Maternal Streptococcus agalactiae colonization in Europe: data from the multi-center DEVANI study.

INTRODUCTION: Despite national guidelines and use of intrapartum antibiotic prophylaxis (IAP), Streptococcus agalactiae (group B streptococci (GBS)) is still a leading cause of morbidity and mortality in newborns in Europe and the United States. The European DEVANI (Design of a Vaccine Against Neonatal Infections) program assessed the neonatal GBS infection burden in Europe, the clinical characteristics of colonized women and microbiological data of GBS strains in colonized women and their infants with early-onset disease (EOD). METHODS: Overall, 1083 pregnant women with a GBS-positive culture result from eight European countries were included in the study. Clinical obstetrical information was collected by a standardized questionnaire. GBS strains were characterized by serological and molecular methods. RESULTS: Among GBS carriers included in this study after testing positive for GBS by vaginal or recto-vaginal sampling, 13.4% had at least one additional obstetrical risk factor for EOD. The five most common capsular types (i.e., Ia, Ib, II, III and V) comprised ~ 93% of GBS carried. Of the colonized women, 77.8% received any IAP, and in 49.5% the IAP was considered appropriate. In our cohort, nine neonates presented with GBS early-onset disease (EOD) with significant regional heterogeneity. CONCLUSIONS: Screening methods and IAP rates need to be harmonized across Europe in order to reduce the rates of EOD. The epidemiological data from eight different European countries provides important information for the development of a successful GBS vaccine.

Neonatal invasive disease caused by Streptococcus agalactiae in Europe: the DEVANI multi-center study.

PURPOSE: Group B streptococcus (GBS) remains a leading cause of invasive disease, mainly sepsis and meningitis, in infants < 3 months of age and of mortality among neonates. This study, a major component of the European DEVANI project (Design of a Vaccine Against Neonatal Infections) describes clinical and important microbiological characteristics of neonatal GBS diseases. It quantifies the rate of antenatal screening and intrapartum antibiotic prophylaxis among cases and identifies risk factors associated with an adverse outcome. METHODS: Clinical and microbiological data from 153 invasive neonatal cases (82 early-onset [EOD], 71 late-onset disease [LOD] cases) were collected in eight European countries from mid-2008 to end-2010. RESULTS: Respiratory distress was the most frequent clinical sign at onset of EOD, while meningitis is found in > 30% of LOD. The study revealed that 59% of mothers of EOD cases had not received antenatal screening, whilst GBS was detected in 48.5% of screened cases. Meningitis was associated with an adverse outcome in LOD cases, while prematurity and the presence of cardiocirculatory symptoms were associated with an adverse outcome in EOD cases. Capsular-polysaccharide type III was the most frequent in both EOD and LOD cases with regional differences in the clonal complex distribution. CONCLUSIONS: Standardizing recommendations related to neonatal GBS disease and increasing compliance might improve clinical care and the prevention of GBS EOD. But even full adherence to antenatal screening would miss a relevant number of EOD cases, thus, the most promising prophylactic approach against GBS EOD and LOD would be a vaccine for maternal immunization.

Antibiotics inhibit tumor and disease activity in cutaneous T-cell lymphoma.

It has been proposed that CD4 T-cell responses to Staphylococcus aureus (SA) can inadvertently enhance neoplastic progression in models of skin cancer and cutaneous T-cell lymphoma (CTCL). In this prospective study, we explored the effect of transient antibiotic treatment on tumor cells and disease activity in 8 patients with advanced-stage CTCL. All patients experienced significant decrease in clinical symptoms in response to aggressive, transient antibiotic treatment. In some patients, clinical improvements lasted for more than 8 months. In 6 of 8 patients, a malignant T-cell clone could be identified in lesional skin, and a significant decrease in the fraction of malignant T cells was observed following antibiotics but an otherwise unchanged treatment regimen. Immunohistochemistry, global messenger RNA expression, and cell-signaling pathway analysis indicated that transient aggressive antibiotic therapy was associated with decreased expression of interleukin-2 high-affinity receptors (CD25), STAT3 signaling, and cell proliferation in lesional skin. In conclusion, this study provides novel evidence suggesting that aggressive antibiotic treatment inhibits malignant T cells in lesional skin. Thus, we provide a novel rationale for treatment of SA in advanced CTCL.

