
9.5
9.7
10.8
10.8
10.8
11.5
11.5
11.7
10.1
10.4
10.6
10.4
10.4
11.5
10.4
9.7
9.1
11.3
11.3
14.6
13.7
0.0
0.0
—
100.0
100.0
99.8
90.9
91.7
92.0
90.9
91.7
92.2
92.0
91.8
92.0
92.8
93.4
92.2
91.8
88.0
31
M.  bovis
32
9.5
9.5
9.5
9.7
10.6
10.0
8.0
9.5
9.7
10.8
10.8
10.8
11.5
11.5
11.7
10.1
10.4
10.6
10.4
10.4
11.5
10.4
9.7
9.1
11.3
11.3
14.6
13.7
0.0
0.0
0.0
—
100.0
99.8
90.9
91.7
92.0
90.9
91.7
92.2
92.0
91.8
92.0
92.8
93.4
92.2
91.8
88.0
32
M.  africanum
33
9.5
9.5
9.5
9.7
10.6
10.0
8.0
9.5
9.7
10.8
10.8
10.8
11.5
11.5
11.7
10.1
10.4
10.6
10.4
10.4
11.5
10.4
9.7
9.1
11.3
11.3
14.6
13.7
0.0
0.0
0.0
0.0
—
99.8
90.9
91.7
92.0
90.9
91.7
92.2
92.0
91.8
92.0
92.8
93.4
92.2
91.8
88.0
33
M.  bovis
34
9.3
9.3
9.3
9.5
10.4
9.7
7.8
9.3
9.5
10.6
10.6
10.6
11.2
11.2
11.5
9.9
10.2
10.4
10.2
10.2
11.2
10.2
9.5
8.9
11.1
11.0
14.4
13.5
0.2
0.2
0.2
0.2
0.2
—
91.1
91.8
92.2
90.7
91.8
92.4
91.8
91.7
91.8
92.6
93.2
92.0
91.7
88.0
34
M.  canettii
35
9.1
9.1
8.4
8.4
9.5
9.1
7.6
8.2
8.2
9.1
9.1
9.3
9.9
9.9
12.2
8.9
9.3
9.3
9.1
9.1
10.4
10.2
8.2
9.1
10.2
11.0
12.6
12.1
9.8
9.8
9.8
9.8
9.8
9.5
—
92.2
92.4
90.7
91.7
92.0
91.1
91.1
90.5
91.8
93.0
91.5
90.9
87.7
35
M.  xenopi
36
6.3
6.3
5.7
5.5
6.7
5.9
5.3
6.5
5.9
7.8
7.8
7.2
8.4
8.4
10.6
7.2
7.4
7.6
7.4
7.4
9.1
6.5
6.5
6.5
5.7
8.2
12.6
12.1
8.9
8.9
8.9
8.9
8.9
8.7
8.2
—
99.4
93.0
95.6
94.7
93.9
94.3
94.1
95.1
95.8
94.7
93.7
88.6
36
M.  avium
37
6.1
6.1
5.5
5.3
6.5
6.1
5.5
6.7
5.7
8.0
8.0
7.6
8.6
8.6
10.1
7.4
7.2
7.8
7.6
7.6
8.9
6.3
6.3
6.1
5.7
7.8
12.1
11.7
8.4
8.4
8.4
8.4
8.4
8.2
8.0
0.6
—
93.5
95.4
95.1
94.5
94.9
94.5
95.4
96.0
95.3
93.9
88.8
37
M.  avium
38
9.3
9.3
8.4
9.3
9.3
9.9
7.4
8.4
8.0
9.3
9.3
10.8
9.5
9.5
9.1
9.5
8.4
9.3
9.1
9.1
10.6
10.4
8.9
8.2
9.9
11.5
12.6
12.1
9.7
9.7
9.7
9.7
9.7
10.0
9.9
7.4
6.7
—
94.3
93.7
93.5
93.4
92.4
93.9
93.0
93.7
93.0
89.0
38
M.  malmoense
39
7.4
7.4
7.0
7.2
7.0
7.2
6.1
5.9
6.3
7.2
7.2
8.4
7.4
7.4
10.2
7.8
7.6
8.0
7.8
7.8
9.7
7.8
7.2
7.4
7.4
10.2
12.3
11.9
8.9
8.9
8.9
8.9
8.9
8.7
8.9
4.5
4.7
5.9
—
94.5
94.1
94.1
93.4
95.1
94.7
94.3
94.3
90.7
39
M.  intracellulare
40
7.6
7.6
6.7
7.2
7.4
8.4
5.3
6.7
7.6
8.4
8.4
8.4
8.6
8.6
10.1
8.0
7.2
7.6
7.4
7.4
9.7
7.6
7.8
6.7
7.6
9.5
11.5
11.0
8.2
8.2
8.2
8.2
8.2
8.0
8.4
5.5
5.1
6.5
5.7
—
94.3
94.3
94.9
95.1
95.4
93.9
93.4
89.6
40
M.  scrofulaceum
41
7.8
7.8
7.2
7.4
6.8
8.6
6.5
7.2
7.0
9.5
9.5
8.9
9.1
9.1
11.2
7.8
7.8
8.4
8.2
8.2
10.8
8.2
7.0
7.6
8.7
9.9
11.2
10.8
8.4
8.4
8.4
8.4
8.4
8.7
9.5
6.3
5.7
6.8
6.1
5.9
—
99.6
94.5
96.0
95.6
95.6
93.7
89.6
41
M.  marinum
42
7.8
7.8
7.2
7.0
6.8
8.2
6.5
7.2
7.0
9.5
9.5
8.9
9.1
9.1
11.0
7.4
7.4
8.4
8.2
8.2
10.6
8.2
6.5
7.4
8.2
9.5
11.2
10.8
8.7
8.7
8.7
8.7
8.7
8.9
9.5
5.9
5.3
7.0
6.1
5.9
0.4
—
94.3
96.0
95.6
95.4
93.9
89.4
42
M.  ulcerans
43
8.0
8.0
7.6
7.6
7.4
9.1
6.7
7.4
7.2
9.3
9.3
9.5
9.3
9.3
11.0
9.3
8.9
9.3
9.1
9.1
11.2
8.6
8.2
7.4
8.4
11.0
12.6
11.9
8.4
8.4
8.4
8.4
8.4
8.7
10.1
6.1
5.7
8.0
7.0
5.3
5.7
5.9
—
94.3
95.3
95.1
93.4
89.0
43
M.  gordonae
44
7.0
7.0
6.5
6.7
6.5
7.4
6.3
7.0
6.3
8.6
8.6
8.6
8.9
8.9
11.7
8.0
7.6
8.4
8.2
8.2
9.9
7.8
6.5
7.0
8.0
8.4
10.6
10.1
7.6
7.6
7.6
7.6
7.6
7.8
8.6
5.1
4.7
6.3
5.1
5.1
4.1
4.1
5.9
—
95.6
95.4
94.5
90.1
44
M.  szulgai
45
7.0
7.0
6.5
6.5
7.0
7.2
5.9
6.7
6.7
9.1
9.1
8.0
8.6
8.6
10.6
6.7
7.6
8.0
7.8
7.8
9.3
7.4
6.3
6.1
7.0
8.9
11.0
10.6
7.0
7.0
7.0
7.0
7.0
7.2
7.4
4.3
4.1
7.4
5.5
4.7
4.5
4.5
4.9
4.5
—
96.4
94.7
88.8
45
M.  asiaticum
46
8.0
8.0
7.4
7.6
7.4
8.6
7.8
8.0
7.6
10.4
10.4
9.7
9.3
9.3
11.2
9.1
9.1
9.3
9.1
9.1
9.7
8.9
8.0
7.2
8.2
9.9
12.8
12.3
8.2
8.2
8.2
8.2
8.2
8.5
9.1
5.5
4.9
6.5
5.9
6.3
4.5
4.7
5.1
4.7
3.7
—
95.8
88.8
46
M.  kansasii
47
7.6
7.6
7.6
7.4
8.4
9.1
7.0
8.2
7.6
8.9
8.9
10.8
10.1
10.1
11.9
9.7
9.5
8.4
8.2
8.2
10.8
9.7
8.9
8.0
9.1
10.4
14.2
13.7
8.7
8.7
8.7
8.7
8.7
8.9
9.7
6.5
6.3
7.4
5.9
7.0
6.6
6.3
7.0
5.7
5.5
4.3
—
87.9
47
M.  gastri
48
11.7
11.7
11.5
11.7
11.2
13.0
10.4
9.7
11.7
11.9
11.9
12.8
11.0
11.0
13.5
12.6
10.6
11.7
11.9
11.9
11.2
11.7
11.9
11.7
12.8
14.2
14.2
13.5
13.0
13.0
13.0
13.0
13.0
13.0
13.5
12.4
12.1
11.9
9.9
11.2
11.2
11.5
11.9
10.6
12.1
12.1
13.2
—
48
Rhodococcus equi
