| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
vec4
1 Laboratory of Microbiology, Ghent University, K. L. Ledeganckstraat 35, Ghent 9000, Belgium
2 BCCM/LMGTM Bacteria Collection, Ghent University, K. L. Ledeganckstraat 35, Ghent 9000, Belgium
3 Department of Pathology, Bacteriology and Poultry Diseases, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium
4 Czech Collection of Microorganisms, Faculty of Science, Masaryk University, Tvrdého 14, 602 00 Brno, Czech Republic
Correspondence
Sabri M. Naser
Sabri.Naser{at}Ugent.be
 |
ABSTRACT |
|---|
 TOP ABSTRACT MAIN TEXTREFERENCES |
|---|
Published online ahead of print on 27 May 2005 as DOI 10.1099/ijs.0.63752-0.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of LMG 13590T is AJ888906.
A distance matrix tree showing the phylogenetic relationships of E. canintestini sp. nov. and other enterococcal reference species based on 16S rRNA gene sequence comparisons is available as supplementary material in IJSEM Online.
|
MAIN TEXT |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
; Klein, 2003). However, enterococci are ubiquitous and can be found free-living in soil, on plants or in dairy products (Baele et al., 2000; Devriese et al., 1995; Franz et al., 1999). Enterococcus dispar was originally described by Collins et al. (1991) on the basis of two strains from human clinical sources, one from synovial fluid (LMG 13521T) and one from a stool culture. In 1993, an additional strain, LMG 13590, isolated from the faeces of a dog, was assigned to the species based on similar characteristics. A recent biodiversity study of the intestinal flora of dogs revealed that one enterococcal taxon was commonly occurring in dog faeces. These isolates were biochemically most similar to E. dispar but differed in a number of tests which were considered typical for the species (De Graef et al., 2005). In the present study, the taxonomic relationships between E. dispar strains and the novel dog faecal isolates were investigated. Two culture collection strains, E. dispar LMG 13521T and LMG 13590, and the following 12 isolates from faecal samples of healthy dogs from different owners distributed over Flanders were included in the study: LMG 22752 (eve 16a), LMG 22753 (eve 21a), LMG 22754 (eve 117b), LMG 22755 (eve 115a), LMG 22976 (eve 2b), LMG 22977 (eve 3b), LMG 22978 (eve 10a), LMG 22979 (eve 15b), LMG 22980 (eve 19c), LMG 22981 (eve 23b), LMG 22982 (eve 109c) and LMG 22983 (eve 125a). Cultivation and maintenance conditions for the isolates and reference strains were MRS agar (Oxoid) and incubation at 30 °C for 24 h, unless indicated otherwise.
Initially the two culture collection strains, E. dispar LMG 13521T and LMG 13590, were included in a multilocus sequence analysis (MLSA) study applied for the reliable identification of all currently described enterococcal species. MLSA compares the primary DNA sequences from multiple conserved protein-coding loci to assess the diversity and relationships of different isolates across related taxa, thereby using an appropriate phylogenetic or cladistic approach. The criteria used for selecting the multiple protein-coding loci (housekeeping genes) are: (i) widely distributed among lactic acid bacterial (LAB) whole genome sequences; (ii) present in only one copy per organism; and (iii) mutually unlinked in location. Partial sequences for the genes encoding the alpha subunit of ATP synthase (atpA), RNA polymerase alpha subunit (rpoA) and phenylalanyl-tRNA synthase alpha subunit (pheS) were determined. Amplification and sequencing reactions were performed as described by Naser et al. (2005a, b). The designed primers enabled the amplification and final comparison of atpA (1102 bp), rpoA (533 bp) and pheS (455 bp) fragments of all Enterococcus species. The simultaneous use of several housekeeping genes in bacterial taxonomy gives higher taxonomic resolution between Enterococcus species and allows the discrimination of all currently recognized species of Enterococcus to a greater degree than the use of 16S rRNA gene sequences. The MLSA scheme is best suited for analysis at the species and genus levels as it integrates the information from different molecular markers from the bacterial chromosome (Stackebrandt et al., 2002; Zeigler, 2003). At the interspecies level, all enterococcal species were clearly differentiated on the basis of atpA, rpoA and pheS gene sequences, with a maximum of 92, 97 and 86 % similarity, respectively (Naser et al., 2005a, b). Evaluation of the intraspecies variation showed that atpA, rpoA and pheS genes had a high degree of homogeneity among strains of the same species. The MLSA approach for the analysis of the genus Enterococcus provides a reliable, highly reproducible, low-cost and rapid identification tool for the currently known species.