Pneumococci Can Become Virulent by Acquiring a New Capsule From Oral Streptococci.

Pneumococcal conjugate vaccines have been successful, but their use has increased infections by nonvaccine serotypes. Oral streptococci often harbor capsular polysaccharide (PS) synthesis loci (cps). Although this has not been observed in nature, if pneumococcus can replace its cps with oral streptococcal cps, it may increase its serotype repertoire. In the current study, we showed that oral Streptococcus strain SK95 and pneumococcal strain D39 both produce structurally identical capsular PS, and their genetic backgrounds influence the amount of capsule production and shielding from nonspecific killing. SK95 is avirulent in a well-established in vivo mouse model. When acapsular pneumococcus was transformed with SK95 cps, the transformant became virulent and killed all mice. Thus, cps from oral Streptococcus strains can make acapsular pneumococcus virulent, and interspecies cps transfer should be considered a potential mechanism of serotype replacement. Our findings, along with publications from the US Centers for Disease Control and Prevention, highlight potential limitations of the 2013 World Health Organization criterion for studying pneumococcal serotypes carried without isolating bacteria. We show that an oral streptococcal strain, SK95, and a pneumococcal strain, D39, both produce chemically identical capsular PS. We also show that transferring SK95 cps into noncapsulated, avirulent pneumococcus gave it the capacity for virulence in a mouse model.

Capsule Typing of Haemophilus influenzae by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry.

Encapsulated Haemophilus influenzae strains belong to type-specific genetic lineages. Reliable capsule typing requires PCR, but a more efficient method would be useful. We evaluated capsule typing by using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Isolates of all capsule types (a-f and nontypeable; n = 258) and isogenic capsule transformants (types a-d) were investigated. Principal component and biomarker analyses of mass spectra showed clustering, and mass peaks correlated with capsule type-specific genetic lineages. We used 31 selected isolates to construct a capsule typing database. Validation with the remaining isolates (n = 227) showed 100% sensitivity and 92.2% specificity for encapsulated strains (a-f; n = 61). Blinded validation of a supplemented database (n = 50) using clinical isolates (n = 126) showed 100% sensitivity and 100% specificity for encapsulated strains (b, e, and f; n = 28). MALDI-TOF mass spectrometry is an accurate method for capsule typing of H. influenzae.

Evolution of the ß-lactam-resistant Streptococcus pneumoniae PMEN3 clone over a 30 year period in Barcelona, Spain.

Objectives: To analyse the epidemiology and genetic evolution of PMEN3 (Spain9V-156), a penicillin-non-susceptible clone of Streptococcus pneumoniae, causing invasive pneumococcal disease (IPD) in Barcelona during 1987-2016. Methods: WGS was performed on 46 representative isolates and the data were used to design additional molecular typing methods including partial MLST, PCR-RFLP and detection of surface-exposed proteins and prophages, to assign the remaining isolates to lineages. The isolates were also subjected to antimicrobial susceptibility testing. Results: Two hundred and twenty-seven adult cases of IPD caused by PMEN3 were identified. PMEN3 caused mainly pneumonia (84%) and the 30 day mortality rate was 23.1%. Evidence of recombination events was found, mostly in three regions, namely the capsular operon (associated with capsular switching) and adjacent regions containing pbp2x and pbp1a, the murM gene and the pbp2b-ddl region. Some of these genetic changes generated successful new variant serotype lineages, including one of serotype 11A that is not included in the current PCV13 vaccine. Other genetic changes led to increased MICs of ß-lactams. Notably, most isolates also harboured prophages coding for PblB-like proteins. Despite these adaptations, the ability of this clone to cause IPD remained unchanged over time, highlighting the importance of its core genetic background. Conclusions: Our study demonstrated successful adaptation of PMEN3 to persist over time despite the introduction of broader antibiotics and conjugate vaccines. In addition to enhancing understanding of the molecular evolution of PMEN3, these findings highlight the need for the development of non-serotype-based vaccines to fight pneumococcal infection.

Staphylococcal enterotoxin A (SEA) stimulates STAT3 activation and IL-17 expression in cutaneous T-cell lymphoma.

