Postbiotic mediators derived from Lactobacillus species enhance riboflavin-mediated antimicrobial photodynamic therapy for eradication of Streptococcus mutans planktonic and biofilm growth.

BACKGROUND: Streptococcus mutans has been implicated as a primary causative agent of dental caries and one of its important virulence properties is an ability to form biofilm on tooth surfaces. Thus, strategies to prevent and control S. mutans biofilms are requested. The present study aimed to examine the eradication of S. mutans planktonic and biofilm cells using riboflavin (Rib)-mediated antimicrobial photodynamic therapy (aPDT) enhanced by postbiotic mediators derived from Lactobacillus species. MATERIALS AND METHODS: Minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of Rib and postbiotic mediators were determined. The antimicrobial and anti-biofilm effects of Rib-mediated aPDT (Rib plus blue light), Rib-mediated aPDT in combination with postbiotic mediators derived from Lactobacillus casei (LC) (aPDT+ LC), and Rib-mediated aPDT in combination with postbiotic mediators derived from Lactobacillus plantarum (LP) (aPDT+ LP) were evaluated. The anti-virulence potential of Rib-mediated aPDT, aPDT+ LC, and aPDT+ LP were assessed by measuring the expression of the gtfB gene using quantitative real-time polymerase chain reaction (qRT-PCR) at the highest concentrations of Rib, LC, and LP, at which the S. mutans had proliferation as the same as in the control (non-treated) group. RESULTS: According to the results, the MIC doses of LC, LP, and Rib were 64 µg/mL, 128 µg/mL, and 128 µg/mL, respectively, while the MBC values of LC, LP, and Rib were 128 µg/mL, 256 µg/mL, and 256 µg/mL, respectively. Rib-mediated aPDT, aPDT+ LP, and aPDT+ LC showed a significant reduction in Log10 CFU/mL of S. mutans compared to the control group (4.2, 4.9, and 5.2 Log10 CFU/mL, respectively; all P < 0.05). The most destruction of S. mutans biofilms was observed after treatment with aPDT+ LC followed by aPDT+ LP and Rib-mediated aPDT (77.5%, 73.3%, and 67.6%, respectively; all P < 0.05). The concentrations of 31.2 µg/mL, 62.5 µg/mL, and 62.5 µg/mL were considered as the highest concentrations of LC, LP, and Rib, respectively, at which S. mutans replicates as same as the control group and were used for gtfB gene expression assay using qRT-PCR during Rib-mediated aPDT, aPDT+ LP, and aPDT+ LC treatments. Gene expression results revealed that aPDT+ LP and aPDT+ LC could decrease the gene expression level of gtfB by 6.3- and 5.7-fold, respectively (P < 0.05), while only 5.1-fold reduction was observed after Rib-mediated aPDT (P < 0.05). CONCLUSION: Our findings indicate that aPDT+ LP and aPDT+ LC hold promise for use as a treatment to combat S. mutans planktonic and biofilms growth as well as anti-virulence as a preventive strategy to inhibit biofilms development via reduction of gtfB gene expression.

Photoinactivation of Enterococcus faecalis Biofilm: In Vitro Antimicrobial Effect of Photoexcited Rutin-Gallium(III) Complex via Visible Blue Light.

INTRODUCTION: Endodontic infection is a common problem that can result in tooth loss if not effectively treated. This study focused on investigating the use of rutin-gallium (Ga)(III) complex-mediated antimicrobial photodynamic therapy (aPDT) for the photoinactivation of Enterococcus faecalis biofilm. METHODS: The minimum biofilm eradication concentration of the rutin-Ga(III) complex and the minimum biofilm eradication dose of light-emitting diode against E. faecalis were evaluated. The antimicrobial effect of rutin-Ga(III) complex-mediated aPDT against E. faecalis was assessed. Additionally, the expression of genes associated with E. faecalis virulence, such as ace, gelE, and esp, as well as the production of reactive oxygen species within the cells were evaluated. RESULTS: The minimum biofilm eradication concentration of the rutin-Ga(III) complex was determined to be 25 µmol/L, whereas the minimum biofilm eradication dose of light-emitting diode irradiation was defined as 5 minutes with an energy density of 300-420 J/cm2. Rutin-Ga(III) complex-mediated aPDT demonstrated a significant dose-dependent reduction in the growth of E. faecalis biofilms. Moreover, aPDT led to increased intracellular reactive oxygen species generation in treated E. faecalis cells. Furthermore, the messenger RNA levels of ace, gelE, and esp genes were significantly down-regulated in E. faecalis treated with rutin-Ga(III) complex-mediated aPDT (P < .05). CONCLUSIONS: Rutin-Ga(III) complex-mediated aPDT effectively reduces E. faecalis biofilm growth by disrupting biofilm structure and down-regulating virulence genes. These findings highlight the potential of aPDT with the rutin-Ga(III) complex as an adjuvant therapeutic approach against E. faecalis biofilms.

