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1 Centre National de Référence des Streptocoques, Service de Microbiologie, Hôtel Dieu, AP-HP, Université Paris VI, 1 place du Parvis Notre-Dame, F-75181 Paris 04, France
2 Unité Biodiversité des Bactéries Pathogènes Emergentes, INSERM U389, Institut Pasteur, Paris, France
Correspondence
Anne Bouvet
anne.bouvet{at}htd.ap-hop-paris.fr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The GenBank accession numbers for the 16S rRNA gene sequences determined in this study are AF429762–AF429766.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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reported a strong association between Streptococcus bovis bacteraemia and colonic tumours in humans. This association led to great interest in the identification of group D streptococci, which are common inhabitants of the intestinal flora of humans and animals (Facklam, 1972; Farrow et al., 1984; Osawa et al., 1995). Since the initial antigen-based description of the group D streptococci by Lancefield (1933), Streptococcus equinus and S. bovis have been separated from Streptococcus suis, which may possess this antigen (Bentley et al., 1991; Chatellier et al., 1998; Farrow et al., 1984; Jones et al., 1972; Kawamura et al., 1995, Kilpper-Bälz et al., 1982).
A genetic classification of the group D streptococci was proposed by Farrow et al. (1984), who delineated six different DNA groups in the ‘S. equinus/S. bovis’ complex. DNA group 1 included S. equinus and S. bovis. DNA groups 2, 3 and 4 corresponded to three different unnamed genospecies. DNA group 5 was named Streptococcus saccharolyticus and group 6 was named Streptococcus alactolyticus. Phylogenetic analysis of 16S rDNA sequences reinforced the separation of S. equinus and S. bovis from other streptococcal species (Bentley et al., 1991) and assigned S. saccharolyticus to the genus Enterococcus (Rodriguez & Collins, 1990). S. equinus, S. bovis and S. alactolyticus are commonly identified according to the biochemical schemes proposed by Facklam (1972), Farrow et al. (1984) and Coykendall & Gustafson (1985). Human S. bovis strains are delineated into two biotypes according to their ability (biotype I) or inability (biotype II) to ferment mannitol. Furthermore, Osawa (1990) demonstrated that the S. bovis biotype I strains produce tannase. Indeed, hydrolysis of tannins and decarboxylation of gallic acid are characteristic of strains of DNA group 2 of Farrow et al. (1984), which Osawa et al. (1995) called Streptococcus gallolyticus. Simultaneously, Brooker et al. (1994) proposed that tannin-tolerant strains isolated from goats should be named Streptococcus caprinus. Phenotypic, genotypic and phylogenetic results have since indicated that S. gallolyticus and S. caprinus belong to the same species (Sly et al., 1997).
Another medically important species, Streptococcus infantarius (Bouvet et al., 1997; Schlegel et al., 2000), two other novel species isolated from dairy products, Streptococcus macedonicus (Tsakalidou et al., 1998) and Streptococcus waius (Flint et al., 1999), and S. suis (Kilpper-Bälz & Schleifer, 1987) have not been included in these conventional schemes. Our laboratory has conducted genotypic studies on strains of S. bovis biotype II.1 (mannitol- and 
-glucuronidase-negative and 
-galactosidase-positive). Our results have shown that S. bovis biotype II.1 includes strains belonging to DNA group 1 of Farrow et al. (1984), whereas the strains of S. infantarius subsp. infantarius, which have a closely related phenotype, belong to DNA group 4 (Bouvet et al., 1997; Schlegel et al., 2000). Strains of S. bovis/S. equinus DNA group 1 have been isolated from humans, animals and food products, whereas strains of S. infantarius have mostly been obtained from human infections. S. macedonicus (Tsakalidou et al., 1998) and S. waius (Flint et al., 1999) have been included in the ‘S. bovis/S. equinus’ complex on the basis of their 16S rDNA sequences. However, hybridization experiments have been carried out between the DNAs of the type strains of these two species and the DNA of only a limited number of reference strains (Manachini et al., 2002). These considerations indicate that the relationships between these two species and their position in the ‘S. bovis/S. equinus’ complex remain questionable.
