Taxonomy of the Anginosus group of the genus Streptococcus and description of Streptococcus anginosus subsp . whileyi subsp . nov . and Streptococcus constellatus subsp . viborgensis subsp . nov

The Anginosus group of the genus Streptococcus has been the subject of much taxonomic confusion, which has hampered the full appreciation of its clinical significance. The purpose of this study was to critically re-examine the taxonomy of the Anginosus group, with special attention to b-haemolytic, Lancefield group C strains, using multilocus sequence analysis (MLSA) combined with 16S rRNA gene sequence and phenotypic analyses. Phylogenetic analysis of concatenated sequences of seven housekeeping genes previously used for examination of viridans streptococci distinguished seven distinct and coherent clusters in the Anginosus group. Analyses of 16S rRNA gene sequences and phenotypic characters supported the MLSA clustering and currently recognized taxa of the Anginosus group. Single gene analyses showed considerable allele sharing between species, thereby invalidating identification based on single-locus sequencing. Two novel clusters of b-haemolytic, Lancefield group C strains within the Streptococcus constellatus and Streptococcus anginosus species and isolated from patients with sore throat showed sufficient phylogenetic distances from other clusters to warrant status as novel subspecies. The novel cluster within S. anginosus was identified as the previously recognized DNA homology cluster, DNA group 2. The names S. anginosus subsp. whileyi subsp. nov. (type strain CCUG 391595DSM 258185SK1267) and S. constellatus subsp. viborgensis subsp. nov. (type strain SK13595CCUG 623875DSM 25819) are proposed.


INTRODUCTION
The Anginosus group of the genus Streptococcus has been the subject of much taxonomic confusion, partly due to lack of international consensus on nomenclature (Facklam, 1984(Facklam, , 2002) ) and partly due to lack of reliable distinguishing phenotypic markers (Jones, 1978;Kilian et al., 1989;Whiley et al., 1990Whiley et al., , 1999)).The terms 'Streptococcus milleri' or 'Streptococcus milleri group' have been used by European and Japanese microbiologist to embrace all members of the group, whereas North American microbiologist have been using terms such as Streptococcus MG-intermedius and Streptococcus anginosus-constellatus (Facklam, 1984).Although the term 'Streptococcus milleri group' is still widely used, the term 'Anginosus group' proposed by Kawamura (1995) is preferred as it may eliminate much of the nomenclatural confusion.Currently, three species are formally recognized within the Anginosus group: Streptococcus anginosus, Streptococcus intermedius and Streptococcus constellatus, of which the lastnamed is divided into two subspecies, subsp.constellatus and subsp.pharyngis (Bentley et al., 1991;Jacobs et al., 2000a;Whiley, et al., 1999;Whiley & Hardie, 1989).This classification is primarily based on results of DNA-DNA hybridization and 16S rRNA gene sequence analyses.However, two studies using whole-cell protein electrophoretic analysis and DNA-DNA hybridization suggested that the Downloaded from www.microbiologyresearch.orgby IP: 54.70.40.11On: Fri, 09 Nov 2018 13:35:30 group constitutes a single species, S. anginosus (Coykendall et al., 1987;Vandamme et al., 1998).Other studies have suggested that the Anginosus group exhibits further taxonomic heterogeneity than today recognized (Bergman et al., 1995;Jacobs et al., 2000a, b;Whiley et al., 1999).In particular, b-haemolytic, Lancefield group C strains were in previous studies shown to constitute genetically distinct groups within the Anginosus group (Sultana et al., 1998;Whiley et al., 1997Whiley et al., , 1999)).
Members of the Anginosus group are considered commensals of the human mouth, throat, and gastrointestinal and genital tracts (Frandsen et al., 1991;Paster et al., 2001;Poole & Wilson, 1979;Whiley et al., 1993), but may cause opportunistic infections such as pharyngitis, bacteraemia, and serious purulent infections in soft tissues of the neck and in internal organs such as the brain, lungs and liver, often in association with other bacteria (Broyles et al., 2009;Clarridge et al., 2001;Jacobs et al., 1994;Reißmann et al., 2010;Whiley et al., 1992Whiley et al., , 1999).In addition, recent studies identified members of the Anginosus group as aetiologically important in pulmonary exacerbations in patients with cystic fibrosis (Parkins et al., 2008;Sibley et al., 2008).
The three species of the Anginosus group show surprising antigenic heterogeneity and include strains that react with Lancefield groups A, C, F and G antisera.In addition, strains can be b-haemolytic, a-haemolytic or non-haemolytic.These bacteria are often referred to as minute colony type opposite to their Pyogenic group counterparts in humans of large colony type b-haemolytic group C and G strains belonging to Streptococcus dysgalactiae subsp.equisimilis.
Due to lack of reliable phenotypic differences, isolates of the Anginosus group streptococci are rarely identified to species level in the clinical microbiological laboratory.However, in recent studies real-time PCR, mass spectrometry (MALDI-TOF-MS) as well as conventional PCR assays have shown their potential in discriminating between the individual species of the Anginosus group and could be usable in the clinical microbiology laboratory (Desar et al., 2008;Friedrichs et al., 2007;Olson et al., 2010;Reißmann et al., 2010;Takao et al., 2004).
Recently, Bishop et al. (2009) used multilocus sequence analysis (MLSA) of concatenated sequences of seven housekeeping genes to describe the phylogenetic relationship between bacteria of the genus Streptococcus with special attention to the phylogenetically diverse 'viridans group'.Although few strains from the Anginosus group were included in the study, the MLSA scheme showed its potential to define and circumscribe bacterial species within the Anginosus group.
The purpose of this study was to critically re-examine the taxonomy of the Anginosus group with special attention to b-haemolytic, Lancefield group C strains using MLSA combined with 16S rRNA gene sequence analysis and phenotypic characterization.The study eliminates some of the existing taxonomic confusion within the Anginosus group of streptococci.MLSA data supported by 16S rRNA gene sequence and phenotypic analyses demonstrated seven distinct clusters, two of which were novel clusters of b-haemolytic, Lancefield group C strains within the S. constellatus and S. anginosus branches.These clusters showed sufficient phylogenetic distance from other clusters of the Anginosus group to warrant status as novel subspecies of S. anginosus and of S. constellatus.

