Actinomycetemcomitans Biofilm Research Articles

N-acyl homoserine lactones lactonase est816 suppresses biofilm formation and periodontitis in rats mediated by Aggregatibacter actinomycetemcomitans

ABSTRACT Aims The current study aimed to explore the adjuvant therapeutic effect of N-acyl homoserine lactones (AHLs)-lactonase est816 on Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) biological behaviors and periodontitis progression. Methods The inhibitory properties of est816 were detected by live/dead bacterial staining, scanning electron microscope (SEM), crystal-violet staining and reverse-transcription quantitative PCR (RT-qPCR). Biocompatibility of est816 on human gingival fibroblasts (HGFs) and human gingival epithelial cells (HGEs) was evaluated by CCK8 and ELISA. The ligature-induced periodontitis model was established in rats. Micro computed tomography and immunohistochemical and histological staining served to evaluate the effect of est816 on the prevention of periodontitis in vivo. Results est816 significantly attenuated biofilm formation, reduced the mRNA expression of cytolethal distending toxin, leukotoxin and poly-N-acetyl glucosamine (PNAG) and downregulated expressions of interleukin-6 and tumor necrosis factor-α with low cell toxicity. In vivo investigations revealed est816 decreased alveolar bone resorption, suppressed matrix metalloproteinase-9 expression and increased osteoprotegerin expression. Conclusion est816 inhibited A. actinomycetemcomitans biofilm formation and virulence release, resulting in anti-inflammation and soothing of periodontitis in rats, indicating that est816 could be investigated in further research on periodontal diseases.

Antibiofilm Activity of Probiotic Strains against Aggregatibacter actinomycetemcomitans

Aggregatibacter actinomycetemcomitans is a significant agent for periodontal disease. It develops biofilms on gingival plaque and promotes chronic pro-inflammatory response to periodontal tissues. As bacterial cells within the biofilm, this pathogen successfully eluded the human immune system by preventing the uptake of biofilm cells by phagocytic cells. Antibiotic-resistant biofilms might harm the environment, increase mortality, and add to the cost of healthcare. Anti-biofilms are necessary to prevent the growth of biofilms. It has been demonstrated that probiotics change how immune system cells produce cytokines. Therefore, the efficacy of five probiotic lactobacillus strains to prevent A. actinomycetemcomitans from growing bacteria and forming biofilms was evaluated. The organisms were incubated in anaerobic conditions at 37°C for a certain period. The antimicrobial test was done using the lawn method and measuring the size of the inhibition zone produced by the probiotic spots for each probiotic strain. Tetracycline antibiotic disc was used as a positive control to compare the size of the inhibition zone. The antibiofilm activity was performed using probiotic strains at their exponential and stationary phases against the exponential phase of A. actinomycetemcomitans. Our finding showed the probiotic lactobacillus could inhibit the growth of A. actinomycetemcomitans. The highest inhibition was demonstrated by L. johnsonii NBRC 13952. Besides, all probiotic lactobacillus used for this study significantly (p<0.05) inhibited the A. actinomycetemcomitans biofilm using both stationary and exponential phases. In conclusion, probiotic lactobacillus might be a potential agent that can be used to counteract the periodontal pathogen biofilm. Further study should be done to elucidate the antibiofilm mechanism, and several trials of the probiotic lactobacilli against multiple species of biofilms might be helpful in evaluating their effectiveness.

Evaluation of the effect of photodynamic therapy with Curcumin and Riboflavin on implant surface contaminated with Aggregatibacter actinomycetemcomitans

