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1 School of Food Biosciences, University of Reading, Reading, UK
2 SAC Veterinary Services, Inverness, UK
3 Culture Collection, Department of Clinical Bacteriology, University of Göteborg, Sweden
4 VLA Truro, Polwhele, Truro, UK
Correspondence
Matthew D. Collins
m.d.collins{at}reading.ac.uk
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Published online ahead of print on 13 April 2004 as DOI 10.1099/ijs.0.63082-0.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain CCUG 48324T is AJ606046.
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). Despite the considerable increase in the number of newly described streptococcal species, only Streptococcus phocae and Streptococcus iniae have originated from marine mammals. S. phocae has been reported only from seals (Skaar et al., 1994) and cetaceans (G. Foster, unpublished), whereas S. iniae, although originally recovered from freshwater dolphins (Pier & Madin, 1976), has subsequently been found in aquacultures of fish and has even been isolated from humans (Weinstein et al., 1997). During the course of a study of taxonomically problematic, catalase-negative, Gram-positive cocci from marine mammals, we have characterized a novel Streptococcus-like organism from grey seals (Halichoerus grypus). Based on the presented findings, we describe a novel species, Streptococcus halichoeri sp. nov.
Six bacterial isolates were recovered from different grey seals. Three strains designated M512/02/1T (=CCUG 48324T), M72/03/04 (=CCUG 48325) and M2279/96/5 were recovered from animals following post-mortem in Inverness, UK, whereas strains M188/00/1, M159/01/2 and M466/02/1 (=CCUG 48323) were isolated from grey seals sampled at a rehabilitation centre in Cornwall, UK. The unidentified strains were cultured on Columbia Blood agar base supplemented with 5 % sheep blood at 37 °C, under aerobic conditions. Organisms were characterized biochemically using the API Rapid ID 32Strep and API 20Strep systems (API bioMérieux) according to the manufacturer's instructions. To assess the overall phenotypic resemblance of the novel isolates and reference species, a comparative analysis of whole-cell protein profiles by SDS-PAGE was performed. SDS-PAGE of whole-cell proteins was performed as described by Pot et al. (1994) and Vandamme et al. (1998). For densitometric analysis, normalization and interpretation of protein patterns, the GCW 3.0 software package (Applied Maths) was used. The similarity between all pairs of traces was expressed by using the Pearson product moment correlation coefficient, converted for convenience to a percentage similarity. The G+C content (mol%) of the DNA of strain CCUG 48324T was determined by HPLC as described by Mesbah et al. (1989). For phylogenetic analysis, 16S rRNA genes were amplified by PCR and sequenced directly using a Taq dye-deoxy terminator cycle sequencing kit (Applied Biosystems) and an automatic DNA sequencer (model 373A; Applied Biosystems). The closest known relatives of the novel isolates were determined by performing database searches. Closely related sequences were retrieved from EMBL and aligned with the newly determined sequences using the program DNATools (Rasmussen, 1995). The resulting multiple sequence alignment had approximately 100 bases at the 5' end of the rRNA omitted from further analysis, because of alignment uncertainties due to the highly variable region V1, using the program GeneDoc (Nicholas et al., 1997). A phylogenetic tree was constructed according to the neighbour-joining method (Saitou & Nei, 1987) with the programs DNATools and TREEVIEW (Page, 1996) and the stability of the groupings was estimated by bootstrap analysis (1000 replications).
The unidentified organisms recovered from grey seals were Gram-positive cocci that occurred in pairs or short chains. The organisms were facultatively anaerobic and catalase-negative. Using the API Rapid ID 32Strep system, the unknown cocci closely resembled each other, producing acid from cyclodextrin, maltose, mannitol, ribose and pullulan. Some strains produced acid from lactose, but acid was not formed from any of the other carbohydrates in this test system. All of the isolates were Voges–Proskauer-positive and all showed activity for arginine dihydrolase, alkaline phosphatase, alanine phenylalanine proline arylamidase and pyroglutamic acid arylamidase. All other enzyme tests gave negative results. Using the API 20Strep system, all of the strains produced acid from mannitol and ribose, but variable results were obtained with amygdalin. They were all Voges–Proskauer-positive and displayed activity for arginine dihydrolase, alkaline phosphatase, leucine arylamidase and pyrrolidonyl arylamidase. All other tests were negative with the API 20Strep system. The morphological and general biochemical characteristics of the unidentified cocci from seals were consistent with their tentative assignment to the genus Streptococcus, but they did not correspond to any recognized species. To investigate further their phenotypic resemblance to streptococci, whole-cell protein profiling studies were performed on the novel strains. Three of the seal isolates were examined and were found to possess closely related protein patterns, forming a tight cluster (>90 % intragroup similarity) that was distinct from those of all other reference streptococcal species (data not shown). The nearest neighbours to the unknown species from the PAGE analysis corresponded to Streptococcus thoraltensis and Streptococcus hyovaginalis, joining the cluster formed by the seal organisms at approximately 65 % similarity. 16S rRNA gene sequencing studies were performed to ascertain the phylogenetic affinities of the unidentified Streptococcus-like organisms. Almost-complete gene sequences (>1400 bases) of three of the seal strains (M512/02/1T, M72/03/04 and M466/02/1) were determined and pair-wise analysis revealed 100 % sequence similarity, showing that the organisms were genetically highly related. Searches of the GenBank database revealed streptococci to be the nearest phylogenetic relatives of the unidentified bacterium. Phylogenetic analysis confirmed the association of the unidentified seal bacterium with the genus Streptococcus, with the unknown bacterium showing an affinity with a number of species within the ‘pyogenic’ subcluster. A tree based on a reduced dataset showing the nearest phylogenetic relatives of the unknown bacterium is depicted in Fig. 1. The G+C content of the DNA of a representative strain (M512/02/1T) of the unknown bacterium was determined and corresponded to 39 mol%. This value is similar to those of other close phylogenetic relatives of the seal bacterium (e.g. Streptococcus agalactiae, 34–36 mol%; Streptococcus dysgalactiae, 40 mol%; Streptococcus porcinus, 37 mol%; S. iniae, 33 mol%; Streptococcus uberis, 37·5 mol%; Streptococcus parauberis, 36 mol%).
