The expression levels of all the tested genes for real-time RT-PCR were Akt activation normalized using the 16S rRNA gene of S. mutans (Acc. No. X58303) as an internal standard (Additional file 2, Table S1). Each assay was performed with
at least two independent RNA samples in duplicate. Autoinducer-2 (AI-2) assay It has been suggested [27, 28] that AI-2 signaling may play an important role in the biofilm formation of S. mutans. It is conceivable that, the challenge of stressful condition during the transition to a new surface may alter the quorum sensing (QS) process in the bacteria. Consequently, we tested the secretion of AI-2 signal molecule by S. mutans immobilized in biofilms formed on the different surfaces to determine the impact of the tested material surfaces on the physiology of the attached bacteria. The AI-2 luminescence reporter assay was performed  to check details detect AI-2 secretion levels, in cell-free conditioned
Salubrinal concentration medium of S. mutans biofilms formed on the four tested surfaces. At the end of the biofilm incubation period, a supernatant fluid was collected and filtered through a 0.22 μm-pore size filter (Millipore). The cell-free conditioned medium was either used immediately or stored at -20°C. To determine the amount of AI-2, an overnight culture of Vibrio harveyi MM77, a mutant strain which does not produce either AI-1 nor AI-2, was diluted 1:5,000 in a mixture of 90% (v/v) fresh AB medium and 10% (v/v) conditioned medium to a total volume of 200 μl per well. The negative control contained bacteria in fresh AB medium alone and the positive control Tideglusib contained bacteria, fresh AB medium and 10% v/v spent medium containing AI-2 of V. harveyi BB152 (AI-1-, AI-2+). Readings were performed in triplicate in white 96-well plates with an optic bottom (NUNC) in a GENios reader (TECAN) at 30°C. Luminescence measurements were recorded every 30 min in parallel with optical density absorbance (A 595) readings. The value of each reading
(biofilm on various materials) was divided by the absorbance values to normalize the luminescence value of each sample to its cell density and to avoid dissimilarities caused by differences in growth rates. Fold induction above the non-specific luminescence background of the negative control was determined at the end of bacterial growth after approximately 15 hrs of growth. Fold induction in luminescence of each sample was normalized by the value of total fluorescence of live bacteria within the relevant biofilm as detected by CLSM. Results Using DNA-microarray technology we identified the differentially expressed genes of S. mutans (Figure 1), reflecting the physiological state of biofilms formed on the different biomaterials tested. An empirical Bayesian method (B-test) was applied to test for differential expression in biofilms on various surfaces.