BackgroundPeri-implantitis is a destructive inflammatory disease affecting both hard and soft tissues of the osseointegrated implant and causing bone loss and envelope surrounding the implant. The study aimed at evaluating the effect of Photodynamic therapy with Curcumin and Riboflavin on the level of decontamination of implant surface impregnated with Aggregatibacter actinomycetemcomitans (A.a) biofilm. Materials and methodsIn this experimental and laboratory study, 42 implants (4.3 mm in diameter and 8 mm in length) were infected with A.a. bacterial suspension. Then, the implants carrying A.a biofilm were randomly divided into seven groups (n = 6). The groups included: 1- a negative control group (without treatment), 2- a positive control group of Chlorhexidine 0.12 %, 3- a Curcumin (5 mg/ ml) group, 4- a Riboflavin (0.5 %) group, 5- an LED irradiation group (390–480 nm), 6- a photodynamic therapy with Curcumin group, and 7- a photodynamic therapy with Riboflavin group. Then, the implants were sonicated and the amount of CFU/mL of each sample was calculated. One-way ANOVA and Tamhane tests were used to analyze the data. ResultsThe lowest mean number of colonies of A.a (CFU/ mL) were seen in the following groups, respectively: the positive control group of Chlorhexidine 0.12 %, the photodynamic therapy with Curcumin group, the photodynamic therapy with Riboflavin group, the Curcumin (5 mg/ ml) group, the Riboflavin (0.5 %) group, the LED radiation group, and the negative control group. The use of photodynamic therapy with Curcumin significantly reduced the number of colonies of A.a (CFU/ mL) in comparison with the photodynamic therapy with Riboflavin group (p = 0.004), the Riboflavin group (p = 0.045), the LED radiation group (p = 0.012), and the negative control group (p = 0.007). ConclusionaPDT with Curcumin and LED can reduce A.a biofilm on implant surfaces and can be used as a safe and non-invasive disinfection method to reduce A.a biofilm on implant surfaces.

Effectiveness of katuk leaf chlorophyll (Sauropus androgynus (L) Merr) with blue and red laser a ctivation to reduce Aggregatibacter actinomycetemcomitans and Enterococcus faecalis biofilm

In this study, the efficacy of using Sauropus androgynus (L) Merr, a katuk leaf chlorophyll photosensitizer, to reduce Aggregatibacter actinomycetemcomitans and Enterococcus faecalis biofilm was investigated. A red and blue diode laser is used as the light source. The sample was split into four groups: a negative control group, a positive control group, a blue laser treatment group (B), and a red laser treatment group (R), both with and without the addition of katuk leaf chlorophyll 1.6 mg/ml, and with varying densities of laser energy exposure of 2.5 J/cm2, 5 J/cm2, 7.5 J/cm2, and 10 J/cm2. Laser exposure and chlorophyll photosensitizer were tested using ELISA and ANOVA. At an energy density of 10 J/cm2, the optimal bacterial mortality rate was obtained in each treatment group. Namely, in the Aggregatibacter actinomycetemcomitans biofilm, the negative group, the number of deaths was 73.30% using a blue diode laser and 63.25% using a red diode laser. In the positive group, the number of deaths was 86.12% using a blue diode laser and 83.29% using a red diode laser. In the Enterococcus faecalis biofilm, in the negative group, the number of deaths was 67.78% using the blue diode laser and 75.33% using the red diode laser, and in the positive group, the number of deaths was 71.71% using the blue diode laser and 86.41 using a red diode laser. Exposure to blue and red diode lasers activates chlorophyll in katuk leaves, killing bacteria and reducing biofilms.

The Surface Structure, Chemical Molecules, and Thickness of Aggregatibacter actinomycetemcomitans biofilms by Different Inducers Agent

Aggregatibacter actinomycetemcomitans caused oral infection in recent years. It requires advancements in health technology, such as infection and sickness detection kits. This study purposed to provide data to aid in the detection kits. Aggregatibacter actinomycetemcomitans was prepared to be biofilm cultured sample. Then the samples were induced by glucose, lactose, soy protein, and iron. Scanning Electron Microscopy Energy Dispersive X-Ray, Confocal Laser Scanning Microscopy, and Olympus FluoView ver 4.2a were used to analyze the ultrastructural dimension of biofilms. The surface structure of biofilm induced by iron differed from that of biofilms induced by other inducers. O(78.65±10.32) and Cl(1.28±0.21) were the most abundant chemical compounds induced by glucose; S(14.67±4.56) and Fe(37.46±59.62) were induced by lactose; C(25.41±16.36) and N(13.49±9.37) were induced by soy protein; and P(58.53±17.80) was induced by iron. Iron induced the highest amount of polysaccharides (605.641±71.66). Lactose (857.877±70.86) induced the greatest number of bacterium cells. Iron (11.000±1000)nm caused the thickest layer. The biofilm of Aggregatibacter actinomycetemcomitans induced by glucose, lactose, soy protein, and iron are all different.

3-Phenyllactic acid generated in medicinal plant extracts fermented with plant-derived lactic acid bacteria inhibits the biofilm synthesis of Aggregatibacter actinomycetemcomitans.