Cutaneous T-cell lymphoma (CTCL) is characterized by proliferation of malignant T cells in a chronic inflammatory environment. With disease progression, bacteria colonize the compromised skin barrier and half of CTCL patients die of infection rather than from direct organ involvement by the malignancy. Clinical data indicate that bacteria play a direct role in disease progression, but little is known about the mechanisms involved. Here, we demonstrate that bacterial isolates containing staphylococcal enterotoxin A (SEA) from the affected skin of CTCL patients, as well as recombinant SEA, stimulate activation of signal transducer and activator of transcription 3 (STAT3) and upregulation of interleukin (IL)-17 in immortalized and primary patient-derived malignant and nonmalignant T cells. Importantly, SEA induces STAT3 activation and IL-17 expression in malignant T cells when cocultured with nonmalignant T cells, indicating an indirect mode of action. In accordance, malignant T cells expressing an SEA-nonresponsive T-cell receptor variable region ß chain are nonresponsive to SEA in monoculture but display strong STAT3 activation and IL-17 expression in cocultures with SEA-responsive nonmalignant T cells. The response is induced via IL-2 receptor common γ chain cytokines and a Janus kinase 3 (JAK3)-dependent pathway in malignant T cells, and blocked by tofacitinib, a clinical-grade JAK3 inhibitor. In conclusion, we demonstrate that SEA induces cell cross talk-dependent activation of STAT3 and expression of IL-17 in malignant T cells, suggesting a mechanism whereby SEA-producing bacteria promote activation of an established oncogenic pathway previously implicated in carcinogenesis.

The oral microbiome - friend or foe?

The microbiome and the human body constitute an integrated superorganism, which is the result of millions of years of coevolution with mutual adaptation and functional integration, and confers significant benefits for both parties. This evolutionary process has resulted in a highly diverse oral microbiome, which covers the full spectrum of acidogenic, aciduric, inflammatory, and anti-inflammatory properties. The relative proportions of members of the microbiome are affected by factors associated with modern life, such as general diet patterns, sugar consumption, tobacco smoking, oral hygiene, use of antibiotics and other antimicrobials, and vaccines. A perturbed balance in the oral microbiome may result in caries, periodontal disease, or candidiasis, and oral bacteria passively transferred to normally sterile parts of the body may cause extra-oral infections. Nevertheless, it should never be our goal to eliminate the oral microbiome, but rather we have to develop ways to re-establish a harmonious coexistence that is lost because of the modern lifestyle. With regard to oral diseases, this goal can normally be achieved by optimal oral hygiene, exposure to fluoride, reduction of sucrose consumption, stimulation of our innate immune defense, smoking cessation, and control of diabetes.

Antagonism between Staphylococcus epidermidis and Propionibacterium acnes and its genomic basis.

BACKGROUND: Propionibacterium acnes and Staphylococcus epidermidis live in close proximity on human skin, and both bacterial species can be isolated from normal and acne vulgaris-affected skin sites. The antagonistic interactions between the two species are poorly understood, as well as the potential significance of bacterial interferences for the skin microbiota. Here, we performed simultaneous antagonism assays to detect inhibitory activities between multiple isolates of the two species. Selected strains were sequenced to identify the genomic basis of their antimicrobial phenotypes. RESULTS: First, we screened 77 P. acnes strains isolated from healthy and acne-affected skin, and representing all known phylogenetic clades (I, II, and III), for their antimicrobial activities against 12 S. epidermidis isolates. One particular phylogroup (I-2) exhibited a higher antimicrobial activity than other P. acnes phylogroups. All genomes of type I-2 strains carry an island encoding the biosynthesis of a thiopeptide with possible antimicrobial activity against S. epidermidis. Second, 20 S. epidermidis isolates were examined for inhibitory activity against 25 P. acnes strains. The majority of S. epidermidis strains were able to inhibit P. acnes. Genomes of S. epidermidis strains with strong, medium and no inhibitory activities against P. acnes were sequenced. Genome comparison underlined the diversity of S. epidermidis and detected multiple clade- or strain-specific mobile genetic elements encoding a variety of functions important in antibiotic and stress resistance, biofilm formation and interbacterial competition, including bacteriocins such as epidermin. One isolate with an extraordinary antimicrobial activity against P. acnes harbors a functional ESAT-6 secretion system that might be involved in the antimicrobial activity against P. acnes via the secretion of polymorphic toxins. CONCLUSIONS: Taken together, our study suggests that interspecies interactions could potentially jeopardize balances in the skin microbiota. In particular, S. epidermidis strains possess an arsenal of different mechanisms to inhibit P. acnes. However, if such interactions are relevant in skin disorders such as acne vulgaris remains questionable, since no difference in the antimicrobial activity against, or the sensitivity towards S. epidermidis could be detected between health- and acne-associated strains of P. acnes.

