Dual probiotic intervention with Lactiplantibacillus plantarum Kmiate-6 and Pediococcus acidilactici Kmiate-15 improves periodontal health and oral microbiota balance in dogs

Abstract

Background

Periodontal disease is highly prevalent in dogs, yet current treatments such as dental scaling and antibiotics carry limitations including anesthesia risks, high recurrence, and antibiotic resistance. Probiotics have emerged as a promising strategy to modulate oral microbiota and host inflammation. This study evaluated the effects of dual probiotic intervention with Lactiplantibacillus plantarum Kmiate-6 and Pediococcus acidilactici Kmiate-15 on canine oral health through in vitro and in vivo assays.

Results

Both strains exhibited antimicrobial activity against common oral pathogens, with Lactiplantibacillus plantarum Kmiate-6 showing stronger inhibition against Streptococcus mutans, Fusobacterium nucleatum, and Streptococcus mitis, while Pediococcus acidilactici Kmiate-15 demonstrated superior anti-inflammatory effects by reducing tumor necrosis factor-alpha and increasing interleukin-10 production. In vivo, 21-day oral administration of the probiotic combination significantly reduced dental plaque area and plaque index in dogs, along with decreased salivary tumor necrosis factor-alpha and elevated interleukin-10 levels. Microbiota analysis showed that probiotic intervention decreased the abundance of pathogenic genera such as Fusobacterium and Porphyromonas, and enriched beneficial taxa including Actinomyces and Weissella. Functional predictions based on 16S ribosomal RNA gene data indicated that probiotic treatment enhanced pathways related to detoxification, antioxidation, and propanoate metabolism, while reducing reliance on vitamin biosynthesis pathways typically exploited by pathogens.

Conclusions

Combined oral administration of Lactiplantibacillus plantarum Kmiate-6 and Pediococcus acidilactici Kmiate-15 effectively improved canine periodontal health by inhibiting plaque formation, modulating inflammatory responses, and reshaping oral microbial composition and function. These findings support the potential of probiotic-based strategies as safe and effective alternatives for maintaining oral health in dogs.

Supplementary Information

The online version contains supplementary material available at 10.1186/s42523-026-00524-1.

Introduction

Periodontal disease is among the most prevalent oral conditions in dogs and represents a major health concern in veterinary clinical practice, substantially affecting both canine quality of life and clinical management. Epidemiological studies indicate that over 80% of dogs older than three years suffer from some degree of periodontal disease [1]. Beyond causing localized oral damage, including gingival inflammation, tooth mobility, and tooth loss, periodontal disease may also contribute to systemic health complications through chronic inflammation and bacteremia [2]. The association between oral and systemic health has garnered increasing attention, with severe periodontal infections in dogs being implicated in systemic disorders such as endocarditis and renal pathology [3, 4]. Accordingly, maintaining oral health is critical not only for preventing oral diseases but also for mitigating the risk of associated systemic conditions.

Conventional treatments for canine periodontal disease, including mechanical scaling (professional dental cleaning) and antibiotic therapy, exhibit notable limitations and adverse effects. Mechanical scaling often necessitates general anesthesia, which carries inherent risks, particularly for elderly dogs or those with pre-existing health conditions. Additionally, dental plaque tends to reaccumulate following cleaning, complicating efforts to achieve sustained oral health [5]. Although antibiotics are effective in managing acute infections, prolonged or repeated administration may disrupt the microbial balance, promote antibiotic resistance, and induce gastrointestinal disturbances, among other side effects [6–8]. In light of these challenges, there is a critical need for safer, more effective, and sustainable strategies to maintain oral health in veterinary clinical practice.

Probiotic intervention has emerged as a promising strategy for modulating the oral microbiome and has attracted growing scientific interest [9]. Probiotics are beneficial microorganisms capable of colonizing the host and promoting oral health through multiple mechanisms, including competitively inhibiting pathogenic bacterial adhesion, secreting organic acids and antimicrobial compounds to suppress pathogen proliferation, and modulating host immune responses to alleviate inflammation [10]. Compared to antibiotics, probiotics are less likely to induce antimicrobial resistance and offer the potential to simultaneously suppress harmful bacteria while facilitating the colonization of beneficial microbes, thereby contributing to the maintenance of a balanced oral microbiota [11]. Recent studies in humans and animal models have preliminarily demonstrated the potential benefits of probiotics in periodontal health, including reducing dental plaque accumulation, lowering pathogenic bacterial abundance, and decreasing inflammatory mediator levels [12]. Accordingly, the application of probiotics in the prevention and treatment of canine oral diseases represents a highly valuable research direction with promising clinical implications.

Building upon the aforementioned background, this study aimed to evaluate the effects and underlying mechanisms of combined probiotic intervention in improving canine oral health. Two lactic acid bacterial strains with strong probiotic potential, Lactiplantibacillus plantarum Kmiate-6 and Pediococcus acidilactici Kmiate-15, were selected and used in combination to manage canine periodontal health. Unlike previous studies that primarily focused on single-strain interventions, this study employed a dual-strain approach and systematically assessed the effects across multiple levels, from in vitro to in vivo experiments. Specifically, we evaluated the capacity of the probiotic combination to inhibit common oral pathogens and modulate inflammatory responses in vitro, as well as its impact on dental plaque formation, oral microbiota composition, inflammatory marker expression, and microbial functional homeostasis in vivo. Through this series of investigations, we sought to verify the efficacy of the combined probiotics in restoring oral microbiota balance, alleviating inflammation, and preventing periodontal disease in dogs, thereby providing scientific evidence to support the application of probiotics in veterinary oral healthcare.

