Several ecological hypotheses (e.g., specific plaque, non-specific plaque and keystone pathogen) regarding the etiology of periodontitis have been proposed since the 1990s, most of which have been centered on the concept of dysbiosis associated with periodontitis. Nevertheless, none of the existing hypotheses have presented mechanistic interpretations on how and why dysbiosis actually occurs. Hubbell's neutral theory of biodiversity offers a powerful null model to test hypothesis regarding the mechanism of community assembly and diversity maintenance from the metagenomic sequencing data, which can help to understand the forces that shape the community dynamics such as dysbiosis. Here we reanalyze the dataset from Abusleme et al.'s comparative study of the oral microbial communities from periodontitis patients and healthy individuals. Our study demonstrates that 14 out of 61 communities (23%) passed the neutrality test, a percentage significantly higher than the previous reported neutrality rate of 1% in human microbiome (Li & Ma 2016, Scientific Reports). This suggests that, while the niche selection may play a predominant role in the assembly and diversity maintenance in oral microbiome, the effect of neutral dynamics may not be ignored. However, no statistically significant differences in the neutrality passing rates were detected between the periodontitis and healthy treatments with Fisher's exact probability test and multiple testing corrections, suggesting that the mechanism of community assembly is robust against disturbances such as periodontitis. In addition, our study confirmed previous finding that periodontitis patients exhibited higher biodiversity. These findings suggest that while periodontitis may significantly change the community composition measured by diversity (i.e., the exhibition or 'phenotype' of community assembly), it does not seem to cause the 'mutation' of the 'genotype" (mechanism) of community assembly. We argue that the 'phenotypic' changes explain the observed link (not necessarily causal) between periodontitis and community dysbiosis, which is certainly worthy of further investigation.

The oral fissure is immensely inhabited with a number of polymicrobial colonies similar to the intestinal system. Periodontitis is a dysbiotic disease resulting from deviation in subgingival Gram-positive bacteria to Gram-negative bacteria shift from Gram-positive bacteria. The development of periodontal dysbiosis occurs over a broadened timeframe, which slowly turns the symbiotic association of host and microbe to pathogenic. This review highlights a recent paradigm of periodontitis progression has been postulated which challenges the traditional concept of periodontitis being induced by few particular periopathogens such as belonging to red complex, but by a more comprehensive dysbiotic-synergistic community.

The overall health of the oral cavity is dependent on proper homeostasis between health-associated bacterial colonizers and bacteria known to promote dental caries. Streptococcus sanguinis is a health-associated commensal organism, a known early colonizer of the acquired tooth pellicle, and is naturally competent. We have shown that LytF, a competence-controlled murein hydrolase, is capable of inducing the release of extracellular DNA (eDNA) from oral bacteria. Precipitated LytF and purified LytF were used as treatments against planktonic cultures and biofilms. Larger amounts of eDNA were released from cultures treated with protein samples containing LytF. Additionally, LytF could affect biofilm formation and cellular morphology. Biofilm formation was significantly decreased in the lytF-complemented strain, in which increased amounts of LytF are present. The same strain also exhibited cell morphology defects in both planktonic cultures and biofilms. Furthermore, the LytF cell morphology phenotype was reproducible in wild-type cells using purified LytF protein. In sum, our findings demonstrate that LytF can induce the release of eDNA from oral bacteria, and they suggest that, without proper regulation of LytF, cells display morphological abnormalities that contribute to biofilm malformation. In the context of the oral biofilm, LytF may play important roles as part of the competence and biofilm development programs, as well as increasing the availability of eDNA.IMPORTANCEStreptococcus sanguinis, a commensal organism in the oral cavity and one of the pioneer colonizers of the tooth surface, is associated with the overall health of the oral environment. Our laboratory showed previously that, under aerobic conditions, S. sanguinis can produce H₂O₂ to inhibit the growth of bacterial species that promote dental caries. This production of H₂O₂ by S. sanguinis also induces the release of eDNA, which is essential for proper biofilm formation. Under anaerobic conditions, S. sanguinis does not produce H₂O₂ but DNA is still released. Determining how S. sanguinis releases DNA is thus essential to understand biofilm formation in the oral cavity.

Dysbiosis in the periodontal microbiota is associated with the development of periodontal diseases. Little is known about the initiation of dysbiosis. It was hypothesized that some commensal bacteria suppress the outgrowth of pathobionts by H₂O₂ production. However, serum and blood components released due to inflammation can neutralize this suppressive effect, leading to the initiation of dysbiosis. Agar plate, dual-species and multi-species ecology experiments showed that H₂O₂ production by commensal bacteria decreases pathobiont growth and colonization. Peroxidase and blood components neutralize this inhibitory effect primarily by an exogenous peroxidase activity without stimulating growth and biofilm formation of pathobionts directly. In multi-species environments, neutralization of H₂O₂ resulted in 2 to 3 log increases in pathobionts, a hallmark for dysbiosis. Our data show that in oral biofilms, commensal species suppress the amounts of pathobionts by H₂O₂ production. Inflammation can neutralize this effect and thereby initiates dysbiosis by allowing the outgrowth of pathobionts.

Little is known about the initiation of dysbiosis in oral biofilms, a topic of prime importance for understanding the etiology of, and preventing, periodontitis. The aim of this study was to evaluate the effect of different concentrations of crevicular and salivary peroxidase and catalase on dysbiosis in multispecies biofilms in vitro.

