Enterococcus faecalis rnc gene modulates its susceptibility to disinfection agents: a novel approach against biofilm

Background Enterococcus faecalis (E. faecalis) plays an important role in the failure of root canal treatment and refractory periapical periodontitis. As an important virulence factor of E. faecalis, extracellular polysaccharide (EPS) serves as a matrix to wrap bacteria and form biofilms. The homologous rnc gene, encoding Ribonuclease III, has been reported as a regulator of EPS synthesis. In order to develop novel anti-biofilm targets, we investigated the effects of the rnc gene on the biological characteristics of E. faecalis, and compared the biofilm tolerance towards the typical root canal irrigation agents and traditional Chinese medicine fluid Pudilan. Methods E. faecalis rnc gene overexpression (rnc+) and low-expression (rnc−) strains were constructed. The growth curves of E. faecalis ATCC29212, rnc+, and rnc− strains were obtained to study the regulatory effect of the rnc gene on E. faecalis. Scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM), and crystal violet staining assays were performed to evaluate the morphology and composition of E. faecalis biofilms. Furthermore, the wild-type and mutant biofilms were treated with 5% sodium hypochlorite (NaOCl), 2% chlorhexidine (CHX), and Pudilan. The residual viabilities of E. faecalis biofilms were evaluated using crystal violet staining and colony counting assays. Results The results demonstrated that the rnc gene could promote bacterial growth and EPS synthesis, causing the EPS-barren biofilm morphology and low EPS/bacteria ratio. Both the rnc+ and rnc− biofilms showed increased susceptibility to the root canal irrigation agents. The 5% NaOCl group showed the highest biofilm removing effect followed by Pudilan and 2% CHX. The colony counting results showed almost complete removal of bacteria in the 5% NaOCl, 2% CHX, and Chinese medicine agents’ groups. Conclusions This study concluded that the rnc gene could positively regulate bacterial proliferation, EPS synthesis, and biofilm formation in E. faecalis. The rnc mutation caused an increase in the disinfectant sensitivity of biofilm, indicating a potential anti-biofilm target. In addition, Pudilan exhibited an excellent ability to remove E. faecalis biofilm. Supplementary Information The online version contains supplementary material available at 10.1186/s12903-022-02462-1.

