Experimental Inoculation of Aggregatibacter actinomycetemcomitans and Streptococcus gordonii and Its Impact on Alveolar Bone Loss and Oral and Gut Microbiomes

Oral bacteria are implicated not only in oral diseases but also in gut dysbiosis and inflammatory conditions throughout the body. The periodontal pathogen Aggregatibacter actinomycetemcomitans (Aa) often occurs in complex oral biofilms with Streptococcus gordonii (Sg), and this interaction might influence the pathogenic potential of this pathogen. This study aims to assess the impact of oral inoculation with Aa, Sg, and their association (Aa+Sg) on alveolar bone loss, oral microbiome, and their potential effects on intestinal health in a murine model. Sg and/or Aa were orally administered to C57Bl/6 mice, three times per week, for 4 weeks. Aa was also injected into the gingiva three times during the initial experimental week. After 30 days, alveolar bone loss, expression of genes related to inflammation and mucosal permeability in the intestine, serum LPS levels, and the composition of oral and intestinal microbiomes were determined. Alveolar bone resorption was detected in Aa, Sg, and Aa+Sg groups, although Aa bone levels did not differ from that of the SHAM-inoculated group. Il-1β expression was upregulated in the Aa group relative to the other infected groups, while Il-6 expression was downregulated in infected groups. Aa or Sg downregulated the expression of tight junction genes Cldn 1, Cldn 2, Ocdn, and Zo-1 whereas infection with Aa+Sg led to their upregulation, except for Cldn 1. Aa was detected in the oral biofilm of the Aa+Sg group but not in the gut. Infections altered oral and gut microbiomes. The oral biofilm of the Aa group showed increased abundance of Gammaproteobacteria, Enterobacterales, and Alloprevotella, while Sg administration enhanced the abundance of Alloprevotella and Rothia. The gut microbiome of infected groups showed reduced abundance of Erysipelotrichaceae. Infection with Aa or Sg disrupts both oral and gut microbiomes, impacting oral and gut homeostasis. While the combination of Aa with Sg promotes Aa survival in the oral cavity, it mitigates the adverse effects of Aa in the gut, suggesting a beneficial role of Sg associations in gut health.


Introduction
Periodontitis involves multifactorial inflammatory processes that result in the destruction of tooth-support tissues, driven by dysbiotic microbiota present in supra-and subgingival biofilms [1].Specific periodontopathogenic bacteria play a key role in the composition of dental biofilm, leading to heightened proportions of inflammatory organisms and increased virulence potential [2].
Aggregatibacter actinomycetemcomitans (Aa) is a Gram-negative, facultative anaerobic rod primarily associated with rapidly progressing periodontitis in young individuals, previously known as aggressive periodontitis (AP) [3].This organism is able to subvert host response due to the production of a leukotoxin and a cytolethal distending toxin (CDT) [4].Leukotoxin deactivates neutrophils, monocytes, and endothelial cells in humans and certain non-human primates but does not affect experimental rodent models [5,6].AaCDT disrupts the cell cycle of epithelial cells and fibroblasts, induces apoptosis in T lymphocytes, and impairs macrophages' phagocytic activity [7][8][9].Additionally AaLPS triggers the release of inflammatory cytokines such as IL-1, IL-6, and TNFα [10,11], while the outer membrane protein OMP29 facilitates invasion of non-phagocytic cells [12] and exerts inhibitory effects on the expression of several inflammatory mediators [13].
Periodontitis, including the rapidly progressing form associated with Aa, is marked not only by oral dysbiosis but also by changes in the fecal microbiome [14][15][16].Experimental evidence suggests that gut dysbiosis may arise following the ingestion of oral bacteria, resulting in intestinal barrier disruption and systemic inflammation [17][18][19][20][21].
During homeostasis, the integrity of the gut mucosa relies on the expression of transmembrane molecular complexes like tight junction (TJ) proteins and mucus production [22,23].Alterations in the gut microbiota have been linked to changes in intestinal permeability originated from inflammation-induced damage and shifts in the transcriptional profile of TJ proteins [24].Prior studies have shown that oral pathogens like P. gingivalis and Aa can compromise the oral mucosa barrier [25][26][27] since P. gingivalis proteases degrade epithelial adhesion molecules [28] whereas AaCDT induces redistribution of E-cadherin and of proteins of the adherens junction complex within the epithelial cells and causes detachment of basal cells from the basement membrane [24].
The virulence and survival of bacteria from multispecies biofilms in the oral cavity are influenced by host factors and neighboring microorganisms.Periodontitis arises from the synergistic effect of the entire microbiota and a disease-associated bacterial consortium including Aa and Streptococcus gordonii (Sg) was reported in aggressive periodontitis [29].The interaction between Aa and H 2 O 2 -producing streptococci results in altered virulence and increased survival of Aa, as demonstrated by in vitro biofilm studies and in a murine abscess model [30][31][32].However, the potential of the Aa-streptococci consortium to induce experimental periodontitis and its role in in promoting microbial dysbiosis in the oral cavity and gut have not been evaluated.
Therefore, we aimed to assess the impact of the oral administration of Aa, Sg, and a consortium of Aa and Sg on alveolar bone resorption, expression of genes related to gut mucosa integrity, and oral and gut microbiomes, in a murine experimental model.

Side Effects and Weight Gain Induced by Different Treatments
Four groups were evaluated, Aa-, Sg-, and Aa+Sg-infected groups along with the SHAM-inoculated group (negative control) (Figure 1).No changes in skin, fur, behavior, or mobility were observed in the animals throughout the 30-day experimental period.Initial weight was similar among groups.However, infection with Aa seemed to pose a detrimental effect on mice, since the Aa = infected groups (Aa and Aa+Sg) showed lower final weights compared to the Sg group (Figure 2A).Study design: C57BL/6 mice were randomly assigned into four groups: the SHAM inoculated group, the group infected with Aa, the group infected with Sg, and the group infected with both species, Aa+Sg.After a 30-day experimental period, the mice were euthanized, and samples were collected.

