Inhibition of adhesion of viridans streptococci to fibronectin-coated hydroxyapatite beads by lipoteichoic acid

Fibronectin-coated hydroxyapatite (FnHA) beads were used in a model adhesion assay to isolate the lipoteichoic acid (LTA) mediated adhesion of oral streptococci. Representative strains of the commonly isolated viridans streptococci were incubated with FnHA beads in the presence and absence of exogenous LTA. The LTA inhibited the adhesion of all strains to a greater or lesser extent, but only a very few strains were inhibited by more than 90%. Strains of Streptococcus sanguis Type II and Streptococcus mitis which synthesize an amphiphile other than LTA were also inhibited. The findings provided circumstantial evidence for the involvement of LTA in the adhesion of this group of oral bacteria.     

Determination of viridans streptococci surface lipoteichoic acid by enzyme linked immune sorbent assay

An enzyme linked immune sorbent assay (ELISA) was developed to measure bacterial surface lipoteichoic acid (LTA). Numerous strains of oral streptococci belonging to the 'viridans' group were examined on three separate occasions. The results show that, under these cultural conditions, oral streptococci do not normally express LTA on the cell surface. Occasionally strains produced amounts of LTA detectable using the ELISA but this was not a reproducible phenomenon.     

Comparative adhesion of seven species of streptococci isolated from the blood of patients with sub-acute bacterial endocarditis to fibrin-platelet clots in vitro

The adhesion to fibrin-platelet clots in vitro of 21 strains of streptococci isolated from the blood of patients with sub-acute bacterial endocarditis (SABE) was measured. The species, in order of greatest adhesion, were Streptococcus faecalis, Streptococcus mutans, Streptococcus milleri, Streptococcus sanguis , dextran-positive Streptococcus mitior , dextran-negative Streptococcus mitior and Streptococcus salivarius. Individual strains within species, however, cannot be assumed to be representative of their species and may exhibit unusually high or low adhesion. Adhesion depended upon both bacterial concentration and period of contact. There was no simple relationship between ability to adhere and liability to cause endocarditis. Formation of dextran did not increase adhesion. The streptococci were more adhesive than strains of Escherichia coli and Neisseria sicca and less adhesive than strains of Staphylococcus aureus and Streptococcus pyogenes.     

Comparative adhesion of seven species of streptococci isolated from the blood of patients with sub-acute bacterial endocarditis to fibrin-platelet clots in vitro

The adhesion to fibrin-platelet clots in vitro of 21 strains of streptococci isolated from the blood of patients with sub-acute bacterial endocarditis (SABE) was measured. The species, in order of greatest adhesion, were Streptococcus faecalis, Streptococcus mutans, Streptococcus milleri, Streptococcus sanguis, dextran-positive Streptococcus mitior, dextran-negative Streptococcus mitior and Streptococcus salivarius. Individual strains within species, however, cannot be assumed to be representative of their species and may exhibit unusually high or low adhesion. Adhesion depended upon both bacterial concentration and period of contact. There was no simple relationship between ability to adhere and liability to cause endocarditis. Formation of dextran did not increase adhesion. The streptococci were more adhesive than strains of Escherichia coli and Neisseria sicca and less adhesive than strains of Staphylococcus aureus and Streptococcus pyogenes.     

The hydrophobicity of 'viridans' streptococci isolated from the human mouth

The hydrophobicity of human oral streptococci was measured with the hexadecane assay modified by the incorporation of polyethylene glycol 6000. Large variability in the hydrophobicity between cultures of some strains grown on different occasions was observed whereas other strains were less variable. The variation in hydrophobicity was significantly reduced by growing the cells in continuous culture in a chemostat under glucose-limiting conditions. The Streptococcus mutans strains used all had low hydrophobicity and the mean hydrophobicity of this species was significantly lower (P less than 0.05) than the mean hydrophobicity of Strep. salivarius, Strep. sanguis Type I and Strep. sanguis Type II strains. This finding supports the view that hydrophobicity is a contributing factor in the adhesion of viridans streptococci to oral surfaces.     

The hydrophobicity of 'viridans' streptococci isolated from the human mouth

The hydrophobicity of human oral streptococci was measured with the hexadecane assay modified by the incorporation of polyethylene glycol 6000. Large variability in the hydrophobicity between cultures of some strains grown on different occasions was observed whereas other strains were less variable. The variation in hydrophobicity was significantly reduced by growing the cells in continuous culture in a chemostat under glucose-limiting conditions. The Streptococcus mutans strains used all had low hydrophobicity and the mean hydrophobicity of this species was significantly lower ( P < 0.05) than the mean hydrophobicity of Strep. salivarius, Strep. sanguis Type I and Strep. sanguis Type II strains. This finding supports the view that hydrophobicity is a contributing factor in the adhesion of viridans streptococci to oral surfaces.     

