Sue Shin; Eui Chong Kim; Jong-Hyun Yoon

doi:10.3343/kjlm.2006.26.3.153

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Shin, Kim, and Yoon: Identification of Nontuberculous Mycobacteria by Sequence Analysis of the 16S Ribosomal RNA, the Heat-shock Protein 65 and the RNA Polymerase β-Subunit Genes

Original Article

Korean J Leg Med 2006;26(3):153-160.

Published online: 10 February 2006

DOI: https://doi.org/10.3343/kjlm.2006.26.3.153

Identification of Nontuberculous Mycobacteria by Sequence Analysis of the 16S Ribosomal RNA, the Heat-shock Protein 65 and the RNA Polymerase β-Subunit Genes

Sue Shin, M.D.^1,³, Eui Chong Kim, M.D.^2,³, Jong-Hyun Yoon, M.D.^1,³

¹Department of Laboratory Medicine, Boramae Hospital, Seoul, Korea

²Department of Seoul National University Hospital, Seoul, Korea

³Department of Seoul National University College of Medicine, Seoul, Korea

Received 23 September 2005 Revised 12 December 2005

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

The diagnosis of diseases caused by nontuberculous mycobacteria (NTM) is difficult, because NTM are prevalent in the environment such as soil and water, and because they have fastidious properties. In this study we investigated clinical isolates of NTM for their distribution pattern and accurate species identification.

Methods

We selected presumptive NTM isolates negative for probe hybridization for M. tuberculosis complex, cultured in a third referral hospital from 21 January 2003 to 20 January 2004. Ninety seven-isolates were identified to the species level by direct sequencing of fragments of 16S rRNA, hsp65 and rpoB genes. A total of 120 isolates were studied for the distribution analysis.

Results

Frequently identified NTM species were M. avium (30.8%), M. intracellulare (23.3%) and M. abscessus (18.3%). Others were M. gordonae, M. senegalense, M. fortuitum, M. peregrinum, M. kansasii, M. terrae complex, M. lentiflavum, M. chelonae, and M. szulgai. Three M. tuberculosis complex (2.5%) were also identified among the presumptive NTM isolates. The identification rate by sequencing of 16S rRNA, rpoB, and hsp65 were 65%, 82% and 87%, respectively. The hsp65 or rpoB gene was more efficient than 16S rRNA for the identification of NTM by sequencing.

Conclusions

Some NTM are increasingly considered to be the causative organisms in clinical diseases. Thus, direct sequencing could be adapted to routine work of clinical laboratories for accurate identification of NTM to the species level.

Keywords: Sequence analysis, Atypical mycobacteria, rpoB, 16S rRNA, Heat-shock protein

References

1. Tenholder MF, Moser RJ 3rd, Tellis CJ. Mycobacteria other than tuberculosis. Pulmonary involvement in patients with acquired immunodeficiency syndrome. Arch Intern Med. 1988; 148:953–5.

2. Falkinham JO 3rd. Epidemiology of infection by nontuberculous mycobacteria. Clin Microbiol Rev. 1996; 9:177–215.

3. Debrunner M, Salfinger M, Brandli O, von Graevenitz A. Epidemiology and clinical significance of nontuberculous mycobacteria in patients negative for human immunodeficiency virus in Switzerland. Clin Infect Dis. 1992; 15:330–45.

4. Dobos KM, Quinn FD, Ashford DA, Horsburgh CR, King CH. Emergence of a unique group of necrotizing mycobacterial diseases. Emerg Infect Dis. 1999; 5:367–78.

5. Collins MT, Lisby G, Moser C, Chicks D, Christensen S, Reichelderfer M, et al. Results of multiple diagnostic tests for Mycobacterium avium subsp. paratuberculosis in patients with inflammatory bowel disease and in controls. J Clin Microbiol. 2000; 38:4373–81.

6. Kim SJ, Hong YP, Kim SC, Bai GH, Jin BW, Park CD. A case of pulmonary disease due to M. avium-intracellulare complex. Tuberc Respir Dis. 1981; 28:121–4.

7. Bai GH, Park KS, Kim SJ. Clinically isolated mycobacteria other than mycobacterium tuberculosis from 1980 to 1990 in Korea. J Korean Soc Microbiol. 1993; 28:1–6.

8. American Thoracic Society. Diagnosis and treatment of disease caused by nontuberculous mycobacteria. Am J Respir Crit Care Med. 1997; 156:S1–25.

