Biosafety characteristics and antibacterial activity of probiotic strains against Streptococcus mutans, Aggregatibacter actinomycetemcomitans, and Porphyromonas gingivalis

Abstract

Background

Oral diseases with high prevalence worldwide are recognized as severe health problems. Probiotics are used to prevent oral diseases, including dental caries, oral malodor, periodontitis, and subgingival plaque. In this study, we aimed to confirm the antibacterial effect of probiotics on oral pathogens and to assess their characterization and safety as probiotics.

Methods

The antibacterial effects of Lacticaseibacillus rhamnosus MG4706, Lacticaseibacillus paracasei MG4715, and Limosilactobacillus reuteri MG4722 on the growth biofilm formation of Streptococcus mutans, Aggregatibacter actinomycetemcomitans, and Porphyromonas gingivalis were evaluated. We also investigated the production of antibacterial substances (H₂O₂ and reuterin) by these strains and their ability to adhere to oral epithelial cells. The safety of L. reuteri MG4722 was verified through whole-genome sequencing analysis and antibiotic susceptibility, lactate dehydrogenase activity, hemolytic activity, and bile acid hydrolase activity. The reuterin biosynthesis genes of L. reuteri MG4722 were identified using genomic analysis.

Results

L. reuteri MG4722 significantly inhibited the growth of S. mutans, A. actinomycetemcomitans, and P. gingivalis and suppressed the biofilm formation by A. actinomycetemcomitans. In addition, it showed considerable adhesion ability to oral epithelial cells. L. reuteri MG4722 produced H₂O₂ and reuterin as antibacterial substances, as confirmed by the presence of genes encoding the antibacterial compounds reuterin, reuteran, and reutericyclin. L. reuteri MG4722 showed no hemolysis, bile salt hydrolase activity, antibiotic resistance or toxicity to HT-29 cells, and no antibiotic-resistance genes were identified.

Conclusion

L. reuteri MG4722 demonstrated antibacterial effects on oral pathogens by producing antibacterial substances and adhering to oral epithelial cells. These results suggest that L. reuteri MG4722 could be an effective probiotic for oral health.

Background

Oral diseases are highly prevalent worldwide and are recognized as major health issues (Peres et al. 2019). According to the Centers for Disease Control and Prevention (CDC), more than 40% of adults experience oral discomfort and spend billions of dollars annually on treatment (Mann et al. 2021). More than 600 species of microorganisms are present in the oral cavity, and bacterial infections are responsible for most oral diseases. Various bacteria present in the oral cavity directly infiltrate vascular endothelial cells or damaged blood vessels and attach to specific organs, ultimately causing systemic diseases (Buchbauer et al. 1991).

Oral diseases include periodontal disease, dental caries, and halitosis (Haraguchi et al. 2014; Izidoro et al. 2022). Periodontal disease is a bacterial-induced inflammatory disease that destroys the tissue around teeth and is the leading cause of tooth loss during adulthood (Sang-Ngoen et al. 2021). The main bacteria associated with periodontal diseases are Actinobacillus actinomycetemcomitans (A. actinomycetemcomitans), Porphyromonas gingivalis (P. gingivalis), and Treponema denticola. A. actinomycetemcomitans is a gram-negative bacterium closely related to periodontitis, tooth loss, and neoplastic lesions (Damgaard et al. 2017). P. gingivalis is a gram-negative anaerobic bacterium, which initiates and progresses periodontal disease by decomposing proteins. Dental caries is a highly preventable disease worldwide that damages the calcified structure of tooth enamel (Chen et al. 2020). Streptococcus mutans (S. mutans) is a gram-positive facultative anaerobic bacterium that has long been considered pathogenic in dental caries and destroys tissues by excreting toxins or secondary products into periodontal tissues (Palombo 2011; Kulik et al. 2019). Halitosis is a disagreeable smell discharged from oral or nasal passages (Murata et al. 2002). Approximately 90% of cases of halitosis are attributed to conditions within the oral region, nasal cavity, upper respiratory tract, and upper digestive tract (Renvert et al. 2020). The main cause of halitosis is methyl mercaptan (CH₃SH), and the proportion of CH₃SH is relatively high in individuals with periodontal disease (Loesche and Kazor 2002). These compounds are synthesized by P. gingivalis (Kang et al. 2006).

