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Department of Microbiology and Immunology, Nihon University School of Dentistry at Matsudo, 2-870-1 Sakaecho-nishi, Matsudo, Chiba 271-8587, Japan
Correspondence
Masatomo Hirasawa
hirasawa.masatomo{at}nihon-u.ac.jp
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; Hamada & Slade, 1980; Whiley et al., 1988). Recently, a novel species displaying close affinity with mutans streptococci from equine teeth was reported (Collins et al., 2004). The colonization of the oral cavity by mutans streptococci is assumed to be caused by sucrose intake. As pigs are omnivorous animals, they often have the opportunity to ingest sucrose. We investigated the microflora of the porcine oral cavity, focusing on mutans streptococci. To investigate the oral microflora of pigs, a group of Gram-positive cocci were characterized. Mitis salivarius agar (Difco) is widely used to isolate S. mutans as well as other oral streptococcal species. The five mutans streptococcus-like strains selected for this study were randomly chosen from among 117 streptococcal isolates obtained on mitis salivarius agar from the oral cavities of 39 pigs (from two different farms). The strains formed small, raised, adherent colonies with irregular margins. The strains were grown at 37 °C under anaerobic conditions on brain-heart infusion agar (Difco) supplemented with 5 % horse blood. A biochemical analysis was conducted using the Rapid ID32 Strep, API 50 CH and API ZYM (bioMérieux) and Streptogram (Wako) systems according to the manufacturers' instructions. The colonial formation and biochemical characteristics resembled those of mutans streptococci. Isolates NUM 1001T, NUM 1002, NUM 1003, NUM 1004 and NUM 1005 were subjected to further taxonomic study.
DNA was extracted from bacterial cultures by using the Promega Genome kit according to the manufacturer's instructions. The G+C contents of the DNAs were determined by HPLC using a method described previously (Hirasawa & Takada, 1994). The DNA G+C contents of strains NUM 1001T, NUM 1002, NUM 1003, NUM 1004 and NUM 1005 ranged from 42 to 44 mol %, which is similar to that for S. criceti. DNA–DNA hybridization was performed according to the microtitration plate method (Ezaki et al., 1989), with minor modifications. A heat-denatured sample of DNA (1 µg) was immobilized in each well of a microplate (Immuno plate II; Nunc) at 30 °C for 2 h. The microplate was dried at 45 °C for 2 h and then photobiotin-labelled heat-denatured probe DNA (0.125 µg per well) was used for the hybridization (incubated at 42.5 °C for 3 h). Other procedures were conducted according to the original instructions. Levels of DNA–DNA hybridization were examined using labelled DNA from strain NUM 1001T and S. criceti ATCC 19642T against other strains of mutans streptococci. There were very high levels of hybridization among the five isolates and between S. criceti strains ATCC 19642T and OMZ 61 relative to each labelled strain, confirming a relationship at the species level. The highest level of DNA–DNA hybridization was found with S. criceti ATCC 19642T, but the level of relatedness was only 42.5–58.1 %. DNAs from strains of mutans streptococci, namely S. downei NCTC 11391T, S. sobrinus NIDR6715, S. ratti ATCC 19645T and S. mutans JC2, exhibited relatedness levels of 21.4, 12.8, 7.4 and 7.2 %, respectively, to strain NUM 1001T. To determine the phylogenetic affinity of each of the clinical isolates, the almost-complete 16S rRNA gene was sequenced and subjected to a comparative analysis. The 16S rRNA gene was amplified by using a PCR with primers 27f (5'-AGAGTTTGATCCTGGCTCAG-3'; Escherichia coli positions 8–27) and 1525r (5'-AAAGGAGGTGATCCAGCC-3'; E. coli positions 1543–1525) according to the method described by Shinoda et al. (2000). The PCR products were purified with a Mini Elute gel extraction kit (Qiagen) and directly sequenced using a BigDye Terminator v1.1 cycle sequencing kit (Applied Biosystems) and an automatic DNA sequencer (ABI PRISM, model 373A; Applied Biosystems) by Takara Bio (Mie, Japan). The closest known relatives of the novel isolates were identified by performing database searches. Their 16S rRNA gene sequences were compared with those available in the DNA Data Bank of Japan (Mishima, Japan) using BLAST software. Nucleotide substitution rates (Knuc values) were calculated (Kimura, 1980) and phylogenetic trees were constructed by using the neighbour-joining method (Saitou & Nei, 1987). The topologies of the trees were evaluated by performing a bootstrap analysis of the sequence data, using CLUSTAL W software (Thompson et al., 1994). Sequence similarity values were calculated manually. The highest levels of 16S rRNA gene sequence similarity to strain NUM 1001T were obtained with S. criceti ATCC 19642T and S. downei NCTC 11391T (97.8 and 94.1 %, respectively). Despite the relatively high levels of sequence similarity among S. criceti ATCC 19642T, S. downei NCTC 11391T and NUM 1001T compared with the levels of similarity among representatives of the other species within the mutans streptococci, the results show that the pig strains are sufficiently dissimilar from S. criceti and S. downei to warrant separate species status. A tree constructed by neighbour-joining, depicting the phylogenetic affinity of strain NUM 1001T with members of the genus Streptococcus, is shown in Fig. 1.
