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1 BCCM/LMG Bacteria Collection, Faculty of Sciences, Ghent University, Ledeganckstraat 35, B-9000 Ghent, Belgium
2 Institute of Biology, Romanian Academy, Cell Biology Department, Splaiul Independentei 296, 060042 Bucharest, Romania
3 Laboratory of Veterinary Bacteriology and Mycology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium
4 Laboratoire de Microbiologie et de Biologie Moléculaire, CNRST (Centre National de la Recherche Scientifique et Technique), 52 bd Omar Ibn Khatab, BP 8027-10102 Agdal, Rabat, Morocco
5 Research Group of Industrial Microbiology, Fermentation Technology and Downstream Processing (IMDO), Department of Applied Biological Sciences, Vrije Universiteit Brussel (VUB), Pleinlaan 2, B-1050 Brussels, Belgium
6 Laboratory of Microbiology, Faculty of Sciences, Ghent University, Ledeganckstraat 35, B-9000 Ghent, Belgium
Correspondence
M. Vancanneyt
Marc.Vancanneyt{at}UGent.be
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of LMG 21727T, LMG 22195 and LMG 22197 are AJ626902, AJ626903 and AJ626904, respectively.
A figure showing an extended phylogenetic tree is available as supplementary material in IJSEM Online.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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). Numerical analysis showed that four LAB strains, isolated from dairy sources from three different countries, occupied a homogeneous but separate position in the dendrogram. In the present study, the taxonomic position of the four strains is investigated.
In an ongoing study, strains were isolated on different occasions from LAB in dairy products. One Belgian strain, LMG 21727T, was obtained in 2002 from a dairy factory where the strain was isolated as a contaminant of pasteurized cow's milk on plate count agar (Oxoid) at 30 °C under aerobic conditions. Two Romanian strains, LMG 22195 (=R-19307) and LMG 22196 (=R-19653), were obtained in 2002 from a commercial fermented milk product and raw cow's milk, respectively. Strain LMG 22195 was isolated and purified after direct plating on de Man–Rogosa–Sharpe agar (MRS; Oxoid) and incubation at 37 °C under aerobic conditions. Strain LMG 22196 was isolated and purified on Streptococcus thermophilus agar (Dave & Shah, 1996) and aerobic incubation at 42 °C. A Moroccan strain, LMG 22197 (=R-7861=CCMM B208), was isolated in 1999 from white soft cheese after enrichment in MRS broth for 24 h at 30 °C under aerobic conditions and purification on MRS agar.
Basic microbiological tests demonstrated that the strains were Gram-positive, catalase- and oxidase-negative, non-motile cocci. Cultivation conditions for further experiments and for maintenance of the dairy isolates and reference strains listed in Table 1 were MRS agar and incubation at 30 °C for 24–48 h, unless indicated otherwise.
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. 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). Whole-cell protein profiles of the dairy isolates were initially compared with an in-house database, comprising profiles of nearly all LAB species with validly published names. The strains constituted a homogeneous and separate cluster among profiles of enterococci (Pot & Janssens, 1993; data not shown). Fig. 1 shows a dendrogram that was obtained after UPGMA linkage cluster analysis of all four dairy isolates and the type strains of Enterococcus saccharolyticus and Enterococcus sulfureus (closest phylogenetic neighbours; see below).
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. 16S rRNA gene amplification, purification and sequencing were 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 by using a BigDye Terminator cycle sequencing kit (Applied Biosystems) and purified by using a Montage SEQ96 sequencing reaction cleanup kit (Millipore). Sequencing was performed by using an ABI Prism 3100 genetic analyser (Applied Biosystems). Sequence assembly was done by using the program AutoAssembler (Applied Biosystems). The 16S rRNA gene sequences (continuous stretches of 1509 bp) and sequences of strains retrieved from GenBank/EMBL were aligned, and a phylogenetic tree was constructed by using the neighbour-joining method using the Bionumerics software package, version 3.50 (Applied Maths). Unknown bases were discarded in the analyses. Bootstrapping analysis was undertaken to test the statistical reliability of the topology of the neighbour-joining tree using 500 bootstrap resamplings of the data (Fig. 2; an extended version of Fig. 2 can be viewed as supplementary material in IJSEM Online). The complete determined sequences of the three dairy isolates revealed similarities higher than 99·9 %. Comparison with deposited sequences available in GenBank/EMBL classified the strains in the genus Enterococcus with nearest neighbours E. sulfureus and E. saccharolyticus (sequence similarities of 98·6 and 98·0 %, respectively). The latter reference taxa and the dairy isolates constituted a separate branch in the phylogenetic tree. Lower sequence similarities (97·6–97·5 %) were obtained with Enterococcus gallinarum and Enterococcus casseliflavus.
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), the strains showed similar electrophoretic patterns differing from all described enterococcal species (data not shown).
DNA G+C contents were determined for three dairy isolates, LMG 21727T, LMG 22195 and LMG 22197, and the reference strains E. saccharolyticus LMG 11427T and E. sulfureus LMG 13084T. Cells were cultivated in MRS broth at 37 °C for 24 h. DNA was extracted from 0·5–0·75 g (wet wt) by using the protocol described by Marmur (1961) with the following modifications: (i) cells were suspended overnight in Tris/HCl buffer pH 8·0 that contained lysozyme (8 mg ml–1) before 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 
-phage DNA (Sigma) was used as the calibration reference. DNA G+C contents of strains LMG 21727T, LMG 22195 and LMG 22197 were 39, 39 and 38 mol%, respectively. These values are similar to the DNA G+C contents determined for the type strain of E. saccharolyticus LMG 11427T (37 mol%) and of E. sulfureus LMG 13084T (38 mol%), results which confirm the data described in literature (Devriese & Pot, 1995).
