Systemic antibiotics increase microbiota pathogenicity and oral bone loss

Long-term use of antibiotics caused gut dysbiosis and increased periodontitis-related pathogens in the oral microbiota

Mice in the Abs group were treated with a four-antibiotic cocktail (cefoxitin, gentamicin, metronidazole, and vancomycin, 1 mg·mL−1 of each) in their drinking water while mice in the N group received normal drinking water; both treatments lasted for four weeks (Fig S1a). The results indicated that the community evenness and community diversity of the gut microbiota in the Abs group were lower than those in the N group after antibiotic treatment (Fig. 1b). In the principal coordinate analysis (PCoA), the N and Abs groups were significantly distinguished on the genus level, suggesting differences in community composition between these groups (Fig. 1c). At the phylum level, the abundance of Proteobacteria, a potential factor in gut microbiota dysbiosis and gut disease,24 was sharply increased in the Abs group (Fig. 1a). At the genus level, the probiotics Lachnospiraceae_NK4A136_group and Alistipes25 were decreased in the Abs group (Fig. 1d, S2b).

Fig. 1figure 1

Long-term use of antibiotics caused gut dysbiosis. Community bar plot and Wilcoxon rank-sum test bar plot of the gut microbiota at the phylum (a) and genus level (d). Alpha diversity of the gut microbiota (b). PCoA analysis of the gut microbiota (c). *P < 0.05, **P < 0.01, ***P < 0.001

Different from the gut microbiota results, antibiotics increased the community evenness and diversity of the oral microbiota (Fig. 2b). Antibiotics also changed the composition (Fig. 2a, c–d, S2c, d) and increased the pathogenicity of the oral microbiota. Bacteria associated with periodontal health were decreased, including Streptococcus, Neisseria, and Corynebacterium26 (Fig. 2d). Moreover, bacteria associated with periodontitis were increased, such as Enterococcus and Dysgnomonas27 (Fig. 2d). These results demonstrate that long-term use of antibiotics causes gut dysbiosis, increases periodontitis-related pathogens, and decreases periodontal health-related probiotics in the oral microbiota.

Fig. 2figure 2

Long-term use of antibiotics increased periodontitis-related pathogens in the oral microbiota. Community bar plot and Wilcoxon rank-sum test bar plot of the oral microbiota at the phylum (a) and genus level (d). Alpha diversity of the oral microbiota (b). PCoA analysis of the oral microbiota (c). *P < 0.05, **P < 0.01, ***P < 0.001

Gut microbiota dysbiosis did not recover and oral pathogenicity in mice with experimental periodontitis increased two weeks after withdrawal of antibiotics

After four weeks, the antibiotic water was removed from the Abs group and then mice in the N and Abs groups were divided into two groups, respectively (n = 12): N + N group, N + Lig (Ligature) group, Abs+N group, and Abs+Lig group. An experimental periodontitis model was established in the N + Lig and Abs+Lig groups using a silk ligature for two weeks (Fig. S1a). As for the gut microbiota, the Sobs index revealed that 2 weeks after withdrawal of the antibiotics, the species richness in the Abs+N group was sharply decreased compared with the N + N group (Fig. 3b). Previous reductions in community evenness and community diversity caused by antibiotics (Abs+N and Abs+Lig groups) did not recover. It was worth mentioning that in the ligature groups (N + Lig and Abs+Lig), community evenness and community diversity were increased (Fig. 3b). Both antibiotics and periodontitis changed the community composition (Fig. 3c, S3a, b). In the Abs+N group, Proteobacteria, a potential factor in gut dysbiosis, were still abundant while the abundance of Actinobacteriota was significantly decreased (Fig. 3a, S4a). Actinobacteriota plays a vital role in modulating gut permeability, the immune system, metabolism, and the gut-brain axis.28 At the genus level, pathogenic bacteria were increased significantly, including Blautia, Parasutterella, and Moganella29 (Fig. 3d, S3b). However, the abundance of the gut health-related bacteria norank_Muribaculaceae30 was decreased in the Abs+N group (Fig. 3d, S3b). These data suggest that the gut microbiota could not recover after a 2-week withdrawal of antibiotics. A previous study also reported that antibiotics can disturb the gut microbiota with long-lasting effects.31

Fig. 3figure 3

Gut microbiota dysbiosis did not recover after 2-week withdrawal of antibiotics. Community bar plot and Wilcoxon rank-sum test bar plot of the gut microbiota at the phylum (a) and genus level (d). Alpha diversity of the gut microbiota (b). PCoA analysis of the gut microbiota (c) *P < 0.05, **P < 0.01, ***P < 0.001