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48


### 3.2. Phylogenetic Tree Construction

A phylogenetic tree, which provided the basis for species differentiation in the genusMycobacterium, was constructed (Figure 1). The absolute majority tested species showed good separation. The rapid-growing species were well defined from the slow-growing species in the tree.M. chelonae,M. abscessus subspeciesmassiliense, andM. abscessus, which are categorized in the pathogenic taxonomic group of rapid-growing mycobacteria, formed a distinctive cluster which was much closer to the slow-growing species compared with the nonpathogenic group of rapid-growing mycobacteria. The reliability of the phylogenetic tree was verified by the bootstrap method, usingR. equi as the outgroup.Figure 1
Phylogenetic tree based onrpsA gene sequences shows the relationship of the 47 type strains of mycobacteria and 1 outgroup strain. This tree was constructed by the neighbor-joining method. Topology was also evaluated by bootstrap analysis (MEGA program, 1000 repeats, withR. equi as the outgroup). The numerical values in the tree represent bootstrap results. The distance between two strains is the sum of the branch lengths between them.


### 3.3. Species Identification Outcomes of the Clinical Isolates

rpsA sequence and alignment efficiently identified clinical isolates representing 8 mycobacterial species. We found that a criterion of first distinctrpsA sequence match >97% confirmed the identification of 183 of 188 (97.3%) clinical isolates. 20M. abscessus strains, 2M. fortuitum strains, and 10M. avium strains were identified to the subspecies level. Identification discrepancies betweenrpsA and other sequences were only encountered with 4M. gordonae strains and 1M. kansasii strain (see Table 3). No discrepancies were found in the species submitted asM. intracellulare,M. avium,M. abscessus, andM. tuberculosis complex.Table 3
Comparison of mycobacteria identified by 16S rDNA/ITS/hsp65/rpoB gene sequencing and by rpsA sequencing among the 188 clinical isolatesa.
Primary identificationb (n)
Primary identificationb (n)
First choice
Second choice
Identification (n)
%match
Identification (n)
%match
M.  intracellulare (75)
M.  intracellulare (4)
100%
M.  avium (4)
95.6%
M.  intracellulare (1)
99.8%
M.  avium (1)
95.3%
M.  intracellulare (53)
98.9%
M.  avium (53)
95.1%
M.  intracellulare (10)
98.7%
M.  avium (10)
94.9%
M.  intracellulare (1)
98.5%
M.  avium (1)
95.3%
M.  intracellulare (3)
98.1%
M.  avium (3)
94.7%
M.  intracellulare (1)
97.5%
M.  avium (1)
93.2%
M.  intracellulare (2)
97.1%
M.  avium (2)
96.1%
M.  abscessus (39)
M.  abscessus (9)
100%
M.  abscessussubspeciesmassiliense (9)
98.7%
M.  abscessus (10)
99.8%
M.  abscessussubspeciesmassiliense (10)
98.5%
M.  abscessussubspecies massiliense (20)
99.4%
M.  abscessus (20)
99.1%
M.  kansasii (23)
M.  kansasii (22)
100%
M.  asiaticum (22)
96.3%
M.  szulgai (1)
96.6%
M.  marinum/M.  ulcerans (1)
96.0%
M.  avium (10)
M.  aviumsubspeciesparatuberculosis (5)
99.6%
M.  avium (5)
99.4%
M.  aviumsubspeciesparatuberculosis (4)
99.8%
M.  avium (4)
99.2%
M.  aviumsubspeciesparatuberculosis (1)
97.5%
M.  avium (1)
97.0%
M.  fortuitumcomplex (17)
M.  fortuitum (2)
100%
M.  senegalense/M.  thermoresistibile (2)
98.1%
M.  fortuitum (11)
99.8%
M.  porcinum (11)
98.5%
M.  fortuitum (1)
99.6%
M.  porcinum (1)
98.3%
M.  fortuitum/M.  porcinum (1)
98.9%
M.  senegalense/M.  thermoresistibile (1)
98.3%
M.  thermoresistibile/M.  senegalense (2)
99.6%
M.  fortuitum (2)
98.1%
M.  gordonae (7)
M.  gordonae (1)
99.2%
M.  asiaticum (1)
95.5%
M.  gordonae (2)
98.5%
M.  asiaticum (2)
96.3%
M.  gordonae (3)
95.9%
M.  asiaticum (3)
95.5%
M.  szulgai (1)
95.9%
M.  gordonae (1)
94.7%
M.  tuberculosiscomplex (16)
M.  tuberculosis (2)
100%
M.  canetti (2)
99.8%
M.  tuberculosis (12)
99.8%
M.  canetti (12)
99.6%
M.  tuberculosis (2)
99.6%
M.  canetti (2)
99.4%
M.  neoaurum (1)
M.  neoaurum (1)
98.7%
M.  smegmatis (1)
95.4%
aNumbers in parentheses (n) represent the numbers of isolates identified as a particular species.
