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1 Virginia Institute of Marine Science, College of William and Mary, Gloucester Point, VA 23062, USA
2 Laboratory Services, British Columbia Centre for Disease Control, Vancouver BC, Canada V5Z 4R4
3 Avenir Group, Inserm U570, Avenir Group, Faculté de Médecine Necker-Enfants Malades, 75730 Paris Cedex 15, France
4 Mycobacteria/Nocardia Laboratory, University of Texas Health Center, Tyler, TX 75708, USA
5 Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA
6 Office of Laboratory Animal Care, University of California, Berkeley, CA 94720, USA
7 Department of Comparative Medicine, Stanford University, Stanford, CA 94305, USA
8 MIDI, Inc. Research and Development, Newark, DE 19713, USA
9 US Geological Survey, Leetown Science Center, National Fish Health Research Laboratory, Kearneysville, WV 25430, USA
Correspondence
Martha W. Rhodes
martha{at}vims.edu
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The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA and hsp65 gene sequences of Mycobacterium pseudoshottsii ATCC BAA-883T are AY570988 and AY571788, respectively.
Aligned erp gene sequences of Mycobacterium pseudoshottsii sp. nov., Mycobacterium marinum, Mycobacterium shottsii, Mycobacterium ulcerans and Mycobacterium tuberculosis are available as a supplementary figure in IJSEM Online.
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). These new taxa comprise isolates of both clinical and environmental origin. DNA-based methods are particularly valuable for characterizing the latter, which typically have lower growth-temperature preferences and biochemical characteristics that often do not allow conclusive species identification using diagnostic techniques developed almost exclusively for clinical isolates.
The primary pathogens of mycobacteriosis in fish are reported to be Mycobacterium abscessus (formerly Mycobacterium chelonae subsp. abscessus), Mycobacterium chelonae, Mycobacterium fortuitum and Mycobacterium marinum (Belas et al., 1995). Recently, three previously unrecognized species were isolated: ‘Mycobacterium chesapeaki’ (Heckert et al., 2001) and Mycobacterium shottsii from an epizootic in striped bass (Morone saxatilis) (Rhodes et al., 2003), and Mycobacterium montefiorense from captive moray eels (Gymnothorax funebris and Gymnothorax moringa) (Levi et al., 2003). Characterization using molecular methods allowed discrimination of Mycobacterium shottsii and Mycobacterium montefiorense from the clinically significant related species Mycobacterium marinum (Dobos et al., 1999) and Mycobacterium triplex, respectively (Floyd et al., 1996; Hazra et al., 2001).
During an epizootic of mycobacteriosis in Chesapeake Bay striped bass, a variety of mycobacteria were isolated and then characterized using traditional biochemical and growth features (Rhodes et al., 2004). In this study, we report the isolation, from striped bass, of a group of mycobacteria phenotypically resembling Mycobacterium shottsii, which shares with Mycobacterium marinum a cladic relationship with Mycobacterium tuberculosis, Mycobacterium bovis and several other species (Goodfellow & Magee, 1998). Polyphasic studies involving biochemical testing, mycolic acid analysis, sequencing of the 16S rRNA gene, the 65 kDa heat-shock-protein gene (hsp65) and the exported repetitive protein (erp) gene, and gene amplification of Mycobacterium ulcerans-specific insertion sequences (IS2404 and IS2606) indicate that these organisms belong to a novel species.
All of the mycobacterial isolates (L1, L6, L9, L15T, L23, L25, L28, L31, L32, L37, L39, L43, L50, L58, L62, R97, R129, R174, R177 and R179) examined in this study were isolated from Morone saxatilis collected from Chesapeake Bay (the largest estuary in the US and bordered by the states of Maryland and Virginia) or one of its tributaries (i.e. York and Rappahannock rivers). Splenic tissue was dissected aseptically, homogenized in phosphate buffer, spread-plated on Middlebrook 7H10 agar (Difco/Becton Dickinson Diagnostic Systems) with oleic acid/albumin/dextrose/catalase (OADC) enrichment, then incubated at 23 °C for 3 months. Growth and biochemical testing included reference strains of Mycobacterium avium (M1), Mycobacterium fortuitum (M6), Mycobacterium gordonae (M8), Mycobacterium kansasii (M10) and Mycobacterium marinum (M11) (all of which were obtained from the Environmental Protection Agency, Cincinnati, OH, USA, and Consolidated Laboratory Services, Commonwealth of Virginia, Richmond, VA, USA), Mycobacterium ulcerans (ATCC 19423T) and Mycobacterium shottsii M175T (previously deposited, from our laboratory, in the American Type Culture Collection, Manassas, VA, USA as ATCC 700981T).