Mouthwashes are commonly used to suppress pathogens in the oral cavity (Lee et al. 2021). However, these antibacterial substances can lead to an imbalance in the oral microbiome, eliminating beneficial bacteria and potentially leading to resistance (Kim et al. 2020). Probiotics can reduce and prevent oral diseases while resolving these side effects (How and Yeo 2021). Probiotic bacteria, such as Lacticaseibacillus rhamnosus, Limosilactobacillus reuteri, and Lacticaseibacillus paracasei, can rapidly colonize the oral cavity (Jiang et al. 2020). L. rhamnosus is safe for teeth and has been extensively studied as an oral probiotics (Elgamily et al. 2018). Studies on probiotics, such as L. paracasei and L. reuteri, have been conducted to prevent oral diseases or alleviate symptoms such as dental caries, oral malodor, periodontitis, and subgingival plaque, and L. reuteri has been reported to reduce the proportion of anaerobic bacteria in patients with chronic periodontitis (How and Yeo 2021).

In this study, we investigated the antibacterial effects and biological safety of L. rhamnosus, L. paracasei, and L. reuteri strains of oral origin against oral pathogens.

Materials and methods

Preparation of cell-free supernatant (CFS) of probiotic strains

L. rhamnosus MG4706, L. paracasei MG4715, and L. reuteri MG4722 were isolated from the oral cavity of a healthy human. Probiotic strains were confirmed by 16S rRNA gene sequencing (SolGent Co., Ltd., Daejeon, Republic of Korea) and registered on the NCBI database using BLAST (Table 1). Probiotics were cultured in de Man, Rogosa, and Sharp (MRS) broth (BD Bioscience, Franklin Lakes, NJ, USA) at 37°C for 24 h. Subsequently, the microbial load of probiotics was adjusted to an OD₆₀₀ of 1.0 (10⁸ CFU/mL) and subcultured at 37°C for 24 h. Cell-free supernatant (CFS) was obtained via centrifugation at 4,000 × g for 15 min at 4°C, adjusted to pH 7.4, and filtered using a 0.22-µm polytetrafluoroethylene membrane filter (ADVANTEC, Tokyo, Japan).

Table 1 Accession numbers and origins of probiotic strains used in this study

Bacterial cultures and antibacterial activity against oral pathogens

S. mutans KCTC3065, A. actinomycetemcomitans KCTC2581, and P. gingivalis KCTC5352 were purchased from the Korean Collection for Type Cultures (KCTC, Republic of Korea). S. mutans and A. actinomycetemcomitans were spread on a brain heart infusion (BHI) agar (Difco) plate and cultured at 37°C for 48 h. Single colony formed on the plate of S. mutans was cultured in BHI broth (Difco) for 24 h, adjusted to OD₆₀₀ of 1.0 (1 × 10⁸ CFU/mL), inoculated onto a 96-well plate (2 × 10⁵ CFU/180 µL/well), treated with 10% CFS (20 µL), and incubated at 37°C for 24 h. A single colony formed on the plate of A. actinomycetemcomitans was cultured in BHI broth for 24 h, adjusted to an OD₆₀₀ of 1.0 (1 × 10⁸ CFU/mL), inoculated onto a 96-well plate (2 × 10⁶ CFU/180 µL/well), treated with 10% CFS (20 µL), and incubated at 37°C for 48 h. P. gingivalis was spread on Tryptic soy agar (TSA) containing 5 µg/mL hemin, 1 µg/mL vitamin K₁, and 5% sheep blood plate and cultured at 37°C for 7 days. Colonies formed on the plate were transferred on half-BHI medium containing yeast extract (5 mg/mL), hemin (5 µg/mL), and vitamin K₁ (1 µg/mL), adjusted to an OD₆₀₀ of 1.0 (10⁸ CFU/mL), inoculated onto 96-well plates (2 × 10⁶ CFU/180 µL/well), treated with 10% CFS (20 µL), and incubated at 37°C for 4 days. Culture conditions for each strain were established through previous studies, and all strains were cultured under anaerobic conditions. The inhibitory effect of oral pathogens was assessed by measuring the absorbance at 600 nm using a microplate reader (BioTek, Winooski, VT, USA).

