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1 Institute of Medical Microbiology and Hygiene, University of Regensburg, Regensburg, Germany
2 DSMZ – Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany
3 Institute of Medical Microbiology and Hygiene, Technical University of Munich, Munich, Germany
4 Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit (LGL), Oberschleißheim, Germany
5 Mycobacteriology Reference Center, Microbiology Laboratory, Careggi Hospital, Florence, Italy
6 Cytogenetics and Genetics Unit, Careggi Hospital, Florence, Italy
7 Microbiological and Virological Serum-Immunology Laboratory, Careggi Hospital, Florence, Italy
Correspondence
Enrico Tortoli
e.tortoli{at}libero.it
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of B9-21-178T is AF107039. The GenBank/EMBL/DDBJ accession numbers for the hsp65 gene sequences of strains FI-00234, FI-03115 and FI-05352 are DQ381731, DQ381730 and DQ381732, respectively. Those for the ITS sequences of B9-21-178T, FI-03115, the six sequence variants of FI-00234 and the seven sequence variants of FI-05352 are DQ473393, DQ381726, DQ466891–DQ466896 and DQ466897–DQ466903, respectively.
A phylogenetic tree based on hsp65 gene sequences of selected mycobacterial species and a table giving the fatty acid contents of strain B9-21-178T and related mycobacteria are available as supplementary material in IJSEM Online.
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). In the last few years, a new scenario has appeared, with many novel rapidly growing species being described that are often responsible for disease in humans (Brown-Elliott & Wallace, 2002). The majority of such novel mycobacteria are not pigmented and have emerged from the splitting of the heterogeneous group known, until a few years ago, as the M. fortuitum complex (Wallace et al., 2004; Adékambi et al., 2006). Here, four strains of a scotochromogenic rapidly growing mycobacterium are described. Their unique phenotypic and genotypic features justify their classification in a novel species. The first strain (B9-21-178T) was isolated 1998 in Germany from a bronchial lavage of an 80-year-old patient hospitalized to treat multifocal lung carcinoma and insulin-dependent diabetes mellitus. Tuberculostatic treatment did not change the clinical symptoms. Three other strains were isolated between 2000 and 2005 from unrelated patients hospitalized in different cities in Italy. Of particular interest is strain FI-00234, which was isolated from the biopsy material of an 11-year-old boy presenting with a fistula on his right thigh as a consequence of accidental deep penetration of a screwdriver. Various minor surgical operations had no effect until a complete emptying of the wound, which deepened to the bone, resolved the problem. The other two strains, of uncertain clinical significance, were FI-03115, grown from the sputum of a 31-year-old HIV-positive woman with bronchopneumonitis, and FI-05352, obtained from the sputum of an 82-year-old patient suspected to have lung cancer.
Strains were isolated from clinical specimens, subjected to standard N-acetyl-L-cysteine-NaOH decontamination, on Lowenstein–Jensen medium at 37 °C. Strains were maintained at –80 °C in Middlebrook 7H9 broth supplemented with 15 % glycerol.
Genetic sequencing was carried out using standard procedures (Reischl et al., 1998; Roth et al., 1998; McNabb et al., 2004) on three different species markers. The almost complete 16S rRNA gene sequences (1450 bp) of the four investigated strains were identical and differed by 14 bp from the 16S rRNA gene sequence of Mycobacterium doricum, the most closely related species (99 % similarity). Of the bulk of sequences from as yet unnamed mycobacterial isolates deposited in GenBank so far, entry AF387804 (Mycobacterium sp. JLS; environmental isolate) was 100 % identical, whereas entries AY083217 and AF387803 (Mycobacterium sp. KMS and MCS, respectively; environmental isolates) differed by only 2 nt from the novel strains described herein.
In the sequence of the hypervariable region within the hsp65 gene, determined as reported previously (McNabb et al., 2004), two sequevars were detected that differed by 4 bp, with the first shared by FI-00234 and FI-05352 and the second by B9-21-178T and FI-03115 (Fig. 1). Again, the sequence of M. doricum was the most closely related (13–15 bp diversity within the analysed region of 424 bp; 96.5–96.9 % similarity).
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). To investigate nucleotide ambiguities within the ITS sequences of strains FI-00234 and FI-05352 in more detail, the corresponding ITS-derived amplicons were cloned into DH5
cells using the TOPO-TA cloning kit for sequencing (Invitrogen). From each of the two cloning experiments (strains FI-00234 and FI-05352), 12 colonies were selected for plasmid preparation and subsequent DNA sequencing using BigDye Terminator chemistry and an AB3730 DNA sequencer (Applied Biosystems). Clear and unambiguous sequences were observed for every cloned ITS-derived amplicon. Of the 12 plasmid preparations investigated from each strain, seven different sequence variants were identified in strain FI-05352 and six sequence variants were found in strain FI-00234. These precisely determined ITS sequences of the four novel strains were clearly divergent from those of other mycobacterial species. Sequence alignments of the ITS region revealed Mycobacterium vanbaalenii as the most closely related species. From a practical point of view, the alignment process was only possible for two stretches of approximately 125 and 60 bp within the ITS sequence, whereas only weak similarity was observed with the ITS sequence of M. vanbaalenii and those of other mycobacterial species. Such data may support the suggestion that the usefulness of ITS sequencing for phylogenetic studies of rapidly growing mycobacteria is somewhat limited.