Alveolar Bone Loss Computed Microtomography (micro-CT)
Analysis of bone volume and porosity in the interproximal region between the firs and second molars in the left hemimaxillae revealed that infection with Sg resulted in a lower percentage of bone volume and higher bone porosity compared to the other groups However, alveolar bone loss values for the Sg group were similar to those observed for the Aa+Sg group.Infection with Aa alone did not significantly alter bone volume and porosity compared to the SHAM group, although the Aa group exhibited similar alveolar bone loss to the Aa+Sg group (Figures 2B, 2C).

LPS Levels in Serum
Serum LPS levels did not vary significantly among the groups, although the mean values of endotoxin units (EUs) were higher in the Aa-infected groups (Aa+Sg and Aa compared to the Sg and SHAM groups.

Gene Expression Related to Gut Inflammation and Barrier Permeability
Aa infection resulted in higher expression of Il-1β compared to infection with Sg or Aa+Sg.Expression of Il-6 was downregulated in the Aa-and Sg-infected groups but not in the Aa+Sg group compared to SHAM (Figure 3A,B).There were no differences in the tran scription levels of Il-10 and Tnfα among the groups.Study design: C57BL/6 mice were randomly assigned into four groups: the SHAMinoculated group, the group infected with Aa, the group infected with Sg, and the group infected with both species, Aa+Sg.After a 30-day experimental period, the mice were euthanized, and samples were collected.
Figure 1.Study design: C57BL/6 mice were randomly assigned into four groups: the SHAM inoculated group, the group infected with Aa, the group infected with Sg, and the group infecte with both species, Aa+Sg.After a 30-day experimental period, the mice were euthanized, an samples were collected.

Alveolar Bone Loss Computed Microtomography (micro-CT)
Analysis of bone volume and porosity in the interproximal region between the firs and second molars in the left hemimaxillae revealed that infection with Sg resulted in lower percentage of bone volume and higher bone porosity compared to the other groups However, alveolar bone loss values for the Sg group were similar to those observed fo the Aa+Sg group.Infection with Aa alone did not significantly alter bone volume an porosity compared to the SHAM group, although the Aa group exhibited similar alveola bone loss to the Aa+Sg group (Figures 2B, 2C).

LPS Levels in Serum
Serum LPS levels did not vary significantly among the groups, although the mea values of endotoxin units (EUs) were higher in the Aa-infected groups (Aa+Sg and Aa compared to the Sg and SHAM groups.

Gene Expression Related to Gut Inflammation and Barrier Permeability
Aa infection resulted in higher expression of Il-1β compared to infection with Sg o Aa+Sg.Expression of Il-6 was downregulated in the Aa-and Sg-infected groups but not i the Aa+Sg group compared to SHAM (Figure 3A,B).There were no differences in the tran scription levels of Il-10 and Tnfα among the groups.

Alveolar Bone Loss Computed Microtomography (micro-CT)
Analysis of bone volume and porosity in the interproximal region between the first and second molars in the left hemimaxillae revealed that infection with Sg resulted in a lower percentage of bone volume and higher bone porosity compared to the other groups.However, alveolar bone loss values for the Sg group were similar to those observed for the Aa+Sg group.Infection with Aa alone did not significantly alter bone volume and porosity compared to the SHAM group, although the Aa group exhibited similar alveolar bone loss to the Aa+Sg group (Figure 2B,C).

LPS Levels in Serum
Serum LPS levels did not vary significantly among the groups, although the mean values of endotoxin units (EUs) were higher in the Aa-infected groups (Aa+Sg and Aa) compared to the Sg and SHAM groups.

Gene Expression Related to Gut Inflammation and Barrier Permeability
Aa infection resulted in higher expression of Il-1β compared to infection with Sg or Aa+Sg.Expression of Il-6 was downregulated in the Aaand Sg-infected groups but not in the Aa+Sg group compared to SHAM (Figure 3A,B).There were no differences in the transcription levels of Il-10 and Tnfα among the groups.Infection with Aa and/or Sg induced changes in the transcriptional profile of gene encoding TJ proteins claudin 1, claudin 2, and occludin and the anchoring protein zonul in the intestinal barrier.Overall, infection with Aa or Sg downregulated the transcriptio of these genes, whereas infection with both organisms in the Aa+Sg group increased the mRNA levels, similar to the levels of the SHAM group, except for Cldn1, which achieve lower mRNA levels in Aa and Aa+Sg groups (Figure 3C-F).Transcription of muc encoding genes was anot altered by the treatments.

Oral and Gut Bacterial Community Structure
The oral biofilm and gut samples underwent 16S rRNA sequencing analysis.DN was successfully extracted from oral and gut samples of 24 animals (6 per group However, two gut samples from the SHAM group failed to yield amplicons for analysi resulting in microbiome data of only four SHAM samples.A total of 7,207,606 sequence were obtained (ranging from 97,250 to 246,890).After filtering and removing chimera 2,719,335 sequences remained for oral samples (ranging from 87,139 to 150,712) an 390,140 for gut samples (ranging from 4854 to 32,775).Rarefaction curves based on th number of operational taxonomic units (OTUs) indicated that all samples reached plateau when oral biofilm samples were normalized to 10,000 amplicon sequence varian (ASVs), and gut samples were normalized to 4000 sequences.Infection with Aa and/or Sg induced changes in the transcriptional profile of genes encoding TJ proteins claudin 1, claudin 2, and occludin and the anchoring protein zonulin in the intestinal barrier.Overall, infection with Aa or Sg downregulated the transcription of these genes, whereas infection with both organisms in the Aa+Sg group increased their mRNA levels, similar to the levels of the SHAM group, except for Cldn1, which achieved lower mRNA levels in Aa and Aa+Sg groups (Figure 3C-F).Transcription of mucin encoding genes was anot altered by the treatments.