METHODS
Bacterial isolates.A total of 123 isolates from the Anginosus group of streptococci were included in the study.Fifty-nine of these were clinical isolates consecutively collected from 2006 to 2007 at Viborg Hospital, Denmark.Forty-six of these were b-haemolytic, Lancefield antigen group C isolates from patients with sore throats, while the remaining 13 isolates were isolated from blood.The blood isolates were either a-or b-haemolytic and expressed a variety of Lancefield antigens.Twenty-nine isolates from the CCUG culture collection (CCUG, Gothenburg, Sweden), 19 oral clinical isolates from Japan and 11 non-oral clinical isolates from the Streptococcus reference laboratory at Statens Serum Institut (SSI), Copenhagen, Denmark (supplied by the late Jørgen Henrichsen), were also included.The designated type strain of the four species and subspecies presently recognized in the Anginosus group, S. anginosus ATCC 33397 T , S. intermedius ATCC 27335 T , S. constellatus subsp.constellatus ATCC 27823 T , S. constellatus subsp.pharyngis CCUG 46377 T , and the strain MAS 624 representing the DNA group 2 described by Whiley et al. (1997Whiley et al. ( , 1999) ) were included as references.A full list of the strains included in the study is provided in Table 1.
Phenotypic characterization.Colony size and b-haemolysis were determined on 5 % horse blood agar plates (Statens Serum Institut, Copenhagen, Denmark) after incubation in an anaerobic chamber for 24 h at 37 uC.All isolates with no haemolysis on blood agar were incubated for 24 h at 37 uC on chocolate agar plates for detection of a-haemolysis (liberation of hydrogen peroxide) observed as green zones around colonies due to oxidation of haemoglobin.Growth patterns in Todd-Hewitt broth were observed after 24 h incubation at 37 uC and were recorded as either flocculent or diffuse.The Lancefield group antigen was identified using the Streptex method (Oxoid A/S) according to the manufacturer's instructions.Hyaluronidase activity was demonstrated as described by Smith and Willett (1968).Acid production from sorbitol and salicin, as well as glucose, lactose, raffinose, and amygdalin for clusters 4 and 7, hydrolysis of aesculin and D- arginine, and production of acetoin from glucose (Voges-Proskauer test) were detected as described by Kilian et al. (1989), with the exception that aesculin and D-arginine hydrolysis were detected after anaerobic growth for 7 days, as some isolates did not grow in these substrates under aerobic conditions.The enzymes b- glucuronidase, a-galactosidase, neuraminidase, b-D-glucosidase, b-D-galactosidase (ONPG), N-acetyl-b-galactosaminidase, a-D-glucuronidase and b-fucosidase were detected with synthetic fluorogenic substrates as described by Whiley et al. (1990).As our results for b- D-galactosidase for the DNA group 2 strains, including strain MAS 624, differed from those reported by Whiley et al. (1999), the presence of b-D-galactosidase was also tested using 2-nitrophenyl-b-D-galactopyranoside as substrate.The test was carried out in a test tube with 0.3 ml of a 0.1 % solution of 2-nitrophenyl-b-Dgalactopyranoside.The formation of a strong yellow colour after 4 h of incubation indicates the presence b-D-galactosidase PCR and sequencing of housekeeping genes.The MLSA scheme originally developed by Bishop et al. (2009) based on sequences of seven housekeeping genes to study the phylogenetic relationships and to enable online identification of isolates of the genus Streptococcus with particular focus on Mitis group streptococci was used.
Fragments of the housekeeping genes map, pfl, ppaC, pyk, rpoB, sodA and tuf were amplified by PCR and sequenced using degenerate primers as described (Bishop et al., 2009).Briefly, 1 ml of each primer (10 mM), 10 ml of DNA and 13 ml of H 2 O were added to illustra PuReTaq Ready-To-Go PCR Beads (GE Healthcare).The annealing temperature was 55 uC for map, pfl and ppaC and 50 uC for pyk, rpoB, sodA and tuf with 30 cycles of amplification for both annealing temperatures.Purified PCR products were sequenced from both directions at the Beijing Genomics Institute, China, using standard procedures.
Data analysis.Sequences of the seven housekeeping genes were edited, aligned and subjected to phylogenetic analysis using MEGA5.05(Tamura et al., 2011).Trimmed sequences from Bishop et al. (2009) were used as templates for trimming and alignment.After alignment, the trimmed sequences for each of the housekeeping genes as well as a concatenated sequence of all seven genes were subjected to phylogenetic analysis using the minimum-evolution algorithm.
Bootstrap tests were performed with 500 replicates for minimumevolution trees based on each of the seven housekeeping genes and on the concatenated sequence.The sequences of 420 streptococcus strains from the study of Bishop et al. (2009) and 95 sequences from strains of the Pyogenic group (Jensen & Kilian, 2012) were included in the phylogenetic analysis.In addition, sequences of the corresponding housekeeping genes from the genomes of S. anginosus 1_2 62CV (ADME01000013), S. anginosus F0211 (AECT01000037), S. intermedius F0395 (AFXN01000001), and S. intermedius F0413 (AFXO1000003) were extracted and included in the analysis.
Genetic distances were calculated in MEGA 5.05 using the Kimura twoparameter model.The average frequencies of synonymous substitutions per potential synonymous site (d s ) and non-synonymous substitutions per potential non-synonymous site (d n ) were calculated by the method of Nei and Gojobori in MEGA 5.05.
16S rRNA gene analyses.16S rRNA gene sequences were amplified and sequenced using the universal eubacteria primers 27f, 59-AGAGTTTGATCMTGGCTCAG-39, and 1492r, 59-TACGGYTAC-CTTGTTACGACTT-39.The reaction mix consisted of 3 ml of each primer (10 mM), 5 ml DNA and 14 ml H 2 O added to illustra PuReTaq Ready-To-Go PCR Beads (GE Healthcare).The annealing temperature was 55 uC and 30 cycles were run.Purified PCR products were sequenced from both ends using the same primers at GATC-Biotech, Konstanz, Germany, using standard procedures.A total of 1401 bases were included for phylogenetic analysis.
PCR specific for DNA group 2. To confirm the identity of one cluster to the DNA group 2 described by Whiley et al. (1997Whiley et al. ( , 1999)), two different PCR assays were employed.The first PCR assay is a multiplex assay developed by Takao et al. (2004) and is based on primers targeting the hyaluronate lyase gene, the 16S rRNA gene and the intermedilysin gene, which, by specific band patterns, can separate the species and subspecies as well as DNA group 2 of the Anginosus group.Five isolates and the type strain from each of seven MLSA clusters were analysed.The second PCR assay targeted the 16S-23S rRNA intergenic spacer region, which has shown size polymorphisms between species and subspecies within the Anginosus group (Whiley et al., 1995).Specifically, DNA group 2 strains were shown to give a band with a size of 600 bp, unique to DNA group 2 among the Anginosus group streptococci.The assay was performed as previously described (Whiley et al., 1995).