In the oral microbiota, although no significant differences in species richness were found between the different groups, the community evenness and community diversity were much lower in the antibiotic groups (Abs+N and Abs+Lig) compared to the groups without antibiotics (N + N, N + Lig) (Fig. 4b). In the groups with experimental periodontitis (N + Lig and Abs+Lig), antibiotic treatment increased the pathogenicity of the oral microbiota. Compared with the N + Lig group, the abundance of probiotics such as Streptococcus, Neisseria, Bergeyella, Lactococcus, and Weissella were significantly decreased, and the abundance of the oral pathological bacteria Klebsiella was increased in the Abs+Lig group14,26,32 (Fig. 4d, S3d). The results above reveal that, under the condition of periodontitis, antibiotic treatment can increase the pathogenicity of the oral microbiota.

Fig. 4figure 4

Oral pathogenicity in mice with experimental periodontitis increased after 2-week withdrawal of antibiotics. Community bar plot and Wilcoxon rank-sum test bar plot of the oral microbiota at the phylum (a) and genus level (d). Alpha diversity of the oral microbiota (b). PCoA analysis of the oral microbiota (c). *P < 0.05, **P < 0.01, ***P < 0.001

Antibiotics led to intestinal damage and aggravated alveolar bone loss

The body weights of mice were recorded throughout the experiment. An obvious reduction in body weight was observed in the Abs group from D1 to D9. Later, the weights of these mice increased rapidly to a level higher than that of mice in the N group. After D30, Mice with ligatures (N + Lig, Abs+Lig) exhibited significant reductions in body weight, especially in the Abs+Lig group (Fig. 5b). Obvious intestinal damage was observed in the groups administered antibiotics (Abs+N and Abs+Lig), especially in the ileum and cecum, as evidenced by hematoxylin-eosin (HE) staining and histological score analysis (Fig. 5a). Less goblet cells (Fig. 5c) and less positive expression of tight junction-related proteins (Fig. 5d) were observed in the groups administered antibiotics (Abs+N and Abs+Lig), as compared to the groups without antibiotics. Moreover, antibiotics aggravated periodontitis in mice with ligatures. According to the micro-CT and HE staining analyses, the Abs+Lig group exhibited greater alveolar bone loss and neutrophil infiltration than the N + Lig group (Fig. 6c, d). In addition, there were more TRAP-positive osteoclasts and greater expression of Th17 cell-related proinflammatory cytokines (IL-17A, IL-6) in the Abs+Lig group than in the N + Lig group (Fig. 6a, b). Antibiotics also decreased the expression of Treg cell-related proinflammatory cytokines (Foxp3 and IL-10) in the Abs+Lig group compared with the N + Lig group (Fig. 6a).

Fig. 5figure 5

Antibiotics use led to intestinal damage. a HE staining and histological scores of the ileum, cecum, and colon (Scale bar = 200 μm). b Weight record of mice (*Abs+N vs N + N, #Abs+N vs Abs+Lig). c AB-PAS staining and relative area of goblet cells of the colon (Scale bar = 200 μm). d Immunohistochemical analysis of occluding and ZO-1 positive area (Scale bar = 250 μm, ROI: 80 μm × 80 μm). *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 6figure 6

Antibiotics use aggravated alveolar bone loss. a Immunohistochemical analysis of IL-17A, IL-6, Foxp3, and IL-10 (Scale bar = 250 μm, ROI: 80 μm × 80 μm). b Trap staining and analysis (Scale bar = 250 μm). c Micro-CT analysis. d HE staining of maxillae (Scale bar = 250 μm), CEJ-ABC measurement, and neutrophil count analysis. *P < 0.05, **P < 0.01, ***P < 0.001

FMT with normal mice feces improved the gut dysbiosis caused by antibiotics but had no obvious effect on the oral microbiota

FMT, a new form of treatment, rebuilds the species composition and physiological function of the normal gut microbiota by transplanting the fecal microbiota of healthy donors to diseased recipients. Another 30 mice were provided with the four-antibiotic cocktail in drinking water for four weeks, the same as described above for the Abs group. Then, the antibiotic water was replaced with regular drinking water and the mice were divided into FMT-N and FMT-Abs groups. The fecal microbiota of mice in the N + N and Abs+N groups were transferred to the FMT-N and FMT-Abs mice, respectively (Fig. S1b). Two weeks after FMT, the gut microbiota of the recipient mice in the FMT-N and FMT-Abs groups were clearly distinguished (Fig. S5a. c, d, S7a, b). There were more Bacteroidota and Actinobacteriota and fewer Firmicutes in the gut microbiota of the FMT-N group compared to the FMT-Abs group (Fig. S5a). The recipient mice in each group showed similar gut microbiota to the donor mice, which indicates the success of FMT. Although the composition of the oral microbiota was different (Fig. S6a, c, d, S7c, d), there were no statistically significant differences in alpha and beta diversity between the FMT-Abs and FMT-N groups (Fig. S6b, c). These results indicate that FMT did not directly alter the oral microbiota as it did the gut microbiota.