Colony morphology and the ability to grow at temperatures ranging from 23 to 37 °C were determined after 1 and 2 months incubation on Middlebrook 7H10 agar with OADC enrichment. The following characters were assessed at 23 °C: production of acid phosphatase, arylsulfatase after 3 and 14 days, catalase after heating at 68 °C, 
-galactosidase, nitrate reductase, niacin, pyrazinamidase, semi-quantitative catalase, Tween 80 hydrolysis, urease and growth on Löwenstein–Jensen medium containing NaCl (50 mg ml–1) (Kent & Kubica, 1985; Lutz, 1995). The proportion method (Kent & Kubica, 1985; Lévy-Frébault & Portaels, 1992) was used to determine drug susceptibility. Testing media were prepared by adding hydroxylamine, isoniazid, p-nitrobenzoic acid, thiacetone and thiophene-2-carboxylic hydrazide to Middlebrook 7H10 agar with OADC; alternatively, the desired drug concentration was achieved by elution from submerged discs (BBL/Becton Dickinson Diagnostic Systems). Oleate resistance was determined in the same manner using Dubos oleic acid agar (control) (BBL/Becton Dickinson Diagnostic Systems) and the same base medium supplemented with oleic acid.
The results of HPLC analysis of mycolic acids from L15T and Mycobacterium shottsii M175T were compared using the Sherlock Mycobacteria Identification System (SMIS; MIDI). Cells from cultures grown on Middlebrook 7H10 agar with OADC were saponified, extracted and derivatized according to the manufacturer's instructions and loaded onto an Agilent ChemStation HPLC (Agilent Technologies). Separation of the mycolic acids was achieved by using a gradient of methanol and 2-propanol. Results were analysed using MIDI Sherlock software (version 1.0).
The 16S rRNA genes of isolates L15T and Mycobacterium shottsii M175T were sequenced by using published methods (Lane, 1991; Turenne et al., 2003); the BLASTN tool (http://www.ncbi.nlm.nih.gov/blast) was then used to compare the sequences with those in the GenBank database. The sequence for L15T was submitted to the GenBank database under accession number AY570988. The 16S rRNA gene sequence for Mycobacterium shottsii was modified and the original sequence updated in GenBank (AY005147). Phylogenetic analysis of the 16S rRNA gene was performed by neighbour-joining based on the uncorrected (P) distances and by maximum parsimony and maximum likelihood using PAUP version 4.0b10 (Swofford, 2002). Parsimony was performed with the gaps treated as missing. The model for the maximum-likelihood analyses was determined by MODELTEST 3.06 (Posada & Crandall, 1998) to determine the model which best fit the data based on hierarchical tests of models by calculating the likelihood ratio statistic for each model and its associated P value. We suspected a serious error at the 3' end of the GenBank-deposited ‘Mycobacterium chesapeaki’ sequence. An approximately 320 bp fragment of the 3' end of the sequence is unlikely to be a valid Mycobacterium species sequence, but rather is corrupted by either severe errors in sequence determination or contamination by foreign DNA. Therefore, the distance and maximum-likelihood analyses were performed on a dataset of 1120 aligned nucleotide positions from the 5' end of the Mycobacterium species 16S rRNA gene sequences.
The DNA sequence of the central region of the erp gene, corresponding to amino acid residues 81–174 (de Mendonça-Lima et al., 2001), was obtained by sequencing PCR fragments generated from genomic DNA recovered from Mycobacterium shottsii M175T and L1, L6 and L15T as described previously (Pelicic et al., 1996) but with minor modifications. The primers used for amplification by Pfu Turbo Taq polymerase (Stratagene) (30 cycles) were P1 (5'-atgaccgcgctgtcgcagggt-3') and P2 (5'-ggggtcacccaggatcggata-3'); the annealing temperature was 55 °C. PCR fragments were sequenced and subsequently analysed with DNA Strider (Marck, 1988). The sequences were submitted to GenBank under the following accession numbers: Mycobacterium shottsii M175T, AY496288; L1, AY496289; L6, AY496290; and L15T, AY496291. The accession numbers of the erp gene sequences of other mycobacterial species used for the phylogenetic analyses were as follows: Mycobacterium tuberculosis, L38851; Mycobacterium ulcerans, AF213154; and Mycobacterium marinum, AF213153. Protein sequences were generated by virtual translations using MacVector version 7.2 (Accelrys) and aligned using the CLUSTAL W alignment program in the MacVector package. DNA sequences were also aligned using CLUSTAL W and gap positions were adjusted to correspond with those in the amino acid alignment. Phylogenetic analysis was performed as described above for the 16S rRNA gene except that parsimony was performed with the gap informative feature (gaps treated as a fifth nucleotide state).
