Sanguis Biofilms Research Articles

A Self-Supplying H2O2 Modified Nanozyme-Loaded Hydrogel for Root Canal Biofilm Eradication.

The success of root canal therapy depends mainly on the complete elimination of the root canal bacterial biofilm. The validity and biocompatibility of root canal disinfectant materials are imperative for the success of root canal treatment. However, the insufficiency of the currently available root canal disinfectant materials highlights that more advanced materials are still needed. In this study, a nanozyme-loaded hydrogel (Fe3O4-CaO2-Hydrogel) was modified and analyzed as a root canal disinfectant material. Fe3O4-CaO2-Hydrogel was fabricated and examined for its release profile, biocompatibility, and antibacterial activity against E. faecalis and S. sanguis biofilms in vitro. Furthermore, its efficiency in eliminating the root canal bacterial biofilm removal in SD rat teeth was also evaluated. The results in vitro showed that Fe3O4-CaO2-Hydrogel could release reactive oxygen species (ROS). Moreover, it showed good biocompatibility, disrupting bacterial cell membranes, and inhibiting exopolysaccharide production (p < 0.0001). In addition, in vivo results showed that Fe3O4-CaO2-Hydrogel strongly scavenged on root canal biofilm infection and prevented further inflammation expansion (p < 0.05). Altogether, suggesting that Fe3O4-CaO2-Hydrogel can be used as a new effective biocompatible root canal disinfectant material. Our research provides a broad prospect for clinical root canal disinfection, even extended to other refractory infections in deep sites.

Selective activities of STAMPs against Streptococcus mutans.

The present study aimed to design, synthesize and screen specifically targeted antimicrobial peptides (STAMPs) that can selectively kill Streptococcus mutans (S. mutans) in the biofilm, and to detect protein metabolism, in order to investigate the mechanism of the antibacterial functions of STAMPs against S. mutans. A series of STAMPs were synthesized, and their effects on the selective antibacterial activity of S. mutans on single species and multi-species biofilms under the condition of the planktonic state were studied. The total protein of S. mutans was extracted before and after C11H, and matrix-assisted laser adsorption ionization-time of flight mass spectrometry identification was performed. The antibacterial activity on planktonic S. mutans was increased 3- to 4-fold via C8H, C11H, C12H, C13H, and C14H compared with hLF1-11 (H) alone, and there was no difference between Streptococcus gordonii (S. gordonii) and Streptococcus sanguis (S. sanguis). C8H, C11H, C12H, C13H, and C14H had significant inhibitory effects on the growth of S. mutans biofilm, but there were no significant effects on S. gordonii and S. sanguis biofilms. The number of S. mutans in biofilm decreased at 4 h after C8H, C11H, C12H, C13H and C14H and C8, C11, C12, C13 and C14 had no effect on the growth of planktonic and biofilm states of S. mutans, S. gordonii and S. sanguis species. C11H and C12H exhibited the most obvious effects, followed by C13H and C14H, and then C8H. A total of 21 protein spots with a mean change ratio of 1.5 were identified, all of which were downregulated after C11H. A total of 19 proteins were successfully identified, including cell cycle-relative proteins, nucleic acid metabolism-related enzymes and proteins, virulence factors, protein biosynthesis and regulation, proteins involved in energy metabolism, and proteins with unknown function. In the present study, STAMPs with selective antibacterial activity against S. mutans grown in planktonic or biofilm states but without obvious effects on oral Streptococci and multi-species biofilm were successfully designed and synthesized. Differential protein expression before and after C11H was identified. The mechanism of the antibacterial function was also discussed. Results of the present study laid the foundation for application of STAMPs in the prevention and treatment of dental caries.

