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1 Department of Pathology, Bacteriology and Poultry Diseases, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium
2 BCCM/LMG Bacteria Collection, Laboratory of Microbiology, Faculty of Sciences, Ghent University, K. L. Ledeganckstraat 35, B-9000 Gent, Belgium
3 Department of Clinical Chemistry, Microbiology and Immunology, Ghent University Hospital, Ghent University, De Pintelaan 185, B-9000 Gent, Belgium
Correspondence
Margo Baele
Margo.Baele{at}rug.ac.be
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of S. ferus strains LMG 19829 and LMG 16520T are AF336367 and AY058218.
A dendrogram based on tDNA-PCR fingerprints including a wider sample of Streptococcus species is available as supplementary material in IJSEM Online (http://ijs.sgmjournals.org/).
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MAIN TEXT |
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). Streptococcus sobrinus is also a colonizer of the human mouth and can be very cariogenic (Coykendall, 1983). Streptococcus downei and Streptococcus macacae were isolated from the dental plaque of monkeys (Coykendall, 1977; Beighton et al., 1984).
During an investigation of the tonsillar and nasal flora of piglets with tRNA-intergenic spacer length polymorphism analysis (tDNA-PCR) (Baele et al., 2001a), a group of Gram-positive coccal isolates was isolated and characterized further. Representative strains were studied extensively and were found to belong to the poorly described species Streptococcus ferus.
Ten atypical streptococcus-like isolates were isolated on Columbia CNA blood agar (Oxoid) from the tonsils or nasal conchae of pigs. The strains were isolated from three different farms in Belgium. Six isolates originated from the same farm. Three of them were isolated from the nasal conchae of weaned piglets, one from the tonsils of one of those piglets and two from the tonsils of two other piglets from the same litter. On a second farm, three similar cultures were obtained from the tonsils of weaned piglets. A tenth isolate was received from another laboratory and was isolated on a third farm from the nose of a pig. The following isolates were subjected to further taxonomic study: KOMA 81 (=LMG 19829), KOMA 188 (=LMG 19830), COR 146 (=LMG 19831), KOMA 101, KOMA 115, KOMA 206, KOMA 215, KOMA 229 and KOMA 248, together with the type strain of S. ferus, LMG 16520T (=ATCC 33477T), isolated from a rat.
DNA extraction, tDNA-PCR, capillary electrophoresis and data analysis were done as described before (Baele et al., 2000). tDNA-PCR has been shown to be discriminative at the species level for all enterococcal and most streptococcal species (Baele et al., 2000, 2001b; De Gheldre et al., 1999). The tDNA-PCR fingerprints of the 10 isolates from the upper respiratory tract of piglets and of the type strain of S. ferus were composed of fragments with lengths of 71·5, 81·5, 157, 255·4 and 262·5 bp. A distance matrix was obtained with the in-house software. Cluster analysis of tDNA-PCR fingerprints was done using the UPGMA algorithm with the NEIGHBOR software of PHYLIP (http://evolution.genetics.washington.edu/phylip.html) and the dendrogram was visualized using the program TREEVIEW (http://taxonomy.zoology.gla.ac.uk/rod/treeview.html). The ten isolates grouped in a single cluster that was clearly separated from other streptococcal species (Fig. 1). A version of the dendrogram including a wider sample of Streptococcus species is available as supplementary material in IJSEM Online (http://ijs.sgmjournals.org/).
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-NOT (5'-TCAAACTAGGACCGAGTC) and 
MB (5'-TACCTTGTTACTTCACCCCA) and the Taq Mastermix (Qiagen). Sequencing was performed using the BigDye Terminator sequencing kit with primers pD, Gamma*, 3 and O* and an ABI PRISM 310 Genetic Analyzer (Coenye et al., 1999). Phylogenetic analysis was performed using the software Bionumerics (Applied Maths) after including the consensus sequence in an alignment of small ribosomal subunit sequences collected from GenBank. Multiple alignment was calculated using an open gap penalty of 100 % and a unit gap penalty of 0 %. A similarity matrix was created by homology calculation with a gap penalty of 0 % and after discarding unknown bases. The resulting tree was constructed using the neighbour-joining method. The two strains represented a separate phylogenetic branch in the S. mutans group (Fig. 2), which contains several other ‘oral’ streptococci (Bentley et al., 1991). Similarities of 94 and 93·9 %, respectively, were obtained with the closest relatives S. mutans and S. macacae.
