Streptococcus mutans extracellular DNA levels depend on the number of bacteria in a biofilm

Streptococcus mutans is a component of oral plaque biofilm that accumulates on the surface of teeth. The biofilm consists of extracellular components including extracellular DNA (eDNA). This study was conducted to investigate the factors that may affect the eDNA levels of S. mutans in biofilms. For the study, S. mutans UA159 biofilms were formed for 52 h on hydroxyapatite (HA) discs in 0% (w/v) sucrose +0% glucose, 0.5% sucrose, 1% sucrose, 0.5% glucose, 1% glucose, or 0.5% sucrose +0.5% glucose. Acidogenicity of S. mutans in the biofilms was measured after biofilm formation (22 h) up to 52 h. eDNA was collected after 52 h biofilm formation and measured using DNA binding fluorescent dye, SYBR Green I. Biofilms cultured in 0.5% sucrose or glucose had more eDNA and colony forming units (CFUs) and less exopolysaccharides (EPSs) than the biofilms cultured in 1% sucrose or glucose at 52 h, respectively. The biofilms formed in 0% sucrose +0% glucose maintained pH around 7, while the biofilms grown in 0.5% sucrose had more acidogenicity than those grown in 1% sucrose, and the same pattern was shown in glucose. In conclusion, the results of this study show that the number of S. mutans in biofilms affects the concentrations of eDNA as well as the acidogenicity of S. mutans in the biofilms. In addition, the thickness of EPS is irrelevant to eDNA aggregation within biofilms.

glucose or sucrose over 15 h after media change (at 22 h and 46 h). The biofilms had various pH values, but were higher in sucrose than glucose when they were in the media less than 9 h after media change (at 31 h and 52 h).

Viability and EPS of S. mutans in biofilms formed in different media. The viability and EPSs of
S. mutans in the 52-h-old biofilms were analyzed via bio-volume (Fig. 2a) and mean thickness (Fig. 2b) using confocal laser scanning microscopy (CLSM). Bio-volume and mean thickness of bacteria were higher in 0.5% sucrose than 1% sucrose, and the same pattern was shown in glucose, whereas EPSs had lower bio-volume and mean thickness in 0.5% sucrose than 1% sucrose. In 0.5% sucrose +0.5% glucose, bio-volume and mean thickness of bacteria were higher than those in 1% sucrose and lower than those in 0.5% sucrose, and bio-volume and mean thickness of EPS were lower than those in 0.5% and in 1% sucrose. According to the representative bacterial and EPS images from the CLSM study shown in Fig. 2 (1-6), while S. mutans produced abundant EPSs in sucrose media, there were almost no EPSs detected in 0% sucrose +0% glucose media or in glucose media. In addition, S. mutans showed more EPSs in 0.1% sucrose than in 0.5% sucrose or 0.5% sucrose +0.5% glucose.
The concentrations of S. mutans eDNA within biofilms are cell-number dependent. The levels of S. mutans eDNA in the 52-h-old biofilms (Fig. 3a) coincided with the numbers of CFUs in the biofilms (Fig. 3b). The biofilms grown in 0.5% glucose and sucrose had more S. mutans than those formed in 1% concentrations, and eDNA levels were approximately 1.8 fold higher in 0.5% sucrose than 1% sucrose and about 3 fold higher in 0.5% glucose than 1% glucose. S. mutans in biofilms had more CFUs and eDNA in 0.5% sucrose +0.5% glucose than in 1% sucrose and less than in 0.5% sucrose. To investigate the relationship between eDNA levels in biofilms and the bio-volume of bacteria or EPS in the biofilms, relative eDNA levels were calculated and compared (Fig. 4).

