Antibacterial and Antibiofilm Effect of Cell-Free Supernatant of Lactobacillus brevis KCCM 202399 Isolated from Korean Fermented Food against Streptococcus mutans KCTC 5458

This study aims to determine the antibiofilm effect of cell-free supernatant (CFS) of Lactobacillus brevis strains against Streptococcus mutans strains. To study the antibiofilm mechanism against S. mutans strains, antibacterial effects, cell surface properties (auto-aggregation and cell surface hydrophobicity), exopolysaccharide (EPS) production, and morphological changes were examined. The antibiofilm effect of L. brevis KCCM 202399 CFS as morphological changes were evaluated by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM), compared with the control treatment. Among the L. brevis strains, L. brevis KCCM 202399 showed the highest antibiofilm effect on S. mutans KCTC 5458. The antibacterial effect of L. brevis KCCM 202399 against S. mutans KCTC 5458 was investigated using the deferred method (16.00 mm). The minimum inhibitory concentration of L. brevis KCCM 202399 against S. mutans KCTC 5458 was 25.00%. Compared with the control treatment, L. brevis KCCM 202399 CFS inhibited the bacterial adhesion of S. mutans KCTC 5458 by decreasing auto-aggregation, cell surface hydrophobicity, and EPS production (45.91%, 40.51%, and 67.44%, respectively). L. brevis KCCM 202399 CFS inhibited and eradicated the S. mutans KCTC 5458 biofilm. Therefore, these results suggest that L. brevis KCCM 202399 CFS may be used to develop oral health in the probiotic industry.

After washing the cells, the absorbance at 600 nm (OD Initial ) was adjusted to 0.5 ± 0. Chloroform (0.5 ml) was added to each cell suspension (2 ml) and pre-incubated for 10 min at 37°C. Thereafter, the mixtures were vortexed for 2 min and incubated for 15 min at 37°C. The aqueous phase was measured at 600 nm (OD Treatment ). The cell surface hydrophobicity was calculated using the following formula: Cell surface hydrophobicity (%) =

Analysis of Total EPS Production Rate
EPS production by S. mutans KCTC 5458 was measured by the phenol-sulfuric acid method with some modifications [16]. Five milliliters of S. mutans KCTC 5458 diluted to 10 7 CFU/ml in BHI broth containing 3% sucrose was mixed with 5 ml of CFS diluted to 1/2 × MIC in BHI broth containing 3% sucrose and incubated at 37°C for 24 h under anaerobic conditions; non-treated cells were used as a control. After incubation, the treated mixtures were centrifuged at 12,000 ×g at 4°C for 10 min, and 1 ml of supernatant was mixed with 2 ml of 99% ethyl alcohol and incubated for 24 h at 4°C. After incubation, the mixture was centrifuged at 14,240 ×g for 15 min, and the pellets were resuspended in 500 μl of distilled water. In the cell suspension (100 μl), 5% phenol (100 μl), and 95% sulfuric acid (4 ml) were mixed; the mixture was vortexed, and incubated at 30°C for 10 min. The absorbance was calculated using the following formula: EPS production rate (%) =

Biofilm Assay
Biofilm inhibition and eradication were measured using a crystal violet assay, with some modifications [17]. Overnight cultured S. mutans KCTC 5458 was diluted to 10 7 CFU/ml in BHI broth containing 3% sucrose. To determine the inhibitory effect of L. brevis CFS on the formation of S. mutans biofilm, 50 μl of bacterial cultures and 50 μl of CFS diluted to 1/2 × MIC in BHI broth containing 3% sucrose were transferred to a 96 well plate and incubated at 37°C for 24 h; untreated cells were used as a control. After biofilm formation, the cell suspensions were removed using a micropipette. The plates were washed twice with 150 μl of PBS. Plates were dried at 37°C for 20 min. Thereafter, 1% crystal violet was added to each well to stain the biofilm-forming cells for 30 min at room temperature. After dyeing, the plate was rinsed and dissolved in a solution of 30% methanol and 10% acetic acid. The OD of each sample was measured at 570 nm using a microplate reader (Molecular Devices, USA).
To investigate the effect of eradication on the formation of S. mutans biofilms, the cell density was adjusted to 10 7 CFU/ml in BHI broth containing 3% sucrose, and 100 μl of cell suspension was inoculated into 96 well plate and incubated at 37°C for 24 h under anaerobic conditions; non-treated cells were used as a control. After incubation, each well was washed twice with 150 μl PBS. One hundred microliters of L. brevis CFS (1/2 × MIC and MIC) was added to each well and incubated at 37°C for 24 h. Non-treated cells were used as a control. The results were quantified as follows.