Characterization of Streptococcus sanguis molecular receptors for Streptococcus mutans binding molecules

Background: Dental caries is a major problem in oral cavity. If dental caries causes cavity, the structure of dental hard tissue will not be reversible because of damage in the structure of the hard tissue. The early pathogenesis mechanism of dental caries is an adhesion interaction between cariogenic Streptococcus mutans microorganisms and tooth surface pellicles. The attachment involves a specific molecular component interaction between the bacterial complement molecules and the surface of the host. Streptococcus sanguis as a dominant ecology at the beginning of bacterial plaque aggregation will colonize the tooth surface earlier than S. mutans. The surface of bacterial cells can express some adesin. The bacteria also can express receptors for adhesins of other bacteria. Specific receptors for adhesions of S. Mutans bacteria are not only found in the pellicles, but also present in pioneer bacteria, such as S. sanguis. Adhesion between those bacteria is called as coagregation. Purpose: This study aimed to analyze the characterization of Streptococcus sanguis molecular receptors for Streptococcus mutans binding molecules. Method: This study used a sonication method for protein isolation of S. mutans and S. sanguis bacterial biofilms, as well as electrophoresis method using 12 % SDS-PAGE gel and Western Blot analysis. Result: Results of the protein profile analysis of S. mutans biofilms using 12% SDS-PAGE showed that there were 17 bands, each of which molecular weights was 212, 140, 81, 65, 61, 48, 45, 44, 40, 39, 33 , 25, 23, 19, 17, 12, and 11 kDa. On the other hand, results of the protein profile analysis of S. sanguis biofilms using 12% SDS-PAGE showed that there were 15 bands, each of which molecular weight was 130, 85, 65, 61, 48, 46, 40, 37, 29, 25, 23, 21, 17, 15, and 12 kDa. And, results of the analysis of S. sanguis receptor molecules using Western blot showed that there were three bands, each of which molecular weight was 130, 85, and 40 kDa. Conclusion: S. sanguis bacteria have specific receptor molecules for S. mutans bacteria with the molecular weight of 130, 85, and 40 kDa.

In vitro anti‐biofilm activity of macelignan isolated from Myristica fragrans Houtt. against oral primary colonizer bacteria

In early dental plaque formation, oral primary colonizers such as Streptococcus mutans, Streptococcus sanguis and Actinomyces viscosus are initially attached to the pellicle-coated tooth surface to form a biofilm. The study aimed to determine the efficacy of macelignan, isolated from nutmeg (Myristica fragrans Houtt.), in removing each single oral primary biofilm in vitro on a polystyrene 96-well microtiter plate. Four biofilm growth phases (4, 12, 20 and 24 h) were evaluated in this study after treatment with macelignan at various concentrations (0.2, 2 and 10 microg/mL) and exposure times (5, 10 and 30 min). Anti-biofilm activity of macelignan was measured as the percentage of the remaining biofilm absorbance after macelignan treatment in comparison with the untreated control. At 24 h of biofilm growth, S. mutans, A. viscosus and S. sanguis biofilms were reduced by up to 30%, 30% and 38%, respectively, after treatment with 10 microg/mL macelignan for 5 min. Increasing the treatment time to 30 min resulted in a reduction of more than 50% of each of the single primary biofilms. The results indicate that macelignan is a potent natural anti-biofilm agent against oral primary colonizers.

Interaction between GIC and S. sanguis biofilms: Antibacterial properties and changes of surface hardness