Biofilm formation

Biofilm formation by pathogens was assessed using crystal violet staining, as previously described, with some modifications (Zanetta et al. 2023). S. mutans was cultured in BHI broth for 24 h, adjusted to an OD₆₀₀ of 1.0 (1 × 10⁸ CFU/mL), inoculated onto a 96-well plate (1 × 10⁴ CFU/180 µL/well), and cultured under anaerobic conditions for 12 h. Subsequently, 10% CFS (20 µL) was treated for an additional 24 h. A. actinomycetemcomitans was cultured in BHI broth for 24 h, adjusted to OD₆₀₀ of 1.0 (1 × 10⁸ CFU/mL), inoculated onto a 96-well plate (1 × 10⁷ CFU/180 µL/well), and cultured under anaerobic conditions for 24 h. Subsequently, 10% CFS (20 µL) was added for an additional 24 h. P. gingivalis was suspended in half-BHI broth, adjusted to an OD₆₀₀ of 1.0 (1 × 10⁸ CFU/mL), and inoculated onto a 96-well plate (2 × 10⁶ CFU/180 µL/well). After 5 days, the cells were treated with 10% CFS (20 µL) for 24 h. After cultivation, the pathogens were washed at least twice with distilled water and allowed to air dry. The pathogens were stained with 0.1% crystal violet for 2 min, washed thrice with distilled water, air-dried, and dissolved in 95% ethanol. The absorbance at 575 nm was measured using a microplate reader. Biofilm formation was calculated using the following equation: $\:Biofilm\:formation\:\left(\%\right)=100-\:\left[\frac{\left(ODcontrol-ODblank\right)-\:\left(ODsample-ODblank\right)}{ODcontrol-ODblank}\times\:100\right]$

OD_blank, Microbial culture medium; OD_control, Microbial suspension; OD_sample, Sample treated microbiological suspension.

Hydrogen peroxide (H2O2) production

The H₂O₂ production by the strains was evaluated using 3,3’,5,5’-tetramethyl-benzidine (TMB)-MRS agar plates. The plates were prepared by autoclaving and drying MRS agar supplemented with 1.0 mM TMB and 10 µg/mL peroxidase (Sigma-Aldrich, St. Louis, MO, USA). The probiotics were suspended in MRS broth, spread on TMB-MRS agar plates, and then incubated under anaerobic conditions at 37°C for 48 h. Then, the plates were exposed to ambient air for 2 h to verify H₂O₂ production by the probiotic strains. The presence of H₂O₂ was indicated by the consumption of peroxidase, which catalyzes the oxidation of TMB, resulting in a blue coloration (Park et al. 2023).

Reuterin production

Reuterin was quantified using a colorimetric method, as previously reported, with some modifications (Cadieux et al. 2008). L. reuteri MG4722 was cultured at 37°C for 24 h and transferred to 300 mM glycerol under anaerobic conditions for 3 h. CFS was obtained by centrifugation at 4000 × g for 15 min at 4°C and filtered using a 0.22-µm polytetrafluoroethylene membrane filter. The supernatant (300 µL) was mixed with 10 mM tryptophan (225 µL), and 12 N HCl (900 µL) was added. After 30 min of incubation at 37°C, the absorbance at 450 nm was measured using a microplate reader. A standard curve was prepared using acrolein (AccuStandard, Inc., New Haven, CT, USA).

