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1 Food Microbial Sciences Unit, School of Food Biosciences, University of Reading, Whiteknights, PO Box 226, Reading RG6 6AP, UK
2 Waltham Centre for Pet Nutrition, Waltham-on-the-Wolds, Melton Mowbray, UK
3 Culture Collection, Department of Clinical Microbiology, University of Göteborg, Sweden
Correspondence
Paul A. Lawson
p.a.lawson{at}reading.ac.uk
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; Davis et al., 1977; Benno & Mitsuoka, 1989; Benno & Mitsuoka, 1992; Benno et al., 1992a, b). Hence detailed information on the range of species that reside in the canine gut is very poor. In recent years 16S rRNA gene sequencing has greatly improved the phylogenetic identification of bacteria. In particular, this tool, when used in concert with phenotypic approaches, has provided a powerful aid for the recognition of novel diversity. During the course of a study of taxonomically problematic organisms from the canine gut (Greetham et al., 2002), we have used this strategy to characterize a hitherto unknown species within the Gram-positive, high G+C content genus Slackia. Currently only two species of this genus are known, Slackia exigua, which is associated with human oral lesions, and Slackia heliotrinireducens, from the rumen of sheep (Wade et al., 1999). Based on the presented findings, we propose a novel species of this genus from canine faeces, Slackia faecicanis.
Strain 5WC12T was isolated from the faeces of a healthy male Labrador dog. The faecal sample was collected immediately following defecation and was used to prepare a 10 % (w/v) slurry using pre-reduced 0·1 M PBS (0·138 M NaCl, 0·0027 M KCl; pH 7). The slurry was transferred to an anaerobic cabinet (10 % H2, 10 % CO2, 80 % N2) and homogenized for 10 min. Serial tenfold dilutions were prepared using half-strength peptone water and cysteine/HCl (0·5 g l–1). Strain 5WC12T was isolated from a 10–5 dilution on Bacteroides Agar (Holdeman et al., 1997), which had been incubated at 37 °C for 48 h.
The strain was characterized biochemically by using a combination of conventional tests, as described previously in the VPI anaerobic manual (Holdeman et al., 1997), and the API ZYM system, according to the manufacturer's instructions (API bioMérieux). All biochemical tests were performed in duplicate. End products of glucose metabolism were determined by using GLC. Cells were cultured on Chocolate agar with Columbia agar base for 48 h at 37 °C, and centrifuged. Saponification, methanolysis, extraction and identification of the fatty acid methyl esters were performed by using the Microbial Identification System (Microbial ID) as described by Jousimies-Somer et al. (2002) and Moore et al. (1994). The mol% G+C content of DNA was determined by HPLC, according to Mesbah et al. (1989), except that the methanol content of the chromatographic buffer was decreased to 8 % and the temperature was increased to 37 °C. The 16S rRNA genes of the isolate were amplified by PCR by using universal primers pA (positions 8–28, Escherichia coli numbering) and pH (positions 1542–1522). The amplified product was purified by using a QIAquick PCR purification kit (QIAgen) and directly sequenced by using primers directed towards conserved positions of the rRNA gene, 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 (Lipman & Pearson, 1985). These sequences and those of other known related strains were retrieved from GenBank and aligned with the newly determined sequence by using the program SEQtools (Rasmussen, 2002). The resulting multiple sequence alignment was corrected manually by using the program GeneDoc (Nicholas et al., 1997) and a phylogenetic tree was constructed according to the neighbour-joining method with the programs SEQtools and TreeView (Page, 1996). The stability of the groupings was estimated by bootstrap analysis (1000 replications) by using the same program.