Oral and Gut Bacterial Community Structure
The oral biofilm and gut samples underwent 16S rRNA sequencing analysis.DNA was successfully extracted from oral and gut samples of 24 animals (6 per group).However, two gut samples from the SHAM group failed to yield amplicons for analysis, resulting in microbiome data of only four SHAM samples.A total of 7,207,606 sequences were obtained (ranging from 97,250 to 246,890).After filtering and removing chimeras, 2,719,335 sequences remained for oral samples (ranging from 87,139 to 150,712) and 390,140 for gut samples (ranging from 4854 to 32,775).Rarefaction curves based on the number of operational taxonomic units (OTUs) indicated that all samples reached a plateau when oral biofilm samples were normalized to 10,000 amplicon sequence variants (ASVs), and gut samples were normalized to 4000 sequences.
Faith's phylogenetic diversity index analysis revealed that inoculation with Sg increased the diversity of the oral microbiome compared to Aa [33], although they did not differ from SHAM.In contrast, the concomitant administration of Aa+Sg reversed this effect (Figure 4A).Pielou (evenness) and Shannon (richness) diversity index analyses for the oral microbiome did not result in significant differences among the groups.Faith's phylogenetic diversity index analysis revealed that inoculation with Sg increased the diversity of the oral microbiome compared to Aa [33], although they did not differ from SHAM.In contrast, the concomitant administration of Aa+Sg reversed this effect (Figure 4A).Pielou (evenness) and Shannon (richness) diversity index analyses for the oral microbiome did not result in significant differences among the groups.Analysis of the gut microbiome revealed differences in evenness index (Pielou) among the three infected groups [34] but not from the SHAM-inoculated group (Figure 4B).Inoculation with Aa or Sg resulted in reduced diversity compared to inoculation with Analysis of the gut microbiome revealed differences in evenness index (Pielou) among the three infected groups [34] but not from the SHAM-inoculated group (Figure 4B).Inoculation with Aa or Sg resulted in reduced diversity compared to inoculation with both organisms in the Aa+Sg group.There were no differences in Faith and Shannon indexes among the groups for the gut microbiome.
Beta diversity analysis based on weighted and unweighted UniFrac distances revealed that the treatments affected both the oral and gut microbiomes.Infection with Aa or Sg altered the structure of the oral microbiome, while inoculation with Aa+Sg did not result in significant changes compared to SHAM (Figure 5A(I,II)).Analysis of gut samples showed clear segregation among the infected groups and SHAM.However, the gut microbiomes of Aa, Sg, and Aa+Sg did not differ from each other, except for differences between Aa+Sg and Sg based on unweighted UniFrac distances (Figure 5B(I,II)).
both organisms in the Aa+Sg group.There were no differences in Faith and Shannon indexes among the groups for the gut microbiome.
Beta diversity analysis based on weighted and unweighted UniFrac distances revealed that the treatments affected both the oral and gut microbiomes.Infection with Aa or Sg altered the structure of the oral microbiome, while inoculation with Aa+Sg did not result in significant changes compared to SHAM (Figure 5A (I,II)).Analysis of gut samples showed clear segregation among the infected groups and SHAM.However, the gut microbiomes of Aa, Sg, and Aa+Sg did not differ from each other, except for differences between Aa+Sg and Sg based on unweighted UniFrac distances (Figure 5B  There were no differences in the abundance of different phyla in the oral or gut microbiomes among the groups (Figures 6 and 7, respectively).Firmicutes was the most abundant phylum in the oral microbiome, followed by Pseudomonadota.ANCOM Sham Aa Sg Aa+Sg There were no differences in the abundance of different phyla in the oral or gut microbiomes among the groups (Figures 6 and 7, respectively).Firmicutes was the most abundant phylum in the oral microbiome, followed by Pseudomonadota.ANCOM revealed that infection with Aa led to an increased abundance of Gammaproteobacteria, Enterobacterales, and the genus Alloprevotella, while infection with Sg resulted in increased abundance of the genera Alloprevotella and Rothia in the oral cavity.Infection with both organisms did not lead to significant changes in the oral microbiome (Figure 6).revealed that infection with Aa led to an increased abundance of Gammaproteobacteria, Enterobacterales, and the genus Alloprevotella, while infection with Sg resulted in increased abundance of the genera Alloprevotella and Rothia in the oral cavity.Infection with both organisms did not lead to significant changes in the oral microbiome (Figure 6).Firmicutes was the most abundant phylum in the gut microbiome (Figure 7).Inoculation of Aa, Sg, or both microorganisms in the oral cavity resulted in decreased abundance of the order Erysipelotrichales and the family Erysipelotrichaceae compared to SHAM in the gut.
Firmicutes was the most abundant phylum in the gut microbiome (Figure 7).Inoculation of Aa, Sg, or both microorganisms in the oral cavity resulted in decreased abundance of the order Erysipelotrichales and the family Erysipelotrichaceae compared to SHAM in the gut.eHOMD database analysis revealed that Aa was detected in three out of six oral biofilm samples from the Aa+Sg group, but not in the Aa group, at an abundance lower than 0.001.Sg was not detected in the oral cavity of any mice.None of the inoculated species was detected in the gut.

Discussion
Periodontitis is associated with a dysbiotic microbiome.The microbial community within any host is influenced not only by the neighboring microorganisms but also by host defense, environmental, genetic, and epigenetic factors [35].However, not all these factors can be replicated in a murine model.Additionally, animal models for studying periodontitis are limited by the specificity of certain organisms and their virulence factors to humans.eHOMD database analysis revealed that Aa was detected in three out of six oral biofilm samples from the Aa+Sg group, but not in the Aa group, at an abundance lower than 0.001.Sg was not detected in the oral cavity of any mice.None of the inoculated species was detected in the gut.