In parallel with our investigations, Poyart et al. (2002) undertook sequence analysis and phylogenetic studies of the sodAint gene in group D streptococci. They propose the recognition of two additional species, which they called Streptococcus lutetiensis and Streptococcus pasteurianus, for S. bovis biotype II.1 and biotype II.2 strains, respectively. However, S. lutetiensis exhibits both phenotypic and genetic similarity to S. infantarius subsp. coli (Schlegel et al., 2000), and preliminary results show a close relationship between S. pasteurianus and S. gallolyticus.
In the present study, we examined isolates of S. bovis biotype II.2 (mannitol-negative, -glucuronidase- and -mannosidase-positive) from various human infections. We have reappraised the classification of the ‘S. bovis/S. equinus’ complex proposed by Farrow et al. (1984). Using DNA–DNA hybridizations and 16S rDNA sequences, we demonstrate that S. macedonicus and S. waius are subjective synonyms and that S. bovis biotype II.2 strains, S. gallolyticus and S. macedonicus are subspecies of a single species. The name S. gallolyticus has nomenclatural priority according to Rule 24b(2) of the International Code of Nomenclature of Bacteria (Lapage et al., 1992). We propose to designate these three subspecies S. gallolyticus subsp. pasteurianus subsp. nov., S. gallolyticus subsp. gallolyticus subsp. nov. and S. gallolyticus subsp. macedonicus subsp. nov., respectively.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Pure cultures were stored at -80 °C in brain heart infusion broth supplemented with 15 % (w/v) glycerol. All subcultures were carried out on Columbia blood agar (bioMérieux) or in buffered glucose broth (Bio-Rad).
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). Production of enzymes and fermentation of carbohydrates were determined using both the API 20 Strep and Rapid ID32 STREP strips, according to the instructions of the manufacturer (bioMérieux). Production of tannase was assayed by the colorimetric test of Osawa et al. (1995). Investigation of this enzyme was preferred to the assessment of gallate decarboxylase, since the former test is easier to carry out and because both enzymes are present in most strains of S. gallolyticus (Osawa et al., 1995). Moreover, the hydrolysis of tannin is known to be the first step of this decarboxylation pathway (Chamka et al., 2002). Isolates were investigated for the presence of the Lancefield group D antigen by using a latex-bead agglutination reagent after enzymic lysis at 37 °C for 30 min (Pastorex; Bio-Rad).
Preparation of DNA.
Bacteria were grown in 1·5 l buffered glucose broth for 5–7 h to late exponential phase at 37 °C. One hour before harvesting of the bacteria, glycine (30 g l-1; Sigma) was added to the culture broth to facilitate cell lysis. Bacteria were then harvested by centrifugation, washed in TE buffer (10 mM Tris/HCl, 1 mM disodium EDTA, pH 8) and suspended in lysis buffer (10 mM Tris/HCl, 1 mM disodium EDTA, 1 M sucrose, pH 8) (Bouvet et al., 1991). Mutanolysin (5 U ml-1; Sigma Aldrich) and lysozyme (10 mg ml-1; Boehringer Mannheim) were added and the mixture was incubated overnight at 37 °C (Grimont & Grimont, 1995). Bacterial membrane disruption was achieved by adding proteinase K (0·4 mg ml-1) and SDS (1 %, w/v). For several strains, lysis was completed with an additional 24 h incubation of the mixture at 37 °C. DNA was extracted and purified using the phenol/chloroform method (Grimont & Grimont, 1995).
Quantitative DNA–DNA hybridization.
