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1 Laboratoire de biologie, Centre Hospitalier du Bassin de Thau, 34207 Sète, France
2 Université Montpellier 1, Faculté de Pharmacie, Laboratoire de Bactériologie-Virologie, EA 3755, 34060 Montpellier, France
3 Service de chirurgie orthopédique, Centre Hospitalier du Bassin de Thau, Sète, France
4 UMR CNRS 5557, Center for Microbial Ecology, Opportunistic Pathogens and Environment Research Group, Faculté de Médecine, Université Claude Bernard, Lyon, France
5 Laboratoire de Bactériologie, Hôpital de la Croix Rousse, Hospices Civils de Lyon, France
Correspondence
Brigitte Lamy
blamy{at}ch-bassindethau.fr
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ABSTRACT |
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Supplementary figures showing the cell wall fatty acid analysis and phylogenetic trees are available with the online version of this paper.
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TOPABSTRACTMAIN TEXTREFERENCES |
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, 2004; Brown-Elliott & Wallace, 2002; Adékambi et al., 2006). Debate still persists on whether Mycobacterium mageritense belongs to the M. fortuitum group (Brown-Elliott & Wallace, 2002; Adékambi & Drancourt, 2004).
M. fortuitum-group members cause a variety of human infections (Brown-Elliott & Wallace, 2002, 2005). Accurate bacterial identification, rising from a relevant classification, is crucial as antimicrobial resistance is species-dependent (Wallace et al., 1991).
Here we describe a new member of the M. fortuitum group. The isolate, designated ABO-M06T, was obtained from a patient with post-traumatic soft tissue infection and osteitis. A polyphasic study showed a unique genotype and phenotype, suggesting that this strain is representative of a novel species.
Strain ABO-M06T was isolated from a 52-year-old patient admitted to Bassin de Thau Hospital, Sète, France, for a chronic soft tissue infection of the left foot that occurred after he stepped on a nail. The local infection was later complicated with osteitis and tenosynovitis. The organism was initially isolated after a 21 day culture on Löwenstein–Jensen slants incubated at 37 and 30 °C. It was subcultured on Löwenstein–Jensen slants and blood agar plates, and formed colonies in less than 7 days at 20, 25 and 37 °C. Strain ABO-M06T was a Gram-positive, acid-fast bacterium initially identified as M. peregrinum with the INNO-LiPA method (Innogenetics) and the patient was treated with clarithromycin (1 g per day for 30 days). However, treatment failure led us to go further into bacterial identification. The initial isolate was maintained in glycerol suspension (10 % v/v) at –80 °C.
The isolate was examined for pigmentation and morphological characteristics as described previously (Schinsky et al., 2004). Arylsulfatase (3 day) and catalase activities, iron uptake and growth on Löwenstein–Jensen medium containing 5 % NaCl were tested as described by Vincent et al. (2003). We also inoculated API Coryne, API 20E and API 20NE strips (bioMérieux) as recommended by the manufacturer, and incubated them for 5 days at 30 and 37 °C in a highly humidified atmosphere (Adékambi et al., 2006). Standard fatty acid and mycolic acid analyses were performed by the Deutsche Sammlung von Mikroorganismen und Zellkulturen by means of gas chromatography and HPLC, respectively, as previously described (Butler et al., 1992), using standard Microbial Identification System software (MIDI). The broth microdilution method was used to test susceptibility to amikacin, clarithromycin, cefoxitin, doxycycline, gatifloxacin, moxifloxacin, sulfamethoxazole and tobramycin, as recommended by the Clinical Laboratory Standards Institute (NCCLS, 2002, 2003). Imipenem susceptibility was tested with the disc diffusion method on 5 % sheep blood agar plates incubated for 3 days at 30 °C.
A multigene sequencing approach was used, according to Adékambi & Drancourt (2004) and Devulder et al. (2005). Double-strand partial sequences were obtained for four genes amplified as described elsewhere, namely (i) 16S rRNA gene [near-complete sequence of 1361 nt, corresponding to positions 46–1400 of the Escherichia coli numbering system (Rodríguez-Nava et al., 2004)], (ii) rpoB gene [encoding the β-subunit of bacterial RNA polymerase; 764 nt corresponding to positions 2573–3337 of Mycobacterium smegmatis ATCC 14468 (Adékambi et al., 2003)], (iii) hsp65 gene [65 kDa heat-shock protein; 441 nt corresponding to positions 396–836 of Mycobacterium tuberculosis CIP 105795 (Ringuet et al., 1999)], and (iv) sodA gene [superoxide dismutase, 442 nt corresponding to positions 82–523 of Nocardia farcinica IFM 10152 (Zolg & Philippi-Schulz, 1994)]. For phylogenetic analysis, these sequences (excluding the primer sequences used for amplification) were compared with sequences in the GenBank and EMBL databases using the BLAST (Altschul et al., 1997), LALIGN (www.expasy.org) and leBIBI programs (Devulder et al., 2003). Gene sequences were selected from GenBank for the type strains of the 13 or 14 most closely related Mycobacterium species and aligned with CLUSTAL_X 1.83 (Thompson et al., 1997). The alignment was checked and corrected manually before reconstruction of phylogenetic trees. The phylogenetic trees were constructed with the software packages MEGA (Kumar et al., 2004) and PHYLO_WIN (Galtier et al., 1996), using the maximum-likelihood (Felsenstein, 1981), maximum-parsimony (Kluge & Farris, 1969) and neighbour-joining (Saitou & Nei, 1987) treeing algorithms. Tree robustness was determined by using 1000 bootstrapped datasets.