In the present study, the effect of PLA on a periodontic pathogen, Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans), the biofilm, and virulence-related genes was investigated. We confirmed that two lactic acid bacteria (LAB) strains isolated from plant sources, Lactiplantibacillus plantarum MSC-C2 and Pediococcus pentosaceus K40, secrete PLA into the de Man, Rogosa & Sharpe (MRS) broth when supplemented with phenyl pyruvic acid (PPA) as a precursor to PLA. Moreover, PLA was generated in the fermentation broths of two medicinal plant extracts, Paeonia lactiflora Pall (PR) and Carthamus tinctorius (CT), when used by each LAB strain and each extract supplemented with PPA. We determined that the minimum inhibitory concentration (MIC) of PLA against A. actinomycetemcomitans was 20 mM. PLA significantly decreased biofilm formation and suppressed the transcription of pgA, ltxA, and cdtB genes, which encode the poly-N-acetylglucosamine (PGA) polysaccharide of biofilm matrix and exotoxins leukotoxin and cytolethal distending toxin (CDT), respectively. The PLA produced by the MSC-C2 and K40 strains was increased several times by the addition of PPA to the MRS broth. The anti-biofilm effect of the extracts from the fermentation broth was proportional to the increasing PLA concentration, while a cumulatively higher effect than that of PLA alone suggested a combinational effect of PLA and the other metabolites, such as lactic acid (LA). Among the two medicinal plants, PLA, produced after the addition of PPA, was higher in PR extract in case of both the LAB strains. PLA production by the MSC-C2 strain in the PR extract reached 4.8 ± 0.23 mM, which was obviously higher than that in the MRS broth (3.88 ± 0.12 mM) supplemented with 1 mg/ml PPA. The activity to inhibit biofilm formation in the fermented PR extract was clearly high. PLA formed in the fermented PR extract downregulated the dispersin B encoding the dspB gene together with pgA, ltxA, and cdtB. In conclusion, this study shows a promising activity of PLA against the A. actinomycetemcomitans biofilm and virulence genes. In addition, the combinational effect of PLA and the medicinal plant extract can be achieved by fermentation with a specific plant-derived LAB strain.

Hybrid system with stable structure of hard/soft tissue substitutes induces re-osseointegration in a rat model of biofilm-mediated peri-implantitis.

Re-osseointegration of an infected/contaminated dental implant poses major clinical challenges. We tested the hypothesis that the application of an antibiotic-releasing construct, combined with hard/soft tissue replacement, increases the efficacy of reconstructive therapy. We initially fabricated semi-flexible hybrid constructs of β-TCP/PHBHHx, with tetracycline (TC) (TC amounts: 5%, 10%, and 15%). Thereafter, using in vitro assays, TC release profile, attachment to rat bone marrow-derived stem cells (rBMSCs) and their viability as well as anti-bacterial activity were determined. Thereafter, regenerative efficacies of the three hybrid constructs were assessed in a rat model of peri-implantitis induced by Aggregatibacter actinomycetemcomitans biofilm; control animals received β-TCP/Bio-Gide and TC injection. Eight weeks later, maxillae were obtained for radiological, histological, and histomorphometric analyses of peri-implant tissues. Sulcus bleeding index was chronologically recorded. Serum cytokines levels of IL-6 and IL-1β were also evaluated by enzyme-linked immunosorbent assay. Substantial amounts of tetracycline, from hybrid constructs, were released for 2 weeks. The medium containing the released tetracycline did not affect the adhesion or viability of rBMSCs; however, it inhibited the proliferation of A. actinomycetemcomitans. Osteogenesis and osseointegration were more marked for the 15% hybrid construct group than the other two groups. The height of attachment and infiltration of inflammatory cells within fibrous tissue was significantly reduced in the experimental groups than the control group. Our protocol resulted in re-osseointegration on a biofilm-contaminated implant. Thus, an antibiotic releasing inorganic/organic construct may offer a therapeutic option to suppress infection and promote guided tissue regeneration thereby serving as an integrated multi-layer substitute for both hard/soft tissues.