bIdentification based on sequencing of at least two of the following: 16S rDNA, 16-23S rRNA gene internal transcribed spacer (ITS), and rpoB and hsp65 genes.The sequence divergence among MTC members was 0.4%. The intraspecies divergence among the 188 clinical strains ranged from 0.6% to 5.3% (Table3). The sequence diversity among theM. abscessus was 0.6%, while that among theM. gordonae clinical isolates was 5.3%. NoM. gordonae clinical strain had an identical sequence with that of theM. gordonae reference strain (ATCC14470). Interestingly, therpsA sequence of one strain among the 23M. kansasii isolates exhibited a relatively low level of sequence similarity (95.6%) to that of the reference strain (ATCC 12478), while those of all the otherM. kansasii isolates were identical. Since the strain was confirmed asM. kansasii byrpoB andhsp65 gene alignment, it might suggest a distant variant or a new subtype.


## 3.1.rpsA Sequence Alignment of the Reference Strains

Between 85.4% and 100% sequence homology (interspecies divergence, 0% to 14.6%) was observed among the 42 tested reference strains and the 5 additional mycobacterial species whose sequences were obtained from the GenBank database (Table2). All therpsA gene sequences of the analyzed mycobacteria strains were distinct from the outgroup strainR. equi. Among the 19 reference strains of the slow-growingMycobacterium genus, 11 strains were greater than 97% homology, including 5M. tuberculosis complex strains. Among the 28 reference strains of the rapid-growingMycobacterium genus, 14 strains were greater than 97% homology (Table 1). The pathogenicM. kansasii was easily differentiated from the nonpathogenicM. gastri (95.8% homology), while those two species were not distinguishable by the 16S rDNA sequence alignment. The sequence homologies between various species were 91.8% betweenM. chelonae andM. abscessus, 95.6% betweenM. avium andM. intracellulare, and 93.9% betweenM. szulgai andM. malmoense. However, the sequence homology between other species was higher; for example, it was 99.6% betweenM. ulcerans andM. marinum and 98.7% betweenM. abscessus subspeciesmassiliense andM. abscessus. All members of theM. tuberculosis complex had identical sequences as didM. senegalense andM. thermoresistibile,M. parafortuitum andM. trivial,M. diernhoferi andM. duvalii, andM. austroafricanum andM. terrae.Table 2
Sequence pair distances of 47 type strains of mycobacteria and 1 out-group strain determined byusing the multiple alignment algorithm in the MegAlign package (version 7.1.0; DNASTAR, Madison, WI).
Percent identity
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
1
—
100.0
98.7
98.1
96.0
95.3
95.4
96.4
96.2
94.5
94.5
95.1
94.5
94.5
92.0
93.9
94.1
94.3
94.5
94.5
92.8
95.6
93.7
94.7
93.2
92.4
88.8
89.2
91.1
91.1
91.1
91.1
91.1
91.3
91.5
93.9
94.1
91.3
93.0
92.8
92.6
92.6
92.4
93.4
93.4
92.4
92.8
89.2
1
M.  thermoresistibile
2
0.0
—
98.7
98.1
96.0
95.3
95.4
96.4
96.2
94.5
94.5
95.1
94.5
94.5
92.0
93.9
94.1
94.3
94.5
94.5
92.8
95.6
93.7
94.7
93.2
92.4
88.8
89.2
91.1
91.1
91.1
91.1
91.1
91.3
91.5
93.9
94.1
91.3
93.0
92.8
92.6
92.6
92.4
93.4
93.4
92.4
92.8
89.2
2
M.  senegalense
3
1.3
1.3
—
98.3
96.6
95.8
95.8
96.8
96.8
94.7
94.7
95.6
95.4
95.4
92.0
94.1
95.4
94.9
95.1
95.1
93.0
96.2
94.7
95.6
94.5
93.0
89.4
89.8
91.1
91.1
91.1
91.1
91.1
91.3
92.0
94.5
94.7
92.0
93.4
93.5
93.2
93.2
92.8
93.7
93.7
93.0
92.8
89.4
3
M.  porcinum
4
1.9
1.9
1.7
—
96.2
95.8
95.8
96.2
96.2
94.1
94.1
95.4
94.5
94.5
92.0
94.3
94.5
94.3
94.5
94.5
93.9
96.2
94.9
94.9
93.7
93.7
90.1
90.5
90.9
90.9
90.9
90.9
90.9
91.1
92.0
94.7
94.9
91.3
93.2
93.2
93.0
93.4