Adhesion assay on oral epithelial cells

The ability of the probiotic strains to adhere to mouth epidermal carcinoma (KB) cells was assessed as previously described, with some modifications (Park et al. 2023). Briefly, KB cells (Korea Cell Line Bank) were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin-streptomycin (P/S; Gibco). Briefly, KB cells were seeded in 24-well plates (2.0 × 10⁵ cells/well) and incubated for 48 h to form a monolayer. Subsequently, cells were treated with probiotic strains (1 × 10⁸ CFU/mL) for 2 h, washed thrice, and lysed with 10 mM phosphate-buffered saline (PBS, pH 7.4). The adhesion rate (%) was determined through colony counts on the MRS agar plates and calculated using the following equation:

$$\:Adhesion\:rate\:\left(\%\right)=\:\frac{\text{log}\left(adherent\:counts\right)CFU/mL}{\text{log}\left(initial\:counts\right)CFU/mL}\times\:100$$

Initial count, initial bacterial count before attachment to cells; adherent counts, bacterial count after washing.

Antibiotic susceptibility

The antibiotic resistance of L. reuteri MG4722 was determined using the minimum inhibitory concentrations (MICs) of antibiotics (ampicillin, chloramphenicol, clindamycin, erythromycin, gentamicin, kanamycin, streptomycin, and tetracycline). L. reuteri MG4722 was cultured in MRS broth at 37°C for 18 h, harvested by centrifugation at 4000 × g for 10 min at 4°C, and washed twice with 10 mM PBS (pH 7.4). Cells were resuspended in PBS at a McFarland standard turbidity of 0.5 and inoculated onto Mueller-Hinton agar and LAB susceptibility test medium (LSM; 90% Iso-Sensitest broth, 10% MRS broth, and 1.7% agar). MIC test strips (Liofilchem, Inc., Roseto degli Abruzzi, Italy) were placed on the plate according to the manufacturer’s instructions and incubated at 37°C for 24 h. Antibiotic susceptibility was determined according to the European Food Safety Authority (EFSA) guidelines (FEEDAP et al. 2018).

Cytotoxicity

HT-29 cells (Korea Cell Line Bank) were cultured in 96-well plates (2.5 × 10⁴ cells/well) in DMEM with 10% FBS and 1% P/S at 37°C under 5% CO₂ for 24 h. L. reuteri MG4722 (10⁶−10⁸ CFU/mL) was treated for 24 h. Cytotoxicity of L. reuteri MG4722 was determined using a Quanti-LDH PLUS cytotoxicity assay kit (Biomax, Seoul, Republic of Korea) following the manufacturer’s instruction. Cytotoxicity was calculated using the following equation:

$$\:Cytotoxicity\:\left(\%\right)=100-\left[\frac{\left(Sample-Low\:control\right)}{\left(High\:control-Background\:control\right)}\times\:100\right]$$

Low control, cell culture supernatant of cells only; High control, cell culture supernatant of cells after lysis; Background control, medium only.

Hemolytic activity

Hemolytic activity was determined using TSA containing 5% sheep blood (MBcell, Seoul, Republic of Korea). L. reuteri MG4722 was grown in MRS broth, streaked onto a TSA plate, and incubated for 48 h at 37°C. After 24 h, hemolytic activity was determined by evaluating the presence or absence of hemolysis around the colonies (Yasmin et al. 2020).

Bile salt hydrolase (BSH) activity

BSH activity was determined as previously described (Lee et al. 2023). L. reuteri MG4722 was grown in MRS broth, streaked onto a taurodeoxycholic acid hydrate (Sigma-Aldrich) agar plate, and incubated for 48 h at 37°C. BSH activity was assessed by examining the appearance of colonies surrounded by precipitated zones.

