Mitis group streptococci express variable pilus islet 2 pili

Background

Streptococcus oralis, Streptococcus mitis, and Streptococcus sanguinis are members of the Mitis group of streptococci and agents of oral biofilm, dental plaque and infective endocarditis, disease processes that involve bacteria-bacteria and bacteria-host interactions. Their close relative, the human pathogen S. pneumoniae uses pilus-islet 2 (PI-2)-encoded pili to facilitate adhesion to eukaryotic cells.

Methodology/Principal Findings

PI-2 pilus-encoding genetic islets were identified in S. oralis, S. mitis, and S. sanguinis, but were absent from other isolates of these species. The PI-2 islets resembled the genetic organization of the PI-2 islet of S. pneumoniae, but differed in the genes encoding the structural pilus proteins PitA and PitB. Two and three variants of pitA (a pseudogene in S. pneumoniae) and pitB, respectively, were identified that showed ≈20% difference in nucleotide as well as corresponding protein sequence. Species-independent combinations of pitA and pitB variants indicated prior intra- and interspecies horizontal gene transfer events. Polyclonal antisera developed against PitA and PitB of S. oralis type strain ATCC35037 revealed that PI-2 pili in oral streptococci were composed of PitA and PitB. Electronmicrographs showed pilus structures radiating >700 nm from the bacterial surface in the wild type strain, but not in an isogenic PI-2 deletion mutant. Anti-PitB-antiserum only reacted with pili containing the same PitB variant, whereas anti-PitA antiserum was cross-reactive with the other PitA variant. Electronic multilocus sequence analysis revealed that all PI-2-encoding oral streptococci were closely-related and cluster with non-PI-2-encoding S. oralis strains.

Conclusions/Significance

This is the first identification of PI-2 pili in Mitis group oral streptococci. The findings provide a striking example of intra- and interspecies horizontal gene transfer. The PI-2 pilus diversity provides a possible key to link strain-specific bacterial interactions and/or tissue tropisms with pathogenic traits in the Mitis group streptococci.     

Evolution of Streptococcus pneumoniae and its close commensal relatives

Streptococcus pneumoniae is a member of the Mitis group of streptococci which, according to 16S rRNA-sequence based phylogenetic reconstruction, includes 12 species. While other species of this group are considered prototypes of commensal bacteria, S. pneumoniae is among the most frequent microbial killers worldwide. Population genetic analysis of 118 strains, supported by demonstration of a distinct cell wall carbohydrate structure and competence pheromone sequence signature, shows that S. pneumoniae is one of several hundred evolutionary lineages forming a cluster separate from Streptococcus oralis and Streptococcus infantis. The remaining lineages of this distinct cluster are commensals previously collectively referred to as Streptococcus mitis and each represent separate species by traditional taxonomic standard. Virulence genes including the operon for capsule polysaccharide synthesis and genes encoding IgA1 protease, pneumolysin, and autolysin were randomly distributed among S. mitis lineages. Estimates of the evolutionary age of the lineages, the identical location of remnants of virulence genes in the genomes of commensal strains, the pattern of genome reductions, and the proportion of unique genes and their origin support the model that the entire cluster of S. pneumoniae, S. pseudopneumoniae, and S. mitis lineages evolved from pneumococcus-like bacteria presumably pathogenic to the common immediate ancestor of hominoids. During their adaptation to a commensal life style, most of the lineages gradually lost the majority of genes determining virulence and became genetically distinct due to sexual isolation in their respective hosts.     

Proteolytic activity of oral streptococci

Streptococcus mutans and Streptococcus sobrinus were the least proteolytic of 8 species of oral streptococci while Streptococcus oralis and Streptococcus sanguis were the most proteolytic. Degradation of FITC-BSA was significantly correlated with the hydrolysis of synthetic endopeptidase substrates. As S. oralis strains proliferate in dental plaque in the absence of dietary food their success, in vivo, might be due partially to their greater proteolytic activity compared to other oral streptococci.     