DNA from type or reference strains S. bovis HDP 89505T, S. equinus HDP 89506T, S. gallolyticus HDP 98035T, S. bovis biotype II.2 HDP 90084, S. infantarius subsp. infantarius HDP 90056T, S. infantarius subsp. coli HDP 90246T, S. alactolyticus HDP 90057T, S. macedonicus HDP 98362T and S. waius HDP 99442T was labelled in vitro with [3H]ATP, [3H]TTP, [3H]GTP and [3H]CTP using the Megaprime DNA labelling reaction kit (all from Amersham). Hybridizations of these labelled DNAs with DNA of representative strains of the ‘S. bovis/S. equinus’ complex were carried out in a liquid medium under stringent conditions consisting of 60 °C for 16 h, according to a modification of the S1 nuclease/trichloracetic acid precipitation method (Crosa et al., 1973; Grimont et al., 1980). The temperature at which 50 % of the reassociated DNAs were hydrolysed by S1 nuclease (Tm) was determined. The difference between the melting temperatures of homoduplexes and heteroduplexes (
Tm) allowed the estimation of DNA divergence between strains with high levels of DNA relatedness (Grimont et al., 1980).
16S rDNA sequence determination and phylogenetic analysis.
PCR amplification of 16S rRNA-encoding DNA and sequencing of amplified fragments were carried out as described previously (Janvier & Grimont, 1995) for S. equinus HDP 89506T, S. bovis biotype II.2 HDP 90084, S. infantarius subsp. infantarius HDP 90056T, S. infantarius subsp. coli HDP 90246T and the reference strain of DNA group 3, HDP 90058. Alignment with a selection of the available sequences of 16S rDNA from GenBank and phylogenetic analysis of the 16S rDNA data were performed with the MEGALIGN program from the DNAstar package. Sequences were aligned by using the CLUSTAL multiple-sequence method. A distance matrix was then computed using a Kimura model for nucleotide substitution. Phylogenetic trees were generated from the distance matrices by using the neighbour-joining method.
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RESULTS |
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- or non-haemolytic on sheep-blood agar in an aerobic atmosphere. They were tellurite-negative. None of the strains, except the type strain of Enterococcus saccharolyticus, grew on bile/aesculin agar or in 6·5 % NaCl broth. All the strains produced leucine aminopeptidase and alanyl-phenylalanyl-proline arylamidase. Table 2 shows the reactions obtained with the 84 strains representative of all species of group D streptococci sensu stricto, the two strains of DNA group 3 of Farrow et al. (1984) and the type strains of S. suis and E. saccharolyticus. Except for S. suis and E. saccharolyticus, they did not produce pyrrolidonyl arylamidase. Most strains produced acetoin according to the Voges–Proskauer test, and did not produce arginine dihydrolase. However, the strains of DNA group 3 and E. saccharolyticus were Voges–Proskauer-negative and the strain of S. suis was Voges–Proskauer-negative and arginine dihydrolase-positive. A limited number of biochemical tests allowed further assignment to different species within the ‘S. bovis/S. equinus’ complex (Table 2). These included acidification of mannitol and hydrolysis of gallate (production of tannase) by S. gallolyticus, production of -glucuronidase by S. bovis biotype II.2 and S. suis, production of -mannosidase by S. bovis biotype II.2, absence of production of -glucuronidase and production of -galactosidase (-GAR test) by S. macedonicus and S. waius and the absence of -glucosidase by S. macedonicus, S. waius and by most of the strains of S. infantarius subsp. infantarius. The association of the production of -glucuronidase, -galactosidase (-GAL test) and -mannosidase with the acidification of trehalose clearly distinguished the S. bovis biotype II.2 strains from other species (Table 2).