Scanning Electron Microscopy (SEM) Analysis
SEM was performed to investigate the biofilm inhibition effect of L. brevis KCCM 202399 CFS on S. mutans KCTC 5458 biofilm using a modified method [18]. Overnight cultured S. mutans KCTC 5458 was diluted to 10 7 CFU/ml using BHI broth containing 3% sucrose. Two milliliters of bacterial suspension and 2 ml of CFS diluted to 1/2 × MIC in BHI broth containing 3% sucrose were cultured in each well of a six-well plate containing glass coupons and incubated at 37°C for 24 h under anaerobic conditions. The control group was treated with BHI broth containing 3% sucrose. The biofilms formed on the glass coupons were fixed with 2.5% glutaraldehyde in PBS at 4°C for 1 h. The fixed samples were washed twice with PBS and dehydrated for 30 min using gradually increasing concentrations of ethanol solutions (50%, 70%, 80%, 90%, and 100%). Ethanol was replaced with isoamyl acetate, and the coupons were dried in a freeze dryer and then coated with platinum particles (15 mV for 1.5 min). The S. mutans KCTC 5458 biofilm was observed using a field-emission scanning electron microscope (FESEM; SU8010; Hitachi High-Technologies Co., Japan).

Confocal Laser Scanning Microscopy (CLSM) Analysis
CLSM was performed to evaluate the biofilm inhibition effect of L. brevis KCCM 202399 CFS on S. mutans KCTC 5458 biofilm. Biofilms of S. mutans KCTC 5458 were prepared using the same protocol as described in section 2.8. After biofilm formation, the glass coupons were washed twice with PBS. Live and dead cells were stained with 1 μM SYTO9 and propidium iodide (PI) for 20 min in the dark at room temperature. After staining, the glass coupons were washed twice with PBS and observed under a Zeiss LSM 800 microscope (Carl Zeiss, Germany) using a 10 × objective lens and an appropriate standard filter.

Statistical Analysis
All experiments were repeated three times with duplicate samples, and the results are presented as the mean ± standard deviation. All statistical analyses were performed using SPSS 18.0. Significant differences among means were determined using one-way analysis of variance (ANOVA).

Antibacterial Effect against S. mutans Strains
The antibacterial effects of Lactobacillus strains against S. mutans strains are presented in Table 1. Among S. mutans strains, L. brevis strains showed a higher antibacterial effect against S. mutans KCTC 5458 than against S. mutans KCTC 5124 and S. mutans KCTC 5316, except for L. rhamnosus GG. Antibacterial effects of L. rhamnosus GG were 7.55 ± 1.3 mm and 9.11 ± 1.0 mm against S. mutans KCTC 5124 and S. mutans KCTC 5316, respectively (Table 1; p < 0.05). L. brevis strains showed a high antibacterial effect against S. mutans KCTC 5458 (all Lactobacillus strains examined had an inhibition zone over 10 mm). L. rhamnosus GG and L. brevis KCCM 202399 showed higher antibacterial effect against S. mutans KCTC 5458 than other L. brevis strains (16.66 ± 1.0 mm and

Cell Surface Properties
The effects of L. brevis strains CFS on auto-aggregation and cell surface hydrophobicity of S. mutans KCTC 5458 are shown in Table 3. Treatment with L. brevis strains CFS decreased auto-aggregation (p < 0.05) and cell-surface hydrophobicity (p < 0.05) of S. mutans KCTC 5458, compared with the negative control. The auto-aggregation ability of S. mutans KCTC 5458 treated with L. brevis KCCM 202399 and L. brevis KU15147 decreased by 45.91% and 49.11%, respectively. Additionally, S. mutans KCTC 5458 treated with L. rhamnosus GG was 46.35%. The cell surface hydrophobicity of S. mutans KCTC 5458 treated with L. rhamnosus GG CFS and L. brevis KCCM 202399 CFS was 32.97% and 40.51%, respectively. Our results showed that Lactobacillus strains CFS inhibited bacterial adhesion by decreasing the auto-aggregation and cell-surface hydrophobicity of S. mutans KCTC 5458.

EPS Production Rate
The EPS production rate of S. mutans KCTC 5458 treated with L. brevis strains CFS was evaluated using a modified phenol-sulfuric acid method [16]. Fig. 1 presents the inhibitory effect of L. brevis strain CFS on EPS production rate (p < 0.05). Treatment with L. brevis KCCM 202399 CFS resulted in the lowest EPS production rate (67.44%). For treatment with L. rhamnosus GG CFS, the EPS production rate was 72.35%, followed by treatment with L. brevis KU15147, and L. brevis KCCM 200019 (73.66% and 76.44%, respectively). As shown in Fig. 1 and Supplementary Tables 1, 2, and 3, L. brevis KCCM 202399, L. brevis KU15159, and L. brevis KU15147 CFS had a greater inhibitory effect on S. mutans growth and EPS production than other L. brevis strains.