Recently, E. faecalis has been paid more attention due to its dominant role in the formation of extra radicular biofilm and periapical lesions [6]. According to the study by Barbosa-Ribeiro et al., E. faecalis was the most abundant bacteria in the teeth with endodontic treatment failure and was also associated with the periapical lesions of over 3-mm size [7]. It colonizes the biofilms, invades the dentinal tubules, and resists nutritional deprivation, thereby causing therapeutic failure and heavy economic burdens [4,8].
E. faecalis is a Gram-positive coccus, which is homologous with the dental caries pathogen Streptococcus mutans (S. mutans). The formation of biofilms results in the adhesion and aggregation of bacteria cells as well as increased resistance to root canal irrigants. The VicRK two-component signal transduction system is a key regulator in the synthesis of exopolysaccharide (EPS) in S. mutans. A previous study reported that the rnc gene, encoding ribonuclease III (RNase III), could promote the EPS synthesis and alter the morphology of biofilm [9]. However, the rnc gene function has rarely been detected in E. faecalis. Our previous study showed that rnc could repress vicRKX expressions at the post-transcriptional level via microRNA-size small RNAs (msRNAs) [10]. The WalRK signal transduction system in E. faecalis, which is homologous to VicRK, could also regulate EPS synthesis. It was reported that inhibiting the biofilm formation-related gene walR could reduce EPS synthesis and enhance the susceptibility of E. faecalis biofilms to chlorhexidine (CHX) [11]. Therefore, regulating the metabolism of biofilms might be a feasible way for eliminating E. faecalis biofilm infections. Due to the homology of the rnc gene in E. faecalis with that in S. mutans, it was speculated that the rnc gene could regulate the morphology of biofilms by promoting the EPS synthesis in E. faecalis.
Sodium hypochlorite (NaOCl) has been widely used in the irrigation of root canal due to its excellent antibacterial properties and ability to remove organic components and tissue remnants [12]. However, it has also raised concerns due to its cytotoxic effects on the periapical and pulp tissues [13]. CHX has also been widely used for the irrigation of root canal due to its excellent antibacterial activity. However, it is unable to dissolve the tissue remnants, which restricts its applications as a standard irrigation agent [14]. The current irrigation agents cannot be considered an ideal choice individually. Therefore, exploring new irrigation agents is needed.
Traditional Chinese medicine (TCM) has a history of thousands of years. These natural medicines are increasingly applied for the treatment of oral diseases. Pudilan is a TCM fluid, which has anti-inflammatory and antibacterial effects. It is made up of the extracts of multiple cold and calm herbs, including Scutellaria baicalensis root, Taraxacum mongolicum, Bunge corydalis herb, and Isatis indigotica [15]. Its anti-inflammatory effects have been confirmed in several classic inflammatory models [16]. It has also been applied to cure oral diseases, such as mild recurrent aphthous ulcers and chronic gingivitis [17,18]. The active ingredient in Pudilan has proved to inhibit the production of varies inflammatory factors, such as periodontitis target IL-1β [19,20]. Nevertheless, the antibacterial effects of Pudilan on E. faecalis or periapical periodontitis have not been investigated yet. Therefore, this study was aimed to explore the potential targets for the disinfection of E. faecalis biofilms and also explore the clinical alternative drugs. The main objectives of this study were as follows: (1) to construct and verify the rnc overexpression and low-expression mutant strains of E. faecalis; (2) to detect the regulatory effect of rnc on the morphology of biofilm and EPS production; and (3) to evaluate the rnc modulated susceptibility of E. faecalis biofilms to root canal irrigation agents and Pudilan.

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 1 μg/mL competence-stimulating peptides (CSP) and the rnc overexpression mutant strain (rnc+) was established [10]. 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][22][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% N 2 , 10% H 2 , 10% CO 2 ). 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 (14-16 h). Then, the cultures were diluted to 1:20 with BHI medium and grown under anaerobic conditions for 2.5-3 h until the cells reached the mid-log phase (OD 600nm = 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 midlog phases (OD 600nm = 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 1000×, 5000×, 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 min. The OD 575 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), and PBS were added respectively to the biofilm samples and incubated for 10 min. Pudilan keyanning antibacterial mouthwash is a product mainly contains extracts of herbs in Pudilan formula. Therefore, we selected it to represent the Pudilan and detected its antibiofilm effect. 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). TB Green ® Premix Ex Taq ™ (Tli RNaseH Plus) (Takara, JAPAN) was used in the experiment according to the manual. The reaction process is as follows: 95 °C 30 s in the holding stage, then 95 °C 5 s and 60 °C 30 s for 40 cycles in the cycling stage, followed by melt curve stage and cool down. The reactions were carried out in triplicate. 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.

Antibacterial assays
The 24-h E. faecalis ATCC 29212 parent and mutant biofilm samples were prepared and the planktonic bacteria were removed using PBS. Each group of the biofilms were incubated with 1 mL 5% NaOCl, 2%CHX, Pudilan, and PBS respectively for 10 min. Then, the drugs were washed gently. PBS solution (1 mL) was added to each sample to form a uniform bacteria suspension. Then the bacteria suspension was diluted to different concentrations by PBS according to the antibiofilm ability of the drugs. The bacteria was diluted to 10 -2 folds in the 5% NaOCl group, 10 -3 folds in the 2%CHX and Pudilan groups and 10 -5 folds in the PBS group. After mixing, 10 μL diluted bacterial suspension was dropped on BHI plate [24].