9. Pae HH, Lee JH, Yoo CG, Lee CT, Chung HS, Kim YW, et al. Study for clinical characteristics of nontuberculous mycobacterial pulmonary disease. Tuberc Respir Dis. 1999; 47:735–46.

10. Lew WJ, Ahn DI, Yoon YJ, Cho JS, Kwon DW, Kim SJ, et al. Clinical experience on mycobacterial disease other than tuberculosis. Tuberc Respir Dis. 1992; 39:425–32.

11. Lee HW, Kim MN, Shim TS, Bai GH, Pai CH. Nontuberculous mycobacterial pulmonary infection in immunocompetent patients. Tuberc Respir Dis. 2002; 53:173–82.

12. Koh WJ, Kwon OJ, Kang EH, Jeon IS, Pyun YJ, Ham HS, et al. Clinical and radiographic characteristics of 12 patients with Mycobacterium abscessus pulmonary disease. Tuberc Respir Dis. 2003; 54:45–56.

13. Springer B, Stockman L, Teschner K, Roberts GD, Bottger EC. Two-laboratory collaborative study on identification of mycobacteria: molecular versus phenotypic methods. J Clin Microbiol. 1996; 34:296–303.

14. Brown-Elliott BA, Griffith DE, Wallace RJ Jr. Newly described or emerging human species of nontuberculous mycobacteria. Infect Dis Clin North Am. 2002; 16:187–220.

15. Patel JB, Leonard DG, Pan X, Musser JM, Berman RE, Nachamkin I. Sequence-based identification of Mycobacterium species using the MicroSeq 500 16S rDNA bacterial identification system. J Clin Microbiol. 2000; 38:246–51.

16. Kim BJ, Lee SH, Lyu MA, Kim SJ, Bai GH, Chae GT, et al. Identification of mycobacterial species by comparative sequence analysis of the RNA polymerase gene (rpoB). J Clin Microbiol. 1999; 37:1714–20.

17. Turenne CY, Tschetter L, Wolfe J, Kabani A. Necessity of quality-controlled 16S rRNA gene sequence databases: identifying nontuberculous Mycobacterium species. J Clin Microbiol. 2001; 39:3637–48.

18. Ringuet H, Akoua-Koffi C, Honore S, Varnerot A, Vincent V, Berche P, et al. hsp65 sequencing for identification of rapidly growing mycobacteria. J Clin Microbiol. 1999; 37:852–7.

19. Harmsen D, Rothganger J, Frosch M, Albert J. RIDOM: Ribosomal Differentiation of Medical Micro-organisms Database. Nucleic Acids Res. 2002; 30:416–7.

20. Telenti A, Marchesi F, Balz M, Bally F, Bottger EC, Bodmer T. Rapid identification of mycobacteria to the species level by polymerase chain reaction and restriction enzyme analysis. J Clin Microbiol. 1993; 31:175–8.

21. Pai S, Esen N, Pan X, Musser JM. Routine rapid Mycobacterium species assignment based on species-specific allelic variation in the 65-kilodalton heat shock protein gene (hsp65). Arch Pathol Lab Med. 1997; 121:859–64.

22. Kim BJ, Lee KH, Park BN, Kim SJ, Bai GH, Kim SJ, et al. Differentiation of mycobacterial species by PCR-restriction analysis of DNA (342 base pairs) of the RNA polymerase gene (rpoB). J Clin Microbiol. 2001; 39:2102–9.

23. Adekambi T, Colson P, Drancourt M. rpoB-based identification of nonpigmented and late-pigmenting rapidly growing mycobacteria. J Clin Microbiol. 2003; 41:5699–708.

24. Koh WJ, Kwon OJ, Yu CM, Jeon K, Suh GY, Chung MP, et al. Recovery rate of nontuberculous mycobacteria from acid-fast-bacilli smear-positive sputum specimen. Tuberc Respir Dis. 2003; 54:22–32.

25. Good RC, Snider DE Jr. Isolation of nontuberculous mycobacteria in the United States, 1980. J Infect Dis. 1982; 146:829–33.

26. O'Brien RJ, Geiter LJ, Snider DE Jr. The epidemiology of nontuberculous mycobacterial disease in the United States. Results from a national survey. Am Rev Respir Dis. 1987; 135:1007–14.

