In Vitro Preventive Effect and Mechanism of Action of Weissella cibaria CMU against Streptococcus mutans Biofilm Formation and Periodontal Pathogens

In this study, we evaluated the in vitro anti-biofilm, antibacterial, and anti-inflammatory activity of Weissella cibaria CMU (CMU), an oral probiotic, against periodontopathogens. Compared to other oral probiotics, CMU showed a superior inhibitory effect on the biofilm formation and growth of Streptococcus mutans on orthodontic wires and artificial teeth (p < 0.05). CMU exerted potent antibacterial effects against S. mutans and Porphyromonas gingivalis according to a line test. In human gingival fibroblasts (HGFs) stimulated by P. gingivalis, Fusobacterium nucleatum, or Prevotella intermedia, CMU suppressed the gene expression of pro-inflammatory cytokines [interleukin (IL)-6, IL-1β, IL-8, and tumor necrosis factor-α] in a dose-dependent manner (p < 0.05). CMU restored the production of the tissue inhibitor of metalloproteinase-1 following its inhibition by P. gingivalis, and it suppressed the expression of matrix metalloproteinase (MMP)-1 and -3 induced by periodontopathogens (p < 0.05). Moreover, CMU needed direct contact with HGFs to exert their anti-inflammatory function, indicating that they act directly on gingival cells to modulate local inflammation. Our preclinical study provides evidence for the potential benefits of topical CMU treatments in preventing the development of caries and periodontitis caused by the dysbiosis of the dental plaque microbiome.


Introduction
Dental caries and periodontal disease are the most common oral diseases worldwide [1][2][3]. Dental caries particularly occurs in childhood due to unhealthy oral hygiene habits, eating habits, and oral bacterial alterations which are considered risk factors for caries [4]. In the formation of dental caries, the tooth enamel is damaged and tooth decay is caused by acids produced by the bacterial breakdown of sugars [5]. Plaque is a major direct cause of dental caries and is characterized by a persistent biofilm. Therefore, the prevention of dental caries can be achieved by inhibiting the plaque-related biofilm formation due to various cariogenic bacteria including Streptococcus mutans [5].
Periodontitis-a chronic disease that destroys the alveolar bone supporting the gums and teeth through exacerbated local inflammation-is one of the leading causes of tooth loss in adults [2,6]. Periodontitis is caused by periodontopathogens, such as Porphyromonas gingivalis [7,8]. Chronic inflammation due to repeated bacterial infection promotes the secretion of pro-inflammatory cytokines and matrix metalloproteinases (MMPs) [9]. Interleukin (IL)-6, IL-1β, IL-8, and tumor necrosis factor (TNF)-α are prominent pro-inflammatory cytokines associated with periodontal tissue destruction [10,11].

Effects of Oral Probiotics on S. mutans Biofilm Formation on Orthodontic Wires
To determine the effects of oral probiotics on biofilm formation by S. mutans, we performed a tube wire test as previously described with minor modifications [30]. Briefly, equal amounts (5 × 10 6 CFU/mL) of S. mutans and each oral probiotic were cultivated in a tube containing 30 mL of the test medium {pH 6.5; equal amounts of BHI and MRS with 5% sucrose, 0.5% yeast extract [MB cell], and 0.1 M MES [2-(N-Morpholino) ethanesulfonic acid monohydrate; MB cell]}. One orthodontic wire with a length of 4 cm and a diameter of 0.8 mm (Remanium, Dentaurum, Pforzheim, Germany) was suspended from a conical tube and immersed in the test medium. After gentle shaking (50 rpm) at 37 • C for 24 h, the weight of the S. mutans biofilm formed on each wire was measured. S. mutans inoculated alone was used as a control. We confirmed the dose-dependent effects of W. cibaria in 10-fold serial dilutions relative to the S. mutans concentration.
To measure the effects of oral probiotics on the growth of S. mutans after orthodontic wire removal, all cultures were serially diluted and inoculated onto Mitis Salivarius Bactiracin (MSB) agar (Difco).