The classification of streptococci and human streptococcal diseases

The modern nomenclature, phenotypic, medical, ecological and phylogenetic classification of streptococci and different classification of streptococcal human diseases are presented. All phylogenetic groups of streptococci have been shown to contain species causing diseases in man. The most medically significant groups are the phylogenetic groups Pyogenes and Mitis. Directions of the improvement of the classification of streptococci and streptococcal human diseases on the basis of modern concepts on the taxonomy of streptococci, the biological properties and ecology of the infective agents, as well as the genesis and clinical picture of diseases induced by them, have been determined.     

Phenotypic and molecular methods used for identification of oral streptococci and related microorganisms

The oral cavity contains the greatest biodiversity, over 70 species being isolated from mouth mucosa, saliva, denture surfaces and/or dental-plaque. The oral streptococci, representing over 80% of the mouth micro flora, are able to synthesize glucosyl-transferases, enzymes involved in glucans production. Glucans are involved in production of an extracellular slime layer promoting adhesion and formation of a dental plaque biofilm. The 43 isolates studied obtained from partially and/or totally edentulous, were identified by VITEK system using gram-positive identification cards. Species-specific regions within the genes coding for glucosyl-transferases (gtf genes) were targeted for PCR identification of isolates. Sequencing of 16S rRNA was used as gold standard for strain confirmation. VITEK system identified a number of 11 strains as S. mitis/oralis, 12 strains as S. anginosus/gordonii, 12 strains as S. sanguinis/parasanguinis, 3 strains as S. salivarius, 3 strains as S. plurianimalium, 1 strain as S. cristatus and 1 strain as S. alactolyticus, respectively. The PCR system targeting gtf genes was able to identify S. oralis, S. salivarius and S. gordonii strains. Sequence of 16S rRNA discriminated among streptococci species and revealed 16 strains of Leuconostoc mesenteroides. Many studies are needed in order to select the most reliable phenotypic and genotypic methods in order to improve the identification algorithm for oral streptococci used by clinical laboratories. Their accurate identification is mandatory for better understanding their role in human infections.     

Relationship between the ability of oral streptococci to interact with platelet glycoprotein Ibalpha and with the salivary low-molecular-weight mucin, MG2

The oral streptococci Streptococcus sanguinis, Streptococcus gordonii and Streptococcus oralis are common aetiological agents of infective endocarditis, and their ability to adhere to and induce the aggregation of platelets is thought to be a virulence trait. The platelet glycoprotein GPIbalpha has been implicated as the adhesion receptor for S. sanguinis and S. gordonii, but it is not known if this is the case for S. oralis and other species. The aim of this study was to determine the GPIbalpha-interactive capability of a range of oral streptococci and to determine the relationship between this capability and their ability to interact with the salivary constituents that they would encounter in their normal habitat. All platelet-adhesive S. sanguinis strains and most S. gordonii strains adhered in a GPIbalpha-dependent manner, but strains of S. oralis, Streptococcus cristatus, Streptococcus parasanguinis and Streptococcus mitis had no direct affinity for platelets. Those strains that were able to bind GPIbalpha also bound to the low-molecular-weight submandibular salivary mucin, MG2, and this interaction was sialic acid-dependent. The data suggest that S. sanguinis and S. gordonii may be efficient colonizers of platelet vegetations because of their adaptation to recognize sialylated salivary mucins. In contrast, S. oralis does not interact with platelets and so is likely to colonize vegetations through an as yet unidentified mechanism.     

Differentiation of α-hemolytic Streptococci by direct mass spectrometry profiling

The modern phenotypic and genetic methods except for Multi Locus Sequence Typing do not allow the reliable differentiation within Mitis group of α-hemolytic streptococci. During this study the MALDI mass spectra were acquired for 28 clinical isolates initially identified as S. pneumoniae by routine bacteriological tests. Due to Multi Locus Sequence Typing these isolates were found to belong to two closely related species - S. pneumoniae (n = 22) and S. mitis (n = 6). Distribution of those isolates in accordance with cluster analysis of collected mass spectra matched to Multi Locus Sequence Typing data. The diagnostic model based on Genetic Algorithm classifier demonstrated the differentiation of α-hemolytic streptococci with 100% sensitivity and 94.6% accuracy. Statistical analysis of MS peak areas revealed 2 peaks which are different for S. mitis and S. pneumoniae groups.     