Dosage Effectiveness of the Combination Calcium Hydroxide-Propolis as an Inhibitor for Aggregatibacter actinomycetemcomitans Biofilm

Objective: Calcium hydroxide is widely used as pulp-capping material, but it is resistant to Aggregatibacteractinomycetemcomitans bacteria. Because of this deficiency, many researchers are considering usingalternative natural ingredients, such as propolis. Propolis has been widely used in dentistry due to itsproperties and because it has active ingredients that can act as antibacterial agents. It is hoped that whencalcium hydroxide and propolis are combined, the combination can overcome the disadvantages. The aim ofthis study was to determine the dosage effectiveness of the combination calcium hydroxide-propolis as aninhibitor for A. actinomycetemcomitans biofilm. Material and methods: The sample was divided into fivegroups. The positive control group is a combination of calcium hydroxide-sterile aquadest, and the negativecontrol group is A. actinomycetemcomitans bacteria. Group I is a combination of calcium hydroxidepropolis with a ratio of 1:1, group II has a ratio of 1:1.5, and group III has a ratio of 1:2. Each group consistsof five replications. Combination of calcium hydroxide-propolis and A. actinomycetemcomitans bacterialsuspension was incubated for 2x24 hours then washed three times using PBS. Furthermore, 2% of crystalviolet was used for staining and covered with aluminum foil and then incubated for 15 minutes. Isopropanol200µL was added, followed by a measurement of OD with an ELISA reader, and the OD values werecalculated using an inhibition biofilm formula. Results: Group II has the largest mean inhibition with 76%compared to groups one and three. Conclusion: A combination of calcium hydroxide and propolis with aratio of 1: 1.5 is more effective than 1:1 or 1:2 to inhibit A. actinomycetemcomitans biofilm.

Antibiofilm effect of Theobroma cacao (cacao pod) extract on Aggregatibacter actinomycetemcomitans biofilm in vitro

Successful of periodontal treatment is to eradicate biofilm of bacteria. Aggregatibacter actinomycetemcomitans is a Gram-negative bacterium that have been suggested to be the main causes of periodontal disease. Theobroma cacao (cacao pod) is a medicinal plant that has a broad range of pharmacological effects. The aim of this study was to assess the antibiofilm effect of cacao pod extract against A. actinomycetemcomitans biofilm in vitro. A. actinomycetemcomitans were cultured in Brain Heart Infusion broth. Crystal-violet staining in biofilm assays were used to evaluate the cacao pod extract effect on A. actinomycetemcomitans ATCC 33384 biofilms and 0.2% chlorhexidine-gluconate was used as a positive control. After 24 hours of incubation, the optical density of each well in microtiter plates was measured. The results showed that the biofilm density after incubation with the cacao pod extract was significantly decreased in all concentrations and all incubation times (p<0.05). The most effective concentration for inhibiting biofilm A. actinomycetemcomitans was 100% cacao pod extract and 3 hrs of incubation time (p<0.05) with a 98.9% reduction of biofilm compared to negative control. Cacao pod extract is effective in inhibiting the growth of A. actinomycetemcomitans biofilm.

Enhanced efficacy against bacterial biofilms via host:guest cyclodextrin‐doxycycline inclusion complexes

Periodontal disease is characterized by a microbial infection and it is one of the major causes of teeth loss. The growth of pathogenic bacteria in oral cavity, such as Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans), provides a favorable enviromment for the biofilm formation, which result in periodontal diseases. The development of new technologies to potentiate existing drugs, avoiding bacterial biofilms resistance and improving chemical stability is highly desirable. Here, we report the use of host–guest chemistry to enhance the activity of antibacterial doxycycline (DOX) in A. actinomycetemcomitans bacterial strain suspension and biofilm in vitro through complexation with hydroxypropyl-β-cyclodextrin (HPβCD) using different molar ratios of DOX/HPβCD. 2D 1H NMR confirmed the host–guest complexation of DOX and HPβCD. We assessed the colloidal characteristics of the complex DOX/HPβCD via Dynamic Light Scattering (DLS) and Zeta Potential (PZ). The mixing ratio 1:2 DOX/HPβCD significantly decreased the minimum inhibitory concentration (MIC) and improved efficacy against A. actinomycetemcomitans suspensions and biofilms, respectively, when compared to free DOX and other DOX/HPβCD complexes. Further, the interaction of different molar ratio proportions of DOX/HPβCD complex with bacterial membrane was demonstrated via Isothermal Titration Calorimetry (ITC). Thus, we suggested the enhanced efficacy of the DOX/HPβCD complexes, at molar ratio 1:2, is due the higher cyclodextrin ratio, which potentiate the interaction between drug and bacterial membrane through nonionic interactions, such as hydrogen bonding or other van der Waals interactions. Collectively, the development of these complexes enables increased efficacy against bacterial biofilms, which hold promise for the treatment of aggressive and non-responsive forms of periodontitis.