Effects of Oral Probiotics on S. mutans Biofilm Formation on Artificial Teeth
We assessed the effects of oral probiotics on S. mutans biofilm formation on artificial teeth as previously described, with minor modifications [45]. In summary, 1.5 mL of the test medium (see Section 2.2.1.) was added to a 24-well plate containing resin-based artificial teeth (VIPI-DENT plus, Madespa, Toledo, Spain) and inoculated with equal amounts (5 × 10 6 CFU/mL) of S. mutans and oral probiotics. After culturing for 24 h at 37 • C, the supernatant was completely removed and wells containing teeth were rinsed twice with phosphate-buffered saline. To measure the amount of biofilm formed, each tooth was stained with 0.1% safranin (BD Biosciences, Sparks, MD, USA) for 15 min, rinsed with distilled water three times, and treated with 30% acetic acid to release bound safranin from the stained cells; the absorbance of the solution was measured at 530 nm. S. mutans inoculated alone was used as a control. We confirmed the dose-dependent effects of W. cibaria using 10-fold serial dilutions relative to the S. mutans concentration.

Evaluation of Oral Probiotic Antagonism against Oral Pathogens
We determined the antagonistic activity between oral probiotics and pathogens using line tests (a conventional antagonism test) as previously described, with minor modifications [46]. In summary, 20 µL S. mutans culture diluted to 10 6 CFU/mL was first dropped onto agar mixed with equal amounts of BHI and MRS or BHI alone and allowed to flow vertically. P. gingivalis was inoculated on TSB hemin menadione agar containing 5% (v/v) sterile defibrinated sheep blood (MB cell) at 20 µL to achieve 10 7 CFU/mL. After drying the pathogens, equal amounts and 10-fold dilutions of oral probiotics were vertically dropped across the pathogens from the edge of the plate. Agar plates for S. mutans and P. gingivalis were incubated aerobically and anaerobically at 37 • C for 3-7 d, respectively. The antioxidant activity of oral probiotics was tested with cell-free supernatants (CFSs) and evaluated using a 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay (Sigma-Aldrich, St. Louis, MO, USA), as previously described, with some modifications [47]. To prepare the bacterial CFSs, cells were removed by centrifugation (5000× g, 10 min, 4 • C) and the CFS was filtrated (0.22 µm; JET BIOFIL, Guangzhou, China). We mixed 400 µL of CFS and 800 µL of freshly prepared DPPH solution (0.2 mM in ethanol), which we left to react for 25 min at 25 • C and then centrifuged (5000× g, 5 min, 25 • C). We transferred 200 µL of the supernatants to 96-well plates. The mixture of MRS broth and DPPH was used as a blank treatment. The level of scavenged DPPH was measured at 517 nm using a microplate reader (VersaMax, Molecular Devices, San Jose, CA, USA).

Human Gingival Fibroblast Culture
HGFs (Lifeline Cell Technology, Walkersville, MD, USA) were provided by the Laboratory of Oral Anatomy (School of Dentistry, Wonkwang University, Iksan, Korea) and grown in Dulbecco's modified Eagle's medium (DMEM; Gibco, Thermo Fisher Scientific, Gaithersburg, MD, USA), supplemented with 10% heat-inactivated fetal bovine serum (Gibco) and 1% antibiotic-antimycotic solution (GenDepot, Katy, TX, USA) at 37 • C in a humidified atmosphere containing 5% CO 2 . Experiments were carried out on HGFs of passages 3 to 9. The cells were sub-cultured and plated at 80% confluency. Serum and antibiotic-free DMEM were used for the co-culture of HGFs and live bacteria.

ELISA Analysis
The concentrations of secreted inflammatory mediators were quantified using humanspecific ELISA kits (DuoSet system, R&D Systems, Minneapolis, MN, USA). The 96-well plates were coated with anti-human MMP-1, MMP-3, MMP-8, MMP-9, or TIMP-1 monoclonal antibodies at 4 • C overnight. All assays were performed according to the manufacturer's instructions, and the level of each inflammatory mediator was determined using the standard curve prepared for each assay. The optical density at 450 nm was measured for each well using the microplate reader, with wavelength correction set at 540 nm.

Cell Viability Assay
To measure cell viability after treatment with live oral probiotics, we used a viability assay kit (Cellrix, MediFab, Seoul, Korea). HGFs were seeded on 96-well plates at a density of 10 4 cells/well in only DMEM or DMEM containing 2% FBS. The cells were treated with various concentrations of oral probiotics (MOI = 0.1, 1, 10, and 100) for 24 h at 37 • C in a 5% CO 2 atmosphere. The medium was then carefully replaced with a fresh medium containing water-soluble tetrazolium-8 (WST-8) salt solution, and the plates were incubated at 37 • C in a 5% CO 2 atmosphere for 4 h. Cell viability was measured at 450 nm using the microplate reader.

