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1 Laboratory of Veterinary Bacteriology and Mycology, Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium
2 BCCM/LMG Bacteria Collection, Laboratory of Microbiology, Faculty of Sciences, Ghent University, Ledeganckstraat 35, B-9000 Ghent, Belgium
3 School of Food Biosciences, University of Reading, Reading RG6 6AP, UK
Correspondence
E. M. De Graef
evelyne.degraef{at}ugent.be
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Published online ahead of print on 13 December 2002 as DOI 10.1099/ijs.0.02549-0.
The GenBank/EMBL/DDBJ accession numbers for the 16S DNA sequences reported in this paper are X76177 and AY156090.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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, 2002). Many strains do not possess the Lancefield group D antigen, several species do not grow on commonly used enterococci-selective media or at 10 or 45 °C, and characteristics such as growth in 6·5 % NaCl and on aesculin bile agar or production of pyrrolidonyl arylamidase have become less useful as distinguishing characteristics. Two other easy reactions have been proposed to this end: ribose acidification and acetoin production (Voges–Proskauer; VP) (Devriese & Pot, 1995). However, these tests have also proved not to be universally applicable. In the present study, a group of mostly VP-negative, enterococcal-like strains isolated from dogs was shown to belong to a novel species of the genus Enterococcus. In addition, synonymy of Enterococcus porcinus with Enterococcus villorum was demonstrated. |
METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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, 2001, 2002).
Phenotypic analysis.
Growth tests were carried out as described by 
vec et al. (2001). Acidification of carbohydrates was recorded after 3 days incubation in API 50 CH galleries (bioMérieux) under paraffin cover. Lancefield antigens were detected by using the Streptococcal Grouping kit (Oxoid). Biochemical reactions were determined by using the BBL Crystal Gram-positive ID kit (Becton Dickinson) and the API 20 STREP system (bioMérieux). VP tests were duplicated using Voges–Proskauer diagnostic tablets (Rosco).
SDS-PAGE of whole-cell proteins.
After incubation of cells for 24 h on MRS agar medium (Oxoid), whole-cell protein extracts were prepared and SDS-PAGE was performed as described by Pot et al. (1994). Densitometric analysis, normalization and interpolation of the protein profiles and numerical analysis were performed by using the GelCompar software package, versions 3.1 and 4.0, respectively (Applied Maths).
tRNA intergenic length polymorphism analysis (tDNA-PCR).
Genomic DNA was extracted by suspending a bacterial colony in 20 µl lysis buffer (0·25 % SDS, 0·05 M NaOH). After heating at 95 °C for 5 min, 180 µl sterile distilled water was added and the lysate was centrifuged at 13 000 g for 5 min. The spacers between the tRNA genes were amplified using primers T3B (5'-aggtcgcgggttcgaatcc-3') and T5A (5'-AGTCCGGTGCTCTAACCAACTGAG-3'), which are directed against the conserved edges of the tRNA genes. PCR mixtures and cycle conditions were the same as previously described (Baele et al., 2000). One primer (T3B) was fluorescently labelled to enable detection during capillary electrophoresis of the DNA fragments, which was done with an ABI PRISM 310 Genetic Analyzer (Applied Biosystems). Electrophoretograms obtained with GENESCAN software (Applied Biosystems) were compared to our database by using in-house software (Baele et al., 2000).
16S rRNA gene sequence analysis.
To amplify the 16S rRNA gene, PCR was performed by using the conserved primers 
-NOT (5'-TCAAACTAGGACCGAGTC-3') and 
MB (5'-TACCTTGTTACTTCACCCCA-3') and the Taq PCR Master Mix kit (Qiagen). A QiaQuick PCR Purification kit (Qiagen) was used to purify the PCR product. Subsequent sequencing reactions were performed by using the BigDye Terminator Sequencing kit (Applied Biosystems) and the primers *Gamma, *O, PD and 3, as described by Coenye et al. (1999); the sequences were determined with an ABI PRISM 310 Genetic Analyzer (Applied Biosystems). Phylogenetic analysis was done by using the program GENEBASE (Applied Maths). Pairwise alignment homologies were calculated and a dendrogram was constructed using the neighbour-joining method.