Methods

Antibacterial testing of Kmiate-6 and Kmiate-15

The Lactiplantibacillus plantarum strain Kmiate-6 and Pediococcus acidilactici strain Kmiate-15 used in this study were originally isolated from the feces of healthy police dogs and are preserved in the strain collection of our laboratory. Prior to experimentation, the strains were activated by anaerobic cultivation at 37 °C in a modified de Man, Rogosa, and Sharpe (MRS) medium, and bacterial suspensions were prepared for subsequent assays. To evaluate their antibacterial activity against common oral pathogens, an agar well diffusion assay was performed. Four representative oral pathogens were selected: Streptococcus mitis BNCC354378, Streptococcus mutans BNCC373993, Fusobacterium nucleatum BNCC361670, and Porphyromonas gingivalis BNCC363426. Each pathogen was cultured to the logarithmic growth phase and evenly spread onto LB agar plates to establish a uniform bacterial lawn. Sterile punches (8 mm diameter) were used to create three equidistant wells in each plate, into which 50 µL of Kmiate-6 suspension (1 × 10⁸ CFU/mL), 50 µL of Kmiate-15 suspension (1 × 10⁸ CFU/mL), and 50 µL of sterile physiological saline (blank control) were respectively added. Each experimental condition was independently replicated three times. After 24 h of anaerobic incubation at 37 °C, the diameters of the inhibition zones surrounding each well were measured in millimeters using a vernier caliper, and the inhibitory effects of Kmiate-6 and Kmiate-15 against each pathogen were quantitatively assessed based on the measured inhibition zone diameters.

Evaluation of the anti-inflammatory effects of Kmiate-6 and Kmiate-15 in RAW 264.7 cells

The mouse macrophage cell line RAW 264.7 was cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics at 37 °C in a 5% CO₂ atmosphere until reaching 80–90% confluence. Cells were then detached using 0.25% trypsin-EDTA, resuspended in fresh complete medium, and adjusted to a concentration of 5 × 10⁵ cells/mL. A total of 100 µL of the cell suspension (approximately 5 × 10⁴ cells) was seeded into each well of a 96-well plate and incubated for 24 h under the same conditions to allow adherence. After adherence, the medium was removed, and cells were stimulated with lipopolysaccharide (LPS) at a final concentration of 500 ng/mL to induce an inflammatory response. Simultaneously, bacterial suspensions of Lactiplantibacillus plantarum Kmiate-6 or Pediococcus acidilactici Kmiate-15 were added to the respective treatment groups at a final concentration of 1 × 10⁵ CFU/mL, establishing the LPS+Kmiate-6 group and the LPS+Kmiate-15 group. The blank control group received neither LPS nor probiotics, whereas the LPS model group was treated with LPS alone without probiotic intervention. All groups were incubated for an additional 12 h, after which the cell culture supernatants were collected and stored at − 80 °C for subsequent analysis. The concentration of tumor necrosis factor-alpha (TNF-α) in the supernatants was measured using a commercial enzyme-linked immunosorbent assay (ELISA) kit (Meilian Biotech, Shanghai, China) to evaluate the regulatory effects of the probiotics on LPS-induced inflammatory responses in RAW 264.7 cells.

Separately, RAW 264.7 cells were cultured and allowed to adhere under the same conditions. After removing the culture medium, DMEM containing 1 × 10⁵ CFU/mL of Kmiate-6 or Kmiate-15 was added to the treatment groups without LPS stimulation, while the control group received an equal volume of DMEM alone. Following a 12-hour incubation at 37 °C in a 5% CO₂ atmosphere, the supernatants were collected and stored at − 80 °C. The concentration of the anti-inflammatory cytokine interleukin-10 (IL-10) in the supernatants was determined using an ELISA kit (Meilian Biotech, Shanghai, China) to assess the effects of Kmiate-6 and Kmiate-15 on the anti-inflammatory responses of RAW 264.7 macrophages.

Oral administration of Kmiate-6 and Kmiate-15 in Beagle dogs

Sixteen healthy adult Beagle dogs were randomly assigned to either a control group (Con group, n = 8) or a Kmiate-6 and Kmiate-15 treatment group (K6 + K15 group, n = 8). All dogs were fed an identical standardized basal diet throughout the experimental period. On Day 0 (prior to intervention), baseline saliva samples were collected from each dog and stored at − 80 °C for subsequent inflammatory marker analysis. Baseline dental plaque staining and scoring were also performed at this time. Following baseline assessments, the dogs underwent a 21-day intervention period. Dogs in the treatment group received a daily oral spray containing a probiotic formulation of Kmiate-6 and Kmiate-15 (each at a concentration of 1 × 10⁹ CFU/mL). Specifically, 2 mL of the probiotic suspension (1 mL applied to each dental arch, containing approximately 1 × 10⁹ CFU of each probiotic strain) was sprayed onto the tooth surfaces once daily. After spraying, the dogs were fasted for 30 min to allow the probiotics to exert their effects in the oral cavity. Dogs in the control group received an equal volume of sterile physiological saline following the same administration procedure. This treatment was selected as a negative control to eliminate any potential biological interference from live microorganisms or their metabolites, thereby ensuring that the observed effects could be attributed solely to the probiotic intervention. At the end of the 21-day intervention, dental plaque staining and scoring were repeated, and dental plaque swabs, oral swabs, saliva, and fecal samples were collected and stored at − 80 °C for subsequent inflammatory marker detection and microbiome analysis. The aim of these procedures was to evaluate the effects of probiotic intervention on dental plaque formation, oral inflammatory responses, and the composition of oral and gut microbiota. All experimental protocols were approved by the Animal Ethics Committee of Nanjing Agricultural University (Approval No.: NJAU.No20240313047).