Differentiation of α-hemolytic streptococci by direct mass spectrometric profiling

Modern phenotypic and genetic methods (with the exception of multilocus sequence typing) do not provide a reliable differentiation of α-hemolytic streptococci of the Mitis group. In this study, we obtained MALDI mass spectra for 28 clinical isolates that were initially identified by standard methods as Streptococcus pheumoniae. The isolate distribution that was obtained in the course of clustering correlated well with the data on multilocus typing. These studied isolates were attributed by this typing to two closely related species: S. pneumoniae and S. mitis. A classification model that differentiated α-hemolytic streptococci with 100% sensitivity and 94.6% specificity was developed on the basis of a genetic algorithm. Two peaks that were different for the S. mitis and S. pneumoniae isolates were revealed by the analysis of statistical diagrams of peak areas.     

Complete structure of the cell surface polysaccharide of Streptococcus oralis C104: a 600-MHz NMR study

Specific lectin-carbohydrate interactions between certain oral streptococci and actinomyces contribute to the microbial colonization of teeth. The receptor molecules of Streptococcus oralis, 34, ATCC 10557, and Streptococcus mitis J22 for the galactose and N-acetylgalactosamine reactive fimbrial lectins of Actinomyces viscosus and Actinomyces naeslundii are antigenically distinct polysaccharides, each formed by a different phosphodiester-linked oligosaccharide repeating unit. These streptococci all coaggregated strongly with both A. viscosus and A. naesludii strains, whereas S. oralis C104 interacted preferentially with certain strains of the latter species. Receptor polysaccharide was isolated from S. oralis C104 cells and was shown to contain galactose, N-acetylgalactosamine, ribitol, and phosphate with molar ratios of 4:1:1:1. The 1H NMR spectrum of the polysaccharide shows that it contains a repeating structure. The individual sugars in the repeating unit were identified by 1H coupling constants observed in E-COSY and DQF-COSY spectra. NMR methods included complete resonance assignments (1H and 13C) by various homonuclear and heteronuclear correlation experiments that utilize scalar couplings. Sequence and linkage assignments were obtained from the heteronuclear multiple-bond correlation (HMBC) spectrum. This analysis shows that the receptor polysaccharide of S. oralis C104 is a ribitol teichoic acid polymer composed of a linear hexasaccharide repeating unit containing two residues each of galactopyranose and galactofuranose and a residue each of GalNAc and ribitol joined end to end by phosphodiester linkages with the following structure. [----6)Galf(beta 1----3)Galp(beta 1----6)Galf(beta 1----6)GalpNAc(beta 1----3) Galp(alpha 1----1)ribitol(5----PO4-]n     

New serotype of mutans streptococci isolated from pig oral cavity

Gram-positive streptococcal mutans-like strains, but with clearly different colony formation than S. orisuis on Mitis Salivarius agar, were isolated from the pig oral cavity and identified by 16S rRNA sequencing, G+C content, DNA-DNA homology and extensive biochemical and serological testing. The phenotypic data showed that the strains were similar to S. orisuis except for susceptibility to bacitracin. DNA-DNA homology between the isolates and S. orisuis was 72∼81%. However, serological data showed that they have a different sero-specific antigen from S. orisuis and other mutans streptococci. A new serotype, designated p, strains are classified in a serovar of S. orisuis, one of mutans streptococci.     