DNA base composition.
Cells were cultivated in MRS broth (Oxoid) for 24 h at 37 °C. DNA was extracted from 0·5–0·75 g cells (wet weight), using the protocol described by Marmur (1961) with the following modifications: (i) cells suspended in Tris/HCl buffer were treated with lysozyme overnight (8 mg ml-1) before addition of SDS; (ii) lysed cells were treated with proteinase K (360 mg l-1; Merck) for 2 h at 37 °C. For determination of DNA base composition, DNA was enzymically degraded into nucleosides as described by Mesbah et al. (1989). The obtained nucleoside mixture was then separated by HPLC, using a Waters SymmetryShield C8 column maintained at 37 °C. The solvent was 0·02 M NH4H2PO4 (pH 4·0) with 1·5 % acetonitrile. Non-methylated lambda phage DNA (Sigma) was used as the calibration reference.
DNA–DNA hybridization.
High-molecular-weight native DNA was prepared as described for the determination of DNA base composition. DNA–DNA hybridizations were performed by using a modification of the microplate method described by Ezaki et al. (1989) and Goris et al. (1998), using an HTS 7000 Bio Assay Reader (PerkinElmer) for the fluorescence measurements. Biotinylated DNA was hybridized with single-stranded unlabelled DNA that was non-covalently bound to the microplate wells. Hybridizations were performed under stringent conditions at 34 °C in hybridization solution (2x SSC, 5x Denhardt's solution, 2·5 % dextran sulphate, 50 % formamide, 100 µg denaturated salmon sperm DNA ml-1, 1250 ng biotinylated probe DNA ml-1).
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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.
16S rRNA gene sequences.
The 16S rDNA sequences of strains LMG 12316T and LMG 21553, isolated respectively from chronic otitis externa and an anal swab from dogs, demonstrated a similarity of 99·6 %. Highest inter-species 16S rDNA homologies were shown with the type strains of the Enterococcus faecium species group (98·4–99·0 %). This close phylogenetic similarity is also reflected by their phenotypic resemblance to the unidentified dog strains (Table 1). Somewhat lower similarities were shown between the canine strains and members of the Enterococcus avium species group (97·9–98·3 %).
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). The description of E. villorum (Vancanneyt et al., 2001) was based on the study of this strain (among others). As the name E. villorum was published earlier than the name E. porcinus, the latter name is to be considered to be a junior synonym of E. villorum.
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, 1986; Schleifer & Kilpper-Bälz, 1984; Farrow & Collins, 1985; Vancanneyt et al., 2001).
DNA–DNA hybridization experiments
The level of DNA–DNA binding between the canine strains LMG 12316T and LMG 12315 was 86 %. Despite the fact that 16S rDNA sequence homologies between the unidentified dog strains and reference enterococcal species were relatively high, very low DNA–DNA reassociation levels were observed. Homology levels of 7–13 % between the dog strains and members of the E. faecium species group (E. faecium LMG 11423T, Enterococcus durans LMG 10746T, Enterococcus hirae LMG 6399T, Enterococcus mundtii LMG 10748T and E. villorum LMG 12287T) clearly demonstrate that the isolates from dogs represent a separate genospecies and are distinct from other recognized members of the E. faecium group.
SDS-PAGE of whole-cell proteins
Duplicate protein extracts were prepared, to check the reproducibility of the growth conditions and the extract preparation. The level of correlation between duplicate protein patterns was r
0·95. The whole-cell protein profiles of the dog strains were initially compared with patterns of >800 enterococcal strains, representing all currently described Enterococcus species; the unidentified isolates formed a separate cluster (Pot & Janssens, 1993; data not shown). A dendrogram obtained after average linkage cluster analysis, showing the separateness of the dog isolates from members of the E. faecium species group, is shown in Fig. 2. The dog strains formed a single and separate cluster (r0·91) and displayed almost-identical electrophoretic patterns. The pattern of the E. porcinus strain was highly similar to that of the E. villorum strain.