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and 4 summarize the results obtained in hybridization experiments with radiolabelled DNA of type and reference strains. The existence of four DNA homology groups within the ‘S. bovis/S. equinus’ complex was demonstrated. We designated these DNA clusters using Roman numerals, instead of the Arabic numerals used for the DNA–DNA homology groups of Farrow et al. (1984). DNA cluster I is formed by the type strains of S. equinus and S. bovis (Table 3). S. gallolyticus HDP 98035T, S. macedonicus HDP 98362T, S. waius HDP 99422T and a strain of S. bovis biotype II.2, HDP 90084, appeared to belong to the same DNA cluster, cluster II (Table 4). According to the DNA–DNA relatedness analysis, this cluster contains three different subgroups. Within each subgroup, the level of relatedness between strains was over 77 % (Tm
2 °C), whereas the relatedness between the different subgroups ranged from 48 to 93 % (Tm6 °C). The first subgroup in this DNA cluster, cluster II, included the reference strain HDP 90055 of DNA homology group 2 of Farrow et al. (1984), the type strains of S. gallolyticus and S. caprinus and strain HDP 90299, identified biochemically as S. gallolyticus (81–100 % DNA relatedness). The second subgroup included the type strains of S. macedonicus and S. waius (77–100 % DNA relatedness, Tm2 °C). The last subgroup was formed by strains of S. bovis II.2, which were biochemically and genetically homogeneous (100 % DNA relatedness among the strains tested). A high level of DNA reassociation was also found for S. infantarius subsp. infantarius and S. infantarius subsp. coli, which constitute DNA cluster III (64–67 % between strains of the two subspecies). The strain of S. alactolyticus appeared to belong to another relatedness group (DNA cluster IV) within the ‘S. bovis/S. equinus’ complex.
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, HDP 90058. 16S rDNA sequences of other representative streptococcal strains were obtained from GenBank. We selected long (>1350 bp) and high-quality (<1 % undetermined positions) sequences of type or reference strains. The length of the alignment was further limited to 1350 sites to reduce the weight of gaps and mismatches at the beginning or end of the sequences. The corresponding phylogenetic tree (Fig. 1) may be divided into seven major clusters: the ‘S. bovis/S. equinus’ complex, the ‘thermophilic’ streptococci, the ‘milleri’ group, the pyogenic streptococci, the S. suis species, the Streptococcus mitis group and the ‘mutans’ group. This 16S rDNA-based analysis led to classification of the DNA group 3 reference strain within the cluster of S. suis, as demonstrated by the high level of similarity of the sequence of strain HDP 90058 to S. suis type 22 (98·9 %). S. bovis, S. equinus, S. gallolyticus, S. infantarius subsp. infantarius, S. infantarius subsp. coli, S. alactolyticus, S. macedonicus and S. waius were grouped together within the ‘S. bovis/S. equinus’ complex (Fig. 1).
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; Fig. 2). Three major divisions corresponding to four DNA clusters were observed. The first division included S. infantarius (DNA cluster III) and ‘S. bovis/S. equinus’ (DNA cluster I). The second division corresponded to S. gallolyticus, S. macedonicus, S. waius and S. bovis biotype II.2 (DNA cluster II). The third division included S. alactolyticus and Streptococcus intestinalis (DNA cluster IV). The 99·5 % similarity between S. waius and S. macedonicus favours the notion of a single species. In DNA cluster II, the divergence between the 16S rDNA sequences from S. bovis biotype II.2 HDP 90084, S. gallolyticus HDP 98035T, S. macedonicus HDP 98362T and S. waius HDP 99422T ranged from 2·6 to 7·1 %.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Facklam & Washington, 1991; Farrow et al., 1984; Hardie, 1986; Seeley & Dain, 1960). However, phenotypic heterogeneity was recognized among isolates in terms of carbohydrate fermentation, starch hydrolysis and production of glucan from sucrose. These variations led to the description of ‘variant strains' of S. bovis by different authors (Coykendall & Gustafson, 1985; Facklam, 1972; Knight & Shlaes, 1985; Nelms et al., 1995). The species S. alactolyticus, S. suis, S. gallolyticus, S. macedonicus, S. waius and S. infantarius were further described. In this study, we have collected a large number of strains to improve the classification of the ‘S. bovis/S. equinus’ complex and related species. Most of these strains have already been subjected to genetic analyses such as DNA–DNA hybridization or 16S rDNA sequencing. Some additional fresh clinical isolates were included when necessary to establish the phenotypic description of species.