Discussion
Periodontitis is associated with a dysbiotic microbiome.The microbial community within any host is influenced not only by the neighboring microorganisms but also by host defense, environmental, genetic, and epigenetic factors [35].However, not all these factors can be replicated in a murine model.Additionally, animal models for studying periodontitis are limited by the specificity of certain organisms and their virulence factors to humans.
Therefore, our findings should be interpreted within the constraints of the animal model utilized.Colonization represents a crucial phase in infectious diseases, yet Aa oral colonization in mice is hindered due to the inability of Aa adhesins Aae and OMP100 to bind to murine epithelial cells [36,37].In addition, Aa leukotoxin targets only human and certain non-human primate monocytes, macrophages, and neutrophils [38].It is also important to notice that the resident microbiota of rodents can be easily influenced by mouse lineage but also by various factors, including breeding sources and conditions [39], altering the results of assays performed at different animal facilities.
The mice infected with Aa present a lower weight at the end of the experimental period when compared with those infected with Sg or SHAM-inoculated mice.This observation differs from studies in humans where detection of Aa in the oral microbiome was associated with obesity [40].Experimental administration of Aa in mice fed a high-fat diet also resulted in increased weight gain compared to non-infected mice, whereas infection had no effect in weight gain in animals under a normal chow diet [21].However, any comparison between our data and the latter study is prevented by different tested protocols, especially by the use of norfloxacin, an antimicrobial that modulates the gut microbiota and leads to improved fasting glycemia and oral glucose tolerance [41].
Despite these limitations, our data revealed that the interaction with Sg increased the ability of Aa to colonize the oral biofilm of mice, since half of the animals of the Aa+Sg group harbored Aa in their oral cavity after 30 days of the experimental period, whereas this species was not detected in any mouse of the Aa group.Sg increases Aa fitness by providing lactate for Aa and producing H 2 O 2 , enhancing oxygen bioavailability which alters the metabolism from fermentation to respiration, changes the transcription profile of the pathogen [6,32], and promotes its virulence in an abscess model [31,42].Moreover, Aa dispersal in biofilms is dependent on dispersin B, whose expression is upregulated in co-infection with Sg, as shown by in vitro data [31].
Our data evidenced the relevance of the interaction with Sg to increase Aa survival in the oral biofilm of rodents.A single study in rats reported that the oral inoculation of Aa resulted in successful colonization of the oral biofilm and induction of periodontitis in wild type rodents [43].Therefore, most studies on experimental alveolar bone loss in rodents induced by Aa inoculated the living organism or its products in the gingival tissue [44][45][46][47][48], resulting in Aa persistent colonization of the gingival tissues but not of the oral biofilm [46].This association of Aa with lactate producers in the oral cavity is not only relevant for the murine model but has already been reported in experimental infection of Aa in primates.However, Aa recovery was low even in these primates after 4 weeks [49].
We have also shown that the three infected groups exhibited alveolar bone resorption, notwithstanding the similar bone volume and porosity observed in the Aa group compared to SHAM and to Aa+Sg.Therefore, although the lactate/peroxide producer streptococci provided increased survival of Aa in the oral biofilm, this association did not enhance the destruction of the periodontal tissues.It is worth mentioning that the bottom of deep periodontal pockets in humans represents a highly anaerobic environment where peroxide production is limited by the low concentration of oxygen, and Aa metabolism is anaerobic [32].Furthermore, microbiome data indicated that the increased abundance of Aa in deep periodontal pockets of periodontitis patients is accompanied by reduced levels of streptococci and other lactate producers [16].
The oral administration of Sg also resulted in alveolar bone loss, either independently or in conjunction with Aa in the murine model.Sg is considered an accessory pathogen in the oral cavity due to its interactions with pathogens [50], despite its relatively low prevalence compared to other H 2 O 2 producers streptococci in the oral cavity [51].This species can activate osteoclasts [52] and induce an inflammatory response in experimental apical periodontitis [53], stimulating the production of chemokines such as CXCL8 by periodontal ligament fibroblasts [54] as well as the maturation of dendritic cells [55].Thus, under the limitations of the murine model, it seems that Sg may be important for Aa survival in the supragingival biofilm, but the co-infection may not be relevant for Aainduced bone resorption.
Although Sg facilitated the recovery of Aa in the oral cavity, the impact of oral administration of Aa in the gut was more pronounced compared to that promoted by the Aa+Sg consortium.Limited knowledge exists regarding the role of Aa and/or Sg as inducers of intestinal dysbiosis and alteration of the intestinal barrier.Aa infection induced the expression of Il-1β in the gut, while Sg or the Aa+Sg consortium did not.Il-1β is commonly associated with heightened intestinal permeability [56].Conversely, Sg mitigated the Aainduced inflammatory response in the gut, evidenced by the low transcriptional levels of Il-1β in the Aa+Sg group.Previous data have indicated that H 2 O 2 produced by streptococci activates the Nrf2 pathway in macrophages, inhibiting the NFκB transcription factor and reducing the production of proinflammatory cytokines [57].Other factors may also have contributed to this disparity.Aa mono-infection, but not the dual infection with Aa and Sg, resulted in the expression of virulence factors such as the cytolethal distending toxin (CDT) [32], which is known to activate the inflammasome leading to IL-1β release [58].Furthermore, H 2 O 2 acts as a signal to trigger the expression of catalase and OMP100 (ApiA) by Aa, which are involved in defense against the host's innate immune system [59].In contrast, the IL-1β pathway may hinder Aa survival in the gut [60].
We have also demonstrated downregulation of Il-6 in the Aa, Sg, and Aa+Sg groups compared to the SHAM group.IL-6 plays a role in leukocyte recruitment during the acute inflammatory response by selectively regulating inflammatory chemokines and apoptotic events [17,61,62].Therefore, it appears that infection with Aa and Sg contributed to the control of the inflammatory process, likely facilitated by the release of H 2 O 2 by the accessory pathogen Sg [50], although other factors, such as additional regulatory proteins such as Aa outer membrane protein OMP29, may also play a role [13].
