Inhibition of biofilm formation by Limosilactobacillus fermentum supernatant against Porphyromonas gingivalis and Fusobacterium nucleatum: an in-vitro study.

BACKGROUND

This study aims to investigate the inhibitory effect of the supernatant of Limosilactobacillus fermentum CCFM1139 on the LuxS/AI-2 quorum sensing system in dual-species biofilms of Porphyromonas gingivalis and Fusobacterium nucleatum, and its anti-biofilm formation efficacy.

METHODS

Construct an in vitro dual-species biofilm model of P. gingivalis and F. nucleatum; after adding the supernatant of L. fermentum CCFM1139 at four stages of biofilm formation (0 h, 12 h, 24 h, 36 h), the following experiments were conducted: (1) Biofilm mass changes were assessed using crystal violet staining; (2) Morphological changes were observed via scanning electron microscopy (SEM); (3) Quantitative Real-Time Reverse Transcription Polymerase Chain Reaction(qRT-PCR) was employed to detect luxS gene expression levels in the biofilm; (4) AI-2 signaling molecule activity was quantified using Vibrio harveyi BB170 bioluminescence assay.

RESULTS

The supernatant of L. fermentum CCFM1139 significantly inhibited the formation of the dual-bacterial biofilm in a time-dependent manner: the inhibition rate peaked at 0 h (65.54%), followed by 60.20% at 12 h, decreasing to 47.09% at 24 h and 26.67% at 36 h. The inhibitory effect during the early phase (0-12 h) was significantly superior to that in the late phase (24-36 h) (p < 0.05). SEM observation revealed that the experimental group biofilm exhibited a loose monolayer structure with increased bacterial spacing, whereas the control group displayed a dense multilayer structure with tightly connected bacteria. qRT-PCR results indicated that the supernatant downregulated luxS gene expression in both P. gingivalis and F. nucleatum: inhibition of P. gingivalis was primarily observed between 0 and 24 h, while inhibition of F. nucleatum was stronger and more persistent. The Vibrio harveyi BB170 bioluminescence assay revealed that the supernatant from L. fermentum CCFM1139 significantly reduced the activity of the AI-2 signaling molecule in the dual-bacterial biofilm (p < 0.05).

CONCLUSION

The supernatant of L. fermentum CCFM1139 effectively reduced biofilm formation by suppressing the LuxS/AI-2 quorum sensing system in P. gingivalis and F. nucleatum, demonstrating its potential as an antimicrobial agent.