Refractory Periodontitis Research Articles

Research progress in pathogenesis and control of Enterococcus faecalis with persistent infection in root canals

Root canal therapy is the common treatment for endodontic infections. Successful root canal therapy depends on favorable root canal preparation, root canal medication and three-dimensional obturation of the root canal system. The key to successful root canal therapy is to prevent re-infection of the highly complex root canal systems by removing infecious biofilms and bacterial toxins in the root canal system. The present paper reviews the pathogenic mechanism of the Enterococcus faecalis in the harsh environment of root canal system, the inflammation and immunity of refractory periapical periodontitis and the progress of infection control methods.

Detection of Immuno Dominant Peptides against pgaB of Acinetobacter baumannii

Background: Acinetobacter baumannii is a gram negative non-motile coccobacillus, which was considered as a low priority pathogen with low virulence. Recently, it was declared as the priority pathogen under the critical category of the most dangerous pathogen by WHO. Acinetobacter Baumannii is an inhabitant of oral biofilms, and it also increases the risk of refractory periodontitis. It causes nosocomial infections with pgaB, a part of pgaABCD operon which is involved in the biofilm formation.
 Aim: The aim of the present study was to detect the immunodominant peptides against pgaB of Acinetobacter baumannii using bioinformatic tools and databases.
 Materials and Methods: The present study was carried out using immune informatics. The protein sequence of the pgaB protein from A.baumannii was subjected to assess allergenicity, secondary structure, antigenicity and stability prediction of selected T cell epitopes, physico-chemical analysis, Identification of MHC class 2 binders, Final selection of B-cell epitopes was done with IEDB B-cell epitope tool
 Results: Final docking of the peptides were interpreted by hydrogen bonds and interac- tion scores with TLR-2. Promising scores on antigenicity, instability were obtained. Based on the combinatorial scores, one vaccine peptide (LNLTLGLAL) was suggested to be a promising vaccine candidate against pgaB of A.baumannii.
 Conclusion: The findings of the present study suggest epitope LNLTLGLAL as a promising vaccine candidate against pgaB of A.baumannii. The vaccine peptides targeting the pgaB Gene in A.baumannii using an immune-informatics approach suggests promising results in the present study. However, the predicted epitope peptides need further experimentation in animal models for its application against A.baumannii.

Cyanobacteria-based self-oxygenated photodynamic therapy for anaerobic infection treatment and tissue repair

Photodynamic therapy (PDT) is an important technique to deal with drug-resistant bacterial infections in the post-antibiotic era. However, the hypoxic environment in intractable infections such as refractory keratitis and periodontitis, makes PDT more difficult. In this work, spontaneous oxygen-producing cyanobacteria were used as the carrier of photosensitizer (Ce6), and ultrasmall Cu5.4O nanoparticles (Cu5.4O USNPs) with catalase activity for infection and inflammation elimination and rapid tissue repair (CeCycn-Cu5.4O). The loading of Ce6 and Cu5.4O USNPs onto cyanobacteria surface were confirmed by transmission electron microscopy, nano particle size analyzer, scanning electron microscopy. In vitro sterilization and biofilm removal experiments demonstrated that the restriction of hypoxic environment to PDT was significantly alleviated due to the oxygen production of cyanobacteria. Under laser irradiation, the close transfer of energy photons to oxygen produced by cyanobacteria reduced more than 90% of Ce6 dosages (660 nm, 200 mW/cm2, 2 min). It is worth mentioning that both rapid sterilization through PDT and long-term oxidized free radicals elimination were achieved by adjusting the ratio of Ce6 and Cu5.4O USNPs. Both periodontitis and refractory keratitis animal models proved the excellent self-oxygenation enhanced antibacterial property and promotion of tissue repair.

Periodontal referral patterns in Norway: 2003 versus 2018.

