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1 Virginia Institute of Marine Science, College of William and Mary, PO Box 1346, Gloucester Point, VA 23062, USA
2 United States Department of Agriculture, Beltsville, MD, USA
3 Tuberculosis/Mycobacteriology Branch, Centers for Disease Control and Prevention, Atlanta, GA, USA
4 US Geological Survey, Leetown Science Center, National Fish Health Research Laboratory, Kearneysville, WV, USA
Correspondence
Martha W. Rhodes
martha{at}vims.edu
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ABSTRACT |
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The GenBank accession number for the 16S rRNA sequence of Mycobacterium shottsii M175T is AY005147.
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; Falkinham, 1996; Dailloux et al., 1999). An increased awareness of the diversity and complexity within the genus has resulted from the application of molecular techniques to the analysis of isolates from environmental sources and clinical specimens (Springer et al., 1993). During a recent epizootic of mycobacteriosis in striped bass, Morone saxatilis, from the Chesapeake Bay, various mycobacteria were isolated including a homogeneous group that, on the basis of traditional biochemical tests, mycolic acid analyses and a distinct 16S rRNA gene sequence, could not be assigned to any recognized species (Rhodes et al., 2001). More extensive characterization using additional biochemical tests, antimicrobial susceptibility and HPLC mycolic acid pattern analysis of additional isolates indicated that these isolates belong to a novel taxon. In this report, we describe the results of a taxonomic study of these isolates and propose that they are representative of a novel species, Mycobacterium shottsii sp. nov.
Isolate M175T and 20 similar isolates were recovered from granulomatous lesions in striped bass (Rhodes et al., 2001). One isolate each (M23 and M216) was recovered from kidney and skin lesions and all others were from the spleen. Additional isolates included M115, M120, M121, M148, M177, M179, M182, M200, M202, M203, M205, M208, M210, M211 and M217–M220. Growth and biochemical testing included reference strains of Mycobacterium avium (M1), Mycobacterium chelonae (M3), Mycobacterium flavescens (M4), Mycobacterium fortuitum (M6), Mycobacterium gordonae (M8), Mycobacterium kansasii (M10), Mycobacterium marinum (M11–M13), Mycobacterium nonchromogenicum (M14), Mycobacterium phlei (M15), Mycobacterium scrofulaceum (M17), Mycobacterium simiae (M19, M20) and Mycobacterium terrae (M21), obtained from the Environmental Protection Agency, Cincinnati, OH, USA, and Consolidated Laboratory Services, Commonwealth of Virginia, Richmond, VA, USA.
Colony morphology and the ability to grow at temperatures ranging from 23 to 42 °C were determined after 1 and 2 months incubation on Middlebrook 7H10 agar with albumin-dextrose-catalase (ADC) enrichment. The following tests were performed at 23 °C using accepted methods (Kent & Kubica, 1985; Vincent Lévy-Frébault & Portaels, 1992): production of acid phosphatase, arylsulfatase, catalase, 
-galactosidase, nitrate reductase, niacin, pyrazinamidase, Tween 80 hydrolysis, urease and growth on media containing hydroxylamine (500 µg ml-1), isoniazid (1 and 10 µg ml-1), p-nitrobenzoic acid (500 µg ml-1), NaCl (50 mg ml-1), thiacetone (10 µg ml-1) and thiophene-2-carboxylic hydrazide (TCH; 2 µg ml-1). Drug susceptibility tests were performed using the disc method (Kent & Kubica, 1985).
HPLC was used to analyse mycolic acids from four isolates (M115, M121, M148 and M175T) as p-bromophenacyl esters as described by Butler et al. (1986). Specimens were processed as described previously using the standard method for sample preparation and UV analysis (Butler et al., 1996). Differentiation of specific Mycobacterium species was by a visual decision method using an internal size standard, an identification process reviewed recently by Butler & Guthertz (2001).
Sequencing of 140 bp for the signature region ‘A’ of the 16S rRNA gene from four isolates (M115, M121, M148 and M175T) was completed using standard protocols (Rhodes et al., 2001).
