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1 Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE 68198-6495, USA
2 Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE 68198-6495, USA
Correspondence
Steven H. Hinrichs
shinrich{at}unmc.edu
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The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene and ITS1 sequences of M. nebraskense UNMC-MY1349T are AY368456 and AY368458, respectively.
An alignment of hypervariable regions of the 16S rRNA gene and the ITS1 of the novel species and related mycobacteria and a fuller phylogenetic tree are available as supplementary material in IJSEM Online.
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). Although members of the Mycobacterium tuberculosis complex are the most frequent species to cause pulmonary infection worldwide, many non-tuberculous Mycobacterium (NTM) species have been found capable of causing similar infections (El-Solh et al., 1998; Koh et al., 2002).
The differential identification of Mycobacterium species is important for the control and treatment of infection. Currently, most clinical laboratories use conventional phenotypic assays to identify suspected mycobacterial pathogens. These assays were developed primarily for the identification of the more common clinically associated mycobacterial species and frequently are unable to identify uncommon or newly described Mycobacterium species due to the lack of discriminatory power (Kirschner et al., 1993; Tortoli et al., 1999).
To overcome the limitation of phenotypic assays, genetic methods of analysis have been developed (Roth et al., 2000). The combination of molecular assays and conventional methods creates a conclusive identification approach that can provide for the identification of unusual mycobacterial species and also for the description of novel taxa (Floyd et al., 2000). In the present study, a novel NTM species isolated from five different patients with general symptoms of pulmonary infections is described.
Five acid-fast-positive isolates from sputum previously identified as Mycobacterium species were submitted to our regional laboratory for species identification (strains identified as UNMC-MY1349T, UNMC-MY1293, UNMC-MY1174, UNMC-MY2873 and UNMC-MY3257). These isolates were collected from individual patients representing multiple geographical regions throughout Nebraska. A lung biopsy sample that was collected and archived at –70 °C for further analysis was also obtained.
Bacterial cell morphology for each isolate was determined by acid-fast staining using the Ziehl–Neelsen method and by the auramine O fluorescent stain method. Colony morphology, pigment production under dark and light conditions and ability to grow at temperatures ranging from 25 to 42 °C were examined following inoculation to both Lowenstein–Jensen (LJ) and Middlebrook 7H11 media. Conventional biochemical testing was performed as described previously by Kent & Kubica (1985). Tests included niacin accumulation, nitrate reductase, arylsulfatase on day 3, semi-quantitative catalase (45 mm), 68 °C catalase, Tween 80 hydrolysis, urease activity, tolerance to 5 % sodium chloride, growth on MacConkey agar without crystal violet and pyrazinamidase production.
HPLC mycolic acid analysis using procedures as described by Miller (1997) was performed on each isolate following growth on Middlebrook 7H10 medium. An internal calibration standard spiked with low- (LMIS) and high-molecular-mass internal standards (HMIS) (Ribi Immunochem) was included with each HPLC run. The mycolic acid compositions of the isolates were matched with those in a library of known mycobacterial profiles and expressed numerically using a similarity index (SI). The SI expresses how closely the mycolic acid composition of an unknown isolate matches those profiles within the library. An exact match of the mycolic acid of an unknown with the mean of a library profile results in an SI of 1·000. As each mycolic acid varies from the mean percentage, the SI decreases in proportion to the cumulative variance between the composition of the unknown and the library entry.
Genomic DNA was extracted from cultured isolates by the glass bead agitation method as described previously by Plikaytis et al. (1990). The lung tissue biopsy was homogenized using a Brinkmann Polytron P10/35 homogenizer (Diversified Equipment Company, Inc.) prior to extraction of the DNA using the glass bead agitation method. The crude DNA extract was purified using the QIAmp blood kit (Qiagen) according to protocols provided by the manufacturer. Two different DNA complex genetic targets were analysed to include the 16S rRNA gene and the internal transcribed spacer 1 (ITS1) region located between the 16S and 23S rRNA genes. The complete 16S rRNA gene (1506 bp) was amplified by PCR as described by Turenne et al. (2001) and the hypervariable region of the ITS1 target (209 bp) was amplified as described by Roth et al. (2000). PCR products of all targets were cloned directly following amplification using the standard protocol of the Original TA cloning kit (Invitrogen). Purified plasmids from selected colonies were screened for the correct insert by digestion with endonuclease EcoRI (New England Biolabs) and analysed by gel electrophoresis for recovery of the approximately sized product. Selected plasmids were sequenced at the Eppley Molecular Biology Core Laboratory (University of Nebraska Medical Center) using the universal M13 forward and reverse sequencing primers. The analysis was repeated three times for each of the five isolates from separate single colonies to ensure the accuracy of the generated sequences. The 16S rRNA gene and ITS1 sequences of each studied isolate were compared with the GenBank information using the BLAST analysis program (http://www.ncbi.nlm.nih.gov/blast/).