It is well recognized that dysregulation of the mucosal barrier facilitates the translocation of bacterial components or living cells [56] from the intestinal lumen through the epithelium, triggering immune activation and leading to systemic inflammation [63,64].Alterations in the expression of TJ proteins can compromise the integrity of the barrier, promoting increased intestinal permeability, thus resulting in a condition often referred to as "leaky gut" [63,65,66].
Oral infection with Aa and/or Sg resulted in altered transcription of several genes involved in gut barrier integrity [67].Generally, Aa or Sg downregulated the expression of Cldn 1 and Cldn 2, Ocdn, and Zo-1, whereas the Aa+Sg consortium upregulated the expression of Cldn 2, Ocdn, and Zo-1.The elevated levels of Il-1β transcripts and low levels of Il-6 transcripts in the gut of Aa-infected mice may partially account for this regulation induced by the pathogen [68].Microbial insults and the resulting inflammatory response are known to contribute to the regulation of TJ expression and localization.Expression of Claudin 1 diminishes upon chronic exposure to LPS challenge [69], whilst Claudin 2 expression is associated with protection of microbial-induced colitis [38].IL-6 upregulates expression of Claudin 2, while TNF and IL-1β indirectly trigger occludin removal from the tight junction and increase leaky pathway permeability.On the other hand, IL-10 and TGFβ promote epithelial proliferation and restitution, leading to barrier restoration [70].Thus, the attenuation of Il-1β and Il-6 regulation observed when Aa was combined with Sg may contribute to the enhanced expression of TJs in the gut barrier.Previous data reported that Sg exerts a beneficial effect on the epithelial barrier function, particularly in gingival cells, by inhibiting cytokine secretion and enhancing the expression of key tight junction components, thus reinforcing the oral barrier against microbial invasion [71].However, it should be noticed that the changes in TJ transcription profiles induced by Aa and/or Sg were discreet, since we could not detect endotoxemia in any of the studied groups, an indicator of altered gut permeability [72].
Oral inoculation of Aa, Sg, or Aa+Sg led to alterations in both oral and gut microbiomes.The oral microbiota plays a critical role in maintaining oral health and can influence intestinal dysbiosis.In periodontitis, dysbiosis has been observed not only in the oral cavity but also in the intestine [14][15][16].The mechanism behind this phenomenon involves the ingestion of oral bacteria, which may persist in the gut for at least 24 h [73], potentially inducing dysbiosis [74].
Our data indicate that oral administration of Sg increased the diversity of the oral microbiome (Faith phylogenetic diversity index), whereas Aa or the consortium Aa+Sg reduced the diversity when compared to mono-infection with Sg.Phylogenetic diversity accounts for the phylogenetic relatedness of the community members [33] and reduced diversity induced by Aa means that this organism puts pressure on the oral microbiome in order to select a subset of organisms.These findings are consistent with studies showing a decrease in the microbial diversity of the oral cavity in periodontitis patients [75,76], indicating a role for Aa in oral dysbiosis.
The population structure of the oral microbiome differed between the Aa-infected group and the Sg-infected group compared with the SHAM group, with all infected groups differing from each other.The oral microbiome of Aa-infected mice exhibited increased abundance of Pseudomonadota, class Gammaproteobacteria, order Enterobacterales, and of the genus Alloprevotella.Sg administration also increased the abundance of Alloprevotella but additionally led to higher levels of Rothia.The implications of these microbial shifts are not fully understood, as members of Enterobacterales and the family Prevotellaceae comprise species associated with both disease and health [77,78].However, Enterobacterales has previously been associated with Crohn's disease [79][80][81][82], whereas Rothia is typically associated with health [83].
Animals infected with Aa, Sg, or both organisms exhibited a reduced abundance of Erysipelotrichaceae in the gut, reaching non-detectable levels.Erysipelotrichaceae is considered a beneficial organism to the gut (97) and previous data have shown that Aa oral inoculation resulted in lower abundance of Turicibacter, a genus of the family Erysipelotrichaceae, in feces [21].Oral administration of streptococci in mice, such as S. mitis or S. salivarius, also reduced the levels of Turicibacter in stool samples [84].The beneficial role of Erysipelotrichaceae in the gut appears to be due to the production of butyric acid, a fatty acid that provides energy to colonocytes and regulates the immune response [85].Thus, our data suggest that infection with Aa, Sg, or both organisms results in depletion of beneficial organisms, in agreement with reduced transcription of genes associated with gut permeability.
Periodontitis has been linked to chronic inflammatory bowel disease (IBD) and colon cancer [86].It is noteworthy that gut dysbiosis in patients with periodontitis, particularly the molar-incisor pattern associated with high abundance of Aa, was characterized by a greater abundance of sulfate-reducing bacteria (SRB), which are potential risk factors for colon cancer [16,87].Aa can be found in low abundance in healthy individuals [88], but the oral microbiota in health harbors a higher proportion of H 2 O 2 producers, such as S. sanguinis and S. gordonii, compared to disease [51].Thus, the low transcriptional levels of Il-1β in the gut of mice of the Aa+Sg group compared to the Aa group suggest that these streptococci may limit the inflammatory potential of Aa in the gut.On the other hand, damage not only to the periodontium but also to the gut barrier appears to increase when the abundance of Aa rises due to host and bacterial factors, concurrent with decreased levels of lactate/H 2 O 2 producers [89].
Taken altogether, our data indicate that Aa can disrupt both oral and gut microbiomes.The pathogen may disturb gut homeostasis by inducing the expression of inflammatory mediators such as IL-1β and by altering the expression profile of proteins related to intestinal barrier integrity.Aa co-exists in the oral cavity with other species such as Sg, which enhances its survival in the oral biofilm.However, co-infection of Aa with Sg seems to mitigate the effects of Aa on the gut.