The aim of this study was to investigate the interaction of Streptococcus sanguis with two glass-ionomer formulations (GIC:A containing fluoride and GIC:B without fluoride) with particular reference to bacterial growth and changes in hardness of the cement with respect to time. Discs of two water activated glass-ionomer cements were prepared according to the manufacturer's instruction. Hydroxyapatite discs (HA) were used as controls. 3D laser scanning technique was used to characterize surface roughness and area of the substrate prior to growing biofilms. Surface hardness was evaluated before and after biofilm growth. A constant depth film fermenter system (CDFF) was used to grow S. sanguis biofilms on the specimens in a similar manner to that described previously by Wilson et al. in 1995. For susceptibility measurement, specimens were removed from CDFF aseptically over periods up to 14d after the first colonization with bacteria. Counts of viable bacterial in the accumulating biofilm layer on each surface were measured and converted to colony forming units per unit surface area. To determine the effect of storage media, hardness discs were exposed to distilled water, lactic acid pH 4, lactic acid pH 5, citric acid pH 5, artificial saliva and S. sanguis biofilms. Twenty-four hours after preparing and subsequent autoclaving, specimens were transferred to a vessel containing 40 ml storage medium. The specimens were investigated for periods up to 7d. The viable counts of S. sanguis per mm2 on GIC:A were significantly less than those on HA and GIC:B during the first 5d (p<0.05). The viable counts of bacteria on the surface of GIC:B were lower during the initial 5d when compared to HA. Exposure of GIC:A and GIC:B to different medium produced softening to the surface of cement. It is apparent that the effects of the biofilms are significantly greater than storage in water but similar to storage in lactic acid pH 5. This investigation showed that the growth of S. sanguis biofilms were significantly affected by both glass-ionomer formulations, the greater reduction being noted on the surface of the fluoride containing GIC. S. sanguis biofilms produced reduction on the surface hardness of the cement equivalent to that seen after immersion in lactic acid at pH 5. This indicates that while S. sanguis biofilm is affected by the GIC, there is also a decrease in hardness of the cement indicating some cement degradation.

Early biofilm formation and the effects of antimicrobial agents on orthodontic bonding materials in a parallel plate flow chamber

Decalcification is a commonly recognized complication of orthodontic treatment with fixed appliances. A technology, based on a parallel plate flow chamber, was developed to investigate early biofilm formation of a strain of Streptococcus sanguis on the surface of four orthodontic bonding materials: glass ionomer cement (Ketac Cem), resin-modified glass ionomer cement (Fuji Ortho LC), chemically-cured composite resin (Concise) and light-cured composite resin (Transbond XT). S. sanguis was used as it is one of the primary colonizers of dental hard surfaces. Artificial saliva was supplied as a source of nutrients for the biofilms. The effects of two commercially available mouthrinses (i.e. a fluoride containing rinse and chlorhexidine) were evaluated. Initial colonization of the bacterium was assessed after 6 hours of growth by the percentage surface coverage (PSC) of the biofilm on the disc surfaces. There were statistically significant differences in bacterial accumulation between different bonding materials (P < 0.05), Concise being the least colonized and Transbond XT being the most colonized by S. sanguis biofilms. All materials pre-treated with 0.05 per cent sodium fluoride mouthrinse showed more than 50 per cent reduction in biofilm formation. The 0.2 per cent chlorhexidine gluconate mouthrinse caused significant reduction of biofilm formation on all materials except Ketac Cem. This in vitro study showed that the use of a chemically-cured composite resin (Concise) reduced early S. sanguis biofilm formation. Also, fluoride had a greater effect in reducing the PSC by S. sanguis biofilms than chlorhexidine. Rinsing with 0.05 per cent sodium fluoride prior to placement of orthodontic appliances is effective in reducing early biofilm formation.

Single species biofilm‐forming ability of root canal isolates on gutta‐percha points

The participation of bacterial biofilms in the over-filled gutta-percha points associated with refractory periapical periodontitis has recently been reported. This study investigated the initial biofilm-forming ability of root canal isolates (Enterococcus faecalis, Streptococcus sanguis, Strep. intermedius, Strep. pyogenes, Staphylococcus aureus, Fusobacterium nucleatum, Propionibacterium acnes, Porphyromonas gingivalis and Prevotella intermedia) on gutta-percha points in vitro. Each bacterial strain was suspended in 100% cell culture medium or in culture medium containing 4.5, 45 or 90% (vol/vol) serum. The bacterial suspensions were then co-incubated anaerobically with gutta-percha points for 7 d. The gutta-percha points were processed for scanning electron microscopic observation and examined for biofilm presence and thickness. E. faecalis, Strep. sanguis, Strep. intermedius, Strep. pyogenes and Staph. aureus biofilms were generated on the surfaces of the specimens incubated in culture medium supplemented with 45 or 90% (vol/vol) serum. The E. faecalis and Strep. sanguis biofilms were significantly thicker than those of Strep. intermedius, Strep. pyogenes and Staph. aureus. No biofilms were detected on the specimens incubated with F. nucleatum, Prop. acnes, Porph. gingivalis and Prev. intermedia. These findings suggest that Gram-positive facultative anaerobes have the ability to colonize and form extracellular matrices on gutta-percha points, while serum plays a crucial role in biofilm formation.