ObjectivesChanges in periodontal referral patterns over time have been reported from the United States and Australia. To date, comparable studies have not been published from Europe. The objectives of the present study were to examine changes in periodontal referral patterns in Norway in 2003 versus 2018 and to compare these with trends observed in the United States and Australia using universal criteria for grading of periodontal severity.Materials and methodsA retrospective analysis of 369 charts from four Norwegian periodontics clinics was completed. Data on year of referral, gender, age, tobacco smoking, periodontal status and missing teeth at initial examination, teeth planned for extraction, and periodontal case type were collected using a survey format; case type I, II, III, and IV representing increasing severity of periodontitis, case type V representing referral for other periodontal conditions (peri‐implantitis, refractory periodontitis, etc.). Chi‐square, t‐tests, and negative binomial regression were used for the statistical analysis.ResultsCompared with 2003, the 2018 data showed an increase in mean age at referral (p < 0.05), overall distribution of case type III and V (p = 0.047), and number of missing teeth (p = 0.001). Further, a decrease in prevalence of smokers (p < 0.05), but no change in number of teeth planned for extraction (p = 0.104), were observed.ConclusionsDuring a period of 15 years, changes in periodontal referral patterns in Norway are similar to those in the United States and Australia. The adoption of a guideline‐based referral practice might be beneficial for both the dental profession and patients.

Photodynamic Inactivation of an Endodontic Bacteria Using Diode Laser and Indocyanine Green-Loaded Nanosphere.

Apical periodontitis, an inflammatory lesion causing bone resorption around the apex of teeth, is treated by eradicating infectious bacteria from the root canal. However, it has a high recurrence rate and often requires retreatment. We investigated the bactericidal effect of antimicrobial photodynamic therapy (aPDT)/photodynamic antimicrobial chemotherapy (PACT) using indocyanine green (ICG)-loaded nanospheres coated with chitosan and a diode laser on a biofilm of Enterococcus faecalis, a pathogen of refractory apical periodontitis. Biofilm of E. faecalis was cultured in a porcine infected root canal model. ICG solution was injected into the root canal, which was then irradiated with a laser (810 nm wavelength) from outside the root canal. The bactericidal effect was evaluated by colony counts and scanning electron microscopy. The result of the colony counts showed a maximum 1.89 log reduction after irradiation at 2.1 W for 5 min. The temperature rise during aPDT/PACT was confirmed to be within a safe range. Furthermore, the light energy transmittance through the root was at a peak approximately 1 min after the start of irradiation, indicating that most of the ICG in the root canal was consumed. This study shows that aPDT/PACT can suppress E. faecalis in infected root canals with high efficiency.

The feasibility of phage therapy for periodontitis.

Periodontitis, a chronic progressive inflammation caused by plaque biofilm, is the main cause of tooth loss in adults. For certain refractory periodontitis cases, it is difficult to achieve a good curative effect using the existing periodontal treatment approaches, which may be due to periodontal pathogenic mechanism in the affected periodontal tissue that the host cannot resist and eliminate. Various pieces of evidence collectively revealed that most studies are focusing on phages in periodontal disease. Several studies have reported periodontitis treatment using phage therapy, highlighting its features including specificity, rapid propagation, and effectiveness on bacteriophage biofilms. In this study, we focus on these reports, aiming to lay the foundation for improved periodontal treatment approaches.

Effects of ClpP protease on biofilm formation of Enterococcus faecalis.

Enterococcus faecalis (E. faecalis), one of the main pathogens responsible for refractory periapical periodontitis and nosocomial infections, exhibits markedly higher pathogenicity in biofilms. Objectives Studies have shown that caseinolytic protease P (ClpP) is involved in biofilm formation. However, to date, few studies have investigated the role of ClpP in the survival of E. faecalis, and in enhancing biofilm formation. Therefore, we investigated the role of ClpP in the formation of E. faecalis biofilms.Methodology In our study, we used homologous recombination to construct clpP deleted and clpP complement strains of E. faecalis ATCC 29212. A viable colony counting method was used to analyze the growth patterns of E. faecalis. Crystal violet staining (CV) and confocal scanning laser microscopy (CLSM) were used to characterize biofilm mass formation and scanning electron microscopy (SEM) was used to observe the biofilm microstructure. Data was statistically analyzed via Student’s t-test or one-way analysis of variance (ANOVA).Results The results exhibited altered growth patterns for the clpP deletion strains and depleted polysaccharide matrix, resulting in reduced biofilm formation capacity compared to the standard strains. Moreover, ClpP was observed to increase biofilm formation in E. faecalis.Conclusion Our study shows that ClpP can increase biofilm formation in E. faecalis and emphasizes the importance of ClpP as a potential target against E. faecalis.