Staphylococcal enterotoxins stimulate lymphoma-associated immune dysregulation.

Patients with cutaneous T-cell lymphoma (CTCL) are frequently colonized with Staphylococcus aureus (SA). Eradication of SA is, importantly, associated with significant clinical improvement, suggesting that SA promotes the disease activity, but the underlying mechanisms remain poorly characterized. Here, we show that SA isolates from involved skin express staphylococcal enterotoxins (SEs) that induce crosstalk between malignant and benign T cells leading to Stat3-mediated interleukin-10 (IL-10) production by the malignant T cells. The SEs did not stimulate the malignant T cells directly. Instead, SEs triggered a cascade of events involving cell-cell and asymmetric cytokine interactions between malignant and benign T cells, which stimulated the malignant T cells to express high levels of IL-10. Much evidence supports that malignant activation of the Stat3/IL-10 axis plays a key role in driving the immune dysregulation and severe immunodeficiency that characteristically develops in CTCL patients. The present findings thereby establish a novel link between SEs and immune dysregulation in CTCL, strengthening the rationale for antibiotic treatment of colonized patients with severe or progressive disease.

The natural history of cutaneous propionibacteria, and reclassification of selected species within the genus Propionibacterium to the proposed novel genera Acidipropionibacterium gen. nov., Cutibacterium gen. nov. and Pseudopropionibacterium gen. nov.

The genus Propionibacterium in the family Propionibacteriaceaeconsists of species of various habitats, including mature cheese, cattle rumen and human skin. Traditionally, these species have been grouped as either classical or cutaneous propionibacteria based on characteristic phenotypes and source of isolation. To re-evaluate the taxonomy of the family and to elucidate the interspecies relatedness we compared 162 public whole-genome sequences of strains representing species of the family Propionibacteriaceae. We found substantial discrepancies between the phylogenetic signals of 16S rRNA gene sequence analysis and our high-resolution core-genome analysis. To accommodate these discrepancies, and to address the long-standing issue of the taxonomically problematic Propionibacterium propionicum, we propose three novel genera, Acidipropionibacterium gen. nov., Cutibacterium gen. nov. and Pseudopropionibacterium gen. nov., and an amended description of the genus Propionibacterium. Furthermore, our genome-based analyses support the amounting evidence that the subdivision of Propionibacterium freudenreichii into subspecies is not warranted. Our proposals are supported by phylogenetic analyses, DNA G+C content, peptidoglycan composition and patterns of the gene losses and acquisitions in the cutaneous propionibacteria during their adaptation to the human host.

Re-evaluation of the taxonomy of the Mitis group of the genus Streptococcus based on whole genome phylogenetic analyses, and proposed reclassification of Streptococcus dentisani as Streptococcus oralis subsp. dentisani comb. nov., Streptococcus tigurinus as Streptococcus oralis subsp. tigurinus comb. nov., and Streptococcus oligofermentans as a later synonym of Streptococcus cristatus.

