Effects of combined oleic acid and fluoride at sub-MIC levels on EPS formation and viability of Streptococcus mutans UA159 biofilms

Despite the widespread use of fluoride, dental caries, a biofilm-related disease, remains an important health problem. This study investigated whether oleic acid, a monounsaturated fatty acid, can enhance the effect of fluoride on extracellular polysaccharide (EPS) formation by Streptococcus mutans UA159 biofilms at sub-minimum inhibitory concentration levels, via microbiological and biochemical methods, confocal fluorescence microscopy, and real-time PCR. The combination of oleic acid with fluoride inhibited EPS formation more strongly than did fluoride or oleic acid alone. The superior inhibition of EPS formation was due to the combination of the inhibitory effects of oleic acid and fluoride against glucosyltransferases (GTFs) and GTF-related gene (gtfB, gtfC, and gtfD) expression, respectively. In addition, the combination of oleic acid with fluoride altered the bacterial biovolume of the biofilms without bactericidal activity. These results suggest that oleic acid may be useful for enhancing fluoride inhibition of EPS formation by S. mutans biofilms, without killing the bacterium.     

Cytotoxicity and the effect of cationic peptide fragments against cariogenic bacteria under planktonic and biofilm conditions

This study evaluated the cytotoxicity and effect of fragments derived from three oral cationic peptides (CP): LL-37, D6-17 and D1-23 against cariogenic bacteria under planktonic and biofilm conditions. For cytotoxicity analysis, two epithelial cell lines were used. The minimum inhibitory concentration and the minimal bactericidal concentration were determined for the CP fragments and the control (chlorhexidine-CHX) against cariogenic bacteria. The fractional inhibitory concentration was obtained for the combinations of CP fragments on Streptococcus mutans. Biofilm assays were conducted with the best antimicrobial CP fragment against S. mutans. The results indicated that D6-17 was not cytotoxic. D1-23, LL-37 and CHX were not cytotoxic in low concentrations. D1-23 presented the best bactericidal activity against S. mutans, S. mitis and S. salivarius. Combinations of CP fragments did not show a synergic effect. D1-23 presented a higher activity against S. mutans biofilm than CHX. It was concluded that D1-23 showed a substantial effect against cariogenic bacteria and low cytotoxicity.     

Anti‐biofilm and bactericidal effects of magnolia bark‐derived magnolol and honokiol on Streptococcus mutans

Dental caries affects people of all ages and is a worldwide health concern. Streptococcus mutans is a major cariogenic bacterium because of its ability to form biofilm and induce an acidic environment. In this study, the antibacterial activities of magnolol and honokiol, the main constituents of the bark of magnolia plants, toward planktonic cell and biofilm of S. mutans were examined and compared with those of chlorhexidine. The minimal inhibitory concentrations of magnolol, honokiol and chlorhexidine for S. mutans were 10, 10 and 0.25 µg/mL, respectively. In addition, each agent showed bactericidal activity against S. mutans planktonic cells and inhibited biofilm formation in a dose‐ and time‐dependent manner. Magnolol (50 µg/mL) had greater bactericidal activity against S. mutans biofilm than honokiol (50 µg/mL) and chlorhexidine (500 µg/mL) at 5 min after exposure, while all showed scant activity against biofilm at 30 s. Furthermore; chlorhexidine (0.5–500 µg/mL) exhibited high cellular toxicity for the gingival epithelial cell line Ca9‐22 at 1 hr, whereas magnolol (50 µg/mL) and honokiol (50 µg/mL) did not. Thus; it was found that magnolol has antimicrobial activities against planktonic and biofilm cells of S. mutans. Magnolol may be a candidate for prevention and management of dental caries.     

Effect of brief periodic fluoride treatments on the virulence and composition of a cariogenic biofilm

The present study investigated the effect of periodic 1-min fluoride treatments on Streptococcus mutans biofilms and then determined the relationship between anti-biofilm activity, treatment frequency, and fluoride concentration using a linear-fitting procedure. S. mutans biofilms were periodically treated (1-min/treatment) with fluoride during biofilm formation and analyzed using microbiological methods, confocal microscopy, and real-time PCR. The results indicated that reductions in the dry weight and acidogenicity of biofilms due to periodic fluoride treatment occurred in a concentration dependent manner. The reduction in dry weight without affecting bacterial cell viability was observed mainly due to the inhibitory effect of fluoride on gtfB and gtfC gene expression, which suppresses EPS production and avoids reduction of the pH below the critical point on the tooth surface. This study suggests that brief periodic exposure to appropriate fluoride concentrations through mouthwashes and toothpastes may affect the virulence and composition of cariogenic biofilms and subsequently prevent dental caries.     