92.8
93.5
93.7
92.8
93.0
89.2
4
M.  fortuitum
5
4.1
4.1
3.5
3.9
—
94.3
95.4
96.4
96.0
93.9
93.9
93.4
94.3
94.3
91.5
94.3
94.3
94.5
94.7
94.7
91.5
94.3
94.1
94.9
92.8
91.1
89.8
90.3
90.1
90.1
90.1
90.1
90.1
90.3
91.1
93.5
93.7
91.3
93.4
93.0
93.5
93.5
93.0
93.7
93.4
93.0
92.0
89.6
5
M.  phlei
6
4.9
4.9
4.3
4.3
5.9
—
95.1
95.3
94.1
93.7
93.7
94.7
94.1
94.1
91.3
93.7
93.5
93.4
93.5
93.5
93.2
93.7
93.7
93.7
93.0
92.4
88.6
89.0
90.7
90.7
90.7
90.7
90.7
90.9
91.5
94.3
94.1
90.7
93.2
92.0
91.8
92.2
91.5
93.0
93.2
91.8
91.5
88.0
6
M.  gadium
7
4.7
4.7
4.3
4.3
4.7
5.1
—
96.6
96.0
95.4
95.4
94.7
94.7
94.7
93.0
95.1
95.3
95.6
95.8
95.8
93.2
94.1
94.9
94.7
92.4
92.0
90.1
90.5
92.4
92.4
92.4
92.4
92.4
92.6
92.8
94.9
94.7
93.0
94.1
94.9
93.7
93.7
93.5
93.9
94.3
92.6
93.4
90.3
7
M.  flavescens
8
3.7
3.7
3.3
3.9
3.7
4.9
3.5
—
96.6
96.6
96.6
95.3
96.2
96.2
92.0
96.2
96.2
96.4
96.2
96.2
93.5
94.7
94.5
94.5
93.9
91.5
90.5
90.9
91.1
91.1
91.1
91.1
91.1
91.3
92.2
93.7
93.5
92.0
94.3
93.5
93.2
93.2
93.0
93.4
93.5
92.4
92.2
90.9
8
M.  smegmatis
9
3.9
3.9
3.3
3.9
4.1
6.1
4.1
3.5
—
96.6
96.6
95.1
96.8
96.8
92.4
95.8
95.6
96.2
96.4
96.4
93.0
95.6
94.9
94.7
92.4
92.0
91.5
91.8
90.9
90.9
90.9
90.9
90.9
91.1
92.2
94.3
94.5
92.4
93.9
92.8
93.4
93.4
93.2
93.9
93.5
92.8
92.8
89.2
9
M.  vaccae
10
5.7
5.7
5.5
6.1
6.3
6.5
4.7
3.5
3.5
—
100.0
94.9
96.2
96.2
93.0
94.9
95.8
96.4
96.6
96.6
93.0
94.3
92.8
93.0
92.4
91.7
89.8
90.1
89.9
89.9
89.9
89.9
89.9
90.1
91.5
92.6
92.4
91.3
93.2
92.0
91.1
91.1
91.3
91.8
91.5
90.3
91.7
89.0
10
M.  parafortuitum
11
5.7
5.7
5.5
6.1
6.3
6.5
4.7
3.5
3.5
0.0
—
94.9
96.2
96.2
93.0
94.9
95.8
96.4
96.6
96.6
93.0
94.3
92.8
93.0
92.4
91.7
89.8
90.1
89.9
89.9
89.9
89.9
89.9
90.1
91.5
92.6
92.4
91.3
93.2
92.0
91.1
91.1
91.3
91.8
91.5
90.3
91.7
89.0
11
M.  triviale
12
5.1
5.1
4.5
4.7
7.0
5.5
5.5
4.9
5.1
5.3
5.3
—
96.4
96.4
91.8
94.5
95.3
95.1
95.3
95.3
94.1
95.1
92.8
92.2
92.6
93.0
90.3
90.7
89.9
89.9
89.9
89.9
89.9
90.1
91.3
93.2
92.8
89.9
92.0
92.0
91.7
91.7
91.1
91.8
92.4
90.9
89.9
88.2
12
M.  neoaurum
13
5.7
5.7
4.7
5.7
5.9
6.1
5.5
3.9
3.3
3.9
3.9
3.7
—
100.0
92.6
95.3
95.8
95.6
95.8
95.8
94.1
94.9
94.1
93.7
92.8
92.2
91.3
91.7
89.4
89.4
89.4
89.4
89.4
89.6
90.7
92.0
91.8
91.1
93.0
91.8
91.5
91.5
91.3
91.7
91.8
91.3
90.5
89.8
13
M.  diernhoferi
14
5.7
5.7
4.7
5.7
5.9
6.1
5.5
3.9
3.3
3.9
3.9
3.7
0.0
—
92.6
95.3
95.8
95.6
95.8
95.8
94.1
94.9
94.1
93.7
92.8
92.2
91.3
91.7
89.4
89.4
89.4
89.4
89.4
89.6
90.7
92.0
91.8
91.1
93.0
91.8
91.5
91.5
91.3
91.7
91.8
91.3
90.5
89.8
14
M.  duvalii
15
8.4
8.4
8.4
8.4
9.1
9.3
7.4
8.5
8.0
7.4
7.4
8.6
7.8
7.8
—
93.0
93.9
93.0
93.2
93.2
91.7
91.8
91.1
91.1
89.4
91.1
89.8
90.1
89.2
89.2
89.2
89.2
89.2
89.4
88.8
90.1
90.5
91.5
90.5
90.5
89.6
89.8
89.8
89.2
90.1
89.6
89.0
87.7
15
M.  aurum
16
6.3
6.3
6.1
5.9
5.9
6.5
5.1
3.9
4.3
5.3
5.3
5.7
4.9
4.9
7.4
—
96.6
96.0
95.8
95.8
93.9
94.7
94.7
93.5
92.8
92.4
91.8
92.4
90.5
90.5
90.5
90.5
90.5
90.7
91.7
93.2
93.0
91.1
92.6
92.4
92.6
93.0
91.3
92.4
93.5
91.5
90.9
88.4
16
M.  chubuense
17
6.1
6.1
4.7
5.7
5.9
6.8
4.9
3.9
4.5
4.3
4.3
4.9
4.3
4.3
6.3
3.5
—
97.2
97.0
97.0
94.1
95.6
94.5
94.1
93.5
93.4
91.3
91.7
90.3
90.3
90.3
90.3
90.3
90.5
91.3
93.0
93.2
92.0
92.8
93.2
92.6
93.0
91.7
92.8
92.8
91.5
91.1
90.1
17
M.  gilvum
18
5.9
5.9
5.3
5.9
5.7
7.0
4.5
3.7
3.9
3.7
3.7
5.1
4.5
4.5
7.4
4.1
2.9
—
99.8
99.8
94.1
94.9
92.8
93.4
92.6
93.0
90.7
91.1
90.1
90.1
90.1
90.1
90.1
90.3
91.3
92.8
92.6
91.3
92.4
92.8
92.0
92.0
91.3
92.0
92.4
91.3
92.0
89.2
18
M.  vanbaalenii
19
5.7
5.7
5.1
5.7
5.5
6.8
4.3
3.9
3.7
3.5
3.5
4.9
4.3
4.3
7.2
4.3
3.1
0.2
—
100.0
93.9
95.1
93.0
93.5
92.8
93.2
90.9
91.3
90.3
90.3
90.3
90.3
90.3
90.5
91.5