Periodontal diseases originate from a dysbiosis within the oral microbiota, which is associated with a deregulation of the host immune response. Although little is known about the initiation of dysbiosis, it has been shown that H₂O₂ production is one of the main mechanisms by which some commensal bacteria suppress the outgrowth of pathobionts. Current models emphasize the critical nature of complex microbial biofilms that form unique microbial ecologies and of their change during transition from health (homeostatic) to disease (dysbiotic). However, very little is known on how this alters their virulence and host responses. The objective of this study was to determine differences in virulence gene expression by pathobionts and the inflammatory host response in homeostatic and dysbiotic biofilms originating from the same ecology. Quantitative polymerase chain reaction was performed to quantify the pathobiont outgrowth. Expression analysis of bacterial virulence and cellular inflammatory genes together with cytokine enzyme-linked immunosorbent assays were used to detect differences in bacterial virulence and to analyze potential differences in inflammatory response. An increase in pathobionts in induced dysbiotic biofilms was observed compared to homeostatic biofilms. The main virulence genes of all pathobionts were upregulated in dysbiotic biofilms. Exposure of these dysbiotic biofilms to epithelial and fibroblast cultures increased the expression of interleukin (IL)-6, IL-1β, tumor necrosis factor-α, and matrix metalloprotease 8, but especially the chemokine CXCL8 (IL-8). Conversely, homeostatic and beneficial biofilms had a minor immune response at the messenger RNA and protein level. Overall, induced dysbiotic biofilms enriched in pathobionts and virulence factors significantly increased the inflammatory response compared to homeostatic and commensal biofilms.

Periodontitis is a dysbiotic inflammatory disease with an adverse impact on systemic health. Recent studies have provided insights into the emergence and persistence of dysbiotic oral microbial communities that can mediate inflammatory pathology at local as well as distant sites. This Review discusses the mechanisms of microbial immune subversion that tip the balance from homeostasis to disease in oral or extra-oral sites.

The human body supports the growth of a wide array of microbial communities in various niches such as the oral cavity, gastro-intestinal and urogenital tracts, and on the surface of the skin. These host associated microbial communities include yet-un-cultivable bacteria and are influenced by various factors. Together, these communities of bacteria are referred to as the human microbiome. Human oral microbiome consists of both symbionts and pathobionts. Deviation from symbiosis among the bacterial community leads to “dysbiosis”, a state of community disturbance. Dysbiosis occurs due to many confounding factors that predispose a shift in the composition and relative abundance of microbial communities. Dysbiotic communities have been a major cause for many microbiome related systemic infections. Such dysbiosis is directed by certain important pathogens called the “keystone pathogens”, which can modulate community microbiome variations. One such persistent infection is oral infection, mainly periodontitis, where a wide array of causal organisms have been implied to systemic infections such as cardio vascular disease, diabetes mellitus, rheumatoid arthritis, and Alzheimer’s disease. The keystone pathogens co-occur with many yet-cultivable bacteria and their interactions lead to dysbiosis. This has been the focus of recent research. While immune evasion is one of the major modes that leads to dysbiosis, new processes and new virulence factors of bacteria have been shown to be involved in this important process that determines a disease or health state. This review focuses on such dysbiotic communities, their interactions, and their virulence factors that predispose the host to other systemic implications.

In China, chronic periodontitis (CP) is common in patients with type 2 diabetes mellitus (T2DM). The purpose of this study is to identify the sociodemographic characteristics associated with such patients and to assess the periodontal health status and subgingival microbiota of patients with CP and T2DM (T2DMCP) in the Chinese population.

Dysbiosis, or the imbalance in the structural and/or functional properties of the microbiome, is at the origin of important infectious inflammatory diseases such as inflammatory bowel disease (IBD) and periodontal disease. Periodontitis is a polymicrobial inflammatory disease that affects a large proportion of the world's population and has been associated with a wide variety of systemic health conditions, such as diabetes, cardiovascular and respiratory diseases. Dysbiosis has been identified as a key element in the development of the disease. However, the precise mechanisms and environmental signals that lead to the initiation of dysbiosis in the human microbiome are largely unknown. In a series of previous in vivo studies using metatranscriptomic analysis of periodontitis and its progression we identified several functional signatures that were highly associated with the disease. Among them, potassium ion transport appeared to be key in the process of pathogenesis. To confirm its importance we performed a series of in vitro experiments, in which we demonstrated that potassium levels a increased the virulence of the oral community as a whole and at the same time altering the immune response of gingival epithelium, increasing the production of TNF-α and reducing the expression of IL-6 and the antimicrobial peptide human β-defensin 3 (hBD-3). These results indicate that levels of potassium in the periodontal pocket could be an important element in of dysbiosis in the oral microbiome. They are a starting point for the identification of key environmental signals that modify the behavior of the oral microbiome from a symbiotic community to a dysbiotic one.

Periodontitis is a common inflammatory disease that leads to tooth loss and has been linked to cardiovascular disease and diabetes mellitus. The periodontal microbiome is highly diverse, and metatranscriptomic studies have indicated that the genes that are expressed by the microbiota are more relevant than the microbial composition in the pathogenesis of periodontitis. A recent study of early metabolic activities in the dysbiotic microbiome reveals a functional signature that distinguishes periodontal sites that will become inflamed, supporting the idea that microbial communities as a whole drive disease progression.