The Mitis group of the genus Streptococcus currently comprises 20 species with validly published names, including the pathogen S. pneumoniae. They have been the subject of much taxonomic confusion, due to phenotypic overlap and genetic heterogeneity, which has hampered a full appreciation of their clinical significance. The purpose of this study was to critically re-examine the taxonomy of the Mitis group using 195 publicly available genomes, including designated type strains for phylogenetic analyses based on core genomes, multilocus sequences and 16S rRNA gene sequences, combined with estimates of average nucleotide identity (ANI) and in silico and in vitro analyses of specific phenotypic characteristics. Our core genomic phylogenetic analyses revealed distinct clades that, to some extent, and from the clustering of type strains represent known species. However, many of the genomes have been incorrectly identified adding to the current confusion. Furthermore, our data show that 16S rRNA gene sequences and ANI are unsuitable for identifying and circumscribing new species of the Mitis group of the genus Streptococci. Based on the clustering patterns resulting from core genome phylogenetic analysis, we conclude that S. oligofermentans is a later synonym of S. cristatus. The recently described strains of the species Streptococcus dentisani includes one previously referred to as 'S. mitis biovar 2'. Together with S. oralis, S. dentisani and S. tigurinus form subclusters within a coherent phylogenetic clade. We propose that the species S. oralis consists of three subspecies: S. oralis subsp. oralis subsp. nov., S. oralis subsp. tigurinus comb. nov., and S. oralis subsp. dentisani comb. nov.

Commensal streptococci serve as a reservoir for ß-lactam resistance genes in Streptococcus pneumoniae.

Streptococcus pneumoniae is a leading cause of pneumonia, meningitis, septicemia, and middle ear infections. The incidence of S. pneumoniae isolates that are not susceptible to penicillin has risen worldwide and may be above 20% in some countries. Beta-lactam antibiotic resistance in pneumococci is associated with significant sequence polymorphism in penicillin-binding proteins (PBPs). Commensal streptococci, especially S. mitis and S. oralis, have been identified as putative donors of mutated gene fragments. However, no studies have compared sequences of the involved pbp genes in large collections of commensal streptococci with those of S. pneumoniae. We therefore investigated the sequence diversity of the transpeptidase region of the three pbp genes, pbp2x, pbp2b, and pbp1a in 107, 96, and 88 susceptible and nonsusceptible strains of commensal streptococci, respectively, at the nucleotide and amino acid levels to determine to what extent homologous recombination between commensal streptococci and S. pneumoniae plays a role in the development of beta-lactam resistance in S. pneumoniae. In contrast to pneumococci, extensive sequence variation in the transpeptidase region of pbp2x, pbp2b, and pbp1a was observed in both susceptible and nonsusceptible strains of commensal streptococci, conceivably reflecting the genetic diversity of the many evolutionary lineages of commensal streptococci combined with the recombination events occurring with intra- and interspecies homologues. Our data support the notion that resistance to beta-lactam antibiotics in pneumococci is due to sequences acquired from commensal Mitis group streptococci, especially S. mitis. However, several amino acid alterations previously linked to beta-lactam resistance in pneumococci appear to represent species signatures of the donor strain rather than being causal of resistance.

Stable analogues of nojirimycin--synthesis and biological evaluation of nojiristegine and manno-nojiristegine.

Two novel iminosugars called nojiristegines, being structural hybrids between nor-tropane alkaloid calystegine and nojirimycins, have been synthesised and found to be stable molecules despite the presence of a hemiaminal functionality. The synthesised iminosugars were evaluated against a panel of glycosidases and the best inhibition (IC50), found against α-glucosidases, was in the micromolar region. The compounds were also evaluated as potential antibiotics but no useful level of activity was observed.

Clonality and anatomic distribution on the skin of antibiotic resistant and sensitive Propionibacterium acnes.

Increasing antibiotic resistance in the population of Propionibacterium acnes is a major concern. Our aims were to examine the clonal relationships and anatomical distribution of resistant and sensitive P. acnes. A collection of 350 P. acnes isolates was therefore used to determine the minimum inhibitory concentration of tetracycline, erythro-mycin and clindamycin, multilocus sequence type, and the identity of genetic resistance markers. Two hitherto unknown resistance mutations were detected. Resistant P. acnes mainly belonged to clonal clusters in division I-1a frequently isolated from skin and associated with moderate to severe acne. All high-level tetracycline resistant strains were members of a single clone. Multiple isolates from distinct anatomic areas of surface skin and follicles of 2 acne patients revealed substantial clonal diversity between areas and co-existence of resistant and sensitive clones. Fifty-two percent of Danish acne patients and 43% of controls carried at least one resistant P. acnes strain, resistance to clindamycin being most frequent followed by tetracycline and erythromycin. Resistance to tetracycline was detected exclusively among isolates from acne patients. In conclusion, antibiotic resistance is associated with particular evolutionary clades of P. acnes and a substantial part is due to a single geographically widespread clone (ST3). Individuals carry a strikingly complex population of P. acnes with distinct virulence potential and antibiotic resistance.