Effectiveness of bacterial biofilms photodynamic inactivation mediated by curcumin extract, nanodoxycycline and laser diode

Biofilms have higher levels of antibiotic resistance compared to bacteria, so the alternatives are needed as therapy for diseases caused by biofilm infections. Photodynamic Therapy (PDT) has the advantage of being a safe alternative that involves molecular-level photochemical reactions. The use of different types of exogenous photosensitizers (PS) was done to compare their effectiveness. Turmeric extract containing curcumin has good effectiveness in PDT, whereas nanodoxycycline as an antibiotic has a fairly broad absorption spectrum and is effective as PS. The purpose of this study is to compare the effectiveness of photodynamic therapy on infections by Aggregatibacter actinomycetemcomitans causing periodontitis using exogenous organic and non-organic photosensitisers (PS). The A. actinomycetemcomitans biofilm had been grown on 96-well microplate for 72 hours incubation time. The samples were divided into three groups, treated with Laser diode, Laser + Turmeric Extract 0.5%, and Laser + Nanodoxycycline 0.1%. Treatment was done with a variety of exposure times: 30, 60, 90, 120, and 150 seconds. The data were analyzed using ANOVA test. The results of data analysis showed that diode laser irradiation treatment with endogenous porphyrin, diode laser with Curcumin and diode laser with nanodoxycycline produced significantly different biofilm reductions. Treatment with diode laser irradiation at various energy densities (4.15, 8.28, 12.44, 16.59, and 20.73 J/cm2) showed no significant difference in reducing bacterial biofilm. The treatment with diode and curcumin, and the treatment with diode laser irradiation and nanodoxycyclin showed a significant difference. Diode laser irradiation of 20.73 J/cm2 with irradiation time of 150 seconds resulted in the greatest reduction of biofilm 14.94%, diode laser irradiation + Curcumin 47.82%, and diode laser irradiation + nanodoxycyclin 53.76%. Therefore, PDT using a diode laser combined with exogenous PS extract of curcumin and nanodoxycycline is more effective to reduce bacterial biofilms.

In vitro effect of antimicrobial photodynamic therapy with phycocyanin on Aggregatibacter actinomycetemcomitans biofilm on SLA titanium discs.

The aim of this in vitro study was to evaluate antimicrobial photodynamic therapy (aPDT) with phycocyanin on Aggregatibacter actinomycetemcomitans biofilm formed on sandblasted, large-grit, and acid-etched (SLA) titanium discs. In this in vitro experimental study, the minimum inhibitory concentration (MIC), sublethal dose of diode laser irradiation time, and sublethal dose of aPDT were first determined. Next, 30 SLA titanium discs with 8 mm diameter and 2 mm thickness were incubated with A. actinomycetemcomitans in order for the bacterial biofilm to form, and were randomly divided into 5 groups (n = 6): (I) negative control with no treatment, (II) positive control, immersed in 0.2 % chlorhexidine (CHX) for 5 min, (III) phycocyanin alone with ×2 MIC concentration for 5 min, (IV) diode laser alone (635 nm wavelength, 220 mW power), and (V) PDT with diode laser and phycocyanin. The samples were then sonicated, and the number of colony-forming units (CFUs) on each disc was calculated. Data were analyzed using one-way ANOVA, t-test, and a post-hoc test. aPDT with 125 μg/mL phycocyanin and 4 min irradiation of 635 nm diode laser decreasedA. actinomycetemcomitans biofilm by 40.07 %. The lowest mean colony count (CFUs/mL) was noted in 0.2 % CHX group (0.0 × 105 CFU/mL) while the highest mean was observed in the negative control group (4.55 ± 0.08 × 105 CFUs/mL). Using phycocyanin alone significantly decreased the A. actinomycetemcomitans count by 27.54 % (3.29 ± 0.06 × 105 CFUs/mL) compared with the negative control group (P < 0.0001). Significant differences were noted between the negative control and other groups (P < 0.0001). aPDT with phycocyanin and diode laser can decrease the A. actinomycetemcomitans biofilm on SLA titanium implant surfaces and can be used as a safe and non-invasive decontamination method for reduction of A. actinomycetemcomitans biofilm on implant surfaces.

Aggregatibacter actinomycetemcomitans Biofilm Reduces Gingival Epithelial Cell Keratin Expression in an Organotypic Gingival Tissue Culture Model.

