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1 Department of Botany and Microbiology, University of Oklahoma, Norman, OK 73019, USA
2 Culture Collection, Department of Clinical Bacteriology, University of Göteborg, S-41346 Göteborg, Sweden
3 Fermentation Biotechnology Research Unit, National Center for Agricultural Utilization Research, USDA, Agricultural Research Service, 1815 N. University Street, Peoria, IL 61604, USA
Correspondence
Terence R. Whitehead
whitehtr{at}ncaur.usda.gov
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to accommodate a phylogenetically distinct group of Gram-positive, motile, coccus-shaped organisms that resembled lactococci in reacting with Lancefield group N antisera. The genus was originally monospecific, comprising the species Vagococcus fluvialis, isolated from chicken faeces and river water (Hashimoto et al., 1974). V. fluvialis has since been recovered from diverse sources, including human clinical specimens (blood, peritoneal fluid and wounds; Teixeira et al., 1997) and various domestic animals (chickens, pigs, cattle, horses and cats; Pot et al., 1994). Four further species have subsequently been assigned to the genus. Vagococcus salmoninarum has been recovered from diseased fish (e.g. Atlantic salmon, rainbow trout and brown trout with peritonitis; Schmidtke & Carson, 1994), Vagococcus fessus (Hoyles et al., 2000) has been isolated from a seal and a harbour porpoise, Vagococcus lutrae originated from the common otter (Lawson et al., 1999) while Vagococcus carniphilus was isolated from ground beef (Shewmaker et al., 2004). Here we report the results of a polyphasic taxonomic study on a strain of a Vagococcus-like organism isolated from a swine-manure storage pit. On the basis of the findings presented we consider that this bacterium represents a novel species of the genus Vagococcus.
An unknown, elongated coccus-shaped organism, designated strain PPC9T, was isolated from a swine-manure storage pit. The isolate stained Gram-positive and was catalase-negative. The unidentified organism was cultured on basic media containing macrominerals, microminerals, buffers, reducing agents and other components as in the routine growth medium described by Hespell et al. (1987), with 0.2 % carbohydrate added as a carbon source. All media used in these experiments were prepared anaerobically by using the method of Hungate as modified by Bryant (1972). Strain PPC9T was characterized biochemically by using the API Rapid ID32S and API ZYM systems at 37 °C for 4 h aerobically, according to the manufacturer's instructions (API bioMérieux). Determination of the DNA G+C content was carried out by thermal denaturation of chromosomal DNA by using a Beckman model DU 640 spectrophotometer equipped with a high-performance temperature controller and Tm analysis software (Johnson, 1994). 16S rRNA gene fragments were generated by PCR with universal primers pA (positions 8–28 according to the Escherichia coli numbering system) and pH* (1542–1522). The amplified product was purified by using a QIAquick PCR purification kit and sequenced directly by using primers to conserved regions of the 16S rRNA gene. Sequencing was performed by using a PRISM Taq Dye Deoxy Terminator cycle sequencing kit (Applied Biosystems) and an automatic DNA sequencer (model 373A; Applied Biosystems). The closest known relatives of the new isolate were determined by performing database searches with the program FASTA (Pearson & Lipman, 1985). These sequences and those of strains of related recognized species were retrieved from GenBank and aligned with the newly determined sequence by using the program SEQtools (Rasmussen, 2002). The resulting multiple sequence alignment was corrected manually with the program GeneDoc (Nicholas et al., 1997) and a phylogenetic tree was constructed according to the neighbour-joining method (Saitou & Nei, 1987) with the programs SEQtools and TreeView (Page, 1996). The stability of the groupings was estimated by bootstrap analysis (1000 replications) with the same programs.