MLSA
The seven primer pairs, initially developed for amplification of housekeeping genes from 'viridans' streptococci (Mitis, Anginosus, Mutans and Salivarius groups), successfully amplified the relevant sequences from all isolates included in this study.The trimmed sequences were all of the same length, as is the case for other 'viridans' streptococci.A minimum-evolution tree based on concatenated sequences determined in this study combined with sequences of a selection of species of the genus Streptococcus represented in the MLSA Viridans streptococcus database (http://viridans.emlsa.net/) is shown in Fig. 1.In agreement with the proposal of Kawamura (1995) the tree based on concatenated sequences showed that the Anginosus group constitutes a phylogenetically separate branch within the genus Streptococcus.As demonstrated in the more detailed tree in Fig. 2, the Anginosus group can be further subdivided into seven well-resolved clusters as indicated by the bootstrap values.A neighbour-joining tree gave similar topology to the minimum-evolution tree shown in Fig. 2 (not shown).
Cluster 1 represents S. intermedius and includes the designated type strain of this species, although S. intermedius ATCC 27335 T separates from the other isolates of cluster 1 by having an rpoB allele that is similar to alleles found in isolates from cluster 5 (S.anginosus).Cluster 1 is genetically distant from the other clusters within the Anginosus group (Fig. 2 and Table 2).Cluster 2 includes classical isolates of S. constellatus including the type strain of S. constellatus subsp.constellatus, while cluster 3, which is genetically homogeneous and only includes b-haemolytic isolates with Lancefield group C antigen represents S. constellatus subsp.pharyngis.Cluster 4 is, like cluster 3, genetically homogeneous and potentially represents a novel taxon consisting of b-haemolytic, Lancefield group C isolates from patients with a sore throat.Cluster 5 comprises four very closely related and three more distantly related isolates (in particular strain CCUG 35181, the association of which to cluster 5 was supported only be a bootstrap value of 65).Cluster 5 clearly represents a separate taxon but its exact status relative to existing species depends on studies of additional strains.We tentatively refer to the cluster as S. anginosus genomosubspecies AJ1.Cluster 6 is genetically heterogeneous compared with the other clusters and includes mostly invasive, non-haemolytic isolates with Lancefield group F antigen.Interestingly, the type strain of S. anginosus forms a separate lineage with SK534 only distantly related to isolates in cluster 6, mainly because of divergent alleles in the pyk and tuf genes.The last cluster, cluster 7, is genetically homogeneous and is not closely related to other clusters within the Anginosus group.The cluster consists exclusively of b-haemolytic, Lancefield antigen group C isolates.Most isolates were collected at Viborg Hospital, Denmark, but three CCUG isolates from Sweden and the United States as well as the DNA group 2 strain MAS 624 from the Netherlands were part of the cluster.Most isolates were from sore throats, two isolates from blood and MAS 624 was from an abdominal infection.
MLSA revealed several CCUG strains that have been incorrectly assigned to species in the database (Table 1 and Fig. 2).According to MLSA, four isolates listed as S. anginosus clustered with S. constellatus (cluster 2) while two isolates listed as S. intermedius clustered with S. anginosus (cluster 6) or with S. constellatus subsp.pharyngis (cluster 3).Furthermore, strain F0395, for which the genome sequence has been released under the designation S. intermedius, clustered with S. constellatus subsp.constellatus (cluster 2).These findings illustrate the difficulties in identifying isolates from the Anginosus group correctly to species level using traditional criteria.

