Anti-multispecies microbial biofilms and anti-inflammatory effects of antimicrobial photo-sonodynamic therapy based on acrylic resin containing nano-resveratrol.

BACKGROUND: Polymethylmethacrylate (PMMA)-based removable orthodontic appliances are susceptible to microbial colonization due to the surface porosity, and accumulating the biofilms causes denture stomatitis. the present study evaluated the anti-biofilm and antiinflammatory effects of antimicrobial photo-sonodynamic therapy (aPSDT) against multispecies microbial biofilms (Candida albicans, Staphylococcus aureus, Streptococcus sobrinus, and Actinomyces naeslundii) formed on acrylic resin modified with nanoresveratrol (NR). MATERIALS AND METHODS: Following the determination of the minimum biofilm inhibitory concentration (MBIC) of NR, in vitro anti-biofilm activity of NR was evaluated. The antibiofilm efficacy against multispecies microbial biofilm including C. albicans, S. aureus, S. sobrinus, and A. naeslundii, were assessed by biofilm inhibition test and the results were measured. To reveal the anti-inflammatory effects of aPSDT on human gingival fibroblast (HGF) cells, the gene expression levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were evaluated via quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: According to the results, the MBIC dose of NR against multispecies microbial biofilm was considered 512 µg/mL. The highest biofilm reduction activity was observed in MBIC treated with aPSDT and 2 × MBIC exposed to light emitting diode (LED) and ultrasound waves (UW). The expression level of TNF-α and IL-6 genes were significantly increased when HGF cells were exposed to multispecies microbial biofilms (P<0.05), while after treatment with aPSDT, the expression levels of genes were significantly downregulated in all groups (P<0.05). CONCLUSION: Overall, NR-mediated aPSDT reduced the growth of the multispecies microbial biofilm and downregulated the expression of TNF-α and IL-6 genes. Therefore, modified PMMA with NR can be serving as a promising new orthodontic acrylic resin against multispecies microbial biofilms and the effect of this new material is amplified when exposed to LED and UW.

In silico and in vitro insights into the prediction and analysis of natural photosensitive compounds targeting Acinetobacter baumannii biofilm-associated protein.

BACKGROUND: The spread of Acinetobacter baumannii strains has become a global concern due to its extensive antibiotic resistance and biofilm formation. To overcome it, new antimicrobial strategies have been needed. Among them, antimicrobial photodynamic therapy (aPDT) is an efficient approach against various microorganisms. This study was focused on the use of curcumin (Cur) and quercetin (Qct) as natural photosensitive compounds to improve the activity of aPDT against A. baumannii biofilm-associated protein (Bap). MATERIALS AND METHODS: In this in silico and in vitro study, after determining drug-likeness property, ADME/Toxicity profile, and pharmacological activity of Cur and Qct, virtual screening and molecular docking were assessed to determine the potential binding modes of Cur and Qct to Bap. Then, the anti-biofilm potential of natural photosensitizers-mediated aPDT against A. baumannii was evaluated after the determination of minimum inhibitory concentration (MIC). Subsequently, reverse transcription-quantitative real-time PCR (RT-qPCR) was used to exhibit the anti-virulent effect of aPDT against the gene involved in the biofilm formation of A. baumannii RESULTS: Cur and Qct showed almost similar pharmacokinetic and pharmacodynamics properties. These natural photosensitizers obeyed all the criteria of Lipinski's rule of five principles. According to the molecular docking analysis of protein-ligand complexes, Qct and Cur with a high affinity for Bap showed binding affinity of -6.34 and -6.98 kcal/mol, respectively. According to the findings, aPDT using 4 ×, and 8 × MIC of Cur and Qct could significantly reduce A. baumannii growth in biofilm structures in comparison with the control group (P < 0.05). Also, a significant downregulation by 3.7-, and 5.2-fold in gene expression of bap was observed after treatment with sub-MIC doses of Cur- and Qct-mediated aPDT, respectively (P < 0.05). CONCLUSIONS: In summary, the in silico analysis showed that Cur and Qct had strong binding affinity with Bap as a stable protein of A. baumannii. Furthermore, in vitro results displayed that targeted aPDT based on these natural photosensitizers can be considered a treatment against A. baumannii infections by reducing the growth of microbial biofilm and reducing the expression of bap as a gene involved in A. baumannii biofilm formation.