Cells of strain PPC9T occurred in short chains and were elongated in the direction of the chain. This elongation was so pronounced that some cells gave the appearance of short, fat rods. The new isolate was facultatively anaerobic and produced lactic acid but not gas from glucose. Cells appeared to be non-motile based on a 0.4 % agar tube test (Bailey & Scott, 1974, pp. 376–377) and when observed microscopically. The organism grew optimally at 37 °C. When grown for 3 days under CO2 on trypticase soy agar supplemented with 5 % defibrinated horse blood, it produced colonies that were grey, convex and approximately 0.5–2 mm in diameter. Growth under strictly anaerobic conditions was slower and incubation continued for 5–7 days. No haemolysis was observed. With the API Rapid ID32S system the organism was relatively inactive, producing positive reactions only for 
-glucosidase (weak), 
-galactosidase and glycyl tryptophan arylamidase. All other tests were negative. With the API ZYM system the unknown organism showed weak activity for alkaline phosphatase, esterase C4 and esterase lipase C8. No other enzymic activities were detected. The DNA G+C content of strain PPC9T was 44.5 mol%.
Comparative 16S rRNA gene sequencing was performed in order to determine the phylogenetic affinities of the unknown bacterium. Searches of GenBank revealed that the 16S rRNA gene sequence of strain PPC9T was phylogenetically most closely related to those of the lactic acid group of bacteria and in particular was most highly related to those of vagococci (94.0–96.7 % sequence similarity). A neighbour-joining tree depicting the phylogenetic relationships of strain PPC9T is shown in Fig. 1. The treeing analysis confirmed the close association of the organism with members of the genus Vagococcus. Strain PPC9T formed a distinct subline based on this comparative 16S rRNA gene sequence analysis sharing a branching node with V. lutrae CCUG 39187T (96.7 % similarity). Phylogenetically, strain PPC9T formed a robust cluster with the vagococci; the association was statistically significant, although sequence divergence values of approximately 4.1–6 % between the unknown bacterium and the five recognized Vagococcus species clearly demonstrate that strain PPC9T represents a genetically distinct species. There is no precise correlation between level of 16S rRNA gene sequence divergence and species delineation, but it is generally recognized that divergence values of 3 % or more are significant (Stackebrandt & Goebel, 1994). Support for the distinctiveness of the unknown manure bacterium was also evident from phenotypic analyses. Biochemically, the unknown species most closely resembled V. fessus isolated from a seal and harbour porpoise based on their shared generally unreactive biochemical properties. However, strain PPC9T could be distinguished from V. fessus in not producing the enzymes -chymotrypsin or pyroglutamic acid arylamidase and by its production of -glucosidase (weak) and -galactosidase. In addition to 16S rRNA gene sequence analysis data, strain PPC9T could be readily distinguished from the other recognized Vagococcus species based on the traits given in Table 1. In particular, the production of API Rapid ID32S numerical profile 22000400000 served to distinguish the novel bacterium from all recognized Vagococcus species. Thus, based on the phylogenetic and phenotypic distinctiveness of strain PPC9T we consider that it should be classified as representing a novel species of the genus Vagococcus, for which the name Vagococcus elongatus sp. nov. is proposed. Although only a single strain of V. elongatus is currently known, we consider that the formal description of this species, together with 16S rRNA gene sequence data and biochemical criteria to aid its identification, will facilitate its recognition in routine laboratory investigations, thereby permitting the recovery of additional strains.
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Cells are Gram-positive cocci occurring singly or in short chains, with cells elongated in the direction of the chain, and some giving the appearance of short, fat rods. Facultatively anaerobic and catalase-negative. Non-haemolytic and non-motile. Lactic acid but no gas is produced from glucose metabolism. No growth in broth containing 6.5 % NaCl. No growth at 10 or 45 °C. Using the API ID32S system, -galactosidase, -glucosidase (weak reaction) and glycyl tryptophan arylamidase are detected. Acid is not produced from N-acetyl--glucosaminidase, alkaline phosphatase, alanyl phenylalanine proline arylamidase, arginine dihydrolase, D-arabitol, L-arabinose, cyclodextrin, -glucuronidase, -galactosidase, glycogen, lactose, maltose, mannitol, -mannosidase, melibiose, methyl -D-glucopyranoside, melezitose, pyroglutamic acid arylamidase, pullulan, D-raffinose, D-ribose, sorbitol, sucrose, tagatose, trehalose or urease. Using the API ZYM system, weak reactions are obtained for alkaline phosphatase, esterase C4 and esterase lipase C8. No activity is detected for acid phosphatase, naphthol-AS-BI-phosphohydrolase, N-acetyl--glucosaminidase, -chymotrypsin, cystine arylamidase, -galactosidase, -galactosidase, -glucosidase, -glucosidase, -glucuronidase, lipase (C14), leucine arylamidase, -mannosidase, -fucosidase, trypsin or valine arylamidase. Nitrate is not reduced. Voges–Proskauer test is negative. Aesculin is hydrolysed but hippurate and gelatin are not. The DNA G+C content is 44.5 mol%.