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), have a number of common characteristics that are useful for phenotypic identification. These differential characteristics can also be used to differentiate the dog strains from other groups of enterococcal species (Table 1
). However, the dog strains are unusual in that they do not grow on commonly used enterococcal selective media that contain 0·04 % sodium azide (NaN3) and in that most strains give a negative VP reaction. These characteristics can be used along with other tests, notably certain carbohydrate acidifications, to differentiate the unidentified dog strains from related enterococcal species (Table 2). E. porcinus, described by Teixeira et al. (2001), is not included in this table because its biochemical activity is identical to that of E. villorum.
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; vec et al., 2001). DNA–DNA pairing studies showed unequivocally that the canine isolates represent a different genomic species from other members of the E. faecium group. Further support for the distinctiveness of the novel species comes from phenotypic evidence; in particular, protein profiling confirms the separateness of the dog isolates from all currently defined enterococcal species. The novel species can also be readily distinguished from other enterococci by using a combination of physiological and biochemical tests. Therefore, based on both phylogenetic and phenotypic evidence, we propose that the isolates from dogs are assigned to Enterococcus canis sp. nov. To date, E. canis has only been isolated from dogs. Although this species may occur in chronic and complicated otitis externa cases in this host, it probably does not play a pathogenic role. This type of lesion is often contaminated by faecal flora.
Description of Enterococcus canis sp. nov.
Enterococcus canis (ca'nis. L. gen. n. canis of a dog).
Cells are non-motile, facultatively anaerobic, Gram-positive, slightly elongated cocci that occur in pairs, short chains or small groups. Colonies on blood agar are circular, smooth and surrounded by narrow, sharply demarcated zones of -haemolysis. Growth is optimal at 37 °C, slower at 42, 30 and 25 °C and unaffected by the absence or presence of 5 % CO2. Strains grow in 6·5 % NaCl broth. On Edward's Streptococcus selective medium, brownish aesculin-degrading colonies are formed. No growth is seen after 1 day on Slanetz–Bartley medium, but after 2 days, pinpoint-sized colonies are formed without any evidence of tetrazoliumchloride reduction. Abundant growth and blackening are observed in aesculin bile agar. Starch is not digested. Positive results are obtained in API tests for leucine arylamidase and pyrrolidonyl arylamidase (hydrolysis of L-pyrroglutamic acid-AMC in Crystal, with some weak reactions) and in the following Crystal reactions: hydrolysis of L-valine-AMC, L-phenylalanine-AMC, 4MU--D-glucoside, L-tryptophan-AMC, p-nitrophenyl--D-glucoside, p-nitrophenyl phosphate and p-nitrophenyl--D-cellobioside. Acid is produced in the API 20 STREP and/or API 50 CH kits in tests with glycerol (often delayed), L-arabinose, ribose, galactose, D-glucose, D-fructose, D-mannose, mannitol, N-acetylglucosamine, amygdalin, arbutin, salicin, cellobiose, maltose, lactose, -gentiobiose, gluconate and 2-ketogluconate (often weak). Most strains react positively in tests with -methyl-D-glucoside (10/13 strains), D-arabinose (11/13 strains) and D-xylose (8/13 strains). Few strains are positive for acid production from sucrose (2/13 positive), trehalose (1/13 positive) and starch (4/13 strains with weak reactions). Strain- or method-dependent reactions are observed with arginine (negative in API 20 STREP and Rosco; 6/13 strains positive in Crystal galleries) and VP (negative in API 20 STREP, 2/13 strains positive with Rosco tablets). Strains are negative in API tests for hippurate, -glucuronidase, -galactosidase and alkaline phosphatase, and in Crystal tests for hydrolysis of L-valine-AMC, 4MU-phosphate, 4MU--D-glucuronide, L-isoleucine-AMC and urease. Acid is produced from erythritol, L-xylose, adonitol, L-sorbose, rhamnose, dulcitol, sorbitol, -methyl-D-mannoside, melibiose, inulin, melezitose, D-raffinose, glycogen, xylitol, D-turanose, D-lyxose, D- and L-fucose, D-tagatose, D- and L-arabitol and 5-ketogluconate. The DNA G+C content is 41·7–43·0 mol%.
The type strain, LMG 12316T (=CCUG 46666T), was isolated from anal swabs and chronic otitis externa in dogs.
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REFERENCES |
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