Preparation and characterisation of a gellan gum-based hydrogel enabling osteogenesis and inhibiting Enterococcus faecalis

Infections are the leading cause of failure of osteogenic material implantation. Antibiotic treatment, treatment with bone cement, or collagen sponge placement can result in drug resistance and difficulties in operation. To address this, gellan gum (GG) was selected in this study and prepared as an injectable hydrogel containing chlorhexidine (CHX) and nanohydroxyapatite (nHA) that overcomes these intractable problems. Scanning electron microscopy and micro-computed tomography revealed a three-dimensional polymeric network of the hydrogel. The hydrogel had excellent biocompatibility, as detected by cell counting kit-8 and Live/Dead assay. Bone marrow mesenchymal stem cells could be encapsulated into the network, showing that the structure was suitable for cell growth. Additionally, loading the hydrogel with nHA improved its mechanical, biodegradable, and osteogenic properties. Quantitative alkaline phosphatase and Alizarin Red S staining validated its osteogenic ability. Furthermore, antibacterial activity assessment showed that the hydrogel loaded with 50 μg/mL CHX inhibited Enterococcus faecalis in a concentration-dependent manner. Thus, we report an injectable GG-based hydrogel with superior antibacterial effect against E. faecalis and osteogenesis, which holds promise for treating infectious bone defects caused by refractory periradicular periodontitis.

MiR-200a contributes to the migration of BMSCs induced by the secretions of E. faecalis via FOXJ1/NF\u03baB/MMPs axis

BackgroundUpon migrating to the injured sites, bone marrow mesenchymal stem cells (BMSCs) play critical roles in the repair of bone lesion caused by chronic apical periodontitis. Emerging evidences have shown that Enterococcus faecalis is always associated with apical periodontitis, especially refractory apical periodontitis. But the mechanism underlying how Enterococcus faecalis affects the migration of BMSCs remains unclear.MethodsThe effects of Enterococcus faecalis supernatants on the migration of BMSCs were determined by transwell migration assays. miRNA sequencing was performed to detect the significantly differentially expressed miRNAs of BMSCs. Proteomics analysis was used to detect the protein expression alterations of BMSCs. Luciferase report assays were deployed to verify the targets of miRNA. Western blot analysis was performed to examine the expressions of matrix metalloproteinases-3, matrix metalloproteinases-9, Forkhead Box Protein J1 (FOXJ1), and nuclear factor kappa B (NFκB). The activations of NFκB were detected by luciferase assays with NFκBluc reporter.ResultsWe found that Enterococcus faecalis supernatants could promote the migration of BMSCs. The upregulation of miR-200a-3p in this process contributed to BMSC migration through downregulating its target Forkhead Box Protein J1. Moreover, FOXJ1/ NFκB axis was found to regulate matrix metalloproteinases (MMPs) in this process.ConclusionsThese results above suggest that miR-200a contributes to the migration of BMSCs induced by the secretions of E. faecalis via FOXJ1/NFκB/MMPs axis.

Enterococcus faecalis induces necroptosis in human osteoblastic MG63 cells through the RIPK3 / MLKL signalling pathway.

