Abstract：
Antimicrobial Photodynamic Therapy (aPDT) based on the action of visible light and photosensitizers has emerged as a promising microbial reduction and alternative to antibiotics resistance to cariogenic pathogens. The present research aims to evaluate the antimicrobial effect of aPDT mediated by a new photosensitizer (amino acid porphyrin conjugate 4i) on Streptococcus mutans (S. mutans) biofilm. Qualitative morphologic characteristics of S. mutans biofilms are shown by scanning electron microscopy (SEM). The colony plate counting method is used to measure the dark toxicity and the phototoxicity of different concentrations of 4i-aPDT to S. mutans biofilms. MTT assay is conducted to investigate the effect of 4i mediated aPDT on the metabolic activity of S. mutans biofilm. Changes in structure morphology, bacterial density and extracellular matrix of S. mutans biofilm are observed by SEM. The distribution of living and dead bacteria in biofilm is detected using Confocal laser microscopy (CLSM). The results indicate that single laser irradiation has no antibacterial effect on S. mutans biofilms. With the increase of 4i concentration or the prolongation of laser irradiation time, the antibacterial effect of 4i-mediated aPDT on S. mutans biofilm is more statistically significant compared to the control. When the concentration of 62.5 µmol/L 4i is continuously illuminated for 10 min, the logarithm of the colonies in the biofilm shows a reduction of 3.4 log10. MTT assay detected absorbance values of biofilm by 4i-mediated aPDT are the lowest, indicating a significant decrease in biofilm metabolic activity. SEM analysis shows that 4i mediated aPDT reduced the quantity and density of S. mutans. A dense red fluorescence image of the 4i-aPDT treated biofilm is observed under CLSM, indicating that the dead bacteria are widely distributed.
摘要：
基于可见光和光敏剂作用的抗微生物光动力疗法（aPDT）已经成为一种有希望的微生物减少和替代抗生素对致龋病原体的抗性。本研究旨在评估由新型光敏剂（氨基酸卟啉缀合物4i）介导的aPDT对变异链球菌（S.mutans)生物膜。通过扫描电子显微镜（SEM）显示了变异链球菌生物膜的定性形态特征。菌落平板计数法用于测量不同浓度4i-aPDT对变形链球菌生物膜的暗毒性和光毒性。进行MTT测定以研究4i介导的aPDT对变形链球菌生物膜代谢活性的影响。结构形态的变化，通过SEM观察变异链球菌生物膜的细菌密度和细胞外基质。使用共聚焦激光显微镜（CLSM）检测生物膜中活细菌和死细菌的分布。结果表明，单次激光照射对变形链球菌生物膜没有抗菌作用。随着4i浓度的增加或激光照射时间的延长,与对照相比,4i介导的aPDT对变形链球菌生物膜的抗菌作用更具统计学意义。当浓度为62.5μmol/L的4i连续照射10分钟时，生物膜中菌落的对数显示减少3.4log10。MTT法检测4i介导的aPDT对生物膜的吸光度值最低,表明生物膜代谢活性显著降低。SEM分析显示4i介导的aPDT降低了变形链球菌的数量和密度。在CLSM下观察到4i-aPDT处理的生物膜的密集红色荧光图像，表明死亡细菌分布广泛。