Morphology

L. reuteri MG4722 was cultured in MRS broth for 24 h, washed twice with PBS, and lyophilized for use. The morphology of L. reuteri MG4722 was assessed using a field emission-scanning electron microscope (SU5000 FE-SEM; Hitachi, Tokyo, Japan) as previously reported, with some modifications (Green Buzhor et al. 2024). The samples were prepared by vacuum-coating with a platinum bilayer. The surface and cross-sectional images of the strains were obtained at an acceleration voltage of 3.0 kV.

Whole genome sequencing (WGS)

The genomic DNA of L. reuteri MG4722 was extracted using a PureLink™ Microbiome DNA purification kit (Invitrogen, MA, USA) according to the manufacturer’s instructions. A DNA library was prepared using a TruSeq Nano DNA library prep kit (Illumina, Inc., San Diego, CA, USA). WGS was performed using an Illumina Novaseq6000 instrument (Illumina, Inc.) for 2 × 150-bp sequencing on an Illumina platform by a certified service provider (DNA Link, Inc., Republic of Korea). The gene prediction of the coding sequences (CDS), ribosomal RNA (rRNA), and transfer RNA (tRNA) in the assembled gene was performed using Prokka v1.13. Gene annotation was performed using Blast2GO (BioBam Bioinformatics, Valencia, Spain).

Gene annotation was also reanalyzed according to the prokaryotic genome annotation pipeline. To identify the species based on genomic sequences, average nucleotide identity (ANI) values between L. reuteri MG4722 and several reference-type strains were compared using JSpecies v.1.2.1. Additionally, virulence factors were identified through homology searches using the Virulence Factor Database (VFDB) as a reference. The secondary metabolite biosynthetic gene cluster of L. reuteri MG4722 was identified using the antiSMASH ver. 7.0 database.

Statistical analysis

All results are expressed as mean ± standard error of the mean (SEM) of three independent measurements. Normal distribution was verified using the Shapiro-Wilk test before further statistical analysis. In case the groups were normally distributed, a one-way analysis of variance (ANOVA) followed with the Dunnett’s multiple comparisons test was performed. In case the groups were not normally distributed, the results were analyzed using the Kruskal–Wallis test followed with the Dunn’s multiple comparisons to compare more than two group calculations. Statistical analysis was performed using Prism (ver. 10.4.0; GraphPad Software, San Diego, CA, USA). Statistical significance was set at p < 0.05.

Results

Growth and biofilm inhibitory effect of probiotic strains against oral pathogens

We investigated whether CFS of probiotic strains affected the growth of S. mutans, A. actinomycetemcomitans, and P. gingivalis. The growth of S. mutans was significantly inhibited by 31.4–42.2% by all strains, whereas the growth of A. actinomycetemcomitans and P. gingivalis was inhibited by 19.6% and 40%, respectively, by L. reuteri MG4722 (Fig. 1A).

Fig. 1

Antimicrobial activity of LAB strains against S. mutans, A. actinomycetemcomitans, and P. gingivalis. Growth rate (A) and Biofilm formation (B). All values are represented as mean ± SEM (n = 3). Significant differences indicate the means at ^* p < 0.05, ^** p < 0.01, and ^*** p < 0.001 with Dunnett’s multiple comparisons test

We confirmed the antibiofilm activity of probiotic strains, showing that L. rhamnosus MG4706 and L. paracasei MG4715 significantly inhibited the biofilm formation by S. mutans (p < 0.05). All of the tested strains considerably inhibited biofilm formation by more than 50%, with L. reuteri MG4722 showing the highest inhibition of 80% (p < 0.001) against A. actinomycetemcomitans. L. reuteri MG4722 reduced the biofilm formation by P. gingivalis; however, the difference was not statistically significant (Fig. 1B).