Statistical Analysis
The results are presented as the mean ± standard deviation of triplicate measurements. Statistical analyses were performed using SPSS Statistics version 21.0 for Windows (IBM, Armonk, NY, USA). A one-way analysis of variance (ANOVA) with Duncan's multiple range test was used to compare the differences between group means. Statistical significance was set at p < 0.05.

Antibiofilm Activity against S. mutans on Orthodontic Wires
We performed a tube wire test to determine the effects of oral probiotics against S. mutans antibiofilm formation on orthodontic wires. L. reuteri, W. cibaria CMU, and CMS1 Microorganisms 2023, 11, 962 6 of 18 strongly inhibited S. mutans biofilm formation, whereas other commercial oral probiotics had little inhibitory effects (Figure 1a-c). Both W. cibaria CMU and CMS1 inhibited S. mutans biofilm formation and growth in a dose-dependent manner (Figure 1d,e). The growth of S. mutans was inhibited by 99.99% by both W. cibaria CMU as well as CMS1 (p < 0.05) ( Table 1).  Data are presented as the mean ± standard deviation. Different letters indicate significant differences at p < 0.05.

Antibiofilm Activity against S. mutans on Artificial Teeth
We determined the effects of oral probiotics against S. mutans biofilm formation on resin-based artificial teeth. W. cibaria CMU and CMS1 inhibited S. mutans biofilm formation by 96.8% and 94.6%, respectively, whereas other commercial oral probiotics had

Antibacterial Activity against S. mutans and P. gingivalis
We performed a line test to elucidate the antibacterial activity of oral probiotics against S. mutans, the representative cariogenic bacterium (Figure 3a-f), and P. gingivalis, a periodontopathic bacterium (Figure 3g-l). W. cibaria CMU and CMS1 showed a strong direct inhibition against both S. mutans (Figure 3e,f) and P. gingivalis (Figure 3k,l).

Cytotoxic Effects of Oral Probiotics on HGFs
We evaluated the cytotoxic effects of live L. reuteri, W. cibaria CMU, and CMS1 according to the viability of HGFs. No cytotoxic effects were detected after the 24 h challenge with various bacterial concentrations in either test medium (Table 2).

Antibacterial Activity against S. mutans and P. gingivalis
We performed a line test to elucidate the antibacterial activity of oral probiotics against S. mutans, the representative cariogenic bacterium (Figure 3a-f), and P. gingivalis, a periodontopathic bacterium (Figure 3g-l). W. cibaria CMU and CMS1 showed a strong direct inhibition against both S. mutans (Figure 3e,f) and P. gingivalis (Figure 3k,l).

Inhibitory Effect of W. cibaria on Periodontopathogen-Induced Pro-Inflammatory Cytokine Expression
Based on the above results indicating that L. reuteri, W. cibaria CMU, and CMS1 showed antibacterial activity against P. gingivalis, we compared the effects of these bacteria on the mRNA expression of pro-inflammatory cytokines. L. reuteri had no inhibitory effect on most pro-inflammatory cytokines (IL-6, IL-8, IL-1β, and TNF-α), and it dramatically promoted their expression compared to periodontal bacteria alone (p < 0.05) (Figure 4). We assessed the mRNA expression of pro-inflammatory cytokines in HGFs pre-treated with various concentrations of W. cibaria and stimulated by P. gingivalis (Figure 5a), F. nucleatum (Figure 5b), or P. intermedia (Figure 5c). W. cibaria CMU suppressed the mRNA expression of most pro-inflammatory cytokines in a dose-dependent manner compared to that in treatments with the periodontopathogen alone, and it showed a potent inhibitory effect at higher doses (p < 0.05).