Dental plaque staining and scoring

To detect dental plaque on the surface of the dogs’ teeth, Erythrosine (Red No. 3) was employed as a disclosing agent. A sufficient quantity of Erythrosine solution was prepared, and a cotton swab was dipped into the solution for application. The dye was carefully and evenly applied to the surface of the dogs’ left canine teeth. After application, the dye was left on the teeth for 30 s to 1 min to allow the plaque to absorb the dye and become visible. The oral cavity was then gently rinsed with water to remove excess dye. Stained plaque appeared as a distinct red or pink color, and photographs were taken to document the stained areas. ImageJ software was used to label and measure the plaque area for statistical analysis.

To provide a more precise evaluation of plaque accumulation, a modified version of the Silness plaque index was employed, utilizing a 5-point scoring system [13]. The scoring criteria were as follows: 0 indicated no plaque, 1 indicated a small amount of plaque near the gingival margin, 2 indicated visible plaque on the gingival margin and tooth surface, covering a small area, 3 indicated moderate plaque coverage over a wider area of the tooth surface, 4 indicated heavy plaque coverage over most of the tooth surface, and 5 indicated that the tooth surface was almost entirely covered with thick plaque, obscuring the tooth surface. Each tooth was independently scored by two observers using the aforementioned criteria. The scores were then used for further analysis of plaque distribution and the effects of probiotic intervention.

Detection of inflammatory markers in canine saliva

Saliva samples were used to evaluate local oral inflammation levels in dogs. The concentrations of tumor necrosis factor-alpha (TNF-α) and interleukin-10 (IL-10) in canine saliva were quantitatively measured using commercial enzyme-linked immunosorbent assay (ELISA) kits (Meilian Biotech, Shanghai, China). All assays were conducted strictly in accordance with the manufacturer’s instructions. Each sample was analyzed in triplicate, and the mean value of the three measurements was used for subsequent statistical analysis.

DNA extraction and 16S sequencing

DNA was extracted from 0.3 g of samples using the QIAamp Fast DNA fecal Mini Kit (Qiagen, Valencia, California, USA) following the manufacturer’s instructions. The quantity and purity of the extracted DNA were determined using the NanoDrop 2000 (Thermo Fisher Scientific, USA). The 218 F and 806R primers were used to amplify the region V3-V4 of the bacterial S rRNA gene using the TransGen AP221-02 Kit (TransGen, Beijing, China). For bacterial sequencing, the library was sequenced using the Illumina Novaseq 6000 platform by a 250 bp paired-end approach according to the standard protocols.

Sequencing Data Processing: Paired-end reads was assigned to samples based on their unique barcode and truncated by cutting off the barcode and primer sequence. Low-quality sequences were removed using Trimmomatic v. 0.40. The original fragments with paired-end reads were merged by FLASH v.1.2.11. Chimeric sequences were truncated by UCHIME. Then, Sequences with ≥ 97% similarity were assigned to the same operational taxonomic units (OTUs). Representative sequence for each OTU was screened for further annotation. Taxonomy was assigned against the SILVA 138.1 reference database for bacterial 16S rRNA OTUs. To investigate the functional potential of microbial communities based on 16S rRNA sequencing data, we employed PICRUSt2 (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States), an advanced software designed for predicting functional abundances based only on marker gene sequences.

Microbial community analysis

The microbiota diversity analyses (including alpha and beta diversity) were conducted and visualized using the vegan and ggplot2 packages in R (version 4.2.1). Specifically, alpha diversity metrics, such as Shannon and Simpson Indices were calculated with the vegan package. The differences in alpha diversity indexes between groups were tested by Wilcoxon rank sum test. Principal coordinate analysis (PCoA) based on Bray-Curtis distances to examine the differences in the structures of the microbial communities. Differences in PCoA ordination between groups were tested by permutational multivariate analysis of variance (PERMANOVA). Linear discriminant analysis (LDA) coupled with effect size measurements (LEfSe) analysis was utilized to search for statistically different biomarkers between groups. In our study, the discriminative microbial taxonomic between the groups were identified using LEfSe with a LDA score > 2.5.

Statistical analysis

Statistical analyses were performed using SPSS 26.0 (IBM, USA) and GraphPad Prism 8.0 (GraphPad Software, USA). For normally distributed data, comparisons between two groups were performed using Student’s t-test. For non-normally distributed or ordinal data (e.g., diversity indices, relative abundances), the Wilcoxon rank-sum test was applied. Microbial community structure differences were assessed by permutational multivariate analysis of variance (PERMANOVA). Differential taxa were identified using linear discriminant analysis effect size (LEfSe) with an LDA score threshold > 2.0. Correlations between microbial taxa and functional pathways were evaluated using Spearman correlation. P < 0.05 was considered statistically significant.

Results

Antimicrobial and Immunomodulatory activities of Kmiate-6 and Kmiate-15 in vitro

The antimicrobial and immunomodulatory activities of Lactiplantibacillus plantarum Kmiate-6 and Pediococcus acidilactici Kmiate-15 were evaluated through in vitro assays. The agar well diffusion method was employed to assess their inhibitory effects against four common oral pathogens. The results demonstrated that Kmiate-6 exhibited significantly stronger inhibition against Streptococcus mutans (Fig. 1A, P < 0.01), Fusobacterium nucleatum (Fig. 1B, P < 0.01), and Streptococcus mitis (Fig. 1C, P < 0.01) compared to Kmiate-15. However, no significant difference was observed between the two strains in their inhibitory effects against Porphyromonas gingivalis (Fig. 1D, P ≥ 0.05). Additionally, an in vitro inflammatory model using RAW 264.7 macrophages was established to investigate the regulatory effects of Kmiate-6 and Kmiate-15 on cytokine expression following lipopolysaccharide (LPS) stimulation. Kmiate-15 significantly reduced TNF-α secretion relative to Kmiate-6 (Fig. 1E, P < 0.05) and markedly enhanced IL-10 secretion compared to Kmiate-6 (Fig. 1F, P < 0.001), indicating its superior anti-inflammatory potential.