DNA amplification of the hsp65 gene was accomplished by using the method described by Telenti et al. (1993). Sequencing of the hsp65 fragment was performed on an ABI Prism 310 or 3100 genetic analyser (Applied Biosystems) with the Big Dye Terminator Cycle Sequencing Ready Reaction kit, version 3.0 or version 3.1 (Applied Biosystems) as described previously, with the modification that 10 ng DNA was used with the Tb11 (forward) primer and 30 ng DNA was used with the Tb12 (reverse) primer in all sequencing reactions (Kapur et al., 1995). hsp65 sequences were determined for L1, L6, L15T and Mycobacterium shottsii M175T and compared with existing sequences in GenBank and the hsp65 gene database of the British Columbia CDC Laboratory Services, Vancouver, Canada. The GenBank accession number for the hsp65 sequence of L15T is AY571788. Phylogenetic analysis was performed using PAUP*, version 4.0b10 (Swofford, 2002). Sequences were aligned and phylogenetic analyses were as described above for the 16S rRNA gene.
Isolate L15T, Mycobacterium shottsii M175T and Mycobacterium ulcerans ATCC 19423T were evaluated by using PCR amplification and restriction endonuclease analysis of the 441 bp hsp65 gene sequence as described previously (Steingrube et al., 1995; Brown et al., 1999; Telenti et al., 1993). Cells were grown and harvested on Middlebrook 7H10 agar and ground cell supernatants were used as DNA templates (Steingrube et al., 1995). Appropriate positive and negative controls, as recommended by Telenti et al. (1993), were included. Restriction endonucleases BstEII and HaeIII (Promega) were selected (Steingrube et al., 1995) and used according to the manufacturer's recommendations. Restriction fragments were electrophoresed on 3 % Metaphor agarose (4 bp resolution; FMC Bioproducts) containing ethidium bromide in a Mini-Sub cell electrophoresis system (Bio-Rad) (Brown et al., 1999). Fragment sizes (in bp) were estimated on a computerized Bio Image system (Millipore).
DNA amplification of the Mycobacterium ulcerans-specific insertion sequences, IS2404 and IS2606, was performed with primer sets MU5/MU6 and MU7/MU8, respectively, as described by Stinear et al. (1999). DNA was extracted from cultures of L15T, Mycobacterium shottsii and Mycobacterium ulcerans and PCR amplifications were performed as described previously (Trott et al., 2004). PCRs were performed using a variety of DNA polymerases: AmpliTaq Gold DNA polymerase (Applied Biosystems), Taq DNA polymerase (Invitrogen), Taq DNA polymerase (Roche) and Platinum Taq DNA polymerase High Fidelity (Invitrogen).
Mycobacterial cells grown on Middlebrook 7H10 agar were acid-fast coccobacilli, small (approx. 0·4–0·6 µm by 0·8–1 µm) and tended to clump. Neither cell branching nor spores was observed. On Middlebrook 7H10 agar, isolates grew as rough colonies that were approximately 1–3 mm in diameter after incubation for 2 months at 23 °C and were not emulsifiable in water. Colonies were rough, initially flat with a slightly irregular margin, becoming umbonate upon continued incubation. Little or no growth occurred at 30 °C and none occurred at 37 °C. Colonies slowly became gold following exposure to light. Phenotypic features that varied between isolates are presented in Table 1. Other characteristics are listed in the species description below.
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; Springer et al., 1996) and differences near the 3' end have been used to distinguish species (Tønjum, 1998; Portaels et al., 1996). 16S rRNA gene sequences of closely related species may be identical or may differ by only a few nucleotides (Tortoli, 2003). In the phylogenetic analyses, trees of similar topology were obtained by neighbour-joining (Fig. 2), maximum parsimony and maximum likelihood. Although the neighbour-joining and maximum-likelihood analyses were done on a dataset with aligned truncated (approx. 320 bp shorter) sequences, the topology of the trees was similar to that when the complete dataset with the suspected corrupted portion of the ‘Mycobacterium chesapeaki’ 16S sequence was included, except that branch length on the ‘Mycobacterium chesapeaki’ sequence was considerably shorter with the revised dataset.
