Periodontitis, a prevalent oral disease, significantly threatens oral health. Its development is closely associated with imbalances in the oral microbiome. The oral microbial community, typically in a dynamic balance, can be disrupted by various factors, leading to the excessive growth of pathogenic bacteria and triggering inflammatory responses in periodontal tissues.

Systemic antibiotics are commonly used to treat various infectious diseases, but they can disrupt gut microbiota balance. Studies indicate that the gut microbiome might be a potential link between overall health and periodontitis. An abnormal oral microbiome can cause oral pathogens to migrate to the gut, inducing colonic inflammation and activating immune and inflammatory responses, thereby worsening periodontitis. Moreover, the imbalance of Th17/Treg cells may be related to the microbiome, further aggravating periodontitis. It is currently unclear whether antibiotic-induced dysbiosis of the gut microbiota adversely affects periodontitis.

Yuan X. et al. published an article titled "Systemic antibiotics increase microbiota pathogenicity and oral bone loss' in International Journal of Oral Science. This study exposed mice to drinking water containing a mixture of four antibiotics to explore how systemic antibiotics affect microbial pathogenicity and oral bone loss. The results demonstrated for the first time that long-term antibiotic-induced gut microbiota dysbiosis can disrupt the oral microbiome and exacerbate periodontitis.

Materials and Methods

Four-week-old male C57 mice were randomly split into two primary groups: the N group with regular drinking water and the Abs group given antibiotic-containing water (cefoxitin, gentamicin, metronidazole, and vancomycin at 1 mg·mL⁻¹ each). After 4 weeks, their feces and mouth swabs were collected. Subsequently, some mice were further divided for periodontitis induction via ligature placement. Additionally, 30 mice underwent similar antibiotic treatment before fecal microbiota transplantation (FMT). 16S rRNA gene sequences, DNA analysis, and micro-CT and histologic analyses of relevant tissues were carried out. Data analysis was performed using SPSS software.

Results

Analysis of the two treatment groups showed that after antibiotic treatment, the Abs group had lower gut microbiota community evenness and diversity than the N group (Fig. 1a-b). PCoA analysis at the genus level significantly distinguished the two groups, indicating different community compositions (Fig. 1c). At the phylum level, the Abs group sharply increased Proteobacteria, linked to gut microbiota dysbiosis and disease. At the genus level, probiotics like Lachnospiraceae_NK4A136_group and Alistipes decreased in the Abs group (Fig. 1d).

Fig. 1 Long-term use of antibiotics caused gut dysbiosis.

Antibiotics increased community evenness and diversity of oral microbiota, altering the composition and boosting its pathogenicity (Fig. 2a-d). Bacteria beneficial for periodontal health, like Streptococcus, Neisseria, and Corynebacterium, decreased. Conversely, bacteria linked to periodontitis, such as Enterococcus and Dysgnomonas, increased (Fig. 2d).

Fig. 2 Long-term use of antibiotics increased periodontitis-related pathogens in the oral microbiota.

Four weeks after treatment, the N and Abs groups were split into N+N, N+Lig, Abs+N, and Abs+Lig. Periodontitis was induced in N+Lig and Abs+Lig with silk ligatures for two weeks. Two weeks after stopping antibiotics, Abs+N had a sharp drop in species richness compared to N+N (Fig. 3b). The reduced community evenness and diversity in Abs+N and Abs+Lig didn't recover, but increased in the ligature groups (Fig. 3b).

Antibiotics and periodontitis changed the community composition (Fig. 3c). In Abs+N, Proteobacteria remained abundant while Actinobacteriota decreased (Fig. 3a). Pathogenic bacteria increased, and beneficial ones decreased at the genus level (Fig. 3d).

Fig. 3 Gut microbiota dysbiosis did not recover after 2-week withdrawal of antibiotics.

In oral microbiota, species richness didn't vary significantly among groups. But Abs+N and Abs+Lig groups had lower community evenness and diversity than N+N and N+Lig (Fig. 4a-b). For periodontitis-induced groups, antibiotics increased pathogenicity. In Abs+Lig relative to N+Lig, probiotics like Streptococcus, Neisseria, Bergeyella, Lactococcus, and Weissella decreased, while the pathogen Klebsiella increased (Fig. 4d).