To explore the activation of necroptosis triggered by Enterococcus faecalis in human osteoblastic MG63 cells and provide new insights into the pathogenesis of refractory apical periodontitis. The viability of MG63 cells exposed to live E.faecalis was investigated using the cell counting kit-8 assay. The relative expressions of specific markers for necroptosis, namely p-RIPK3 and p-MLKL, were determined by western blotting. Cells pretreated with necrosulfonamide and GSK'872, which are specific inhibitors for MLKL and RIPK3, respectively, were then subjected to lactate dehydrogenase (LDH) cytotoxicity assay, flow cytometry analysis and Hoechst 33342/PI double fluorescence staining. Lentiviral-delivered short hairpin RNA (shRNA) targeting MLKL was employed to further confirm the activation of necroptosis in MG63 cells infected with E.faecalis. Transmission electron microscopy was additionally used to observe the morphological characteristics. Statistical analysis was conducted using Student's t-tests or one-way ANOVA followed by the Student-Newman-Keuls test. The infection with E.faecalis significantly inhibited the viability of MG63 cells in a multiplicity of infection- and infection time-dependent manners (P<0.05). In line with this, the expression levels of necroptosis-related markers, p-RIPK3 and p-MLKL, were significantly increased postinfection (P<0.05). Significant reductions in death rate were detected in the case of E.faecalis-infected MG63 cells following pretreatment with the inhibitors of RIPK3 and MLKL (P<0.01). Furthermore, silencing of MLKL by shRNA significantly decreased LDH release (P<0.01) and resulted in less mitochondrial swelling and vacuole-like changes, as well as reduced endoplasmic reticulum expansion. Enterococcus faecalis infection-induced necroptosis of MG63 cells via the RIPK3/MLKL signalling pathway, which may exert a negative influence on the healing process of refractory apical periodontitis. This study may offer novel insights into the pathogenesis and potential therapeutic targets of refractory apical periodontitis.

Biological characteristics and whole-genome analysis of the Enterococcus faecalis phage PEf771.

Enterococcus faecalis is a common pathogen causing refractory periapical periodontitis and secondary intraradicular infections. In this study, E. faecalis YN771 isolated from a re-treated root canal at a stomatology department was used as the host bacterium and was co-cultured with wastewater from the same department and patient samples to isolate a phage that lyses E. faecalis. We studied the biological and genomic characteristics of this phage. Transmission electron microscopy showed that this phage's head is icosahedral in structure, with a head diameter of around 98.4 nm, and a contractile tail of around 228.5 nm in length and a diameter of 17.3 nm. The phage was identified as a member of the Myoviridae family and named PEf771. It is sensitive to proteinase K but resistant to chloroform and Triton X-100. Its lytic cycle is 45 min, burst size is 78, optimal multiplicity of infection is 0.1, lysis spectrum is narrow, and host strain specificity is strong. Its optimal growth temperature is 37 °C, most suitable pH is 6.0, and is sensitive to ultraviolet radiation. Whole-genome sequencing of PEf771 indicated it has a genome size of 151 052 bp, with a GC content of 36.97%, and encodes 197 proteins plus 26 tRNAs. PEf771 is most closely related to E. faecalis phage EFDG1. Phage PEf771 has strong host specificity and lytic ability, so it is important to further characterize this phage and its interaction with E. faecalis.

Applications of Lasers in Refractory Periodontitis: A Narrative Review.

ABSTRACTObjective:The purpose of the study is to evaluate the various lasers in dentistry and to investigate if it can be used for treatment of refractory periodontitis.Methods:The study followed partially the PRISMA guidelines as it is a narrative review. A number of articles were selected from a period of 1980 to 2020 from databases, PubMed, PubMed central, Cochrane and Scopus. Articles related to the effects of lasers on periodontitis both refractory and aggressive were investigated.Results:After reviewing the literature, 70 articles were found, related to application of lasers in periodontal diseases. Out of the 70, 11 articles pertained to the effect of laser for the treatment of Refractory and inflammatory periodontitis. 5 articles related to experimental animal models, one pertaining to in-vitro and six studies related to in-vivo in human cohorts.Discussion:It was found that lasers if used in controlled parameters by incorporating laser assisted treatment such as Photodynamic therapy and low level laser therapy can be of use as an adjunct therapy for treatment of refractory periodontitis. The use of different wavelengths in the initial and maintenance phase of periodontal disease plays a positive role. The presence of in-vitro and animal model studies is one of the limitation to this study. The available studies have shown marked reduction in inflammation and better clinical and microbiological parameters. The drawback of this study is the limited literature involving laser management for refractory periodontitis in human cohorts.Conclusion:Different wavelengths of laser and choice of laser assisted periodontal treatment plays an important role in the overall progress and prognosis of periodontal disease activity.

A New NLRP3 Inflammasome Inhibitor, Dioscin, Promotes Osteogenesis.

