| Background:In the mouth there are a number of microbial species and the oral cavity provides a unique habitat for colonization of amount of various kinds of microbial species.As a Gram-negative oral anaerobe,P.gingivalis is an opportunistic human pathogen that is strongly associated with periodontal disease and peri-implantitis.Biofilm adhesion-mediated infection is regarded of a contributing factor to the occurrence of periodontitis and peri-implantitis.P.gingivalis has specialized virulence,there have been several studies demonstrating that the P.gingivalis which resides within biofilms is one of the main causative factors in the pathogenesis of implant failures.As the extracellular matrices protect biofilm-associated bacteria from antimicrobials and the host immune system,biofilm-derived bacteria are inherently more tolerant to antimicrobial treat.Thus,discovery of new agents to prevent biofilm formation and/or disrupt established biofilms is of considerable interest for the prevention and treatment of peri-implantitis.D-Amino acids are fundamental in microbial physiology where they are key constituents of the peptidoglycan(PG),an essential part of the bacterial cell wall.D-amino acids are synthesized and released by bacteria from diverse phyla,at up to millimolar concentrations.Released D-amino acids function to regulate cell wall chemistry and architecture as well as biofilm development in bacteria.The findings of some studies have been showed that not only pure D-amino acid but also D-amino acid mixture can effectively trigger biofilm disassembly and be active in inhibiting bacterial biofilm formation.For instance,a mixture of D-amino acids could prevent biofilm formation and trigger disassembly of already existing biofilms in Bacillus subtilis;Biofilm formation was inhibited in A.baumannii and in P.aeruginosa by D-amino acids;The growth of S.aureus and S.aureus biofilm formation were efficiently inhibited by either D-Proline r,D-Tyrosine or D-Phenylalanine,while a mixture of these three D-amino acids was also able to disassemble already existing S.aureus biofilms.However,the mechanism of action of D-amino acids on biofilms has not been completely solved at this stage.It has been proposed that D-amino acids inhibit biofilm formation by inhibiting bacterial adhesion,reducing the content of extracellular matrix,regulating the bacterial motility,interfering with the formation of proteins and recombinant bacterial cell walls.In the recent years,the relationship of D-amino acids and bacterial biofilm as well as the idea of using D-amino acids to combat biofilm adhesion-mediated infections have been getting attention and are studied by many scholars.To date,there has been not any studies demonstrated that D-His displays specific inhibition of biofilm formation against strains of P.gingivalis and has a specific capability of triggering the existing P.gingivalis biofilm disassembly.Objectives:The aim of this study is to evaluate the effects of D-Histidine for the biofilm formation and dispersion of the P.gingivalis.And hope the results will provide further rationale for future clinical drug treatment of periodontitis and peri-implantitisMethods:P.gingivalis ATCC 33277 Strain was used in this study and was cultured in agar plate,next a single P.gingivalis colony was inoculated into BHI broth liquid medium for growth.Antimicrobial activity testing of D-His was evaluated by determining the.The effects of D-His on the viability of P.gingivalis was assessed by growth curves and MTT staining method.Crystal violet biofilm biomass assay was used to evaluate the potential activity of D-His effectively having impact on biofilm biomass.Polysaccharide measurement was performed to evaluate the influence of D-His on the water-insoluble extracellular polymeric substance(EPS)of P.gingivalis biofilm.The scanning electron microscopy(SEM)examination was used to observe morphology of P.gingivalis and the structure of biofilm.Confocal laser scanning microscopy(CLSM)imagines were used to observe that whether D-His was able to kill the P.gingivalis in the developing biofilm and mature biofilm.Results:1.D-His at concentrations≥1%could have significant effects on the viability and metabolism of P.gingivalis.2.D-His effectively inhibited P.gingivalis biofilm formation2.1 D-His at concentrations≥10%effectively inhibited P.gingivalis biofilm biomass and disrupted the ability of P.gingivalis to synthesize water-insoluble EPS in a dose-dependent manner.2.2 Scanning electron microscopy observations showed that 25%、50%、75%、100%D-Histidine treated groups,the bacterial aggregation amount declined with increasing concentrations of D-His,and bacteria also didn’t connect to the pellicle,the presence of extravasated bacterial components.2.3 The Confocal laser scanning microscopy(CLSM)was used to observe thegingivalis with the presence of D-His.50%、75%、100%D-Histidine treated groups the bacterial adhesion and aggregation amount were affected by exposure to D_Histidine and the number of both of live bacteria and dead bacteria dramatically reduced.when treated D-His at concentration of 10%to 25%,the two groups had mainly live bacteria and P.gingivalis also formed large aggregates,however these aggregates can’t subsequently connect to the pellicle3.Biofilm-inhibitory concentrations of D-His could trigger mature P.gingivalis biofilm disassembly.3.1 Treating the existing.gingivalis biofilms with 25%、50%、75%and 100%D-His caused a concentration-dependent reduction of the biomass values and polysaccharide amounts.3.2 When D-His was at 25%、50%、75%and 100%,these data demonstrated that D-His resulted in effective disassembly of mature P.gingivalis biofilm,and the number of dead bacteria was also increased.Conclusions:1.The low concentrations of D-His can inhibit the viability and metabolism of P.gingivalis and effectively inhibits the growth of planktonic P.gingivalis in a dose-dependent manner.2.D-His effectively inhibits P.gingivalis biofilm formation and biofilm-inhibitory concentrations of D-Histidine has potent activity at triggering existing P.gingivalis biofilm. |
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