Our results are consistent with the data of Farrow et al. (1984) on group D streptococci. The type strain and isolates of S. bovis (identified as S. bovis biotype II.1) differed from S. equinus by a small number of positive biochemical reactions, such as the production of -galactosidase and the acidification of starch, lactose and raffinose. DNA–DNA hybridization experiments using total DNA from S. bovis and S. equinus type strains displayed between 91 and 100 % relatedness (Table 3
; DNA cluster I). Our results are in accordance with previous studies (Garvie & Farrow, 1981; Kilpper-Bälz et al., 1982; Farrow et al., 1984, Knight & Shlaes, 1985; Nelms et al., 1995) that have demonstrated that S. bovis and S. equinus form a single genospecies (DNA group 1 of Farrow et al., 1984). In addition, we have determined the 16S rDNA sequence of S. equinus HDP 89506T and compared it with those of the other type strains of the ‘S. bovis/S. equinus’ complex. The 16S rDNA sequence of S. bovis HDP 89505T differs from that of S. equinus HDP 89506T by only 15 sites along a 1455-base fragment (99·0 % similarity). These high degrees of similarity both in total DNA–DNA hybridization and in 16S rDNA sequence confirm that S. bovis and S. equinus belong to a single species, according to the overall criteria of Stackebrandt & Goebel (1994), Drancourt et al. (2000) and Stackebrandt et al. (2002). Therefore, S. equinus and S. bovis can be considered definitively as a single species. The name S. equinus has nomenclatural priority, though S. bovis is widely used to designate these species in the medical literature.
DNA group 2 of Farrow et al. (1984) included mannitol-hydrolysing strains isolated from bovine mastitis specimens and from human blood cultures. The existence of tannase and gallate decarboxylase activities has been demonstrated in these strains, and it was proposed that they be designated as S. gallolyticus (Osawa, 1990; Osawa et al., 1995). The initial report by these authors indicated the existence of atypical strains of S. bovis that lacked tannase activity and that yielded significant relatedness with S. gallolyticus (60–66 % DNA–DNA hybridization with the type strain ACM 3611T). From this study, DNA cluster II includes S. gallolyticus, S. bovis biotype II.2, S. macedonicus and S. waius in three separate subgroups. The DNA relatedness ranged from 48 to 93 % (Tm<6 °C) between different subgroups. The levels of hybridization of total DNA between S. gallolyticus and S. bovis biotype II.2 are similar to the results of Coykendall & Gustafson (1985), Garvie & Bramley (1979) and Osawa et al. (1995). Only a few biochemical characteristics (acidification of arbutin and melibiose) distinguish S. macedonicus from S. waius. Both the DNA–DNA relatedness of the type strains, HDP 98362T and HDP 99422T, and the phylogeny of the 16S rDNA sequences indicate a close relationship (Fig. 2; Table 5). Our results are in agreement with the proposition of Manachini et al. (2002) to group the two species together. The name S. macedonicus, which was the first to be validly published, must be maintained, according to the Bacteriological Code (Lapage et al., 1992).