A phylogenetic tree was constructed using the neighbour-joining method with Jukes–Cantor distances (data not shown), aligning the entire 16S rRNA gene sequence of strain M175T with 23 selected Mycobacterium species. Nocardia asteroides and Nocardia farcinica were used as outgroups in the reconstruction of the evolutionary relationships among M175T and other species within the genus Mycobacterium. The sequences were aligned using the program PILEUP in the Wisconsin package of the Genetics Computer Group (Madison, WI, USA). Aligned sequences were analysed using the Molecular Evolutionary Genetics Analysis (MEGA) package version 1.01 (Kumar et al., 1993), which was also used to generate bootstrap confidence values using 500 permutations of the datasets.
Cells grown on Middlebrook 7H10 agar were acid-fast coccobacilli (0·4–0·6x0·8–1 µm) that tended to occur in cell aggregates. Cell branching and spores were not observed. On Middlebrook 7H10 agar, isolates grew as dysgonic, rough and non-pigmented colonies of 0·5–1 mm after 4–6 weeks at 23 °C. The dominant colony morphology was initially flat with a slightly irregular margin, becoming umbonate upon continued incubation. A second colony type was smooth, slightly raised and with a more entire margin. Little or no growth occurred at 30 °C and none at 37 °C or above. Colonies did not produce pigment following exposure to light for several hours or upon prolonged exposure for several days. Phenotypic characteristics that gave varying results for the novel isolates are presented in Table 1. Other characteristics are given in the species description below.
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. Although isolate M175T exhibited a single cluster of eight peaks that resembled reference patterns for species of the M. tuberculosis complex, peak elution times differed. Shorter elution times indicated that isolate M175T contained more polar, shorter carbon chain-length mycolic acids than species of the M. tuberculosis complex. A profile comparable to that obtained with isolate M175T was not available in the Mycobacterium HPLC mycolic acid database at the Centers for Disease Control and Prevention.
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) and between our isolates and Mycobacterium ulcerans or M. marinum, differed by only a single base pair. A phylogenetic tree reconstructed from the aligned 16S rRNA gene sequences of isolate M175T and 23 other Mycobacterium species (Fig. 2) also indicated a close relationship with M. ulcerans and M. marinum within the slow-growing Mycobacterium species. The 16S rRNA gene of isolate M175T differed from M. ulcerans by three insertions and eight substitutions (one base of the M. ulcerans sequence in GenBank is N) and from M. marinum by four insertions and seven substitutions (one base of the M. marinum sequence in GenBank is N). Most of these differences were located at the 3' end of the gene. We reported this close relationship previously (Rhodes et al., 2001) based on the high sequence similarity in 16S rRNA gene sequences between isolate M175T and M. marinum (99·2 %), M. ulcerans (99·2 %), Mycobacterium bovis (98·7 %) and M. tuberculosis (98·7 %). Similar differences of a few nucleotides in 16S rRNA gene sequences between other Mycobacterium species have been reported previously (Tønjum et al., 1998).
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. Accumulation of niacin in culture media easily distinguishes the novel isolates from other slowly growing mycobacteria that grow optimally at temperatures 
30 °C. In addition, M. marinum and Mycobacterium cookii differ from the novel isolates by producing pigment. The presence of urease activity in the novel isolates distinguishes them from M. ulcerans and Mycobacterium haemophilum. Other slowly growing mycobacteria that may accumulate niacin are differentiated from the novel isolates by growth at 37 °C and resistance profiles to inhibitory agents. The novel isolates differ from ‘Mycobacterium chesapeaki’, a proposed species also isolated from striped bass (Heckert et al., 2001), by tests for growth at 37 °C, niacin production, pyrazinamidase activity (7 days) and 16S rRNA gene sequence. These results indicate that isolate M175T represents a novel member of the genus Mycobacterium, for which the name Mycobacterium shottsii sp. nov. is proposed.
