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1 Department of Microbiology and Immunology, Cancer Research Institute and Liver Research Institute, College of Medicine, Seoul National University, Seoul 110-799, Korea
2 The Korean Institute of Tuberculosis, The Korean National Tuberculosis Association, Seoul 137-140, Korea
Correspondence
Bum-Joon Kim
kbumjoon{at}snu.ac.kr
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the partial 16S rRNA gene, hsp65 and rpoB sequences of strain 03-19T are respectively DQ536403, DQ536401 and DQ536405.
Sequence alignments and neighbour-joining trees based on hsp65 and rpoB sequences are available as supplementary material in IJSEM Online.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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). Conventional biochemical tests have been performed extensively to detect and identify Mycobacterium tuberculosis and clinically important non-tuberculous mycobacteria (Goodfellow & Magee, 1998; Wayne & Kubica, 1986). However, because of the increasing number of newly defined taxa and the recognition of ‘difficult-to-identify’ variants of known species, tests sometimes fail to provide precise identification (Kirschner et al., 1993). To overcome the limitations of conventional methods, sequencing methods targeting several chronometer molecules have been developed (Kim et al., 1999, 2005; Roth et al., 1998; Stahl & Urbance, 1990; Stone et al., 1995). Moreover, the combination of molecular assays and conventional methods provides conclusive identification of infrequently encountered species and allows the delimitation of novel taxa. In the present study, a novel non-tuberculous mycobacterial species was isolated from a patient with general symptoms of pulmonary infection. The organism was discovered during an exercise designed to identify Korean slowly growing mycobacteria (SGM) clinical isolates using the recently developed hsp65 sequencing method (Kim et al., 2005). The subject strain of the present study, strain 03-19T, was one of the ‘difficult-to-identify’ isolates submitted to the Korean Institute of Tuberculosis by mycobacteriology laboratories in Korea during 2003. This strain was isolated from sputum samples of a 52-year-old female who had been experiencing general pulmonary symptoms. The same strain was isolated from sputum specimens obtained from this patient on three successive occasions; no other mycobacterium was observed.
The phenetic characteristics of strain 03-19T and eight mycobacteria reference strains were analysed and compared (Table 1). Colony morphology, pigment production in the dark, photoinduction and the ability to grow at temperatures ranging from 25 to 45 °C were examined during a 6 week incubation on Lowenstein–Jensen (LJ) medium and Middlebrook 7H10 agar. Acid–alcohol-fastness was determined by Ziehl–Neelsen and auramine O staining. The following biochemical features (Kent & Kubica, 1985) were investigated: niacin accumulation, nitrate reductase, arylsulfatase on days 3 and 14, heat-stable catalase (pH 7, 68 °C), tellurite reductase, Tween 80 hydrolysis, urease and pyrazinamidase. Inhibition tests included tolerance of thiophene-2-carboxylic acid hydrazide (TCH), p-nitrobenzoate (PNB), 5 % sodium chloride, ethambutol (EMB) and picric acid and ability to grow on MacConkey agar without crystal violet.
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or as described in the guidelines of the CDC National Center for HIV, STD, and TB Prevention Division of Tuberculosis Elimination (http://www.cdc.gov/nchstp/tb/Laboratory_Services/Liquid_Chroma.htm). Low- and high-molecular-mass standards (Ribi ImmunoChem) were added for peak identification. To identify and quantify mycolic acids and assign these to Mycobacterium species based on mycolic acid patterns, the Microbial Identification system (MIDI Inc.) was used.
Chromosomal DNA for molecular taxonomy was extracted using the bead-beater phenol extraction method as reported previously (Kim et al., 2005). Purified DNA was used as a template for PCR amplifications of three independent genes, the 16S rRNA gene, hsp65 (encoding heat-shock protein 65) and rpoB (encoding a subunit of RNA polymerase). The nearly complete 16S rRNA gene sequence (1523 bp) and partial sequences of hsp65 (644 bp) and rpoB (352 bp) were amplified as described previously (Springer et al., 1996; Kim et al., 1999, 2005). PCR amplicons of all target genes were cloned directly using Topo TA cloning kits (Invitrogen) and sequenced (Kim et al., 2005). To obtain sequence information on the rpoB and hsp65 genes of M. nebraskense, which were not available in GenBank, these sequences were also analysed from M. nebraskense ATCC BAA-837T, purchased from the ATCC. The 16S rRNA gene sequence of 03-19T obtained in the present study was compared with sequences from GenBank using the BLAST analysis program (http://www.ncbi.nlm.nih.gov/blast/).