Genotypic heterogeneity of Streptococcus oralis and distinct aciduric subpopulations in human dental plaque

The genotypic heterogeneity of Streptococcus oralis isolated from the oral cavity was investigated using repetitive extragenic palindromic PCR. Unrelated subjects harbored unique genotypes, with numerous genotypes being isolated from an individual. S. oralis is the predominant aciduric bacterium isolated from noncarious tooth sites. Genotypic comparison of the aciduric populations isolated at pH 5.2 with those isolated from mitis-salivarius agar (MSA) (pH 7.0) indicated that the aciduric populations were genotypically distinct in the majority of subjects (chi(2) = 13.09; P = 0.0031). Neither the aciduric nor the MSA-isolated strains were stable, with no strains isolated at baseline being isolated 4 or 12 weeks later in the majority of subjects. The basis of this instability is unknown but is similar to that reported for Streptococcus mitis. Examination of S. oralis strains isolated from cohabiting couples demonstrated that in three of five couples, genotypically identical strains were isolated from both partners and this was confirmed by using Salmonella enteritidis repetitive element PCR and enterobacterial PCR typing. These data provide further evidence of the physiological and genotypic heterogeneity of non-mutans streptococci. The demonstration of distinct aciduric populations of S. oralis implies that the role of these and other non-mutans streptococci in the caries process requires reevaluation.     

Inhibition by yeast killer toxin-like antibodies of oral Streptococci adhesion to tooth surfaces in an ex vivo model

BACKGROUND: Monoclonal (KTmAb) and recombinant (KTscFv) anti-idiotypic antibodies, representing the internal image of a yeast killer toxin, proved to be microbicidal in vitro against important eukaryotic and prokaryotic pathogens such as Candida albicans, Pneumocystis carinii, Mycobacterium tuberculosis, Staphylococcus aureus, S. haemolyticus, Enterococcus faecalis, E. faecium, and Streptococcus pneumoniae, including multidrug-resistant strains. KTmAb and KTscFv exerted a strong therapeutic effect in well-established animal models of candidiasis and pneumocystosis. Streptococcus mutans is the most important etiologic agent of dental caries that might result from the metabolic end products of dental plaque. Effective strategies to reduce the disease potential of dental plaque have considered the possibility of using antibiotics or antibodies against oral streptococci in general and S. mutans in particular. In this study, the activity of KTmAb and KTscFv against S. mutans and the inhibition and reduction by KTmAb of dental colonization by S. mutans and other oral streptococci in an ex vivo model of human teeth were investigated. MATERIALS AND METHODS: KTscFv and KTmAb were used in a conventional colony forming unit (CFU) assay against a serotype C strain of S. mutans, and other oral streptococci (S. intermedius, S. mitis, S. oralis, S. salivarius). An ex vivo model of human teeth submerged in saliva was used to establish KTmAb potential of inhibiting or reducing the adhesion to dental surfaces by S. mutans and other oral streptococci. RESULTS: KTmAb and KTscFv kill in vitro S. mutans and other oral streptococci. KTmAb inhibit colonization of dental surfaces by S. mutans and oral streptococci in the ex vivo model. CONCLUSIONS: Killer antibodies with antibiotic activity or their engineered derivatives may have a potential in the prevention of dental caries in vivo.     

Selectivity of Mitis Salivarius agar and a new selective medium for oral streptococci in dogs

An evaluation on the applicability of Mitis Salivarius agar (MS) medium, commonly used for the detection of oral streptococci in human and animals, to dog specimens and the development of a new selective medium for isolating streptococci from the canine oral cavity are described. Oral samples from dogs were cultured on MS medium under anaerobic conditions. The predominant facultative anaerobic bacteria on MS plates were gram-negative rods. Selectivity of streptococci on MS medium was 21.2%. A new selective medium, designated MS-CAN-AE, was developed for the isolation of streptococci from the canine oral cavity. The average growth recovery of laboratory and clinically isolated strains of streptococci on MS-CAN-AE medium was 84.1% of that on MS medium. Gram-positive rods and gram-negative rods and cocci rarely grew on the MS-CAN-AE. The selectivity of MS-CAN-AE was 95.0% for clinical samples. MS-CAN-AE medium will be helpful for investigations of streptococci in the canine oral cavity.     

Horizontal spread of an altered penicillin-binding protein 2B gene between Streptococcus pneumoniae and Streptococcus oralis

Abstract The region encoding the transpeptidase domain of the penicillin-binding protein 2B (PBP 2B) gene of two penicillin-resistant clinical isolates of Streptococcus oralis was > 99.6% identical in nucleotide sequence to that of a penicillin-resistant serotype 6 isolate of Streptococcus pneumoniae . The downstream 849 base pairs of these genes were identical. Analysis of the data indicates that the PBP gene has probably been transferred from S. pneumoniae into S. oralis , rather than vice versa, and shows that one region of this resistance gene has been distributed horizontally both within S. pneumoniae and into two different viridans group streptococci.     