Inhibitory Effect of W. cibaria on Periodontopathogen-Induced MMP Expression
L. reuteri stimulation increased the mRNA expression of MMP-1 compared to the treatment with periodontal bacteria alone (p < 0.05) (Figure 6a). We measured the mRNA expression of MMPs in HGFs pre-treated with various concentrations of W. cibaria and stimulated by each periodontopathogen. W. cibaria CMU altered the mRNA expression of MMP-1, MMP-3, and TIMP-1 in a dose-dependent manner compared to P. gingivalis treatment alone (Figure 6b-d). W. cibaria CMU also suppressed the mRNA expression of MMP-1, MMP-8, and MMP-9 in a dose-dependent manner compared to F. nucleatum or P. intermedia alone (p < 0.05) (Figure 7a,b). After stimulation with F. nucleatum or P. intermedia for 24 h, W. cibaria CMU suppressed the mRNA expression of MMP-1 and MMP-3 in a dose-dependent manner (p < 0.05) (Figure 7c,d). Pre-treatment with W. cibaria CMU increased TIMP-1 protein levels in a dose-dependent manner in HGFs infected with P. gingivalis (Figure 8a). W. cibaria CMU reduced the protein levels of MMP-1 and MMP-3 induced by F. nucleatum or P. intermedia in a dose-dependent manner. After stimulation with F. nucleatum and P. intermedia for 24 h, high concentrations of W. cibaria CMU reduced MMP-1 and MMP-3 protein levels by 95.4% and 98.2% (Figure 8b) as well as 88.9% and 96.5%, respectively (p < 0.05) (Figure 8c). 5 Figure 5. Dose-dependent effects of W. cibaria CMU on the mRNA expression of pro-inflammatory cytokines stimulated by P. gingivalis (a), F. nucleatum (b), and P. intermedia (c). HGFs were pretreated with W. cibaria CMU for 30 min at various doses (0.1, 1, and 10) and then incubated with each periodontal pathogen (MOI = 100) for 4 h. CMU, W. cibaria CMU. Data are presented as the mean ± standard deviation. * p < 0.05, in comparison to the untreated group. # p < 0.05, in comparison to the control group.

Immunomodulatory Mechanisms of W. cibaria
We investigated the effect of direct contact between W. cibaria CMU and HGFs on the regulation of periodontopathogen-induced inflammation by separating W. cibaria CMU from HGFs and periodontal bacteria using cell culture inserts in a Transwell system. After separating W. cibaria CMU, no inhibition of mRNA expression (Figure 9a-c) or protein secretion (Figure 9d-f) was observed for most of the inflammatory mediators.     gingivalis (a,d), F. nucleatum (b,e), and P. intermedia (c,f). W. cibaria CMU was pre-treated for 30 min at 10-fold the dose of P. gingivalis, F. nucleatum, or P. intermedia and then incubated with each periodontopathogen (MOI = 100) for 4 h (a-c) or 24 h (d-f). CMU, W. cibaria CMU; CI, cell culture inserts in the Transwell system. Data are presented as the mean ± standard deviation. * p < 0.05, compared to the untreated group. # p < 0.05, compared to the control group.

