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

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.

1. Introduction

Members of the genus Mycobacterium 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, in M. celatum and M. terrae complex [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 genus Mycobacterium.

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 that RpsA is the target of pyrazinoic acid, the active form of the antituberculosis drug pyrazinamide [5]. Mycobacterium tuberculosis has a single copy of the rpsA 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 that rpsA may be suitable for phylogenetic study of the genus Mycobacterium. In this paper, we report an evaluation of the rpsA 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 genus Mycobacterium (Table 1), 172 clinical NTM isolates, and 16 M. 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 of M. avium, 75 strains of M. intracellulare, 23 strains of M. kansasii, 39 strains of M. abscessus, 17 strains of M. fortuitum complex, 7 strains of M. gordonae, and 1 strain of M. neoaurum.

Table 1

Percentage rpsA 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.  abscessus  subspecies massiliense	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.  tuberculosis  H37Rv	ATCC27294	M.  tuberculosis H37Ra/M.  africanum/M.  bovis BCG/M.  bovis (100%)	M.  canettii (99.8%)
M.  tuberculosis  H37Ra	ATCC25177	M.  tuberculosis H37Rv/M.  africanum/M.  bovis BCG/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.  bovis BCG	ATCC35735	M.  tuberculosis H37Ra /M.  tuberculosis H37Rv /M.  africanum/M.  bovis (100%)	M.  canettii (99.8%)
M.  bovis	ATCC19210	M.  tuberculosis H37Ra/M.  tuberculosis H37Rv/M.  africanum/M.  bovis BCG (100%)	M.  canettii (99.8%)
M.  canettii	CIPT140010059	M.  tuberculosis/M.  africanum/M.  bovis BCG/M.  bovis (99.8%)	M.  asiaticum (93.2%)
M.  xenopi	ATCC19250	M.  asiaticum (93.0%)	M.  avium subspecies paratuberculosis (92.4%)
M.  avium	ATCC25291	M.  avium subspecies paratuberculosis (99.6%)	M.  asiaticum (95.8%)
M.  avium subspecies paratuberculosis	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.  avium subspecies paratuberculosis (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 the rpsA gene of Mycobacterium tuberculosis, GenBank accession number NC_000962.2, and reverse primer, 5′-TGTCGATGACCTTGACCATC-3′, position 1032–1051 in the rpsA gene of Mycobacterium 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, the rpsA sequences of 5 other mycobacterial species were obtained directly from GenBank, including M. avium subspecies paratuberculosis (GenBank accession number NC_002944.2), M. ulcerans (GenBank accession number NC_008611.1), M. vanbaalenii (GenBank accession number NC_008726.1), M. abscessus subspecies massiliense (GenBank accession number NC_018150.1), and M. 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) using Rhodococcus 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 (Table 2). All the rpsA gene sequences of the analyzed mycobacteria strains were distinct from the outgroup strain R. equi. Among the 19 reference strains of the slow-growing Mycobacterium genus, 11 strains were greater than 97% homology, including 5 M. tuberculosis complex strains. Among the 28 reference strains of the rapid-growing Mycobacterium genus, 14 strains were greater than 97% homology (Table 1). The pathogenic M. kansasii was easily differentiated from the nonpathogenic M. 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% between M. chelonae and M. abscessus, 95.6% between M. avium and M. intracellulare, and 93.9% between M. szulgai and M. malmoense. However, the sequence homology between other species was higher; for example, it was 99.6% between M. ulcerans and M. marinum and 98.7% between M. abscessus subspecies massiliense and M. abscessus. All members of the M. tuberculosis complex had identical sequences as did M. senegalense and M. thermoresistibile, M. parafortuitum and M. trivial, M. diernhoferi and M. duvalii, and M. austroafricanum and M. 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 genus Mycobacterium, 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 subspecies massiliense, and M. 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, using R. equi as the outgroup.

Figure 1

Phylogenetic tree based on rpsA 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, with R. 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 distinct rpsA sequence match >97% confirmed the identification of 183 of 188 (97.3%) clinical isolates. 20 M. abscessus strains, 2 M. fortuitum strains, and 10 M. avium strains were identified to the subspecies level. Identification discrepancies between rpsA and other sequences were only encountered with 4 M. gordonae strains and 1 M. kansasii strain (see Table 3). No discrepancies were found in the species submitted as M. intracellulare, M. avium, M. abscessus, and M. 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.  abscessus  subspecies  massiliense (9)	98.7%
	M.  abscessus (10)	99.8%	M.  abscessus  subspecies  massiliense (10)	98.5%
	M.  abscessus  subspecies  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.  avium  subspecies paratuberculosis (5)	99.6%	M.  avium (5)	99.4%
	M.  avium subspecies paratuberculosis (4)	99.8%	M.  avium (4)	99.2%
	M.  avium subspecies paratuberculosis (1)	97.5%	M.  avium (1)	97.0%
M.  fortuitum  complex (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.  tuberculosis  complex (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% (Table 3). The sequence diversity among the M. abscessus was 0.6%, while that among the M. gordonae clinical isolates was 5.3%. No M. gordonae clinical strain had an identical sequence with that of the M. gordonae reference strain (ATCC14470). Interestingly, the rpsA sequence of one strain among the 23 M. 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 other M. kansasii isolates were identical. Since the strain was confirmed as M. kansasii by rpoB and hsp65 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 of Mycobacterium, whose members are much more closely related to each other. Furthermore, some mycobacterial strains shared uniform sequence of 16S rDNA gene, such as M. intracellulare and M. avium, M. kansasii and M. gastri, M. abscessus and M. chelonae, and M. szulgai and M. malmoense.

In this study, we found that the rpsA 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 = [na 1 + n(n − 1)d/2]; a 1 = 1, n = 46, d = 1}, the rpsA 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, the rpsA resolution might increase. For the most commonly seen pathogenic NTM species such as M. intracellulare, M. avium, M. abscessus, M. chelonae, and M. 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 by rpsA gene blast. Discrepancies of identification were only encountered with 4 M. gordonae strains and 1 M. kansasii strain. Both these species have reportedly more intraspecies sequence divergence [9, 14–16]. Three out of the 4 M. gordonae strains with discrepant identification still had the first distinction as M. gordonae by rpsA, 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 the M. tuberculosis complex, between M. senegalense and M. thermoresistibile, between M. parafortuitum and M. trivial, between M. diernhoferi and M. duvalii, and between M. austroafricanum and M. 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 of rpoB, hsp65, and sodA in clinical isolates of the Mycobacterium 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 the rpsA 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 for Mycobacterium 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 than 16s rDNA but had similar resolution as ITS, rpoB, and hsp65.

Due to the high resolution power of rpsA 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 as ITS, rpoB, hsp65, and rpsA. 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 of rpsA sequencing as a routine means of identifying mycobacteria reside in the need for a comprehensive database. Therefore, we constructed our own rpsA sequence database by including as many type strains as possible and integrating rpsA sequences deposited in GenBank such as M. avium subspecies paratuberculosis, M. ulcerans, M. vanbaalenii, M. abscessus subspecies massiliense, and M. 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.