An orthodontic acrylic resin containing seaweed Ulva lactuca as a photoactive phytocompound in antimicrobial photodynamic therapy: Assessment of anti-biofilm activities and mechanical properties.

BACKGROUND: Uncontrolled accumulation of microbial plaque and formation of biofilm on the surface orthodontic acrylic removable appliances increases the risk of enamel decalcification and periodontal diseases. The purpose of the present study was to evaluate antimicrobial activities, anti-virulence potencies, and mechanical properties of orthodontic acrylic resin containing different concentrations of Ulva lactuca (a green marine macroalga) following photo-activation against Streptococcus mutans. MATERIALS AND METHODS: Minimum inhibitory concentration (MIC) of U. lactuca was determined against S. mutans. Acrylic resin specimens with different concentrations of U. lactuca (0.2 %, 0.5 %, 1%, 2.5 %, 5%, and 10 % weight/weight) were fabricated. Flexural strength values, antimicrobial effects, and anti-biofilm activities of samples were assessed in comparison with original acrylic resin as the control group. Also, the expression of the virulence-associated genes was assessed by quantitative real-time polymerase chain reaction. RESULTS: U. lactuca at concentrations of 1-10% significantly reduced the S. mutans growth rate by 20.3%-63.3% in comparison to the control group (P < 0.05). Therefore, the concentration of 1% of U. lactuca was considered as a MIC. The highest and lowest flexural strength values were observed in the control group (43.5 ± 2.4 MPa) and the group with a 10 % concentration of U. lactuca (19.2 ± 1.8 MPa), respectively. Flexural strength values decreased in samples containing 2.5 %, 5%, and 10 % concentrations of U. lactuca in comparison to the control group significantly (P > 0.05). In the disc agar diffusion test, the growth inhabitation zones around samples containing different concentrations of photo-activated U. lactuca ranged from 13 mm to 25 mm in diameter. Interestingly, the anti-biofilm activity of U. lactuca-mediated aPDT against S. mutans was dose-dependent. Additionally, the sub-MIC dose of U. lactuca (0.5 %) following photo-activation could significantly decrease the expression levels of gtfB, gtfC, and gtfD to 4.1-, 5.3-, and 7.4-fold, respectively. CONCLUSIONS: Adding photo-activated U. lactuca to the orthodontic acrylic resin at a concentration of 1% increases its antibacterial and anti-biofilm activities besides not detrimentally affects its flexural strength.

Anti-biofilm activity of Chlorella-mediated light activated disinfection: Ex vivo inhibition of intracanal mature Enterococcus faecalis biofilms via application of natural product.

BACKGROUND: This study aimed to evaluate the effects of light-activated disinfection (LAD) as a strategy for optimizing root canal disinfection using Chlorella (Chlo) as a natural photosensitizer against Enterococcus faecalis biofilms ex vivo. MATERIALS AND METHODS: The physical and chemical stability and absorption spectra of Chlo were determined. The mature microbial biofilm of E. faecalis was formed in root canals of 70 freshly extracted single-rooted premolars. After determining the minimum inhibitory concentration (MIC) of Chlo using the agar dilution testing, E. faecalis biofilms were exposed in the following groups (n = 10): 1- Chlo at a concentration of 2× MIC, 1- Chlo at a concentration of 4× MIC, 3- Diode laser, 4-2× MIC dose of Chlo-mediated LAD, 5-4× MIC dose of Chlo-mediated LAD, 6-0.2 % chlorohexidine (CHX), and 7- control group (E. faecalis biofilms without exposure to any photosensitizer and light source). The quantitative and qualitative evaluations of E. faecalis biofilms were done using counts of colony forming units (CFUs) and scanning electron microscope (SEM) and fluorescence microscope analysis, respectively. RESULTS: According to the results, the MIC of Chlo was 125 µg/mL, which inhibited the growth of E. faecalis. To evaluate the anti-biofilm effects of Chlo, the 2× and 4× MICs of Chlo (250 and 500 µg/mL, respectively) were used in the current study. The 4× MIC dose (500 µg/mL) of Chlo-mediated LAD was significantly more effective compared to other groups (P < 0.05), while the lowest percentage of dead cells was detected in the diode laser irradiation group. In addition, there was no significant difference in the log10 CFU of E. faecalis between the biofilm treated with 500 µg/mL Chlo-mediated LAD (1.27 ±â€¯0.05) versus 0.2 % CHX (1.10 ±â€¯0.06) (P < 0.05). As Moreover, SEM and fluorescence microscope images of the microbial biofilms showed that the highest percentage of dead bacteria was found in the 500 µg/mL Chlo-mediated LAD group. CONCLUSIONS: The results of this study suggest that Chlo-mediated LAD can be used as an adjuvant therapy to eliminate the E. faecalis biofilms in the root canal system.