Periodontitis is an extremely prevalent disease worldwide and is driven by complex dysbiotic microbiota. Here we analyzed the transcriptional activity of the periodontal pocket microbiota from all domains of life as well as the human host in health and chronic periodontitis. Bacteria showed strong enrichment of 18 KEGG functional modules in chronic periodontitis, including bacterial chemotaxis, flagellar assembly, type III secretion system, type III CRISPR-Cas system, and two component system proteins. Upregulation of these functions was driven by the red-complex pathogens and candidate pathogens, e.g. Filifactor alocis, Prevotella intermedia, Fretibacterium fastidiosum and Selenomonas sputigena. Nine virulence factors were strongly up-regulated, among them the arginine deiminase arcA from Porphyromonas gingivalis and Mycoplasma arginini. Viruses and archaea accounted for about 0.1% and 0.22% of total putative mRNA reads, respectively, and a protozoan, Entamoeba gingivalis, was highly enriched in periodontitis. Fourteen human transcripts were enriched in periodontitis, including a gene for a ferric iron binding protein, indicating competition with the microbiota for iron, and genes associated with cancer, namely nucleolar phosphoprotein B23, ankyrin-repeat domain 30B-like protein and beta-enolase. The data provide evidence on the level of gene expression in vivo for the potentially severe impact of the dysbiotic microbiota on human health.

Background: While CRISPR-Cas systems have been identified in bacteria from a wide variety of ecological niches, there are no studies to describe CRISPR-Cas elements in Bacteroides species, the most prevalent anaerobic bacteria in the lower intestinal tract. Microbes of the genus Bacteroides make up ~25% of the total gut microbiome. Bacteroides fragilis comprises only 2% of the total Bacteroides in the gut, yet causes of >70% of Bacteroides infections. The factors causing it to transition from benign resident of the gut microbiome to virulent pathogen are not well understood, but a combination of horizontal gene transfer (HGT) of virulence genes and differential transcription of endogenous genes are clearly involved. The CRISPR-Cas system is a multi-functional system described in prokaryotes that may be involved in control both of HGT and of gene regulation. Results: Clustered regularly interspaced short palindromic repeats (CRISPR) elements in all strains of B. fragilis (n = 109) with publically available genomes were identified. Three different CRISPR-Cas types, corresponding most closely to Type IB, Type IIIB, and Type IIC, were identified. Thirty-five strains had two CRISPR-Cas types, and three strains included all three CRISPR-Cas types in their respective genomes. The cas1 gene in the Type IIIB system encoded a reverse-transcriptase/Cas1 fusion protein rarely found in prokaryotes. We identified a short CRISPR (3 DR) with no associated cas genes present in most of the isolates; these CRISPRs were found immediately upstream of a hipA/hipB operon and we speculate that this element may be involved in regulation of this operon related to formation of persister cells during antimicrobial exposure. Also, blood isolates of B. fragilis did not have Type IIC CRISPR-Cas systems and had atypical Type IIIB CRISPR-Cas systems that were lacking adjacent cas genes. Conclusions: This is the first systematic report of CRISPR-Cas systems in a wide range of B. fragilis strains from a variety of sources. There are four apparent CRISPR-Cas systems in B. fragilis-three systems have adjacent cas genes. Understanding CRISPR/Cas function in B. fragilis will elucidate their role in gene expression, DNA repair and ability to survive exposure to antibiotics. Also, based on their unique CRISPR-Cas arrays, their phylogenetic clustering and their virulence potential, we are proposing that blood isolates of B. fragilis be viewed a separate subgroup.

Periodontitis is a polymicrobial biofilm-induced inflammatory disease that affects 743 million people worldwide. The current model to explain periodontitis progression proposes that changes in the relative abundance of members of the oral microbiome lead to dysbiosis in the host-microbiome crosstalk and then to inflammation and bone loss. Using combined metagenome/metatranscriptome analysis of the subgingival microbiome in progressing and non-progressing sites, we have characterized the distinct molecular signatures of periodontitis progression.

Recent advancements in the periodontal research field are consistent with a new model of pathogenesis according to which periodontitis is initiated by a synergistic and dysbiotic microbial community rather than by select periopathogens, such as the red complex. In this polymicrobial synergy, different members or specific gene combinations within the community fulfill distinct roles that converge to shape and stabilize a disease-provoking microbiota. One of the core requirements for a potentially pathogenic community to arise involves the capacity of certain species, termed keystone pathogens, to modulate the host response in ways that impair immune surveillance and tip the balance from homeostasis to dysbiosis. Keystone pathogens also elevate the virulence of the entire microbial community through interactive communication with accessory pathogens. Other important core functions for pathogenicity require the expression of diverse molecules (e.g. appropriate adhesins, cognate receptors, proteolytic enzymes and proinflammatory surface structures/ligands), which in combination act as community virulence factors to nutritionally sustain a heterotypic, compatible and proinflammatory microbial community that elicits a non-resolving and tissue-destructive host response. On the basis of the fundamental concepts underlying this model of periodontal pathogenesis, that is, polymicrobial synergy and dysbiosis, we term it the PSD model.