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, 2003). The central region of the Erp protein consists of tandem repeats of five amino acids that vary in terms of the numbers of repeats and the sequence of the repeat between mycobacterial species. In this study, sequence analysis of the central region of the erp gene revealed significant differences between isolates L1, L6 and L15T, which were all identical to each other, and Mycobacterium marinum, Mycobacterium shottsii M175T and Mycobacterium ulcerans. L1, L6 and L15T had a reduced number of repeats (30 nt), as well as 13 substitutions, in comparison with Mycobacterium shottsii (erp sequences are available as a supplementary figure in IJSEM Online).
The hsp65 gene, encoding heat-shock protein, has been used to distinguish between different mycobacterial species (Devallois et al., 1997; Leclerc et al., 2000). The sequences of the hsp65 genes (401 nt, primers removed) from L1, L6 and L15T were identical and differed from that of Mycobacterium shottsii M175T by three nucleotides. When hsp65 gene sequence results were compared with those in the database at the British Columbia Centre for Disease Control, L15T was found to have a sequence identical to that of ‘Mycobacterium seriolae’ (Kusuda et al., 1987). Sequences deposited in GenBank for a Mycobacterium ulcerans-like organism (AY500839) (Trott et al., 2004) and Mycobacterium marinum strains eilaticum DL240490 (AF456468), hellenicum DL045 (AF456471) and cyprinum CC240299 (AF456469) (Ucko et al., 2002) were identical to that of L15T. Mycobacterium ulcerans (AY550209 and AF547888) differed by 1 or 3 nt and Mycobacterium marinum (AF547855) differed by 2 nt from L15T. The topologies of the trees generated by neighbour-joining (Fig. 3) and parsimony were similar.
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; Telenti et al., 1993; Devallois et al., 1997; Leclerc et al., 2000; Brunello et al., 2001). BstEII digests of L15T, Mycobacterium shottsii M175T and Mycobacterium ulcerans ATCC 19423T produced identical restriction fragments of 235/210 nt. Digestion with HaeIII yielded identical restriction fragments of 145/105/80 nt with Mycobacterium ulcerans and L15T, while Mycobacterium shottsii yielded restriction fragments of 220/105 nt. Mycobacterium ulcerans-specific insertion sequences, IS2404 and IS2606, were detected in L1, L6, L15T and Mycobacterium ulcerans, but only weakly or not at all in Mycobacterium shottsii M175T when Taq DNA polymerase was used in PCRs. However, use of high-fidelity Taq DNA polymerase enhanced the signal for IS2404 in Mycobacterium shottsii M175T and yielded unequivocal positive results when tested independently in three of the laboratories participating in this study.
On the basis of 16S rRNA and hsp65 gene sequences, L15T is very similar to scotochromogenic species isolated from Mediterranean fish and described as strains of Mycobacterium marinum by Ucko et al. (2002), who postulated that Mycobacterium marinum isolates have genotypes that are unique and specific to a geographical region. The 401 nt sequence of the L15T hsp65 gene PCR product was identical to that of ‘Mycobacterium seriolae’, a photochromogenic mycobacterium isolated from maricultured yellowtail (Seriola quinqueradiata) in Japan (Kusuda et al., 1987), and a Mycobacterium ulcerans-like non-chromogenic mycobacterium isolated from a laboratory colony of frogs (Xenopus tropicalis) and which is also positive for IS2404 and IS2606 (Trott et al., 2004). These results demonstrate the complexity of distinguishing interspecific polymorphisms from intraspecific variation, even when using a molecular approach. van Berkum et al. (2003) recently demonstrated that tree phylogenies based on different loci can be significantly different, i.e. 16S rRNA gene sequences versus those constructed with 23S rRNA gene sequences and internally transcribed spacer region sequences, and postulated that such differences could arise via lateral transfer and recombination. These observations underline the importance of examining different DNA loci to discriminate closely related species.