93.0
92.8
91.5
92.6
93.0
92.2
92.2
91.5
92.2
92.6
91.5
92.2
89.0
19
M.  terrae
20
5.7
5.7
5.1
5.7
5.5
6.8
4.3
3.9
3.7
3.5
3.5
4.9
4.3
4.3
7.2
4.3
3.1
0.2
0.0
—
93.9
95.1
93.0
93.5
92.8
93.2
90.9
91.3
90.3
90.3
90.3
90.3
90.3
90.5
91.5
93.0
92.8
91.5
92.6
93.0
92.2
92.2
91.5
92.2
92.6
91.5
92.2
89.0
20
M.  austroafricanum
21
7.6
7.6
7.4
6.3
9.1
7.2
7.2
6.7
7.4
7.4
7.4
6.1
6.1
6.1
8.9
6.3
6.1
6.1
6.3
6.3
—
93.7
91.8
91.7
91.1
92.6
88.8
89.2
89.4
89.4
89.4
89.4
89.4
89.6
90.3
91.5
91.7
90.1
90.9
90.9
89.9
90.1
89.6
90.7
91.3
90.9
89.9
89.6
21
M.  rhodesiae
22
4.5
4.5
3.9
3.9
5.9
6.6
6.1
5.5
4.5
5.9
5.9
5.1
5.3
5.3
8.7
5.5
4.5
5.3
5.1
5.1
6.5
—
93.7
94.5
93.4
94.1
90.3
90.7
90.3
90.3
90.3
90.3
90.3
90.5
90.5
93.7
93.9
90.3
92.6
92.8
92.2
92.2
91.8
92.6
93.0
91.7
90.9
89.2
22
M.  obuense
23
6.5
6.5
5.5
5.3
6.1
6.5
5.3
5.7
5.3
7.6
7.6
7.6
6.1
6.1
9.5
5.5
5.7
7.6
7.4
7.4
8.6
6.5
—
95.8
93.9
93.0
91.7
92.0
90.9
90.9
90.9
90.9
90.9
91.1
92.2
93.7
93.9
91.7
93.2
92.6
93.4
93.7
92.2
93.7
93.9
92.4
91.7
89.0
23
M.  aichiense
24
5.5
5.5
4.5
5.3
5.3
6.5
5.5
5.7
5.5
7.4
7.4
8.2
6.5
6.5
9.5
6.7
6.1
7.0
6.7
6.7
8.9
5.7
4.3
—
93.7
93.4
91.3
91.8
91.5
91.5
91.5
91.5
91.5
91.7
91.5
93.7
94.1
92.2
93.0
93.5
92.8
93.0
93.0
93.4
94.1
93.2
92.4
89.2
24
M.  sphagni
25
7.2
7.2
5.7
6.5
7.6
7.4
8.0
6.3
8.0
8.0
8.0
7.8
7.6
7.6
11.5
7.6
6.7
7.8
7.6
7.6
9.5
7.0
6.3
6.5
—
92.0
88.8
89.4
89.6
89.6
89.6
89.6
89.6
89.8
90.5
94.5
94.5
90.7
93.0
92.8
91.8
92.2
92.0
92.4
93.4
92.2
91.5
88.2
25
M.  tokaiense
26
8.0
8.0
7.4
6.5
9.5
8.0
8.4
9.1
8.4
8.9
8.9
7.4
8.2
8.2
9.5
8.0
7.0
7.4
7.2
7.2
7.8
6.1
7.4
7.0
8.4
—
91.5
91.8
89.6
89.6
89.6
89.6
89.6
89.8
89.8
92.2
92.6
89.4
90.5
91.1
90.7
91.1
89.8
92.0
91.7
90.7
90.3
87.1
26
M.  chelonae
27
12.1
12.1
11.5
10.6
11.0
12.4
10.6
10.1
9.1
11.0
11.0
10.4
9.3
9.3
11.0
8.6
9.3
9.9
9.7
9.7
12.1
10.4
8.9
9.3
12.1
9.1
—
98.7
86.7
86.7
86.7
86.7
86.7
86.9
88.4
88.4
88.8
88.4
88.6
89.4
89.6
89.6
88.4
90.1
89.8
88.2
87.1
87.1
27
M.  abscessus
28
11.7
11.7
11.0
10.1
10.4
11.9
10.1
9.7
8.6
10.6
10.6
9.9
8.9
8.9
10.6
8.0
8.9
9.5
9.3
9.3
11.7
9.9
8.4
8.6
11.5
8.7
1.3
—
87.5
87.5
87.5
87.5
87.5
87.7
88.8
88.8
89.2
88.8
89.0
89.8
89.9
89.9
89.0
90.5
90.1
88.6
87.5
87.7
28
M.  abscessus
29
9.5
9.5
9.5
9.7
10.6
10.0
8.0
9.5
9.7
10.8
10.8
10.8
11.5
11.5
11.7
10.1
10.4
10.6
10.4
10.4
11.5
10.4
9.7
9.1
11.3
11.3
14.6
13.7
—
100.0
100.0
100.0
100.0
99.8
90.9
91.7
92.0
90.9
91.7
92.2
92.0
91.8
92.0
92.8
93.4
92.2
91.8
88.0
29
M.  tuberculosis
30
9.5
9.5
9.5
9.7
10.6
10.0
8.0
9.5
9.7
10.8
10.8
10.8
11.5
11.5
11.7
10.1
10.4
10.6
10.4
10.4
11.5
10.4
9.7
9.1
11.3
11.3
14.6
13.7
0.0
—
100.0
100.0
100.0
99.8
90.9
91.7
92.0
90.9
91.7
92.2
92.0
91.8
92.0
92.8
93.4
92.2
91.8
88.0
30
M.  tuberculosis
31
9.5
9.5
9.5
9.7
10.6
10.0
8.0
9.5
9.7
10.8
10.8
10.8
11.5
11.5
11.7
10.1
10.4
10.6
10.4
10.4
11.5
10.4
9.7
9.1
11.3
11.3
14.6
13.7
0.0
0.0
—
100.0
100.0
99.8
90.9
91.7
92.0
90.9
91.7
92.2
92.0
91.8
92.0
92.8
93.4
92.2
91.8
88.0
31
M.  bovis
32
9.5
9.5
9.5
9.7
10.6
10.0
8.0
9.5
9.7
10.8
10.8
10.8
11.5
11.5
11.7
10.1
10.4
10.6
10.4
10.4
11.5
10.4
9.7
9.1
11.3
11.3
14.6
13.7
0.0
0.0
0.0
—
100.0
99.8
90.9
91.7
92.0
90.9
91.7
92.2
92.0
91.8
92.0
92.8
93.4
92.2
91.8
88.0
32
M.  africanum
33
9.5
9.5
9.5
9.7
10.6
10.0
8.0
9.5
9.7
10.8
10.8
10.8
11.5
11.5
11.7
10.1
10.4
10.6
10.4
10.4
11.5
10.4
9.7
9.1
11.3
11.3
14.6
13.7
0.0
0.0
0.0
0.0
—
99.8
90.9
91.7
92.0
90.9
91.7
92.2
92.0
91.8
92.0
92.8
93.4
92.2
91.8
88.0
33
M.  bovis
34
9.3
9.3
9.3
9.5
10.4
9.7
7.8
9.3
9.5
10.6
10.6
10.6
11.2
11.2
11.5
9.9
10.2
10.4
10.2
10.2
11.2
10.2
9.5
8.9
11.1
11.0
14.4
13.5
0.2
0.2
0.2
0.2
0.2
—
91.1
91.8
92.2
90.7
91.8
92.4