Response of single species biofilms and microcosm dental plaques to pulsing with chlorhexidine.

The aim of this study was to determine the effect of pulsing chlorhexidine gluconate, at concentrations commonly used in mouthwashes, on Streptococcus sanguis biofilms and microcosm dental plaques in vitro. Biofilms were grown on bovine enamel and nutrients were supplied in the form of artificial saliva. Pulsing experiments were carried out on steady-state biofilms using 0.05 or 0.2% chlorhexidine solutions delivered twice daily for 1 min. In a separate study, the enamel discs on which the biofilms were formed were pre-treated with chlorhexidine and pulsed directly after inoculation and then at regular intervals. With both concentrations of chlorhexidine used, a c.2 log10 reduction in the viable counts of S. sanguis was achieved with the initial pulse, but as pulsing continued, the bacterial population recovered, albeit not to the previous level. A c.1 log10 reduction in the total viable counts of the microcosm plaques was seen after the first pulse with 0.2% chlorhexidine. The total count then recovered rapidly and, after the fifth pulse, the total viable counts were not significantly different from those before pulsing. The total counts then remained at a similar level throughout the course of the experimental runs. Pre-treatment of the enamel discs with 0.2% chlorhexidine before inoculation produced viable counts of c.10(5) cfu/mm2, a 1 log10 reduction compared with untreated discs. After pulsing with 0.2% chlorhexidine at 8 h, a 3 log10 reduction was seen in the total aerobic and anaerobic counts, but again the viable counts subsequently increased despite twice-daily chlorhexidine pulsing. Regardless of the nature of the biofilm, pulsing initially achieved substantial kills, but the viability of the biofilms subsequently increased despite continued pulsing. Chlorhexidine was effective at reducing the viability of microcosm plaques when it was applied to the substratum before exposure to bacteria and subsequently pulsed on to the biofilms.

In vitro studies of the effect of antiseptic-containing mouthwashes on the formation and viability of Streptococcus sanguis biofilms.

The aims of this study were to evaluate the growth of Streptococcus sanguis on hydroxyapatite, bovine enamel and polytetrafluoroethylene substrata in a constant depth film fermentor, and to determine the effects of three antimicrobial-containing mouthwashes on biofilm formation and bacterial viability on hydroxyapatite and enamel. There was little difference in the final cell density (5 x 10(4) cfu mm-2) of the Strep. sanguis biofilm on the three substrata. When hydroxyapatite-grown biofilms were exposed to the mouthwashes for 1 min, the one containing triclosan (T) proved the most effective. The chlorhexidine-containing mouthwash (CX) also achieved significant kills. The T-containing mouthwash was the most effective at killing biofilms grown on enamel. Pre-treatment of hydroxyapatite with CX, cetylpyridium chloride (CPC) or T for 1 min resulted in undetectable biofilm formation after 8 h. After 8 h of growth, only biofilms grown on enamel discs pre-treated with CX showed a reduction in the number of viable organisms. In conclusion, the results of this study have shown that while growth of Strep. sanguis on hydroxyapatite and enamel were similar, the ability of antimicrobial agents to prevent the accumulation of viable bacteria depended on the nature of the substratum.