Discussion
In the present study, we sought to characterize the factors associated with eDNA aggregation in biofilms, and the results of this study show that eDNA levels in biofilms are related to the number of S. mutans in the biofilms.
S. mutans is an oral bacteria that generates glucosyltansferases (Gtf B, Gtf C, and Gtf D) to produce insoluble and soluble glucans, and these glucans promote the formation of biofilms 19 . The oral bacteria that produce acids in these biofilms are considered to acidify the microenvironment by reducing the pH of plaque, enhancing the growth of acidogenic and aciduric bacteria, and leading to dental caries 20,21 .
According to the results in this study, when acidogenicity of biofilms was detected at 52 h, the pH values were inversely proportional to the number of CFUs of S. mutans in the biofilms. Furthermore, the acidogenicity in glucose media that formed no EPS was similar to that in sucrose media after overnight culture. This suggests that acidogenicity of S. mutans is closely associated with number of bacteria in the presence of sugar and does not correlate to the thickness of EPS, a major component of biofilms. This needs further investigation.
The viability of S. mutans was affected by sugar type. We assumed that the bacterium could have better viability in media that had higher concentrations of sugar since S. mutans grows faster and more abundantly in sucrose media than glucose media 22,23 . However, in this study, S. mutans grew more in 0.5% sucrose than in 1% sucrose and had less EPS in 0.5% sucrose than 1% sucrose media, suggesting that EPS thickness in biofilms is related to the concentration of sucrose, whereas CFUs do not coincide with sucrose concentration. However, Cai et al. showed that the thickness of EPS in biofilms can increase in up to 1% sucrose, while that in greater than 1% sucrose media decreased 24 , suggesting that EPS thickness is also not associated with sucrose concentration greater than 1% in culture media. In glucose media, S. mutans also grew more abundantly in 0.5% than 1%. While the biofilms grown in the combination of 0.5% sucrose +0.5% glucose displayed lower CFUs and EPSs than those formed in 0.5% sucrose, they showed higher CFUs and less EPSs than those grown in 1% sucrose. This can be explained as the addition of glucose can reduce the synthesis of EPSs and growth of S. mutans 25,26 . Some researchers suggested that S. mutans produce more eDNA in biofilms than in planktonic culture 15 . However, as shown in 0.5% sucrose, when there were more bacteria in biofilm, the eDNA levels were higher than in 1% sucrose and 0.5% sucrose +0.5% glucose. Interestingly, S. mutans grown on discs in glucose media showed the same pattern, which suggests that eDNA levels of S. mutans are related to the number of bacteria regardless of EPS. The result also showed that S. mutans in biofilms with EPS has more eDNA, as glucans produced by S. mutans in sucrose media provide binding sites to bacteria, allowing S. mutans to aggregate more in biofilms 27,28 . This can be explained by a study that showed the exposure of S. mutans to the nutrient flow was lower when sucrose concentration in culture media was higher and accordingly, adhesion of S. mutans increases as sucrose levels increase up to a certain concentration (0.45%) and then it decreases as sucrose concentration increases. However, water-insoluble EPSs and dry-weight increase in a sucrose-level dependent manner in biofilms formed in the media less than 1% sucrose 24 .  Regarding the single-species biofilms used in this study, this phenomenon could be different in multi-species biofilms. According to Castillo Pedraza et al., UA159 had more biomass and more eDNA in single-species biofilms than multi-species biofilms 29 . Micro-environmental conditions and amounts of carbohydrates also affect EPS and eDNA contents in multi-species biofilms 30,31 .
In summary, the results of this study provide new findings that eDNA levels in biofilms are dependent on the CFUs in the biofilms, that acidogenicity of S. mutans biofilms is related to the CFUs in the biofilms, and eDNA levels in biofilms are not related to the thickness of EPS of the biofilms.

