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1 Food Microbial Sciences Unit, School of Food Biosciences, Whiteknights, PO Box 226, University of Reading, Reading RG6 6AP, UK
2 Waltham Centre for Pet Nutrition, Waltham-on-Wolds, Melton Mowbray, UK
3 DSMZ–Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany
4 Culture Collection, Department of Clinical Microbiology, University of Göteborg, Sweden
Correspondence
Matthew D. Collins
m.d.collins{at}reading.ac.uk
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; Davis et al., 1977). The major categories of organisms reported to be resident in the intestines and faeces of canines include bacteroides, bifidobacteria, clostridia, eubacteria, lactobacilli and anaerobic Gram-positive cocci (Balish et al., 1977; Davis et al., 1977; Benno & Mitsuoka, 1992; Benno et al., 1992a), although, due to limitations of phenotypic methods of characterization, precise information on the nature of species present is often lacking. A large number of bacterial isolates recovered from canines have, in the past, been assigned to species on the basis of poor taxonomic evidence (Balish et al., 1977; Davis et al., 1977) whilst, in other cases, organisms remain unidentified and have simply been assigned to broad groups (e.g. clostridia, anaerobic Gram-positive cocci) (Mitsuoka, 1985; Benno & Mitsuoka, 1989, 1992; Benno et al., 1992a, b). Consequently, it is highly likely that the canine gut harbours many organisms that do not correspond to currently defined species and represent hitherto unknown taxonomic diversity. During the course of a molecular-based taxonomic study of the micro-organisms of canine faeces, we have isolated an unusual Gram-positive, spore-forming, rod-shaped organism that does not appear to correspond to any recognized Clostridium species based on phenotypic criteria. In this article, we report the phenotypic characteristics of this Clostridium-like organism and the results of a phylogenetic study. Based on these findings, we propose that the unknown, spore-forming, rod-shaped bacterium be designated as the type strain of a novel species of the genus Clostridium, Clostridium colicanis sp. nov.
A rod-shaped organism designated 3WC2T was isolated from the faeces of a male Labrador dog. The faecal sample was collected immediately after defecation and was used to prepare a 10 % (w/v) slurry using pre-reduced PBS (0·1 M phosphate, pH 7). The slurry was transferred into an anaerobic cabinet (H2/CO2/N2, 10 : 10 : 80 by vol.) and homogenized for 10 min. Serial 10-fold dilutions were prepared using half-strength peptone water and cysteine hydrochloride (0·5 g l-1). The strain was isolated from Wilkins–Chalgren agar for total anaerobes (Wilkins & Chalgren, 1976) incubated at 37 °C for 4 days, from a 10-4 dilution.
Haemolysis was tested on Columbia sheep-blood agar (BBL). Motility was examined from peptone/yeast extract/glucose (PYG) broth and agar, trypticase-nitrate broth (BBL) and Columbia sheep-blood agar (BBL). Temperature ranges for growth were determined in PYG medium (with 1 g Tween 80 l-1 and 2 g sodium bicarbonate l-1) (Holdeman et al., 1977) using 100 % N2 as the atmosphere in Hungate tubes. Growth was recorded by measuring the OD600 directly in Hungate tubes using an LKB Ultrospec II spectrophotometer. Traditional biochemical tests were performed as described by Holdeman et al. (1977). Hydrolysis of starch and gelatin was, in addition, tested using double-layer starch-agar plates and 0·4 % (w/v) gelatin-agar plates. Biochemical tests were also performed using the commercially available API Rapid ID32AN system (bioMérieux) according to the manufacturer's instructions. Fermentation products formed after growth in peptone/yeast extract (PY) medium with 1 % (w/v) glucose were determined as described by Holdeman et al. (1977). Alcohols and volatile acids were extracted with tert-butyl ethyl ether instead of diethyl ether. A Shimadzu GC-14A gas chromatograph, a Nukol capillary column (30 m) at 170 °C, a flame-ionization detector and hydrogen carrier gas were used for the analysis. For the determination of G+C content, DNA was isolated after disruption of cells using a French pressure cell and then purified in hydroxyapatite. The G+C content (mol%) was determined by HPLC after digestion of DNA with P1 nuclease and alkaline phosphatase as described by Mesbah et al. (1989). The 16S rRNA gene(s) of the isolate was amplified by PCR and sequenced directly using a dRhodamine terminator cycle sequencing kit (PE Applied Biosystems) and an automatic DNA sequencer (model 377; PE Applied Biosystems). The closest known relatives of the novel isolate were determined by performing database searches using the program FASTA. These sequences and those of other known related strains were retrieved from GenBank or the Ribosomal Database Project database and aligned with the newly determined sequence using the program DNATools (Rasmussen, 1995). The resulting multiple sequence alignment was corrected manually using the program GeneDoc (Nicholas et al., 1997). A phylogenetic tree was constructed according to the neighbour-joining method with the program NEIGHBOR and the stability of the groupings was estimated by bootstrap analysis (500 replications) using the programs DNABOOT, DNADIST, NEIGHBOR and CONSENSE (Felsenstein, 1989).