The Interaction of Phospholipid Liposomes with Bacteria and Their Use in the Delivery of Bactericides

Liposomes have been prepared from dipalmitoylphosphatidylcholine (DPPC) incorporating the cationic lipids stearylamine (SA), dimethyldioctadecylammonium bromide (DDAB) and dimethylaminoethane carbamoyl cholesterol (DCchol) and the anionic lipids dipalmitoylphosphatidylglycerol (DPPG) and phosphatidylinositol (PI). Their adsorption to biofilms of skin-associated bacteria (Staphylococcus epidermidis and Proteus vulgaris) and oral bacteria (Streptococcus mutatis and sanguis) has been investigated as a function of mole % cationic and anionic lipid. Targeting (adsorption) was most effective for the systems DPPC-chol-SA, DPPC-DPPG and DPPC-PI liposomes to S. epidermidis. The effect of extracellular mucopolysaccharide on targeting was investigated for S. epidermidis biofilms. It was found that targeting increased with the level of extracellular mucopolysaccharide for all liposome compositions studied. The delivery of the oil-soluble bactericide Triclosan and the water soluble bactericide chlorhexidine was studied for a number of liposomal compositions. Superior delivery of both bactericides relative to the free bactericide occurred for DPPC-chol-SA liposomes and for Triclosan delivery by DPPC-DPPG and DPPC-PI liposomes targeted to S. epidermidis at low bactericide concentrations. DPPC-chol-SA liposomes were also effective for delivery of Triclosan to S. sanguis biofilms. Double labelling experiments using [14C]-chlorhexidine and [3H]-DPPC suggested that there was exchange between adsorbed liposomes which had delivered bactericide to the biofilm and those in the bulk solution implying a diffusion mechanism for bactericide delivery.

Resistance ofStreptococcus sanguisbiofilms to antimicrobial agents

Bacteria living in biofilms as dental plaque on tooth surfaces are generally more resistant to antimicrobial agents than bacteria in batch culture normally used for in vitro susceptibility testing. In order to compare the resistance of free-living and surface-grown oral bacteria, the MIC of Streptococcus sanguis 804 and ATCC 10556 to amoxicillin, doxycycline and chlorhexidine was determined by a broth dilution method. Subsequently, S. sanguis biofilms established in an in vitro flow model were perfused with the antimicrobial agents for 48 h at concentrations equal to and up to 500 times the MIC, and biofilm cell number was determined during this period. The antibiotics at the MIC did not affect the cell number of S. sanguis biofilms compared to the starting point, and only after 48 h at 500 times the MIC were the biofilm bacteria eliminated. At intermediate concentrations biofilm cell number gradually decreased. Chlorhexidine also gradually reduced biofilm cell number, but was inhibitory at concentrations closer to the MIC than was the case for the antibiotics. Thus S. sanguis in biofilms survived up to 500 times the MIC found in batch culture for up to 48 h.

Targeting and delivery of bactericide to adsorbed oral bacteria by use of proteoliposomes

Proteoliposomes having surface-bound succinylated concanavalin A (s-conA) have been prepared from a range of phospholipid mixtures by sonication (SUV) and reverse phase evaporation (REV) covering a range of size ( weight-average diameter ( d w ) ) from approx. 35 to 310 nm and weight-average number of protein molecules per liposomes ( P w ) from approx. 50 to 3000. The targeting of the proteoliposomes to adsorbed biofilms of the bacteria Streptococcus sanguis and Streptococcus mutans has been assessed from the extent of inhibition of an enzyme-linked immunosorbent assay (ELISA) for bacterial cell surface antigens. The surface-bound lectin enhances targeting relative to ‘naked’ liposomes of comparable concentration by factors of 2–50 depending on the liposomal lipid composition and P w . The effect of the bactericide Triclosan® on the thermal properties and permeability characteristics of liposomes has been studied. At and above a molar ratio of Triclosan® to lipid of 0.6, Triclosan® eliminates the gel to liquid-crystalline phase transition in dipalmitoylphosphatidylcholine (DPPC) containing liposomes and increases the bilayer permeability of both liposomes and proteoliposomes to d-glucose. The proteoliposomes have been used to deliver Triclosan® to S. sanguis biofilms and the inhibition of growth of the bacteria after treatment with liposomally delivered Triclosan® has been determined using a microtitre plate re-growth assay and compared with growth inhibition by ‘free’ Triclosan®. It is shown that for short exposure times (1 to 2 min) proteoliposomally delivered Triclosan® is a more effective growth inhibitor than free Triclosan®. The results are discussed in terms of the targeting, retention and subsequent release of Triclosan® into the bacterial biofilms.