Fig. 1.

In vitro antimicrobial and immunomodulatory activities of Lactiplantibacillus plantarum Kmiate-6 and Pediococcus acidilactici Kmiate-15. (A–D) Antibacterial effects of Kmiate-6 and Kmiate-15 against common oral pathogens were assessed using the agar well diffusion method. Representative images of inhibition zones (left panels) and corresponding quantitative measurements (right panels) are shown for (A) Streptococcus mutans, (B) Fusobacterium nucleatum, (C) Streptococcus mitis, and (D) Porphyromonas gingivalis. (E–F) Immunomodulatory activities were evaluated in LPS-induced RAW 264.7 macrophages. (E) TNF-α production and (F) IL-10 secretion were measured using ELISA kits. Data are presented as mean ± SEM (n = 3). Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparisons test. * P < 0.05, * P < 0.01, *** P < 0.001

Effects of Kmiate-6 and Kmiate-15 on canine dental plaque and oral inflammatory markers

Following 21 days of probiotic treatment, the growth rate of dental plaque area and plaque scores were compared between the probiotic-treated group and the control group. The data revealed that the plaque area in the K6 + K15 group exhibited a negative growth rate, whereas the control group demonstrated a significant positive growth rate. Statistical analysis confirmed a significant difference between the two groups (Fig. 2B, P < 0.01). Additionally, the plaque scores in the K6 + K15 group were significantly lower than those in the control group (Fig. 2C, P < 0.05). Further investigation showed that TNF-α levels in the saliva of the K6 + K15 group were significantly reduced compared to the control group (Fig. 2D, P < 0.05), while IL-10 levels were significantly elevated in the K6 + K15 group (Fig. 2E, P < 0.05).

Fig. 2.

Effects of Kmiate-6 and Kmiate-15 on canine dental plaque accumulation and oral inflammatory markers. (A) Experimental design schematic. (B) Representative images of dental plaque staining at baseline (Day 0) and after 21 days (Day 21) in both groups. The right panel shows the quantified growth rate of dental plaque area, with a significant reduction observed in the K6 + K15 group compared to the control (P < 0.01). (C) Comparison of plaque index scores between groups at baseline and Day 21. (D) TNF-α levels. (E) IL-10 levels. Data are presented as mean ± SEM (n = 8). Statistical analysis was performed using two-way ANOVA followed by Tukey’s multiple comparisons test. P < 0.05, * P < 0.01

Modulatory effects of Kmiate-6 and Kmiate-15 on oral microbiota composition in dogs

Following 21 days of probiotic intervention, significant alterations in the oral microbiota composition were observed between the K6 + K15 group and the control group. Alpha diversity analysis demonstrated that the Shannon index (Fig. 3A, P < 0.05), Simpson index (Fig. 3B, P < 0.05), ACE index (Fig. 3C, P < 0.05), and Chao1 index (Fig. 3D, P < 0.05) were significantly decreased in the K6 + K15 group compared to the control group, indicating reduced microbial diversity and richness. Principal coordinate analysis (PCoA) based on Bray–Curtis distances revealed a distinct separation between the two groups (Fig. 3E), although no significant difference was detected in intra-group Bray–Curtis distances (Fig. 3F, P > 0.05). At the phylum level, compositional changes were observed, with the K6 + K15 group showing a decreased relative abundance of Bacteroidota compared to the control group, while Firmicutes remained dominant in both groups (Fig. 3G). LEfSe analysis at the order level identified Bacteroidales, Fusobacteriales, Peptostreptococcales-Tissierellales, Lachnospirales, Kapabacteriales, Veillonellales-Selenomonadales, Spirochaetales, and Absconditabacteriales (SR1) as significantly enriched in the control group, whereas Sphingobacteriales and Synergistales were more abundant in the K6 + K15 group (Fig. 3H). At the genus level, stacked bar plots revealed that the relative abundance of Fusobacterium, Porphyromonas, and Moraxella decreased in the K6 + K15 group, while an increase in Weissella was observed (Fig. 3I). LEfSe analysis further confirmed that Fusobacterium, Porphyromonas, Moraxella, Peptoanaerobacter, Fusibacter, Kapabacteriales-g-unclassified, Lachnospiraceae-g-unclassified, g-F0058, Treponema, Absconditabacteriales (SR1)-g-unclassified, Proteocatella, Tannerella, Johnsonella, and Family XI-g-unclassified were significantly enriched in the control group, whereas Kocuria and Sphingobacterium were significantly enriched in the K6 + K15 group (Fig. 3J). Collectively, these results suggest that probiotic supplementation with Lactiplantibacillus plantarum Kmiate-6 and Pediococcus acidilactici Kmiate-15 effectively modulated the oral microbiota by reducing microbial diversity, decreasing the abundance of potential pathogenic taxa, and altering the overall community composition.

Fig. 3.