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with the following modifications: the washed cell pellet was resuspended and lysed in a buffer (10 mM Tris/HCl, 100 mM EDTA, pH 8·0) containing RNase (200 µg ml-1; Sigma), mutanolysin (100 U ml-1; Sigma) and lysozyme (25 mg ml-1; Serva) for 1 h at 37 °C. Before addition of GES reagent, proteinase K (200 µg ml-1; Merck) was added and the mixture was incubated for 15 min. DNA was enzymically degraded into nucleosides and separated by HPLC as described previously (Vancanneyt et al., 2001). The G+C contents of strains LMG 19829 and LMG 19830 were respectively 43·0 and 42·7 mol%.
Isolates LMG 19829, LMG 19830 and LMG 19831 and LMG 16520T were included in SDS-PAGE analysis of whole-cell proteins. Cells were cultivated as indicated for determination of DNA base compositions. Whole-cell protein extracts were prepared and PAGE was performed as described before (Pot et al., 1994). Registration of the protein patterns, normalization of the densitometric traces, pattern storage, grouping of the strains using Pearson's product-moment correlation coefficient (r) and UPGMA cluster analysis were performed as described by Pot et al. (1994) using the software GelCompar (Applied Maths). This yielded highly similar patterns, confirming that the isolates represent a single species. The profiles were different from all patterns of lactic acid bacteria in the database (data not shown), confirming their separate species status. The patterns of the three strains studied and their phylogenetically closest neighbours are shown in Fig. 3.
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summarizes the characteristics that can be used for the differentiation of the species from phylogenetically related species. It should be noted that the tests indicated in the original description of S. ferus (Coykendall, 1977) and in related papers (Freedman et al., 1982; Coykendall, 1983) as well as in reviews (Hardie & Whiley, 1995) cannot be used. Notably, failure to produce acid from raffinose, as described by Coykendall (1977, 1983), is not a distinguishing characteristic, because the test was found to be positive for all strains from pigs.
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) Coykendall 1983
Acid is produced (API 50 CH, bioMérieux) from N-acetylglucosamine, aesculin, amygdalin, cellobiose, D-fructose, galactose, D-glucose, glycogen, -gentiobiose, lactose, D-mannose, maltose, maltotriose, sucrose, salicin, starch and trehalose. Variable acidification of D-tagatose, arbutin, inulin, mannitol, melibiose, D-raffinose and sorbitol. No acid is produced from adonitol, D- or L-arabinose, D- or L-arabitol, dulcitol, erythritol, D- or L-fucose, gluconate, glycerol, inositol, 2- or 5-ketogluconate, D-lyxose, melezitose, methyl -glycoside, methyl -D-mannoside, methyl -D-glycoside, rhamnose, ribose, L-sorbose, D-turanose, xylitol or D- or L-xylose.
S. ferus strains are positive in tests (API 20 STREP, bioMérieux; BBL Crystal Gram-positive ID kits, Becton Dickinson) for aesculin, leucine arylamidase, enzymic hydrolysis of 4-methylumbelliferyl (4MU) -D-glucoside, L-valine 7-amido-4-methylcoumarin (AMC), L-phenylalanine AMC, 4MU -D-glucoside, L-tryptophan AMC, methyl - and -glucoside, p-nitrophenyl (pNP) -D-glucoside, pNP -D-cellobioside, proline and leucine p-nitroanilide, pNP phosphate and pNP -D-maltoside. They are usually positive in tests for acetoin, L-arginine AMC and L-isoleucine AMC and variable for alkaline phosphatase and -galactosidase. Negative for hippurate, pyrrolidonyl arylamidase, -glucuronidase, -galactosidase and hydrolysis of arginine (in API 20 Strep), L-pyroglutamic acid AMC, 4MU N-acetyl -D-glucosaminide, 4MU phosphate and 4MU -D-glucuronide. An adhesive glucan is produced from sucrose.
