Effect of D-cysteine on dual-species biofilms of Streptococcus mutans and Streptococcus sanguinis

Dental caries is a highly prevalent disease worldwide. It is caused by the cariogenic biofilms composed of multiple dynamic bacteria on dental surface. Streptococcus mutans and Streptococcus sanguinis are resident members within the biofilms and an antagonistic relationship has been shown between these two species. S. mutans, as the major causative microorganism of dental caries, has been reported to be inhibited by free D-cysteine (D-Cys). However, whether D-Cys could affect S. sanguinis and the interspecies relationship between S. mutans and S. sanguinis remains unknown. The aim of the current study was to investigate the effect of D-Cys on the growth and cariogenicity of dual-species biofilms formed by S. mutans and S. sanguinis. We measured dual-species biofilms biomass, metabolic activity, lactate production. We also detected the biofilms structure, the ratio of live/dead bacteria, extracellular polysaccharide (EPS) synthesis and bacterial composition in the dual-species biofilms. We found that D-Cys could reduce the metabolic activity and lactic acid production of dual-species biofilms (p < 0.05). In addition, biofilms formation, the proportion of S. mutans in dual-species biofilms, and EPS synthesis were decreased with D-Cys treatment. The results suggested that D-Cys could inhibit the growth and cariogenic virulence of dual-species biofilms formed by S. mutans and S. sanguinis, indicating the potential of D-Cys in clinical application for caries prevention and treatment.


Results
Growth of the S. mutans and S. sanguinis affected by D-cysteine. As

D-Cys inhibited Biofilms formation and metabolic activity.
Biofilm biomass of the single species was measured. As the data shown in Fig. 2A, D-Cys significantly inhibited biofilms formation of S. mutans. Biofilm biomass decreased in all D-Cys treatment groups. However, D-Cys slightly influenced biofilms formation of S. sanguinis. Biofilm biomass decreased a bit in 20 mM group, and no significant difference was found in 40 mM and 60 mM groups compared to the non-treated control group. The result of metabolic activity of dual-species biofilms affected by D-Cys was shown in Fig. 2B. Biofilms in control group displayed a relatively high metabolic activity. Biofilms metabolic activity were significantly decreased with D-Cys treatment, especially in 40 mM and 60 mM groups.

D-Cys inhibited lactic acid production in dual-species biofilms. Acid excreted by cariogenic bacteria
is the direct cause of tooth demineralization. We examined lactic acid production in dual-species biofilms (Fig. 3). 20 mM and 40 mM of D-Cys markedly inhibited acid production. Lactic acid production in these two groups was approximately 1/3 of that in the control group. Besides, lactic acid was hardly to be detected in 60 mM of D-Cys group.

D-Cys decreased the ratio of live/dead cells in dual-species biofilms.
Live bacteria were stained green and dead bacteria were stained red. The ratio of live/dead cells was decreased in 40 mM and 60 mM groups (Fig. 4), indicating that the proportion of live cells was up-regulated by D-Cys. However, the bacterial composition of the live cells was unclear.

D-Cys inhibited the EPS synthesis in dual-species biofilms.
It was reported that the EPS are the key cariogenic virulence of S. mutans 15 . We measured EPS synthesis by confocal laser scanning microscopy (CLSM). Both bacterial cell (green) numbers and EPS (red) synthesis were decreased by D-Cys (Fig. 5B). Biofilms were looser and less EPS were around the bacteria in three D-Cys-treated groups (Fig. 5A). D-Cys disassembled bacterial biofilms. The structure of biofilms was imaged by a scanning electron microscopy (SEM). D-Cys disassembled bacterial biofilms (Fig. 6). Biofilms structures were more and more loose  The data are presented as the means + standard deviation from three independent experiments, and the asterisks represent significant differences compared with the non-treated control group (*P < 0.05, ***P < 0.001, ns: no significance).
www.nature.com/scientificreports www.nature.com/scientificreports/ and less bacterial clusters were observed along with the rise of D-Cys concentration. The complete structure of biofilms disappeared and there were many aperture gaps in 60 mM group.

D-Cys up-regulated the proportion of S. sanguinis cells in dual-species biofilms.
The species-specific fluorescent in situ hybridization (FISH) labeled biofilms were imaged and quantitative real-time polymerase chain reaction (q-PCR) was performed to analyze the bacterial proportion in dual-species biofilms. S. mutans cells were labeled green and S. sanguinis cells were labeled red. As shown in Fig. 7A, the integrated green fluorescence  The data are presented as the means + standard deviation. In each plot, different letters mean significance between the two groups ( a&a no significance, a&b P < 0.05). The volume of EPS and bacteria, calculated according to 3 random sights of biofilms (mean + sd). In each plot, different letters mean significance between the two groups ( a&b P < 0.05, a&c P < 0.001).  www.nature.com/scientificreports www.nature.com/scientificreports/ intensity was much weaker and integrated red fluorescence intensity was much stronger in D-Cys-treated groups, indicating a down-regulation of S. mutans and an up-regulation of S. sanguinis in dual-species biofilms. According to the results of q-PCR, the ratio of S. mutans to S. sanguinis was appreciably decreased by D-Cys (Fig. 7B). D-Cys altered the bacterial composition in dual-species biofilms, making the proportion of S. sanguinis higher.