27. Ostroff S, Hutwagner L, Collin S. Mycobacterial species and drug resistance patterns reported by state laboratories-1992. 93rd American Society for Microbiology General Meeting. May 16, 1993. Atlanta, GA. Abstract U-9, P.170.

28. Scientific committee in Korean academy of tuberculosis and respiratory disease. National survey of mycobacterial disease other than tuberculosis in Korea. Tuberc Respir Dis. 1995; 42:277–94.

29. Wong DA, Yip PC, Tse DL, Tung VW, Cheung DT, Kam KM. Routine use of a simple low-cost genotypic assay for the identification of mycobacteria in a high throughput laboratory. Diagn Microbiol Infect Dis. 2003; 47:421–6.

Fig. 1.

Nucleotide sequences of the partial hsp65 gene from 15 M. abscessus(HSP2-HSP107) and one M. chelonae (HSP74) strains aligned with reference sequences from Genebank (AF071128 and AF071139, M. abscessus strains; AF071130, AF071141 and AF 071142, M. chelonae strains).

Table 1.

Primers for amplification of 16S rRNA, rpoB and hsp65 genes

Genes	Primers (5′ → 3′)	Amplicon	Ta^* (°C)	Reference
16S rRNA	F:AGT TTG ATC CTG GCT CAG	527 bp	53	17
	R:GTA TTG CCG CGG CTG CTG			19
rpoB	F:CGA CCA CTT CGG CAA CCG	351 bp	60	16
	R:TCG ATC GGG CAC ATC CGG
hsp65	F:ACC AAC GAT GGT GTG TCC AT	441 bp	53	20
	R:CTT GTC GAA CCG CAT ACC CT

^* Ta, annealing temperature.

Table 2.

Similarity search results using 16S rRNA gene sequence

First rank	N	Overlap (bp)	Identity (%)	Second rank	Inter-species difference (bp)
		Identified: 62 isolates
M. avium	26	415–434	99.0–100	M. lepraemurium	1
M. fortuitum	3	352–404	99.4–100	M. mucogenicum	1
M. gordonae	3	419–424	100	M. kansasii	15
M. intracellulare	24	417–431	99.3–100	M. avium	2–9
M. lentiflavum	1	411	100	M. simiae	5
M. szulgai	1	420	100	M. malmoense	5
M. terrae complex	2	399–423	99.3–99.7	M. hiberniae	5–7
MTBC	2
		Unidentified: 33 isolates
M. abscessus^*	14	401–420	99.0–100	M. fortuitum	8–11
M. kansasii^†	2	416–420	99.8–100	M. malmoense	10
M. peregrinum^‡	3	407–423	99.8–100	M. alvei	2
M. senegalense^§	3	404–406	100	M. mucogenicum	1
Bacillus sp.	3
Signal unsatisfied	8

^* M. abscessus and M. chelonae share an identical sequence in the 5′ −16S rDNA sequence;

^† M kansasii and M. gastri share an identical 16S rDNA;

^‡ M peregrinum and M. septicum showed an identical 5′ −16S rDNA sequence;

^§ M. senegalense and M. farcinogenes and M fortuitum third biovar (sorbitol +) share an identical 5′-16S rDNA sequence.

Table 3.

Final identification of the isolates difficult to identify with a single gene fragment

Final identification/Genes	16S rDNA	rpoB	hsp65
M. avium	Bacillus sp	M. avium	NA^†
M. abscessus	Bacillus sp	M. abscessus	M. abscessus
M. avium	Bacillus sp	M. avium	M. avium
M. abscessus	M. abscessus	NA^†	M. abscessus
M. abscessus	M. abscessus	NA^†	M. abscessus
M. abscessus	M. abscessus	NA^†	M. abscessus
M. abscessus	M. abscessus	NA^†	M. abscessus
M. abscessus	M. abscessus	NA^†	M. abscessus
M. abscessus	M. abscessus	NA^†	M. abscessus
M. abscessus	M. abscessus	NA^†	M. abscessus
M. abscessus	MS^*	NA^†	M. abscessus
unidentifiable	MS^*	M. gordonae^‡	NA^†
M. gordonae	MS^*	M. gordonae	M. gordonae
M. avium	MS^*	M. avium	M. avium
M. avium	MS^*	M. avium	M. avium
M. fortuitum	MS^*	M. fortuitum	M. fortuitum
M. fortuitum	MS^*	M. fortuitum	M. fortuitum
M. peregrinum	MS^*	M. peregrinum	M. peregrinum
M. gordonae	M. gordonae	NA^†	M. gordonae
M. gordonae	M. gordonae	NA^†	M. gordonae
M. intracellulare	M. intracellulare	NA^†	MS^*
M. kansasii	M. kansasii	NA^†	NA^†
M. avium	M. avium	M. avium	NA^†
M. avium	M. avium	M. avium	MS^*

^* MS, mixed sequence;

^† NA, no amplicon;

^‡ M. gordonae like organism with 96.7% similarity.