Effects of Kmiate-6 and Kmiate-15 on the diversity and composition of the oral microbiota in dogs. (A) Shannon index of oral microbiota. (B) Simpson index of oral microbiota. (C) ACE index of oral microbiota. (D) Chao1 index of oral microbiota. (E) Principal coordinate analysis (PCoA) based on Bray–Curtis distances. (F) Within-group Bray–Curtis distances. (G) Taxonomic composition at the phylum level. (H) LEfSe analysis of differentially abundant taxa at the order level. (I) Taxonomic composition at the genus level. (J) LEfSe analysis of differentially abundant taxa at the genus level. Statistical analysis was performed using the Wilcoxon rank-sum test for alpha diversity and LEfSe analysis for differential taxa identification. P < 0.05 was considered statistically significant. * P < 0.05

Effects of Kmiate-6 and Kmiate-15 on functional profiles of oral microbiota in dogs

Functional prediction analysis based on 16S rRNA gene sequencing data revealed significant differences (P < 0.05) in microbial metabolic pathways between the K6 + K15 and control groups. Compared to the K6 + K15 group, the control group exhibited higher predicted abundances of pathways involved in thiamine metabolism (Fig. 4A), one-carbon pool by folate (Fig. 4B), porphyrin and chlorophyll metabolism (Fig. 4C), pantothenate and CoA biosynthesis (Fig. 4D), and zeatin biosynthesis (Fig. 4E). In contrast, the K6 + K15 group showed significantly higher levels of chloroalkane and chloroalkene degradation (Fig. 4F), benzoate degradation (Fig. 4G), glutathione metabolism (Fig. 4H), fatty acid degradation (Fig. 4I), geraniol degradation (Fig. 4J), betalain biosynthesis (Fig. 4K), tyrosine metabolism (Fig. 4L), phenylalanine metabolism (Fig. 4M), chlorocyclohexane and chlorobenzene degradation (Fig. 4N), propanoate metabolism (Fig. 4O), and caprolactam degradation (Fig. 4P) pathways ( P < 0.05 for all). These results suggest that probiotic intervention with Lactiplantibacillus plantarum Kmiate-6 and Pediococcus acidilactici Kmiate-15 significantly altered the predicted functional profiles of the oral microbiota, promoting the upregulation of pathways associated with the degradation of xenobiotic compounds and amino acid metabolism.

Fig. 4.

Effects of Kmiate-6 and Kmiate-15 on functional profiles of oral microbiota in dogs. (A) Thiamine metabolism. (B) One carbon pool by folate. (C) Porphyrin and chlorophyll metabolism. (D) Pantothenate and CoA biosynthesis. (E) Zeatin biosynthesis. (F) Chloroalkane and chloroalkene degradation. (G) Benzoate degradation. (H) Glutathione metabolism. (I) Fatty acid degradation. (J) Geraniol degradation. (K) Betalain biosynthesis. (L) Tyrosine metabolism. (M) Phenylalanine metabolism. (N) Chlorocyclohexane and chlorobenzene degradation. (O) Propanoate metabolism. (P) Caprolactam degradation. Statistical analysis was performed using the Wilcoxon rank-sum test. P < 0.05 was considered statistically significant. * P < 0.05, * P < 0.01

Modulatory effects of Kmiate-6 and Kmiate-15 on dental plaque microbiota composition in dogs

Following 21 days of probiotic intervention, analysis of the dental plaque microbiota revealed that the Shannon index (Fig. 5A), Simpson index (Fig. 5B). ACE index was significantly reduced in the K6 + K15 group compared to the control group (Fig. 5C, P < 0.05), suggesting a decrease in microbial richness. Chao1 index (Fig. 5D) showed no significant differences between the K6 + K15 and control groups (P > 0.05) Principal coordinate analysis (PCoA) based on Bray–Curtis distances indicated a distinct separation trend between groups (Fig. 5E), although no significant difference in intra-group Bray–Curtis distances was observed (Fig. 5F, P > 0.05). At the phylum level, the dominant phyla in both groups included Fusobacteriota, Bacteroidota, Proteobacteria, and Firmicutes, with a slight reduction in Fusobacteriota observed in the K6 + K15 group (Fig. 5G). LEfSe analysis at the order level showed that Staphylococcales was significantly enriched in the control group (Fig. 5H). At the genus level, stacked bar plots indicated a decrease in the relative abundance of Fusobacterium and Moraxella following probiotic intervention (Fig. 5I). Further LEfSe analysis at the genus level identified that Cardiobacteriaceae-g-unclassified, g-TM7x, Comamonadaceae-g-unclassified, Gemella, Corticibacter, Candidatus Saccharimonas, Abiotrophia, and Proteiniphilum were significantly enriched in the control group, while Chryseobacterium was relatively enriched in the K6 + K15 group (Fig. 5J). These results suggest that probiotic supplementation with Lactiplantibacillus plantarum Kmiate-6 and Pediococcus acidilactici Kmiate-15 modulated the dental plaque microbiota composition by reducing microbial richness and selectively altering the abundance of specific bacterial taxa.

Fig. 5.

Modulatory effects of Kmiate-6 and Kmiate-15 on dental plaque microbiota composition in dogs. (A) Shannon index of dental plaque microbiota. (B) Simpson index of dental plaque microbiota. (C) ACE index of dental plaque microbiota. (D) Chao1 index of dental plaque microbiota. (E) Principal coordinate analysis (PCoA) based on Bray–Curtis distances. (F) Within-group Bray–Curtis distances. (G) Taxonomic composition at the phylum level. (H) LEfSe analysis of differentially abundant taxa at the order level. (I) Taxonomic composition at the genus level. (J) LEfSe analysis of differentially abundant taxa at the genus level. Statistical analysis was performed using the Wilcoxon rank-sum test for alpha diversity and LEfSe analysis for differential taxa identification. P < 0.05 was considered statistically significant. * P < 0.05

Effects of Kmiate-6 and Kmiate-15 on predicted functional pathways of dental plaque microbiota

Functional prediction based on 16S rRNA gene sequencing of dental plaque samples revealed significant alterations in microbial metabolic pathways between the K6 + K15 and control groups. Specifically, the K6 + K15 group exhibited a significantly higher predicted abundance of pathways involved in pentose and glucuronate interconversions (Fig. 6A) and nitrogen metabolism (Fig. 6B) compared to the control group (P < 0.05 for both). These results suggest that probiotic intervention modulated the metabolic potential of the dental plaque microbiota, particularly enhancing pathways related to carbohydrate processing and nitrogen utilization.