Epithelial cells express keratins, which are essential for the structural integrity and mechanical strength of the cells. In the junctional epithelium (JE) of the tooth, keratins such as K16, K18, and K19, are expressed, which is typical for non-differentiated and rapidly dividing cells. The expression of K17, K4, and K13 keratins can be induced by injury, bacterial irritation, smoking, and inflammation. In addition, these keratins can be found in the sulcular epithelium and in the JE. Our aim was to estimate the changes in K4, K13, K17, and K19 expression in gingival epithelial cells exposed to Aggregatibacter actinomycetemcomitans. An organotypic gingival mucosa and biofilm co-culture was used as a model system. The effect of the biofilm after 24 h was assessed using immunohistochemistry. The structure of the epithelium was also studied with transmission electron microscopy (TEM). The expression of K17 and K19, as well as total keratin expression, decreased in the suprabasal layers of epithelium, which were in close contact with the A. actinomycetemcomitans biofilm. The effect on keratin expression was biofilm specific. The expression of K4 and K13 was low in all of the tested conditions. When stimulated with the A. actinomycetemcomitans biofilm, the epithelial contact site displayed a thick necrotic layer on the top of the epithelium. The A. actinomycetemcomitans biofilm released vesicles, which were found in close contact with the epithelium. After A. actinomycetemcomitans irritation, gingival epithelial cells may lose their resistance and become more vulnerable to bacterial infection.

Efficacy of antimicrobial photodynamic therapy for elimination of Aggregatibacter actinomycetemcomitans biofilm on Laser-Lok titanium discs.

Antimicrobial Photodynamic therapy (aPDT) is a novel modality suggested for treatment of peri-implantitis. This study aimed to assess the effect of aPDT with toluidine blue (TBO) and indocyanine green (ICG) and 635 nm and 808 nm diode laser on Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) biofilm formed on Laser-Lok titanium discs. Eighty sterile Laser-Lok titanium discs were inoculated with A. actinomycetemcomitans to form biofilm and were randomly divided into 8 groups (n = 10) of control, chlorhexidine (CHX), TBO, ICG, 635 nm diode laser with 220 mW power, 808 nm diode laser with 250 mW power, 100 μg/mL TBO+635 nm diode laser and ICG+808 nm diode laser. Number of colony forming units (CFUs) on the surface of each disc was counted after the intervention. Data were analyzed using the Kruskal-Wallis test. Significant differences were noted in colony count among the eight groups after the intervention (P = 0.001). Pairwise comparisons with adjusted P value test showed that aPDT with TBO+635 nm laser and ICG+808 nm laser caused significant reduction of bacterial biofilm compared to the control group (P = 0.0001). TBO alone caused significant reduction of biofilm compared to the control group (P = 0.004). No other significant differences were noted (P > 0.05). Within the limitations of this study, the results showed that aPDT is a potential modality for decontamination of implant surface and reduction of A. actinomycetemcomitans biofilm in vitro. In this study, aPDT with TBO+635 nm diode laser and ICG+808 nm diode laser decreased the bacterial load on titanium discs.

The Microflora Diversity and Profiles in Dental Plaque Biofilms on Brackets and Tooth Surfaces of Orthodontic Patients

Objective: Fixed orthodontic appliances may influence the oral environment through accumulation of plaque, decreased plaque pH, and increased gingival inflammation. These changes in the oral cavity can potentially lead to periodontal disease, demineralization, and other infectious diseases. Materials and Methods: To investigate the changes in biofilm throughout the initial 2 weeks, we placed a stainless steel bracket on the upper second premolar and collected plaque samples on the bracket and on the tooth surface at different time points (0, 24, 48 h and 1 and 2 weeks) and plated on tryptic soy agar blood agar plate, and kept at 37°C in an anaerobic chamber for 5 days to determine the CFUs of bacteria. At the end of 2 weeks, we removed the bracket and elastomeric module, and we isolated genomic DNA from the bacterial biofilm for identification of bacteria by 16S rRNA PCR analysis. We also analyzed the morphology of biofilm on the bracket by scanning electron microscope. Results: Our results show that the bacterial biofilm was significantly increased on the bracket in all the subjects, whereas on the tooth surface, the CFUs were not significantly increased. PCR assay showed that biofilm on orthodontic brackets from all subjects showed colonization by Streptococcus gordonii, Porphyromonas gingivalis and Streptococcus mutans were observed on some of the subjects after 48 h, whereas Aggregatibacter actinomycetemcomitans biofilm was observed in all the time points except 24 h. Conclusion: This study demonstrated that both periodontal and cariogenic bacterial biofilms were formed on the bracket as early as 24 h.