Peptide Inhibitors of Streptococcus mutans in the Control of Dental Caries

Peptides have been investigated as potential inhibitors of Streptococcus mutans, the main cause of dental caries, and have demonstrated considerable promise. In a human trial, topical application to tooth surfaces of a synthetic peptide inhibitor (p1025) of S. mutans adhesion prevented recolonisation with the oral pathogen following treatment with chlorhexidine gluconate (a broad spectrum antiseptic compound). An important feature of this treatment is that the duration of protection extends well beyond the period in which p1025 is applied. The specific targeting of S. mutans which allows other members of the oral flora associated with health to recolonise the oral cavity and competitively exclude S. mutans may explain the extended protection. Further in vitro studies have identified several other peptides which may have potential as inhibitors of S. mutans. Of particular interest are studies that demonstrate that competence stimulating peptides of S. mutans act as inhibitors of S. mutans growth and that peptides derived from the competence stimulating peptides can be used as a means of specifically targeting broad spectrum antimicrobial peptides.     

KR-12-a5 is a non-cytotoxic agent with potent antimicrobial effects against oral pathogens

This study evaluated the cytotoxicity and antimicrobial activity of analogs of cationic peptides against microorganisms associated with endodontic infections. L-929 fibroblasts were exposed to LL-37, KR-12-a5 and hBD-3–1C^V and chlorhexidine (CHX, control), and cell metabolism was evaluated with MTT. The minimal inhibitory concentration (MIC) and the minimal bactericidal/fungicidal concentration (MBC/MFC) of the peptides and CHX were determined against oral pathogens associated with endodontic infections. Enterococcus faecalis and Streptococcus mutans biofilms were cultivated in bovine dentin blocks, exposed to different concentrations of the most efficient antimicrobial peptide and analyzed by confocal laser scanning microscopy. CHX and peptides affected the metabolism of L-929 at concentrations > 31.25 and 500 μg ml^?1, respectively. Among the peptides, KR-12-a5 inhibited growth of both the microorganisms tested with the lowest MIC/MBC/MFC values. In addition, KR-12-a5 significantly reduced E. faecalis and S. mutans biofilms inside dentin tubules. In conclusion, KR-12-a5 is a non-cytotoxic agent with potent antimicrobial and anti-biofilm activity against oral pathogens associated with endodontic infections.     

Antimicrobial activity of nisin against the swine pathogen Streptococcus suis and its synergistic interaction with antibiotics

Streptococcus suis serotype 2 is known to cause severe infections in pigs, including meningitis, endocarditis and pneumonia. Furthermore, this bacterium is considered an emerging zoonotic agent. Recently, increased antibiotic resistance in S. suis has been reported worldwide. The objective of this study was to evaluate the potential of nisin, a bacteriocin of the lantibiotic class, as an antibacterial agent against the pathogen S. suis serotype 2. In addition, the synergistic activity of nisin in combination with conventional antibiotics was assessed. Using a plate assay, the nisin-producing strain Lactococcus lactis ATCC 11454 proved to be capable of inhibiting the growth of S. suis (n = 18) belonging to either sequence type (ST)1, ST25, or ST28. In a microdilution broth assay, the minimum inhibitory concentration (MIC) of purified nisin ranged between 1.25 and 5 μg/mL while the minimum bactericidal concentration (MBC) was between 5 and 10 μg/mL toward S. suis. The use of a capsule-deficient mutant of S. suis indicated that the presence of this polysaccharidic structure has no marked impact on susceptibility to nisin. Following treatment of S. suis with nisin, transmission electron microscopy observations revealed lysis of bacteria resulting from breakdown of the cell membrane. A time-killing curve showed a rapid bactericidal activity of nisin. Lastly, synergistic effects of nisin were observed in combination with several antibiotics, including penicillin, amoxicillin, tetracycline, streptomycin and ceftiofur. This study brought clear evidence supporting the potential of nisin for the prevention and treatment of S. suis infections in pigs.     