L15T-like isolates, previously described as phenotypic Group F (Rhodes et al., 2004), have been isolated from approximately 12 % of Chesapeake Bay striped bass sampled (n=24/192), but the pathogenicity of these isolates has not been assessed. Despite sharing certain phenotypic similarities and a common host, i.e. striped bass, L15T has been clearly differentiated from Mycobacterium shottsii, in our study, on the basis of three genetic loci. The taxonomic relationship between L15T and other mycobacteria reported in the literature as having different chromogenic characteristics but similar 16S rRNA and hsp65 sequences is not known. It is possible that the aforementioned isolates, which have been described previously but whose names are not taxonomically valid, belong to the same taxon as the novel mycobacterium described in this study, for which we propose the name Mycobacterium pseudoshottsii sp. nov. These and previous observations (Chemlal et al., 2002; Ucko et al., 2002; Rhodes et al., 2003) demonstrate the continuum of mycobacterial strains that are intermediate between Mycobacterium marinum and Mycobacterium ulcerans. Additional molecular studies, e.g. DNA–DNA homology and comparison of disease pathologies in human and animal hosts, are necessary for the taxonomic classification of species that are transitional between Mycobacterium marinum and Mycobacterium ulcerans.
Although IS2606 has been reported in Mycobacterium lentiflavum (Stinear et al., 1999), the presence of both IS2404 and IS2606 is considered specific to Mycobacterium ulcerans. Recently, both insertion sequences were identified in a mycobacterium isolated during an infection of laboratory-reared frogs that was characterized as being intermediate between Mycobacterium ulcerans and Mycobacterium marinum (Trott et al., 2004). Chemlal et al. (2002) characterized a mycobacterium isolated from a human infection as resembling Mycobacterium marinum but having a low copy number for IS2404. Our finding of both insertion sequences in mycobacteria isolated from a euryhaline fish in a temperate climate implies that the use of IS2404 and IS2606 for the specific detection of Mycobacterium ulcerans in aquatic environments (Stinear et al., 1999, 2000) may not be universally applicable. Sequencing of the PCR products generated by the primers designed for these insertion sequences would be informative regarding the similarity between human and environmental isolates.
Description of Mycobacterium pseudoshottsii sp. nov.
Mycobacterium pseudoshottsii (pseu.do.shott'si.i. Gr. adj. pseudes false; N.L. gen. n. shottsii name of a species; N.L. gen. n. pseudoshottsii a false Mycobacterium shottsii, not the true Mycobacterium shottsii).
Acid-fast coccobacillus (0·4–0·6x0·8–1 µm) that forms cell aggregates in culture. Spores and cell branching are not present. Colonies on Middlebrook 7H10 agar are rough, photochromogenic and, typically, flat with an irregular margin, becoming umbonate with age. A pale-yellow to gold pigment develops following exposure to light and intensifies with age. Visible colonies are observed from a dilute inoculum after 4–6 weeks incubation at 23 °C. Little or no growth occurs at 30 °C and none occurs at 37 °C. Isolates do not grow on MacConkey agar without crystal violet or on Löwenstein–Jensen medium with 5 % NaCl, are negative for arylsulfatase (at 3 and 14 days), -galactosidase, nitrate reductase, pyrazinamidase (at 7 days), semi-quantitative catalase and Tween 80 hydrolysis, and have variable reactions for acid phosphatase and catalase at 68 °C. Positive pyrazinamidase reactions do not occur when incubation is extended to 14–21 days. Isolates are typically positive for urease production and niacin accumulation. These mycobacteria tolerate isoniazid at 1 µg ml–1 (but not at 10 µg ml–1), thiacetazone and thiophene-2-carboxylic hydrazide. Growth is inhibited in media containing hydroxylamine and oleate. Isolates are resistant to p-aminosalicylic acid and isoniazid but susceptible to ethambutol, kanamycin, rifampicin and streptomycin in disc-based antibiotic-susceptibility tests. The mycolic acid HPLC pattern consists of a single cluster of peaks resembling that of Mycobacterium shottsii. The 16S rRNA, erp and hsp65 gene sequences are unique among recognized species of Mycobacterium and are most similar to those of Mycobacterium shottsii, Mycobacterium ulcerans and Mycobacterium marinum.
The type strain, L15T (=ATCC BAA-883T=NCTC 13318T), was isolated from granulomatous lesions in splenic tissue from a striped bass (Morone saxatilis).
Slowly growing mycobacteria, which grow either poorly or not at all at 37 °C, or produce niacin, are compared with Mycobacterium pseudoshottsii in Table 2. Although the combined traits of photochromogenicity and niacin production distinguish Mycobacterium pseudoshottsii from other species that grow optimally at 
30 °C, expression of both features is dependent on sufficient incubation, i.e. 2–3 months. Other slowly growing mycobacteria that may accumulate niacin are differentiated from Mycobacterium pseudoshottsii by growth at 37 °C and by the profiles for resistance to inhibitory agents.
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