Ethical Considerations
This study was conducted after obtaining approval from the Institutional Animal Care and Use Committee (ICB USP approval number: 4828281020), in accordance with the ethical principles of animal experimentation outlined by the Brazilian College of Animal Experimentation (COBEA).

Animals and Maintenance Conditions
C57BL/6 specific pathogen-free (SPF) male mice, aged 6-8 weeks, were obtained from the Animal Facility at the Faculty of Medicine and subsequently transferred to the Department of Microbiology and Parasitology at the Institute of Biomedical Sciences, University of São Paulo.They were housed in cages with a maximum of four mice per cage, fitted with microisolator filters, and placed on ventilated shelves to maintain pathogen-free conditions, with food (chow diet) provided ad libitum.

Sample Calculation and Group Allocation
Sample calculation was conducted for alveolar bone loss as the outcome, with a significance level (alpha) of 5%, power of 80%, and considering a 10% loss of animals during the experiment, based on our previous pilot data [90,91].After a 7-day acclimation period in the facility, forty-eight (48) animals were randomly assigned to four groups, with twelve animals per group: SHAM, infected with Aa, infected with Sg, and infected with both species (Aa+Sg).The animals were then monitored for an experimental period of 30 days.Animals showing abnormalities in growth, weight, or physical characteristics at baseline were excluded from the study.Throughout the experiment, animals were monitored for changes in weight, mobility, coat condition, and presence of skin lesions.

Blinding
Each animal was assigned a temporary random number within its respective group.Cages were numbered based on their placement on the rack.A cage was randomly selected from each group from the available pool.Blinding procedures were maintained throughout the allocation process, evaluation of results, and data analysis.However, blinding was not possible during the experiment due to the same researcher being involved in both the preparation and inoculation of organisms.Additionally, the bacterial suspensions differed in color from the vehicles used.

Strains and Culture Conditions
Aa JP2 [38] and Sg DL1 [92] strains were utilized.All strains were maintained in 20% glycerol in brain-heart broth at −80 • C.
Aa was cultured in tryptone soy broth (Difco Laboratories, Detroit, MI, USA) supplemented with 0.5% yeast extract (Difco Laboratories) while Sg was cultured in BHI broth (Difco Laboratories), both incubated at 37 • C, in 5-10% CO 2 .For the assays, liquid medium was utilized to grow bacteria to mid-log phase.Subsequently, the cultures were centrifuged (355× g for 5 min), and the cell pellets were suspended in phosphate-buffered saline solution (1× PBS) to an optical density at 590nm of approximately 1, corresponding to 1 × 10 9 CFU Aa/mL and to 1 × 10 12 CFU Sg/mL.
The lyophilized material was stored in a freezer at −80 • C. The viability of each batch of lyophilized bacteria was assessed by culturing them on agar plates under suitable conditions.Before each day of inoculation, the samples were reconstituted in BHI broth for one hour.Subsequently, the lyophilized bacteria were suspended in PBS at standardized concentrations.

Experimental Infections
Fifty-microliter aliquots containing 1 × 10 9 CFU of Aa and/or 1 × 10 9 CFU of Sg in PBS with 1.5% carboxymethylcellulose were inoculated into the oral cavity of mice using a gavage needle, three times a week, for four weeks, totalizing 12 inoculations [47].On the first, third, and fifth days of the experimental period, the animals underwent intraperitoneal anesthesia with ketamine (100 mg/kg) (10% Dopalen, Vetbrands Laboratory, Rio de Janeiro, Brazil) and xylazine (10 mg/kg) (2% Ansedan, Vertbrands Laboratory), when they received an injection at the interproximal gingiva between the first and second molars in the left hemimaxilla of a volume of 10 µL containing 1 × 10 7 CFU of Aa in PBS [48].The vehicle (PBS with 1.5% carboxymethylcellulose) was orally administered to the SHAM-inoculated group.Both the SHAM and Sg groups were anesthetized and received gingival injections with PBS on the same days and with the same volumes as the other Aa infected groups.After 30 days, the mice were euthanized, and samples were collected.The study design is illustrated in Figure 1.

Euthanasia and Sample Collection
The animals were anesthetized intraperitoneally with ketamine (100 mg/kg) and xylazine (10 mg/kg).Blood was collected via cardiac puncture, and the mice were euthanized by exsanguination.Serum was obtained by centrifugation.The left hemimaxillae were transferred to formaldehyde solution and kept at room temperature for 24 h, then transferred to PBS and stored at 4 • C until analysis.The jejunal portions of the intestine were placed in RNAlater TM Stabilization Solution (Invitrogen by Thermo Fisher Scientific, Vilnius, Lithuania) and stored at −80 • C. Oral biofilms were collected using microbrushes, and jejunal content samples were obtained using a wooden spatula and stored in Tris-EDTA buffer, pH 7.4.

Computerized Microtomography (Micro CT)
Alveolar bone resorption was assessed using microtomography (SkyScan 1174 version 1.1, Kontich, Belgium) operating at 45 kV voltage, 550 µA current, with an 8.71 µm pixel size and a 0.2 mm aluminum filter.Scans were conducted on the left hemimaxillae, and a blinded evaluator identified a standardized area of 60 × 30 pixels (ROI) at the interproximal region between the first and second molars, originating from the cementoenamel junction of the second molar across 15 coronal sections.Bone volume and porosity percentages were determined through image analysis using CT Analyzer software version 1.15.4.0.