Biofilm Inhibition and Eradication Effects of CFS
The inhibitory effect of L. brevis CFS on S. mutans KCTC 5458 biofilm is shown in Fig

SEM Analysis on Glass Coupon
The effects of L. brevis KCCM 202399 CFS on biofilm formation by S. mutans KCTC 5458 on glass coupons were also evaluated by SEM (Fig. 3). In the control group, S. mutans KCTC 5458 formed numerous bacterial cells and a large biofilm on the glass coupons (Fig. 3A). However, biofilm structures of S. mutans KCTC 5458 treated-L. rhamnosus GG CFS and L. brevis KCCM 202399 CFS were spread, resulting in decreased bacterial cells and biofilms on glass coupons (Figs. 3B and 3C). Considering the results of the other experiments, including cell surface properties (Table 3), EPS production ( Fig. 1), biofilm inhibition and degradation effects (Fig. 2), the inhibitory effect of probiotic L. brevis KCCM 202399 CFS from S. mutans biofilm was confirmed.

CLSM Analysis on Glass Coupon
The antibiofilm and antibacterial effects of L. brevis KCCM 202399 CFS against S. mutans KCTC 5458 on glass coupons were observed via CLSM. CLSM images showed S. mutans biofilms with viable and non-viable cells (green and red, respectively). In the control, S. mutans KCTC 5458 showed a dense biofilm structure and biofilm cells (Fig. 4A1, 2). However, the biofilm structures of L. rhamnosus GG CFS-and L. brevis KCCM 202399 CFStreated S. mutans KCTC 5458 were decreased. Furthermore, L. brevis KCCM 202399 CFS showed significantly reduced viability of S. mutans KCTC 5458 biofilm cells compared with L. rhamnosus GG CFS (Figs. 4B and 4C).  represents the mean ± standard deviation, with a-d different letters on each bar representing significant differences (p < 0.05). : × 1,000, × 5,000, and × 10,000)