Antibacterial substance (H₂O₂ and Reuterin) production of probiotic strains

We investigated whether the probiotic strains produce antibacterial substances. L. reuteri MG4722 showed H₂O₂ bioactivity. However, L. rhamnosus MG4706 and L. paracasei MG4715 did not produce H₂O₂ (Fig. 2). In addition, L. reuteri MG4722 showed a colorimetric change to blue, indicating the presence of reuterin. Table 2 shows the contents of H₂O₂ and reuterin produced by L. reuteri MG4722.

Fig. 2

H₂O₂ production of probiotic strains. The blue colonies on the TMB agar would be categorized as H₂O₂-positive

Table 2 Production of antimicrobial substances of L. reuteri MG4722

Adhesion ability of probiotic strains to oral epithelial cells

We confirmed the LAB strain’s ability to adhere to oral epithelial KB cells. All strains showed high adhesion to oral epithelial cells, with a range of 86.05–93.49% (Table 3).

Table 3 Adhesion of LAB strains on oral epithelial KB cells

Antibiotic susceptibility of L. Reuteri MG4722

We confirmed the antibiotic susceptibility of L. reuteri MG4722. As shown in Table 4, the MICs of all eight antibiotics against L. reuteri MG4722 were lower than the cut-off values in the EFSA guidelines. These results indicate that L. reuteri MG4722 is safe as a probiotic.

Table 4 Antibiotic susceptibility of L. reuteri MG4722

Morphology and safety of L. Reuteri MG4722

In terms of morphology, L. reuteri MG4722 had a short rod-shaped form (Fig. 3A). We determined the safety of L. reuteri MG4722 by assessing the cytotoxicity on HT-29 cells, and its hemolytic and BSH activities. L. reuteri MG4722 showed no cytotoxicity on HT‐29 cells (Fig. 3B), no hemolytic activity (γ-hemolysis) on the host, and no BSH activity (Fig. 3C and D).

Fig. 3

Morphology and safety of L. reuteri MG4722. SEM micrographs (A), cytotoxicity (B), hemolysis (C), and BSH activity (D) of L. reuteri MG4722. SEM image showing the surface morphology of L. reuteri MG4722. Imaging was conducted at 10,000 x magnification with an accelerating voltage of 3 kV. The scale bar represents 5 µm. HT‐29 cells were treated with L. reuteri MG4722 (10⁶-10⁸ cells/mL). Data are presented as the mean ± SEM (n = 3)

Genome analysis of L. Reuteri MG4722

Genomic analysis of L. reuteri MG4722 indicated a single circular chromosome of 1,943,662 bp and a GC content of 38.93% (Fig. 4). The chromosome of L. reuteri MG4722 (contig 1) contained 1,925 CDS, 15 rRNA genes (five each of the 5 S, 16 S, and 23 S rRNA operons), and 69 tRNA genes. A DNA plot was used to illustrate the structural and functional features of contig 1 and the chromosome of L. reuteri MG4722. The ANI analysis confirmed that the strain was an L. reuteri species, with 99.81% similarity to L. reuteri JCM1112 as the type strain using Jspecies ver 1.2.1 (Table 5).

Fig. 4

Genomic map of L. reuteri MG4722. Marked genome characteristics are shown from outside to the center: CDS on the forward strand, CDS on the reverse strand, tRNA, rRNA, GC content, and GC skew

Table 5 ANI values between the genome of type strains belong to the genus Limosilactobacillus

In addition, no antibiotic-resistance genes were found in L. reuteri MG4722, as confirmed using ResFinder DB.

The search for secondary metabolite biosynthesis gene clusters using the AntiSmash database did not reveal any gene clusters containing bacteriocin, type 1 polyketide synthases (PKS), non-ribosomal peptide synthetases (NRPS), or post-translationally modified peptides (RiPPs).