We show that the 16S rDNA sequence of strain CIP 105070T, proposed by Poyart et al. (2002) as the type strain of the novel species S. pasteurianus, clusters with that of S. bovis biotype II.2 (strain HDP 90084; Fig. 2). In addition, the biochemical patterns of S. pasteurianus strains, including HDP 90084, and the DNA–DNA hybridization values reported by Poyart et al. (2002) indicate that S. pasteurianus is similar to our subgroup of S. bovis biotype II.2 strains. However, the levels of DNA–DNA hybridization reported by Poyart et al. (2002) and determined in the present study, as well as the values of Tm (Table 4), do not allow these strains to be proposed as distinct species (Wayne et al., 1987; Stackebrandt et al., 2002). Therefore, S. gallolyticus, S. macedonicus, S. waius, S. pasteurianus and S. bovis biotype II.2 can be merged into a single species. In addition, phylogenetic analysis of 16S rDNA does not separate the subgroups into different species at the cut-off level of 97 % similarity. However, the topology of the phylogenetic trees that we have constructed (Figs 1 and 2) confirms the existence of separate subspecies. Taking together the variability of biochemical patterns between subgroups, the differences in reassociation levels between total DNAs of strains and the divergence of the 16S rDNA sequences, we propose the division of DNA cluster II into three subspecies. This cluster contains the reference strain of the unnamed DNA group 2 of Farrow et al. (1984) and the former type strains of S. gallolyticus, S. macedonicus, S. waius and S. pasteurianus. Taxonomic rules (Lapage et al., 1992) imply use of the name of S. gallolyticus, as it was validated in 1996. We propose S. gallolyticus subsp. gallolyticus subsp. nov., S. gallolyticus subsp. pasteurianus subsp. nov. and S. gallolyticus subsp. macedonicus subsp. nov. as the names for the three different taxa, which were respectively formerly designated as S. gallolyticus, S. bovis biotype II.2 (or S. pasteurianus according to Poyart et al., 2002) and S. macedonicus (or S. waius).
DNA cluster III includes the strains of S. infantarius subsp. infantarius and S. infantarius subsp. coli that we described previously (Schlegel et al., 2000). This species demonstrates limited DNA–DNA hybridization with and an elevated percentage of divergence from the subspecies of S. gallolyticus. The analysis of 16S rDNA sequences yielded the phylogenetic relationships of S. infantarius with S. bovis or S. equinus (DNA cluster I). As shown in Fig. 2, the subspecies previously described as S. infantarius subsp. coli includes strains proposed as S. lutetiensis by Poyart et al. (2002). We have previously hybridized one of these strains (NCDO 1600=NEM 1764=HDP 97317) with other strains of S. infantarius (Schlegel et al., 2000); under stringent conditions, we found DNA–DNA relatedness values of 93 % with strain HDP 90246T of S. infantarius subsp. coli and 69 % (Tm=1 °C) with S. infantarius subsp. infantarius HDP 90104. These results are not in agreement with the conclusions of Poyart et al. (2002) based on phylogenetic analysis of sodAint genes and we therefore propose to maintain the two subspecies of S. infantarius. In our original description, no type strain was designated for S. infantarius subsp. coli (Schlegel et al., 2000); we have therefore included descriptions of the two subspecies of S. infantarius below.
The 16S rDNA phylogenetic analysis also confirmed the inclusion of S. alactolyticus (DNA group 6 of Farrow et al., 1984) and Streptococcus intestinalis (Robinson et al., 1988) in the ‘S. bovis/S. equinus’ complex as a single species, S. alactolyticus. Vandamme et al. (1999) had noticed that isolates identified as S. alactolyticus or S. intestinalis were indistinguishable using SDS-PAGE of whole-cell proteins. Our comparison of the 16S rDNA sequences of the two type strains displayed six nucleotide differences along a 1350 bp stretch (i.e. 0·4 % divergence), which is consistent with the existence of a single species. S. alactolyticus is presently the sole species belonging to DNA cluster IV.