DNA–DNA hybridizations were performed between strains LMG 21727T, LMG 22195 and LMG 22197, E. saccharolyticus LMG 11427T and E. sulfureus LMG 13084T (DNA was prepared as described above). The microplate method was used as described by Ezaki et al. (1989) and Goris et al. (1998), using a HTS7000 bio assay reader (Perkin Elmer) for the fluorescence measurements. Biotinylated DNA was hybridized with unlabelled ssDNA, which was bound non-covalently to microplate wells. Hybridizations were performed at 36 °C in hybridization mixture (2x SSC, 5x Denhardt's solution, 2·5 % dextran sulfate, 50 % formamide, 100 µg denaturated salmon sperm DNA ml–1, 1250 ng biotinylated probe DNA ml–1). The DNA relatedness percentages presented are means, based on at least two independent hybridization experiments. Reciprocal reactions (e.g. AxB and BxA) were performed and also considered as independent hybridization experiments.
Hybridization levels of 11–13 % were found between the type strain of E. sulfureus and strains LMG 21727T, LMG 22195 and LMG 22197; and a level of 11 % between the type strain of E. saccharolyticus and strains LMG 21727T, LMG 22195 and LMG 22197. Hybridization levels of 72–79 % were found between LMG 21727T, LMG 22195 and LMG 22197; this indicates that the dairy strains constitute a single species.
Growth tests were carried out as described by 
vec et al. (2001). Acidifications of carbohydrates were recorded after 3 days incubation in API 50 CH galleries under paraffin cover. Lancefield antigens were detected using the Streptococcal grouping kit (Oxoid). Biochemical reactions were determined in the API 20 STREP system (bioMérieux). VP tests were duplicated using Voges–Proskauer diagnostic tablets (Rosco). Although the new taxon evidently belongs to the genus Enterococcus, several phenotypic traits do not conform with the physiological characteristics attributed to this genus. The strains did not tolerate 6·5 % NaCl and growth and aesculin degradation on bile aesculin agar (Oxoid) was poor. They were severely inhibited by 0·4 % sodium azide-containing Slanetz–Bartley agar (Oxoid). Moreover, they did not produce acid from carbohydrates such as ribose, 
-gentiobiose and arbutin, which are commonly degraded by enterococci (Devriese et al., 1993). A detailed description of other phenotypic characteristics of the novel species is given below and characteristics differentiating the novel species from its closest relatives E. sulfureus and E. saccharolyticus are shown in Table 2.
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Description of Enterococcus saccharominimus sp. nov.
Enterococcus saccharominimus (sac.cha.ro.mi'ni.mus Gr. n. sacchar sugar; L. sup. adj. minimus; very least N.L. sup. adj. saccharominimus meaning that this organism differs from other enterococci in that it lyses only few sugars).
Cells are irregularly sized, Gram-positive and coccal or ovoidal, which are predominantly arranged in small groups. Colonies on Columbia sheep blood agar are smaller than commonly seen with the enterococci (up to 2 mm in diameter), unpigmented, regular and translucent, surrounded by zones of semi-transparent 
-haemolysis. The strains grow equally well at 30 and 37 °C. Addition of 5 % CO2 does not enhance growth. They form deposits with clear supernatants in brain heart infusion broth (Oxoid) and react weakly with Lancefield group D antiserum (Streptococcal grouping kit; Oxoid). Not motile. All strains tested positive in API 20 STREP tests with Voges–Proskauer reagents and for pyrrolidonyl arylamidase and leucine arylamidase. Aesculin is degraded weakly. In API 50 CH galleries, acid is produced from galactose, D-glucose, D-fructose, D-mannose, N-acetylglucosamine, maltose, lactose and sucrose. Reactions are strain-dependent for acid production from amygdalin, cellobiose, mannitol, methyl -D-glucoside, salicin, trehalose, D-turanose and D-tagatose. All strains tested negative for activity of hippurate, -glucuronidase, -galactosidase, -galactosidase and alkaline phosphatase, and for acid production from glycerol, erythritol, DL-arabinose, ribose, DL-xylose, adonitol, methyl -xyloside, L-sorbose, rhamnose, dulcitol, inositol, methyl -D-mannoside, arbutin, melibiose, inulin, melezitose, D-raffinose, starch, glycogen, -gentiobiose, xylitol, D-lyxose, DL-fucose, DL-arabitol, gluconate and 2- and 5-ketogluconate. DNA G+C content is 38–39 mol%. Habitat: dairy products.
The type strain is LMG 21727T (=CCM 7220T), which was isolated from contaminated pasteurized cow's milk.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Dave, R. I. & Shah, N. P. (1996). Evaluation of media for selective enumeration of Streptococcus thermophilus, Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus acidophilus and bifidobacteria. J Dairy Sci 79, 1529–1536.[Abstract]
Devriese, L. A. & Pot, B. (1995). The genus Enterococcus. In The Genera of Lactic Acid Bacteria, pp. 327–367. Edited by B. J. B. Wood & W. A. Holzapfel. London: Blackie.
Devriese, L. A., Pot, B. & Collins, M. D. (1993). Phenotypic identification of the genus Enterococcus and differentiation of phylogenetically distinct enterococcal species and species groups. J Appl Bacteriol 75, 399–408.[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.
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]
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.
Niemann, S., Puhler, 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. & Janssens, D. (1993). The potential role of a culture collection for identification and maintenance of lactic acid bacteria. In The Lactic Acid Bacteria, Proceedings of the First Lactic Acid Computer Conference, pp. 81–87. Edited by E.-L. Foo, H. G. Griffin, R. Möllby & C.-G. Hedèn. Norfolk, UK: Horizon Scientific Press.
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, UK: Wiley.
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. & 7 other authors (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.
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