Enhancement of fluoride activity against Streptococcus mutans biofilms by a substance separated from Polygonum cuspidatum

Polygonum cuspidatum is a plant with spreading rhizomes and numerous reddish-brown stems that has been used in Korean folk medicine to improve oral hygiene. Nevertheless, there are no reports related to its possible effect on the virulence of dental biofilms. In this study, the ability of a fraction (F1) separated from P. cuspidatum, alone or in combination with fluoride, to disrupt virulence factors and the composition of Streptococcus mutans biofilms was examined. F1 was mainly composed of resveratrol, emodin and physcion (approximately 16.2%, 18.9% and 2.07% of the weight of F1, respectively). F1 showed inhibitory effects on acid production and F-ATPase activity of S. mutans in biofilms, and could enhance fluoride activity against acid production and acid tolerance of S. mutans in biofilms. When S. mutans biofilms were briefly treated with F1 (10 min, a total of five times), the biomass accumulation, water-insoluble polysaccharides and intracellular iodophilic polysaccharides were reduced. Furthermore, the fluoride activity against biomass accumulation was enhanced by F1. These results suggest that F1 may be useful in the control of dental biofilms and in improving the cariostatic properties of fluoride without increasing its exposure.     

Sterilization effects of a TiO<Subscript>2</Subscript> photocatalytic film against a<Emphasis Type="Italic">Streptococcus mutans</Emphasis> culture

Streptococcus mutans is one of the more significant pathogens involved in the development of dental caries in humans. The purpose of this research was to design a TiO₂-coated dental instrument and to determine the bactericidal effects of the instrument onS. mutants. TiO₂ photocatalytic films were prepared by the low-pressure metal-organic chemical vapor deposition (LPMOCVD) method using titanium tetraisopropoxide (TTIP) as precursor. The photocatalytic reaction was carried out on a TiO₂-coated pyrex petri dish with an ultraviolet (UV) light emitting diode (LED) illuminator or a fluorescent lamp light source. Our data indicates that the relative survival ratio ofS. mutans when plated onto TiO₂ photocatalytic films and under exposure to UV-A light for 15 min was 0.01%. In addition, a fluorescent lamp light source also had bactericidal effects on theS. mutans plated TiO₂ photocatalytic films. These results indicate that TiO₂-coated dental materials or devices may be useful in dental treatments for the prevention of carious or enamel demineralization.     

Comparison between amine fluoride and chlorhexidine with institutionalized elders: a pilot study

doi: 10.1111/j.1741‐2358.2012.00650.x Comparison between amine fluoride and chlorhexidine with institutionalized elders: a pilot study Objective: Compare the efficacy of amine fluoride toothpaste and gel with chlorhexidine spray in an institutionalised population. Background: People who live in nursing homes have poorer oral hygiene because of their dependency for the basic activities of daily living as they rely on caregivers. Methods: Twenty‐six people over 65 years old who had at least four teeth and living in a nursing home. They were assigned to three groups: A: amine fluoride toothpaste and once a week amine fluoride gel (Elmex^®), B: 0.12% spray‐chlorhexidine once a day (Perio‐Aid^®) and C: brush teeth without toothpaste. The plaque and gingival index of Silness and Löe, General Oral Health Assessment Index, McLeran and Pfeiffer index were recorded, and the number of colonies of Streptoccocus mutans and Lactobacillus and the remineralisation of caries were evaluated using Diagnodent^®. Measurements were taken at the beginning of the study and after 6 months. Results: Twenty‐two people finished the study. No group showed a statistical difference in the plaque or gingival index, but there was a tendency to show improvement in the amine fluoride group. There was also no difference between the number of colonies of either S. mutans or lactobacillus. There was a significant difference between the plaque and gingival index and the cognitive status (p = 0.0054), along with their requirement for assistance to perform good oral hygiene (p = 0.0001). Both products remineralised the carious lesions in this period compared with the control group (p = 0.0151). Conclusion: The plaque and gingival indices did not improve during the study, but both products remineralised the previous caries lesions.     

Influence of fluoride on the bacterial composition of a dual-species biofilm composed of Streptococcus mutans and Streptococcus oralis

Despite the widespread use of fluoride for the prevention of dental caries, few studies have demonstrated the effects of fluoride on the bacterial composition of dental biofilms. This study investigated whether fluoride affects the proportion of Streptococcus mutans and S. oralis in mono- and dual-species biofilm models, via microbiological, biochemical, and confocal fluorescence microscope studies. Fluoride did not affect the bacterial count and bio-volume of S. mutans and S. oralis in mono-species biofilms, except for the 24-h-old S. mutans biofilms. However, fluoride reduced the proportion and bio-volume of S. mutans but did not decrease those of S. oralis during both S. oralis and S. mutans dual-species biofilm formation, which may be related to the decrease in extracellular polysaccharide formation by fluoride. These results suggest that fluoride may prevent the shift in the microbial proportion to cariogenic bacteria in dental biofilms, subsequently inhibiting the cariogenic bacteria dominant biofilm formation.     