No strain belonging to DNA group 3 has been identified since the study of Farrow et al. (1984). This is primarily due to the large variability of biochemical characteristics of these strains (Farrow et al., 1984) or to a misidentification of these isolates. The analysis of 16S rDNA sequences leads to a change in the phylogenetic position of DNA group 3 from the ‘S. bovis/S. equinus’ complex to the cluster of S. suis. Some controversial data may be found in the literature about the taxonomic position of S. suis. Because some strains of S. suis may react with antisera of serogroups R, S, T and D (Farrow et al., 1984; Kilpper-Bälz & Schleifer, 1987), this species was initially associated with group D streptococci in the ‘S. bovis/S. equinus’ complex. In the study of Farrow et al. (1984), S. suis exhibited relatively high DNA–DNA relatedness values with seven strains of DNA group 3 isolated from raw milk or from porcine mastitis specimens, although its apparent relationship with S. bovis decreased dramatically under stringent conditions (approx. 40 %). The species S. suis was constructed by successive addition of strains isolated from diseased pigs. These strains were selected because they shared identical biochemical characteristics (i.e. they are Voges–Proskauer-negative, arginine dihydrolase-positive, hydrolyse aesculin, produce -glucuronidase and acidify trehalose and starch). They were distinguished according to a serologically specific scheme (Chatellier et al., 1998; Gottschalk et al., 1989; Kilpper-Bälz & Schleifer, 1987). The phylogenetic studies of Kawamura et al. (1995) and Chatellier et al. (1998) have shown that S. suis is distant from all other streptococcal species. Our 16S rDNA sequence analyses allow the inclusion of the sequence corresponding to the reference strain for DNA group 3, HDP 90058, within the cluster of S. suis (Fig. 1). The biochemical pattern of these strains appeared distinct from that of S. suis HDP 90052T (Table 2). This is in agreement with the phenotypic variability previously recognized among the serotypes of S. suis (Tarradas et al., 1994), which suggests that S. bovis DNA group 3 is similar to S. suis serotype 22, described in 1989 (Gottschalk et al., 1989).
The taxonomic position of E. saccharolyticus, which previously belonged to streptococcal DNA group 5 of Farrow et al. (1984), has been clarified previously by Rodriguez & Collins (1990), who demonstrated these strains as being classified in the genus Enterococcus. The results obtained by reverse transcription base sequencing of cellular RNA were later confirmed by direct sequencing of the 16S rDNA (Kawamura et al., 1995).
Our study provides an update of the classification and identification of streptococci belonging to the ‘S. bovis/S. equinus’ complex sensu stricto. It includes seven species or subspecies: S. equinus, S. gallolyticus subsp. gallolyticus, S. gallolyticus subsp. pasteurianus, S. gallolyticus subsp. macedonicus, S. infantarius subsp. infantarius, S. infantarius subsp. coli and S. alactolyticus. These can be identified according to differential biochemical reactions (Table 6). The successive changes in the classification of the ‘S. bovis/S. equinus’ complex illustrate the usefulness of a polyphasic approach to bacterial identification when phenotypic, genetic or phylogenetic methods alone are insufficient for the recognition of different species and for establishing a classification.
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. The type strain is strain HDP 90056T (=ATCC BAA-102T=CCUG 43820T=CIP 103233T=NCDO 599T).
Description of Streptococcus infantarius subsp. coli subsp. nov.
The description is identical to that effectively published by Schlegel et al. (2000). The type strain is strain HDP 90246T (=CCUG 43822T=NCDO 964T), formerly designated as a reference strain.
Emended description of Streptococcus gallolyticus Osawa et al. 1996
Colonies of about 1 mm in size, when grown on blood agar for 24 h at 37 °C, are circular, unpigmented, -haemolytic or non-haemolytic. They are catalase-negative and facultatively anaerobic, but their growth is enhanced by 5 % CO2. Strains show homogeneous growth in buffered glucose broth and brain heart infusion broth. They grow in MRS broth without gas production. Growth in 6·5 % NaCl broth is variable. Cells are coccoid, Gram-positive and mostly grouped in pairs or in short chains. They are non-motile and non-sporulating. They do not produce exopolysaccharide in 5 % sucrose medium. All strains produce leucine aminopeptidase, alanyl-phenylalanyl-proline arylamidase and acetoin (Voges–Proskauer test is positive). Most of them produce -galactosidase. They do not produce pyrrolidonyl arylamidase or alkaline phosphatase. Arginine, urea and hippurate are not hydrolysed. The production of -galactosidase, -glucuronidase, -mannosidase and -glucosidase is variable, as is the hydrolysis of aesculin. Strains produce acid from lactose, maltose and sucrose, but not from arabitol, tagatose, ribose, sorbitol or cyclodextrin. Variable results are observed with glycogen, inulin, mannitol, melibiose, melezitose, methyl -D-glucopyranoside, pullulan, raffinose, trehalose and starch. Some strains may produce tannase or gallate decarboxylase. The presence of the Lancefield group D antigen is variable. The type strain is ACM 3611T (=CCUG 35224T=CIP 105428T=JCM 10005T=LMG 16802T=HDP 98035T).