Statistical analyses
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. The Shapiro-Wilk test was applied and verified the data are normally distributed. Fisher's least significant difference was performed to compare the means of each group. Two-way ANOVA was applied to assess differences of the growth curves [25]. A P value < 0.05 was considered statistically significant.

Down-regulation of the rnc gene inhibited bacterial growth and EPS synthesis
We tried different methods to reduce rnc expression level. Firstly, polymerase chain reaction ligation mutagenesis [26] was used to construct rnc deletion mutants without success. No colony growth on the antibiotics selective plate. The rnc gene seems to be essential for E. faecalis ATCC29212 viability. Then we introduced plasmids carrying rnc antisense sequences into E. faecalis ATCC29212. This method can effectively hinder the expression of rnc by pairing and forming a rnc− antisense rnc duplex structure. Similarly, the rnc+ mutant strain was made by introducing plasmids carrying rnc sequences [22]. The expression level of the rnc gene was identified using RT-qPCR (Fig. 1A). The results showed that as compared to the E. faecalis ATCC 29212 wildtype, the rnc expression level of the rnc+ strain increased by 40.13 times, while that of the rnc− decreased by 0.22 times. This confirmed the successful construction of rnc+ and rnc− mutant strains. The growth curve of rnc+ and wildtype E. faecalis ATCC 29212 strains were similar; both reached the midlog growth phase nearly the same time (Fig. 1C). However, the rnc− strain showed a slower growth rate under the same culture conditions, indicating its weaker proliferation capability. The rnc− strain spent a longer time reaching the mid-log growth phase and presented a lower OD 600 value at the stationary phase. The average OD value of ATCC29212, rnc+ and rnc− were 0.737, 0.749 and 0.684 respectively. Statistical tests found significant difference between the rnc− strain and the other two species.
Crystal violet assays were performed to determine the differences in the total amount of EPS synthesis in the 24-h biofilms of wildtype, rnc+, and rnc− E. faecalis ATCC 29212 strains. As shown in Fig. 1B, the rnc+ strain showed significantly higher EPS productions as compared to the wildtype strain, while the rnc− strain showed significantly lower EPS contents (both P < 0.0001).
The morphology of the biofilms was evaluated using SEM (Fig. 1D). As compared to the wild-type strain, the Then, under 20,000× magnification, the biofilm looked uneven and the bacterial cells aggregated through the extracellular matrix. On the contrary, the biofilm of rnc− strain contained a sparse matrix with fewer cracks on the surface. Under 20,000× magnification, the rnc− strain showed a loose combination between the matrix and bacterial cells.
The microscopic morphologies of the wildtype, rnc+, and rnc− strains were consistent with their performances under the naked eye. While preparing the samples, the rnc+ biofilms were found to be firmly attached to the glass slide and were more resistant to the water impact, while the rnc− biofilms were fragile. The CLSM showed that both the EPS and bacteria showed a thick accumulation in the rnc+ biofilm, while those in the rnc− biofilm showed decreased production and were scattered and unevenly distributed ( Fig. 2A). Furthermore, the EPS/ bacteria ratio in the rnc+ biofilm was higher than that of the wildtype strain (P < 0.05), while that of the rnc− strain was the lowest (P < 0.05) (Fig. 2B). Overall, the results consistently revealed that the rnc gene could positively regulate bacterial growth and biofilm formation in E. faecalis.

