Bacteria strains and culture conditions
Streptococcus mutans UA159 was commercially obtained from the American Type Culture Collection (ATCC). Bacteria were grown in brain heart infusion broth (BHI; Difco, Sparks, MD, USA) at 37°C in a 5% CO2 atmosphere until the cells reached the mid-logarithmic phase (OD600nm = 0.5). To determine the sub-inhibitory level of hyperosmotic challenge, bacteria were grown in BHI supplemented with 0.05, 0.1, 0.2, 0.4, 0.5, 0.6, 0.8, 1.0 M of sodium chloride respectively. For in vitro biofilm establishment, bacterial cells were grown in BHI supplemented with 1% sucrose (wt/vol).
Bacteria susceptibility assays
The sub-inhibitory concentration of sodium chloride was determined by a microdilution method as described previously . Growth curves of S. mutans UA159 were further constructed by monitoring the optical density (OD600nm) of the cultures for 24 h using a Bioscreen C analyzer (Oy Growth Curves AB Ltd., Finland) . The formation of S. mutans biofilm under increasing concentrations of NaCl was quantified in a 96-well microtiter plate as described previously . Briefly, S. mutans UA159 (1 × 106 CFU/ml) was grown in BHI supplemented with 1% (wt/vol) sucrose and NaCl (0.05 M to 1.0 M) at 37°C for 24 h. The culture supernatant from each well was then decanted, and the adherent biofilm was washed three times with PBS, fixed with methanol for 15 min, and stained with 0.1% (wt/vol) crystal violet (Sigma-Aldrich Corp., St. Louis, MO, USA) for 5 min. Subsequently, the wells were rinsed with deionized water until the blank wells appeared colorless; 200 μl of 95% ethanol was added. The plates were shaken at room temperature for 30 min, and the absorbance at 595 nm was recorded. The short-term effect of hyperosmotic challenge on the pre-established biofilm was also determined by quantification of the biomass of 24 h S. mutans biofilm after exposure to 0.4 M NaCl for 15 min using the same method as described above. All the experiments were performed in three-replicates and the average was calculated.
Biofilm viability assays
24 h pre-established S. mutans biofilms were treated with 0.4 M NaCl for 15 min, gently harvested with a cell scraper, and suspended in PBS to an OD600nm of 1.0. The bacterial cells suspension was then serially diluted and plated in triplicate on BHI agar plates. After 48 hours incubation at 37°C (5% CO2), colony forming unit (CFU) of biofilms was enumerated.
The treated biofilms were also stained with a two-color fluorescence assay kit (LIVE/DEAD BacLight-Bacterial Viability Kit 7012, Invitrogen, Molecular Probes, Inc., Eugene, OR, USA) according to the manufacturer’s instructions. The biofilms images were captured using a Leica TCS SP2 confocal laser scanning microscope (Leica, Germany), and the percentage of viable cells was calculated by Image Pro-Plus 6.0 (Media Cybernetics Inc., Bethesda, MD, USA).
Microbial biofilm configuration
Scanning electron microscopy (SEM) was performed as described previously  to investigate the configuration of S. mutans biofilm under hyperosmotic condition. S. mutans biofilms were either established on glass slides in the presence of 0.4 M of NaCl for 24 h, or pre-established 24 h biofilm on glass slides and then treated with 0.4 M of NaCl for 15 min. Biofilm samples were gently washed two times with sterile PBS to remove planktonic cells and fixed with 2.5% glutaraldehyde at 4°C overnight. The samples then were dehydrated in a graded series of ethanol (50%, 60%, 70%, 80%, 90%, 95% and 100%), dried in a freeze dryer, gold coated and observed under a SEM (FEI, Hillsboro, OR, USA).
The biofilm samples were also double-labeled by the method as described by Koo et al. [27, 28]. In brief, the extracellular polysaccharides matrix of S. mutans biofilm was labeled by incorporating 2.5 μmol l-1 of Alexa Fluor 647-labelled dextran conjugate (10000 MW; absorbance/fluorescence emission maxima of 650/668 nm; Molecular Probes Inc., Eugene, OR, USA) into the newly formed glucan. The bacterial cells in biofilms were labeled by means of SYTO 9 green fluorescent nucleic acid stain (2.5 μmol 1-1, 480/500 nm; Molecular Probes Inc.). The biofilm images were captured using a Leica TCS SP2 confocal laser scanning microscope (Leica, Germany). The confocal image stacks were analyzed by the image-processing software COMSTAT as described previously . The three-dimensional architecture of the biofilms was visualized using AmiraTM5.0.2 (Mercury Computer Systems, Chelmsford, MS, USA).
Mid-logarithmic phase cells of S. mutans (OD600nm = 0.5) were incubated with 0.4 M of NaCl at 37°C for 15 min. Cells were collected and then treated with RNAprotect reagent (Qiagen, Valencia, CA, USA) immediately. Total RNA was extracted using RNeasy Mini kits (Qiagen) as described previously . Rnase-Free DNase Set (Qiagen) was used to remove genome DNA. A Nanodrop ND 1000 spectrophotometer (Thermo Fisher Scientific, Pittsburgh, PA, USA) was used to determine total RNA concentrations, and an Agilent 2100 Bioanalyser (Agilent Technologies, Santa Clara CA, USA) was used to evaluate the RNA quality (see Additional file 2 for RNA quality control). The isolated RNA was stored at −80°C before use.
Streptococcus mutans UA159 (NC004350) NimbleGen Genechip (4*72 K) whole-genome array was employed for transcriptional profiling in this study. The oligoarrays included 1949 S. mutans UA159 open reading frames with twelve 24-mer probe pairs (PM/MM) per gene, and each probe was replicated 3 times. The design also included random GC and other control probes. Array images were scanned by Gene Pix® 4000B Microarray Scanner (Axon Instruments, Union City, CA, USA). Raw data were normalized using RMA algorithm by Roche NimbleScan software version 2.6. We used the average value of each replicate probe as the signal intensity for the corresponding gene, and all the values were log2 transformed for further analysis. The normalized data with annotation information was processed by combining several different R/Bioconductor packages. We conducted a non-parametric statistical method contained in the RanProd package to detect the differentially expressed genes (DEG) . With 100,000 permutation test, genes having a minimum 2-fold change with the false discovery rate (FDR) smaller than 0.1 were considered as DEG, indicating a significant up- or down-regulation under hyperosmotic stress. For the pathway analysis, we firstly constructed the whole S. mutans UA159 pathway database based on the KEGG Pathway. Then gene set enrichment analysis (GSEA) was used to determine the pathways that changed significantly in response to hyperosmotic stress [32, 33].
The microarray results were further validated by quantitative RT-PCR for selected genes (see Additional file 3 for detailed primer sequences for qPCR). Quantitative RT-PCR assays were performed using a SYBR Green reverse transcription-PCR kit (TaKaRa, Dalian, China) according to the manufacturer’s instructions.
We used Student’s T-test to compare the non-treated control groups with treatment groups. All statistical procedures were conducted by R software . Data were considered significantly different if the two-tailed P-value was < 0.05.