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1 Laboratory of Microbiology, Ghent University, K. L. Ledeganckstraat 35, Ghent 9000, Belgium
2 BCCMTM/LMG Bacteria Collection, Ghent University, K. L. Ledeganckstraat 35, Ghent 9000, Belgium
Correspondence
Sabri M. Naser
Sabri.Naser{at}Ugent.be
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ABSTRACT |
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78 %). Data from the present study led to the proposal that E. flavescens should be reclassified as a later synonym of E. casseliflavus and that E. saccharominimus should be reclassified as a later synonym of E. italicus.
Published online ahead of print on 13 October 2005 as DOI 10.1099/ijs.0.63891-0.
The GenBank/EMBL/DDBJ accession numbers for the pheS, rpoA and atpA gene sequences determined in this study are given in Fig. 1
and Supplementary Figs S1 and S2 in IJSEM Online.
Neighbour-joining trees based on rpoA and atpA gene sequences of enterococcal strains are available as supplementary material in IJSEM Online.
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to include the Lancefield group D faecal streptococci Streptococcus faecalis and Streptococcus faecium, as Enterococcus faecalis and Enterococcus faecium (Farrow et al., 1983; Leclerc et al., 1996; Ludwig et al., 1985). E. casseliflavus was originally described as Streptococcus faecium var. casseliflavus (Mundt & Graham, 1968), later elevated to species rank as Streptococcus casseliflavus (Vaughan et al., 1979) and finally transferred to the genus Enterococcus (Schleifer & Kilpper-Bälz, 1984). The species has been isolated from plants, silage and soil (Schleifer & Kilpper-Bälz, 1984), but it is also clinically significant and has been incriminated in blood infections (Nauschuetz et al., 1993). Enterococcus flavescens was described based on four strains of enterococci isolated from humans with severe infections (Pompei et al., 1991). These strains were further investigated and designated E. flavescens (Pompei et al., 1992). Phenotypically, E. casseliflavus and E. flavescens are yellow-pigmented, motile and possess intrinsic low-level resistance to vancomycin; E. flavescens can be differentiated from E. casseliflavus through its inability to produce acid from the fermentation of ribose (Pompei et al., 1992) and its failure to produce 
-haemolysis on sheep blood (Descheemaeker et al., 1997). Since its initial identification, some doubt has remained over the validity of describing E. flavescens as a distinct species. The 16S rRNA gene sequences of the type strains of E. casseliflavus and E. flavescens show about 100 % similarity (Patel et al., 1998). Furthermore, DNA–DNA hybridization experiments confirmed that E. casseliflavus and E. flavescens constitute a single species (Teixeira et al., 1997), in contrast to the data reported by Pompei et al. (1992). Other literature data were in complete accordance with Teixeira et al. (1997), supporting the synonymy between the two taxa. Descheemaeker et al. (1997) were unable to discriminate between the two taxa using PFGE or oligonucleotide D11344-primed PCR. Clark et al. (1998) and Dutta & Reynolds (2003) reported an extensive similarity between the sequences of the vancomycin resistance genes vanC-2 of E. casseliflavus and vanC-3 of E. flavescens. Other techniques, such as (GTG)5-PCR (
vec et al., 2005), randomly amplified polymorphic DNA (RAPD) analysis (Quednau et al., 1998), tRNA intergenic spacer PCR (Baele et al., 2000) and sequence comparison of genes encoding manganese-dependent superoxide dismutase (sodAint) (Poyart et al., 2000) and D-alanine–D-alanine ligase-related enzymes (ddl) (Navarro & Courvalin, 1994) and vanC genes (Dutka-Malen et al., 1995) were also unable to differentiate between E. casseliflavus and E. flavescens. Despite these data supporting the synonymy between E. casseliflavus and E. flavescens, no formal reclassification has been proposed to date.