Discussion
Dental caries is a major oral disease that is multi-species biofilm-mediated. Dental plaque, which is a multispecies biofilm, is transformed from cariogenic to non-cariogenic plaque. S. mutans is a cariogenic bacteria in dental plaque that colonizes the tooth surface and forms biofilms [5]. Once S. mutans forms a biofilm, it is difficult to remove; therefore, its early control is important. This study was aimed at investigating the antibacterial and antibiofilm effects of L. brevis strains isolated from kimchi.
In this study, methods were developed to screen the antibacterial effect of L. brevis strains against S. mutans strains. The results showed that L. brevis strains showed a higher antibacterial effect against S. mutans KCTC 5458 than against S. mutans KCTC 5124 and S. mutans KCTC 5316. Thereafter, antibacterial effect of L. brevis strains against S. mutans strains was investigated by using L. brevis CFS; it was confirmed that L. brevis strains have a higher antibacterial effect on S. mutans KCTC 5458 than on S. mutans KCTC 5124 and S. mutans KCTC 5316. Some Lactobacilli strains can metabolize sucrose, co-aggregate with S. mutans, and are often tolerant to fluoride [19]. Therefore, probiotic supernatant could be safely used as an antibacterial agent for treating dental plaque. Taku et al. [20] reported that the "expression" or "sensitivity" of gtf gene, which synthesizes water in-soluble or soluble glucans from sucrose, would be affected differently in different S. mutans strains. CFSs of all L. brevis strains showed greater antibacterial effect against S. mutans KCTC 5458 than against S. mutans KCTC 5124 and S. mutans KCTC 5316. In particular, L. brevis KCCM 202399 CFS showed an antibacterial effect at the lowest concentration among the L. brevis strains (Table 2). Therefore, we focused on the antibacterial effect of L. brevis CFS against S. mutans KCTC 5458.
Auto-aggregation, cell surface hydrophobicity, and EPS production changes in S. mutans are important to prevent S. mutans adhesion, colonization, and early biofilm formation [21]. Auto-aggregation of S. mutans is beneficial for its adhesion to tooth surface, as the resulting biofilm formed prevents this bacterium from an adverse external environment [22]. S. mutans has a high overall proportion of hydrophobic bacteria, and its cell surface hydrophobicity may play a role in the adherence of oral bacteria to the tooth surface [23]. Therefore, we investigated the effect of L. brevis CFS on the auto-aggregation ability and cell surface physiochemical properties of S. mutans KCTC 5458. In this study, L. brevis KCCM 202399 CFS showed the highest reduction in autoaggregation and cell surface hydrophobicity of S. mutans KCTC 5458. In another study, auto-aggregation of S. mutans ATCC 25175 treated with L. brevis BBE-Y52 was higher than that in the presence of other Lactobacillus strains [24]. In addition, Bacillus velezensis K68-treated L. brevis strains exhibited increased cell surface hydrophobicity, compared with the untreated control [25]. EPS produced by S. mutans is a major factor in biofilm formation. As sucrose exists in oral conditions, gtfs from S. mutans plays critical roles in the development of virulent dental plaque [26]. In the presence of L. brevis CFS, EPS production by S. mutans KCTC 5458 decreased. In particular, L. brevis KCCM 202399 CFS showed the highest reduction in EPS production by S. mutans. These results suggest that L. brevis KCCM 202399 reduces sucrose-dependent EPS production by downregulating gtfs. In our previous study, L. brevis KU15153 CFS decreased EPS production by approximately 41% (p < 0.05) [27]. A previous study reported that biosurfactants produced by probiotics have antibacterial and anti-adhesive properties [28]. The biosurfactants in metabolites exuded by Lactobacilli interfere with the adhesion of cells. These decrease the hydrophobicity of the cell surface substratum and interfere with the progression of microbial adhesion ability [29]. In addition, Tahmourespour et al. [30] reported that Lactobacillus acidophilus-derived biosurfactant down-regulated gtfs B and C genes, and virulence factors were associated with glucan in dental plaque.
Changes in cell surface properties and EPS production by L. brevis KCCM 202399 CFS also affected biofilm formation by S. mutans KCTC 5458. In this study, 1/2 MIC and MIC concentrations of L. brevis CFS were used for treatment and the inhibition rate of S. mutans biofilm as a function of CFS concentration was investigated. The biofilm formed by S. mutans in the presence of L. brevis strains CFS exhibited a dose-dependent reduction in biomass compared to the control group that did not receive CFS. In particular, L. brevis KCCM 202399 CFS showed the highest inhibitory effect against S. mutans KCTC 5458 at 1/2 MIC and MIC levels. In our previous study, L. rhamnosus GG was reported to have a significant antibacterial effect against S. mutans [4]. Ahn et al. [31] reported that lipoteichoic acid of probiotics could inhibit biofilm formation by S. mutans. Additionally, bacteriocin, an antibacterial substance produced by probiotic Lactobacilli, can kill gram-positive bacteria by disrupting their cell membranes, inhibit their growth by lowering pH and hamper bacterial DNA synthesis by producing hydrogen peroxide [32]. We also conducted SEM and CLSM analyses to investigate the reduction in biofilm formation and viability of biofilm cells as imaging. S. mutans KCTC 5458 treated with L. brevis KCCM 202399 CFS showed that the biofilm was dispersed with little aggregation, and the number of cells on glass coupons was decreased compared with that for the control (Fig. 3). Compared with L. rhamnosus GG, L. brevis KCCM 202399 showed a higher number of dead biofilm cells, as evidenced by PI staining (Fig. 4). Generally, mature biofilms are more difficult to remove than early biofilms. Biofilms can protect oral bacteria, and S. mutans can induce membrane proteins to migrate and overcome cellular damage caused by environmental stress [5,33]. The biofilm degradation effect is also important to examine the antibiofilm effect against S. mutans; however, eradication effect of probiotics on mature biofilms have rarely been reported. L. brevis KCCM 202399 CFS showed the highest biofilm eradication effect against S. mutans KCTC 5458 at 1/2 MIC and MIC levels, demonstrating its action through biofilm formation and disruption of mature biofilm.
Six L. brevis strains isolated from kimchi were tested for antibacterial effects against S. mutans strains. The results showed that L. brevis KCCM 202399 had the highest antibacterial and antibiofilm effects among the L. brevis strains. Furthermore, L. brevis KCCM 202399 showed more antibacterial effects against S. mutans KCTC 5458 than against S. mutans KCTC 5124 and S. mutans KCTC 5316. L. brevis KCCM 202399 CFS inhibited and eradicated the biofilm of S. mutans KCTC 5458 by decreasing its auto-aggregation, cell-surface hydrophobicity, and EPS production. The antibiofilm effects of CFS against S. mutans KCTC 5458 were also confirmed by SEM and CLSM. Therefore, this study suggests that L. brevis KCCM 202399 could be used as a functional food in the food industry.