Refractory periapical periodontitis, which is a persistent infection after root canal treatment, still has no effective treatment. Its most common pathogen is Enterococcus faecalis. Here, the precursor of phytosteroids, dioscin, is introduced to fight against the inflammation induced by Enterococcus faecalis. The findings suggest that dioscin inhibits the nuclear transport of NF-κB and the expression of reactive oxygen species (ROS) induced by lipoteichoic acid from the Enterococcus faecalis. The decrease in mRNA and protein levels of NLRP3, Caspase-1, and IL-1β is observed in dioscin treated mouse macrophages. In the MC3T3-E1 cells, dioscin also promotes the expression of osteogenic-related factors, ALP, Runx2, and OCN. The increased formation of mineralized nodules after the application of dioscin further indicates that dioscin has the potential to promote osteogenesis. The above results suggest dioscin can be a potential root canal irrigation or root canal sealant for the treatment of refractory apical periodontitis.

Clinical outcomes of adjunctive indocyanine green-diode lasers therapy for treating refractory periodontitis: A randomized controlled trial with in vitro assessment

It is still challengeable to treat periodontal pockets refractory to mechanical debridement. This study is to evaluate the potential of indocyanine green (ICG)-diode laser-based photothermal therapy (PTT) for solving this dilemma. Bone marrow-derived mesenchymal stem cells (BMSCs) and periodontal ligament cells (PDLCs) were incubated with phosphate-buffered saline, chlorhexidine, or ICG, non-irradiated or irradiated with 810-nm diode lasers, and the cell viability was evaluated. Patients with teeth refractory to mechanical periodontal debridement on different quadrants were recruited. At baseline (T0), all examined teeth received scaling and root planing, and those on the test quadrant (PTT group) received ICG-diode laser treatment. The outcome was evaluated using clinical parameters and cytokines in the gingival crevicular fluids at 4-6 weeks (T1) and 6 months (T2). In ICG-treated cultures, the viability of BMSCs and PDLCs was recovered on day 4, and laser irradiation inhibited the metabolic activities of BMSCs. 22 patients with 30 control teeth and 35 PTT-treated teeth were examined. All examined teeth showed modest reductions in probing pocket depth (PPD), clinical attachment loss (CAL), bleeding upon probing (BOP), and plaque score at T1 and T2 and significant reductions in IL-1β and MMP-8 at T2. Compared with controls, BOP was reduced more prominently, IL-1β and MMP-8 were significantly lower, and reductions in PPD and CAL were slightly greater in the PTT group at T1 (0.05-0.19mm). ICG-diode laser-based PTT is compatible to periodontium and assists in faster resolution of gingival inflammation in periodontal pockets refractory to mechanical debridement.

Disturbed bone remodelling activity varies in different stages of experimental, gradually progressive apical periodontitis in rats

Bone remodelling keeps going through the lifespan of human by bone formation and bone resorption. In the craniofacial region, mandibles act as the main force for biting and chewing, and also become susceptible to a common bone-loss disease, namely, apical periodontitis, once infected dental pulp is not treated timely, during which bone resorption occurs from the apical foramen to the apical bone area. Although conventional root canal treatment (RCT) can remove the most of the infection, chronical apical periodontitis due to incomplete removal of dental pulp and subsequent microleakage will become refractory and more challenging, and this process has scarcely been specifically studied as a bone remodelling issue in rat models. Therefore, to study chronical and refractory apical periodontitis owing to incomplete cleaning of infected dental pulp and microleackage in vivo, we establish a modified rat model of gradually progressive apical periodontitis by sealing residual necrotic dental pulp and introducing limited saliva, which simulates gradually progressive apical periodontitis, as observed in the clinical treatment of chronical and refractory apical periodontitis. We show that bone-loss is inevitable and progressive in this case of apical periodontitis, which confirms again that complete and sound root canal treatment is crucial to halt the progression of chronical and refractory apical periodontitis and promote bone formation. Interestingly, bone remodelling was enhanced at the initial stage of apical periodontitis in this model while reduced with a high osteoblast number afterwards, as shown by the time course study of the modified model. Suggesting that the pathological apical microenvironment reserve its hard tissue formation ability to some degree but in a disturbed manner. Hopefully, our findings can provide insights for future bone regenerative treatment for apical periodontitis-associated bone loss.