Natural Antimicrobials ε-Poly-l-lysine and Nisin A for Control of Oral Microflora

The present study aims to examine the efficacy of the natural antimicrobial ε-poly-l-lysine against Streptococcus mutans and against total aerobic oral microflora, alone and in combination with the natural antimicrobial peptide nisin. In in vitro studies, natural antimicrobials nisin A and ε-poly-l-lysine synergized in their action against S. mutans, leading to the microorganism’s full inhibition, while having a less inhibitory effect on total aerobic oral microbiota.     

Influences of naturally occurring agents in combination with fluoride on gene expression and structural organization of Streptococcus mutans in biofilms

Background  

The association of specific bioactive flavonoids and terpenoids with fluoride can modulate the development of cariogenic biofilms by simultaneously affecting the synthesis of exopolysaccharides (EPS) and acid production by Streptococcus mutans, which enhanced the cariostatic effectiveness of fluoride in vivo. In the present study, we further investigated whether the biological actions of combinations of myricetin (flavonoid), tt-farnesol (terpenoid) and fluoride can influence the expression of specific genes of S. mutans within biofilms and their structural organization using real-time PCR and confocal fluorescence microscopy.     

In vitro bactericidal activity of the N-terminal fragment of the frog peptide esculentin-1b (Esc 1–18) in combination with conventional antibiotics against Stenotrophomonas maltophilia

In this study the bactericidal effect of the N-terminal fragment of the frog skin peptide esculentin-1b [Esc(1–18)] in combination with clinically used antimicrobial agents was evaluated against Stenotrophomonas maltophilia, either in standard conditions (phosphate buffer) or in the presence of human serum. A synergistic bactericidal effect was observed after a 24 h incubation when combinations of Esc(1–18) and amikacin or colistin were used against clinical strains of S. maltophilia with or without resistance to these antibiotics, both in buffer and in the presence of serum. An indifferent effect was observed when the peptide was combined with levofloxacin or ceftazidime. A synergistic effect was also observed at earlier time points when the peptide was used in combination with colistin. Sequential exposure of bacterial cells to Esc(1–18) and amikacin or colistin, or vice versa, indicated that while Esc(1–18) and colistin cooperated in enhancing the bactericidal effect of their combination, when Esc(1–18) was combined with amikacin, the peptide had a major role in initiating the bactericidal effect, while amikacin was required for the subsequent effector phase. Altogether, the results obtained indicate that exposure of S. maltophilia to sub-bactericidal concentrations of Esc(1–18) increases its susceptibility to amikacin or colistin and may also render resistant strains susceptible to these antibiotics.     

Antibacterial activity of phellodendron bark against Streptococcus mutans

Streptococcus mutans is a major cause of tooth decay due to its promotion of biofilm formation and acid production. Several plant extracts have been reported to have multiple biological activities such as anti-inflammation and antibacterial effects. This study investigated the antibacterial activity of three plant extracts, phellodendron bark (PB), yucca, and black ginger, and found that PB had a stronger effect than the other extracts. Then, the minimum inhibitory concentration (MIC) of PB against 100 S. mutans strains was investigated. The MIC range of PB was 9.8–312.5 µg/mL. PB suppressed the growth kinetics of S. mutans in a dose-dependent manner, even at sub-MICs of PB. Then, we investigated the effect of PB on S. mutans virulence. The PB suppressed biofilm formation at high concentrations, although PB did not affect the expression of glucosyltransferase genes. Additionally, PB suppressed the decrease in pH from adding an excess of glucose. The expression of genes responsible for acid production was increased by the addition of excess glucose without PB, whereas their expression levels were not increased in the presence of 1× and 2× MIC of PB. Although PB showed a bacteriostatic effect on planktonic S. mutans cells, it was found that more than 2× MIC of PB showed a partial bactericidal effect on biofilm cells. In conclusion, PB not only showed antibacterial activity against S. mutans but also decreased the cariogenic activity in S. mutans.     