Multiple sequence alignments of the type strain and reference 16S rRNA gene sequences of a wide range of both slowly and rapidly growing mycobacterial species were created using the PILEUP program from the GCG Wisconsin Package for Sequence Analysis (Accelrys Inc.). Regions of uncertain alignment were truncated from each end of the gene. Evolutionary analysis of the aligned sequences was performed using PHYLIP version 3.5c. A tree was constructed by using the neighbour-joining method (Saitou & Nei, 1987). The analysis included calculation of the distance matrix using DNADIST that was applied to distances corrected for multiple hits and for unequal transition and transversion rates according to Kimura's two-parameter model (Kimura, 1980). Due to the high similarity of mycobacterial 16S rRNA genes, bootstrapping was not applied. However, the tree positions were confirmed by parsimony analysis for generation of a majority rule consensus tree using CONSENSE. The tree was rooted with Nocardia asteroides as an outgroup.
The results showed that all five strains were acid-fast-positive, non-motile and non-spore-forming bacilli. Mature colonies developed at 3 weeks on Middlebrook 7H11 medium and required 4 or more weeks on LJ medium to mature. The growth temperature range was 25–35 °C with optimal growth obtained at 30–35 °C and no growth at 42 °C. Mature colonies were rough in appearance with an elevated centre and yellow pigmentation under both dark and photoinduction conditions. No growth occurred on MacConkey agar without crystal violet or by the addition of 5 % NaCl to the culture medium.
All five strains were negative for nitrate reductase, urease activity, arylsulfatase activity, niacin accumulation, semi-quantitative catalase and pyrazinamidase production and positive for heat stable catalase (Table 1). The five unknown strains showed variable results for Tween 80 hydrolysis. The phenotypic characteristics of the five unknown strains did not match those of any of the other slowly growing mycobacterial species.
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). This pattern did not match any of the previously reported mycobacterial mycolic acid profiles (Butler & Guthertz, 2001). The closest match was with the Mycobacterium intracellulare–avium–scrofulaceum complex, with an SI of 0·552. The most significant qualitative difference was apparent in peak number one, which eluted before the LMIS in the unknown profile and after the LMIS in the M. intracellulare–avium–scrofulaceum complex. A quantitative difference between the two profiles was also apparent in the different peak numbers and heights.
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A phylogenetic tree was created to show the position of the novel species with regard to both slowly and rapidly growing mycobacteria using the entire 16S rRNA gene sequence (Fig. 2). The result of the phylogenetic analysis grouped strain UNMC-MY1349T within the slowly growing Mycobacterium species, close to Mycobacterium kansasii and Mycobacterium gastri. This unknown species was grouped closest to the uncharacterized Mycobacterium sp. IWGMT-90160. A fuller phylogenetic tree is available as Supplementary Fig. B in IJSEM Online.
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Description of Mycobacterium nebraskense sp. nov.
Mycobacterium nebraskense (ne.bras.ken'se. N.L. neut. adj. nebraskense referring to the State of Nebraska, USA).
Cells are non-spore-forming rods that stain acid-fast-positive. Mature growth is produced in 3 weeks on Middlebrook 7H11 medium and in 4 or more weeks on LJ medium at 25–35 °C, with optimal growth at 30–35 °C and no growth at 42 °C. Mature colonies are rough with an elevated centre and produce strong yellow pigmentation in the dark. The organism is positive for heat-stable catalase and negative for nitrate reductase, urease activity, niacin accumulation, arylsulfatase activity at 3 days and pyrazinamidase production. Less than 45 mm foam is produced in the semi-quantitative catalase test and variable results are indicated for Tween 80 hydrolysis. Growth is inhibited on MacConkey agar without crystal violet and by the addition of 5 % NaCl to the culture medium. Exhibits a unique mycolic acid profile by HPLC analysis characterized by a widely separated double peak clusters pattern that is typical for genus Mycobacterium. Genetically, the organism has unique Mycobacterium sequences for the 16S rRNA gene and ITS1 region targets. Phylogenetic analysis using 16S rRNA gene sequences shows that M. nebraskense belongs to the slowly growing mycobacteria and is closely related to M. kansasii, M. scrofulaceum, Mycobacterium malmoense and M. avium. The sequences of the 16S rRNA gene (AY368456) and the partial ITS1 region (AY368458) of the type strain have been deposited in GenBank.