Neighbour-joining trees of the housekeeping genes investigated showed that strain LMG 13590 represents a separate lineage distantly related to the other enterococcal species and to E. dispar LMG 13521T (90, 98 and 89 % atpA, rpoA and pheS gene sequence similarities, respectively). The other closest type strains were Enterococcus asini LMG 18727T (83 and 84 % atpA and rpoA gene sequence similarities, respectively), Enterococcus canis LMG 12316T (83 % atpA and rpoA gene sequence similarities, respectively) and Enterococcus saccharolyticus LMG 11427T (83, 84 and 84 % atpA, rpoA and pheS gene sequence similarities, respectively) (Figs 1 and 2 ). In a further study, the pheS gene sequences of the 12 isolates from dog faeces were determined and, together with strain LMG 13590, they occupied a homogeneous and separate position in the pheS tree (98·6 % pheS gene sequence similarity) (Fig. 2).
|
|
. 16S rRNA gene amplification, purification and sequencing was performed as described by Vancanneyt et al. (2004) with the following modifications: PCR-amplified 16S rRNA genes were purified by using a NucleoFast 96 PCR clean-up kit (Macherey-Nagel). Sequencing reactions were performed with a BigDye terminator cycle sequencing kit (Applied Biosystems) and purified by using a Montage SEQ96 sequencing reaction clean-up kit (Millipore). Sequencing was performed with an ABI Prism 3100 Genetic Analyzer (Applied Biosystems). Sequence assembly was done by using the GENEBUILDER program (Applied Maths). The 16S rRNA gene sequences of strain LMG 13590 and those of strains retrieved from GenBank/EMBL were aligned and a phylogenetic tree was constructed from the global alignment by using the neighbour-joining method using the BioNumerics software package, version 3.50 (Applied Maths). Bootstrap analysis was undertaken to test the statistical reliability of the topology of the neighbour-joining tree using 500 bootstrap resamplings of the data (Supplementary Fig. S1 available in IJSEM Online). The strain was classified in the genus Enterococcus with E. dispar LMG 13521T, E. asini LMG 18727T and E. canis LMG 12316T as the nearest neighbours (99, 98 and 98 % 16S rRNA gene sequence similarities, respectively).
E. dispar LMG 13521T, E. canis 12316T, E. asini LMG 18727T, strain LMG 13590 and the 12 dog faecal isolates were investigated using PAGE of whole-cell proteins. Whole-cell protein extracts were prepared and SDS-PAGE was performed as described by Pot et al. (1994). Densitometric analysis, normalization and interpolation of protein profiles and numerical analysis were performed by using the GelCompar software package, versions 3.1 and 4.0, respectively (Applied Maths). The 12 dog strains and LMG 13590 constituted a homogeneous and separate cluster (Fig. 3).
