褪黑素联合盐酸米诺环素对牙周致病菌混合生物膜的抑制作用

Inhibition effect of melatonin combined with minocycline hydrochloride against mixed biofilms of periodontal pathogens

目的:探究褪黑素与盐酸米诺环素联合使用对牙周致病菌混合生物膜的抑制作用,探索牙周抗菌斑药物治疗的新方法。方法:通过微量液体稀释法分别测定褪黑素和盐酸米诺环素对牙周主要致病菌,戈登链球菌、具核梭杆菌、牙龈卟啉单胞菌、伴放线聚集杆菌的最小抑菌浓度(MIC)和最小杀菌浓度(MBC),应用微量棋盘稀释法检测药物联合应用对各致病菌的抑菌浓度分数指数(FICI),采用结晶紫染色法评估药物联合应用对单菌及混合菌生物膜的影响。应用激光共聚焦扫描显微镜评估药物联合应用对混合菌生物膜的抑制及离散作用。结果:褪黑素对戈登链球菌、具核梭杆菌、牙龈卟啉单胞菌、伴放线聚集杆菌均具有显著抑制作用,其MIC值均为1%,MBC值均为2%;对上述混合菌的MIC值为2%,MBC值为4%。褪黑素联合盐酸米诺环素对牙龈卟啉单胞菌和伴放线聚集杆菌生长的抑制表现为协同作用,对戈登链球菌、具核梭杆菌和混合菌悬液均表现为相加作用。与对照组相比,褪黑素与盐酸米诺环素联合应用可显著提高对牙周各致病菌和混合菌种生物膜的抑制和离散效果,其中实验组浓度为1%+1/2MIC时,对单菌种和混合多菌种生物膜的抑制作用提高最显著(P<0.05),当药物浓度为1%+MIC时,对生物膜的离散作用增长最显著(P<0.05),其抑制和清除效果均高于单独应用浓度为2MIC的盐酸米诺环素。结论:褪黑素联合盐酸米诺环素可显著提高对牙周致病菌斑生物膜的抑制能力,有望在牙周炎的辅助治疗中发挥作用。

Objective:To explore the inhibitory effect of melatonin combined with minocycline hydrochloride on mixed biofilm of periodontal pathogens,and to explore new methods for drug treatment of periodontal antimicrobial spots.Methods:The minimal inhibitory concentration(MIC),minimum bactericidal concentration(MBC)of melatonin and minocycline hydrochloride against the main periodontal pathogens.Streptococcus gordon,Fusobacterium nucleatum,Porphyromonas gingivalis,and Actinobacillus actinomycetes were measured by microliquid dilution method,and the bacteriostatic concentration index(FICI)of the combination of drugs on the main periodontal pathogens was detected by microcheckerboard dilution method.The effect of drug combination on biofilm of single and mixed strains was evaluated by crystal violet staining.The inhibition and discrete effects of combined drugs on mixed bacterial biofilms was assessed by confocal laser scanning microscopy.Results:The MIC values of melatonin to Streptococcus gordonii,Fusobacterium nucleatum,Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans were all 1%,MBC values were 2%,MIC values of the above mixed bacteria were 2%,MBC values were 4%.Melatonin combined with minocycline hydrochloride showed a synergistic effect on the growth inhibition of Porphyromonas gingivalis and Actinobacillus actinomycetes,and an additive effect on Streptococcus gordonii,Fusobacterium nucleatum and mixed bacteria suspension.Compared with the control group,the combination of melatonin and minocycline hydrochloride could significantly improve the inhibition and discrete effect of various periodontal pathogens and mixed bacteria.When the experimental groupwas 1%+1/2MIC,the inhibition of single species andmixedmultispecies biofilms increasedmost significantly(P<0.05).When the drug concentration was 1%+MIC,the discrete effect of the biofilm increased significantly(P<0.05),and the inhibition and clearance effect were higher than that of minocycline hydrochloride with a pure concentration of 2MIC.Conclusion:Melatonin combined with minocycline hydrochloride can significantly improve the inhibition ability of periodontal plaque biofilm,which is expected to play a role in the adjuvant treatment of periodontitis.