Fig. 6.

Effects of Kmiate-6 and Kmiate-15 on predicted functional pathways of dental plaque microbiota. (A) Pentose and glucuronate interconversions. (B) Nitrogen metabolism. Statistical analysis was performed using the Wilcoxon rank-sum test. P < 0.05 was considered statistically significant. * P < 0.05

Associations between differential microbial taxa and differential metabolic pathways following probiotic intervention

Spearman correlation analysis was conducted to explore the associations between the significantly altered microbial taxa and the predicted metabolic pathways. In the oral microbiota, several differential genera were significantly correlated with functional pathways (Fig. 7A). Notably, Fusobacterium, Porphyromonas, and Moraxella were negatively correlated with pathways such as chloroalkane and chloroalkene degradation, fatty acid degradation, and propanoate metabolism, whereas Kocuria and Sphingobacterium showed positive correlations with multiple degradation pathways. In contrast, pathways including thiamine metabolism and one-carbon pool by folate were positively correlated with Fusobacterium and Porphyromonas. In the dental plaque microbiota, significant correlations were also observed (Fig. 7B). Cardiobacteriaceae-g-unclassified and Comamonadaceae-g-unclassified exhibited positive correlations with nitrogen metabolism and pentose and glucuronate interconversions, while Chryseobacterium and TM7x showed negative correlations with these pathways.

Fig. 7.

Associations between differential microbial taxa and differential metabolic pathways following probiotic intervention. (A) Correlation heatmap between differential genera in the oral microbiota and altered predicted metabolic pathways. (B) Correlation heatmap between differential genera in the dental plaque microbiota and altered predicted metabolic pathways. The color gradient represents Spearman correlation coefficients, with red indicating positive correlations and blue indicating negative correlations. Statistical analysis was performed using Spearman correlation. P < 0.05 was considered statistically significant. * P < 0.05, * P < 0.01

Effects of oral probiotic spray on fecal microbiota composition

To assess the potential systemic effects of oral probiotic spray, shotgun metagenomic sequencing was performed on fecal samples. No significant differences were observed between the K6 + K15 group and the control group in terms of α-diversity, as measured by the Shannon and inverse Simpson indices (Supplementary Figure S1A, B, p > 0.05). Principal coordinate analysis (PCoA) based on Bray–Curtis distances revealed no significant separation between groups (Supplementary Figure S1C). Taxonomic profiling at the species level showed no notable shifts in dominant species (Supplementary Figure S1D), although LEfSe analysis identified minor differences, with Sarcina ventriculi enriched in the control group and Turicibacter bilis, Turicibacter sp. 1E2, Bifidobacterium pseudocatenulatum, and Lactobacillus acidophilus enriched in the K6 + K15 group (Supplementary Figure S1E). Overall, these findings suggest that local application of Lactiplantibacillus plantarum Kmiate-6 and Pediococcus acidilactici Kmiate-15 via oral spray did not exert adverse effects on the gut microbiota composition.

Discussion

This study, through a combination of in vitro and in vivo experiments in a canine model, demonstrated the multifaceted probiotic effects of an oral spray containing L. plantarum Kmiate-6 and P. acidilactici Kmiate-15. These effects encompassed direct antibacterial activity, reduction of dental plaque accumulation, remodeling of the oral microbial community structure and function, and modulation of host immune and inflammatory responses, thereby collectively contributing to periodontal health maintenance.

The in vitro assays showed that both strains significantly inhibited Streptococcus mutans, S. mitis, Fusobacterium nucleatum, and Porphyromonas gingivalis; Kmiate-6 produced consistently larger inhibition zones than Kmiate-15. This observation is broadly in line with previous reports on Lactobacillus salivarius, L. rhamnosus and L. paracasei, which possess wide-spectrum antagonism against oral pathogens [14]. Because the well-characterised strain L. plantarum 14,917 secretes plantaricins, we speculate that Kmiate-6 may rely on a comparable bacteriocin repertoire, whereas Kmiate-15 could inhibit pathogens through organic-acid accumulation, H₂O₂ release or other peptides such as nisin analogues. Although true synergism between the two strains was not formally tested here, their complementary inhibition profiles suggest a potential additive or cooperative effect that merits dedicated follow-up experiments [15–17].

It is noteworthy that the in vivo experiments further substantiated the direct benefits derived from the antibacterial mechanisms. Compared to the control group, dogs receiving the oral spray containing L. plantarum Kmiate-6 and P. acidilactici Kmiate-15 exhibited a significant reduction in dental plaque area and lower plaque index scores, as demonstrated by image analysis (P < 0.01). The formation of dental plaque relies on the adhesion of pathogenic bacteria and the aggregation of biofilms. Probiotics can disrupt plaque formation and maturation through multiple mechanisms, including acid production that lowers local pH, bacteriocin secretion, and competitive adhesion, thereby inhibiting major cariogenic and periodontal pathogens [18, 19]. These findings are consistent with previous clinical observations indicating that probiotic mouthwashes significantly reduced plaque indices over short periods, achieving effects comparable to chlorhexidine mouthwash but with fewer adverse effects [20]. Collectively, this study provides strong experimental evidence that the antibacterial activity of L. plantarum Kmiate-6 and P. acidilactici Kmiate-15 directly translates into effective control of dental plaque formation, supporting the application of probiotics in the prevention of periodontal disease.