Table 4.

Similarity search results using rpoB sequence

First rank	N	Overlap (bp)	Identity (%)	Second rank	Inter-species difference (bp)
		Identified: 78 isolates
M. abscessus	7	277–300	98.3–100	M. immunogen	3–7
M. avium	30	232–294	99.0–100	M. scrofulaceum	3–15
M. chelonae	1	277	100	M. abscessus	7
M. fortuitum	5	279–296	98.9–100	M. farcinogenes	5
M. gordonae	2	285	98.6–99.5	M. asiaticum	12
M. intracellulare	22	277–289	98.2–100	M. asiaticum	9–18
M. kansasii	1	277	100	M. haemophilum	8
M. peregrinum	4	277–296	99.3–100	M. porcinum	7
M. senegalense	3	274–277	99.6	M. porcinum	3
M. szulgai	1	279	98.9	M. gordonae	13
MTB complex	2
		Unidentified: 17 isolates
M. avium	1	313	92.0
M. intracellulare	1	283	95.8	M. avium	9–18
M. gordonae	1	210	96.7	M. asiaticum	8
M. nonchromogenicum	2	282	96.5	M. terrae complex	1
No amplicon	12

Table 5.

Similarity search results using hsp65 sequence

First rank	N	Overlap (bp)	Identity (%)	Second rank	Inter-species difference (bp)
		Identified: 83 isolates
M. abscessus	15	374–396	99.2–100	M. chelonae	25–28
M. avium	27	352–406	99.7–100	M. intracellulare	3–10
M. chelonae	1	396	99.7	M. abscessus	28
M. fortuitum	5	381–392	100	M. senegalense	3
M. gordonae	4	389–403	97.5–99.5	M. asiaticum	8–16
M. intracellulare	22	380–406	99.2–100	M. avium	7–16
M. kansasii	1	386	100	M. gastri	8
M. lentiflavum	1	382	100	M. triplex	9
M. peregrinum	4	374–381	100	M. septicum	3
M. szulgai	1	383	100	M. lentiflavum	14
MTB complex	2
		Unidentified: 12 isolates
M. senegalense^*	3	374–396	98.2–98.5	M. fortuitum	1
M. avium	1	396	98.0	M. intracellulare	3
M. terrae complex	2	398	97.0	M. abscessus	8–16
No amplicon	4
Signal unsatisfied	2

^* M. senegalense and M. farcinogenes showed an identical hsp65 sequence.

Table 6.

Distribution of MTBC probe negative, presumptive NTM isolates

Organism	No. case	Percent (%)
M. avium	37	30.8
M. intracellulare	28	23.3
M. abscessus	22	18.3
M. fortuitum	8	6.7
M. peregrinum	4	3.3
M. gordonae	3	2.5
M. senegalense	3	2.5
M. kansasii	2	1.7
M. terrae complex	3	1.7
M. chelonae	1	0.8
M. celatum	1	0.8
M. lentiflavum	1	0.8
M. szulgai	1	0.8
MTB complex	3	2.5
Oral contaminant	2	1.7
unidentifiable^*	1	0.8
Total	120	100

^* M. gordonae like organism (similarity 96.7%).

Abbreviations: MTBC, Mycobacteriun tuberculosis complex; NTM, nontuberculous mycobacteria.

Table 7.

Comparison of the three gene segments used in identification of presumptive NTM isolates

Genes	Cases^*	Presence of amplicon (%)	Identification of NTM (%)
16S rRNA	95	100	65
rpoB	95	87	82
hsp65	95	96	71^†
			87^‡

^* Of the studied 97 isolates, two oral contaminants were excluded because they were not mycobacteria;

^† The percent was lowered to 70% when the two MTBC isolates were also excluded. But other values were not changed;

^‡ We could get the percentage when the sequences were compared with those in reference 18.

Abbreviation: NTM, nontuberculous mycobacteria.

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