|
, the 12 strains isolated from dog faecal samples, were subjected to tRNA intergenic length polymorphism analysis (tDNA-PCR). PCR was carried out using the tRNA-gene consensus primers T5A and fluorescently labelled T3B (Welsh & McClelland, 1991), which are directed outwardly against the conserved edges of the tRNA genes. PCR mixtures and cycle conditions were as described previously (Baele et al., 2000). Capillary electrophoresis of the DNA fragments was performed with an ABI Prism 310 Genetic Analyzer (Applied Biosystems). Electrophoretograms obtained with GENESCAN software were compared with a database developed at the Faculty of Veterinary Medicine, Ghent University, using a software program developed at our laboratory (Baele et al., 2000). The pattern of the 12 dog isolates was composed of mean peak positions at 56·9, 62·6, 64·4, 79·4, 157, 230·1, 249·5 and 265·5 bp, which corresponded to the peak values of LMG 13590 included in our database as one of the E. dispar reference strains. A comparison of this pattern with the fragment lengths of all known LAB species in our database, including E. dispar type strain LMG 13521T, revealed a separate grouping for the 12 dog faecal isolates and LMG 13590.
DNA G+C content was determined for E. dispar LMG 13521T and three dog faecal isolates, LMG 13590, LMG 22752 and LMG 22753. Cells were cultivated in MRS broth at 37 °C for 24 h. DNA was extracted from 0·5–0·75 g (wet weight) using the protocol described by Marmur (1961) with the following modifications: (i) cells were suspended overnight in Tris/HCl buffer that contained lysozyme (8 mg ml–1) before the addition of SDS and (ii) lysed cells were treated with proteinase K (360 mg l–1; Merck) at 37 °C for 2 h. For determination of the DNA G+C content, DNA was degraded enzymically into nucleosides as described by Mesbah et al. (1989). The nucleoside mixture was then separated by HPLC using a Waters SymmetryShield C8 column maintained at a temperature of 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. The DNA G+C content of the dog faecal strains was in a range from 36 to 37 mol%. For E. dispar LMG 13521T, a slightly higher value of 38 mol% was observed.
DNA–DNA hybridizations were performed between strains E. dispar LMG 13521T and LMG 13590, LMG 22752 and LMG 22753. Genomic DNA was prepared according to the protocol of Niemann et al. (1997). The microplate method was used as described by Ezaki et al. (1989) and Goris et al. (1998), using an HTS7000 Bio Assay Reader (Perkin Elmer) for the fluorescence measurements. Biotinylated DNA was hybridized with unlabelled single-stranded DNA, which was bound non-covalently to microplate wells. Hybridizations were performed at 36 °C with the hybridization mixture (2x SSC, 5x Denhardt's solution, 2·5 % dextran sulfate, 50 % formamide, 100 µg denatured salmon sperm DNA ml–1, 1250 ng biotinylated probe DNA ml–1). The DNA–DNA relatedness percentages presented are means based on at least two independent hybridization experiments. Reciprocal reactions were performed and also considered as independent hybridization experiments. Hybridization values of 51·5 % were found with the type strain of E. dispar. Hybridization values of 93–96 % were found between LMG 13590, LMG 22752 and LMG 22753, indicating that these three dog faecal strains constitute a single novel species.
Growth tests were carried out as described by vec et al. (2001). Lancefield antigens were detected by using a Streptococcal grouping kit (Oxoid). Biochemical reactions were determined in API 50 CH galleries under paraffin cover and in the API 20 STREP system (bioMérieux). The unknown dog faecal isolates present a number of characteristics in common with their closest phylogenetic relative, E. dispar, but, as outlined in Table 1, several phenotypic differences were noted compared with the E. dispar species description. These differences can easily be used to differentiate the unidentified dog faecal isolates from this related enterococcal species.
|
Description of Enterococcus canintestini sp. nov.
Enterococcus canintestini (can.in.tes'ti.ni. L. gen. n. canis of a dog; L. neut. n. intestinum gut; N.L. gen. n. canintestini of the gut of a dog).