Evaluation of anti-biofilm effect of antimicrobial sonodynamic therapy-based periodontal ligament stem cell-derived exosome-loaded kojic acid on Enterococcus faecalis biofilm.

Introduction. Antimicrobial sonodynamic therapy (aSDT) is an approach that uses ultrasound waves (UWs) and a sonosensitizer to generate reactive oxygen species (ROS) to damage microbial cells in biofilms. Using nano-carriers, such as exosomes (Exos), to deliver the sonosensitizer can potentially enhance the effectiveness of aSDT.Hypothesis/Gap Statement. aSDT can downregulate the expression of gelE and sprE genes, increasing the production of endogenous ROS and degradation of pre-formed Enterococcus faecalis biofilms.Aim. This study investigated the anti-biofilm effect of aSDT-based periodontal ligament stem cell-derived exosome-loaded kojic acid (KA@PDL-Exo) on pre-formed E. faecalis biofilms in root canals.Methodology. Following the isolation and characterization of PDL-Exo, KA@PDL-Exo was prepared and confirmed. The minimal biofilm inhibitory concentration (MBIC) of KA, PDL-Exo, KA@PDL-Exo and sodium hypochlorite (NaOCl) was determined, and their anti-biofilm effects were assessed with and without UWs. The binding affinity of KA with GelE and SprE proteins was evaluated using in silico molecular docking. Additionally, the study measured the generation of endogenous ROS and evaluated changes in the gene expression levels of gelE and sprE.Results. The results revealed a dose-dependent decrease in the viability of E. faecalis cells within biofilms. KA@PDL-Exo was the most effective, with an MBIC of 62.5 µg ml-1, while NaOCl, KA and PDL-Exo had MBIC values of 125, 250 and 500 µg ml-1, respectively. The use of KA@PDL-Exo-mediated aSDT resulted in a significant reduction of the E. faecalis biofilm (3.22±0.36 log10 c.f.u. ml-1; P<0.05). The molecular docking analysis revealed docking scores of -5.3 and -5.2 kcal mol-1 for GelE-KA an SprE-KA, respectively. The findings observed the most significant reduction in gene expression of gelE and sprE in the KA@PDL-Exo group, with a decrease of 7.9- and 9.3-fold, respectively, compared to the control group (P<0.05).Conclusion. The KA@PDL-Exo-mediated aSDT was able to significantly reduce the E. faecalis load in pre-formed biofilms, decrease the expression of gelE and srpE mRNA, and increase the generation of endogenous ROS. These findings imply that KA@PDL-Exo-mediated aSDT could be a promising anti-biofilm strategy that requires additional in vitro and in vivo investigations.

Ex-vivo effects of propolis quantum dots-nisin-nanoquercetin-mediated photodynamic therapy on Streptococcus mutans biofilms and white spot lesions.