Porphyromonas gingivalis and Filifactor alocis are fastidious anaerobic bacteria strongly associated with chronic forms of periodontitis. Our understanding of the growth activities of these microorganisms in situ is very limited. Previous studies have shown that copy numbers of ribosomal-RNA precursor (pre-rRNA) of specific pathogen species relative to genomic-DNA (gDNA) of the same species (P:G ratios) are greater in actively growing bacterial cells than in resting cells. The method, so-called steady-state pre-rRNA-analysis, represents a novel culture-independent approach to study bacteria. This study employed this technique to examine the in situ growth activities of oral bacteria in periodontitis before and after non-surgical periodontal therapy. Sub-gingival paper-point samples were taken at initial and re-evaluation appointments. Pre-rRNA and gDNA levels of P. gingivalis and F. alocis were quantified and compared using reverse-transcriptase qPCR. The results indicate significantly reduced growth activity of P. gingivalis, but not F. alocis, after therapy. The P:G ratios of P. gingivalis and F. alocis were compared and a low-strength, but statistically significant inter-species correlation was detected. Our study demonstrates that steady-state pre-rRNA-analysis can be a valuable culture-independent approach to studying opportunistic bacteria in periodontitis.

Filifactor alocis, a previously unrecognized Gram-positive anaerobic rod, is now considered a new emerging pathogen that may play a significant role in periodontal disease. F. alocis' unique characteristics and variations at the molecular level that may be responsible for the functional changes required to mediate the pathogenic process are discussed.

Infection-induced periodontal disease has been primarily focused on a small group of periodontal pathogens. A paradigm shift, based on data emerging from the oral microbiome project, now suggests the involvement of as-yet-unculturable and fastidious organisms. Collectively, these studies have demonstrated that there are changes in the periodontal status associated with shifts in the composition of the bacterial community in the periodontal pocket. In addition, it is likely that the emerging new pathogens may play a more significant role in the disease. One of the organisms previously unrecognized is Filifactor alocis. While this Gram-positive anaerobic rod has been identified in peri-implantitis, in endodontic infections, and in patients with localized aggressive periodontitis, its presence is now observed at significantly higher levels in patients with adult periodontitis or refractory periodontitis. Its colonization properties and its potential virulence attributes support the proposal that F. alocis should be included as a diagnostic indicator of periodontal disease. Moreover, these emerging characteristics would be consistent with the polymicrobial synergy and dysbiosis (PSD) periodontal pathogenesis model. Here, unique characteristics of F. alocis are discussed. F. alocis has specific factors that can modulate multiple changes in the microbial community and host cell proteome. It is likely that such variations at the molecular level are responsible for the functional changes required to mediate the pathogenic process.

Streptococcus gordonii, an accessory pathogen and early colonizer of plaque, co-aggregates with many oral species including Porphyromonas gingivalis. It causes α-hemolysis on blood agar, a process mediated by H₂ O₂ and thought to involve concomitant oxidation of hemoglobin (Hb). Porphyromonas gingivalis has a growth requirement for heme, which is acquired mainly from Hb. The paradigm for Hb heme acquisition involves the initial oxidation of oxyhemoglobin (oxyHb) to methemoglobin (metHb), followed by heme release and extraction through the actions of K-gingipain protease and/or the HmuY hemophore-like protein. The ability of S. gordonii to mediate Hb oxidation may potentially aid heme capture during co-aggregation with P. gingivalis. Hemoglobin derived from zones of S. gordonii α-hemolysis was found to be metHb. Generation of metHb from oxyHb by S. gordonii cells was inhibited by catalase, and correlated with levels of cellular H₂ O₂ production. Generation of metHb by S. gordonii occurred through the higher Hb oxidation state of ferrylhemoglobin. Heme complexation by the P. gingivalis HmuY was employed as a measure of the ease of heme capture from metHb. HmuY was able to extract iron(III)protoporphyrin IX from metHb derived from zones of S. gordonii α-hemolysis and from metHb generated by the action of S. gordonii cells on isolated oxyHb. The rate of HmuY-Fe(III)heme complex formation from S. gordonii-mediated metHb was greater than from an equivalent concentration of auto-oxidized metHb. It is concluded that S. gordonii may potentially aid heme acquisition by P. gingivalis by facilitating metHb formation in the presence of oxyHb.

Many human infections are polymicrobial in origin, and interactions among community inhabitants shape colonization patterns and pathogenic potential ¹ . Periodontitis, which is the sixth most prevalent infectious disease worldwide ² , ensues from the action of dysbiotic polymicrobial communities ³ . The keystone pathogen Porphyromonas gingivalis and the accessory pathogen Streptococcus gordonii interact to form communities in vitro and exhibit increased fitness in vivo ^3,4 . The mechanistic basis of this polymicrobial synergy, however, has not been fully elucidated. Here we show that streptococcal 4-aminobenzoate/para-amino benzoic acid (pABA) is required for maximal accumulation of P. gingivalis in dual-species communities. Metabolomic and proteomic data showed that exogenous pABA is used for folate biosynthesis, and leads to decreased stress and elevated expression of fimbrial adhesins. Moreover, pABA increased the colonization and survival of P. gingivalis in a murine oral infection model. However, pABA also caused a reduction in virulence in vivo and suppressed extracellular polysaccharide production by P. gingivalis. Collectively, these data reveal a multidimensional aspect to P. gingivalis-S. gordonii interactions and establish pABA as a critical cue produced by a partner species that enhances the fitness of P. gingivalis while diminishing its virulence.