Cells are non-motile, small, Gram-positive cocci. They are predominantly arranged in small groups. On Columbia sheep blood agar, colonies are circular, non-pigmented, smooth, shiny and milky, with diameters up to 3 mm. Strains show homogeneous growth in brain-heart infusion broth (Oxoid). Growth is not enhanced by the addition of 5 % CO2. Growth is slow at 25 °C, better at 30 °C and optimal at 37 °C; growth at 42 °C is equal to or slower than at 37 °C. Strains grow in 6·5 % NaCl broth. On Edward's Streptococcus selective medium, aesculin-degrading colonies are formed. Blackening occurs on aesculin bile agar. Strains grow and tetrazolium chloride is reduced on Slanetz & Bartley agar. The strains do not react with Lancefield groups A, B, C, D, G or F antisera. Negative reactions in API tests for hippurate, 
-galactosidase, -glucuronidase and alkaline phosphatase. All strains are positive for arginine, aesculin and the Voges–Proskauer test. Few strains are positive in tests for leucine arylamidase, pyrrolidonyl arylamidase or 
-galactosidase (4/13, 1/13 and 3/13 positive, respectively). Acid is produced in the API 20 STREP and/or API 50 CH kits in tests with ribose, D-galactose, D-glucose, D-fructose, D-mannose, methyl -D-glucopyranoside, N-acetylglucosamine, amygdalin (sometimes weak), arbutin, salicin, D-cellobiose, D-maltose, D-sucrose, trehalose, gentiobiose and D-tagatose. Most strains react positively in tests with D-turanose (11/13 positive with two weak reactions), 2-ketogluconate (8/13 strains with often weak reactions) and lactose (12/13 positive in API 50 CH and 10/13 positive in API 20 STREP). Few strains produce acid from methyl -D-mannopyranoside (2/13 strains) and only one of 13 strains produces acid from glycerol. Reactions are also strain-dependent for acid production from D-lyxose (5/13 positive, 7/13 negative and one weak reaction). No acid is produced from erythritol, D- or L-arabinose, D- or L-xylose, D-adonitol, methyl -D-xylopyranoside, L-sorbose, L-rhamnose, dulcitol, inositol, mannitol, sorbitol, D-melibiose, inulin, D-melezitose, raffinose, starch, glycogen, xylitol, D- or L-fucose, D- or L-arabitol, gluconate or 5-ketogluconate. DNA G+C content is 36–37 mol%.
The type strain, LMG 13590T(=CCM 7285T), was isolated from dog faeces.
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
Collins, M. D., Rodrigues, U. M., Pigott, N. E. & Facklam, R. R. (1991). Enterococcus dispar sp. nov. a new Enterococcus species from human sources. Lett Appl Microbiol 12, 95–98.[Medline]
De Graef, E. M., Devriese, L. A., Baele, M., Vancanneyt, M., Swings, J., Haesebrouck, F. & Decostere, A. (2005). Identification of enterococcal, streptococcal and Weissella species in the faecal flora of individually owned dogs. J Appl Microbiol 99, 348–353.[CrossRef][Medline]
Devriese, L. A., Collins, M. D. & Wirth, R. (1992). The genus Enterococcus. In The Prokaryotes: A Handbook on the Biology of Bacteria: Ecophysiology, Isolation, Identification, Applications, 2nd edn, pp. 1465–1481. Edited by A. Balows, H. G. Trüper, M. Dworkin, W. Harder & K. H. Schleifer. New York: Springer.
Devriese, L. A., Pot, B., Van Damme, P., Kersters, K. & Haesebrouck, F. (1995). Identification of Enterococcus species isolated from foods of animal origin. Int J Food Microbiol 26, 187–197.[CrossRef][Medline]
Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorometric deoxyribonucleic acid-deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane filter hybridization in which radioisotopes are used to determine genetic relatedness among bacterial strains. Int J Syst Bacteriol 39, 224–229.
Franz, C. M. A. D., Holzapfel, W. H. & Stiles, M. E. (1999). Enterococci at the crossroads of food safety? Int J Food Microbiol 47, 1–24.[CrossRef][Medline]
Goris, J., Suzuki, K., De Vos, P., Nakase, T. & Kersters, K. (1998). Evaluation of a microplate DNA–DNA hybridization method compared with the initial renaturation method. Can J Microbiol 44, 1148–1153.[CrossRef]
Klein, G. (2003). Taxonomy, ecology and antibiotic resistance of enterococci from food and the gastro-intestinal tract. Int J Food Microbiol 88, 23–31.