Single locus phylogenetic analysis
While the phylogenetic tree of the concatenated sequences clearly separates the isolates into clusters that correlate with    the present taxonomic outline of the Anginosus group, the topologies of the phylogenetic trees reconstructed using sequences of individual genes were inconsistent, indicating extensive recombination between members of the Anginosus group (Fig. S1).The ppaC locus was the only locus that yielded the approximately same phylogenetic pattern as the concatenated sequences with only a few exceptions including strain SK1366 from cluster 7, which clustered with isolates from cluster 2 (Fig. S1c).Other examples are mentioned above.While these findings illustrate extensive recombination between species within the Anginosus group in all seven housekeeping genes, no evidence of homologous recombination with other species of viridans streptococci was observed (data not shown).

Sequence characteristics of housekeeping genes
Sequence characteristics for concatenated sequences and the individual genes for the whole Anginosus group as well as the seven clusters of the Anginosus group are shown in Tables S2-S5.The ratios (d n /d s ) of non-synonymous (d n ) to synonymous (d s ) substitutions were well below 1 for all genes, indicating purification as normally observed for housekeeping genes.In general, the number of alleles and the genetic distance within the three clusters 3, 4 and 7, which exclusively contain b-haemolytic isolates with Lancefield group C antigen, were considerably lower than observed for the other clusters, indicating a recent clonal origin of these clusters.