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; Takada et al., 1984). Rantz-Randle extract antigens were prepared from cells cultured overnight on brain-heart infusion agar as described previously. Fig. 2 shows the double immunodiffusion patterns obtained with the Rantz-Randle extract antigens and various antibodies. In immunodiffusion experiments with strain NUM 1001T, cross-reactive precipitin bands were formed with typing sera prepared against mutans streptococci strains of serotype a, d or g (Fig. 2, left panel) but not c, e or f. The specific antiserum obtained by absorbing antiserum raised against novel isolate NUM 1001T with cells of serotype a and g reacted with NUM 1001T and a serotype d strain (Fig. 2, centre panel). The antiserum raised against NUM 1001T or serotype d cells reacted identically with NUM 1001T and serotype d (Fig. 2, right panel). The antiserum obtained by absorbing antiserum raised against NUM 1001T with cells of serotype d did not react with NUM 1001T. These results suggest that isolate NUM 1001T should be grouped within serotype d.
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). The phenotypic characteristics that differentiate strain NUM 1001T from other mutans streptococci are shown in Table 1. Therefore, on the basis of the distinct phenotypic characteristics and biochemical and molecular genetic data obtained in this study, strain NUM 1001T warrants classification within a novel species of the genus Streptococcus, and a member of the mutans streptococci, for which the name Streptococcus orisuis sp. nov. is proposed.
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Cells are Gram-positive, non-spore-forming cocci, 0.5–0.75 µm in diameter, that occur in pairs or in short chains. Colonies on blood agar are small and white, 0.75–1.0 mm in diameter and non-haemolytic at 37 °C. On mitis salivarius agar, colonies are small, dark blue and crinkled. Facultatively anaerobic and catalase-negative. No Lancefield carbohydrate antigens (streptococcal grouping kit; Oxoid) are detected. According to API systems, strains produce acid from D-glucose, galactose, D-fructose, D-mannose, mannitol, sorbitol, N-acetylglucosamine, amygdalin, arbutin, aesculin, salicin, cellobiose, maltose, lactose, melibiose, sucrose, trehalose, inulin, D-raffinose, 
-gentiobiose, methyl -D-glucopyranoside and D-tagatose. Alanine phenylalanine proline arylamidase, -glucosidase, 
-glucosidase, -galactosidase, acid phosphatase, leucine arylamidase, valine arylamidase, cystine arylamidase, naphthol-AS-BI-phosphohydrolase and glucosyltransferase are produced. Arginine dihydrolase, -glucuronidase, alkaline phosphatase, -galactosidase and N-acetyl--glucosaminidase are not produced. Voges–Proskauer test is positive. Hippurate hydrolysis test is negative. Resistant to bacitracin. An adhesive, insoluble glucan is produced from sucrose. The bacterium is cariogenic when monoassociated with specific pathogen-free rats. The serotype is type d. The DNA G+C content of the type strain is 43 mol %.
The type strain, NUM 1001T (=JCM 14035T=DSM 18307T), was isolated from clinical specimens from the oral cavities of pigs.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Collins, M. D., Lundstrom, T., Welinder-Olsson, C., Hansson, I., Wattle, O., Hudson, R. A. & Falsen, E. (2004). Streptococcus devriesei sp. nov., from equine teeth. Syst Appl Microbiol 27, 146–150.[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.
Hamada, S. & Slade, H. D. (1980). Biology, immunology, and cariogenicity of Streptococcus mutans. Microbiol Rev 44, 331–384.
Hirasawa, M. & Takada, K. (1994). Porphyromonas gingivicanis sp. nov. and Porphyromonas crevioricanis sp. nov., isolated from beagles. Int J Syst Bacteriol 44, 637–640.
Hirasawa, M., Kiyono, H., Shiota, T., Michalek, S. M. & McGhee, J. R. (1980). Virulence of Streptococcus mutans: immunochemical characterization of a serotype-g-defective mutant (C307). Infect Immun 27, 697–699.
Kimura, M. (1980). A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16, 111–120.[CrossRef][Medline]
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Shinoda, Y., Kato, N. & Morita, N. (2000). Phylogenetic analysis of a bacterium by sequencing its 16S ribosomal RNA gene. Shimazu Hyouron 57, 121–132 (in Japanese, with English abstract).
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Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673–4680.
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Whiley, R. A., Russell, R. R. B., Hardie, J. M. & Beighton, D. (1988). Streptococcus downei sp. nov. for strains previously described as Streptococcus mutans serotype h. Int J Syst Bacteriol 38, 25–29.
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