The potential effect of Bruton’s tyrosine kinase in refractory periapical periodontitis

To determine the expression of Bruton’s tyrosine kinase (BTK) in refractory periapical periodontitis and analyze the relationship between BTK and bone resorption in refractory periapical periodontitis. The mechanism of bone resorption is also discussed. The OneArray Plus expression microarray was used to screen for genes related to refractory periapical periodontitis. Real-time PCR was used to detect the expression of BTK in refractory periapical periodontitis tissues. A model of periapical periodontitis was established by sealing E.faecalis into the pulp of rats. To establish a model of E.faecalis LTA infection of osteoclasts, the relationship between BTK and bone destruction during refractory periapical periodontitis was analyzed. OneArray Plus expression microarray results showed that we found that the expression of 1787 genes in the two samples was different. After validating these samples, we found that BTK was closely related to refractory periapical periodontitis. The results showed that the expression of BTK in refractory periapical periodontitis tissues was higher than that in normal tissues. Immunohistochemistry, enzyme histochemistry and real-time PCR showed that the BTK expression curve in the experimental model resembled a reverse V shape from week 1 to week 4. Osteoclasts were cultured in vitro and treated with E. faecalis LTA. The expression of BTK in the E. faecalis model was greater than that in the control group. BTK played an important role in the progression of refractory periapical periodontitis.

Clinical and Biological Effects of Adjunctive Photodynamic Therapy in Refractory Periodontitis.

Introduction: To date, no novel treatment approach is available for optimum outcomes regarding refractory periodontitis. The aim of the present study was to assess the efficiency of photodynamic therapy (PDT) in treating patients diagnosed with refractory periodontitis and compare the clinical and biological outcomes of conventional periodontal treatment with or without adjunctive PDT in these patients, by assessing clinical parameters (plaque index [PI], gingival recession [GR], bleeding on probing [BOP], periodontal probing depth [PPD] and clinical attachment level [CAL]) as well as biological parameters (IL-1β) in the gingival crevicular fluid (GCF). Methods: Sixteen patients within the age of 30 to 60 years, with a mean age of 40 years old, diagnosed with refractory periodontitis were included. In this split mouth design study, 2 quads (1 upper + 1 lower) from the same patient were randomly treated with (scaling and root planing [SRP]+PDT) together. The other 2 quadrants (1 upper + 1 lower) were treated by SRP only and selected to serve as controls. Clinical parameters including PI, GR, BOP, PPD and CAL and biological parameters (IL-1β) in the GCF were measured at baseline, then at, 2 and 6 months after therapy. Results: A statistically significant reduction in several clinical parameters as, BOP (P < 0.001), PI (P < 0.001), PPD (P < 0.001) and CAL (P < 0.001) in quadrant treated with SRP and adjunctive PDT when compared to control group treated with SRP alone was observed and both therapies showed non-statistically significant differences in the reduction of IL-1β level. Conclusion: The inclusion of PDT as an adjunctive measure to nonsurgical conventional periodontal treatment seems to be a useful therapeutic measure in refractory periodontitis treatment.

Survival study on teeth after successful endodontic surgical retreatment: influence of crown height, root length, crown-to-root ratio and tooth type