Coaggregation of Porphyromonas gingivalis and oral streptococci is thought to play an important role in P. gingivalis colonization. Previously, we reported that P. gingivalis major fimbriae interacted with Streptococcus oralis glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and that amino acid residues 166 to 183 of GAPDH exhibited strong binding activity toward P. gingivalis fimbriae (H. Nagata, M. Iwasaki, K. Maeda, M. Kuboniwa, E. Hashino, M. Toe, N. Minamino, H. Kuwahara, and S. Shizukuishi, Infect. Immun. 77:5130-5138, 2009). The present study aimed to identify and characterize P. gingivalis components other than fimbriae that interact with S. oralis GAPDH. A pulldown assay was performed to detect potential interactions between P. gingivalis client proteins and S. oralis recombinant GAPDH with amino acid residues 166 to 183 deleted by site-directed mutagenesis. Seven proteins, namely, tonB-dependent receptor protein (RagA4), arginine-specific proteinase B, 4-hydroxybutyryl-coenzyme A dehydratase (AbfD), lysine-specific proteinase, GAPDH, NAD-dependent glutamate dehydrogenase (GDH), and malate dehydrogenase (MDH), were identified by two-dimensional gel electrophoresis followed by proteomic analysis using tandem mass spectrometry. Interactions between these client proteins and S. oralis GAPDH were analyzed with a biomolecular interaction analysis system. S. oralis GAPDH showed high affinity for five of the seven client proteins (RagA4, AbfD, GAPDH, GDH, and MDH). Interactions between P. gingivalis and S. oralis were measured by a turbidimetric method and fluorescence microscopy. RagA4, AbfD, and GDH enhanced coaggregation, whereas GAPDH and MDH inhibited coaggregation. Furthermore, the expression of luxS in P. gingivalis was upregulated by RagA4, AbfD, and GDH but was downregulated by MDH. These results indicate that the five P. gingivalis client proteins function as regulators in P. gingivalis biofilm formation with oral streptococci.

Periodontal diseases are polymicrobial diseases that cause the inflammatory destruction of the tooth-supporting (periodontal) tissues. Their initiation is attributed to the formation of subgingival biofilms that stimulate a cascade of chronic inflammatory reactions by the affected tissue. The Gram-negative anaerobes Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola are commonly found as part of the microbiota of subgingival biofilms, and they are associated with the occurrence and severity of the disease. P. gingivalis expresses several virulence factors that may support its survival, regulate its communication with other species in the biofilm, or modulate the inflammatory response of the colonized host tissue. The most prominent of these virulence factors are the gingipains, which are a set of cysteine proteinases (either Arg-specific or Lys-specific). The role of gingipains in the biofilm-forming capacity of P. gingivalis is barely investigated. Hence, this in vitro study employed a biofilm model consisting of 10 "subgingival" bacterial species, incorporating either a wild-type P. gingivalis strain or its derivative Lys-gingipain and Arg-gingipan isogenic mutants, in order to evaluate quantitative and qualitative changes in biofilm composition.

Porphyromonas gingivalis, an important etiological agent of periodontal disease, is frequently found associated with Treponema denticola, an anaerobic spirochete, in pathogenic biofilms. However, interactions between these two bacteria are not well understood at the molecular level. In this study, we seek to link the influence of T. denticola on the expression of P. gingivalis proteases with its capacities to adhere and to form biofilms. The P. gingivalis genes encoding Arg-gingipain A (RgpA), Lys-gingipain (Kgp), and hemagglutinin A (HagA) were more strongly expressed after incubation with T. denticola compared with P. gingivalis alone. The amounts of the three resulting proteins, all of which contain hemagglutinin adhesion domains, were increased in culture supernatants. Moreover, incubation of P. gingivalis with T. denticola promoted static and dynamic biofilm formation, primarily via a time-dependent enhancement of P. gingivalis adhesion capacities on bacterial partners such as Streptococcus gordonii. Adhesion of P. gingivalis to human cells was also increased. These results showed that interactions of P. gingivalis with other bacterial species, such as T. denticola, induce increased adhesive capacities on various substrata by hemagglutinin adhesion domain-containing proteins.

Fusobacterium nucleatum is a Gram-negative oral anaerobe, capable of systemic dissemination causing infections and abscesses, often in mixed-species, at different body sites. We have shown previously that F. nucleatum adheres to and invades host epithelial and endothelial cells via a novel FadA adhesin. In this study, vascular endothelial (VE)-cadherin, a member of the cadherin family and a cell-cell junction molecule, was identified as the endothelial receptor for FadA, required for F. nucleatum binding to the cells. FadA colocalized with VE-cadherin on endothelial cells, causing relocation of VE-cadherin away from the cell-cell junctions. As a result, the endothelial permeability was increased, allowing the bacteria to cross the endothelium through loosened junctions. This crossing mechanism may explain why the organism is able to disseminate systemically to colonize in different body sites and even overcome the placental and blood-brain barriers. Co-incubation of F. nucleatum and Escherichia coli enhanced penetration of the endothelial cells by the latter in the transwell assays, suggesting F. nucleatum may serve as an 'enabler' for other microorganisms to spread systemically. This may explain why F. nucleatum is often found in mixed infections. This study reveals a possible novel dissemination mechanism utilized by pathogens.