In-vitro bacteriocin-mediated antagonism by oral streptococci against human carrier strains of staphylococci

All strains of oral streptococci tested and specially those of Streptococcus mutans, Strep. sanguis and Strep. mitior produced more than one distinct bacteriocin-like substance with variable inhibitory activity on 20 indicator staphylococci. Inhibitory activity was comparatively higher on nasal strains of Staph. aureus and Staph. epidermidis than on strains of both species isolated from the mouth. Nineteen of 20 staphylococcal indicators were inhibited by 1-12 of the 12 effector streptococci. Sensitivity of nasal staphylococci to bacteriocins (frequency of positive inhibitory tests and total inhibition zone diameters) was significantly higher (P less than 0.001, chi 2 test and P less than 0.05, t test respectively) than that of oral ones. The sensitivity of nasal over oral Staph. aureus (P less than 0.001 and P less than 0.01) and of oral Staph. epidermidis over oral Staph. aureus (P less than 0.01 and P less than 0.05) was also significantly higher. The evaluation of variability of inhibitory patterns of bacteriocins produced by streptococci (p-typing), of sensitivity patterns of staphylococci to bacteriocins (s-typing) and of the significantly higher sensitivity of nasal over oral staphylococci to bacteriocins from the epidemiological and ecological viewpoints are discussed.     

In-vitro bacteriocin-mediated antagonism by oral streptococci against human carrier strains of staphylococci

All strains of oral streptococci tested and specially those of Streptococcus mutans, Strep. sanguis and Strep. minor produced more than one distinct bacteriocin-like substance with variable inhibitory activity on 20 indicator staphylococci. Inhibitory activity was comparatively higher on nasal strains of Staph. aureus and Staph. epidermidis than on strains of both species isolated from the mouth. Nineteen of 20 staphylococcal indicators were inhibited by 1–12 of the 12 effector streptococci. Sensitivity of nasal staphylococci to bacteriocins (frequency of positive inhibitory tests and total inhibition zone diameters) was significantly higher ( P < 0·001, χ² test and P < 0·05, t test respectively) than that of oral ones. The sensitivity of nasal over oral Staph. aureus ( P < 0·001 and P < 0·01) and of oral Staph. epidermidis over oral Staph. aureus ( P < 0·01 and P < 0·05) was also significantly higher. The evaluation of variability of inhibitory patterns of bacteriocins produced by streptococci (p-typing), of sensitivity patterns of staphylococci to bacteriocins (s-typing) and of the significantly higher sensitivity of nasal over oral staphylococci to bacteriocins from the epidemiological and ecological veiwpoints are discussed.     

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.     

A high molecular mass cranberry constituent reduces mutans streptococci level in saliva and inhibits in vitro adhesion to hydroxyapatite

Previous investigations showed that a high molecular mass, non-dialyzable material (NDM) from cranberries inhibits the adhesion of a number of bacterial species and prevents the co-aggregation of many oral bacterial pairs. In the present study we determined the effect of mouthwash supplemented with NDM on oral hygiene. Following 6 weeks of daily usage of cranberry-containing mouthwash by an experimental group (n = 29), we found that salivary mutans streptococci count as well as the total bacterial count were reduced significantly (ANOVA, P < 0.01) compared with those of the control (n = 30) using placebo mouthwash. No change in the plaque and gingival indices was observed. In vitro, the cranberry constituent inhibited the adhesion of Streptococcus sobrinus to saliva-coated hydroxyapatite. The data suggest that the ability to reduce mutans streptococci counts in vivo is due to the anti-adhesion activity of the cranberry constituent.     

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.     

Isolation and study of fosfomycin-resistant mutants of group A and B streptococci

Eighty-nine fosfomycin-resistant mutants of group A and B streptococci were isolated. The mutants differed essentially from the parent strains in cell-wall characteristics, such as morphology of cocci and chains, sensitivity to detergents and hydrophobic properties. At the same time, the recipient activity of the investigated mutants was not changed. It is supposed that resistance of mutants to fosfomycin is connected with a transport block of glycerol 3-phosphate--important lipoteichoic acid (LTA) structural component. Fosr mutants can be useful for experimental examination of Beachey's hypothesis about the importance of LTA or its complex with the M protein for adhesion.     

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.     