Marmur, J. (1961). A procedure for the isolation of deoxyribonucleic acid from microorganisms. J Mol Biol 3, 208–218.
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.
Naser, S., Thompson, F. L., Hoste, B., Gevers, D., Vandemeulebroecke, K., Cleenwerck, I., Thompson, C. C., Vancanneyt, M. & Swings, J. (2005a). Phylogeny and identification of Enterococci by atpA gene sequence analysis. J Clin Microbiol 43, 2224–2230.
Naser, S. M., Thompson, F. L., Hoste, B., Gevers, D., Dawyndt, P., Vancanneyt, M. & Swings, J. (2005b). Application of multilocus sequence analysis (MLSA) for rapid identification of Enterococcus species based on rpoA and pheS genes. Microbiology 151, 2141–2150.
Niemann, S., Puehler, A., Tichy, H. V., Simon, R. & Selbitschka, W. (1997). Evaluation of the resolving power of three different DNA fingerprinting methods to discriminate among isolates of a natural Rhizobium meliloti population. J Appl Microbiol 82, 477–484.[CrossRef][Medline]
Pot, B., Vandamme, P. & Kersters, K. (1994). Analysis of electrophoretic whole-organism protein fingerprints. In Chemical Methods in Prokaryotic Systematics. Edited by M. Goodfellow & A. G. O'Donnell, pp. 493–521. Chichester: Wiley.
Stackebrandt, E., Frederiksen, W., Garrity, G. M. & 10 other authors (2002). Report of the ad hoc committee for the re-evaluation of the species definition in bacteriology. Int J Syst Evol Microbiol 52, 1043–1047.[Abstract]
vec, P., Devriese, L. A., Sedlá
ek, I., Baele, M., Vancanneyt, M., Haesebrouck, F., Swings, J. & Do
ka
, J. (2001). Enterococcus haemoperoxidus sp. nov. and Enterococcus moraviensis sp. nov., isolated from water. Int J Syst Evol Microbiol 51, 1567–1574.[Abstract]
Vancanneyt, M., Mengaud, J., Cleenwerck, I., Hoste, B., Dawyndt, P., Degivry, M. C., Ringuet, D., Janssens, D. & Swings, J. (2004). Reclassification of Lactobacillus kefirgranum Takizawa et al. 1994 as Lactobacillus kefiranofaciens subsp. kefirgranum subsp. nov., and emended description of L. kefiranofaciens Fujisawa et al. 1988. Int J Syst Evol Microbiol 54, 551–556.
Welsh, J. & McClelland, M. (1991). Genomic fingerprints produced by PCR with consensus tRNA gene primers. Nucleic Acids Res 19, 861–866.
Zeigler, D. R. (2003). Gene sequences useful for predicting relatedness of whole genomes in bacteria. Int J Syst Evol Microbiol 53, 1893–1900.
This article has been cited by other articles:
|
|
 |
|
  S. M. Naser, M. Vancanneyt, C. Snauwaert, G. Vrancken, B. Hoste, L. De Vuyst, and J. Swings Reclassification of Lactobacillus amylophilus LMG 11400 and NRRL B-4435 as Lactobacillus amylotrophicus sp. nov. Int J Syst Evol Microbiol, November 1, 2006; 56(Pt 11): 2523 - 2527. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Vancanneyt, S. M. Naser, K. Engelbeen, M. De Wachter, R. Van der Meulen, I. Cleenwerck, B. Hoste, L. De Vuyst, and J. Swings Reclassification of Lactobacillus brevis strains LMG 11494 and LMG 11984 as Lactobacillus parabrevis sp. nov. Int J Syst Evol Microbiol, July 1, 2006; 56(Pt 7): 1553 - 1557. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
50) after 500 simulations are shown. Bar, 10 % sequence divergence.