Uncontrolled inflammation of the periodontal area may arise when complex microbial communities transition from a commensal to a pathogenic entity. Communication among constituent species leads to polymicrobial synergy between metabolically compatible organisms that acquire functional specialization within the developing community. Keystone pathogens, even at low abundance, elevate community virulence, and the resulting dysbiotic community targets specific aspects of host immunity to further disable immune surveillance while promoting an overall inflammatory response. Inflammophilic organisms benefit from proteinaceous substrates derived from inflammatory tissue breakdown. Inflammation and dysbiosis reinforce each other, and the escalating environmental changes further select for a pathobiotic community. We have synthesized the polymicrobial synergy and dysbiotic components of the process into a new model for inflammatory diseases.

Periodontitis is an infection-induced inflammatory disease that causes loss of the tooth supporting tissues. Much focus has been put on comparison of the microbial biofilm in the healthy periodontium with the diseased one. The information arising from such studies is limited due to difficulties to compare the microbial composition in these two completely different ecological niches. A few longitudinal studies have contributed with information that makes it possible to predict which individuals who might have an increased risk of developing aggressive forms of periodontitis, and the predictors are either microbial or/and host-derived factors. The most conspicuous condition that is associated with disease risk is the presence of Aggregatibacter actinomycetemcomitans at the individual level. This Gram-negative bacterium has a great genetic variation with a number of virulence factors. In this review we focus in particular on the leukotoxin that, based on resent knowledge, might be one of the most important virulence factors of A. actinomycetemcomitans.

The pathogenesis of the chronic periodontal disease is associated with a skewed host inflammatory response to periodontal pathogens, such as Porphyromonas gingivalis, that accounts for the majority of periodontal tissue damage. Neutrophils are the most abundant leukocytes in periodontal pockets and depending on the stage of the disease, also plentiful PMNs are present in the inflamed gingival tissue and the gingival crevice. They are the most efficient phagocytes and eliminate pathogens by a variety of means, which are either oxygen-dependent or -independent. However, these secretory lethal weapons do not strictly discriminate between pathogens and host tissue. Current studies describe conflicting findings about neutrophil involvement in periodontal disease. On one hand literature indicate that hyper-reactive neutrophils are the main immune cell type responsible for this observed tissue damage and disease progression. Deregulation of neutrophil survival and functions, such as chemotaxis, migration, secretion of antimicrobial peptides or enzymes, and production of reactive oxygen species, contribute to observed tissue injury and the clinical signs of periodontal disease. On the other hand neutrophils deficiencies in patients and mice also result in periodontal phenotype. Therefore, P. gingivalis represents a periodontal pathogen that manipulates the immune responses of PMNs, employing several virulence factors, such as gingipains, serine proteases, lipid phosphatases, or fimbriae. This review will sum up studies devoted to understanding different strategies utilized by P. gingivalis to manipulate PMNs survival and functions in order to inhibit killing by a granular content, prolong inflammation, and gain access to nutrient resources.

COPD is recognized as having a series of comorbidities potentially related to common inflammatory processes. Periodontitis is one of the most common human inflammatory diseases and has previously been associated with COPD in numerous observational studies. As periodontitis and COPD are both chronic, progressive conditions characterized by neutrophilic inflammation with subsequent proteolytic destruction of connective tissue, it has been proposed that they share common pathophysiological processes. The mechanisms proposed to link COPD and periodontitis include mechanical aspiration of oral contents into the respiratory tree, overspill of locally produced inflammatory mediators into the systemic circulation or oral or lung-derived bacteremia activating an acute-phase response and also reactive oxygen species (ROS) and cytokine release by systemic neutrophils at distant sites. Studies of systemic neutrophils in COPD and chronic periodontitis describe altered cellular functions that would predispose to inflammation and tissue destruction both in the lung and in the mouth, again potentially connecting these conditions. However, COPD and periodontitis also share risk factors such as age, chronic tobacco smoke exposure, and social deprivation that are not always considered in observational and interventional studies. Furthermore, studies reporting associations have often utilized differing definitions of both COPD and periodontitis. This article reviews the current available evidence supporting the hypothesis that COPD and inflammatory periodontal disease (periodontitis) could be pathologically associated, including a review of shared inflammatory mechanisms. It highlights the potential limitations of previous studies, in particular, the lack of uniformly applied case definitions for both COPD and periodontitis and poor recognition of shared risk factors. Understanding associations between these conditions may inform why patients with COPD suffer such a burden of comorbid illness and new therapeutic strategies for both the diseases. However, further research is needed to clarify factors that may be directly causal as opposed to confounding relationships.

An emerging concept is the tight relationship between dysbiosis (microbiota imbalance) and disease. The increase in knowledge about alterations in microbial communities that reside within the host has made a strong impact not only on dental science, but also on immunology and microbiology as well as on our understanding of several diseases. Periodontitis is a well-characterized human disease associated with dysbiosis, characterized by the accumulation of multiple bacteria that play individual and critical roles in bone loss around the teeth. Dysbiosis is largely dependent on cooperative and competitive interactions among oral microbes during the formation of the pathogenic biofilm community at gingival sites. Oral pathobionts play different and synergistic roles in periodontitis development, depending on their host-damaging and immunostimulatory activities. Host immune responses to oral pathobionts act as a double-edged sword not only by protecting the host against pathobionts, but also by promoting alveolar bone loss. Recent studies have begun to elucidate the roles of individual oral bacteria, including a new type of pathobionts that possess strong immunostimulatory activity, which is critical for alveolar bone loss. Better understanding of the roles of oral pathobionts is expected to lead to a better understanding of periodontitis disease and to the development of novel preventive and therapeutic approaches for the disease.