The impact of Aggregatibacter actinomycetemcomitans biofilm-derived effectors following antimicrobial photodynamic therapy on cytokine production in human gingival fibroblasts.

Antimicrobial photodynamic therapy (aPDT) is an effective adjunctive therapeutic modality for the treatment of local infections, including periodontitis and peri-implantitis. After receiving aPDT, microbial cells in the biofilm structure may produce and/ or release soluble biofilm-derived effectors (BDEs), which may affect the biology of the host cells in the community context of their surrounding microenvironment. Given the fact that no study has investigated the role of BDEs following aPDT in the pathogenesis of infectious diseases, the aim of the current study was to determine the effect of BDEs of Aggregatibacter actinomycetemcomitans following exposure to sub-lethal doses of indocyanine green (ICG)-aPDT on human gingival fibroblasts (HGFs) in terms of cytokines produced. In this study, we evaluated the effect of biofilm-conditioned medium (BCM) resulting from the treatment of A. actinomycetemcomitans biofilm with a sub-lethal dose of aPDT on cytokines production, including IL-6, IL-8, CXCL10, TGF-β, and bFGF of HGFs using enzyme-linked immunoassays (ELISA). The sensitivity of cytokines to BDEs was determined by micro-titer plates. The maximal sub-lethal dose of ICG-PDT was 20.15 μM/mL ICG at a fluence of 31.2 J/cm2. The BCM of ICG-PDT-treated viable A. actinomycetemcomitans significantly reduced IL-6, IL-8, and CXCL10 levels compared to the BCM of untreated viable A. actinomycetemcomitans (78-, 93-, and 61.6-fold reduction, respectively; all P < 0.01). TGF-β and bFGF were strongly induced by BCM of ICG-PDT treated viable A. actinomycetemcomitans (by 57.7 and 36.1 folds, respectively; both P < 0.05). The BCM of untreated viable A. actinomycetemcomitans degraded most of the CxCL10, TGF-β and bFGF (58.8, 61.5, and 71.6%, respectively) in 24 h, while it degraded 9.3% of IL-6 and 15.1% of IL-8 after 24 h. The results of the current study revealed that a sub-lethal dose of ICG-aPDT through the effect of BCM on HGFs could not only significantly reduce the production of pro-inflammatory cytokines but also promoted their role in periodontal regeneration due to increasing the bFGF level. Altogether, ICG-aPDT, with it's antimicrobial effects reduces inflammation and induces of tissue regeneration resulting from BCM, can be considered an efficient adjunctive therapeutic method for the treatment of local infections.

Antimicrobial efficacy of photodynamic therapy using two different light sources on the titanium-adherent biofilms of Aggregatibacter actinomycetemcomitans: An in vitro study.

Antimicrobial photodynamic therapy (aPDT), is a promising approach proposed as an adjunct for the decontamination of dental implant surfaces. This study aimed to investigate the effect of aPDT with laser or light emitting diode (LED) compared with conventional chlorhexidine treatment on the titanium-attached biofilms of Aggregatibacter actinomycetemcomitans. Thirty-six acid-etched and sandblasted (SLA) titanium discs were allocated to six groups and incubated with the titanium-adherent biofilms of A. actinomycetemcomitans as follows. Negative control (no treatment applied), positive control (0.2% chlorhexidine solution), 0.1 mg/mL Toluidine Blue [TBO] group, aPDT-treated groups subjected either to diode laser with a wavelength of 635 nm wavelength or LED with the peak wavelength of 630 nm with TBO as photosensitizer and sterile control (not contaminated). Following sonication and transferring the specimens to the microplate, the number of colony-forming units (CFUs) per disc was calculated. Data were analyzed using one-way analysis of variance (ANOVA) and additional post hoc tests with the level of significance set at P < 0.05. aPDT using TBO + LED was significantly more effective (0.93 ± 0.24 × 104) in the suppression of A. actinomycetemcomitans compared with TBO + Laser (2.65 ± 0.7 × 104). However, the lowest mean of CFU count was found in sterile, and chlorhexidine groups, respectively (P < 0.0001) and the highest bacterial count was observed in the negative control group (P < 0.0001). LEDs and diode lasers have a lower ability to suppress A. actinomycetemcomitans biofilms compared to 0.2% chlorhexidine in vitro. However, the aPDT with the use of LED as a light source and TBO as a photosensitive agent could be an appropriate alternative to conventional chlorhexidine treatment.

The difference in biofilm molecular weight in Streptococcus mutans and Aggregatibacter actinomycetemcomitans induced by sucrose and soy protein (glycine soja).