Erythromycin and penicillin resistance mechanisms among viridans group streptococci isolated from blood cultures of adult patients with underlying diseases

The aim of the study was to evaluate the species distribution, antimicrobial susceptibility and erythromycin-penicillin resistance mechanisms of viridans streptococci (VGS) isolates from blood cultures of adult patients with underlying diseases. Fifty VGS blood culture isolates were screened for their antibiotic susceptibilities against penicillin G, erythromycin and tetracycline by E-test. Clindamycin, cefotaxime, chloramphenicol, levofloxacin, linezolid and vancomycin susceptibility were performed by broth microdilution method. Erythromycin and penicillin resistance genotypes, ermB and mefA/E, pbp1a, pbp2b and pbp2x are amplified using PCR method. The clinical isolates included Streptococcus mitis (n. 19), S.oralis (n. 13), S.sanguinis, S.parasanguinis (n. 6, each), S.salivarius, S.vestibularis (n. 2, each), S.constellatus, S.sobrinus (n. 1, each). The percentage resistance against erythromycin and penicillin was 36% and 30%, respectively. The genotypic carriage rate of erythromycin resistance genes were: 56% ermB, 28% mefE, 8% ermB+mefE. Penicillin-resistant isolates carried pbp2b (33.3%) and pbp2x (20%) genes. Twenty-four VGS isolates were recovered from patients with cancer. S.mitis and S.oralis predominated among patients with cancer who had erythromycin and penicillin resistance isolates. The importance of classical antimicrobial agents like penicillin and erythromycin warrants the continuous surveillance of invasive VGS isolates and can guide better treatment options especially in patients with underlying diseases.     

Genetic relationships among the oral streptococci.

Genetic relationships and species limits among the oral streptococci were determined by an analysis of electrophoretically demonstrable variation in 16 metabolic enzymes. Fifty isolates represented 40 electrophoretic types, among which the mean genetic diversity per locus was 0.857. Mannitol-1-phosphate dehydrogenase was not detected in isolates of the sanguis species complex, and glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were absent in species of the mutans complex. Clustering from a matrix of Gower's coefficient of genetic similarity placed the 40 electrophoretic types in 10 well-defined groups corresponding to the Streptococcus species S. mutans, S. sobrinus, S. cricetus, S. rattus, S. ferus, S. oralis (mitior), two distinct assemblages of S. sanguis strains, and two subdivisions of "S. milleri." The assignments of isolates to these groups were the same as those indicated by DNA hybridization experiments, and the coefficient of correlation between genetic distance estimated by multilocus enzyme electrophoresis and genetic similarity indexed by DNA hybridization was -0.897 (P less than 0.001) for 50 pairwise combinations of isolates. S. ferus, which is widely believed to be a member of the mutans complex, was shown to be phylogenetically closer to species of the sanguis complex.     

Distribution and genetic diversity of the ABC transporter lipoproteins PiuA and PiaA within Streptococcus pneumoniae and related streptococci

Streptococcus pneumoniae is a major cause of morbidity and mortality worldwide. The existence of approximately 90 antigenically distinct capsular serotypes has greatly complicated the development of an effective pneumococcal vaccine. Virulence-associated proteins common and conserved among all capsular types now represent the best strategy to combat pneumococcal infections. PiuA and PiaA are the lipoprotein components of two pneumococcal iron ABC transporters and are required for full virulence in mouse models of infection. Here we describe a study of the distribution and genetic diversity of PiuA and PiaA within typical and atypical S. pneumoniae, Streptococcus oralis, and Streptococcus mitis strains. The genes encoding both PiuA and PiaA were present in all typical pneumococci tested, (covering 20 and 27 serotypes, respectively). The piuA gene was highly conserved within the typical pneumococci (0.3% nucleotide divergence), but was also present in "atypical" pneumococci and the closely related species S. mitis and S. oralis, showing up to 10.4% nucleotide divergence and 7.5% amino acid divergence from the typical pneumococcal alleles. Conversely, the piaA gene was found to be specific to typical pneumococci, 100% conserved, and absent from the oral streptococci, including isolates of S. mitis known to possess pneumolysin and autolysin. These are desirable qualities for a vaccine candidate and as a diagnostic tool for S. pneumoniae.     