The DNA G+C content is 43 mol% and the characteristic tDNA-PCR fingerprint is composed of fragments of 71·5, 81·5, 157, 255·4 and 262·5 bp, as determined by fluorescent capillary electrophoresis.
The type strain, strain LMG 16520T (=ATCC 33477T), was isolated from a rat.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Baele, M., Chiers, K., Devriese, L. A., Smith, H. E., Wisselink, H. J., Vaneechoutte, M. & Haesebrouck, F. (2001a). The Gram-positive tonsillar and nasal flora of piglets before and after weaning. J Appl Microbiol 91, 997–1003.[CrossRef][Medline]
Baele, M., Storms, V., Haesebrouck, F., Devriese, L. A., Gillis, M., Verschraegen, G., De Baere, T. & Vaneechoutte, M. (2001b). Application and evaluation of the interlaboratory reproducibility of tRNA intergenic length polymorphism analysis (tDNA-PCR) for identification of Streptococcus species. J Clin Microbiol 39, 1436–1442.
Beighton, D., Hayday, H., Russell., R. R. B & Whiley, R. A. (1984). Streptococcus macacae sp. nov. from dental plaque of monkeys (Macaca fascicularis). Int J Syst Bacteriol 34, 332–335.
Bentley, R. W., Leigh, J. A. & Collins, M. D. (1991). Intrageneric structure of Streptococcus based on comparative analysis of small-subunit rRNA sequences. Int J Syst Bacteriol 41, 487–494.
Coenye, T., Falsen, E., Vancanneyt, M., Hoste, B., Govan, J. R. W., Kersters, K. & Vandamme, P. (1999). Classification of Alcaligenes faecalis-like isolates from the environment and human clinical samples as Ralstonia gilardii sp. nov. Int J Syst Bacteriol 49, 405–413.
Coykendall, A. L. (1977). Proposal to elevate the subspecies of Streptococcus mutans to species status, based on their molecular composition. Int J Syst Bacteriol 27, 26–30.
Coykendall, A. L. (1983). Streptococcus sobrinus nom. rev. and Streptococcus ferus nom. rev.: habitat of these and other mutans streptococci. Int J Syst Bacteriol 33, 883–885.
De Gheldre, Y., Vandamme, P., Goossens, H. & Struelens, M. J. (1999). Identification of clinically relevant viridans streptococci by analysis of transfer DNA intergenic spacer length polymorphism. Int J Syst Bacteriol 49, 1591–1598.
Freedman, M. L., Coykendall, A. L. & O'Neill, E. M. (1982). Physiology of ‘mutans-like’ Streptococcus ferus from wild rats. Infect Immun 35, 476–482.
Hardie, J. M. & Whiley, R. A. (1995). The genus Streptococcus. In The Genera of the Lactic Acid Bacteria, pp. 55–124. Edited by B. J. B. Wood & W. H. Holzapfel. London: Blackie Academic and Professional.
Pitcher, D. G., Saunders., N. A & Owen, R. J. (1989). Rapid extraction of bacterial genomic DNA with guanidium thiocyanate. Lett Appl Microbiol 8, 151–156.
Pot, B., Vandamme, P. & Kersters, K. (1994). Analysis of electrophoretic whole-organism protein fingerprints. In Chemical Methods in Prokaryotic Systematics, pp. 493–521. Edited by M. Goodfellow & A. G. O'Donnell. Chichester: Wiley.
Vancanneyt, M., Snauwaert, C., Cleenwerck, I. & 8 other authors (2001). Enterococcus villorum sp. nov., an enteroadherent bacterium associated with diarrhoea in piglets. Int J Syst Evol Microbiol 51, 393–400.[Abstract]
Whiley, R. A. & Beighton, D. (1998). Current classification of the oral streptococci. Oral Microbiol Immunol 13, 195–216.[Medline]
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