Role of IgA1 protease-producing bacteria in SARS-CoV-2 infection and transmission: a hypothesis.

Secretory (S) IgA antibodies against severe acute respiratory syndrome (SARS)-CoV-2 are induced in saliva and upper respiratory tract (URT) secretions by natural infection and may be critical in determining the outcome of initial infection. Secretory IgA1 (SIgA1) is the predominant isotype of antibodies in these secretions. Neutralization of SARS-CoV-2 is most effectively accomplished by polymeric antibodies such as SIgA. We hypothesize that cleavage of SIgA1 antibodies against SARS-CoV-2 by unique bacterial IgA1 proteases to univalent Fabα antibody fragments with diminished virus neutralizing activity would facilitate the descent of the virus into the lungs to cause serious disease and also enhance its airborne transmission to others. Recent studies of the nasopharyngeal microbiota of patients with SARS-CoV-2 infection have revealed significant increases in the proportions of IgA1 protease-producing bacteria in comparison with healthy subjects. Similar considerations might apply also to other respiratory viral infections including influenza, possibly explaining the original attribution of influenza to Haemophilus influenzae, which produces IgA1 protease.

Capsular gene typing of Streptococcus agalactiae compared to serotyping by latex agglutination.

We evaluated three different PCR-based capsular gene typing methods applied to 312 human and bovine Streptococcus agalactiae (group B Streptococcus [GBS]) isolates and compared the results to serotyping results obtained by latex agglutination. Among 281 human isolates 27% could not be typed by latex agglutination. All 312 isolates except 5 could be typed by the three PCR methods combined. Two of these methods were multiplex assays. Among the isolates that were typeable by both latex agglutination and capsular gene typing, 94% showed agreement between the two methods. However, each of the PCR methods showed limitations. One of the methods did not include all 10 recognized serotypes, one misidentified eight isolates of serotypes Ib and IV as serotype Ia, and one did not distinguish between serotypes VII and IX. For five isolates that showed aberrant patterns in the capsular gene typing, long-range PCR targeting the cps operon disclosed large insertions or deletions affecting the cps gene cluster. A sensitive flow cytometric assay based on serotype-specific antibodies applied to 76 selected isolates that were nontypeable by latex agglutination revealed that approximately one-half of these did express capsular polysaccharide. A procedure for convenient and reliable capsular gene typing to be included in epidemiological and surveillance studies of S. agalactiae is proposed.

Widespread occurrence of bacterial human virulence determinants in soil and freshwater environments.

The occurrence of 22 bacterial human virulence genes (encoding toxins, adhesins, secretion systems, regulators of virulence, inflammatory mediators, and bacterial resistance) in beech wood soil, roadside soil, organic agricultural soil, and freshwater biofilm was investigated by nested PCR. The presence of clinically relevant bacterial groups known to possess virulence genes was tested by PCR of 16S and 23S rRNA genes. For each of the virulence genes detected in the environments, sequencing and NCBI BLAST analysis confirmed the identity of the PCR products. The virulence genes showed widespread environmental occurrence, as 17 different genes were observed. Sixteen genes were detected in beech wood soil, and 14 were detected in roadside and organic agricultural soils, while 11 were detected in the freshwater biofilm. All types of virulence traits were represented in all environments; however, the frequency at which they were detected was variable. A principal-component analysis suggested that several factors influenced the presence of the virulence genes; however, their distribution was most likely related to the level of contamination by polycyclic aromatic hydrocarbons and pH. The occurrence of the virulence genes in the environments generally did not appear to be the result of the presence of clinically relevant bacteria, indicating an environmental origin of the virulence genes. The widespread occurrence of the virulence traits and the high degree of sequence conservation between the environmental and clinical sequences suggest that soil and freshwater environments may constitute reservoirs of virulence determinants normally associated with human disease.

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 ß-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 ß-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 39159(T) = DSM 25818(T) = SK1267(T)) and S. constellatus subsp. viborgensis subsp. nov. (type strain SK1359(T) = CCUG 62387(T) = DSM 25819(T)) are proposed.