The type strain, PPC9T (=CCUG 51432T=NRRL B-41357T), was isolated from a swine-manure storage pit. Habitat unknown.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Bryant, M. P. (1972). Commentary on the Hungate technique for culture of anaerobic bacteria. Am J Clin Nutr 25, 1324–1328.
Collins, M. D., Ash, C., Farrow, J. A. E., Wallbanks, S. & Williams, A. M. (1989). 16S Ribosomal ribonucleic acid sequence analysis of lactococci and related taxa. Description of Vagococcus fluvialis gen. nov., sp. nov. J Appl Bacteriol 67, 453–460.[Medline]
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Hespell, R. B., Wolf, R. & Bothast, R. J. (1987). Fermentation of xylans by Butyrivibrio fibrisolvens and other ruminal bacteria. Appl Environ Microbiol 53, 2849–2853.
Hoyles, L., Lawson, P. A., Foster, G., Falsen, E., Ohlen, M., Grainger, J. M. & Collins, M. D. (2000). Vagococcus fessus sp. nov., isolated from a seal and a harbour porpoise. Int J Syst Evol Microbiol 50, 1151–1154.[Abstract]
Johnson, J. L. (1994). Similarity analysis of DNAs. In Methods for General and Molecular Bacteriology, pp. 656–682. Edited by P. Gerhardt, R. G. E. Murray, W. A. Wood & N. R. Krieg. Washington, DC: American Society for Microbiology.
Lawson, P. A., Foster, G., Falsen, E., Ohlen, M. & Collins, M. D. (1999). Vagococcus lutrae sp. nov., isolated from the common otter (Lutra lutra). Int J Syst Bacteriol 49, 1251–1254.
Nicholas, K. B., Nicholas, H. B., Jr & Deerfield, D. W., II (1997). GeneDoc: analysis and visualization of genetic variation. EMBnet News 4 (2), 1–4. http://www.embnet.org/download/embnetnews/embnet_news_4_2.pdf
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Pot, B., Devriese, L. A., Hommez, J., Miry, C., Vandemeulebroecke, K., Kersters, K. & Haesebrouck, F. (1994). Characterization and identification of Vagococcus fluvialis strains isolated from domestic animals. J Appl Bacteriol 77, 362–369.[Medline]
Rasmussen, S. W. (2002). SEQtools, a software package for analysis of nucleotide and protein sequences. http://www.seqtools.dk
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Shewmaker, P. L., Steigerwalt, A. G., Morey, R. E., Carvalho Mda, G., Elliott, J. A., Joyce, K., Barrett, T. J., Teixeira, L. M. & Facklam, R. R. (2004). Vagococcus carniphilus sp. nov., isolated from ground beef. Int J Syst Evol Microbiol 54, 1505–1510.
Stackebrandt, E. & Goebel, B. M. (1994). Taxonomic note: a place for DNA-DNA reassociation and 16S rDNA sequence analysis in the present species definition in bacteriology. Int J Syst Bacteriol 44, 846–849.
Teixeira, L. M., Carvalho, M. G. S., Merquior, V. L. C., Steigerwalt, A. G., Brenner, D. J. & Facklam, R. R. (1997). Phenotypic and genotypic characterization of Vagococcus fluvialis, including strains isolated from human sources. J Clin Microbiol 35, 2778–2781.
Wallbanks, S., Martinez-Murcia, A. J., Fryer, J. L., Philips, B. A. & Collins, M. D. (1990). 16S rRNA sequence determination for members of the genus Carnobacterium and related lactic acid bacteria and description of Vagococcus salmoninarum sp. nov. Int J Syst Bacteriol 40, 224–230.
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90 % are shown. Bar, 1 % sequence divergence.