Description of Streptococcus gallolyticus subsp. gallolyticus subsp. nov.
Streptococcus gallolyticus subsp. gallolyticus (gal.lo.ly'ti.cus. N.L. n. gallatum gallate; N.L. adj. lyticus able to loosen; N.L. adj. gallolyticus gallate-digesting).
This subspecies includes the strains identified as S. bovis biotype I or S. gallolyticus according to the results of Osawa et al. (1995). They hydrolyse methyl gallate (tannase activity) and they decarboxylate gallic acid to pyrogallol. Most strains ferment mannitol, trehalose and inulin. They produce acid from starch and glycogen. Most strains have been isolated from the faeces of marsupials, such as koalas, kangaroos, brushtails and possums, as well as from various mammals, such as cows, horses, pigs, dogs and guinea pigs; some strains have been isolated from the sheep rumen and some were shown to be responsible for bovine mastitis (Osawa et al., 1995; Sly et al., 1997). Most of the human strains were isolated from blood or faeces; they were often responsible for endocarditis associated with a colonic cancer. The type strain is ACM 3611T (=CCUG 35224T=CIP 105428T=JCM 10005T=LMG 16802T=HDP 98035T).
Description of Streptococcus gallolyticus subsp. macedonicus subsp. nov.
Streptococcus gallolyticus subsp. macedonicus (ma.ce.do'ni.cus. L. adj. macedonicus of Macedonia, northern Greece, where the bacterium was first isolated).
This subspecies includes strains formerly identified as S. macedonicus Tsakalidou et al. 1998 or S. waius Flint et al. 1999. The strains are positive for -galactosidase (-GAR test), negative for -glucosidase and they do not hydrolyse aesculin. They do not produce acid from glycogen or inulin. They do not produce tannase. They do not produce acid from melibiose. Production of acid from methyl -D-glucopyranoside and starch is variable. Dairy-associated S. macedonicus strains were isolated from the naturally fermented Greek Kasseri cheese, from Italian cheese and from sour mash. Strains previously named S. waius were isolated from biofilms on stainless-steel samples exposed to pasteurized skimmed milk and from dairy products (Manachini et al., 2002). The type strain is ACA-DC 206T (=LAB 617T=ATCC BAA-249T=CCUG 39970T=CIP 105683T=JCM 11119T=LMG 18488T=HDP 98362T).
Description of Streptococcus gallolyticus subsp. pasteurianus subsp. nov.
Streptococcus gallolyticus subsp. pasteurianus (pas.teur'i.a.nus. N.L. masc. adj. pasteurianus of Pasteur, referring to the Pasteur Institute, where the type strain was characterized).
This novel subspecies includes strains formerly identified as S. bovis biotype II.2 or S. pasteurianus Poyart et al. 2002. They produce -glucosidase, -glucuronidase, -mannosidase and -galactosidase (-GAL test). They produce acid from lactose, trehalose and methyl -D-glucopyranoside. Production of acid from melibiose, melezitose, raffinose and starch is variable. Production of acid from glycogen, inulin and mannitol is absent. Strains do not produce tannase, but some strains may yield a gallate decarboxylase activity (Osawa et al., 1995). Strains have been isolated from various human infections, mostly bacteraemia and endocarditis. Some strains were isolated from urinary tract infections or from suppurative infections. The type strain is NEM 1202T (=CIP 107122T).
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