91.8
91.7
91.8
92.6
93.2
92.0
91.7
88.0
34
M.  canettii
35
9.1
9.1
8.4
8.4
9.5
9.1
7.6
8.2
8.2
9.1
9.1
9.3
9.9
9.9
12.2
8.9
9.3
9.3
9.1
9.1
10.4
10.2
8.2
9.1
10.2
11.0
12.6
12.1
9.8
9.8
9.8
9.8
9.8
9.5
—
92.2
92.4
90.7
91.7
92.0
91.1
91.1
90.5
91.8
93.0
91.5
90.9
87.7
35
M.  xenopi
36
6.3
6.3
5.7
5.5
6.7
5.9
5.3
6.5
5.9
7.8
7.8
7.2
8.4
8.4
10.6
7.2
7.4
7.6
7.4
7.4
9.1
6.5
6.5
6.5
5.7
8.2
12.6
12.1
8.9
8.9
8.9
8.9
8.9
8.7
8.2
—
99.4
93.0
95.6
94.7
93.9
94.3
94.1
95.1
95.8
94.7
93.7
88.6
36
M.  avium
37
6.1
6.1
5.5
5.3
6.5
6.1
5.5
6.7
5.7
8.0
8.0
7.6
8.6
8.6
10.1
7.4
7.2
7.8
7.6
7.6
8.9
6.3
6.3
6.1
5.7
7.8
12.1
11.7
8.4
8.4
8.4
8.4
8.4
8.2
8.0
0.6
—
93.5
95.4
95.1
94.5
94.9
94.5
95.4
96.0
95.3
93.9
88.8
37
M.  avium
38
9.3
9.3
8.4
9.3
9.3
9.9
7.4
8.4
8.0
9.3
9.3
10.8
9.5
9.5
9.1
9.5
8.4
9.3
9.1
9.1
10.6
10.4
8.9
8.2
9.9
11.5
12.6
12.1
9.7
9.7
9.7
9.7
9.7
10.0
9.9
7.4
6.7
—
94.3
93.7
93.5
93.4
92.4
93.9
93.0
93.7
93.0
89.0
38
M.  malmoense
39
7.4
7.4
7.0
7.2
7.0
7.2
6.1
5.9
6.3
7.2
7.2
8.4
7.4
7.4
10.2
7.8
7.6
8.0
7.8
7.8
9.7
7.8
7.2
7.4
7.4
10.2
12.3
11.9
8.9
8.9
8.9
8.9
8.9
8.7
8.9
4.5
4.7
5.9
—
94.5
94.1
94.1
93.4
95.1
94.7
94.3
94.3
90.7
39
M.  intracellulare
40
7.6
7.6
6.7
7.2
7.4
8.4
5.3
6.7
7.6
8.4
8.4
8.4
8.6
8.6
10.1
8.0
7.2
7.6
7.4
7.4
9.7
7.6
7.8
6.7
7.6
9.5
11.5
11.0
8.2
8.2
8.2
8.2
8.2
8.0
8.4
5.5
5.1
6.5
5.7
—
94.3
94.3
94.9
95.1
95.4
93.9
93.4
89.6
40
M.  scrofulaceum
41
7.8
7.8
7.2
7.4
6.8
8.6
6.5
7.2
7.0
9.5
9.5
8.9
9.1
9.1
11.2
7.8
7.8
8.4
8.2
8.2
10.8
8.2
7.0
7.6
8.7
9.9
11.2
10.8
8.4
8.4
8.4
8.4
8.4
8.7
9.5
6.3
5.7
6.8
6.1
5.9
—
99.6
94.5
96.0
95.6
95.6
93.7
89.6
41
M.  marinum
42
7.8
7.8
7.2
7.0
6.8
8.2
6.5
7.2
7.0
9.5
9.5
8.9
9.1
9.1
11.0
7.4
7.4
8.4
8.2
8.2
10.6
8.2
6.5
7.4
8.2
9.5
11.2
10.8
8.7
8.7
8.7
8.7
8.7
8.9
9.5
5.9
5.3
7.0
6.1
5.9
0.4
—
94.3
96.0
95.6
95.4
93.9
89.4
42
M.  ulcerans
43
8.0
8.0
7.6
7.6
7.4
9.1
6.7
7.4
7.2
9.3
9.3
9.5
9.3
9.3
11.0
9.3
8.9
9.3
9.1
9.1
11.2
8.6
8.2
7.4
8.4
11.0
12.6
11.9
8.4
8.4
8.4
8.4
8.4
8.7
10.1
6.1
5.7
8.0
7.0
5.3
5.7
5.9
—
94.3
95.3
95.1
93.4
89.0
43
M.  gordonae
44
7.0
7.0
6.5
6.7
6.5
7.4
6.3
7.0
6.3
8.6
8.6
8.6
8.9
8.9
11.7
8.0
7.6
8.4
8.2
8.2
9.9
7.8
6.5
7.0
8.0
8.4
10.6
10.1
7.6
7.6
7.6
7.6
7.6
7.8
8.6
5.1
4.7
6.3
5.1
5.1
4.1
4.1
5.9
—
95.6
95.4
94.5
90.1
44
M.  szulgai
45
7.0
7.0
6.5
6.5
7.0
7.2
5.9
6.7
6.7
9.1
9.1
8.0
8.6
8.6
10.6
6.7
7.6
8.0
7.8
7.8
9.3
7.4
6.3
6.1
7.0
8.9
11.0
10.6
7.0
7.0
7.0
7.0
7.0
7.2
7.4
4.3
4.1
7.4
5.5
4.7
4.5
4.5
4.9
4.5
—
96.4
94.7
88.8
45
M.  asiaticum
46
8.0
8.0
7.4
7.6
7.4
8.6
7.8
8.0
7.6
10.4
10.4
9.7
9.3
9.3
11.2
9.1
9.1
9.3
9.1
9.1
9.7
8.9
8.0
7.2
8.2
9.9
12.8
12.3
8.2
8.2
8.2
8.2
8.2
8.5
9.1
5.5
4.9
6.5
5.9
6.3
4.5
4.7
5.1
4.7
3.7
—
95.8
88.8
46
M.  kansasii
47
7.6
7.6
7.6
7.4
8.4
9.1
7.0
8.2
7.6
8.9
8.9
10.8
10.1
10.1
11.9
9.7
9.5
8.4
8.2
8.2
10.8
9.7
8.9
8.0
9.1
10.4
14.2
13.7
8.7
8.7
8.7
8.7
8.7
8.9
9.7
6.5
6.3
7.4
5.9
7.0
6.6
6.3
7.0
5.7
5.5
4.3
—
87.9
47
M.  gastri
48
11.7
11.7
11.5
11.7
11.2
13.0
10.4
9.7
11.7
11.9
11.9
12.8
11.0
11.0
13.5
12.6
10.6
11.7
11.9
11.9
11.2
11.7
11.9
11.7
12.8
14.2
14.2
13.5
13.0
13.0
13.0
13.0
13.0
13.0
13.5
12.4
12.1
11.9
9.9
11.2
11.2
11.5
11.9
10.6
12.1
12.1
13.2
—
48
Rhodococcus equi
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48


## 3.2. Phylogenetic Tree Construction

A phylogenetic tree, which provided the basis for species differentiation in the genusMycobacterium, was constructed (Figure 1). The absolute majority tested species showed good separation. The rapid-growing species were well defined from the slow-growing species in the tree.M. chelonae,M. abscessus subspeciesmassiliense, andM. abscessus, which are categorized in the pathogenic taxonomic group of rapid-growing mycobacteria, formed a distinctive cluster which was much closer to the slow-growing species compared with the nonpathogenic group of rapid-growing mycobacteria. The reliability of the phylogenetic tree was verified by the bootstrap method, usingR. equi as the outgroup.Figure 1