Serum Lipopolysaccharide Levels
Lipopolysaccharide (LPS) levels were quantified using the Pierce LAL Chromogenic Endotoxin Quantification Kit #88282 (Thermo Scientific™, Rockford, IL, USA).Solutions ranging from 0.1 to 1 endotoxin units (EUs) per milliliter of LPS Endotoxin Standard (E. coli 011: B4) were employed to determine the endotoxin concentration of each sample.Absorbance was measured at 405 nm using the Epoch™ 2 microplate spectrophotometer (BioTek, Winooski, VT, USA).

Gene Expression Linked to Gut Inflammation and Epithelial Barrier Permeability
Gut samples underwent RNA extraction using Trizol LS Reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) with the assistance of a Mini-BeadBeater (BioSpec 3110BX Mini-BeadBeater-1 High Energy Cell Disrupter, Campinas, São Paulo, Brazil) for 20 s, repeated twice.The resulting RNA was treated with desoxyribonuclease (Ambion™ Dnase I, Invitrogen Life Technologies).Subsequently, cDNA was synthesized using the SuperScript TM Vilo TM Synthesis Kit for RT-PCR (Invitrogen Life Technologies).Quantitative PCR was conducted using the StepOne Plus System thermocycler (Applied Biosystems, Foster City, CA, USA).

Gene
Sequence (5 ′ -3 ′ ) Cldn1 The relative expression of target genes was calculated using the ∆∆CT method, utilizing GAPDH as an endogenous control [39] and expressed as fold changes relative to the control group (SHAM).

Oral and Gut Microbiomes
DNA from oral biofilm and gut samples was extracted using the Master PureTM DNA Purification Kit (Epicentre ® Illumina Company, Madison, WI, USA).The quality of purified DNA was assessed using the Qubit 2.0 fluorometer (Thermo-Fisher Scientific, Carlsbad, CA, USA).Unfortunately, DNA from the biofilms of two animals from each group was lost during manipulation, leading to the exclusion of gut samples from these animals, leading to microbiome analyses of six samples from each site per group.
Data were analyzed using Qiime 2 2022.8software [99].Demultiplexed sequences and reads underwent filtering using Dada 2, with a quality threshold set at 25. Subsequently, trimmed sequences were clustered into amplicon sequence variants (ASVs), and taxonomy was assigned using the Silva138 database [100,101].Alpha diversity indices, including Faith, Pielou, and Shannon, were assessed.Beta diversity analyses were conducted using weighted and unweighted UniFrac distance matrices and were visualized through principal coordinate analysis (PCA) [99].Significant differences among groups were determined by PERMANOVA with 999 permutations.The discrepancies in abundances of ASVs among groups were analyzed using ANCOM [102] with a 75% empirical cutoff value for the w statistic [103].To identify the inoculated species in the oral and gut microbiome, the Expanded Human Oral Microbiome Database (eHOMD) was utilized [104].

Data Analyses
The data were assessed for normality using the Kolmogorov-Smirnov statistical test with Lilliefors correction, and the homogeneity of variances was evaluated using the F test.For standard data analysis, the Kruskal-Wallis test followed by Dunn's post hoc test

Figure 1 .
Figure1.Study design: C57BL/6 mice were randomly assigned into four groups: the SHAM inoculated group, the group infected with Aa, the group infected with Sg, and the group infected with both species, Aa+Sg.After a 30-day experimental period, the mice were euthanized, and samples were collected.

Figure 2 .
Figure 2. Analysis reveals alveolar bone resorption in C57BL/6 mice maxilla following various infection protocols (SHAM, Aa, Sg, Aa+Sg).Mice weight (A) and bone porosity (B) and bone volume (C) which were measured at the interproximal area between the first and second molars on the lef hemimaxilla at the end of the experimental period.Distinct letters indicate statistically significan differences (p < 0.05) between groups (Kruskal-Wallis followed by post hoc Dunn test).

Figure 1 .
Figure1.Study design: C57BL/6 mice were randomly assigned into four groups: the SHAMinoculated group, the group infected with Aa, the group infected with Sg, and the group infected with both species, Aa+Sg.After a 30-day experimental period, the mice were euthanized, and samples were collected.

Figure 2 .
Figure 2. Analysis reveals alveolar bone resorption in C57BL/6 mice maxilla following variou infection protocols (SHAM, Aa, Sg, Aa+Sg).Mice weight (A) and bone porosity (B) and bone volum (C) which were measured at the interproximal area between the first and second molars on the le hemimaxilla at the end of the experimental period.Distinct letters indicate statistically significan differences (p < 0.05) between groups (Kruskal-Wallis followed by post hoc Dunn test).

Figure 2 .
Figure 2. Analysis reveals alveolar bone resorption in C57BL/6 mice maxilla following various infection protocols (SHAM, Aa, Sg, Aa+Sg).Mice weight (A) and bone porosity (B) and bone volume (C) which were measured at the interproximal area between the first and second molars on the left hemimaxilla at the end of the experimental period.Distinct letters indicate statistically significant differences (p < 0.05) between groups (Kruskal-Wallis followed by post hoc Dunn test).

Figure 3 .
Figure 3. Relative transcription of genes involved in inflammation and intestinal permeability in th jejunum of C57BL/6 mice subjected to different infection protocols: SHAM (non-infected negativ control), Aa (administered orally and via gingival injections), Sg (administered orally), and Aa+S (Aa and Sg administered orally and Aa via gingival injections), at the end of the experimental perio Il-1β (A), Il-6 (B), Cldn 1 (C), Cldn 2 (D), Ocdn (E), and Zo-1 (F).Relative gene transcription data a presented as fold changes compared to the negative control (SHAM).Distinct letters indica statistically significant differences determined by Kruskal-Wallis followed by Dunn's post hoc te (p < 0.05).

