Abstract: 【Abstract】 Objective To investigate the effect of different incubation time of aminolevulinic acid （ALA） on photodynamic inhibition of Propionibacterium acnes biofilms. Methods Propionibacterium acnes biofilms were formed in 24-well plates with pre-placed cell slides and 96-well plates. The formation of the biofilm structure was observed by confocal laser scanning microscopy （CLSM）, and the growth activity of the biofilm was assessed by the tetrazolium salt XTT assay. The in vitro successfully constructed biofilm models were divided into 6 groups: negative control group receiving neither ALA treatment nor LED radiation, ALA group incubated with ALA alone for 30 minutes, LED group receiving LED radiation alone, ALA-PDT1 group, ALA-PDT2 group and ALA-PDT3 group incubated with ALA for 15, 30 and 60 minutes respectively followed by LED radiation. After the treatment, CLSM was performed to observe the biofilm structure, as well as to determine the dead/living bacteria ratio, and XTT assay to assess the growth activity of the biofilm. Differences among groups were analyzed using one-way analysis of variance and least significant difference-t test. Results CLSM showed that the Propionibacterium acnes biofilm model was successfully constructed in vitro. The dead/living bacteria ratios were 0.90 ± 0.16, 1.75 ± 0.19, and 2.57 ± 0.32 in the ALA-PDT1 group, ALA-PDT2 group and ALA-PDT3 group respectively, which were significantly higher than the dead/living bacteria ratio in the negative control group （0.31 ± 0.01; t = 55.56, 138.62, 74.64, respectively, all P＜0.001）; the biofilm viability value was significantly lower in the ALA-PDT1 group, ALA-PDT2 group and ALA-PDT3 group （0.35 ± 0.02, 0.26 ± 0.02, 0.18 ± 0.01, respectively） than in the negative control group （0.43 ± 0.00; t = 35.66, 2.64, 110.96, respectively, all P < 0.001）. CLSM showed that the structure of the Propionibacterium acnes biofilm was destroyed under the action of ALA-PDT, and the destruction was aggravated with the prolongation of incubation time of ALA. Conclusion The prolongation of incubation time of ALA can enhance the inhibitory effect of ALA-PDT on Propionibacterium acnes biofilms.

Key words: Propionibacterium acnes, Biofilms, Photochemotherapy, Aminolevulinic acid, Incubation time