Fig. 4 Oral pathogenicity in mice with experimental periodontitis increased after 2-week withdrawal of antibiotics.

Antibiotic-treated groups (Abs+N and Abs+Lig) had visible intestinal damage, especially in the ileum and cecum, as shown by HE staining and histological scores (Fig. 5a). These groups also had fewer goblet cells (Fig. 5c) and less positive expression of tight-junction-related proteins compared to non-antibiotic groups (Fig. 5d). After D30, N+Lig and Abs+Lig groups showed weight loss, with Abs+Lig being more significant (Fig. 5b).

Fig. 5 Antibiotics use led to intestinal damage.

Antibiotics aggravated periodontitis in ligated mice. Micro-CT and HE staining showed the Abs+Lig group had more alveolar bone loss and neutrophil infiltration than the N+Lig group (Fig. 6c-d). Abs+Lig also had more TRAP-positive osteoclasts and higher expression of Th17-related pro-inflammatory cytokines (IL-17A, IL-6) (Fig. 6a-b). Meanwhile, it had decreased expression of Treg-related cytokines (Foxp3 and IL-10) compared to N+Lig (Fig. 6a).

Fig. 6 Antibiotics use aggravated alveolar bone loss.

Thirty mice were given a four-antibiotic cocktail in drinking water for 4 weeks as the Abs group. Then, they were divided into FMT-N and FMT-Abs groups, with fecal microbiota from N+N and Abs+N transferred respectively. Two weeks after FMT, the gut microbiota of recipient mice in FMT-N and FMT-Abs groups differed significantly (Fig. 7a-b). After establishing periodontitis models in all mice two weeks later, compared to the FMT-Abs+Lig group, mice in the FMT-N+Lig group had higher gut microbiota α-diversity, associated with a more stable and healthier microbiota (Fig. 7b-c). Probiotics like norank_Muribaculaceae and Prevotellaceae_UCG-001 were significantly more abundant in the FMT-N+Lig group (Fig. 7d).

Fig. 7 FMT with normal mice feces partially restored the gut microbiota disturbed by antibiotics.

The FMT-Abs+Lig and FMT-N+Lig groups showed no significant differences in oral microbiome α-diversity but did exhibit significant β-diversity differences (Fig. 8a-c). The FMT-N+Lig group had reduced levels of the oral pathogen unclassified_Enterobacteriaceae and the opportunistic pathogen Morganella compared to the FMT-Abs+Lig group (Fig. 8d). This suggests that FMT from healthy mice fosters a healthier oral microbiome in mice with induced periodontitis.

Fig. 8 FMT with normal mice feces reduced the pathogenicity of the oral microbiota in antibiotic-treated experimental periodontitis mice.

The FMT-N+Lig group exhibited improved gut health, with lower histological scores, larger goblet cell areas, and elevated tight junction protein levels than FMT-Abs+Lig (Fig. 9a-b, d). Weight differences between groups became more pronounced after ligation (Fig. 9c).

Fig. 9 FMT of normal mice feces alleviated intestinal damage.

Compared to FMT-Abs+Lig, the FMT-N+Lig group had higher expression of Treg markers (Foxp3, IL-10) and lower Th17 cytokines (IL-17A, IL-6), with reduced alveolar bone loss and inflammation (Fig. 10a-d). These results indicate that FMT of normal mice feces alleviated intestinal damage and periodontitis-induced alveolar bone loss in mice administered antibiotics.

Fig. 10 FMT of normal mice feces alleviated alveolar bone loss in mice with experimental periodontitis.

Conclusion

In this study, mice were fed drinking water containing a mixture of four antibiotics to explore how systemic antibiotics affect the pathogenicity of the microbiome and oral bone loss. The results showed that systemic antibiotics induce gut microbiota dysbiosis, leading to Th17/Treg imbalance in periodontal tissues. The findings highlight the potential adverse effects of systemic antibiotics on the oral microbiome and periodontitis, thus suggesting that periodontal status should be regularly assessed in clinical practice when using systemic antibiotics.