In Vitro Effects of Weissella cibaria CMU and CMS1 on Receptor Activator of NF-κB Ligand (RANKL)-Induced Osteoclast Differentiation.

Excessive osteoclast activity can promote periodontitis-associated bone destruction. The inhibitory mechanisms of Weissella cibaria strains CMU and CMS1 against periodontitis have not yet been fully elucidated. In this study, we aimed to investigate whether heat-killed (HK) W. cibaria CMU and CMS1 or their respective cell-free supernatants (CFSs) inhibit osteoclast differentiation and bone resorption in response to receptor activator of nuclear factor kappa-B ligand (RANKL)-treated RAW 264.7 cells. TRAP (tartrate-resistant acid phosphatase) staining and bone resorption assays revealed that both HK bacteria and CFSs significantly suppressed the number of TRAP-positive cells, TRAP activity, and bone pit formation compared to the RANKL-treated control (p < 0.05). HK bacteria dose-dependently inhibited osteoclastogenesis while selectively regulating certain genes in CFSs (p < 0.05). We found that disrupting the direct interaction between HK bacteria and RAW 264.7 cells abolished the inhibitory effect of HK bacteria on the expression of osteoclastogenesis-associated proteins (c-Fos, nuclear factor of activated T cells c1 (NFATc1), and cathepsin K). These results suggest that dead bacteria suppress osteoclast differentiation more effectively than the metabolites and may serve as beneficial agents in preventing periodontitis by inhibiting osteoclast differentiation via direct interaction with cells.

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-1ß, 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.

Therapeutic Efficacy of Weissella cibaria CMU and CMS1 on Allergic Inflammation Exacerbated by Diesel Exhaust Particulate Matter in a Murine Asthma Model.

Background and Objectives: Diesel exhaust particulate matter (DEPM) is an air pollutant that is associated with asthma. In this study, the therapeutic efficacy of Weissella cibaria strains CMU (Chonnam Medical University) and CMS (Chonnam Medical School) 1, together with the drug Synatura, an anti-tussive expectorant, was investigated in a murine asthma model exacerbated by DEPM. Materials and Methods: BALB/c mice were sensitized with ovalbumin (OVA) before intranasal challenge with OVA and DEPM. W. cibaria CMU, CMS1, and Synatura were administered orally for 21 days. Results: Neither Synatura nor W. cibaria strains affected spleen, liver, or lung weights. W. cibaria strains CMU and CMS1 significantly reduced the levels of interleukin (IL)-4, OVA-specific immunoglobulin E (IgE), and total lung collagen in bronchoalveolar lavage fluid (BALF), similar to those with Synatura, regardless of the oral dose concentration (p < 0.05). In addition, the W. cibaria CMU strain significantly alleviated IL-1ß, IL-6, IL-12, monocyte chemotactic protein-1, and tumor necrosis factor-α in BALF, whereas the CMS1 strain significantly alleviated IL-10 and IL-12 in BALF (p < 0.05); however, Synatura did not show any statistical efficacy against them (p > 0.05). All concentrations of W. cibaria CMU and low concentrations of W. cibaria CMS1 significantly reduced lung bronchiolar changes and inflammatory cell infiltration. Conclusions: In conclusion, W. cibaria CMU in asthmatic mice showed better efficacy than W. cibaria CMS1 in improving asthma exacerbated by DEPM exposure, as well as better results than pharmaceuticals.

Toxicological and safety evaluations of Weissella cibaria strain CMU in animal toxicity and genotoxicity.

Weissella cibaria belongs to the Lactobacillaceae family and has been isolated from traditional fermented foods and saliva of children with good oral health. Previous investigations have shown that W. cibaria CMU (Chonnam Medical University) is expected to be safe based on results of in silico and in vitro analyses. However, there is a lack of studies assessing its safety in vivo. A toxicological safety evaluation of W. cibaria CMU was performed using an acute oral safety study in rats, a 14-day oral range finding study, a subsequent 13-week oral toxicity study in rats and a genetic toxicity battery (in vitro bacterial reverse mutation, in vitro chromosome aberration in Chinese Hamster Ovary cells and in vivo micronucleus study in mice). The results of the studies in rats showed that the acute lethal dose of W. cibaria CMU is > 5000 mg/kg body weight (bw)/day (1.8 × 109 CFU/kg bw/day) and the 14-day or 13-week no observed adverse effect level (NOAEL) is 5000 mg/kg bw/day (1.8 × 109 CFU/kg bw/day), the highest dose administered. W. cibaria CMU was non-mutagenic in the bacterial reverse mutation test and non-clastogenic or aneugenic in vitro and in vivo. In conclusion, the toxicological studies performed demonstrated W. cibaria CMU to be a safe strain to consume. This study is the first study examining the potential of a W. cibaria strain to cause genetic toxicity and subchronic toxicity in rats according to the Organization for Economic Cooperation and Development guidelines.