Probiotic intervention not only reduced pathogenic bacterial loads but also induced compositional shifts in both the local dental plaque microbiota and the overall oral microbiome. Regarding the dental plaque microecosystem, 16 S rRNA gene sequencing revealed no significant differences between groups in α-diversity metrics (species richness and evenness) or β-diversity analysis, indicating that short-term probiotic treatment did not markedly alter the overall microbial diversity within dental plaque. However, beneficial directional changes in community composition were observed: the abundance of pathogenic bacteria decreased, whereas the abundance of commensal bacteria increased. Notably, known periodontal pathogens such as Fusobacterium were significantly reduced, while health-associated genera such as Streptococcus and Gemella were significantly enriched. These findings suggest that probiotics suppressed dominant pathogens, thereby creating ecological niches for commensal bacteria and reconstructing a microbial community structure more conducive to oral health. Similar phenomena have been reported in clinical and animal studies. In patients with periodontitis, probiotic interventions were found to reduce the levels of red complex pathogens, including P. gingivalis, T. denticola, and F. nucleatum, while increasing the abundance of health-associated taxa such as Actinomyces and Streptococcus mitis [21]. These findings are consistent with the present study in the canine model and support the hypothesis that targeted probiotic supplementation can selectively suppress pathogenic bacteria and promote beneficial commensals through mechanisms such as competition for nutrients and adhesion sites, as well as the secretion of antimicrobial substances.

Notably, at the whole-oral microbiota level, as assessed via full-mouth swab sampling, significant changes in microbial diversity indices were observed. Compared to the control group, the K6 + K15 group exhibited a significant decrease in α-diversity indices, including the Shannon and ACE indices, indicating a reduction in overall microbial richness across the oral cavity. Although higher microbial diversity is generally considered beneficial for ecosystem stability, this association is not absolute in the context of the oral microbiome [22, 23]. Importantly, the observed decrease in diversity was accompanied by a significant reduction in the abundance of periodontal-associated pathogens. Specifically, pathogenic genera such as Fusobacterium, Porphyromonas, and Treponema were markedly reduced in the probiotic group, while no such changes were observed in the control group. Given that these genera are major contributors to the pathogenesis of periodontal disease, their reduction is typically regarded as a positive indicator of improved periodontal health [24]. Systematic reviews have demonstrated that effective periodontal interventions are often associated with a short-term decrease in microbial diversity alongside compositional remodeling, characterized by consistent reductions in key pathogens such as Porphyromonas, Fusobacterium, and Treponema following treatment [25]. Such microbial shifts are considered markers of a transition toward a healthier oral ecosystem [26]. Therefore, the observed decrease in diversity in the probiotic group likely reflects a transition from a dysbiotic to a healthier and more stable microbiota structure, primarily achieved through the selective depletion of deleterious microbial components.

This broad-scale microbial alteration is relatively uncommon in previous canine oral probiotic studies. For instance, some studies administering probiotics to healthy dogs did not observe significant changes in oral microbial diversity [27]. In contrast, the significant structural shifts detected after only three weeks of probiotic administration in the present study suggest that the selected strain combination and dosage confer strong colonization and modulatory capabilities.

In addition to their direct antibacterial effects, the probiotic-induced remodeling of the microbiota may also be attributed to microecological interactions. Probiotics and their metabolites may function as “cooperative colonizers,” contributing to the reconstruction of a healthier oral microenvironment. Notably, the increased abundance of Streptococcus species, which are recognized as early colonizers in the oral cavity, may play a crucial role. These bacteria are capable of producing hydrogen peroxide and bacteriocins that inhibit the colonization of pathogenic species and promote the stabilization of beneficial microbial communities [28]. Previous studies have demonstrated that certain hydrogen peroxide-producing viridans group streptococci can effectively suppress the growth of cariogenic bacteria such as S. mutans and anaerobic periodontal pathogens, thereby acting as “gatekeepers” of oral health [29]. Thus, the observed increase in commensal genera such as Streptococcus in the present study likely reflects an indirect probiotic effect, wherein probiotics facilitated the expansion of these beneficial taxa, which in turn resisted recolonization by pathogenic microorganisms. This microbial reshaping favors the establishment of a symbiont-dominated and ecologically stable oral microbiota, thereby reducing microbial risks associated with periodontal disease development. Consistent with the decrease in pathogenic burden, the probiotic group also exhibited reductions in dental plaque area and plaque index, suggesting a concomitant decrease in local microbial load and inflammatory stimulation within the periodontal environment [30].

In this study, L. plantarum Kmiate-6 and P. acidilactici Kmiate-15 demonstrated significant regulatory effects on TNF-α and IL-10 production in the RAW 264.7 macrophage cell line, indicating their anti-inflammatory potential. Consistent with these in vitro findings, dogs in the probiotic-treated group exhibited significantly lower levels of the pro-inflammatory cytokine TNF-α and higher levels of the anti-inflammatory cytokine IL-10 compared to the control group. These results suggest that the L. plantarum Kmiate-6 and P. acidilactici Kmiate-15 probiotic combination not only modulated the oral microbiota but also exerted immunomodulatory effects on the host, thereby alleviating periodontal inflammation. TNF-α is a key pro-inflammatory mediator involved in periodontal inflammation, promoting osteoclastogenesis and tissue destruction, whereas IL-10 is a crucial anti-inflammatory cytokine that suppresses the release of inflammatory mediators from macrophages and Th1 cells while enhancing regulatory T cell responses [31, 32]. The opposing changes in TNF-α and IL-10 levels observed in this study imply a shift in the local inflammatory microenvironment from a destructive toward a protective state. Similar findings have been reported in previous studies, where oral or topical administration of probiotics in periodontal disease models reduced the expression of inflammatory cytokines such as TNF-α, IL-1β, and IL-6 in gingival tissues [33].