The morphological and chemotaxonomic characteristics of strain ABO-M06T corresponded to those of the genus Mycobacterium and are detailed in the species description. Phenotype characteristics that differ from closely related species are shown in Table 1. Fatty acid analysis showed straight-chain saturated and unsaturated fatty acids, as expected for a member of the genus Mycobacterium. Major fatty acids included C16 : 0 (37.8 %), C18 : 1
9c (24.1 %), tuberculostearic acid (10-methyl C18 : 0) (9.7 %), C16 : 1 (7.6 %) and C14 : 0 (7.5 %). Cell wall analysis showed mycolic acids typically found in mycobacteria (long-chain mycolic acids sensu stricto), with three major groups of peaks (Supplementary Fig. S1; available in IJSEM Online). The fatty acid and mycolic acid elution profile did not correspond to a specific Mycobacterium species stored in the MIDI databases and was clearly distinct from M. fortuitum (Butler & Kilburn, 1990; Butler & Guthertz, 2001). Although it showed some similarities with patterns from members of the M. fortuitum group, the mycolic acid elution profile is characteristic and strain ABO-M06T can be differentiated from these species by quantitative differences in the mycolic acid composition.
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The gene sequences of strain ABO-M06T differed markedly from those of Mycobacterium species with validly published names. These differences were 0.68 % in the 16S rRNA gene sequence (corresponding to 9 differences among 1336 nucleotides) with M. houstonense ATCC 49403T and M. senegalense CIP 104534T, 4.64 % in rpoB gene sequence (32 differences among 690 nucleotides) with M. conceptionense CIP 108544T, 1.56 % in hsp65 gene sequence (6 differences among 387 nucleotides) with M. houstonense ATCC 49403T and 3.8 % in sodA gene sequence (15 differences among 395 nucleotides) with M. septicum ATCC 700731T. As previously described in the genus Mycobacterium, the sodA and rpoB genes were found to be more discriminatory than the 16S rRNA and hsp65 genes for differentiation of strain ABO-M06T from closely related species. Moreover, partial rpoB gene sequence analysis supported the affiliation of strain ABO-M06T to a novel species according to Adékambi et al. (2003), who have shown that intraspecies and interspecies variabilities in partial rpoB gene sequences are <1.7 % and >3 %, respectively, for rapidly growing mycobacteria. This was confirmed by partial sodA gene sequence analysis. Although data on intraspecies variability are lacking, interspecies variability on this gene fragment was found to be 
0.75 % for rapidly growing mycobacteria.
The evolutionary trees inferred from the three treeing algorithms gave congruent results (data not shown) and the neighbour-joining trees are shown in Figs 1 and 2 and Supplementary Figs S2 and S3. Phylogenetic analysis of the near-complete 16S rRNA gene sequence indicated that strain ABO-M06T belonged to the M. fortuitum group (Fig. 1). A similar result was observed after phylogenetic analysis based on rpoB (Fig. 2), hsp65 (Supplementary Fig. S2) and sodA (Supplementary Fig. S3) gene sequences. Independent lineage was observed in all the trees reconstructed for the strain ABO-M06T. The species most closely related to strain ABO-M06T differed with the phylogenetic marker, but the bootstrap values were too low (below 50 %) to induce much confidence on any phylogenetic relatedness within the M. fortuitum group. The percentages of gene sequence difference, together with the marked evolutionary distances and lack of congruence observed in four-gene sequence analysis, showed that isolate ABO-M06T was clearly distinct from the nearest species. Together with the unique phenotype, these results warranted its classification as a novel Mycobacterium species belonging to the M. fortuitum group, for which we propose the name Mycobacterium setense sp. nov.
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Cells are acid-fast, Gram-positive and pleomorphic rods. Colonies are smooth, convex, round, entire-edged, non-pigmented (beige) and small (approx. 1 mm in diameter). They do not produce aerial hyphae. The cells grow on 5 % sheep blood agar and Löwenstein–Jensen agar within 2 to 4 days at 25 °C, 30 °C (optimum) and 37 °C when subculturing. They also grow in the presence of 5 % NaCl. No growth occurs at 42 °C. Positive for 3 day arylsulfatase production, iron uptake and thermostable catalase activity. It utilizes D-mannitol and D-glucose, but not D-inositol, L-rhamnose or L-arabinose, or citrate as the sole carbon source. It is positive for pyrazinamidase, alkaline phosphatase, nitrate reductase, urease and gelatinase activities. M. setense ABO-M06T sp. nov. belongs to the M. fortuitum group and can be differentiated phenotypically from other species of this group as follows. It differs from M. conceptionense CIP 108544T by D-glucose and D-mannitol utilization, lack of inositol degradation and gelatinase activity. It differs from M. porcinum CIP 105392T by D-glucose utilization and lack of inositol degradation, from M. houstonense ATCC 49403T by the lack of inositol degradation and failure to grow at 42 °C, from M. fortuitum CIP 105534T by failure to grow at 42 °C and by D-mannitol utilization, and from M. septicum ATCC 700731T by lack of inositol degradation. It exhibits unique cellular fatty acid and mycolic acid patterns. Strain ABO-M06T shows 99.32 % 16S rRNA gene similarity with M. houstonense ATCC 49403T and 95.36 % rpoB gene similarity with M. conceptionense CIP 108544T, the phylogenetically closest type strains.
The type strain, ABO-M06T (=CIP 109395T=DSM 45070T), was recovered from an excised skin and soft tissue specimen in a context of osteitis.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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