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Acid-fast coccobacilli (0·4–0·6x0·8–1 µm) that may form cell aggregates in culture. Spores and cell branching are not present. Colonies on Middlebrook 7H10 agar are dysgonic, rough, non-pigmented and typically flat with an irregular margin, becoming umbonate upon ageing. Smooth colonies with an entire margin are seen less frequently. Visible colonies from a dilute inoculum are observed after 4–6 weeks incubation at 23 °C. Little or no growth occurs at 30 °C and none at 37 °C or above. Isolates do not grow on MacConkey agar or Löwenstein–Jensen medium with 5 % NaCl, are negative for arylsulfatase (14 days), -galactosidase, nitrate reductase, pyrazinamidase (7 days), semiquantitative catalase, Tween 80 hydrolysis and Tween opacity and have variable reactions for acid phosphatase and catalase at 68 °C. Positive pyrazinamidase reactions occur when incubation is extended to 14–21 days. Positive for urease and niacin production. Colonies do not produce pigment following exposure to light for several hours or upon prolonged exposure for several days. Tolerates isoniazid at 1 µg ml-1 (but not at 10 µg ml-1), thiacetazone and TCH. Growth is inhibited in media containing hydroxylamine. Resistant to p-aminosalicylic acid and isoniazid but susceptible to ethambutol, ethionamide, kanamycin, rifampicin and streptomycin in disc susceptibility tests. The mycolic acid HPLC pattern consists of a single cluster of eight peaks resembling reference patterns for species of the M. tuberculosis complex but the peaks elute more rapidly. The 16S rRNA gene sequence is unique among species of Mycobacterium and is most similar to those of M. ulcerans and M. marinum. The type strain, isolate M175T (=ATCC 700981T =NCTC 13215T), was isolated from granulomatous lesions in splenic tissue from a striped bass (Morone saxatilis).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Butler, W. R., Ahearn, D. G. & Kilburn, J. O. (1986). High-performance liquid chromatography of mycolic acids as a tool in the identification of Corynebacterium, Nocardia, Rhodococcus, and Mycobacterium species. J Clin Microbiol 23, 182–185.
Butler, W. R., Floyd, M. M., Silcox, V. & 9 other authors (1996). Standardized Method for HPLC Identification of Mycobacteria. Atlanta, GA: Centers for Disease Control and Prevention.
Collins, C. H., Grange, J. M. & Yates, M. D. (1984). Mycobacteria in water. J Appl Bacteriol 57, 193–211.[Medline]
Dailloux, M., Laurain, C., Weber, M. & Hartemann, Ph. (1999). Water and nontuberculous mycobacteria. Water Res 33, 2219–2228.[CrossRef]
Falkinham, J. O., III (1996). Epidemiology of infection by nontuberculous mycobacteria. Clin Microbiol Rev 9, 177–215.[Medline]
Heckert, R. A., Elankumaran, S., Milani, A. & Baya, A. (2001). Detection of a new Mycobacterium species in wild striped bass in the Chesapeake Bay. J Clin Microbiol 39, 710–715.
Kent, P. T. & Kubica, G. P. (1985). Public Health Laboratory. A Guide for the Level III Laboratory. US Department of Health and Human Services Publication no. (CDC) 86-8230. Atlanta, GA: Centers for Disease Control.
Kirschner, P., Meier, K. A. & Böttger, E. C. (1993). Genotypic identification and detection of mycobacteria: facing novel and uncultured pathogens. In Diagnostic Molecular Microbiology, pp. 173–190. Edited by D. H. Persing, T. F. Smith, F. C. Tenover & T. J. White. Washington, DC: American Society for Microbiology.
Kumar, S., Tamura, K. & Nei, M. (1993). MEGA: Molecular Evolutionary Genetics Analysis, version 1.01. University Park, PA: Pennsylvania State University.
Rhodes, M. W., Kator, H., Kotob, S. & 8 other authors (2001). A unique Mycobacterium species isolated from an epizootic of striped bass (Morone saxatilis). Emerg Infect Dis 7, 896–899.[Medline]
Springer, B., Kirschner, P., Rost-Meyer, G., Schröder, K.-H., Kroppenstedt, R. M. & Böttger, E. C. (1993). Mycobacterium interjectum, a new species isolated from a patient with chronic lymphadenitis. J Clin Microbiol 31, 3083–3089.
Tønjum, T., Welty, D. B., Jantzen, E. & Small, P. L. (1998). Differentiation of Mycobacterium ulcerans, M. marinum, and M. haemophilum: mapping of their relationships to M. tuberculosis by fatty acid profile analysis, DNA-DNA hybridization, and 16S rRNA gene sequence analysis. J Clin Microbiol 36, 918–925.
Vincent Lévy-Frébault, V. & Portaels, F. (1992). Proposed minimal standards for the genus Mycobacterium and for description of new slowly growing Mycobacterium species. Int J Syst Bacteriol 42, 315–323.
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85 % strains positive; M, 50–80 % positive; F, 15–49 % positive; -, <15 % positive; ND, not determined.