BACKGROUND: White spot lesions (WSLs) remain one of the most critical adverse sequelae of fixed orthodontic treatment, despite materials and techniques advances in orthodontics. WSLs seem to be a multi-factorial interaction including increased microbial plaque due to intrabuccal appliances that limit the oral-cleansing mechanism and change in the oral microbiome during fixed appliance wear. The aim of this study was to investigate the synergistic effect of propolis quantum dots (PQD), nisin (Nis), and quercetin nanoparticles (nQCT)-mediated photodynamic therapy (PQD-Nis-nQCT-mediated aPDT) in the eradication of Streptococcus mutans biofilms and the remineralization of WSLs ex-vivo. MATERIALS AND METHODS: The cytotoxicity of PQD-Nis-nQCT composite on human gingival fibroblasts was evaluated using neutral red. Intracellular reactive oxygen species (ROS) generation following PQD-Nis-nQCT-mediated aPDT was measured. Enamel slabs were prepared and demineralized using a demineralization solution containing S. mutans. Demineralized enamel slabs were divided into 9 groups (n = 10) and treated in the following groups: 1) Artificial saliva (negative control), 2) 2% neutral sodium fluoride gel (NSF; positive control or treatment control, 3) PQD, 4) Nis, 5) nQCT, 6) Nis-nQCT, 7) PQD-Nis-nQCT 8) Blue laser irradiation (light), 9) PQD-Nis-nQCT with irradiation (PQD-Nis-nQCT-mediated aPDT). Then, the surface changes, microhardness, and surface topography of the demineralized slabs were examined following each treatment using DIAGNOdent Pen reading, digital hardness tester, and SEM, respectively. After the determination of minimum biofilm eradication concentration (MBEC) of PQD, Nis, and nQCT by microtiter plate assay, the synergistic antimicrobial effects of PQD and Nis-nQCT were determined via evaluation of fractional biofilm eradication concentration (FBEC) index. The anti-biofilm effects of each treatment on S. mutans were assessed using a colorimetric assay. The virulence­associated gtfB gene expression was assessed following PQD-Nis-nQCT-mediated aPDT by quantitative real­time PCR. RESULTS: PQD-Nis-nQCT at 2048 µg/mL had no significant cell cytotoxicity on human gingival fibroblasts compared to the control group (P > 0.05). A significantly increased (7.6 fold) in intracellular ROS was observed following PQD-Nis-nQCT-mediated aPDT (13.9 ± 1.41) when compared to the control (1.83 ± 0.13). Following each treatment, the microhardness of the demineralized enamel surface significantly increased except for the artificial saliva (negative) and blue laser irradiation groups. The highest change in microhardness improvement was detected in the PQD-Nis-nQCT-mediated aPDT group (P < 0.05). Also, DIAGNODent Pen reading revealed the highest significant improved change in the level of mineralization degree in the PQD-Nis-nQCT-mediated aPDT group. Nis and blue light irradiation groups, like the artificial saliva-treated demineralized enamel slabs (control group), did not lead to remineralization (P > 0.05). Also, the PQD-Nis-nQCT-mediated aPDT treatment results obtained from SEM revealed that remineralization of demineralized enamel slabs in that group has significantly improved compared to the others. Light-activated nQCT, PQD, Nis-nQCT, and PQD-Nis-nQCT composite significantly reduced pre-formed biofilms of S. mutans compared with unactivated forms of test materials. The relative expression level of the virulence gtfB gene was significantly decreased (7.53-fold) in the presence of PQD-Nis-nQCT-mediated aPDT (P < 0.05). CONCLUSION: PQD-Nis-nQCT-mediated aPDT can be used for the eradication of S. mutans biofilms and remineralization of WSLs. The found in vitro efficacy should be tested further through clinical studies.

Effect of nanomicelle curcumin-based photodynamic therapy on the dynamics of white spot lesions and virulence of Streptococcus mutans in patients undergoing fixed orthodontic treatment: A randomized double-blind clinical trial.