Although increasing knowledge suggests that bacteriophages play important roles in regulating microbial ecosystems, phage-bacteria interaction in human oral cavities remains less understood. Here we performed a metagenomic analysis to explore the composition and variation of oral dsDNA phage populations and potential phage-bacteria interaction. A total of 1,711 contigs assembled with more than 100 Gb shotgun sequencing data were annotated to 104 phages based on their best BLAST matches against the NR database. Bray-Curtis dissimilarities demonstrated that both phage and bacterial composition are highly diverse between periodontally healthy samples but show a trend towards homogenization in diseased gingivae samples. Significantly, according to the CRISPR arrays that record infection relationship between bacteria and phage, we found certain oral phages were able to invade other bacteria besides their putative bacterial hosts. These cross-infective phages were positively correlated with commensal bacteria while were negatively correlated with major periodontal pathogens, suggesting possible connection between these phages and microbial community structure in oral cavities. By characterizing phage-bacteria interaction as networks rather than exclusively pairwise predator-prey relationships, our study provides the first insight into the participation of cross-infective phages in forming human oral microbiota.

Dental diseases are perhaps the most prevalent infection-related diseases in humans. Biofilm is involved in almost every infectious disease compromising oral health, notably caries, periodontal disease, gingivitis, endodontic infections and peri-implantitis. Current therapies of biofilm-derived oral infections lack sensitivity; they are not species-specific and kill pathogenic species as well as commensal species, which are protective against the formation of pathogenic biofilms. Moreover, antibiotics have a limited effect on biofilm and are almost unused in oral diseases. A promising alternative approach is bacteriophage (phage) therapy. Phages play a key role in the natural balance in a predator-prey relationship with bacteria and thus have the potential to be efficient anti-bacterial agents. Phages are highly efficient against biofilm, strain specific and easy to isolate and manipulate. Thus, like in many other medicinal fields, phage therapy offers new horizons to dentistry, both therapeutics and research. The present review presents the etiology of common oral diseases, characterization of the infection and the treatment challenges of phage therapy in dentistry. Recent findings and development in the use of phages for prevention, control, and treatment of oral infections as well as possibilities of engineering the oral microbiome are discussed.

Lactoferrin chimera (LFchimera), a heterodimeric peptide containing lactoferrampin (LFampin265-284) and a part of lactoferricin (LFcin17-30), possesses a broad spectrum of antimicrobial activity. However, there is no report on the inhibitory effects of LFchimera against multispecies oral biofilms. This study aimed to determine the effects of LFchimera in comparison to chlorhexidine digluconate (CHX) and minocycline hydrochloride (MH), on in vitro multispecies biofilms derived from subgingival plaque of periodontitis patients harboring Aggregatibacter actinomycetemcomitans. First the effects of LFchimera against planktonic and an 1-day old biofilm of the periodontopathic bacteria, A. actinomycetemcomitans ATCC 43718 were established. Then, the effects on biofilm formation and bacterial viability in the multispecies biofilm were determined by crystal violet staining and LIVE/DEAD BacLight Bacterial Viability kit, respectively. The results revealed that a significant reduction (P < 0.05) in biofilm formation occurred after 15 min exposure to 20 µM of LFchimera or CHX compared to control. In contrast, MH at concentration up to 100 µM did not inhibit biofilm formation. The ratio of live/dead bacteria in biofilm was also significantly lower after 15 min exposure to 20 µM of LFchimera compared to control and 20-50 µM of CHX and MH. Altogether, the results obtained indicate that LFchimera is able to inhibit in vitro subgingival biofilm formation and reduce viability of multispecies bacteria in biofilm better than CHX and MH.

Periodontitis is a biofilm-induced inflammatory disease characterized by dysbiosis of the commensal periodontal microbiota. It is unclear how natural regulation of inflammation affects the periodontal biofilm. Promoters of active resolution of inflammation, including resolvin E1 (RvE1), effectively treat inflammatory periodontitis in animal models. The goals of this study were 1) to compare periodontal tissue gene expression in different clinical conditions, 2) to determine the impact of local inflammation on the composition of subgingival bacteria, and 3) to understand how inflammation impacts these changes. Two clinically relevant experiments were performed in rats: prevention and treatment of ligature-induced periodontitis with RvE1 topical treatment. The gingival transcriptome was evaluated by RNA sequencing of mRNA. The composition of the subgingival microbiota was characterized by 16S rDNA sequencing. Periodontitis was assessed by bone morphometric measurements and histomorphometry of block sections. H&E and tartrate-resistant acid phosphatase staining were used to characterize and quantify inflammatory changes. RvE1 treatment prevented bone loss in ligature-induced periodontitis. Osteoclast density and inflammatory cell infiltration in the RvE1 groups were lower than those in the placebo group. RvE1 treatment reduced expression of inflammation-related genes, returning the expression profile to one more similar to health. Treatment of established periodontitis with RvE1 reversed bone loss, reversed inflammatory gene expression, and reduced osteoclast density. Assessment of the rat subgingival microbiota after RvE1 treatment revealed marked changes in both prevention and treatment experiments. The data suggest that modulation of local inflammation has a major role in shaping the composition of the subgingival microbiota.

