Figure 1 PCR optimization of nox (A) and fbp (B) genes. To confirm the amplification of nox and fbp genes from each strain, PCR and agarose gel electrophoresis were performed prior to RT-PCR experiments. bp, base pair; Ga, G. adiacens; Ge, G. elegans; Ad, A. defectiva; Sm, S. mutans; Sg, S. gordonii.

Figure 2 Confocal microscopy and crystal violet staining of monospecies biofilms of five oral streptococcal species. For confocal microscopy, bacteria were cultured in brucella broth in the wells of Millicell® EZ slides (Millipore) and stained with Syto9®. Images were acquired on Carl-Zeiss LSM 700 at 630× magnification and 3D view reconstructed using the software ZEN 2012. The bacteria were grown similarly for crystal violet staining except that 24-well cell culture plates (BD Bioscience) were used.

Figure 3 Effect of BITC treatment on the biofilms. The biofilm biomass was quantified by crystal violet staining after treating with BITC for 2 h in 5% CO2 at 37°C. The absorbance was measured at 590 nm wavelength. Ga, G. adiacens; Ge, G. elegans; Ad, A. defectiva; Sm, S. mutans; Sg, S. gordonii.

Figure 4 mRNA expression of nox and fbp genes. Mature biofilms of all strains were treated with BITC (10 μM) for 2 h in 5% CO2 at 37°C. The biofilms were scraped off from the wells, RNA later® was added to the biofilms and preserved at −80°C until used. RNA purified from the samples was converted to cDNA and expression of nox (A) and fbp (B) genes was assessed by Real-time PCR quantification. Ga, G. adiacens; Ge, G. elegans; Ad, A. defectiva; Sm, S. mutans; Sg, S. gordonii; UT, untreated. *P < 0.05.