The faecal organism consisted of non-motile, spore-forming, rod-shaped cells. Typical cells were 0·9–1·0x3–10 µm. Spores were oval to oblong in PY-starch medium; the position of the spores varied from subterminal to almost terminal or occasionally central. The organism stained Gram-negative and was anaerobic, catalase-negative and produced acid from glucose as outlined in the species description. Results of classical biochemical tests and the API Rapid ID32AN test system are given in the species description below. Determination of the G+C content of DNA of strain 3WC2T revealed a value of 31·7 mol%, indicating that it was a member of the low-G+C, Gram-positive Clostridium subphylum.
To ascertain the phylogenetic relationships of the unknown organism, comparative 16S rRNA gene sequencing was conducted. The partial sequence (>1400 nt) of the 16S rRNA gene of the isolate was determined. Sequence database searches revealed that the unknown bacterium from canine faeces was most closely related to clostridial species, in particular members of Clostridium rRNA group I (data not shown). Treeing analysis confirmed these findings, with the unidentified bacterium clustering with a small group of organisms within Clostridium rRNA group I that included Clostridium absonum (95·0 % sequence similarity), Clostridium baratii (96·0 %), Eubacterium budayi (95·5 %), Eubacterium moniliforme (96·4 %), Eubacterium multiforme (95·1 %) and Eubacterium nitritogenes (95·1 %). Fig. 1 depicts a neighbour-joining tree showing the phylogenetic position of the unidentified bacterium within Clostridium rRNA group I.
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). C. butyricum and its close neighbours form a phylogenetically distinct group (designated Clostridium rRNA group I; Collins et al., 1994), and the recovery, from a dog, of the novel bacterium within this rRNA cluster clearly demonstrates that it is, taxonomically, an authentic Clostridium species. Phylogenetically, the unidentified bacterium forms a distinct subline within rRNA group I, displaying a loose association with a small subcluster of species that includes C. absonum, C. baratii, E. budayi, E. moniliforme, E. multiforme and E. nitritogenes. Bootstrap resampling, however, showed that this association was not particularly significant. Sequence divergence values of approximately 3·5–5 % between the faecal isolate and the aforementioned species showed unequivocally that the canine bacterium merits classification as a distinct species. Furthermore, the canine bacterium is phenotypically different from other clostridial species. Based on both phenotypic and phylogenetic considerations, we therefore propose that the unknown, spore-forming, rod-shaped organism from dog faeces be classified in the genus Clostridium, as Clostridium colicanis sp. nov. Tests that are useful in distinguishing C. colicanis from its nearest relatives are shown in Table 1.