Control of biofilm formation by poly-ethylene-co-vinyl acetate films incorporating nisin

The aim of this study was to evaluate the effect of poly-ethylene-co-vinyl acetate (EVA) films incorporating different concentrations (0.1%, 0.5% and 1%) of nisin on the biofilm-forming ability of Listeria monocytogenes ATCC 7644, Staphylococcus aureus 815 and Staphylococcus epidermidis ATCC 35984. Nisin was incorporated into two grades of EVA (EVA14 and EVA28) in the melt during a common film-blowing operation. The efficacy of EVA/nisin films was evaluated by biofilm biomass measurements and Live/Dead staining in combination with fluorescence microscopy. In order to evaluate whether the nisin incorporation could modify the film surface properties, contact angle measurements and scanning electron microscopy were performed. The results revealed the efficacy of EVA14/nisin films in reducing biofilm formation on their surfaces with more evident effect for S. epidermidis than L. monocytogenes and S. aureus strains. In contrast, EVA28/nisin films showed unsatisfactory activity. Fluorescence microscopy confirmed poor biofilm formation on EVA14/nisin films, also characterised by the presence of dead cells. The data presented in this study offer new potential applications for developing strategies aimed to improve the effect of antimicrobial agents.     

醋酸氯己定与乙醇的协同杀菌效果

采用悬液定量杀菌试验,对醋酸氯己定与乙醇的协同杀菌效果进行了实验室研究。研究发现,0.1%醋酸氯己定与75%乙醇混合溶液对金黄色葡萄球菌、大肠埃希菌、白假丝酵母菌和黑曲霉均呈现出显著的协同杀菌作用。结果表明,醋酸氯己定与乙醇具有良好的协同杀菌效果。     

Antimicrobial efficacy of the alkaloid harmaline alone and in combination with chlorhexidine digluconate against clinical isolates of Staphylococcus aureus grown in planktonic and biofilm cultures

Aims: To investigate the antimicrobial efficacy of an alkaloid, harmaline alone and in combination with chlorhexidine digluconate (CHG) against clinical isolates of Staphylococcus aureus (S. aureus) grown in planktonic and biofilm cultures. Methods: Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) were determined for each micro‐organism grown in suspension and in biofilm using microbroth dilution method. Chequerboard assays were used to determine synergistic, indifferent or antagonistic interactions between harmaline and CHG, and the some of results were verified by confocal laser scanning microscopy. Results: Harmaline and CHG showed effective antimicrobial activity against suspensions and biofilm cultures of S. aureus, respectively. As determined by fractional inhibitory concentration index (FICI), synergistic antimicrobial effects between harmaline and CHG were observed in nine and 11 of the 13 S. aureus strains when in suspension and in biofilm, respectively. FICI values were from 0·375 to 1·25 when in suspension and from 0·25 to 1·25 when in biofilm. Conclusions: Synergistic activity of harmaline and CHG against clinical isolates of S. aureus (in suspension and in biofilm) was observed in vitro. Significance and Impact of the Study: This study might provide alternative methods to overcome the problem of drug‐resistance of S. aureus both in suspension and in biofilm.     

Bactericidal activity and oral pathogen inactivation by electromagnetic wave irradiation

Aims: The aim of this work was to clarify the effects of electromagnetic wave irradiation (EMWI) on oral bacterial pathogens. Methods and Results: A Gram‐negative (Porphyromonas gingivalis) or Gram‐positive (Streptococcus mutans, S. intermedius, Enterococcus faecalis) bacterial suspension was irradiated by EMW apparatus (500–1000 kHz, 5–15 times, 1 s time^?1). Quantification of survival bacteria by CFU counting revealed that EMWI exhibited marked bactericidal activity against all tested bacteria and bactericidal activity at 500 kHz increased in an irradiation number‐dependent manner. After EMWI at 500 kHz, scanning electron microscopic observations showed that the chain of S. mutans cells was shortened after 5 irradiations and the outlines of bacterial cells (S. mutans and P. gingivalis) were unclear after 5–10 irradiations. EMWI inhibited the inductive effect of S. mutans on pro‐inflammatory cytokine production in human monocytes and this inhibitory effect was comparable with that of heat‐killed bacteria. Furthermore, using an enzyme activity assay, EMWI partially inactivated the activities of gingipains from P. gingivalis. Conclusions: These findings demonstrated that EMWI has inactivation and bactericidal activities against single microbial species among four kinds of oral pathogens. Significance and Impact of the Study: Electromagnetic wave irradiation may be applicable for medical disinfection and sterilization, such as refractory periapical periodontitis.