Effects of caffeic acid phenethyl ester against multi-species cariogenic biofilms.

Dental caries is a biofilm-related disease, widely perceived to be caused by oral ecological imbalance when cariogenic/aciduric bacteria obtain an ecological advantage. Compared with planktonic bacteria, dental plaques are difficult to remove under extracellular polymeric substance protection. In this study, the effect of caffeic acid phenethyl ester (CAPE) on a preformed cariogenic multi-species biofilm was evaluated, which was comprised of cariogenic bacteria (Streptococcus mutans), commensal bacteria (Streptococcus gordonii), and a pioneer colonizer (Actinomyces naeslundii). Our result revealed that treatment with 0.08 mg/mL CAPE reduced live S. mutans in the preformed multi-species biofilm while not significantly changing the quantification of live S. gordonii. CAPE significantly reduced the production of lactic acid, extracellular polysaccharide, and extracellular DNA and made the biofilm looser. Moreover, CAPE could promote the H2O2 production of S. gordonii and inhibit the expression of SMU.150 encoding mutacin to modulate the interaction among species in biofilms. Overall, our results suggested that CAPE could inhibit the cariogenic properties and change the microbial composition of the multi-species biofilms, indicating its application potential in dental caries prevention and management.

Novel Lactotransferrin-Derived Antimicrobial Peptide LF-1 Inhibits the Cariogenic Virulence Factors of Streptococcus mutans.

We previously developed a novel lactotransferrin-derived antimicrobial peptide, LF-1, with selective antibacterial activity against the characteristic cariogenic bacterium Streptococcus mutans. This study further investigated the effects of LF-1 on the cariogenic virulence factors of S. mutans and evaluated the changes in virulence-associated enzymes and genes; the viability, acidogenicity, and aciduricity of planktonic S. mutans; and initial colonisation and biofilm formation after treatment with LF-1. The method of qRT-PCR was used to evaluate S. mutans virulence-associated gene expression. LF-1 interfered with the cell viability of S. mutans within 6 h. LF-1 inhibited the acidogenicity and aciduricity of S. mutans, with reduced lactic acid production and survival in a lethal acidic environment, and inactivated lactate dehydrogenase and F1F0-ATPase activity. LF-1 decreased surface-adherent S. mutans within 60 min and inhibited S. mutans biofilm formation, where scanning electron microscopy and confocal laser scanning microscopy showed reduced extracellular matrix and bacteria. LF-1 downregulates S. mutans virulence-associated gene expression. LF-1 inhibited the growth and cariogenic virulence factors of S. mutans in vitro with a reduction in key enzymatic activity and downregulation of virulence-associated gene expression. LF-1 has promising application prospects in the fight against S. mutans and dental caries.

Combined Treatment with Fluoride and Antimicrobial Peptide GH12 Efficiently Controls Caries in vitro and in vivo.

Combining fluoride and antimicrobial agents enhances regulation of acid and exopolysaccharide production by biofilms. The combination also weakens the acidogenic and aciduric bacteria that contribute to caries, achieving stronger caries-controlling effects with lower concentrations of fluoride. In previous studies, antimicrobial peptide GH12 has been shown to inhibit lactic acid and exopolysaccharide synthesis in various cariogenic biofilm models, and reduce the proportion of acidogenic bacteria and Keyes caries scores in a rat caries model. The current study aimed to elucidate the effect of a combination of low concentrations of sodium fluoride (NaF) and GH12 and to determine the mechanism by which GH12/NaF combination controls caries. The GH12/NaF combination contained 8 mg/L GH12 and 250 ppm NaF. A rat caries model was built, and rat dental plaque was sampled and cultivated on bovine enamel slabs in vitro and subjected to short-term treatment (5 min, 3 times/day). The caries-controlling effects were evaluated using Keyes scoring and transverse microradiography. The results showed that the GH12/NaF combination significantly decreased the onset and development of dental caries, as well as mineral content loss and lesion depth in vitro (p < 0.05). For the caries-controlling mechanisms, 16S rRNA sequencing of in vivo dental plaque revealed that populations of commensal bacteria Rothia spp. and Streptococcus parasanguinis increased in the GH12/NaF group. In contrast, Veillonella, Lactobacillus, and Streptococcus mutans decreased. Furthermore, the GH12/NaF combination significantly reduced biomass, lactic acid, and exopolysaccharides production of in vitro biofilm (p < 0.05). Overall, fluoride and GH12 efficiently arrested caries development and demineralization by regulating the microbiota and suppressing acid and exopolysaccharide production in biofilms.