Cluster 7
From the MLSA findings based on the concatenated sequences and on single loci, combined with the genetic distances between cluster 7 and the other clusters, it is clear that cluster 7 forms a coherent and phylogenetically distinct population within the Anginosus group.The finding that five out of seven genes show alleles unique to cluster 7 supports this conclusion.
To analyse further the genetic relationship between cluster 7 and recognized taxa within the Anginosus group, we extracted partial sequences of 54 housekeeping genes common to and distributed over the entire genomes of ten strains from the Anginosus group of streptococci.The overall clustering based on the 20 504 bp concatamer was in agreement with the clustering deduced from the MLSA based on seven genes (Fig. S2 and Table S6) and confirmed the separation of the Anginosus group into three distinct species supported by significant bootstrap values.The tree further illustrates the genetic heterogeneity of the S. anginosus cluster.Strain CCUG 39159 representing cluster 7 is clearly part of S. anginosus although separated from the three other strains of S. anginosus, including the type strain, by a distance of 0.04-0.05.The analysis also suggests that strain 1_2 62CV (ADME01000013) of cluster 5 is no more distant from S. anginosus ATCC 33397 T (0.04) than strain F0211 (AECT01000037) (0.05), which is part of cluster 6 in the tree based on seven gene loci.These data show that further analyses based on an extended number of genes are required to disclose the detailed population structure of S. anginosus.
Interestingly, in agreement with the MLSA based on seven loci the extended analysis showed that strain F0395 listed as S. intermedius is closely related to S. constellatus subsp.constellatus ATCC 27823 T , and appears to be misidentified, which is supported also by its 16S rRNA gene sequence (GenBank accession no.GU470908).

Confirmation of cluster 7 as DNA group 2
The phylogenetic analysis of the housekeeping genes suggests that cluster 7 corresponds to DNA group 2 of Whiley et al. (1997Whiley et al. ( , 1999)).To confirm this, two PCR assays reported to be able to differentiate DNA group 2 from other taxa of the Anginosus group were applied.The first PCR, which detects the 16S-23S rRNA spacer size, revealed that all isolates of cluster 7 gave the characteristic unique band of 680 bp.In the second multiplex PCR assay targeting the hyaluronate lyase gene, the 16S rRNA gene and the intermedilysin gene, the expected unique band pattern of DNA group 2 was found in all strains of cluster 7, but not in strains from any of the other clusters (data not shown).Strains from cluster 4 gave the same band pattern as strains of clusters 2 and 3 as expected for strains of S. constellatus.Strains of clusters 1, 5 and 6 gave the band pattern as expected for S. intermedius and S. anginosus, respectively, and consistent with the position of the respective type strains.

16S rRNA gene sequence analysis
Phylogenetic analysis of the 16S rRNA gene sequences of the strains yielded two main branches, of which one consists of isolates from MLSA clusters 5, 6 and 7 while the other contains the isolates from MLSA clusters 1, 2, 3 and 4 (Fig. 3).With a few exceptions, the finer clustering of 16S rRNA gene sequences resembled that based on MLSA.
Strain CCUG 52051 from MLSA cluster 5 has the same 16S rRNA allele as most of the isolates from MLSA cluster 6. Strains from MLSA cluster 2 (S.constellatus subsp.constellatus) split into two 16S rRNA clusters, one of which includes strain SK1327 from MLSA cluster 3. Finally, all isolates from cluster 7 form a distinct 16S rRNA gene cluster, but in contrast to the MLSA results, cluster 7 is very closely related to isolates from cluster 6. Inclusion of all sequences belonging to the Anginosus group from the rdp-10 database in the analysis indicates that the 16S rRNA gene sequences from our study represent the full spectrum of genetic diversity within the Anginosus group detected so far (data not shown).

Phenotypic analysis
The phenotypic characteristics of strains grouped according to the MLSA clusters are summarized in Table 3.In agreement with previous observations, the isolates of S. intermedius in cluster 1 differed from all other clusters by being positive for neuraminidase.Clusters 1 and 3 differed from the other clusters by being positive for Nacetyl-b-galactosaminidase, b-fucosidase and b-galactosidase, although a few isolates from clusters 2 and 4 were also positive for b-galactosidase.Cluster 2, corresponding to S. constellatus subsp.constellatus, was the only cluster that was negative for b-D-glucosidase.Typical isolates of S. anginosus in clusters 5 and 6 could be distinguished from all other clusters by a negative hyaluronidase reaction.Moreover, all isolates from cluster 6 showed diffuse growth in TH-medium in contrast to other taxa, which showed either exclusively flocculent growth or a mixture of growth patterns.Isolates from clusters 3, 4 and 7 were all b- haemolytic and expressed Lancefield group C antigen, but could be differentiated from each other by reactions for Nacetyl-b-galactosaminidase (cluster 3, positive), b-fucosidase (cluster 3, positive) and a-D-glucuronidase (clusters 3 and 4, positive).On blood agar plates, colonies of strains from cluster 7 produced visibly larger colonies and larger haemolytic zones when grown under CO 2 for 24 h than isolates of clusters 3 and 4.

