The Combined Application Of Methylene Blue-mediated Photodynamic Therapy And MTAN To Inhibit Fusobacterium Nucleoli And Its Biofilm In Vitro

ObjectiveTo study the inhibitory effect of methylene blue-mediated photodynamic therapy combined with MTAN on Fusobacterium nucleatum and its biofilm.Methods1 MBC(minimum bactericidal concentration)was obtained by plate counting with different concentrations of MTAN on the suspension of Fusobacterium nucleatum.MBC was obtained by plate counting with different concentrations of MB-PDT(methylene blue mediated photodynamic therapy)on the suspension of Fusobacterium nucleatum.SPSS26.0 software was used for statistical analysis of the results obtained from counting,and α=0.05 was taken as the test level.2 On the basis of the MBC of MTAN and MB-PDT acting separately,the two methods were combined in different order(MTAN treatment or MB-PDT treatment first)to treat the suspension of Fusobacterium nucleatum,and the MBC was obtained by plate counting.SPSS26.0 software was used for statistical analysis of the results obtained from counting,and α=0.05 was taken as the test level.3 On the basis of MBC obtained by MB-PDT and MTAN,MB-PDT and MTAN were combined to treat the biofilm containing Fusobacterium nuclei on titanium plates.The morphological changes of the biofilm were observed under scanning electron microscopy,and the stained biofilm was observed under laser scanning confocal microscopy.The proportion of dead bacteria was calculated by ImageJ software.SPSS26.0 software was used for statistical analysis of the calculated results,and α=0.05 was taken as the test level.Results1 Both MTAN and MB-PDT could inhibit the the suspension of Fusobacterium nucleatum respectively.The antimicrobial activity of MTAN decreased with the concentration decreasing,and the MBC was 4000μg/mL.The bacteriostatic effect of MB-PDT varied with the concentration of MB.When the concentration of MB was 97.6563μg/mL-1.5259μg/mL,the antibacterial effect was weakened,and the MBC was 97.6563μg/mL.2 When MB-PDT and MTAN were combined,MB-PDT treatment followed by MTAN treatment produced better antimicrobial effect than MB-PDT and MTAN treatment alone,the concentration of MBC was 1.5625μg/mL and the concentration of MTAN was 62.5μg/mL.3 MB-PDT treatment followed by MTAN treatment can be used to inhibit the biofilm of Fusobacterium nucleatum.It was found under scanning electron microscope that the higher the concentration of MB-MTAN,the more serious the damage of the biofilm of Fusobacterium nucleatum.Confocal scanning laser microscopy showed that the higher the concentration of MB and MTAN,the higher the proportion of dead bacteria with Fusobacterium nucleoli biofilm was,and the MBC was MB 6.25μg/mL and MTAN 250μg/mLConclusionCombined application of MB-PDT and MTAN can effectively inhibit Fusobacterium nucleatum and its biofilm.In addition,MB-PDT treatment followed by MTAN treatment had better bacteriostatic effect,and its MBC was lower than MBC treatment alone.After MB-PDT treatment and then MTAN treatment,the MBC concentration of Fusobacterium nucleatum suspension was 1.5625μg/mL MB,MTAN concentration was 62.5μg/mL,and the MBC concentration of Fusobacterium nucleoli biofilm was 6.25μg/mL MB,and 250μg/mL MTAN.