Presence of the gene encoding reuterin

We noticed that the gene encoding reuterin was present in the genome of L. reuteri MG4722. The gene for reuterin biosynthesis genes in L. reuteri MG4722 was identified as containing the pdu-cbi-cob-hem cluster (Fig. 5; Table 6). Genomic analysis revealed that L. reuteri MG4722 produces reuterin, which is consistent with the results shown in Table 2.

Fig. 5

Reuterin biosynthesis gene cluster comparison containing pdu-cbi-cob-heme gene cluster in L. reuteri MG4722, L. reuteri JCM1112, and L. reuteri SD2112. The arrows indicate the transcription direction in the pdu-cbi-cob-heme gene cluster, each with the same color. The blue arrows represent genes that are involved in the glycerol and propanediol utilization (pdu); The yellow and green arrows represent genes that are cobalamin biosynthesis (cbi-cob), respectively; The red arrows represent hem genes; The grey arrows are not related to reuterin production

Table 6 Predicted functions of the genes from L. reuteri MG4722 containingpdu-cbi-cob-heme gene cluster

Discussion

Numerous microorganisms are distributed throughout the human body, and several studies have reported a relationship between these microorganisms and human health (Nie et al. 2023). The oral cavity, which contains the second-largest number of microorganisms in the human body, is estimated to accommodate a diverse group of microorganisms, including bacteria, fungi, and viruses (Shoemark and Allen 2015). Oral health is generally affected by health status, nutritional status, lifestyle, and composition of the oral microbiome (Mahasneh and Mahasneh 2017). Biofilm formation is a type of periodontal disease initiated by oral pathogens that form complex structures on the tooth surface and destroy tooth-supporting tissues (How et al. 2016). Gingipain, a proteolytic enzyme of Porphyromonas gingivalis, is a major virulence factor responsible for causing periodontal disease and can help the survival by interacting with other species, including Treponema denticola and Tannerella forsythia (Bao et al. 2014). In the present study, we investigated the antibacterial activities of Lactobacillus strains against oral pathogens. L. reuteri MG4722 significantly inhibited the growth of S. mutans, A. actinomycetemcomitans, and P. gingivalis and biofilm formation by A. actinomycetemcomitans.

Probiotics can help to improve oral health by maintaining homeostasis of the oral microbiota and competing for binding sites with harmful microorganisms, thereby showing preventive and therapeutic effects against pathogenic bacteria (Nie et al. 2023). Probiotics also regulate immune responses and secrete substances with antibacterial properties (Gungor et al. 2015). Probiotics exert their antibacterial effects by producing organic acids that can inhibit pathogens or secreting compounds with antibacterial properties (Lee et al. 2013). Probiotics can produce H₂O₂ through electron transport, causing peroxidation of lipids and increasing membrane permeability, thereby destructing nucleic acids and cellular proteins of bacteria (Naidu et al. 1999). In this study, we confirmed the H₂O₂ production ability of L. reuteri MG4722, suggesting that this strain may have antibacterial properties.

Probiotics prevent the attachment of harmful bacteria and subsequent infections through their ability to adhere (Mann et al. 2021). The ability to adhere to oral epithelial cell monolayers has been used to increase the number of beneficial bacteria [2]. The Lactobacillus genus generally prevents pathogen attachment through the adhesion factors, including cholic acid and surface layer proteins, on the cell surface and may play an important role in subsequent immune regulation (Kaźmierczyk-Winciorek et al. 2021). In this study, L. reuteri MG4722 showed a high ability to adhere to KB epithelial cells, suggesting that its antibacterial activity by inhibiting the adhesion of harmful oral bacteria.

The safety of a strain to be used as a probiotic must be thoroughly evaluated, including by investigating its antibiotic resistance and virulence factors (Ruiz-Ramírez et al. 2023). According to the EFSA guidelines, ingestible probiotic must be assessed for resistance to antibiotics such as gentamicin, kanamycin, streptomycin, tetracycline, erythromycin, clindamycin, chloramphenicol, ampicillin, and vancomycin. In addition, probiotic strains must establish a comprehensive genetic evaluation to confirm the absence of acquired or transferable antibiotic resistance determinants and assess their genomic stability (Campedelli et al. 2019). In our study, L. reuteri MG4722 satisfied the safety requirements of the EFSA cut-off values. In addition, antibiotic-resistance genes were not detected in L. reuteri MG4722.