Figure 3 .
Figure 3. Relative transcription of genes involved in inflammation and intestinal permeability in the jejunum of C57BL/6 mice subjected to different infection protocols: SHAM (non-infected negative control), Aa (administered orally and via gingival injections), Sg (administered orally), and Aa+Sg (Aa and Sg administered orally and Aa via gingival injections), at the end of the experimental period.Il-1β (A), Il-6 (B), Cldn 1 (C), Cldn 2 (D), Ocdn (E), and Zo-1 (F).Relative gene transcription data are presented as fold changes compared to the negative control (SHAM).Distinct letters indicate statistically significant differences determined by Kruskal-Wallis followed by Dunn's post hoc test (p < 0.05).

Figure 4 .
Figure 4. Faith diversity index of the oral microbiome (A) and Pielou diversity index of the gut microbiome (B) in C57BL/6 mice subjected to different infection protocols: SHAM (non-infected negative control), Aa (administered orally and via gingival injections), Sg (administered orally), and Aa+Sg (Aa and Sg administered orally and Aa via gingival injections), at the end of the 30-day experimental period.* Statistically significant differences (p < 0.05) between groups determined by Kruskal-Wallis followed by Dunn's post hoc test.

Figure 4 .
Figure 4. Faith diversity index of the oral microbiome (A) and Pielou diversity index of the gut microbiome (B) in C57BL/6 mice subjected to different infection protocols: SHAM (non-infected negative control), Aa (administered orally and via gingival injections), Sg (administered orally), and Aa+Sg (Aa and Sg administered orally and Aa via gingival injections), at the end of the 30-day experimental period.* Statistically significant differences (p < 0.05) between groups determined by Kruskal-Wallis followed by Dunn's post hoc test.

Figure 5 .
Figure 5. Principal coordinate analysis (PCoA) of oral (A) and gut (B) microbiomes based on weighted (I) and unweighted (II) UniFrac distance of C57BL/6 mice under various infection protocols: SHAM (negative control), Aa (oral and gingival Aa administration), Sg (oral Sg administration), and Aa+Sg (oral Aa and Sg administration and gingival Aa injections), at the end of the 30-day experimental period.Each dot represents one sample.Oral microbiomes showed distinct clustering for each group (PERMANOVA, p < 0.05), except for SHAM and Aa+Sg.In the gut microbiomes, infected groups clustered apart from SHAM (p < 0.05), with Aa+Sg and Sg displaying differences.Ellipses in A show that SHAM and Aa+Sg oral microbiomes cluster apart from Aa-or Sg-infected groups.Ellipses in (B) show that SHAM gut microbiome clustered apart from the infected groups.

Figure 5 .
Figure 5. Principal coordinate analysis (PCoA) of oral (A) and gut (B) microbiomes based on weighted (I) and unweighted (II) UniFrac distance of C57BL/6 mice under various infection protocols: SHAM (negative control), Aa (oral and gingival Aa administration), Sg (oral Sg administration), and Aa+Sg (oral Aa and Sg administration and gingival Aa injections), at the end of the 30-day experimental period.Each dot represents one sample.Oral microbiomes showed distinct clustering for each group (PERMANOVA, p < 0.05), except for SHAM and Aa+Sg.In the gut microbiomes, infected groups clustered apart from SHAM (p < 0.05), with Aa+Sg and Sg displaying differences.Ellipses in A show that SHAM and Aa+Sg oral microbiomes cluster apart from Aaor Sg-infected groups.Ellipses in (B) show that SHAM gut microbiome clustered apart from the infected groups.

Figure 6 .
Figure 6.Oral microbiome of C57BL/6 mice subjected to various infection protocols showed differences in abundance of selected taxa.SHAM (negative control), Aa (oral and gingival Aa administration), Sg (oral Sg administration), and Aa+Sg (oral Aa and Sg administration with gingival Aa injections).Phyla relative abundance (%)-based plots (A).ANCOM revealed increased abundance of Gammaproteobacteria, Enterobacterales, Alloprevotella, and Rothia in each infected group compared to SHAM (B).The 75th percentile of the W distribution was used as the empirical cut-off value, and fold changes in abundances relative to the SHAM group are presented.

Figure 6 .
Figure 6.Oral microbiome of C57BL/6 mice subjected to various infection protocols showed differences in abundance of selected taxa.SHAM (negative control), Aa (oral and gingival Aa administration), Sg (oral Sg administration), and Aa+Sg (oral Aa and Sg administration with gingival Aa injections).Phyla relative abundance (%)-based plots (A).ANCOM revealed increased abundance of Gammaproteobacteria, Enterobacterales, Alloprevotella, and Rothia in each infected group compared to SHAM (B).The 75th percentile of the W distribution was used as the empirical cut-off value, and fold changes in abundances relative to the SHAM group are presented.

Figure 7 .
Figure 7. Gut microbiome of C57BL/6 mice subjected to various infection protocols showed differences in abundance of selected taxa.SHAM (negative control), Aa (oral and gingival Aa administration), Sg (oral Sg administration), and Aa+Sg (oral Aa and Sg administration with gingival Aa injections).Phyla relative abundance (%)-based plots (A).ANCOM revealed that the order Erysipelotrichales and family Erisopelotrichaceae were only detected in SHAM but not in the infected groups (B).

Figure 7 .
Figure 7. Gut microbiome of C57BL/6 mice subjected to various infection protocols showed differences in abundance of selected taxa.SHAM (negative control), Aa (oral and gingival Aa administration), Sg (oral Sg administration), and Aa+Sg (oral Aa and Sg administration with gingival Aa injections).Phyla relative abundance (%)-based plots (A).ANCOM revealed that the order Erysipelotrichales and family Erisopelotrichaceae were only detected in SHAM but not in the infected groups (B).