In the present study, we provide additional evidence that the two taxa represent a single species. Multilocus sequence analysis (MLSA) is a polygenic approach applied for accurate identification of all enterococcal species (Naser et al., 2005a, b). Partial sequences for the genes encoding the phenylalanyl-tRNA synthase alpha subunit (pheS), RNA polymerase alpha subunit (rpoA) and the alpha subunit of ATP synthase (atpA) were determined and compared for E. casseliflavus LMG 10745T, LMG 16286 and LMG 14406 and E. flavescens LMG 13518T, LMG 16313 and LMG 16314. Primer sequences, amplification conditions and sequencing reactions were as described by Naser et al. (2005a, b). In general, the interspecies-level gene sequence similarities of pheS, rpoA and atpA for all enterococcal species tested were at most 86, 97 and 92 %, respectively. Strains of the same species showed at least 97 % pheS, 99 % rpoA and 96·3 % atpA gene sequence similarity. The neighbour-joining trees of pheS, rpoA and atpA gene sequences revealed high relatedness between the investigated strains of E. casseliflavus and E. flavescens, with at least 99 % pheS, 100 % rpoA and 99 % atpA gene sequence similarity, confirming that E. flavescens and E. casseliflavus represent the same species (Fig. 1 and Supplementary Figs S1 and S2 in IJSEM Online).
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with the following modifications: the washed cell pellet was resuspended and lysed in 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. The microplate method was used as described by Ezaki et al. (1989) and Goris et al. (1998), using an HTS7000 Bio Assay reader (Perkin Elmer) for the fluorescence measurements. Biotinylated DNA was hybridized with unlabelled ssDNA, which was bound non-covalently to the microplate wells. Hybridizations were performed at 36 °C in hybridization mixture (2xSSC, 5xDenhardt's solution, 2·5 % dextran sulfate, 50 % formamide, 100 µg denatured salmon sperm DNA ml–1 and 1250 ng biotinylated probe DNA ml–1). Reciprocal reactions (e.g. AxB and BxA) were performed. The DNA–DNA binding values reported are the mean values of a minimum of four hybridization experiments, the reciprocal reactions included. E. casseliflavus LMG 10745T and LMG 14406 and E. flavescens LMG 13518T and LMG 16314 showed high DNA–DNA hybridization values in the range 78–94 %, confirming that the two taxa belong to the same species.
On the basis of the evidence presented, it is proposed that the two species E. casseliflavus and E. flavescens be united under the same name; as a rule of priority (Rules 38 and 42 of the Bacteriological Code; Lapage et al., 1992), the name E. casseliflavus should be retained and strains of E. flavescens should be reclassified as such. The type strain of E. casseliflavus is LMG 10745T (=ATCC 25788T=NCDO 2372T=MUTK 20T). The description of E. casseliflavus remains essentially the same.
In the present study, we also investigated the taxonomic relatedness between Enterococcus italicus and Enterococcus saccharominimus, as the two taxa have a 16S rRNA gene sequence similarity of >99 % and are phenotypically highly similar. The two species were described almost simultaneously in 2004. E. italicus was described by Fortina et al. (2004), who isolated the organism from cows' raw milk used in the production of artisanal Italian cheeses. E. saccharominimus was described by Vancanneyt et al. (2004) and was isolated from Belgian, Morrocan and Romanian dairy products.
MLSA of three housekeeping genes (see above) was used as an initial screening test to investigate the relatedness of the two species. Gene sequences were determined and compared for E. saccharominimus LMG 21727T, LMG 22196 and LMG 22197, E. italicus LMG 22039T and Enterococcus sp. CDC PNS-E1 (=LMG 22681), which was designated as a strain of E. italicus (R. R. Facklam, personal communication). The results confirmed that E. saccharominimus and E. italicus are very highly related, with 100 % pheS, rpoA and atpA gene sequence similarities (Fig. 1 and Supplementary Figs S1 and S2 in IJSEM Online).
Finally, DNA–DNA hybridizations were performed as described above between E. italicus LMG 22039T and LMG 22681 and beween E. saccharominimus LMG 21727T and LMG 22196. The DNA–DNA hybridization level between the four strains was in the range 78–87 %, clearly indicating that the two species constitute a single species.
On the basis of the evidence presented, it is proposed that the two species E. saccharominimus and E. italicus be united under the same name; as a rule of priority (Rules 38 and 42 of the Bacteriological Code; Lapage et al., 1992), the name E. italicus should be retained and strains of E. saccharominimus should be reclassified as such. The type strain of E. italicus is DSM 15952T (=LMG 22039T). The description of E. italicus remains essentially the same.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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