Biofilms of rnc mutant strains showed an increased sensitivity to disinfectants
In order to compare the sensitivities of E. faecalis ATCC 29212 wildtype and rnc mutant strains to the different antibacterial agents, crystal violet assays were performed to quantify the EPS residues in the biofilms after treatment with the respective antibacterial agents. 5% NaOCl was set as a positive control. After incubating for 10 min with 5% NaOCl, all the three biofilms were almost eliminated with no significant differences (Fig. 3A). Interestingly, the rnc+ and rnc− groups showed lower EPS residues as compared to the wildtype strains after treatment with 2% CHX, Pudilan, and PBS, suggesting that the biofilms of rnc mutant strains were more sensitive to these antibacterial agents (Fig. 3B). Particularly, Pudilan showed better anti-biofilm activity as compared to the 2% CHX. Furthermore, the number of active bacteria in the wildtype and rnc mutant biofilms were compared after treatments with different drugs (Fig. 4). Due to the different antibiofilm ability of the agents, we diluted the bacteria suspension to 10 -2 folds in 5% NaOCl group, 10 -3 folds in 2% CHX and Pudilan group, 10 -5 folds in PBS group. As a positive control, 5% NaOCl showed the strongest antibiofilm ability towards the three strains with no significant difference among the ATCC29212, rnc+ and rnc− groups. In the 2% CHX and Pudilan group, there are significantly more colonies of ATCC29212 than rnc+ and rnc−. The PBS treated groups showed similar column number.