Discussion
Probiotics are beneficial bacteria that provide various health benefits to the host [39]. Traditionally, probiotics have been used to improve gut health, especially for the treatment or prevention of gastrointestinal infections and diseases. The mechanisms of the action of probiotics include competitive adhesion inhibition, coaggregation, growth inhibition, bacteriocin production, and immune regulation [19]. Recent studies have proposed that probiotics can improve the regulation of body fat [48], vaginal health [49], and oral health [22]. In particular, many studies have reported effective strategies for the prevention and treatment of oral diseases using probiotics, including tooth decay and periodontal disease [22,30,32,34].
For decades, S. mutans, which ferments sugar, has been considered a major cause of dental caries [5] and has been considered to play an integral role in the etiology and pathogenesis of dental caries. However, recent DNA-and RNA-based studies have reported that S. mutans constitutes only a tiny fraction of the highly diverse bacterial community in carious lesions [50]. Therefore, in consideration of the polymicrobial nature of dental caries, a paradigm shift is needed for the pathogenesis of dental caries. Probiotics have been suggested to prevent dental caries by inhibiting S. mutans activity [23,28]. However, the supporting evidence for this is weak and it has been reported that probiotic bacterial strains may themselves be cariogenic. For example, L. rhamnosus GG, a well-known probiotic bacterium, was found to contribute to rather than inhibit the development of caries in experiments using dental tissue [51].
The current study is the first to confirm the inhibitory effect of oral probiotics against biofilm formation by S. mutans on artificial surfaces. We first compared the inhibitory effects of strains isolated from commercially available oral probiotic products against the growth of S. mutans. Using orthodontic wire as a surface for bacterial growth, we found that L. reuteri, W. cibaria CMU, and CMS1 had the most potent inhibitory effect, whereas L. paracasei and L. gasseri had no effect. Using artificial teeth as a surface for bacterial growth, we found that except for W. cibaria CMU and CMS1, L. reuteri, L. paracasei, L. gasseri, and S. salivarius caused a high degree of tooth discoloration similar to that in cultures with S. mutans alone, indicating no inhibitory effect. Several studies contradict our findings. Many studies have reported that L. reuteri inhibits biofilm formation by S. mutans, but most considered the effect of bacterial CFSs [23,24,32]. In our study, live L. reuteri was shown to inhibit S. mutans-induced biofilm formation on an orthodontic wire, but not on artificial teeth. Several strains of L. paracasei have also been reported to inhibit S. mutans biofilm formation [52,53]. Mann et al. [54] reported that L. gasseri inhibited the formation of biofilm by S. mutans on an orthodontic wire. Moreover, it has been reported that S. salivarius can respond to plaque formation and salivary acidity by producing dextranase and urease after colonizing the oral mucosa of humans [28].
Our study showed that W. cibaria CMU and CMS1 decreased the number of S. mutans cells in a dose-dependent manner. When cultured with S. mutans at the same dose, both W. cibaria CMU and CMS1 reduced the number of S. mutans by 4.1 log CFU/mL; even at a dose 100-times lower than that of S. mutans, W. cibaria CMU and CMS1 reduced the number of S. mutans by 3.4 and 3.5 log CFU/mL, respectively (Table 1). Consistent with our initial results but contrary to expectations, L. reuteri, L. paracasei, L. gasseri, and S. salivarius strains did not show inhibitory activity against S. mutans biofilm and by extension the development of caries.
Several factors are involved in periodontitis. In particular, the highly complex periodontal microbiome is known to play an important role not only in the initiation but also in the progression and establishment of periodontal disease [6][7][8][9][10][11]. Periodontitis results from dysbiosis of the periodontal microbiome, leading to changes in host-microbe crosstalk and initiation of the inflammatory response. P. gingivalis is a key pathogen in the development of periodontitis, along with several major complexes, including F. nucleatum and P. intermedia [6][7][8].
The pathogenic potential of plaque-causing bacteria has been demonstrated in their ability to produce many toxic substances, such as endotoxins, cell wall mucopeptides, fatty and organic acids, hydrogen sulfide, ammonia, indoles, amines, and leukotoxins [8]. Upon stimulation by these pathogens, pro-inflammatory cytokines are released from host cells to recruit immune cells. However, excessive inflammation results in tissue damage, bone resorption, and ultimately, tooth loss. Thus, the modulation of inflammation is a promising strategy for inhibiting disease progression. HGFs are the most abundant cells in gingival connective tissue and are common in periodontal tissues [55]. HGFs stimulated with pathogens have been reported to upregulate the gene expression of the pro-inflammatory cytokines IL-6, IL-1β, IL-8, and TNF-α, which facilitates the inflammatory cascade in periodontitis [10,11,55].