Ex vivo assessment of synergic effect of chlorhexidine for enhancing antimicrobial photodynamic therapy efficiency on expression patterns of biofilm-associated genes of Enterococcus faecalis.

BACKGROUND: It has clearly been demonstrated that Enterococcus faecalis, as a persistent microorganism, is the major agent in the etiopatogeny of endodontic infections. Recently, the limitations of conventional endodontic therapy have given rise to many attempts to introduce antimicrobial photodynamic therapy (aPDT) as an alternative treatment. The aim of this study was to analyze the ex vivo effect of aPDT in combination with 2.0% chlorhexidine (CHX) as a conventional therapy on colony count and expression patterns of genes associated with biofilm formation of E. faecalis. MATERIALS AND METHODS: A total of 125 extracted human single-rooted teeth were divide into six groups (A-F; n = 20) and were incubated with E. faecalis. Group A- photosensitizer (indocyanine green [ICG]); B- diode laser; C- aPDT; D- 2.0% CHX; E- aPDT with photosensitizer modified by 2.0% CHX; and F- control group (no procedure was performed). Five remaining teeth were used to confirm the presence of E. faecalis biofilm via scanning electron microscope. Counts of colony forming units (CFUs) in each group were evaluated separately and quantitative real-time PCR (qRT-PCR) was then applied to monitor genes expression of fsrC, efa, and gelE involved in E. faecalis biofilm. RESULTS: The results showed that none of the tested groups achieved eradication or inhibition of biofilm. On the other hand, aPDT + 2.0% CHX, 2.0% CHX, and ICG- mediated aPDT groups showed significantly less CFU/mL than ICG and diode laser groups. The group with the lowest CFU/mL count was the aPDT + 2.0% CHX, being statistically different from all other groups that could decrease the expression levels of efa, gelE, and fsrC genes 6.8-, 8.3-, and 12.1-fold, respectively. CONCLUSION: Based on the results, the synergism effect of ICG-aPDT with 2.0% CHX leads to modulation of the virulence of E. faecalis strains biofilm model by suppressing the expression of the genes associated with biofilm formation.

Evaluation of photo-activated disinfection effectiveness with methylene blue against Porphyromonas gingivalis involved in endodontic infection: An in vitro study.

BACKGROUND: Eradication or suppression of microbial pathogens is a major goal in endodontic infection therapy. Sub-lethal doses of photo-activated disinfection (sPAD) as a new treatment method might be able to control the microorganisms involved in endodontic infections normally treated with PAD. This study evaluated the effect of sPAD using methylene blue (MB) in combination with diode laser irradiation on the growth and biofilm formation ability of Porphyromonas gingivalis as an endodontic pathogen. MATERIALS AND METHODS: The anti-microbial and anti-biofilm potential of sPAD against P. gingivalis were assessed at sub-lethal doses of MB and irradiation by diode laser on colony forming unit and crystal violet assays, respectively. RESULTS: MB-sPAD using 25µg/mL at a fluency of 117.18J/cm2 and 50-100µg/mL at a fluency of 93.75J/cm2 significantly P. gingivalis growth when compared to the control. MB at 100µg/mL at a fluency of 117.18J/cm2 in MB-mediated PAD showed a significant inhibitory effect on biofilm formation in P. gingivalis compared with MB-sPAD. CONCLUSION: High doses of MB-mediated sPAD exhibited anti-microbial and anti-biofilm potential activity, whereas lower doses of MB-mediated sPAD did not display this ability. Therefore, the dose of PAD used in vivo should be taken into account for endodontic treatment.