DISCUSSION
The taxonomy, including identification, of the Anginosus group of the genus Streptococcus has always been subject to debate and difficulty due to apparently conflicting results obtained with different types of analysis combined with biochemical, antigenic and morphological heterogeneity of the species that were described.This has hampered the identification of these bacteria and resulted in frequent misidentifications and in limited appreciation of their ecology and pathogenic potential.In this study we demonstrated the value of an MLSA scheme, initially developed with focus on the Mitis group within the 'viridans' streptococci (Bishop et al., 2009), for the identification and delineation of Anginosus group streptococci.All 104 isolates and reference strains included in the study yielded useful sequences with the primers for the seven genes involved, and by analysis of concatenated sequences of these genes, we were able to distinguish robust and coherent clusters.Results of this study and our parallel work on the Pyogenic group (Jensen & Kilian, 2012) further emphasize the potential of the MLSA scheme for The MLSA results clearly demonstrate the polygenetic nature of the Anginosus group, with genetic distances between species that are similar to those found in the Mitis and Pyogenic groups (Bishop et al., 2009;Jensen & Kilian, 2012), thereby rejecting suggestions that the Anginosus group consists of a single species as suggested based on whole-cell protein electrophoretic analysis (Vandamme et al., 1998) and DNA-DNA hybridization (Coykendall et al., 1987).DNA-DNA hybridization, long considered the gold standard for species delineation and identification, has limited reproducibility as results may differ according to method and procedure conditions.These problems are illustrated by the considerably different DNA-DNA hybridization values obtained for the type strains of the Anginosus group by different research groups (Coykendall et al., 1987;Whiley et al., 1999).Our results support the more recent suggestion that MLSA is better suited for species identification and delineation (Stackebrandt et al., 2002) as later demonstrated for several genera (Bishop et al., 2009;Nørskov-Lauritsen et al., 2005;Thompson et al., 2007).
Our findings also demonstrate that species identification within the Anginosus group of the genus Streptococcus using a one-gene approach is problematic due to extensive homologous recombination between members of the individual phylogenetic lineages.For example, the genes rpoB and the sodA were suggested as being suitable for identification of streptococci to species level (Drancourt et al., 2004;Poyart et al., 1998).We show that, based on the rpoB gene sequences, the novel cluster 4 belonging to the S. constellatus species was indistinguishable from several strains of S. anginosus in clusters 5 and 6.Likewise, S. constellatus subsp.constellatus and S. constellatus subsp.pharyngis as well as cluster 4 could not be differentiated by rpoB gene sequence analysis.In addition, one strain from the novel cluster 7 would have been identified as S. intermedius if the identification were based on the sodA gene sequence alone.These results, which are analogous to observations reported for streptococci of the Mitis and Pyogenic groups (Hoshino et al., 2005;Jensen & Kilian, 2012;Kilian et al., 2008), also emphasize the problems of evaluating identification schemes with only a few isolates from each species.
Isolates identified as S. anginosus in previous studies and according to phenotypic characters and the 16S rRNA sequences collected in our study were not restricted to a single cluster but were found in cluster 5 as well as in  cluster 6.The type strain of S. anginosus was by MLSA more distantly related to the other strains included in cluster 6 due to divergent alleles, especially in the pyk and tuf genes.
Combined with the unique phenotype of S. anginosus ATCC 33397 T this may indicate that the present type strain of S. anginosus does not adequately represent S. anginosus as generally conceived by bacteriologists.This was also noted by Whiley et al. (1997).Interestingly, according to the MLSA based on seven loci, cluster 5 is almost as closely related to cluster 4, which is part of the S. constellatus lineage, as it is to cluster 6.However, this was not the case in the extended analysis of the 20 504 bp extracted from full genomes.These findings illustrate the genetic diversity of S. anginosus and support the previous findings of S. anginosus as a genetically and phenotypically heterogeneous collection of strains (Jacobs et al., 2000b;Whiley et al., 1997).
Five isolates belonging to cluster 5 (SK443, SK1094, SK1281, SK1416 and CCUG 44049B), three isolated from blood, one from vagina and one from an oral cavit,y were identical in MLSA sequences.The five isolates also had similar 16S rRNA gene sequences, including the same characteristic signatures in the V6 region of the 16S rRNA gene sequences reported for strains of S. anginosus that display a gliding type of motility on chocolate agar plates (Bergman et al., 1995).It is clear from both the MLSA and the 16S rRNA gene sequence analysis that these strains and cluster 5 as a whole represent a distinct evolutionary lineage within S. anginosus that successfully disseminated.However, until studies of additional strains clarify its exact relation to existing species and until distinctive phenotypic characters are found, we refer to this cluster as S. anginosus genomosubspecies AJ1.
In contrast to Denmark, where most b-haemolytic strains within the Anginosus group possess the Lancefield group C antigen (unpublished data), Reißmann et al. (2010) found that all b-haemolytic isolates of the Anginosus group from the Vellore region in India, where the incidences of group C and G streptococcal infections are high, expressed the Lancefield group G antigen.Future MLSA-based analyses may clarify if these b-haemolytic, group G isolates cluster with the type strain of S. anginosus and SK535 as a distinct genomospecies of S. anginosus.et al. (1997, 1999).
Our findings demonstrate that cluster 7 constitutes a distinct and genetically homogeneous cluster within the Anginosus group with a unique phenotypic pattern.Interestingly, strains of cluster 7 were negative for b- galactosidase in our study, in contrast to the findings of Whiley et al. (1999) for DNA group 2. However, it was previously shown that b-galactosidase activity is strongly influenced by the incubation conditions employed (Ahmet et al., 1995).By considering the genetic similarity of strains within cluster 7, the genetic distance to other clusters of the Anginosus group by MLSA, the phenotypic profile, a distinctive 600 bp 16S-23S rRNA intergenic spacer as well as a clear association with sore throats, we conclude that recognition as a novel subspecies of S. anginosus is justified.Furthermore, the level of DNA-DNA hybridization found by Whiley et al. (1999) between strains of DNA group 2 and other strains of S. anginosus is within the criteria for subspecies.
Cluster 4 also constitutes a novel lineage within the Anginosus group but most closely related to S. constellatus subsp.constellatus and S. constellatus subsp.pharyngis.Like S. constellatus subsp.pharyngis (cluster 3), these strains are all b-haemolytic with a Lancefield group C antigen and were isolated from sore throats.Furthermore, strains belonging to cluster 4 have a unique phenotypic pattern distinct from both S. constellatus subsp.pharyngis and S. constellatus subsp.constellatus.Likewise, the 16S rRNA gene sequence in these strains is unique and not identical to any sequence in the GenBank database.Therefore, and in view of the genetic distance between clusters 2, 3 and 4, we conclude that recognition of cluster 4 as a novel subspecies of S. constellatus is warranted.
While clusters 1, 2, 5 and 6 were genetically heterogeneous and contained both non-haemolytic and haemolytic isolates expressing different Lancefield group antigens, clusters 3, 4 and 7 all had a clone-like structure and contained only b-haemolytic, Lancefield group C isolates.
Isolates from all three clusters are mostly isolated from throat infections, and their genetically homogeneous nature suggests that they are successfully disseminating between patients.Based on the isolates from Viborg Hospital, cluster 7 strains seem to be more abundant in sore throats than S. constellatus subsp.pharyngis (cluster 3) and cluster 4 strains.Whether these isolates are associated with tonsillitis, as has been shown for S. constellatus subsp.pharyngis (Whiley et al., 1999), has to be determined.et al., 1992).The description of this subspecies follows the description given by Whiley & Beighton (1991).