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). However, whole-cell-protein profiling showed that the organisms recovered from grey seals in this study were phenotypically quite distinct from S. phocae. Similarly, the unidentified seal bacterium and S. phocae were biochemically very different. The unidentified organisms differed from S. phocae in forming acid from a broader range of carbohydrates, including cyclodextrin, mannitol, pullulan and ribose, and by being arginine dihydrolase-positive. In addition, unlike S. phocae, the unknown organisms did not display 
-haemolytic activity. Phylogenetically it is evident that the unidentified coccus-shaped organisms from seals represent a hitherto unknown species. PAGE analysis of whole-cell proteins confirmed the distinctiveness of the seal organisms and the production of distinct biochemical profiles [API Rapid ID32Strep numerical profile 1430(1)3141001 and API 20Strep profile 1163100(1)] served to distinguish the novel bacterium from all other described streptococcal species. Therefore, based on both phenotypic and phylogenetic criteria, we are of the opinion that the unknown bacterium from grey seals merits assignment to a novel species within the genus Streptococcus, for which the name Streptococcus halichoeri sp. nov. is proposed. Tests that are useful in distinguishing S. halichoeri from other closely related streptococcal species are shown in Table 1.
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Cells stain Gram-positive and are cocci that occur in pairs or short chains. Non-spore-forming. Colonies are white, umbonate, non-haemolytic and 0·5 mm in diameter after 24 h incubation on sheep blood agar. Lancefield serological group B. Facultatively anaerobic and catalase-negative. Using API test kits, acid is produced from cyclodextrin, maltose, mannitol, ribose and pullulan. Acid may or may not be formed from lactose. Acid is not produced from L-arabinose, D-arabitol, glycogen, inulin, mannose, melibiose, melezitose, methyl -D-glucopyranoside, raffinose, sorbitol, starch, sucrose, D-tagatose or trehalose. Arginine dihydrolase, alkaline phosphatase, alanine phenylalanine proline arylamidase, leucine arylamidase and pyroglutamic acid arylamidase are produced. Activity is not detected for 
-galactosidase, -galactosidase, -glucosidase, -glucuronidase, glycyl trytophan arylamidase, -mannosidase, N-acetyl--glucosaminidase or urease. Aesculin and hippurate are not hydrolysed. Acetoin is produced. Isolated from grey seals (Halichoerus grypus).
The type strain is CCUG 48324T (=CIP 108195T). The G+C content of its DNA is 39 mol%.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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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.
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.
Pier, G. B. & Madin, S. H. (1976). Streptococcus iniae sp. nov., a beta-hemolytic streptococcus from an Amazon freshwater dolphin, Inia geoffrensis. Int J Syst Bacteriol 26, 545–553.
Pot, B., Vandamme, P. & Kersters, K. (1994). Analysis of electrophoretic whole-organism protein fingerprints. In Modern Microbial Methods. Chemical Methods in Prokaryotic Systematics, pp. 493–521. Edited by M. Goodfellow & A. G. O'Donnell. Chichester, UK: Wiley & Sons.
Rasmussen, S. W. (1995). DNATools, a Software Package for DNA Sequence Analysis. Carlsberg Laboratory, Copenhagen.
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.[Abstract]
Skaar, I., Gaustad, P., Tønjum, T., Holm, B. & Stenwig, H. (1994). Streptococcus phocae sp. nov., a new species isolated from clinical specimens from seals. Int J Syst Bacteriol 44, 646–650.
Vandamme, P., Torck, U., Falsen, E., Pot, B., Goossens, H. & Kersters, K. (1998). Whole-cell protein electrophoretic analysis of viridans streptococci: evidence for heterogeneity among Streptococcus mitis biovars. Int J Syst Bacteriol 48, 117–125.
Weinstein, M. R., Litt, M., Kertesz, D. A. & 9 other authors (1997). Invasive infections due to a fish pathogen, Streptococcus iniae. S. iniae Study Group. N Engl J Med 337, 589–594.
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