Strains and culture conditions
Enterococcus faecalis ATCC 29212 strain was provided by the State Key Laboratory of Oral Diseases (China) and stored at -80°C. The Rnc gene sequence was acquired from NCBI (Gene ID: 60892348). The rnc overexpression recombinant plasmid was designed and synthesized by adding promoters upstream of the rnc gene and cloning them into a spectinomycin-resistant shuttle vector pDL278. The recombinant plasmids were transformed into the E. faecalis ATCC 29212 strain through the chemical transformation method using 1µg/mL competence-stimulating peptides (CSP) and the rnc overexpression mutant strain (rnc+) was established . In order to establish the rnc low-expression mutant strains (rnc-), the reverse complementary sequences of rnc were designed and introduced into the pDL278 vector with promoter sequences [21–23]. Then, the plasmids were transformed into E. faecalis ATCC 29212 strain similar to that of rnc + strains. The strains were cultured in brain heart infusion (BHI) broth (Difco, Detroit. MI. USA) at 37°C under anaerobic conditions (80% N2, 10% H2, 10% CO2). Spectinomycin was added to BHI plates with a concentration of 1 mg/mL to select the rnc + and rnc- strains as needed.
Growth curve measurement
A single colony of each of the three strains was inoculated into the BHI medium and incubated in anaerobic conditions overnight. Then, the cultures were diluted to 1:20 with BHI medium and grown under anaerobic conditions until the cells reached the mid-log phase (OD600nm = 0.3–0.5) with constant turbidity in each group. The bacterial suspensions were transferred into sterile 96-well microtiter plates at a dilution of 1:100 and covered with sterile mineral oil in each well. Then, the growth of the strains was recorded using a monitoring system (BioTek, USA) for 24 h. Six biological replicates were used for each group in this study.
Biofilm structure imaging and analysis
Scanning electron microscopy (SEM) was used to detect the structures of E. faecalis biofilms. The E. faecalis ATCC 29212 parent and mutant strains in their mid-log phases (OD600nm = 0.3–0.5) were diluted to 1:100 with the BHI medium supplemented with 1% sucrose (BHIS). Bacterial suspensions were then transferred into a 12-well plate (2 mL per well), containing a round glass slide (14 mm in diameter). After 24 h of incubation, the biofilms were gently washed using phosphate buffered saline (PBS), and 2 mL of 2.5% glutaraldehyde was added to each well. The samples were then stored at 4°C overnight. The biofilm samples of each group were serially dehydrated with 30%, 50%, 75%, 85%, 95%, 99% ethanol (v/v) for 15 min each time. There were three biological replicates for each group, which were examined at 1,000×, 5,000×, and 20,000× magnifications using SEM (Inspect Hillsboro, OR, USA).
Confocal laser scanning microscopy (CLSM) was performed to acquire fluorescence images and to determine the EPS/bacteria composition of E. faecalis biofilms. EPS was stained with Alexa Fluor® 647 (Invitrogen, Eugene, OR, USA), and bacteria cells were stained with Syto 9 Nucleic Acid Stain (Invitrogen, Eugene, OR, USA). CLSM (OLYMPUS, JAPAN) in order to observe the fluorescence images under a 20× objective lens. There were three biological replicates for each group, which were observed under three random observation fields. The three-dimensional biofilm images were reconstructed and the EPS/bacteria ratio was analyzed using Imaris 7.0.0 software. (Bitplane, Zurich, Switzerland).
Crystal violet assay
Crystal violet assay was performed to quantitatively analyze the EPS matrix of biofilms. The biofilms of E. faecalis ATCC 29212 parent and mutant strains were incubated for 24 h at 37°C under anaerobic conditions. After gently washing out the planktonic cells twice using PBS, 200 µL of 0.01% crystal violet (v/v) was added to each sample at room temperature for 10 min. After the careful removal of residual dye with running water, 33% acetic acid (v/v) was used to elute crystal violet, 37°C, 150 rpm, 5 minutes. The OD575 values of the eluents were recorded. In order to evaluate the ability of drugs to remove E. faecalis biofilm EPS, 1 mL 5% NaOCl (v/v), 2%CHX (w/v), Pudilan (Pudilan keyanning antibacterial mouthwash, China), or PBS were added respectively to the biofilm samples and incubated for 10 min. Then, the drugs were gently washed using PBS. The procedures of crystal violet assays were the same as mentioned above.
Detection of gene expression level
Total RNA was extracted from the mid-log phase bacteria using a MasterPure™ RNA purification Kit (Epicentre) following the manufacturer’s instructions. NanoDrop™ 2000c Spectrophotometer (Thermo Scientific, USA) was used to measure the concentration and purity of total extracted RNA. PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time) (Takara, JAPAN) was used for the removal of genomic DNA and reverse transcription of RNA to cDNA. Quantitative real-time-PCR (RT-qPCR) was performed using LightCycler 480 (Roche, Switzerland). 16S rRNA was used as an internal standard and the relative expression level of the rnc gene was quantified using the 2−ΔΔCT method. The RT-qPCR primer sequences are listed in Table 1.
The 24-h E. faecalis ATCC 29212 parent and mutant biofilm samples were prepared and the planktonic bacteria were removed using PBS. All the biofilms were incubated with 2% CHX, Chinese medicine agents, and distilled water for 10 min. Then, the drugs were washed gently. PBS solution (1 µL) was added to each sample to form a uniform bacteria suspension. Sterile PBS buffer was used for the serial dilution of bacterial suspension to 105 folds. After mixing, 100 µL diluted bacterial suspension was spread on BHI plate. The number of colonies at 105 folds was counted after incubation for 48 h. Each group had three biological replicates.
Data analyses were performed using SPSS 26.0 (SPSS Inc., Chicago, IL, USA). One-way ANOVA method was used to identify the significance of variables’ effects. Fisher's least significant difference was performed to compare the means of each group. A P-value < 0.05 was considered statistically significant.