Isolated from human sputum in Nebraska, USA. The type strain is UNMC-MY1349T (=ATCC BAA-837T=DSM 44803T).
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ACKNOWLEDGEMENTS |
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El-Solh, A. A., Nopper, J., Abdul-Khoudoud, M. R., Sherif, S. M., Aquilina, A. T. & Grant, B. J. (1998). Clinical and radiographic manifestations of uncommon pulmonary nontuberculous mycobacterial disease in AIDS patients. Chest 114, 138–145.
Falkinham, J. O., III (1996). Epidemiology of infection by nontuberculous mycobacteria. Clin Microbiol Rev 9, 177–215.[Medline]
Floyd, M. M., Gross, W. M., Bonato, D. A., Silcox, V. A., Smithwick, R. W., Metchock, B., Crawford, J. T. & Butler, W. R. (2000). Mycobacterium kubicae sp. nov., a slowly growing, scotochromogenic Mycobacterium. Int J Syst Evol Microbiol 50, 1811–1816.
Kent, P. T. & Kubica, G. P. (1985). Public Health Mycobacteriology. A Guide for the level III Laboratory. US Department of Health and Human Services publication. Atlanta: Centers for Disease Control and Prevention.
Kimura, M. (1980). A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16, 111–120.[CrossRef][Medline]
Kirschner, P., Meier, A. & Bottger, E. C. (1993). Genotypic identification and detection of mycobacteria – facing novel and uncultured pathogens. In Diagnostic Molecular Microbiology: Principles and Application, pp. 173–190. Edited by D. H. Persing, T. F. Smith, F. C. Tenover & T. J. White. Washington DC: American Society for Microbiology.
Koh, W. J., Kwon, O. J. & Lee, K. S. (2002). Nontuberculous mycobacterial pulmonary diseases in immunocompetent patients. Kor J Radiol 3, 145–157.
Kubica, G. P., Gross, W. M., Hawkins, J. E., Sommers, H. M., Vestal, A. L. & Wayne, L. G. (1975). Laboratory services for mycobacterial diseases. Am Rev Respir Dis 112, 773–787.[Medline]
Miller, J. L. (1997). Sherlock Mycobacteria Identification System by High Performance Liquid Chromatography. Operating Manual, version 1. Newark, DE: MIDI Inc.
Plikaytis, B. B., Gelber, R. H. & Shinnick, T. M. (1990). Rapid and sensitive detection of Mycobacterium leprae using a nested-primer gene amplification assay. J Clin Microbiol 28, 1913–1917.
Roth, A., Reischl, U., Streubel, A., Naumann, L., Kroppenstedt, R. M., Habicht, M., Fischer, M. & Mauch, H. (2000). Novel diagnostic algorithm for identification of mycobacteria using genus-specific amplification of the 16S-23S rRNA gene spacer and restriction endonucleases. J Clin Microbiol 38, 1094–1104.
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.[Abstract]
Tortoli, E., Kroppenstedt, R. M., Bartoloni, A., Caroli, G., Jan, I., Pawlowski, J. & Emler, S. (1999). Mycobacterium tusciae sp. nov. Int J Syst Bacteriol 49, 1839–1844.
Turenne, C. Y., Tschetter, L., Wolfe, J. & Kabani, A. (2001). Necessity of quality-controlled 16S rRNA gene sequence databases: identifying nontuberculous Mycobacterium species. J Clin Microbiol 39, 3637–3648.
Wayne, L. G., Good, R. C., Böttger, E. C. & 19 other authors (1996). Semantide- and chemotaxonomy-based analyses of some problematic phenotypic clusters of slowly growing mycobacteria, a cooperative study of the International Working Group on Mycobacterial Taxonomy. Int J Syst Bacteriol 46, 280–297.
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