In vitro inhibition of oral streptococci binding to the acquired pellicle by algal lectins

AIMS: The initial colonization of the tooth by streptococci involves their attachment to adsorbed components of the acquired pellicle. Avoiding this adhesion may be successful in preventing caries at early stages. Salivary mucins are glycoproteins that when absorbed onto hydroxyapatite may provide binding sites for certain bacteria. Algal lectins may be especially interesting for oral antiadhesion trials because of their great stability and high specificity for mucins. This work aimed to evaluate the potential of two algal lectins to inhibit the adherence of five streptococci species to the acquired pellicle in vitro. METHODS AND RESULTS: The lectins used were extracted from Bryothamnion triquetrum (BTL) and Bryothamnion seaforthii (BSL). Fluorescence microscopy was applied to visualize the ability of fluorescein isothiocyanate-labelled lectins to attach to the pellicle and revealed a similar capability for both lectins. Streptococcal adherence assays were performed using saliva-coated microtitre plates. BSL inhibited more than 75% of Streptococcus sanguis, Streptococcus mitis, Streptococcus sobrinus and Streptococcus mutans adherence, achieving 92% to the latter. BTL only obtained statistically significant results on S. mitis and S. sobrinus, whose adherence was decreased by 32.5% and 54.4%, respectively. CONCLUSION: Algal lectins are able to inhibit streptococcal adherence. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results support the proposed application of lectins in antiadhesion therapeutics.     

Surface free energies of oral streptococci and their adhesion to solids

Abstract The adhesion of 3 strains of oral streptococci from a buffered suspension onto 3 different solid substrata was studied. Representative strains of streptococci were selected on the basis of their surface free energy ( γ _b), namely Streptococcus mitis L1 ( γ _b= 37 mJ·m⁻²), Streptococcus sanguis CH3 (95 mJ·m⁻²) and Streptococcus mutans NS (117 mJ·m⁻²). Solid substrata were also selected on basis of their surface free energy ( γ _s), and included polytetrafluorethylene ( γ _s= 20 mJ·m⁻²), polymethylmethacrylate (53 mJ·m⁻²) and glass (109 mJ·m⁻²). Bacterial adhesion was measured as the number of bacteria adhering per cm² at equilibrium. Equilibrium was usually obtained within 20 min. S. sanguis CH3, having an intermediate surface free energy did not show a clear preference for any of the 3 solids. S. mitis L1, however, the lowest surface free energy strain, adhered in highest numbers to the low energy solid PTFE, whereas the highest γ _b strain, S. mutans NS, adhered in highest numbers to the highest γ _s solid, glass. Calculation of the interfacial free energy of adhesion ( ΔF _adh) for each bacterial strain showed that this parameter was predictive of bacterial adhesion to solid substrata.     

Relationship between Expression of the Family of M Proteins and Lipoteichoic Acid to Hydrophobicity and Biofilm Formation in Streptococcus pyogenes

Background

Hydrophobicity is an important attribute of bacteria that contributes to adhesion and biofilm formation. Hydrophobicity of Streptococcus pyogenes is primarily due to lipoteichoic acid (LTA) on the streptococcal surface but the mechanism(s) whereby LTA is retained on the surface is poorly understood. In this study, we sought to determine whether members of the M protein family consisting of Emm (M protein), Mrp (M-related protein), Enn (an M-like protein), and the streptococcal protective antigen (Spa) are involved in anchoring LTA in a manner that contributes to hydrophobicity of the streptococci and its ability to form biofilms.

Methodology/Principal Findings

Isogenic mutants defective in expression of emm, mrp, enn, and/or spa genes of eight different serotypes and their parental strains were tested for differences in LTA bound to surface proteins, LTA released into the culture media, and membrane-bound LTA. The effect of these mutations on the ability of streptococci to form a hydrophobic surface and to generate biofilms was also investigated. A recombinant strain overexpressing Emm1 was also engineered and similarly tested. The serotypes tested ranged from those that express only a single M protein gene to those that express two or three members of the M protein family. Overexpression of Emm1 led to enhanced hydrophobicity and biofilm formation. Inactivation of emm in those serotypes expressing only a single emm gene reduced biofilm formation, and protein-bound LTA on the surface, but did not alter the levels of membrane-bound LTA. The results were more varied in those serotypes that express two to three members of the M protein family.

Conclusions/Significance

Our findings suggest that the formation of complexes with members of the M protein family is a common mechanism for anchoring LTA on the surface in a manner that contributes to hydrophobicity and to biofilm formation in S. pyogenes, but these activities in some serotypes are dependent on a trypsin-sensitive protein(s) that remains to be identified. The need for interactions between LTA and M proteins may impose functional constraints that limit variations in the sequence of the M proteins, major virulence factors of S. pyogenes.     