Multiple alignments of sequences of the three genes of 03-19T and reference strains of a wide range of both slowly and rapidly growing mycobacteria were created using the multiple-alignment algorithm in MEGALIGN as described previously (Kim et al., 1999, 2005). All three trees were inferred by neighbour joining (Saitou & Nei, 1987) and maximum parsimony (Fitch, 1971) using Tsukamurella paurometabola strain NCTC 10741 (16S rRNA gene) or strain KCTC 9821T (hsp65) or Rhodococcus equi ATCC 10146T (rpoB) as an outgroup. Evolutionary distance matrices were generated according to the model described by Jukes & Cantor (1969). The neighbour-joining and maximum-parsimony methods were carried out using MEGA version 2.1 (Kumar et al., 2001) and the resulting trees and topologies were evaluated by bootstrap analyses (Felsenstein, 1985) based on 1000 resamplings.
Acid-fast microscopy showed generally rod-shaped and frequently bent acid-fast bacilli. Occasional coccid forms were noted. Spores and filaments were not present. The optimal growth temperature was 37 °C. No growth was observed at 45 °C. On Middlebrook 7H10 medium, mature growth developed in 3 weeks at 25 and 37 °C. Microcolonies developed in 2 weeks at the same temperature. However, 4 weeks or more was required for mature colonies to form on LJ medium. Colonies grown on Middlebrook 7H10 agar were usually larger, smooth, occasionally rough, and always orange in appearance under both dark and photoinduction conditions. Cells on LJ medium were film-like and produced an orange pigment. No growth was observed on MacConkey agar, after the addition of 5 % NaCl to the culture medium or after adding 5 mg EMB ml–1 or picric acid to the medium. However, strain 03-19T showed tolerance against 10 mg TCH ml–1 and against 500 mg PNB ml–1. The strain was negative for urease activity, arylsulfatase, niacin accumulation and Tween 80 hydrolysis and positive for nitrate reductase, heat-stable catalase, pyrazinamidase and tellurite reductase. Generally, the biochemical profile of 03-19T was most like those of M. nebraskense, another scotochromogenic species. However, since a difference was found between these two species in terms of tolerance against TCH and PNB, such tests might be used to differentiate the two taxa. Cultural and biochemical characteristics that differentiate 03-19T from other closely related SGM species are shown in Table 1
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In HPLC analysis of mycolic acids, the profile of strain 03-19T showed two clusters of peaks that did not overlap with any previously reported mycobacterial profile. The closest match was with the Mycobacterium avium/intracellulare/scrofulaceum complex, with a similarity index of 0.052. In a comparison with the mycolic acids of M. nebraskense ATCC BAA-837T (Fig. 1), differences were found in the relative heights of peaks as follows: peak 1, 2.70 % (strain 03-19T) and 10.45 % (M. nebraskense ATCC BAA-837T); peak 2, 1.53 and 6.64 %; peak 3, 11.16 and 14.21 %; peak 4, 5.67 and 22.20 %; peak 5, 9.74 and 6.23 %; peak 6, 11.74 and 5.06 %, peak 7, 14.88 and 0.84 %; peak 8, 4.48 and 2.27 %; peak 9, 8.79 and 6.64 %, and peak 10, 11.76 and 3.08 %. Such differences in HPLC peak heights have been reported to be species-specific for mycobacteria (CDC, 1996, 1999; Duffey et al., 1996; Floyd et al., 1996). Furthermore, three unique peaks (retention times 4.834, 4.977 and 6.881 min) distinguished strain 03-19T from M. nebraskense ATCC BAA-837T.
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). Perhaps these polymorphisms in the 16S rRNA gene could be used to detect this strain in the future.
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).