Phylogenetic tree based onrpsA gene sequences shows the relationship of the 47 type strains of mycobacteria and 1 outgroup strain. This tree was constructed by the neighbor-joining method. Topology was also evaluated by bootstrap analysis (MEGA program, 1000 repeats, withR. equi as the outgroup). The numerical values in the tree represent bootstrap results. The distance between two strains is the sum of the branch lengths between them.


## 3.3. Species Identification Outcomes of the Clinical Isolates

rpsA sequence and alignment efficiently identified clinical isolates representing 8 mycobacterial species. We found that a criterion of first distinctrpsA sequence match >97% confirmed the identification of 183 of 188 (97.3%) clinical isolates. 20M. abscessus strains, 2M. fortuitum strains, and 10M. avium strains were identified to the subspecies level. Identification discrepancies betweenrpsA and other sequences were only encountered with 4M. gordonae strains and 1M. kansasii strain (see Table 3). No discrepancies were found in the species submitted asM. intracellulare,M. avium,M. abscessus, andM. tuberculosis complex.Table 3
Comparison of mycobacteria identified by 16S rDNA/ITS/hsp65/rpoB gene sequencing and by rpsA sequencing among the 188 clinical isolatesa.
Primary identificationb (n)
Primary identificationb (n)
First choice
Second choice
Identification (n)
%match
Identification (n)
%match
M.  intracellulare (75)
M.  intracellulare (4)
100%
M.  avium (4)
95.6%
M.  intracellulare (1)
99.8%
M.  avium (1)
95.3%
M.  intracellulare (53)
98.9%
M.  avium (53)
95.1%
M.  intracellulare (10)
98.7%
M.  avium (10)
94.9%
M.  intracellulare (1)
98.5%
M.  avium (1)
95.3%
M.  intracellulare (3)
98.1%
M.  avium (3)
94.7%
M.  intracellulare (1)
97.5%
M.  avium (1)
93.2%
M.  intracellulare (2)
97.1%
M.  avium (2)
96.1%
M.  abscessus (39)
M.  abscessus (9)
100%
M.  abscessussubspeciesmassiliense (9)
98.7%
M.  abscessus (10)
99.8%
M.  abscessussubspeciesmassiliense (10)
98.5%
M.  abscessussubspecies massiliense (20)
99.4%
M.  abscessus (20)
99.1%
M.  kansasii (23)
M.  kansasii (22)
100%
M.  asiaticum (22)
96.3%
M.  szulgai (1)
96.6%
M.  marinum/M.  ulcerans (1)
96.0%
M.  avium (10)
M.  aviumsubspeciesparatuberculosis (5)
99.6%
M.  avium (5)
99.4%
M.  aviumsubspeciesparatuberculosis (4)
99.8%
M.  avium (4)
99.2%
M.  aviumsubspeciesparatuberculosis (1)
97.5%
M.  avium (1)
97.0%
M.  fortuitumcomplex (17)
M.  fortuitum (2)
100%
M.  senegalense/M.  thermoresistibile (2)
98.1%
M.  fortuitum (11)
99.8%
M.  porcinum (11)
98.5%
M.  fortuitum (1)
99.6%
M.  porcinum (1)
98.3%
M.  fortuitum/M.  porcinum (1)
98.9%
M.  senegalense/M.  thermoresistibile (1)
98.3%
M.  thermoresistibile/M.  senegalense (2)
99.6%
M.  fortuitum (2)
98.1%
M.  gordonae (7)
M.  gordonae (1)
99.2%
M.  asiaticum (1)
95.5%
M.  gordonae (2)
98.5%
M.  asiaticum (2)
96.3%
M.  gordonae (3)
95.9%
M.  asiaticum (3)
95.5%
M.  szulgai (1)
95.9%
M.  gordonae (1)
94.7%
M.  tuberculosiscomplex (16)
M.  tuberculosis (2)
100%
M.  canetti (2)
99.8%
M.  tuberculosis (12)
99.8%
M.  canetti (12)
99.6%
M.  tuberculosis (2)
99.6%
M.  canetti (2)
99.4%
M.  neoaurum (1)
M.  neoaurum (1)
98.7%
M.  smegmatis (1)
95.4%
aNumbers in parentheses (n) represent the numbers of isolates identified as a particular species.