Description of
The type strain is strain NCTC 10713 T (5ATCC 33397 T ), a b-haemolytic strain with a Lancefield group G antigen isolated from a human throat.
Streptococcus anginosus subsp.whileyi (whi9ley.i.N.L. masc.gen.n. whileyi of Whiley, named in honour of Dr Robert Whiley who has made significant contributions to the understanding of the taxonomy of streptococci and first recognized this taxon).
Small (0.5-1.0 mm in diameter), Gram-positive, nonspore-forming, non-motile cocci in short chains.Catalase-negative.Colonies on blood agar are 1-2.5 mm in diameter, mostly translucent but occasionally dull, and convex with entire margins.Grows well under both aerobic and anaerobic conditions, although growth under aerobic conditions is enhanced by the addition of CO 2 .All strains are b-haemolytic and contain the Lancefield group C antigen, although a strain with Lancefield group G antigen was included in the DNA group 2 by Whiley et al. (1999).
Most strains have been isolated from sore throats and a few strains are isolated from blood, abscesses and abdominal infections.Acetoin (Voges-Proskauer test) is produced.Arginine and aesculin are hydrolysed.Strains produce b-D- glucosidase but not b-D-galactosidase (ONPG), b-Dfucosidase, N-acetyl-b-galactosaminidase, a-galactosidase or neuraminidase.Acid is produced from glucose and lactose.Some strains produce acid from salicin and amygdalin.Acid is not produced from raffinose or sorbitol.
All strains produce hyaluronidase.
The type strain is CCUG 39159 T (5DSM 25818 T 5 SK1267 T ) and was isolated from the human throat.It has all the uniform characteristics described in this study and is salicin fermentation-negative.The DNA G+C content of strain CCUG 39159 T is 38.5 mol%, which was deduced from the assembled genome sequence of CCUG 39159 T .
Small (0.5-1.0 mm in diameter), Gram-positive, nonspore-forming, non-motile cocci in short chains.Catalase-negative.Colonies on blood agar are 0.5-1.5 mm in diameter, mostly translucent but occasionally dull, and convex with entire margins.Grows well under both aerobic and anaerobic conditions, although growth under aerobic conditions is enhanced by the addition of CO 2 .All strains are b-haemolytic and contain the Lancefield group C antigen.All strains have been isolated from throats.Acetoin (Voges-Proskauer test) is produced.Arginine and aesculin are hydrolysed.Strains produce b-D- glucosidase and a-D-glucuronidase, but b-D-galactosidase (ONPG), a-galactosidase, neuraminidase and N-acetyl-bgalactosaminidase are not produced.Strains rarely produce b-D-fucosidase.Acid is produced from glucose and lactose.Some strains produce acid from salicin and amygdalin.Acid is not produced from raffinose and sorbitol.All strains produce hyaluronidase.
The type strain is SK1359 T (5CCUG 62387 T 5DSM 25819 T ) and was isolated from the human throat.It has all the uniform characteristics described above and is b-D- fucosidase-negative.