The pathogenicity of the oral microbiota in antibiotic-treated experimental periodontitis mice decreased with FMT of normal mice feces

Two weeks after FMT, the experimental periodontitis model was established in all mice (FMT-N + Lig and FMT-Abs+Lig groups) (Fig. S1b). In the FMT-N + Lig group, the gut microbiota of mice showed higher alpha diversity, related to a more stable and healthier microbiota, than mice in the FMT-Abs+Lig group (Fig. 7b). The abundances of probiotics, such as norank_Muribaculaceae and Prevotellaceae_UCG-001,30 were much higher in the FMT-N + Lig group (Fig. 7d, S8b). As for the oral microbiota, although no obvious difference in alpha diversity was found between the FMT-Abs+Lig and FMT-N + Lig groups, there was a statistically significant difference in beta diversity (Fig. 8b, c). At the genus level, there was a lower abundance of the oral pathological bacteria unclassified_Enterobacteriaceae15 and the opportunistic pathogen Morganella33 in the FMT-N + Lig group compared to the FMT-Abs+Lig group (Fig. 8d, S8d). These results showed when experimental periodontitis was induced, a healthier oral microbiota composition was formed in mice with FMT of normal mice feces.

Fig. 7figure 7

FMT with normal mice feces partially restored the gut microbiota disturbed by antibiotics. Community bar plot and Wilcoxon rank-sum test bar plot of the gut microbiota at the phylum (a) and genus level (d). Alpha diversity of the gut microbiota (b). PCoA analysis of the gut microbiota (c). *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 8figure 8

FMT with normal mice feces reduced the pathogenicity of the oral microbiota in antibiotic-treated experimental periodontitis mice. Community bar plot and Wilcoxon rank-sum test bar plot of the oral microbiota at the phylum (a) and genus level (d). Alpha diversity of the oral microbiota (b). PCoA analysis of the oral microbiota (c). *P < 0.05, **P < 0.01, ***P < 0.001

FMT of normal mice feces alleviated intestinal damage and alveolar bone loss in mice with experimental periodontitis

The body weights of the mice were recorded throughout the experiment. After D30, mice in the FMT-N group exhibited greater weight gains than mice in the FMT-Abs group. After ligature placement, the weight difference between the FMT-N + Lig and FMT-Abs+Lig groups became even larger (Fig. 9c). With regard to intestinal damage, the FMT-N + Lig group exhibited lower histological scores, a larger area of goblet cells and more positive expression of tight junction proteins than the FMT-Abs+Lig group (Fig. 9a, b, d). This is probably because FMT of normal mice feces rebuilt the antibiotic-disrupted gut microbiota and reduced its pathogenicity. Even so, pathological damage was still observed in the gut tissue, which could be explained by the adverse effects of four weeks of antibiotics use. Additionally, alveolar bone loss, neutrophil infiltration, and TRAP-positive osteoclasts were much lower in the FMT-N + Lig group than in the FMT-Abs+Lig group (Fig. 10b–d). Immunohistochemical staining revealed increased expression of Treg-related transcription factors and cytokines (Foxp3, IL-10) and reduced expression of Th17-related cytokines (IL-17A, IL-6) (Fig. 10a) in the FMT-N + Lig group compared to the FMT-Abs+Lig group. These results indicate that FMT of normal mice feces alleviated intestinal damage and periodontitis-induced alveolar bone loss in mice administered antibiotics.

Fig. 9figure 9

FMT of normal mice feces alleviated intestinal damage. a HE staining and histological scores of the ileum, cecum, and colon (Scale bar = 200 μm). b AB-PAS staining and relative area of goblet cells of the colon (Scale bar = 200 μm). c Weight record of mice. d Immunohistochemical analysis of occludin and ZO-1 (Scale bar = 200 μm, ROI: 200 μm × 200 μm). *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 10figure 10

FMT of normal mice feces alleviated alveolar bone loss in mice with experimental periodontitis. a Immunohistochemical analysis of IL-17A, IL-6, Foxp3, and IL-10 (Scale bar = 250 μm, ROI: 80 μm×80 μm). b HE staining of maxillae (Scale bar = 250μm), CEJ-ABC measurement and neutrophil count analysis. c Trap staining and analysis (Scale bar = 250 μm). d Micro-CT analysis. *P < 0.05, **P < 0.01, ***P < 0.001

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