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1 Centre National de Référence des Bactéries Anaérobies et du Botulisme, Institut Pasteur, 25 rue du Dr Roux, F-75724 Paris Cedex 15, France
2 Section of Clinical Microbiology, The University of Texas M.D. Anderson Cancer, 1515 Holcombe Blvd, Houston, TX 77030, USA
Correspondence
Jean-Philippe Carlier
jcarlier{at}pasteur.fr
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ABSTRACT |
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. Int J Syst Bacteriol 44, 812–826]. The closest recognized relatives were the type strains of Clostridium clostridioforme, Clostridium bolteae and Clostridium asparagiforme (16S rRNA gene sequence similarity values of 90.2–91.4 %). These results suggest that these three clinical isolates represent a novel species of a new genus, for which the name Moryella indoligenes gen. nov., sp. nov. is proposed. The type strain of Moryella indoligenes is AIP 220.04T (=CIP 109174T=CCUG 52648T).
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; Suau et al., 1999) and only 24 % of the molecular species recovered from the human intestinal tract corresponded to recognized species (Suau et al., 1999). Thus, the combination of genetic tools and traditional phenotypic methods of identification should be used whenever possible with the aim of providing greater knowledge of these ‘hidden’ bacteria. Here we report on the characterization of an indole-producing anaerobic bacterium that was recovered from different clinical specimens. Phylogenetically, the strains described represent a hitherto unknown subline within the Clostridium coccoides rRNA group. Based on the data presented, we describe a novel species in a new genus for these strains.
The new isolates were recovered from clinical sources: strain AIP 241.03 from a buttock abscess, strain AIP 220.04T from an intra-abdominal abscess (both from France) and strain MDA2477 from a polymicrobial thigh abscess (from Houston, TX, USA). Thus, these strains might all have originated from the intestinal tract. The strains were maintained in trypticase/glucose/yeast extract medium (TGY) consisting of 3 % (w/v) biotrypcase (bioMérieux), 0.5 % glucose (Prolabo), 2 % yeast extract (Difco), 0.05 % L-cysteine hydrochloride (Prolabo) and 5 µg haemin ml–1 (Calbiochem), under anaerobic conditions at 37 °C for 24 h in an anaerobic jar containing 95 % H2 and 5 % CO2 (v/v). Colony morphology determinations and presumptive identification tests (Engelkirk et al., 1992) were performed on Wilkins–Chalgren agar plates. Biochemical reactions were examined according to the procedures described by Holdeman et al. (1977). Metabolic end products were assayed by quantitative GC as described by Carlier (1985). For electron microscopy, cells were prepared as described by Carlier et al. (2004), and electron microphotographs were taken with a JEOL 1010 transmission electron microscope operating at 80 kV.
Colonies appeared on Wilkins–Chalgren blood agar after 24–48 h incubation. They were circular, convex, about 0.5–1 mm in diameter, non-pigmented and non-haemolytic. Cells were elongated, sometimes warped rods with pointed ends, about 0.8–1.7 µm long and 0.5–0.6 µm wide, usually occurring singly, in pairs or occasionally in short chains (Fig. 1a). They were non-motile, Gram-variable after staining but structurally Gram-positive (Fig. 1b). Spore formation was not observed.
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provides the primary characteristics of these isolates that can be used to differentiate them from several closely related species.
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. Briefly, the three strains were grown anaerobically in 10 ml TGY for 48 h and chromatography of the methyl esters was on a fused-silica capillary column (25 mx0.25 mm ID) coated with 5 % methyl phenyl silicone. Fatty acid methyl ester analysis showed that the strains contained an unknown compound that eluted between C12 : 0 and C13 : 0 (14 %), C14 : 0 (47 %) and C16 : 0 (9 %) as the major components. Minor fatty acids included C12 : 0 (3.4 %), iso-C14 : 0 (2.7 %), iso-C15 : 0 (2.4 %), anteiso-C15 : 0 (7 %), a second unknown compound that eluted between C14 : 0 and iso-C15 : 0 (6.2 %), iso-C16 : 0 (1.4 %), C16 : 1
9c (2.6 %) and C18 : 0 (1.4 %). No hydroxy fatty acids were detected.