Collectively, the findings of this study demonstrate that the L. plantarum Kmiate-6 and P. acidilactici Kmiate-15 probiotic combination can alleviate gingival inflammation through dual modulation of the oral microbiota and host immune responses. This has important implications for the prevention and management of periodontal disease, as chronic inflammation not only leads to destruction of periodontal supporting tissues but also poses risks to systemic health. As safe biological modulators, probiotics offer the potential to mitigate local inflammation while avoiding the risks of antimicrobial resistance and dysbiosis associated with conventional antibiotic therapies [23].

It is noteworthy that the probiotic-induced shifts in microbial composition were accompanied by alterations in the functional metabolic profiles of the microbiota. Functional prediction analysis based on 16 S rRNA gene sequencing revealed that K6 + K15 treatment significantly upregulated pathways involved in the degradation of exogenous pollutants, such as chloroalkane, benzoate, and caprolactam, as well as pathways related to glutathione metabolism, fatty acid metabolism, and propanoate metabolism. These changes suggest that the oral microbiome acquired enhanced detoxification and antioxidative capacities, which could facilitate the elimination of pro-inflammatory small molecules and mitigate oxidative stress [34]. Moreover, an increased potential for SCFA synthesis, particularly propionate, was observed; SCFAs are known to inhibit the NF-κB pathway and exert anti-inflammatory effects. In contrast, the control group exhibited enrichment in pathways associated with the biosynthesis of vitamins such as thiamine, folate, and pantothenate. These cofactor biosynthesis pathways are often exploited by periodontal pathogens, including Fusobacterium and Porphyromonas, to sustain energy metabolism and maintain biofilm homeostasis, reflecting a characteristic pattern of “nutritional scavenging” from the host.

The observed functional reprogramming suggests that L. plantarum Kmiate-6 and P. acidilactici Kmiate-15 may steer the oral microbiome toward greater detoxification capacity, enhanced antioxidant potential and higher SCFA-related pathway abundance, while possibly reducing the community’s dependence on vitamin-B biosynthesis.

The correlation analysis suggests that the probiotic-induced taxonomic shift remodels the metabolic potential of the oral ecosystem in several clinically relevant ways. First, classical periodontal pathogens Fusobacterium, Porphyromonas and Moraxella were inversely associated with chlorinated-alkane/alkene, fatty-acid and propanoate degradation pathways, whereas Kocuria and Sphingobacterium showed the opposite trend. Kocuria spp. are well known for their broad enzymatic repertoire for detoxifying aromatic xenobiotics, including aflatoxin B1, and their enrichment therefore points to an improved community capacity for xenobiotic clearance after probiotic treatment [35]. The positive correlations between Fusobacterium/Porphyromonas and thiamine- as well as one-carbon-pool metabolism echo reports that these pathogens hijack vitamin B₁ to fuel butyrate-rich fermentation and strengthen their biofilms—both recognised hallmarks of dysbiotic periodontal plaque [36, 37]. Within dental plaque, Cardiobacteriaceae-g-unclassified and Comamonadaceae-g-unclassified correlated positively with nitrogen metabolism and pentose/glucuronate interconversions, pathways tied to nitrate reduction and nitric-oxide generation that are protective for periodontal tissues [38]. In contrast, Chryseobacterium and the epibiont TM7x were negatively linked to these functions; TM7x and other Saccharibacteria possess streamlined genomes with limited respiratory and nitrogen-metabolic capacity and can exacerbate plaque dysbiosis when over-represented [39]. Together, these correlation patterns further support that L. plantarum Kmiate-6 and P. acidilactici Kmiate-15 supplementation selectively enriched taxa associated with xenobiotic detoxification and nitrate reduction while suppressing vitamin B1-dependent pathogens. These microbial shifts complement the observed metabolic reprogramming, providing additional mechanistic evidence for the probiotic-induced alleviation of inflammation and stabilization of the oral microenvironment.

In summary, this study demonstrated that the combination of Lactiplantibacillus plantarum Kmiate-6 and Pediococcus acidilactici Kmiate-15 effectively improved periodontal health in dogs through multiple synergistic mechanisms. These findings contribute to the growing body of evidence supporting the application of probiotics in oral medicine. Moreover, the combined use of the two strains did not induce significant alterations in the gut microbiota composition, suggesting a favorable safety profile. Unlike conventional antibiotics or antiseptics, probiotic-based strategies offer natural, safe, and resistance-sparing alternatives, representing a promising approach for the prevention and adjunctive management of periodontal disease. However, this study has certain limitations, including its relatively short intervention period and the lack of a standard probiotic reference strain for comparison, which restrict the generalization of the findings. Nevertheless, further studies are warranted to evaluate the effects of prolonged intervention periods and to determine the durability of probiotic-induced microbial and immunological changes following treatment cessation.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Qinghua Yu, Meiru Chen, and Rui Zhang designed the experiment. Rui Zhang and Weiyang Chen conducted the in vitro and in vivo experiments. Wanjin Hu performed the metagenomic analysis. Wanjin Hu, Rui Zhang, and Saiwei Zhong wrote the manuscript.

Funding

This research received no funding.

Data availability

The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

All experimental procedures were approved by the Animal Ethics Committee of Nanjing Agricultural University (Approval Number: NJAU. NO20240313047).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.