Discussion
Recent studies have revealed that D-Cys could inhibit the biofilm formation of S. mutans. However, dental caries is a disease caused by complex microflora composed of multiple bacteria. S. mutans and S. sanguinis are predominant members in the dental plaque. In the present study, we investigated the effect of D-Cys on the growth and cariogenicity of dual-species biofilms formed by S. mutans and S. sanguinis. The results indicated that D-Cys significantly inhibited biofilms formation, biofilms activity, lactic acid production, and EPS synthesis. Meanwhile, D-Cys altered the proportion of S. mutans and S. sanguinis in dual-species biofilms.
Biofilms provided important living space for bacteria [16][17][18] , and could help bacteria escape from the host defenses and resist the harmful factors [19][20][21] . Considering the importance of biofilms for bacteria, we detected whether D-Cys could affect dual-species biofilms formation. By the SEM observation, we found D-Cys appreciably disassembled bacterial biofilms, biofilm structure was incomplete in 60 mM of D-Cys group. We interestingly found the D-Cys at concentration of 20 mM might slightly promote the growth of planktic bacteria (Fig. 1), but it inhibited the single-species biofilm formation. We guessed the lower concentration might promote the growth of S. mutans and S. sanguinis. When we tested the biofilm formation by crystal violet staining assay, we need to wash the loose bacteria and it led the planktonic bacteria washed away. Although the growth curve of 20 mM seemed higher than the control group, there was no statistical difference.
The bacteria in the dental plaque could metabolize carbohydrates (primarily sucrose) to acids, causing the demineralization of tooth surface 22,23 . Biofilm activity and lactic acid production are associated with the cariogenic capacity. In the present study, we found that D-Cys significantly inhibited biofilms activity and decreased lactate production, indicating a down-regulation of biofilms cariogenicity by D-Cys. The decrease of lactic acid production might result from the inhibition of D-Cys to the dual-species biofilms formation. EPS, the major component in the biofilms, were also the important virulence of the biofilms 24 . EPS can help bacteria adhere to tooth surfaces, provide protection from the outside pressure and provide nutrition, which supply a suitable environment for microbes 25 . The result of confocal laser scanning microscopy assay showed that D-Cys significantly reduced the total synthesis of EPS.
Clinic research has showed that dental caries status in children, the interaction of S. mutans with S. sanguinis was an important factor 26 . Therefore, proportion of S. mutans and S. sanguinis was considered to be a approach to evaluate the caries risk 27 . The species-specific FISH and qPCR results showed D-Cys displayed a down-regulation effect on S. mutans and an up-regulation effect on S. sanguinis in dual-species biofilms. Besides, the ratio of S. sanguinis to S. mutans in multispecies biofilms increased significantly in D-Cys-treated groups compared to the control group. We speculated that the change in bacterial composition might be due to the different effects of D-Cys on the growth of these two strains. According to the result from the growth curve assay (Fig. 1), S. mutans displayed more sensitive than S. sanguinis to the D-Cys. D-Cys significantly inhibited S. mutans growth, while a slight growth inhibition occurred with D-Cys treatment in S. sanguinis group. D-Cys relieved the inhibition effect of S. mutans on S. sanguinis, thus making S. sanguinis more in dual-species biofilms. It has been pointed out that the early colonization of S. sanguinis and its elevated levels in the oral cavity could significantly delay the colonization of S. mutans 9 . Therefore, the elevation of the S. sanguinis proportion in dual-species biofilms by D-Cys indicated an ecological benefit of D-Cys in terms of caries prevention and control.

Conclusion
In conclusion, our results indicated that D-Cys could inhibit the growth and cariogenicity of dual-species biofilms formed by S. mutans and S. sanguinis and altered the dual-species biofilms to a healthier condition. Therefore, D-Cys has potential use as a new drug for dental caries prevention and treatment. However, the biocompatibility of D-Cys needs to be tested in further study. Bacteria inoculation, biofilm formation. S. mutans UA159 and S. sanguinis ATCC10556 were provided by the State Key Laboratory of Oral Diseases (Sichuan University, Chengdu, China). A single colony of S. mutans or S. sanguinis were precultured in BHI medium at 37 °C under anaerobic condition (80% N 2 , 10% CO 2 , 10% H 2 ) overnight. The concentration of S. mutans and S. sanguinis was adjusted to 1 × 10 6 CFU/mL in the study. BHI supplemented with 0.2% sucrose (BHIS) was used to support the biofilms formation [28][29][30] . www.nature.com/scientificreports www.nature.com/scientificreports/ (OD) at 600 nm using a microplate reader (SpectraMax 190; Molecular Devices, Inc., Sunnyvale, CA) every 1 h. There were six replicates of each bacterium for each D-Cys concentration.