To confirm the preventive effect of oral probiotics on the impact of periodontal pathogens, we conducted line tests using L. reuteri, W. cibaria CMU, and CMS1 against periodontopathogens. After pre-treatment of HGFs with these oral probiotics, we found that the expression of IL-6, IL-1β, IL-8, and TNF-α genes induced by the periodontopathogens P. gingivalis, F. nucleatum, and P. intermedia were reduced by the W. cibaria strains, whereas L. reuteri upregulated their gene expression (Figure 4). We confirmed that W. cibaria CMU had a dose-dependent effect on the mRNA expression of pro-inflammatory cytokines, compared to the effect of P. gingivalis alone, especially at higher doses. In response to F. nucleatum and P. intermedia stimulation, W. cibaria CMU reduced the gene expression of most pro-inflammatory cytokines in a dose-dependent manner (except for that of IL-1β), especially at higher doses.
Periodontal lesions are characterized by excessive destruction of gingival connective tissue due to collagen degradation. MMPs secreted by HGFs are involved in the degradation of the extracellular matrix and bone collagen matrix [9,12,13]. Although various MMPs, including MMP-1, -3, -8, and -9, are involved in periodontal tissue remodeling [13], MMP-1 and MMP-3 play a particularly important role because collagen types I and III are predominant in periodontal connective tissues [14]. MMP-3 is expressed in a variety of cells other than gingival fibroblasts, including monocytes, endothelial cells, chondrocytes, and synovial cells, and is known to destroy connective tissue in chronic inflammatory diseases, such as periodontitis, rheumatoid arthritis, and osteoarthritis [13,14]. TIMPs regulate MMP activity, and among the four types of TIMPs, TIMP-1 exerts a strong inhibitory effect on fibroblast-derived MMPs [56].
In the present study, the expression of MMP-1 and -3 genes was increased by stimulation with P. gingivalis, F. nucleatum, and P. intermedia. W. cibaria CMU downregulated the expression of the genes MMP-1 and -3 in a dose-dependent manner, though the degree of downregulation differed slightly between periodontal pathogens. The production of TIMP-1 was decreased by stimulation with P. gingivalis, which was reversed by treatment with W. cibaria CMU in a dose-dependent manner. These results suggest that W. cibaria CMU regulates inflammation by upregulating TIMP-1 and downregulating MMP-3 gene expression induced by P. gingivalis in HGFs.
L. reuteri is known to inhibit the growth of various periodontal bacteria, including P. gingivalis, F. nucleatum, and P. intermedia through the production of reuterin, a bacteriocin [21]. Numerous investigations have also shown that several L. reuteri strains exert their immunomodulatory activity by reducing IL-6, IL-8, and TNF-α levels [22]. L. reuteri may alleviate the inflammatory response and reduce periodontal tissue destruction by regulating the imbalance between MMPs and TIMPs or reducing the production of pro-inflammatory cytokines such as TNF-α and IL-1β [25,26]. Along with W. cibaria CMU and CMS1, we showed that live L. reuteri, a commercial oral probiotic, showed antibacterial activity against P. gingivalis. However, this strain increased the gene expression of P. gingivalis-induced pro-inflammatory cytokines (IL-6, IL-1β, and IL-8) and MMP-1 in gingival cells, as well as in the gene expression of most F. nucleatumand P. intermedia-induced pro-inflammatory cytokines and MMP-1. Therefore, L. reuteri did not have any inhibitory effects on the formation of caries and perio pathogens, indicating that the action of these probiotics is strain-specific.
Oral probiotics are known to function by colonizing the oral cavity and improving the microbiotic balance of the oral environment [38]. Therefore, commercialized products are mainly manufactured in a tablet or lozenge form that is optimized to exert its effects in the oral cavity over an extended period of time. To develop effective probiotics against periodontitis, it is also important to determine whether their anti-inflammatory effects are exerted through direct contact with gingival cells. Previous studies have shown that W. cibaria CMU requires direct contact with oral epithelial cells to inhibit F. nucleatuminduced IL-6 and IL-8 production [31]. In the present study, when using cell culture inserts, W. cibaria CMU was unable to inhibit the gene expression of pro-inflammatory cytokines and MMPs induced by periodontal pathogens when it was not in direct contact with HGFs. In addition, W. cibaria CMU failed to upregulate TIMP-1 production induced by P. gingivalis and did not inhibit MMP-1 and MMP-3 production induced by F. nucleatum and P. intermedia. Through this experiment, we confirmed that oral probiotics required direct contact with HGFs to exert their anti-inflammatory function, thereby regulating local inflammation by acting directly on gingival cells.
Further studies are needed for W. cibaria CMU to modulate the expression of protein mediators of inflammatory responses and signaling pathways. Moreover, the use of biomimetic hydroxyapatite and oral probiotics to reduce the incidence of DMFT (Decayed Missing Filled Teeth) and periodontal risk, as well as the use of ozone and photodynamic therapy to reduce the bacterial load, is yet to be explored [57].

Conclusions
This study is the first to confirm that the oral probiotic W. cibaria CMU inhibits biofilm formation by S. mutans by using an artificial tooth model. In addition, this probiotic strain was shown to act directly on gingival tissue cells to inhibit the gene expression of pro-inflammatory cytokines and MMPs induced by periodontal bacteria. These results underscore the potential use of the oral probiotic W. cibaria CMU in the proactive action against the incidence of oral diseases such as dental caries and periodontitis.

Data Availability Statement:
The data presented in this study are available upon request from the corresponding author.