The isolate originating from the faecal material was strictly anaerobic and consisted of short rod-shaped cells, 0·5x1–2 µm, which stained Gram-positive. Spores were not observed on either Gram- or spore-stains. Colonies on Anaerobic blood agar plates were translucent to grey, circular, with an uneven surface and irregular edges. The isolate was nitrate-positive, but urease- and aesculin-negative. It was catalase- and oxidase-negative. Using the conventional methods, the strain failed to produce acid from glucose, lactose, maltose, mannose, mannitol, melibiose, ribose, starch and sucrose. Using the commercially available API Rapid ID32A and API ZYM test systems, the unidentified isolate was very unreactive, with positive reactions being obtained for only arginine dihydrolase, esterase C-4 and esterase lipase C8. The long-chain cellular fatty acids consisted of C14 : 0 (4·8 %), C16 : 0 (16·6 %), C16 : 1 cis9 (2·1 %), iso-C17 : 1 (6·2 %), C18 : 0 (23·6 %), C18 : 1 cis9 (21·8 %) C18 : 1 cis11 (6·5 %), C18 : 2 cis9 (13·8 %) and iso-C19 : 1 cis9 (3·2 %). Determination of the G+C content of DNA of the unidentified strain revealed a value of 61·2 mol%. The Gram-positive staining reaction together with the relatively high G+C content was strongly indicative that the organism was a member of the class Actinobacteria. To determine the phylogenetic position of the unidentified isolate, its almost complete 16S rRNA gene was amplified by PCR and sequenced. Sequence database searches confirmed that the unknown bacterium was indeed a member of the class Actinobacteria (data not shown). A tree constructed using the neighbour-joining method showing the phylogenetic relationships of the unknown bacterium is depicted in Fig. 1. It shows that the unidentified canine bacterium is a member of the family Coriobacteriaceae. The organism displayed highest sequence similarity with species of the genus Slackia (91·6–92 %), with other members of the family Coriobacteriaceae exhibiting substantially lower relatedness [Atopobium species (83·9–85 %), Collinsella species (87·4–87·8 %), Coriobacterium glomerans (88·5 %), Cryptobacterium curtum (87·4 %), Denitrobacterium detoxificans (90 %), Eggerthella lenta (90·9 %) and Olsenella species (87·2–88·4 %)]. Treeing analysis confirmed the affinity of the unknown bacterium with the genus Slackia (Fig. 1). From the phylogenetic analysis it is evident that the unknown organism represents a hitherto unknown species within the family Coriobacteriaceae. The organism formed a distinct cluster with S. exigua and S. heliotrinireducens within the Coriobacteriaceae clade, and bootstrap resampling showed that the association with the genus Slackia was statistically significant (95 %). Further evidence for the placement of the organism within the family Coriobacteriaceae and, in particular, its affinity with the genus Slackia, comes from an examination of rRNA signatures. The unidentified organism possesses 13 of the 20 16S rRNA base signatures identified by Dewhirst et al. (2001) for defining the family Coriobacteriaceae. Of the seven aberrant positions, five are common to S. exigua and S. heliotrinireducens, whereas two are unique to the canine gut bacterium (Table 1). Although it is not possible to delineate species solely on the basis of 16S rRNA sequence similarities, it is clear that the observed 8 % divergence between the unidentified organism and the two currently recognized Slackia species is consistent with separate species status. It is now generally accepted that organisms displaying 3 % or greater 16S rRNA divergence are not members of the same species. Further evidence for the separate species status of the unidentified organism comes from phenotypic considerations. The novel canine bacterium can be phenotypically readily distinguished from other Slackia species (Table 2). In particular, the API Rapid ID32A system numerical profile 2000100000 serves to distinguish it from S. exigua and S. heliotrinireducens, which produce profiles 2000033705 and 2000023705, respectively. Therefore, based upon phylogenetic and phenotypic criteria, we consider that the unknown bacterium from canine faeces merits classification as a novel species of the genus Slackia, for which the name Slackia faecicanis is proposed.