Phylogenetic analysis within a group of related mycobacterial species, selected on the basis of phenotypic and genotypic properties, was conducted using MEGA version 3.1 software (Kumar et al., 2004) applying the Kimura two-parameter distance correction model. The neighbour-joining method (Saitou & Nei, 1987) was carried out with 1000 bootstrap samples on the largest 16S rRNA gene stretch available in GenBank (positions 49–1448 of Escherichia coli 16S rRNA gene) shared by the species involved. As depicted in Fig. 2, M. doricum was the most closely related species.
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) and the resulting phylogenetic tree (available as Supplementary Fig. S1 in IJSEM Online) confirmed the very close phylogenetic relationship between the test strains and M. doricum. Separation of the respective phyla is supported by a high bootstrap value in each of the determined phylogenetic trees.
Alpha-mycolates, keto-mycolates and wax ester mycolates were detected in B9-21-178T by bi-dimensional TLC, performed as described previously (Schröder et al., 1997). This is a very common mycolic acid pattern shared by a large number of NTM, but nevertheless differs from that of M. doricum, which presents methoxy-mycolates instead of keto-mycolates (Tortoli, 2003).
Major compounds revealed by GLC, determined using the Microbial Identification standard software package (Sasser, 1990), of B9-21-178T included the methyl-branched fatty acid 10-methyl 18 : 0 and the alcohol 2-OH 18 : 0 (the full fatty acid content is available in Supplementary Table S1 in IJSEM Online).
The HPLC pattern of mycolic acids, performed after esterification to bromophenacyl esters (Butler & Kilburn, 1988), was identical in the four investigated strains. It was characterized by the presence of two well-separated clusters of peaks, with the first including four major peaks eluting between 5 and 6 mins and the second, eluting after a 2 min interval, presenting three minor peaks (Fig. 3). Although characterized by a similar motif, the HPLC pattern of M. doricum clearly differed, mainly because of the later emergence of peaks of the first cluster (Fig. 3). A thorough investigation of our HPLC mycobacterium library (available online at http://www.MycobacToscana.it) revealed that Mycobacterium conspicuum, Mycobacterium murale and Mycobacterium terrae present profiles that grossly resemble this pattern.
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), showed uniform susceptibility to amikacin, cefoxitin, ciprofloxacin, clarithromycin, doxycycline and linezolid and variable results for trimethoprim-sulfamethoxazole and imipenem (Table 1).
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), were positive for nitrate reduction, thermostable catalase and Tween 80 hydrolysis, whereas the strains were negative for niacin accumulation, 3-day arylsulfatase and 
-glucosidase. Urease and semi-quantitative catalase activities were variable. All four strains grew in less than a week on solid medium, forming smooth yellow colonies at temperatures of 25–37 °C. Growth, although slower, did occur at 45 °C and on Lowenstein–Jensen medium supplemented with 5 % NaCl, but not on MacConkey medium without crystal violet. It is proposed that the four strains are representatives of a novel species of mycobacteria for which the name Mycobacterium monacense sp. nov. is proposed. The type strain is B9-21-178T (=DSM 44395T=CIP 109237T).
Description of Mycobacterium monacense sp. nov.
Mycobacterium monacense (mo.na.ce'nse. L. neut. adj. monacense from Monacum, the Latin name of the German city Munich where the first strain was isolated).
Gram-positive, acid-fast, non-motile, does not form spores and produces smooth, yellow, scotochromogenic colonies within 7 days at 25–45 °C. Nitrate reduction and Tween 80 hydrolysis are positive. Neither TLC, which reveals the presence of alpha-mycolates, keto-mycolates and wax ester mycolates, nor GLC, whose major compounds include the methyl-branched fatty acid 10-methyl 18 : 0 and the alcohol 2-OH 18 : 0, are suitable for precise differentiation from other species. HPLC may be useful for differentiation as none of the known rapidly growing scotochromogenic strains presents a similar pattern. Susceptible in vitro to amikacin, cefoxitin, ciprofloxacin, doxycycline and linezolid. Differs from every other known mycobacterium in a substantial number of nucleotides in each of the most frequently sequenced regions, 16S rRNA gene, ITS and hsp65. The divergence of the ITS sequence from those of almost all other mycobacteria is noteworthy. Of the rapidly growing mycobacteria, most closely related to M. doricum based on phylogenetic analysis of the 16S rRNA gene sequence.
The type strain, B9-21-178T (=DSM 44395T=CIP 109237T), was isolated in Germany from a bronchial lavage sample. The three other representatives of this species were isolated from clinical specimens and one was responsible for post-traumatic wound infection.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Brown-Elliott, B. A. & Wallace, R. J., Jr (2002). Clinical and taxonomic status of pathogenic nonpigmented or late-pigmenting rapidly growing mycobacteria. Clin Microbiol Rev 15, 716–746.
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McNabb, A., Eisler, D., Adie, K., Amos, M., Rodrigues, M., Stephens, G., Black, W. A. & Isaac-Renton, J. (2004). Assessment of partial sequencing of the 65-kilodalton heat shock protein gene (hsp65) for routine identification of mycobacterium species isolated from clinical sources. J Clin Microbiol 42, 3000–3011.
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