Discussion
The pathogenic biofilms of E. faecalis are closely associated with periapical periodontitis. The removal of E. faecalis biofilms is crucial for avoiding the failure of root canal treatments. Due to the complexity of the root canal system [27], chemical irrigation agents are supposed to disinfect the root canal, especially where the mechanical preparations cannot reach it. The Fig. 2 The rnc gene altered EPS and bacteria biomass ratio of biofilms. A The biofilm morphology was observed by CLSM. Double fluorescent labels marked bacteria (green, SYTO 9) and EPS (red, Alexa Fluor 647) respectively; scale bar = 50 μm. B The EPS/bacteria biomass ratio of E. faecalis ATCC29212, rnc + and rnc − . Three-dimensional reconstruction of the biofilms and quantitative data were performed by Imaris 7.0.0. (*P < 0.05) characteristics of the root canal irrigation agents determine their effects. As irrigation agents, 5% NaOCl and 2% CHX are effective and widely used. However, due to the irritation to the periapical tissue [28], 5% NaOCl should be applied with caution in the clinic. CHX has also shown some side effects, such as irritation to the oral mucosa, causing a burning sensation, and alteration of taste perception [29]. Therefore, the development of alternative drugs and improvement of bacterial susceptibility has been continuously sought. Pudilan is a commercial TCM made up of herbal extract. Pudilan keyanning mouthwash products contain Pudilan extract and 0.03%-0.06% cetylpyridinium chloride (CPC). The CPC (0.03-0.06%) has been reported with little antibacterial effects, which were weaker than 0.12% CHX [15]. In the current study, Pudilan exhibited a stronger EPS removing effect as compared to 2% CHX and was more effective on the rnc+ and rnc− strains. In the colony number assay, Pudilan and 2%CHX also showed stronger antibacterial effect on rnc+ and rnc− strains than ATCC29212. Overall, Pudilan preliminary showed an excellent anti-biofilm effect on E. faecalis but still needs further investigations.
Similar to E. faecalis, another Gram-positive coccus bacteria S. mutans, relies on the formation of stable biofilm to acquire resistance and cause virulence in the mouth. Preliminary studies have shown that the rnc deletion mutation could repress S. mutans cariogenicity in rat models, and the weakness of biofilm was attributed to the reduced EPS production and bacterial adhesion [10]. As rnc is a highly conserved gene, we proposed rnc as a target to eliminate the E. faecalis biofilms. The results demonstrated its excellent regulatory effects on biofilm metabolism and drug sensitivity. The rnc overexpression strain rnc+ and rnc low-expression strain rnc− were established and the effects of its rnc expression level on the growth status of E. faecalis were observed. The rnc+ strain showed a normal growth rate and formed a thriving biofilm, while the rnc− strain showed a delayed growth rate and fragile biofilm. These results revealed that the rnc gene could positively regulate the bacterial growth and formation of biofilm in E. faecalis, which was consistent with our hypothesis. The rnc gene encodes RNase III, which regulates gene expression at the posttranscription level [30]. Therefore, the rnc expression level might have a profound impact on the phenotypes of bacteria and biofilms. On the other hand, antisense walR as a post-transcriptional modulator, has been proven to regulate bacterial growth, virulence and EPS synthesis and aggregation. This is a successful precedent for posttranscriptional level regulation as an anti-biofilm target in E. faecalis [31].
In order to evaluate the ability of the three strains to drug resistance, the EPS residues and their colony number in biofilms after incubation were tested with different drugs. As expected, the 5% NaOCl group showed the least OD 575 absorbance as expected, followed by PBS, Pudilan, and 2% CHX group. The increased OD in the Pudilan and 2% CHX groups as compared to the PBS group might be due to the increased light absorbance by the biofilm pigmentation caused by its color. After treatment, the EPS residues in the biofilms of rnc mutant strains were less than those of the wild-type strain. It was concluded that the rnc− strain showed weakened drug resistance due to its thin and barren extracellular matrix. Interestingly, the thick rnc+ biofilm also showed increased sensitivity to the antibacterial drugs. The SEM and CLSM observation of the thick and uneven rnc+ biofilm suggested that this unevenness of the biofilm allowed the antibacterial drugs to penetrate, thereby showing their antibacterial effects. The rnc interference strategy not only reduced the EPS metabolism of E. faecalis biofilms but also made the biofilms more fragile, resulting in increased drug susceptibility. In order to comprehensively understand the regulatory effects of rnc on biofilm formation and their mechanisms, further studies of related genes, including epaI/epaOX [32], gelE, and esp [33] are required. Approaches to rnc gene regulation rather than the antibiotic use and development of resistance might be more in line with ecological regulation.
However, there are some limitations in this study. First, the biofilm models used in the experiment may not fully reflect the state in the disease. This was an in-vitro experiment and the biofilm samples were 24-h early mature biofilms. Ali et al. showed that the substrate-conditioning substances and biofilm age could affect the components of the cellular and extracellular matrix of E. faecalis biofilms [34]. Moreover, we failed to delete the rnc gene from genomic DNA either through chemical transformation or electroporation method, but the rnc deletion mutant was constructed in E. faecalis V19 [35], which is a plasmid-cured derivative of the vancomycin-resistant clinical isolate V583 [36]. The characteristic differences between type strain ATCC29212 and drug-resistant clinical strain V19 may explain the failure to knock out the rnc gene in ATCC29212. Moreover, the biofilm phenotype and drug resistance changes of the rnc− strain were obvious enough to judge the trend of the results. Therefore, we take the rnc− strain to observe the regulation effect of the rnc gene. The rnc− strain was constructed by transforming a shuttle plasmid loaded with an rnc antisense RNA sequence. Here are other possible hypotheses for failure to construct rnc deletion mutant strain. (1) The exogenous plasmids are abnormally expressed in bacteria; therefore, the mutant strains cannot survive on a selective medium. (2) The thick capsule of membrane shuts long-chain DNA out. (3) The transformation methods need further optimization. Although the rnc− strain showed decreased growth, the copy number variation might cause genetic and expression instability [37]. In brief, more advanced biofilm models and mutant strains are expected to be used in exploring anti E. faecalis targets.

Conclusions
In this study, the rnc overexpression and low-expression mutant strains of E. faecalis were successfully constructed. The biological features of rnc mutant strains and their sensitivity towards typical root canal irrigation agents and TCM fluid Pudilan were evaluated. This study revealed that the overexpression of rnc could promote bacterial growth and EPS synthesis, and vice versa. However, the altered rnc expression level could break the balance, forming a vulnerable biofilm. The altered biofilm structure made it more sensitive to the antibacterial agents, allowing for a decrease in antibiotic use and resistance. Taken together, these data suggested the rnc gene as a biofilm regulatory target and provided evidence for the antibacterial potential of Pudilan, providing a novel strategy for the management of root canal system and apical infection.