| Background:In nature,bacteria exist mainly in both floating and biofilm forms and can convert to each other in order to adapt the environment in these two states,forming a cyclical cycle of bacterial biofilm.Unlike planktonic bacteria,when bacteria grow as biofilms,they have spontaneous resistance to antibacterial agents,host defense mechanisms,and the like,and their metabolic activities and gene expression are also different.Compared to planktonic,when gingivalis grows as a biofilm,about 18%of the genome is differentially expressed.Clinically,5 to 10 years after implant placement,10%of implants and 20%of implanted patients develop peri-implantitis.Implant failures due to inflammation around the implant accounted for 30%of total failures.As a typical biofilm structure,plaque biofilm is the initiating factor for the occurrence and/or development of inflammation around implants.The pathogenic flora that most closely related to inflammation around the implant is called the "red complex" and includes:P.gingivalis,Fusobacterium nucleatum and P.intermedia.Among them,P gingivalis accounts for up to 50%of the "red complex" of the peri-implantitis model,so P.gingivalis is considered to be the main pathogen of inflammation around the implant.However,there is currently no clinically effective method for treating inflammation around the implant without damaging the implant or surrounding tissueAlmost all bacteria form cell walls outside the cell membrane.As a layer of network elastic structure,the cell wall strongly resists external pressure and maintains cell morphology as well as acts as an effective barrier against bacterial,chemical,and biological threats.In Gram-negative bacteria and Gram-positive bacteria,the bacterial cell wall is composed of a peptidoglycan polymer that main component is peptidoglycan.As an important cell wall structure of bacteria,peptidoglycan maintains the structural integrity of cells and reduces mechanical and osmotic damage during cell growth.On the other hand,it can assist cell growth,division and bacterial flagellar movement.The chemical structure of peptidoglycan is dynamic,and many substances exert antibacterial or bactericidal action by destroying peptidoglycan structure or inhibiting peptidoglycan synthesis,such as lysozyme acting on N-acetylmuramic acid,penicillin inhibition Peptidase activity inhibits peptide bridge formation.Peptidoglycans are often modified when bacterial growth enters a stationary phase.D-amino acids and L-amino acids are enantiomeric chiral molecules,have similar physicochemical properties and characteristics,and may have similar or opposite properties in biological functions.The L-amino acid,which plays a leading role in various life activities,inhibits the movement of P.aeruginosa and promotes the formation of biofilm by increasing the level of intracellular c-di-GMP.In recent years,research on D-amino acids has received extensive attention.Kolodkin-Gal and other scholars have found that supernatant extracts from Bacillus subtilis biofilms contain D-tyrosine,D-leucine,D-methionine and D-tryptophan.These extracts are on the one hand.It is possible to inhibit the formation of biofilms and,on the other hand,to promote the dispersion of mature biofilms.Different types of D-amino acids have different effects on different types of bacterial biofilms.In Escherichia coli,Agrobacterium tumefaciens and Bacillus subtilis,D-amino acids inhibit the formation of bacterial biofilms by integration into the fourth or fifth position of the cell wall peptide.However,none of the L-amino acids can remodel bacterial cell walls.At present,the research on D-amino acid on microbial biofilm mainly focuses on Bacillus subtilis and Staphylococcus aureus;however,P.gingivalis is the main pathogen of the occurrence and development of inflammation around implants,there are few studies on the effects of D-amino acids on the biofilm of P.gingivalis.Objectives:In this study,we investigated the effects of D-phenylalanine on the biofilm of P.gingivalis,the main pathogen of periodontal disease,and explored the effective D-phenylalanine concentrations on the biofilm formation of P.gingivalis.Inhibition and the interaction between D-phenylalanine and its enantiomer L-phenylalanine.The experimental results could provide a theoretical basis for the new clinical treatment strategy of peri-implantitis,and it is expected to control the occurrence and development of inflammation around implants and maintain the long-term stability of implants.Methods:In this experiment,a saturated solution of D-phenylalanine was prepared and diluted to 100%,75%,50%,25%,10%,1%,and 0.1%of the saturated concentration,respectively.The P.gingivalis broth was co-cultured with a gradient concentration of D-phenylalanine solution for 72 hours.The effect of D-phenylalanine on the biofilm biomass of P.gingivalis was detected by crystal violet staining.The extracellular polysaccharide of D-phenylalanine on the biofilm of P.gingivalis was detected by phenol-sulfuric acid method.The effect of the content and the effect of D-phenylalanine on the biofilm morphology of P.gingivalis was observed by scanning electron microscopy and laser confocal microscopy.According to the results obtained in the above experiments,the L-phenylalanine solutions of the same concentration as the effective concentration of D-phenylalanine solutions were used for living dead bacteria staining,crystal violet staining,SEM observation of biofilm,phenol-sulfuric acid method.Experiments were conducted to confirm the interaction of D-phenylalanine with its chiral molecule L-phenylalanine in this study.Results:Compared with the control group,when the concentration of D-phenylalanine solution was≥80 mM,the biomass of P.gingivals ATCC 33277 bacterial biofilm was statistically different(P<0.05);the biomass of P.gingivals ATCC 33277 bacterial biofilm was not significantly different from the control group when it was among 0.16 mM and 40 mM(P>0.05).When the concentration of D-phenylalanine solution was>120 mM,the yield of extracellular polysaccharide of P.gingivals ATCC 33277 was statistically different(P<0.05);when the concentration of D-phenylalanine solution was among 0.16 mM and 80 mM.The yield of extracellular polysaccharide of P.gingivals ATCC 33277 was not significantly different from that of the control group(P>0.05).As the concentration of the added D-phenylalanine solution increased,the morphology of the biofilm observed under the scanning electron microscope of P.gingivals ATCC 33277 changed from a dense multilayer network to a sparsely formed single bacteria.With the increase of the concentration of D-phenylalanine solution added the amounts of bacteria in P.gingivals ATCC 33277 stained by live bacteria decreased significantly,and the proportion of dead bacteria increased corresponding to the concentration of L-phenylalanine solution and D-phenylalanine.The corresponding concentrations of L-phenylalanine solution and D-phenylalanine have similar effects on P.gingivals ATCC 33277 in terms of biofilm volume,bacterial activity and biofilm morphology.Conclusions:(1)The concentration of D-phenylalanine≥80 mM has an inhibitory effect on the formation of P.gingivals ATCC 33277 biofilm.(2)When D-phenylalanine concentration≥120 mM,the production of extracellular polysaccharide of P.gingivals ATCC 33277 can be reduced.(3)L-phenylalanine has a similar effect to the corresponding concentration of D-phenylalanine in inhibiting the formation of P.gingivalis ATCC 33277 biofilm;L-phenylalanine inhibits the effect of D-phenylalanine on extracellular polysaccharides... |
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