Measurement of pH, eDNA, and CFU count.
The pH values of biofilms were determined at 22, 31, 46, and 52 h by measuring the old culture media when the media was changed to investigate changes in S. mutans biofilm acidogenicity in the presence of 0% sucrose +0% glucose, 0.5% sucrose, 1% sucrose, 0.5% glucose, 1% glucose, or 0.5% sucrose +0.5% glucose. eDNA of S. mutans was measured as previously described with some modifications 15,33 . Briefly, S. mutans UA159 was cultured overnight in UFTYE supplemented with 1% glucose. On the next day, approximately 6 × 10 5 CFU/ml bacteria was transferred into 24-well plates containing HA discs and UFTYE with 0% sucrose +0% glucose, 0.5% sucrose, 1% sucrose, 0.5% glucose, 1% glucose, or 0.5% sucrose +0.5% glucose and cultured for 52 h. The bacteria on the discs were washed twice with 0.89% NaCl and collected in 0.89% NaCl by scraping them from the discs using a spatula. The bacteria collected from each disc was centrifuged at 10,000 × g at 4 °C for 10 min, and the pellet was re-suspended in 0.89% NaCl and sonicated (10 s, twice at 20% energy level on ice with 1 min interval) to break the biofilms and separate eDNA from the surface of bacteria using a sonifier (VCX 130PB; Sonics and Materials Inc., Newtown, CT, USA). An aliquot (0.1 ml) of the sonicated pellet was serially diluted and plated on BHI agar plates to count the CFUs of the bacteria. The rest were centrifuged at 10,000 × g at 4 °C for 10 min. The supernatant was filtered using 0.22-μm syringe filters (Millipore, USA) to gain pellet-free supernatant. For eDNA measurement, the cell-free and pellet-free supernatants were treated with DNA-binding dye, SYBR Green I (Invitrogen). The samples were excited at 485 nm and emitted at 535 nm. The eDNA concentration was measured using a fluorescence microplate reader (HIDEX). Four independent experiments were performed to measure eDNA and count CFUs.
Confocal laser scanning microscopy. CLSM was performed to determine the changes in S. mutans biofilm formation. One μM of Alexa Fluor 647-labeled dextran conjugate (10,000 MW; absorbance/fluorescence emission maxima 647/668 nm; Molecular Probes Inc., Eugene, OR, USA) was added to 0% sucrose +0% glucose, 0.5% sucrose, 1% sucrose, 0.5% glucose, 1% glucose, or 0.5% sucrose +0.5% glucose BHI broth with S. mutans UA159 (6 × 10 5 CFU/ml) at 0, 22, 31, and 46 h. The fluorescence-labeled dextran serves as a primer for GTFs and can be simultaneously incorporated during the extracellular polysaccharide matrix synthesis over the course of the biofilm development. After 52 h, the bacterial cells in the biofilms were labeled by incubation with 2.5 μM SYTO 9 green fluorescent nucleic acid stain (480/500 nm; Molecular Probes Inc.) for 30 min. CLSM imaging of the biofilms was performed using an LSM 510 META (Carl Zeiss, Jena, Germany) microscope equipped with argon ion and helium-neon lasers. Four independent experiments were performed, and five image stacks per experiment were collected (n = 20). The bio-volume (μm 3 /μm 2 ) and mean thickness (μm) of bacterial micro-colonies and EPSs were quantified from the confocal stacks using COMSTAT.

Evaluation of eDNA production according to bacteria bio-volume or EPS bio-volume.
To compare the eDNA levels in biofilms to bacteria bio-volume or EPS bio-volume of the biofilms, the relative ratio of eDNA level in each biofilm was calculated as follows: among the eDNA levels in biofilms formed in 6 different media, the highest was fixed as 100, and the rest were calculated accordingly. Relative ratios of bacteria bio-volume and EPS bio-volume were calculated in the same way.
= × Relative ratio of eDNA level eDNA in each biofilm The highest eDNA 100 Statistical analysis. All experiments were performed at least three times. The intergroup differences were estimated by one-way analysis of variance (ANOVA). The data are presented as mean ± standard deviation (SD). A p value was considered statistically significant when it was less than 0.05 (p > 0.05).

Data availability
The data generated and analyzed in this study are available from the corresponding author upon request.