The 16S rRNA gene sequences were determined for each strain as described by Carlier et al. (2004). Alignment was via the CLUSTAL W program (Thompson et al., 1994). Regions showing alignment uncertainties and gaps were excluded and 1375 unambiguous nucleotide positions were used. A distance matrix was calculated by using DNADIST with the Jukes–Cantor parameter (Jukes & Cantor, 1969). A phylogenetic tree (Fig. 2) was constructed according to the neighbour-joining method with 100 bootstrap resamplings (Felsenstein, 1993). Sequence comparison revealed that the new isolates shared more than 99.9 % 16S rRNA gene sequence similarity with each other and had no known close relatives among recognized bacteria. Indeed, the nearest phylogenetic neighbours were the type strains of Clostridium clostridioforme, Clostridium bolteae and Clostridium asparagiforme, with sequence similarity values of 90.2–91.4 %. The new isolates formed a distinct lineage within the C. coccoides rRNA group of organisms, and the monophyly of this lineage was strongly supported by the high bootstrap value of 98 %. Thus, these isolates were considered to represent a novel taxon. Within the lineage, an oral clone MCE9_173 branched off; this clone, described by Munson et al. (2002) as ‘Lachnospiraceae’, shared 93.4 % sequence similarity with the novel organism. To determine the genus and species status of the novel organism, its relationship with members of the C. coccoides rRNA cluster was examined. This cluster encompasses a phenotypically heterogeneous collection of organisms, including spore-forming and non-spore-forming genera and species. Several butyrate-producing members are also widely distributed within this cluster (Barcenilla et al., 2000). Although the novel bacterium was phylogenetically closer to the C. clostridioforme group, it differed from these clostridia because it was not a spore-former and produced butyrate. On the other hand, it was phylogenetically too distant from the butyrate-producing members to be assigned to those genera or species. Thus, given the 
10 % 16S rRNA gene sequence divergence from their closest relatives and several distinct phenotypic features, the three strains are considered to represent a novel species of a new genus, for which the name Moryella indoligenes gen. nov., sp. nov. is proposed.
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Cells are Gram-variable after staining but structurally Gram-positive, elongated, non-motile rods with pointed ends. Cells may lose their colour easily. No spores are formed. Strictly anaerobic, without growth under microaerophilic or aerobic conditions. Catalase and urease reactions are negative. Nitrate is not reduced. Indole-positive and weakly saccharolytic. The major metabolic end products are acetic, butyric and lactic acids. Phylogenetically, the genus belongs to the C. coccoides rRNA cluster (rRNA cluster XIVa; Collins et al., 1994). The type species is Moryella indoligenes.
Description of Moryella indoligenes sp. nov.
Moryella indoligenes (in.do.li'ge.nes. N.L. n. indolum indole; N.L. suff. -genes producing from Gr. v. gennaô to produce; N.L. part. adj. indoligenes indole-producing).
Displays the following properties in addition to those given in the genus description. Cells are sometimes warped, about 0.8–1.7 µm long and 0.5–0.6 µm wide, occurring singly, in pairs or occasionally in short chains. Colonies are circular, convex and about 0.5–1 mm in diameter on Wilkins–Chalgren blood agar after 24–48 h incubation, non-pigmented and non-haemolytic. Indole production is the main biochemical characteristic of the species. Glucose, galactose, maltose and ribose fermentation are variable. Acid is not produced from any of the following carbohydrates: raffinose, sucrose, aesculin, arabinose, cellobiose, fructose, glycerol, inositol, lactose, mannitol, mannose, melezitose, melibiose, rhamnose, salicin, sorbitol, starch, trehalose or xylose. Aesculin is not hydrolysed. Gelatin is not liquefied and milk is not modified. The type strain is susceptible to ampicillin, amoxicillin, penicillin G, imipenem, cefalotin, cefotaxim, cefoxitin, latamoxef and metronidazole, moderately resistant to tetracycline and resistant to trimethoprim–sulfamethoxazole, erythromycin and rifampicin. Strains AIP 241.03 and MDA2477 are resistant only to trimethoprim–sulfamethoxazole and erythromycin. The DNA G+C content is 50.2 mol%. Habitat is not known, but likely to be the human gut.
The type strain, AIP 220.04T (=CIP 109174T=CCUG 52648T), was isolated from a clinical specimen from an intra-abdominal abscess. Strains MDA2477 (=ATCC BAA-695) and AIP 241.03 are additional strains of this species.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Bryant, M. P. (1986a). Genus XIII. Lachnospira Bryant and Small 1956, 24. In Bergey's Manual of Systematic Bacteriology, vol. 2, pp. 1375–1376. Edited by P. H. A. Sneath, N. S. Mair, M. E. Sharpe & J. G. Holt. Baltimore: Williams & Wilkins.
Bryant, M. P. (1986b). Genus IV. Butyrivibrio Bryant and Small 1956, 18, emend. Moore, Johnson and Holdeman 1976, 241AL. In Bergey's Manual of Systematic Bacteriology, vol. 2, pp. 1376–1379. Edited by P. H. A. Sneath, N. S. Mair, M. E. Sharpe & J. G. Holt. Baltimore: Williams & Wilkins.
Carlier, J.-P. (1985). Gas chromatography of fermentation products: its application in diagnosis of anaerobic bacteria. Bull Inst Pasteur 83, 57–69.
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Collins, M. D., Lawson, P. A., Willems, A., Cordoba, J. J., Fernandez-Garayzabal, J., Garcia, P., Cai, J., Hippe, H. & Farrow, J. A. (1994). The phylogeny of the genus Clostridium: proposal of five new genera and eleven new species combinations. Int J Syst Bacteriol 44, 812–826.
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