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The distinct sequences of these three gene targets together with the uniqueness of its mycolic acid profile and phenetic traits confirm the taxonomic status of strain 03-19T as a member of a novel mycobacterial species rather than a variant of a previously described species. Moreover, the successive isolations of this strain from sputum samples of a patient at different stages in the absence of other mycobacteria strongly support the possibility that it may be a causative agent of pulmonary disease.
Description of Mycobacterium seoulense sp. nov.
Mycobacterium seoulense (seo.ul.en'se. N.L. neut. adj. seoulense pertaining to Seoul, Republic of Korea, the geographical origin of the type strain).
The bacillus stains acid–alcohol-fast. Cells are generally rod-shaped and frequently bent. Occasional coccid forms are noted. Spores and filaments are not present. The optimal growth temperature is 37 °C. No growth is observed at 45 °C. On Middlebrook 7H10 medium, mature growth develops in 3 weeks at 25 and 37 °C. Microcolonies develop in 2 weeks at the same temperature. However, 4 weeks or more is required for mature colonies to form on LJ medium. Colonies grown on Middlebrook 7H10 agar are usually larger and smooth, although occasionally rough, but always orange in appearance, under both dark and photoinduction conditions. Cells on LJ medium grow in a film-like manner and produce an orange pigment. No growth is observed on MacConkey agar or on culture medium containing 5 % NaCl or 5 mg EMB ml–1. Negative for urease activity, arylsulfatase and Tween 80 hydrolysis and positive for nitrate reductase, heat-stable catalase, pyrazinamidase and tellurite reductase. HPLC analysis shows a unique mycolic acid profile. Genetically, the organism has sequences unique amongst species of Mycobacterium for the 16S rRNA, hsp65 and rpoB genes. Phylogenetic analysis using 16S rRNA gene sequences shows that M. seoulense belongs to the SGM and is closely related to M. nebraskense, M. scrofulaceum and Mycobacterium kansasii.
The type strain is 03-19T (=DSM 44998T=KCTC 19146T), isolated from human sputum samples in Seoul, Republic of Korea.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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CDC (1996). Standardized method for HPLC identification of mycobacteria. Atlanta, GA: Centers for Disease Control and Prevention. http://www.cdc.gov/ncidod/publications/hplc.pdf
CDC (1999). Mycolic acid pattern standards for HPLC identification of mycobacteria. Atlanta, GA: Centers for Disease Control and Prevention. http://www.cdc.gov/nchstp/tb/Laboratory_Services/maps_tagged.pdf
Duffey, P. S., Guthertz, L. S. & Evans, G. C. (1996). Improved rapid identification of mycobacteria by combining solid-phase extraction with high-performance liquid chromatography analysis of BACTEC cultures. J Clin Microbiol 34, 1939–1943.
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Goodfellow, M. & Magee, J. G. (1998). Taxonomy of mycobacteria. In Mycobacteria, vol. 1, Basic Aspects, pp. 1–71. Edited by P. R. J. Gangadharam & P. A. Jenkins. New York & London: Chapman & Hall.
Jukes, T. H. & Cantor, C. R. (1969). Evolution of protein molecules. In Mammalian Protein Metabolism, pp. 21–132. Edited by H. N. Munro. New York: Academic Press.
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Kim, H., Kim, S.-H., Shim, T.-S., Kim, M.-n., Bai, G.-H., Park, Y.-G., Lee, S.-H., Chae, G.-T., Cha, C.-Y. & other authors (2005). Differentiation of Mycobacterium species by analysis of the heat-shock protein 65 gene (hsp65). Int J Syst Evol Microbiol 55, 1649–1656.
Kirschner, P., Springer, B., Vogel, U., Meier, A., Wrede, A., Kiekenbeck, M., Bange, F. C. & Bottger, E. C. (1993). Genotypic identification of mycobacteria by nucleic acid sequence determination: report of a 2-year experience in a clinical laboratory. J Clin Microbiol 31, 2882–2889.
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Wayne, L. G. & Kubica, G. P. (1986). The mycobacteria. In Bergey's Manual of Determinative Bacteriology, 9th edn, pp. 1435–1457. Edited by R. E. Buchanan & N. E. Gibbons. Baltimore: Williams & Wilkins.
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