In Vitro Inactivation of Respiratory Viruses and Rotavirus by the Oral Probiotic Strain Weissella cibaria CMS1.

Weissella cibaria CMS1 (oraCMS1) has been commercially used in Korea as an oral care probiotic for several years. Human respiratory syncytial virus (RSV) and the influenza A virus (H1N1) are representative viruses that cause infantile lower respiratory tract infections. Rotavirus A (RVA) is the most common cause of diarrhea in infants and young children. Here, we aimed to evaluate the efficacy of the cell-free supernatant (CFS) of oraCMS1 in inactivating RSV, H1N1, and RVA in suspension as per ASTM (American Society for Testing and Materials) E1052-20. The mixture of oraCMS1 and these viruses was evaluated at contact times of 1, 2, and 4 h. Virucidal activity was measured using a 50% tissue culture infective dose assay (log10TCID50) after infecting the host cells with the viruses. The CFS of oraCMS1 inactivated RSV by up to 99.0% after 1 h and 99.9% after 2 and 4 h, and H1N1 and RVA were inactivated by up to 99.9% and 99.0% at 2 h, respectively. Although these in vitro results cannot be directly interpreted as implying clinical efficacy, our findings suggest that oraCMS1 provides a protective barrier against RSV, H1N1, and RVA, and therefore, it can help decrease the risk of respiratory tract and intestinal infections.

In Vitro Evaluation of the Effect of Oral Probiotic Weissella cibaria on the Formation of Multi-Species Oral Biofilms on Dental Implant Surfaces.

Oral probiotics are beneficial bacteria that can help prevent periodontal disease. However, little is known about the effects of oral probiotics on the formation of implant biofilms. This study aimed to evaluate the effects of oral probiotics Weissella cibaria CMU and CMS1 in an in vitro complex biofilm model on titanium implant surfaces. First, it was identified through colony biofilm assay that W. cibaria CMU and CMS1 inhibit the formation of multi-species biofilms formed by eight types of bacteria. Two types of saliva-coated titanium discs inoculated with early (Streptococcus gordonii, Streptococcus oralis, Streptococcus sanguinis, Actinomyces naeslundii, and Veillonella parvula), secondary (Fusobacterium nucleatum and Prevotella intermedia), and late (Porphyromonas gingivalis) colonizers were treated with the oral probiotics and then incubated anaerobically for three days. The effects of oral probiotics on titanium disc biofilm formation were analyzed using culture methods, quantitative polymerase chain reaction (qPCR), and microscopic analysis. Both probiotics significantly inhibited the formation of biofilm, and all eight bacterial species were significantly reduced. The effectiveness of both probiotic strains was confirmed by all the methods used. Oral probiotics may have dramatically reduced the biofilm formation of secondary colonizers that act as bridges, thus inhibiting biofilm formation on the titanium surface. Our results suggest that the probiotic W. cibaria offers new possibilities for the prevention of peri-implant mucositis.

Antimicrobial and Antibiofilm Activities of Weissella cibaria against Pathogens of Upper Respiratory Tract Infections.

Recently discovered preventive effects of probiotics on oral health have attracted interest to their use for the prevention and treatment of various diseases. This study aimed to evaluate the antimicrobial and antibiofilm properties of Weissella cibaria against Streptococcus pyogenes, Staphylococcus aureus, S. pneumoniae, and Moraxella catarrhalis, the major pathogens of upper respiratory tract infections (URTIs). The antimicrobial activities of W. cibaria were compared with those of other oral probiotics using a competitive inhibition assay and the determination of the minimum inhibitory concentrations (MICs). In addition, a time-kill assay, spectrophotometry, and confocal laser scanning microscopy were used to confirm the antimicrobial and antibiofilm abilities of W. cibaria CMU (oraCMU) and CMS1 (oraCMS1). Both live cells and cell-free supernatants of all tested probiotics, except Streptococcus salivarius, showed excellent antimicrobial activities. All target pathogens were killed within 4 to 24 h at twice the MIC of oraCMU and oraCMS1, which showed the highest antimicrobial activities against M. catarrhalis. The antimicrobial substances that affected different target pathogens were different. Both oraCMU and oraCMS1 showed excellent abilities to inhibit biofilm formation and remove preformed biofilms. Our results suggest that the W. cibaria probiotics offer new possibilities for the prevention and treatment of bacterial URTIs.