Inhibition of Steptococcus mutans biofilm formation by extracts of Tenacibaculum sp. 20J, a bacterium with wide-spectrum quorum quenching activity

Abstract

Background: Previous studies have suggested the quorum sensing signal AI-2 as a potential target to prevent the biofilm formation by Streptococcus mutans, a pathogen involved in tooth decay. Objective: To obtain inhibition of biofilm formation by S. mutans by extracts obtained from the marine bacterium Tenacibaculum sp. 20J interfering with the AI-2 quorum sensing system. Design: The AI-2 inhibitory activity was tested with the biosensors Vibrio harveyi BB170 and JMH597. S. mutans ATCC25175 biofilm formation was monitored using impedance real-time measurements with the xCELLigence system®, confocal laser microscopy, and the crystal violet quantification method. Results: The addition of the cell extract from Tenacibaculum sp. 20J reduced biofilm formation in S. mutans ATCC25175 by 40-50% compared to the control without significantly affecting growth. A decrease of almost 40% was also observed in S. oralis DSM20627 and S. dentisani 7747 biofilms. Conclusions: The ability of Tenacibaculum sp. 20J to interfere with AI-2 and inhibit biofilm formation in S. mutans was demonstrated. The results indicate that the inhibition of quorum sensing processes may constitute a suitable strategy for inhibiting dental plaque formation, although additional experiments using mixed biofilm models would be required.

Keywords: AI-2; Quorum sensing; biofilms; dental plaque; quorum quenching; streptococci.

Figure 1.

Effect of QS inhibitors and extracts on bioluminescence production by different V. harveyi reporter strains in 96-well cell culture plates. (A) Bioluminescence assays using V. harveyi BB170 (AHL−, AI2+, CAI+) and V. harveyi JMH597 (AHL−, AI2+, CAI−) with furanone (0.02 µg ml⁻¹), the AHL-lactonase Aii20J (20 μg ml⁻¹), and the purified cell extracts from Tenacibaculum sp. 20J (PCE20J) and T. maritimum (PCETm) (100 μg ml⁻¹). (B) Effect of purified cell extract (PCE) and methanolic cell extract on bioluminescence production by V. harvery BB170, JMH597, and the QS independent strain JAF548. Assays were carried out in triplicate. Only one well is shown for each condition.

Figure 2.

Biofilm formation by Streptococcus mutans as measured by xCELLigence® system (■). The effect of purified cell extracts from Tenacibaculum sp. 20J (∆), T. maritimum NCIMB2154^T (◊) (100 μg ml⁻¹); furanone C30 (○)(0.02 μg ml⁻¹) and the AHL lactonase Aii20J (˟) (20 μg ml⁻¹) were tested (n = 3).

Figure 3.

Effect of cell extracts of Tenacibaculum sp. 20J, T. maritimum (100 μg ml⁻¹) and furanone (0.02 μg ml⁻¹) on biofilm formation (A) and cell growth (B). Data were measured as cell index (biofilm growth) and OD₆₀₀ (cell growth) of S. mutans in comparison to control cultures (100%). Experiments were performed in triplicate.

Figure 4.

Effect of the cell extract of Tenacibaculum sp. 20J (100 μg ml⁻¹) on S. mutans cultures inoculated with different sucrose (0.1% and 0.2%) and glucose (0.1% and 0.2%) concentrations. Data were measured as cell index (biofilm growth) of S. mutans in comparison to control cultures (100%). Experiments were performed in triplicate.

Figure 5.

Confocal microscope observation (63 ×) of the effect of the addition of the cell extract if strain 20J (PCE20J) on biofilm formation by S. mutans. The highest and the lowest covered surface fields among the eight fields analysed per sample are shown. A reduction of at least 50% of covered surface is observed in the presence of PCE20J.

Figure 6.

Biofilm formation quantified by crystal violet staining by S. mutans, S. oralis, and S. dentisani in absence or presence of the cell extract of strain 20J (PCE20J). Experiments were performed in triplicate.

Figure 7.

Biofilm formation measured by xCELLigence® (A) and cell growth (B). Measures were done as cell index (biofilm growth) and OD₆₀₀ (cell growth) of S. mutans in comparison to control cultures (100%). Different types of extracts from Tenacibaculum sp. 20J obtained using different solvents such as methanol (MCE), ethylacetate (EA-PCE) and dichlorometane (DM-MCE20J), and dialyzed PCE and ultracentrifuged PCE were tested as anti-biofilm compounds (n = 3).

Figure 8.

Effect of different treatments on the anti-biofilm activity of the cell extract of strain 20J (100 μg ml⁻¹) against S. mutans measured by xCELLigence® system (■). (A) Activity of extracts fractionated by molecular weight <100 kDa (□), the <50 kDa (○), and the <10 kDa (◊). (B) Effect of temperature (●) (100°C, 10 min) and treatment with proteinase K (▲)(1 h at 30°C) on the activity of the PCE. All cultures were carried out in triplicate.