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Cells are rod-shaped, approximately 0·9–1·0x3–10 µm and stain Gram-negative. Spores are observed which are oval to oblong in PY-starch medium; position varies from subterminal to almost terminal or even central. Non-motile. Colonies are 3–5 mm in diameter, round, have an undulate margin and are slightly convex, opaque, greyish-white and glossy. Non-haemolytic on Columbia sheep-blood agar. Growth occurs at 30 and 45 °C but not at 20 or 50 °C; optimum growth temperature approx. 37–40 °C. Anaerobic and catalase-negative. Produces acid from glucose (acidification of PY medium containing 1 % glucose, pH 6·8; after 6 days the pH was 4·8). Using traditional tests, acid is produced from cellobiose, aesculin (weak), fructose, galactose, glucose, lactose, maltose, mannose, ribose, salicin (weak), starch (weak) and sucrose. Acid is not produced from amygdalin, L-arabinose, glycogen, inositol, mannitol, melezitose, melibiose, raffinose, rhamnose, sorbitol, trehalose or xylose. Aesculin and urea, but not gelatin, are hydrolysed. Lecithinase- and lipase-negative. Indole is not produced. Nitrate is reduced to nitrite. Using the commercially available API Rapid 32AN test system, activity is detected for alkaline phosphatase, arginine arylamidase, arginine dihydrolase, 
-galactosidase, glycine arylamidase, histidine arylamidase, leucine arylamidase, leucyl glycine arylamidase and N-acetyl--glucosaminidase. No activity is detected for alanine arylamidase, 
-arabinosidase, -fucosidase, -glucosidase, -glucosidase, -glucuronidase, -galactosidase, -galactosidase-6-phosphate, glutamic acid decarboxylase, glutamyl glutamic acid arylamidase, phenylalanine arylamidase, proline arylamidase, pyroglutamic acid arylamidase, serine arylamidase or tyrosine arylamidase. Nitrate reduction is not detected using the API Rapid ID32AN test system. The G+C content of the DNA is 31·7 mol%. The type strain is 3WC2T (=DSM 13634T =CCUG 44556T). Isolated from faeces of a male Labrador dog.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Benno, Y. & Mitsuoka, T. (1989). Effect of advances in age on intestinal microflora of beagle dogs. Microecol Ther 19, 85–91.
Benno, Y. & Mitsuoka, T. (1992). Evaluation of the anaerobic method for the analysis of fecal microflora of beagle dogs. J Vet Med Sci 54, 1039–1041.[Medline]
Benno, Y., Nakao, H., Uchida, K. & Mitsuoka, T. (1992a). Individual and seasonal variations in the composition of fecal microflora of beagle dogs. Bifidobacteria Microflora 11, 69–76.
Benno, Y., Nakao, H., Uchida, K. & Mitsuoka, T. (1992b). Impact of the advances in age on the gastrointestinal microflora of beagle dogs. J Vet Med Sci 54, 703–706.[Medline]
Collins, M. D., Lawson, P. A., Willems, A., Cordoba, J. J., Fernandez-Garayzabal, J., Garcia, P., Cai, J., Hippe, H. & Farrow, J. A. E. (1994). The phylogeny of the genus Clostridium: proposal of five new genera and eleven new species combinations. Int J Syst Bacteriol 44, 812–826.[CrossRef][Medline]
Davis, C. P., Cleven, D., Balish, E. & Yale, C. E. (1977). Bacterial association in the gastrointestinal tract of beagle dogs. Appl Environ Microbiol 34, 194–206.
Felsenstein, J. (1989). PHYLIP – phylogeny inference package (version 3.2). Cladistics 5, 164–166.
Holdeman, L. V., Cato, E. P. & Moore, W. E. C. (1977). Anaerobe Laboratory Manual, 4th edn. Blacksburg, VA: Virginia Polytechnic Institute and State University.
Mitsuoka, T. (1985). Comparative intestinal microbial ecology and metabolism in man and animals. In Medical and Dental Aspects of Anaerobes, pp. 87–107. Edited by B. I. Duerden, W. G. Wade, J. S. Brazier, A. Eley, B. Wren & M. J. Hudson. Northwood, UK: Science Reviews.
Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precise measurement of the G+C content of deoxyribonucleic acid by high-performance liquid chromatography. Int J Syst Bacteriol 39, 159–167.
Nicholas, K. B., Nicholas, H. B., Jr & Deerfield, D. W., II (1997). GeneDoc: analysis and visualization of genetic variation. EMBNEW News 4, 14.
Rasmussen, S. W. (1995). DNATools, a software package for DNA sequence analysis. Copenhagen: Carlsberg Laboratory.
Wilkins, T. D. & Chalgren, S. (1976). Medium for use in antibiotic susceptibility testing of anaerobic bacteria. Antimicrob Agents Chemother 10, 926–928.
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