Materials and Methods
Crystal violet staining assay. Crystal violet staining assay was performed to measure the biofilm biomass.
Two hundred microliter of the exponential-phase bacteria culture with D-Cys was cultured in the selected wells of a sterile 96-well microtiter plate for 24 h. Biofilms were rinsed by phosphate buffered saline (PBS) twice, then fixed by 100% methyl alcohol for 15 min. The biofilms on the bottom of the microplates were stained with 200 µL of 0.1% crystal violet for 5 min and then washed with sterile distilled water to remove the residual dye. The bounded crystal violet was released by 200 µL 95% ethanol. Before the measurement, the test elution liquid of crystal violet was diluted 1:4 with 95% ethanol to ensure that the readings were within the range of the spectrophotometer. The absorbance of released crystal violet in ethanol was recorded at OD 575 nm by a spectrophotometer. There were six replicates of each bacterium for each D-Cys concentration.
MTT assay. MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was performed to test the biofilms activity 31 . MTT solution was prepared in PBS at a working concentration of 0.5 mg/mL. Discs with 24 h dual-species biofilm were rinsed in PBS and transferred to a new 24-well plate supplemented with 1 mL of MTT solution, and then were incubated for 1 hour at 37 °C in 5% CO 2 in the dark. An hour later, put each disc into a new 24-well plate, and added 1 mL of DMSO (Dimethyl sulfoxide) to cover the biofilm. Then the plate was incubated for 20 min at room temperature on an orbital platform in the dark (covered with foil). After incubation, 200 µL of DMSO solution was transferred to a 96-well plate and the absorbance was detected at OD 540 nm by a spectrophotometer. There were six replicates for each D-Cys concentration.
Lactic acid production measurement. After incubation for 24 h, dual-species biofilms cultured on discs were washed with PBS to remove loose bacteria and then were transferred to a new 24-well plate with 1.5 mL buffered peptone water (BPW) accompanied with 0.2% sucrose 32 . Biofilms were incubated at 37 °C in 5% CO 2 for 3 hours. Lactate concentrations were determined using an enzymatic (lactate dehydrogenase) method. 200 μL of collected BPW solutions were transferred to a new 96-well plate and were measured at the OD 340 nm with a microplate reader 33,34 . There were six replicates for each D-Cys concentration.

Extracellular polysaccharide synthesis measurement by a confocal laser scanning microscope (CLSM).
Dual-species biofilms cultured on sterilized glass discs were grown in BHIS medium and supplemented with 2.5 μM Alexa Fluor 647-labeled dextran conjugate (Molecular Probes) for 24 h. Biofilms were washed with PBS for three times and stained with 2.5 μM SYTO 9 (Molecular Probes) for 15 min. The samples were imaged with a Leica DMIRE 2 confocal laser scanning microscope equipped with a 60× oil immersion objective lens. The 3-dimensional reconstruction was performed with Imaris 7.0.0 (Bitplane, Zürich, Switzerland). The quantification of EPS and bacteria volume was performed with Image-Pro Plus (Media Cybernetics, Silver Spring, MD, USA) and COMSTAT 35,36 . The EPS stained red color and each pixel site could be identified and quantified by this software. We calculated by counting the intensity and presence of color at each pixel site in each picture. In order to keep consistent between images, the parameters were set at beginning and each picture used the same parameters.
Live/Dead assay. Dual-species biofilms cultured on discs were grown in BHIS medium for 24 h. Biofilms were washed with PBS for three times and stained with 2.5 μM SYTO 9 (Molecular Probes, Invitrogen) and propidium iodide (Molecular Probes) for 15 min. The samples were imaged with a DMIRE2 confocal laser scanning microscope (Leica, Wetzlar, Germany) equipped with a 60× oil immersion objective lens 37 . The quantification of live/dead was performed with Image-Pro Plus (Media Cybernetics, Silver Spring, MD, USA).

Biofilms structure observation by a scanning electron microscope (SEM). After culturing in BHIS
medium for 24 h, the biofilms grown on sterilized glass discs were rinsed three times with PBS to remove loose planktonic bacteria and fixed with 2.5% glutaraldehyde overnight at 4 °C. Then, the discs were washed twice in sterile water (immersion in water per washed for 10 mins) and serial dehydrated with graded ethanol (50%, 70%, 75%, 80%, 85%, 90%, 95%, and 100%, immersion in ethanol per dehydrated for 10 mins). Then the samples were sputter-coated with gold for SEM imaging (Quanta 200, FEI, Hillsboro, OR, USA).

Bacterial composition analysis in dual-species biofilms.
The 24h dual-species biofilms grown on sterilized glass discs were washed with PBS for three times, fixed in 4% parafomaldehyde overnight, and investigated by species-specific fluorescent in situ hybridization (FISH) probes as previously described 38 . The labeled biofilms were imaged with an OLYMPUS FV1000 confocal laser scanning microscope (OLYMPUS, TOKYO, JAPAN) equipped with a 100× oil immersion objective lens. The bacterial composition was further quantified by species-specific real time quantitative polymerase chain reaction.