bIdentification based on sequencing of at least two of the following: 16S rDNA, 16-23S rRNA gene internal transcribed spacer (ITS), and rpoB and hsp65 genes.The sequence divergence among MTC members was 0.4%. The intraspecies divergence among the 188 clinical strains ranged from 0.6% to 5.3% (Table3). The sequence diversity among theM. abscessus was 0.6%, while that among theM. gordonae clinical isolates was 5.3%. NoM. gordonae clinical strain had an identical sequence with that of theM. gordonae reference strain (ATCC14470). Interestingly, therpsA sequence of one strain among the 23M. kansasii isolates exhibited a relatively low level of sequence similarity (95.6%) to that of the reference strain (ATCC 12478), while those of all the otherM. kansasii isolates were identical. Since the strain was confirmed asM. kansasii byrpoB andhsp65 gene alignment, it might suggest a distant variant or a new subtype.


## 4. Discussion

16S rDNA gene sequence alignment has been used as the reference method for mycobacterial species identification. However, it has been reported that, by using the 16S rDNA gene alone for species identification of clinical NTM, 37% of such isolates remained unclassified which illustrates the need for additional molecular tools for proper phylogenetic assignment and accurate NTM identification [11]. The common assumption that bacterial isolates belong to the same species if they have fewer than 5–15 bp differences within the 16S rDNA gene sequence [12] or if they have more than 97% 16S rDNA gene sequence identities [13] may not be applicable to genus ofMycobacterium, whose members are much more closely related to each other. Furthermore, some mycobacterial strains shared uniform sequence of 16S rDNA gene, such asM. intracellulare andM. avium,M. kansasii andM. gastri,M. abscessus andM. chelonae, andM. szulgai andM. malmoense.In this study, we found that therpsA gene is useful as a complementary method to the 16S rDNA for mycobacterial species identification. Among the 47 reference strains and totally 1081 paired comparisons {1081 = [n
a
1
+
n
(
n
-
1
)
d
/
2]; a
1
=
1, n
=
46, d
=
1
}, therpsA gene alone can differentiate 97.2% (1051 out of 1081) of them if using 97% sequence homology as cutoff value. When referring to the phylogenetic tree, therpsA resolution might increase. For the most commonly seen pathogenic NTM species such asM. intracellulare, M. avium, M. abscessus, M. chelonae, andM. xenopi,rpsA gene sequence could differentiate these species easily. Furthermore, 183 of the 188 (97.3%) clinical isolates, representing 8 mycobacterial species, were identified correctly byrpsA gene blast. Discrepancies of identification were only encountered with 4M. gordonae strains and 1M. kansasii strain. Both these species have reportedly more intraspecies sequence divergence [9, 14–16]. Three out of the 4M. gordonae strains with discrepant identification still had the first distinction asM. gordonae byrpsA, but the sequence identity was lower than 97%, which means the identification might be correct when a more complete in-house database is being developed.rpsA alone cannot differentiate the following: between members of theM. tuberculosis complex, betweenM. senegalense andM. thermoresistibile, betweenM. parafortuitum andM. trivial, betweenM. diernhoferi andM. duvalii, and betweenM. austroafricanum andM. terrae; however, these species are also difficult to be separated by other markers alone such as 16s rDNA,ITS,rpoB, and hsp65. Several reference strains had similar homology with two or more species which suggests inadequate taxonomy within the currently described species. Based on nucleotide sequences ofrpoB, hsp65, andsodA in clinical isolates of theMycobacterium abscessus group, one-fourth of isolates had discordant identification [16]. Multilocus sequence typing and sequence analysis of several genes facilitate the identification of closely related species or subspecies [17].In our study, we have demonstrated that therpsA homology gene is a promising marker for mycobacterial species identification for both fast- and slow-growing mycobacteria. To be a good marker for species differentiation, the target gene should be stable and sequence variations should occur randomly; additionally, an extremely conserved or highly variable gene may not be adequate. As a single-copy house-keeping gene,rpsA gene could work well as a target gene forMycobacterium species discrimination without ambiguous identification. The 16S rDNA gene has higher homology within the mycobacteria compared to the rpsA gene: 94.3% to 100% compared to 85.4% to 100%. According to our work, when used as sole marker,rpsA had better resolution than16s rDNA but had similar resolution asITS, rpoB, andhsp65.Due to the high resolution power ofrpsA for species identification, we presume it has potential of clinical use. From our own experience, we recommend that when performing species identification by homologue DNA sequence comparison, one should start with 16s RNA gene, plus at least one of the other markers such asITS, rpoB, hsp65, andrpsA. When conflicting outcome or dubious outcomes yield, more markers should be added further. Even though 16s RNA gene is inferior to the other markers considering the capacity, it should be chosen firstly since 16s RNA gene has the most robust sequence database, which can help to avoid big identification errors due to unreliable database of other markers [18].The main deterrents for the primary use ofrpsA sequencing as a routine means of identifying mycobacteria reside in the need for a comprehensive database. Therefore, we constructed our ownrpsA sequence database by including as many type strains as possible and integratingrpsA sequences deposited in GenBank such asM. avium subspeciesparatuberculosis,M. ulcerans, M. vanbaalenii, M. abscessus subspeciesmassiliense, andM. canettii. Besides type strains,rpsA sequences of confirmed clinical strains were also included. We will constantly upgrade our database and the capability to identify the most recent described species.



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*Source: 271728-2015-04-05.xml*