BACKGROUND/PURPOSE: The formation of white spot lesions (WSLs) around fixed orthodontic appliances is a major complication during treatment. The current double-blind, randomized clinical trial (RCT) study aims to investigate the varying effects of nanomicelle curcumin-based photodynamic therapy (NMCur-aPDT) on microbial count and virulence of Streptococcus mutans as well as the number and dynamics of WSLs. MATERIALS AND METHODS: Double-blind prospective RCT, comprised of 48 patients with fixed orthodontic appliances, were recruited for the current study. The patients were divided into four groups according to the type of the treatment (NMCur, LED, NMCur-aPDT or VITIS® anti-caries mouthwash), using block randomization. Antimicrobial and anti-virulence activities of the treatments against isolated S. mutans were assessed via colony counting and quantitative real-time polymerase chain reaction (qRT-PCR), respectively. The visual inspection using the International Caries Detection and Assessment System (ICDAS II) score and laser fluorescence (LF) detection using a DIAGNOdent device were used for the detection and assessment of the dynamics of WSLs, respectively, on the labial surface in four areas (i.e., gingival, incisal, mesial, and distal) of the upper and lower anterior teeth at 30-, 60-, 90-, and 120-days follow-up after bonding of the lower and upper arches. RESULTS: The antimicrobial properties of NMCur, VITIS®, and NMCur-aPDT were time-dependent so the highest reduction in S. mutans population was observed following NMCur-aPDT (99.98%) on day 120 of the study. The gtfB gene expression levels in S. mutans isolates from the NMCur-aPDT group on days 60, 90, and 120 decreased by 2.07-, 2.32-, and 3.01-fold more than in S. mutans isolates from the VITIS® group, respectively (all P < 0.05), while NMCur and LED treatments could not significantly reduce gtfB gene expression up to 120 days of follow-up (P > 0.05). In patients who were treated with LED, an increase in the mean number of WSLs per patient (mean increase, 1.8; P < 0.05) was found, while in NMCur-aPDT and VITIS® groups, not only no increases were observed, but the mean number of WSLs per patient decreased (mean reductions, 0.5 and 0.9, respectively; not significant). LED treatment caused significant increases (P < 0.05) in the mean LF values at 90-and 120-days of follow-up in comparison with the baseline (mean increases, 5.1 and 6.5, respectively) while, in NMCur-aPDT, VITIS®, and NMCur groups 11.8-, 7.1-, and 4.4-reductions in the mean LF values were observed, respectively (all, P < 0.05). CONCLUSIONS: The antimicrobial and anti-virulence activities of NMCur-aPDT against S. mutans were higher than the other treatment groups. In patients who were treated with NMCur-aPDT, the mean number and LF values of WSLs per patient were significantly lower than the other groups in 90-and 120-days of follow-up.

Shear bond strength, adhesive remnant index, and anti-biofilm effects of a photoexcited modified orthodontic adhesive containing curcumin doped poly lactic-co-glycolic acid nanoparticles: An ex-vivo biofilm model of S. mutans on the enamel slab bonded brackets.

BACKGROUND: Potential complications during fixed orthodontic procedures are white spot lesions (WSLs) and tooth decay. This study evaluated the anti-biofilm activity of an orthodontic adhesive (OA) incorporating curcumin (Cur) doped Poly lactic-co-glycolic acid nanoparticles (Cur-PLGA-NPs), which can have the highest concentration of Cur-PLGA-NPs and shear bond strength (SBS) value simultaneously, against cariogenic bacteria i.e., Streptococcus mutans. MATERIALS AND METHODS: Following synthesis and confirmation of Cur-PLGA-NPs, SBS and adhesive remnant index (ARI) of the modified orthodontic adhesives (MOA) containing Cur-PLGA-NPs (3, 5, 7, and 10 % wt.) were measured using universal testing machine and stereomicroscope, respectively. After artificial aging (continuously rinsed up to 180 days), the residual anti-biofilm ability of MOA which can have the highest concentration of Cur-PLGA-NPs and SBS value simultaneously were determined by anti-biofilm assay following photoexcited enamel slab bonded brackets by MOA containing Cur-PLGA-NPs against S. mutans biofilms using crystal violet assay. RESULTS: Adhesive with 7 % wt. Cur-PLGA-NPs revealed the highest concentration of Cur-PLGA-NPs and SBS value (16.19 ± 2.69 MPa, P < 0.05) simultaneously. No statistically significant difference in ARI scores was observed between the MOA and control (Transbond XT without the Cur-PLGA-NPs). On days 15, 30, 60, 90 and 120 there was a considerable decrease in optical density (OD) of preformed S. mutans biofilms on photoexcited enamel slab bonded brackets using MOA containing 7 % wt. Cur-PLGA-NPs, to 94.1 %, 79.6 %, 69.6 %, 69.4 %, and, 55.1 % respectively in comparison to the control group (all, P < 0.05). From days 150 onwards, microbial biofilm formation was progressively increased on enamel slab bonded brackets using MOA containing 7 % wt. Cur-PLGA-NPs compared to the control group (OA). Although chlorhexidine (2 %; as positive control) showed significant activity against pre-formed S. mutans biofilms on enamel slab bonded brackets using OA (99.1 % biofilm reduction; P = 0.001), its activity was slightly higher but not significant than photoexcited enamel slab bonded brackets using MOA containing 7 % wt. Cur-PLGA-NPs on the days 15 and 30 (both, P > 0.05). CONCLUSIONS: The 7 % wt. Cur-PLGA-NPs can serve as an orthodontic adhesive antimicrobial additive as exposure to blue laser provides an acceptable antimicrobial effect against cariogenic bacteria for a considerable time.