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Cells consist of short rods (0·5x1–2 µm) which stain Gram-positive. Colonies after 48 h anaerobic incubation at 37 °C are 1–2 mm in diameter, translucent to grey, with an uneven surface with irregular edges. Strictly anaerobic and catalase-negative. Indole test is negative. Nitrate is not reduced to nitrite. Acid is not produced from glucose, lactose, maltose, mannose, mannitol, melibiose, ribose, starch or sucrose. Using the API Rapid ID32A system, only arginine dihydrolase is positive. Negative results are obtained for acid phosphatase, alkaline phosphatase, alanine arylamidase, 
-arabinosidase, arginine arylamidase, -chymotrypsin, cystine arylamidase, -fucosidase, -glucosidase, 
-glucosidase, -glucuronidase, -galactosidase, -galactosidase, -galactosidase-6-phosphate, glutamic acid decarboxylase, glutamyl glutamic acid arylamidase, histidine arylamidase, leucine arylamidase, leucyl glycine arylamidase, lipase C14, -mannosidase, N-acetyl--glucosaminidase, proline arylamidase, phenyl alanine arylamidase, phosphoamidase, pyroglutamic acid arylamidase, serine arylamidase, trypsin, valine arylamidase, urease or tyrosine arylamidase. With the API ZYM system, weak positive reactions are obtained for esterase C-4, esterase lipase C8, acid phosphatase and naphthol-AS-Bi-phosphohydrolase but alkaline phosphatase, lipase C14, leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, -chymotrypsin, -galactosidase, -galactosidase, -glucuronidase, -glucosidase, -glucosidase, N-acetyl--glucosaminidase, -mannosidase and -fucosidase are not detected. The predominant long-chain cellular fatty acids consist of C16 : 0, C16 : 1 cis9, C18 : 0, C18 : 1 cis9 and iso-C19 : 1 cis9. The G+C content is 61·2 mol%. Isolated from dog faeces. Habitat is not known but probably is a member of the canine gut microbiota.
The type strain is 5WC12T (=CCUG 48399T=CIP 108281T).
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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. Bifidobact. Microflora 11, 69–76.
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Dewhirst, F. E., Paster, B. J., Tzellas, N., Coleman, B., Downes, J., Spratt, D. A. & Wade, W. G. (2001). Characterization of novel human oral isolates and cloned 16S rDNA sequences that fall in the family Coriobacteriaceae: description of Olsenella gen. nov., reclassification of Lactobacillus uli as Olsenella uli comb. nov. and description of Olsenella profuse sp. nov. Int J Syst Evol Microbiol 51, 1797–1804.[Abstract]
Greetham, H. L., Giffard, C., Hutson, R. A., Collins, M. D. & Gibson, G. R. (2002). Bacteriology of the Labrador dog gut: a cultural and genotypic approach. J Appl Microbiol 93, 640–646.[CrossRef][Medline]
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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.
Moore, L. V. H., Bourne, D. M. & Moore, W. E. C. (1994). Comparative distribution and taxonomic value of cellular fatty acids in thirty-three genera of anaerobic gram-negative bacilli. Int J Syst Bacteriol 44, 338–347.
Nicholas, K. B., Nicholas, H. B., Jr & Deerfield, D. W., II (1997). GeneDoc: analysis and visualization of genetic variation. EMBNEW News 4, 14.
Page, R. D. M. (1996). TreeView: an application to display phylogenetic trees on personal computers. Comput Appl Biosci 12, 357–358.
Rasmussen, S. W. (2002). SEQtools, a software package for analysis of nucleotide and protein sequences. http://www.seqtools.dk
Wade, W. G., Downes, J., Dymock, D., Hiom, S. J., Weightman, A. J., Dewhirst, F. E., Paster, B. J., Tzellas, N. & Coleman, B. (1999). The family Coriobacteriaceae: reclassification of Eubacterium exiguum (Poco et al., 1996) and Peptostreptococcus heliotrinreducens (Lanigan 1976) as Slackia exigua gen. nov., comb. nov. and Slackia heliotrinireducens gen. nov., comb. nov., and Eubacterium lentum (Prevot 1938) as Eggerthella lenta gen. nov., comb. nov. Int J Syst Bacteriol 49, 595–600.
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