Biofilms consist of microbial cells and extracellular polymeric substance (EPS). Streptococcus mutans and Aggregatibacter actinomycetemcomtans are bacteria that can form biofilms and generate EPS. Biofilm formation can be induced by specific substances such as sucrose and protein. To identify the molecular weight that determines biofilm protein profile expression of S. mutans and A. actinomycetemcomitans induced by sucrose (carbohydrate) and soy protein (glycine soja). Experimental laboratory study. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to determine the molecular weight. Nil. The results of analysis of protein SDS-PAGE showed the presence of 28 protein bands on A. actinomycetemcomitans biofilm in the media trypticase soy broth (TSB), 20 protein bands on biofilms of S. mutans in the media TSB, 29 protein bands on biofilm A. actinomycetemcomitans in the media brain heart infusion (BHI) + sucrose 2%, and 13 protein bands on biofilms of S. mutans in the media BHI + sucrose 2%. There are differences in biofilm protein profile expression that determine the molecular weight of S. mutans biofilm and A. actinomycetemcomitans induced by sucrose (carbohydrate) and soy protein (glycine soja).

Effect of Antimicrobial Photodynamic Therapy Using Indocyanine Green Doped with Chitosan Nanoparticles on Biofilm Formation-Related Gene Expression of Aggregatibacter actinomycetemcomitans

Objectives:Eradication of Aggregatibacter actinomycetemcomitans (A. actionmycetemcomitans), as an opportunistic periodontopathogen, and inhibition of its virulence factor expression require a new adjunctive therapeutic method. In this study, we accessed the expression level of rcpA gene, as a virulence factor associated with A. actinomycetemcomitans biofilm formation, following treatment by antimicrobial photodynamic therapy (aPDT) using indocyanine green (ICG) doped with chitosan nanoparticles (CS-NPs@ICG).Materials and Methods:CS-NPs@ICG was synthesized and examined using scanning electron microscopy (SEM). A. actinomycetemcomitans ATCC 33384 strain was treated with CS-NPs@ICG, as a photosensitizer, which was excited with a diode laser at the wavelength of 810 nm with the energy density of 31.2 J/cm2. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to determine the changes in rcpA gene expression level.Results:Synthetized CS-NPs@ICG was confirmed via SEM. The results revealed that CS-NPs@ICG-mediated aPDT could significantly decrease rcpA gene expression to 13.2-fold (P<0.05). There was a remarkable difference between aPDT using CS-NPs@ICG and ICG (P<0.05). The diode laser, ICG, and CS-NPs@ICG were unable to significantly downregulate rcpA gene expression (P>0.05).Conclusion:aPDT with CS-NPs@ICG leads to a decrease of the virulence factor of A. actinomycetemcomitans and can be used as an adjunct to routine treatments for successful periodontal therapy in vivo.

Exploration of Chlorella sp. as antibacterial to Aggregatibacter actinomycetemcomitans biofilm

Aggregatibacter actinomycetemcomitans is a facultative anaerobic gram-negative, which is major pathogenic bacteria cause of aggressive periodontitis that has the ability to form biofilm. The therapy of aggressive periodontitis has been done with tetracycline antibiotics, meanwhile it may cause resistance problem. Chlorella sp. has antimicrobial activity against the anaerobic gram-negative bacteria as an adjunctive therapy of aggressive periodontitis. Purpose: This study aimed to determine the antibacterial potency of various concentrations of Chlorella sp. to A actinomycetemcomitans biofilm. Method: The antibacterial potency of Chlorella sp. to A actinomycetemcomitans were examined by biofilm test, divided into 5 groups, each group consisted of 4 samples. The control groups were: K− (aquadest), K+ (tetracycline), and treatment groups were given Chlorella sp. in various concentrations: P1 (0.625%), P2 (1.25%), and P3 (2.5%). The inhibition of biofilm formation were examined on microtiter plate by measure its Optical Density (OD) value on ELISA Reader. Data were analyzed using One Way ANOVA followed by LSD test. Result: Treatment with Chlorella sp in all treatment groups decreased the OD value, as well with tetracycline (p<0.05). The decrease in OD values indicated that more biofilms were inhibited due to its antibacterial potency. Treatment with 2.5% of Chlorella sp. showed the greatest biofilm inhibition among the treatment groups (p<0.05). Conclusion: Chlorella sp. extract has antibacterial potency against A actinomycetemcomitans biofilm