Distinguishing characteristics
Although the taxa of the Anginosus group are very difficult to differentiate using phenotypic characteristics, a few tests are useful in distinguishing S. anginosus subsp.whileyi from the other taxa of the Anginosus group.S. anginosus subsp.whileyi can be differentiated from S. anginosus subsp.anginosus strains by being hyaluronidase-positive and from S. constellatus subsp.viborgensis by lacking a-D-glucosidase.
Distinguishing biochemical characters for the species and subspecies of the Anginosus group of streptococci are summarized in Table 2.In addition to phenotypic tests, S. anginosus subsp.whileyi can also be identified on the basis of unique genotype patterns.S. anginosus subsp.whileyi produces a unique band size in the 16S-23S rRNA spacer PCR assay and shows unique bands of 125 and 752 bp in the multiplex PCR assay targeting the hyaluronate lyase gene, the 16S rRNA gene and the intermedilysin gene.
MLSA provides unequivocal differentiation from other taxa.

Fig. 1 .
Fig.1.Radial minimum-evolution tree based on concatenated sequences of seven housekeeping genes showing the phylogenetic position of individual taxa within the genus Streptococcus.The tree was reconstructed from concatenated sequences of the 127 strains from this study combined with sequences of 420 strains, primarily from the Mitis group, from the study ofBishop et al. (2009), 95 sequences of Pyogenic streptococci from the study ofJensen & Kilian (2012) and 40 sequences of streptococci strains extracted from whole genomes published in the GenBank database.Bar, 0.02 changes per nucleotide position.
Fig.2.Minimum-evolution tree based on concatenated sequences of seven housekeeping genes showing the position of the 127 strains of the Anginosus group.The Anginosus group splits into seven generally well-resolved clusters: cluster 1 (dark green), cluster 2 (turquoise), cluster 3 (blue), cluster 4 (purple), cluster 5 (yellow), cluster 6 (green) and cluster 7 (red).Stars indicate sequences extracted from whole genome sequences.Bootstrap values for major branches are shown.Type of haemolysis and Lancefield antigens are listed for each strain.Bar, 0.01 changes per nucleotide position.The neighbour-joining tree gave a similar topology (not shown).

Fig. 3 .
Fig. 3. Minimum-evolution tree based on 1401 bp sequences of the 16S rRNA gene of 116 strains of the Anginosus group.Colours correspond to the colours of the MLSA cluster from Fig. 2. Bootstrap values for major branches are shown.Bar, 0.005 changes per nucleotide position.

*
The two methods used gave identical results.

Table 3 .
Phenotypic characteristics of the Anginosus group Sultana et al. (1998)supports that both groups of strains belong to the DNA group 2 of Whiley sites.Sultana et al. (1998) also described a group of b- haemolytic, Lancefield group C-positive and hyaluronidase-positive Anginosus group streptococci with a unique 23S rRNA gene sequence.Comparison of 16S rRNA gene sequences of strains belonging to cluster 7 with those described by