Development and evaluation of a chewing gum containing antimicrobial peptide GH12 for caries prevention.

The purpose of this study was to develop a chewing gum containing a novel antimicrobial peptide GH12 and evaluate its biocompatibility, antimicrobial activity, and caries-preventive effects in vivo and in vitro. GH12 chewing gum was developed using a conventional method and its extracts were prepared in artificial saliva. GH12 concentration in the extracts was determined by high-performance liquid chromatography; extracts were used for growth curve assay, time-kill assay, crystal violet staining assay, scanning electron microscopy, and Cell Counting Kit-8 assay. A rat caries model was established, and molars were treated topically with extracts for 5 weeks. Weight gain monitoring, hematoxylin-eosin staining, micro-computed tomography, and Keyes scoring were conducted. Significant inhibition of Streptococcus mutans growth and biofilm formation was observed. Extracts displayed low cytotoxicity against human gingival epithelial cells. No significant differences in weight gain or signs of harm to the mucosal tissues in any of the rats were observed. Keyes scores of caries lesions in the GH12 chewing gum group were lower than those of the negative control group. It was concluded that GH12 chewing gum showed good biocompatibility, antimicrobial activity, and caries-preventive effects, exhibiting great potential to prevent dental caries as an adjuvant to regular oral hygiene.

Selective antibacterial activity of a novel lactotransferrin-derived antimicrobial peptide LF-1 against Streptococcus mutans.

OBJECTIVE: Streptococcus mutans is a key pathogen involved in the development of caries lesions. Previously, we developed a novel lactotransferrin-derived antimicrobial peptide LF-1 with potential selective activity against S. mutans. This study aimed to further confirm the selectivity of LF-1 by investigating its effect on S. mutans membrane. DESIGN: The effects of LF-1 on the viability of human gingival fibroblasts (HGFs) and three common oral Streptococcus (S. mutans, S. sanguinis, and S. gordonii) were evaluated and its structural characteristics were analysed using eukaryotic and prokaryotic membrane-simulated liposomes. Membrane affinity of LF-1 to the three streptococci strains was evaluated using the 3',3'-dipropylthiadicarbocyanine iodide experiment, hydrophobicity assay, and flow cytometry analysis. Transmission electron microscopy (TEM) was used to observe morphological changes in the three streptococcal membranes after LF-1 treatment. RESULTS: LF-1 displayed lower cytotoxicity to HGFs and selective antibacterial activity against S. mutans. LF-1 exhibited a typical α-helix structure and showed a tryptophan fluorescence blue shift in the prokaryotic membrane-simulated model. The most notable LF-1 induced changes occurred in the membrane potential and hydrophobicity of S. mutans among the three streptococci strains. Furthermore, the fluorescence of fluorescein isothiocyanate-labelled LF-1 was higher in S. mutans than in the other species. TEM showed that 16 µmol/L LF-1 could induce mesosome-like structures in S. mutans, whereas no significant morphological changes occurred in the other species. CONCLUSION: LF-1 has selective affinity for and antibacterial activity against S. mutans with strong membrane disrupting ability, highlighting the potential of LF-1 as a crucial antibacterial agent in caries prevention.

Novel lactotransferrin-derived synthetic peptides suppress cariogenic bacteria in vitro and arrest dental caries in vivo: [Novel lactotransferrin-derived anticaries peptides].

Objectives: The aim of the study was to design and synthesise novel lactotransferrin-derived antimicrobial peptides (AMPs) with enhanced antibacterial activity against cariogenic bacteria. Methods: We obtained the LF-1 (WKLLRKAWKLLRKA) and LF-2 (GKLIWKLLRKAWKLLRKA) AMPs, based on the N-terminal functional sequence of lactotransferrin, and characterised their physicochemical properties and secondary structure. Their antibacterial activity against caries-associated bacteria was evaluated using bacterial susceptibility and time-killing assays, as well as transmission electron microscopy (TEM). The antibiofilm activity against Streptococcus mutans biofilms was determined using biofilm susceptibility assays and confocal laser scanning microscopy (CLSM). A rodent model of dental caries was adopted to evaluate their anticaries effectiveness in vivo. Results: Both peptides possessed an α-helical structure with excellent amphipathicity. LF-1 was effective against S. mutans and Actinomyces species, whereas LF-2 showed more potent antibacterial activity than LF-1 against a broader spectrum of tested strains. Both peptides inhibited the formation of S. mutans biofilm starting at 8 µmol/L and exerted effective eradication of S. mutans in preformed biofilms. Both peptides exhibited satisfactory biocompatibility and exerted significant anticaries effects in a rodent model. Conclusion s: Both lactotransferrin-derived peptides displayed strong antimicrobial activity against cariogenic bacteria and S. mutans biofilm in vitro and effectively inhibited dental caries in vivo.