Effect of the environment on genotypic diversity of Actinomyces naeslundii and Streptococcus oralis in the oral biofilm

The genotypic diversity of Actinomyces naeslundii genospecies 2 (424 isolates) and Streptococcus oralis (446 isolates) strains isolated from two sound approximal sites in all subjects who were either caries active (seven subjects) or caries free (seven subjects) was investigated by using the repetitive extragenic palindromic PCR. The plaque from the caries-active subjects harbored significantly greater proportions of mutans streptococci and lactobacilli and a smaller proportion of A. naeslundii organisms than the plaque sampled from the caries-free subjects. These data confirmed that the sites of the two groups of subjects were subjected to different environmental stresses, probably determined by the prevailing or fluctuating acidic pH values. We tested the hypothesis that the microfloras of the sites subjected to greater stresses (the plaque samples from the caries-active subjects) would exhibit reduced genotypic diversity since the sites would be less favorable. We found that the diversity of A. naeslundii strains did not change (chi2 = 0.68; P = 0.41) although the proportional representation of A. naeslundii was significantly reduced (P < 0.05). Conversely, the diversity of the S. oralis strains increased (chi2 = 11.71; P = 0.0006) and the proportional representation of S. oralis did not change. We propose that under these environmental conditions the diversity and number of niches within the oral biofilm that could be exploited by S. oralis increased, resulting in the increased genotypic diversity of this species. Apparently, A. naeslundii was not able to exploit the new niches since the prevailing conditions within the niches may have been deleterious and not supportive of its proliferation. These results suggest that environmental stress may modify a biofilm such that the diversity of the niches is increased and that these niches may be successfully exploited by some, but not necessarily all, members of the microbial community.     

Detection of the aminoglycosidestreptothricin resistance gene cluster ant(6)-sat4-aph(3 ')-III in commensal viridans group streptococci.

High-level aminoglycoside resistance was assessed in 190 commensal erythromycin-resistant alpha-hemolytic streptococcal strains. Of these, seven were also aminoglycoside-resistant: one Streptococcus mitis strain was resistant to high levels of kanamycin and carried the aph(3 ')-III gene, four S. mitis strains were resistant to high levels of streptomycin and lacked aminoglycoside-modifying enzymes, and two S. oralis strains that were resistant to high levels of kanamycin and streptomycin harbored both the aph(3 ')-III and the ant(6) genes. The two S. oralis strains also carried the ant(6)-sat4- aph(3 ' ')-III aminoglycoside-streptothricin resistance gene cluster, but it was not contained in a Tn5405-like structure. The presence of this resistance gene cluster in commensal streptococci suggests an exchange of resistance genes between these bacteria and enterococci or staphylococci.     

Amylase-binding as a discriminition among oral streptococci

The ability of 51 strains, belonging to Streptococcus sanguis, 'S. mitior', S. oralis and related groups, to bind salivary amylase was studied. Most strains were grouped according to their DNA-relatedness and then compared using 14 phenotypic tests. S. mitis, 'S. mitior' and three relatively new groups of strains ('CR', 'MGH' and 'Tufted mitior') bound salivary amylase, while strains of S. sanguis and S. oralis did not. The ability of strains to bind amylase or not was remarkably consistent within groups and the test proved to be reproducible, rapid and easy to perform. Combination of the amylase-binding test with 6 other conventional physiological tests allowed the construction of a dichotomous identification key which correctly identified 95% of strains for which genetic data was available. These findings suggest that the ability of organisms to bind salivary amylase could become a key test in identification schemes for certain oral streptococci.