According to the safety evaluation guidelines of FAO/WHO, probiotic strains must be confirmed for safety, including BSH activity, hemolytic activity, and toxicity (Lee et al. 2023). Hemolytic activity is an important indicator, and the hemolytic properties of bacteria can cause cell lysis and dissolution of hemoglobin (Bitschar et al. 2017; Liu et al. 2021). BSH activity lowers cholesterol; however, excess cholesterol lowering can cause lipid dyspepsia and impair colonic mucosal function, potentially leading to gallstone formation (Lee et al. 2023). In this study, L. reuteri MG4722 did not exhibit hemolytic activity, BSH activity, or cytotoxicity in HT-29 cells.

WGS can be used to study the functional aspects of microorganisms by sequencing their entire genomes and comparing them with previously identified genetic information (Klaenhammer 1988). Genomes with ANI values exceeding 95% are classified as representing the same species (Kim et al. 2014; Greppi et al. 2020). L. reuteri MG4722 was confirmed by comparing with L. reuteri JCM 1112 (ANI 99.81%). Probiotics with high levels of antibiotic resistance may pose safety concerns because antibiotic-resistant genes may be transmitted (Zhang et al. 2018). L. reuteri MG4722 confirmed that no antibiotic-resistance genes were identified and that pathogen transfer is impossible.

Lactobacillus spp. produce various metabolites that protect against colonization by oral periodontal pathogens (Wasfi et al. 2018). L. reuteri has high potential for application as a natural antibacterial agent to prevent pathogenic infections and increase beneficial bacteria through its high persistence and antibacterial activity (Greppi et al. 2020). L. reuteri AN417 inhibits the growth and biofilm formation of oral pathogens (Yang et al. 2021). Additionally, oral tablets containing L. reuteri reduce periodontal pathogens and improve halitosis in clinical trials (Kaźmierczyk-Winciorek et al. 2021). The primary antibacterial compounds produced by L. reuteri are organic acids, hydrogen peroxide, reuterin, reuteran, and reutericyclin (Yang et al. 2021). Reuterin, also known as 3-hydroxy propionaldehyde, is an important compound produced by L. reuteri for inhibiting the growth of pathogens, and its expression is regulated by the pdu-cbi-cob-hem cluster consisting of 58 genes (Lee et al. 2017). In this study, L. reuteri MG4722 was found to have the complete pdu-cbi-cob-hem operon, a reuterin biosynthesis gene. L. reuteri MG4722 did not possess secondary metabolite biosynthetic gene clusters containing bacteriocins, PKS, NRPS, and RiPP. We also confirmed that reuterin was produced in L. reuteri MG4722. In L. reuteri MG4722, only the pdu-cbi-cob-heme gene cluster was found, suggesting that its antibacterial activity may be related to reuterin production.

Conclusion

L. reuteri MG4722 exhibits antibacterial efficacy by attaching to the oral epithelium and secreting antibiotics, such as H₂O₂ and reuterin, into the oral cavity. L. reuteri MG4722 exhibited significant adhesion ability to oral epithelial cells and produced antibacterial substances such as H₂O₂ and reuterin, supported by identifying corresponding biosynthetic genes. Safety assessments confirmed the absence of hemolytic activity, bile salt hydrolase activity, and antibiotic resistance, with no detectable toxicity. Therefore, L. reuteri MG4722 is a potential probiotic candidate for oral hygiene and could be proposed as a functional food or therapeutic agent for oral health. In future studies, the efficacy of L. reuteri MG4722 should be validated in animal models and clinical trials.