The pH-Responsive Property of Antimicrobial Peptide GH12 Enhances Its Anticaries Effects at Acidic pH.

Dental caries is closely related to the acidification of the biofilms on the tooth surface, in which cariogenic bacteria bring about a dramatic pH decrease and disrupt remineralisation equilibrium upon the fermentation of dietary sugars. Thus, approaches targeting the acidified niches with enhanced anticaries activities at acidic pH are highly desirable. In our previous study, a cationic amphipathic α-helical antimicrobial peptide GH12 (Gly-Leu-Leu-Trp-His-Leu-Leu-His-His-Leu-Leu-His-NH2) was designed with good stability, low cytotoxicity, and excellent antibacterial effects. Considering its potent antibacterial activity against the acidogenic bacteria and its histidine-rich sequence, it was speculated that GH12 might show enhanced antimicrobial effects at an acidic pH. In this study, the pH-responsive property of GH12 was determined to evaluate its potential as a smart acid-activated anticaries agent. GH12 possessed much lower minimal inhibitory concentrations and minimal bactericidal concentrations against various kinds of bacteria at pH 5.5 than at pH 7.2. Employing Streptococcus mutans, the principal caries pathogen, as the model system, it was found that GH12 showed much stronger bactericidal effects on both planktonic S. mutans and S. mutans embedded in the biofilm at pH 5.5. In addition, short-term treatment with GH12 showed much more effective inhibitory effects on water-insoluble exopolysaccharides synthesis and lactic acid production of the preformed S. mutans biofilm at pH 5.5. As for the mechanism exploration, it was found that the net positive charge of GH12 increased and the tryptophan fluorescence intensity heightened with the peak shifting towards the short wavelength at pH 5.5, which demonstrated that GH12 could be more easily attracted to the anionic microbial cell membranes and that GH12 showed stronger interactions with the lipid membranes. In conclusion, acidic pH enhanced the antibacterial and antibiofilm activities of GH12, and GH12 is a potential smart anticaries agent targeting the cariogenic acidic microenvironment.

Antimicrobial Peptide GH12 Prevents Dental Caries by Regulating Dental Plaque Microbiota.

Due to the complex microecology and microenvironment of dental plaque, novel caries prevention strategies require modulating the microbial communities ecologically and reducing the cariogenic properties effectively. Antimicrobial peptide GH12 reduced the lactic acid production and exopolysaccharide (EPS) synthesis of a Streptococcus mutans biofilm and a three-species biofilm in vitro in previous studies. However, the anticaries effects and microecological effects of GH12 remained to be investigated in a complex biofilm model in vitro and an animal caries model in vivo In the present study, GH12 at 64 mg/liter showed the most effective inhibition of lactic acid production, EPS synthesis, pH decline, and biofilm integrity of human dental plaque-derived multispecies biofilms in vitro, and GH12 at 64 mg/liter was therefore chosen for use in subsequent in vitro and in vivo assays. When treated with 64-mg/liter GH12, the dental plaque-derived multispecies biofilms sampled from healthy volunteers maintained its microbial diversity and showed a microbial community structure similar to that of the control group. In the rat caries model with a caries-promoting diet, 64-mg/liter GH12 regulated the microbiota of dental plaque, in which the abundance of caries-associated bacteria was decreased and the abundance of commensal bacteria was increased. In addition, 64-mg/liter GH12 significantly reduced the caries scores of sulcal and smooth surface caries in all locations. In conclusion, GH12 inhibited the cariogenic properties of dental plaque without perturbing the dental plaque microbiota of healthy individuals and GH12 regulated the dysbiotic microbial ecology and arrested caries development under cariogenic conditions.IMPORTANCE The anticaries effects and microecological regulation effects of the antimicrobial peptide GH12 were evaluated systematically in vitro and in vivo GH12 inhibited the cariogenic virulence of dental plaque without overintervening in the microbial ecology of healthy individuals in vitro GH12 regulated the microbial ecology of dental plaque to a certain extent in vivo under cariogenic conditions, increased the proportion of commensal bacteria, and decreased the abundance of caries-associated bacteria. GH12 significantly suppressed the incidence and severity of dental caries in vivo This study thus describes an alternative antimicrobial therapy for dental caries.