Aim: To assess the influence of the crown height, root length, crown-to-root ratio, and tooth type on the survival of teeth subjected to surgical endodontic retreatment and classified as periapically healed. Methodology: A single operator performed endodontic microsurgery interventions between 2008 and 2018 on teeth with refractory apical periodontitis. The present analysis selected the teeth classified as ‘‘complete periapical healing’’ according to the scale suggested by Molven. The postoperative periapical radiographs and those taken at the last recall visit were analysed by two independent calibrated examiners, who measured crown height and root length in a blind manner. The crown-to-root ratio was calculated as the ratio of the two variables. The level of inter- and intra-operator agreement was tested with Bland—Altman plots with 95% limits of agreement. An independent statistician conducted a survival analysis using Kaplan—Meier plots and a log-rank test (a = 0.05) to assess the significance of the differences among the subgroups defined by the following criteria: (a) crown height median; (b) root length median; (c) crown-to-root ratio &lt;1 vs. &gt;1; (d) crown-to-root ratio median; (e) single-rooted teeth vs. multi-rooted teeth. Results: At the end of the analysis, 42 patients were evaluated, each one contributing to the study with a single tooth. The mean follow-up period was 4.2 2.4 years. Survival estimates were significantly improved for the teeth with roots longer than 8 mm, in comparison with that with shorter roots ( p &lt; 0.05). There were no statistically significant differences among the remaining considered subgroups. Conclusions: Under the conditions of this retrospective study, teeth with longer residual roots after apical surgery exhibited better chances of survival when compared to teeth with roots shorter than 8 mm. The other considered variables did not seem to affect the survival of apically resected teeth.

Lipoteichoic acids of lactobacilli inhibit Enterococcus faecalis biofilm formation and disrupt the preformed biofilm.

Enterococcus faecalis, a Gram-positive bacterium commonly isolated in patients with refractory apical periodontitis, invades dentin tubules easily and forms biofilms. Bacteria in biofilms, which contribute to recurrent and/or chronic inflammatory diseases, are more resistant to antimicrobial agents than planktonic cells and easily avoid phagocytosis. Although Lactobacillus plantarum lipoteichoic acid (Lp.LTA) is associated with biofilm formation, the effect of Lp.LTA on biofilm formation by E. faecalis is not clearly understood. In this study, we investigated whether Lp.LTA inhibits E. faecalis biofilm formation. The degree of biofilm formation was determined by using crystal violet assay and LIVE/DEAD bacteria staining. The quantification of bacterial growth was determined by measuring the optical density at 600 nm with a spectrophotometer. Formation of biofilms on human dentin slices was observed under a scanning electron microscope. E. faecalis biofilm formation was reduced by Lp.LTA treatment in a dose-dependent manner. Lp.LTA inhibited biofilm development of E. faecalis at the early stage without affecting bacterial growth. LTA from other Lactobacillus species such as Lactobacillus acidophilus, Lactobacillus casei, or Lactobacillus rhamnosus GG also inhibited E. faecalis biofilm formation. In particular, among LTAs from various lactobacilli, Lp.LTA showed the highest inhibitory effect on biofilms formed by E. faecalis. Interestingly, LTAs from lactobacilli could remove the biofilm preformed by E. faecalis. These inhibitory effects were also observed on the surface of human dentin slices. In conclusion, Lactobacillus species LTA inhibits biofilm formation caused by E. faecalis and it could be used as an anti-biofilm agent for prevention or treatment against E. faecalis-associated diseases.

An investigation into the synergistic relationship between lactoferrin and azithromycin with particular reference to periodontopathic bacteria

Abstract The development of treatment strategies for periodontitis that maximise the effectiveness of antibiotics is highly desirable. Azithromycin is proving to be an effective antibiotic for treatment of refractory periodontitis which works by binding to the outer membrane of Gram-negative bacteria and subsequently inhibits protein synthesis. Lactoferrin is a membrane-active host antimicrobial protein and so the objective of this study was to determine whether the effect of azithromycin (AZM) against example periodontopathogens (Porphyromonas gingivalis and Tannerella forsythia) could be potentiated by lactoferrin. Two strains of P. gingivalis and T. forsythia were exposed to lactoferrin (LF; up to 10 mg/ml) and AZM (up to 5 µg/ml) for 0 – 72 h. The MICs for AZM were established using E-Test strips and by agar diffusion. Susceptibility to LF and LF + AZM was evaluated using diffusion assays, with and without iron depletion. The range of MIC values of AZM for P. gingivalis strains and T. forsythia was 0.16 - 0.63 µg/ml and 0.50 - 0.63 µg/ml, respectively. However, no inhibition was observed with iron saturated lactoferrin at any concentration or under iron depletion conditions nor was any effect observed on the AZM MIC by its presence. P. gingivalis and T. forsythia were inhibited by AZM but were not affected by Lf and there was no synergism between AZM and Lf.