The influence of biosurfactants released by S. mitis BMS on the adhesion of pioneer strains and cariogenic bacteria

The influence of Streptococcus mitis BMS biosurfactants on the adhesion of eight pioneer and four cariogenic oral bacterial strains was, for a first screening, examined in a microtiter plate assay. The adhesion to pellicle-coated wells of three cariogenic strains was inhibited >70% by the biosurfactants, while only one pioneer strain showed >70% reduction. The reduction for the other strains did not exceed 50%. Subsequently, adhesion of Streptococcus mutans ATCC 25175 and Streptococcus sobrinus HG 1025, both cariogenic strains, and Actinomyces naeslundii T14V-J1 and Streptococcus oralis J22, two pioneer strains, to biosurfactants-coated enamel with and without a salivary pellicle was studied in a parallel plate flow chamber. A biosurfactants coating to enamel with or without a pellicle caused a reduction in the number of adhering cariogenic organisms, although no such reduction was observed for the pioneer strains. Consequently, it is concluded that S. mitis BMS biosurfactants may play a protective role against adhesion of cariogenic bacteria.     

Physico-chemical and structural properties of the surfaces of Peptostreptococcus micros and Streptococcus mitis as compared to those of mutans streptococci, Streptococcus sanguis and Streptococcus salivarius.

The surface properties of nine Streptococcus mitis and four Peptostreptococcus micros strains from the oral cavity were examined and compared with a large group of oral streptococci. Zeta potential and contact angle measurements were employed to determine physico-chemical cell surface properties. In addition, elemental surface concentration ratios were obtained via X-ray photoelectron spectroscopy, and surface structures were examined with transmission electron microscopy. The S. mitis and P. micros strains were found to have higher isoelectric points, higher hydrophobicities and higher N/C surface concentration ratios than some other oral streptococci. The combined data suggest that both species possess large amounts of surface protein. All the S. mitis strains displayed abundant surface fibrils in negative staining, but the P. micros strains were devoid of surface appendages indicating that surface protein is present in different forms in the two species. The surfaces of S. mitis and P. micros type strains differed significantly from the other strains examined.     

Xylitol and Erythritol Decrease Adherence of Polysaccharide-Producing Oral Streptococci

Xylitol consumption decreases counts of mutans streptococci. However, the mechanism behind this decrease is not well understood. We studied not only type strains and clinical isolates of mutans streptococci, but also other polysaccharide-forming oral streptococci. Growth inhibition and adherence of cells to a smooth glass surface—reflecting synthesis of water-insoluble polysaccharides were studied in the presence of 2% (0.13 mol/l) and 4% (0.26 mol/l) xylitol. The effect of xylitol was compared to a novel polyol sweetener, erythritol. Except for Streptococcus mutans 10449 and S. sobrinus OMZ 176, the glass surface adhesion of most polysaccharide-forming streptococci was reduced by the presence of both 4% xylitol and erythritol. For the S. mutans and S. sobrinus type strains, the growth inhibition with 4% xylitol and erythritol was 36–77% and for the clinical S. mutans isolates 13–73%. Of the other oral streptococci, only S. sanguinis was inhibited with 4% xylitol (45–55%). For both polyols, the magnitude of the growth inhibition observed was not associated with the magnitude of the decrease in adherence (xylitol: r = −0.18; erythritol: r = 0.49). In conclusion, both xylitol and erythritol can decrease polysaccharide-mediated cell adherence contributing to plaque accumulation through a mechanism not dependent on growth inhibition.     

Induction of immune response to Streptococcus pneumoniae by administration of oral viridans streptococci via phosphorylcholine determinant

Expression of the phosphorylcholine (PC) epitope was examined in 48 viridans streptococcal strains, including Streptococcus pneumoniae R36a as the positive control, and their immunogenicity to induce an S. pneumoniae-cross-reactive response was evaluated in mice. Thirteen strains were found to express the PC epitope, while no obvious association was found between the taxonomic categories and PC expression. Serum antibody responses to S. pneumoniae cells were induced in mice by intraperitoneal injection of the PC-positive, but not PC-negative, strains. The cross-reactive antibodies induced by non-pneumococcal oral streptococci were readily inhibited by free hapten PC. IgM was the sole isotype of the anti-pneumococcal and anti-PC antibodies, and the phenomenon of immunological memory was not observed. Since the anti-PC antibody is critically important for resistance against pneumococcal infection in mice, the present results indicate the possibility that PC-expressing oral commensal bacteria have a significant influence on the hosts' responsiveness to S. pneumoniae.