In previous research, to combine the immunogenicity of Fusobacterium nucleatum (F. nucleatum) and the probiotic properties of Lactobacillus acidophilus (L. acidophilus), we constructed a FomA-expressing L. acidophilus strain and assessed its immunogenicity. Our findings indicated that oral administration of the recombinant L. acidophilus strain reduced the risk of periodontal infection by Porphyromonas gingivalis (P. gingivalis) and F. nucleatum. However, because the exogenous FomA is an heterologous protein for the original bacterium, in this study, we assessed whether the biochemical characteristics of the recombinant L. acidophilus strain change due to the expression of the exogenous FomA protein.

Porphyromonas gingivalis alters cytokine expression in gingival epithelial cells, stimulating inflammatory responses that may lead to periodontal disease. This study explored the effect of Lactobacillus acidophilus on the specific expressions of the interleukins (ILs) IL1B, IL6, and IL8 induced by the pathogen. Human gingival epithelial cells were co-cultured with P. gingivalis, L. acidophilus, or L. acidophilus + P. gingivalis; the control group consisted of the cells alone. Protein and gene expression levels of the ILs were detected using ELISA and qRT-PCR, respectively. The supernatant from the P. gingivalis group held significantly higher protein and mRNA levels of IL1B, IL6, and IL8, compared to the control group. In the mixed bacterial group (L. acidophilus + P. gingivalis), the levels of all three ILs decreased with increasing concentrations of L. acidophilus and were significantly different from the P. gingivalis group. This suggests that in gingival cells, L. acidophilus offsets the P. gingivalis-induced secretion of these ILs in a dose-dependent manner.

Dental diseases are now viewed as a consequence of a deleterious shift in the balance of the normally stable resident oral microbiome. It is known that frequent carbohydrate consumption or reduced saliva flow can lead to caries, and excessive plaque accumulation increases the risk of periodontal diseases. However, when these "disease drivers" are present, while some individuals appear to be susceptible, others are more tolerant or resilient to suffering from undesirable changes in their oral microbiome. Health-maintaining mechanisms that limit the effect of disease drivers include the complex set of metabolic and functional interrelationships that develop within dental biofilms and between biofilms and the host. In contrast, "positive feedback loops" can develop within these microbial communities that disrupt resilience and provoke a large and abrupt change in function and structure of the ecosystem (a microbial "regime shift"), which promotes dysbiosis and oral disease. For instance, acidification due to carbohydrate fermentation or inflammation in response to accumulated plaque select for a cariogenic or periopathogenic microbiota, respectively, in a chain of self-reinforcing events. Conversely, in tolerant individuals, health-maintaining mechanisms, including negative feedback to the drivers, can maintain resilience and promote resistance to and recovery from disease drivers. Recently studied health-maintaining mechanisms include ammonia production, limiting a drop in pH that can lead to caries, and denitrification, which could inhibit several stages of disease-associated positive feedback loops. Omics studies comparing the microbiome of, and its interaction with, susceptible and tolerant hosts can detect markers of resilience. The neutralization or inhibition of disease drivers, together with the identification and promotion of health-promoting species and functions, for example, by pre- and probiotics, could enhance microbiome resilience and lead to new strategies to prevent disease.

Probiotics are living microorganisms (e.g., bacteria) that are either the same as or similar to organisms found naturally in the human body and may be beneficial to health. Current researches have shown that the balance between beneficial and pathogenic bacteria is essential in order to maintain the oral health. Therefore, oral cavity has recently been suggested as a relevant target for probiotic applications. Dental caries can be seen as a microbial imbalance where the oral microbiota shift towards community dominance which produces acidogenic and acid-tolerant gram positive bacteria. Similarly, the accumulation of bacteria within the biofilm, facilitated by poor oral hygiene, predisposes to allogenic shifts in the microbial community, leading to the onset of periodontal inflammation. Probiotic bacteria belonging to the genus of Lactobacillus, Bifidobacterium and Streptococcus have been proven effective for preventing caries by reducing the number of cariogenic bacteria in saliva after a short period of consuming the probiotic. In contrast, the effect of probiotics on improving gingivitis and periodontitis has been less investigated. The currently available studies on the effect of probiotics on periodontal pathogens and clinical periodontal parameters showed differing results depending on the strains used and the endpoints analyzed. Many of the clinical studies are pilot in nature and with low quality, therefore, properly conducted clinical trials, using probiotic strains with in vitro proven periodontal probiotic effects, are needed. The putative beneficial effects of probiotics on oral malodour have also been evaluated, but further evidence is needed to fully explore the potential of probiotics for preventing malodour.

Periodontitis is a common chronic inflammatory disease that is initiated by a complex microbial biofilm that poses significant health and financial burdens globally. Porphyromonas gingivalis is a predominant pathogen that maintains chronic inflammatory periodontitis. Toll-like receptors (TLRs) play an important role in periodontitis by recognizing pathogens and maintaining tissue homeostasis. Deficiencies in TLR expression and downstream signaling may reduce the host's innate defenses against pathogens, leading to bacterial persistence and exacerbated inflammation, which are now being better appreciated in disease pathologies. In the case of periodontitis, gingival epithelial cells form the first line of defense against pathogens. Innate immune dysregulation in these cells relates to severe disease pathology. We recently identified a blunted TLR2 expression in certain gingival epithelial cells expressing diminished cytokine signaling upon P. gingivalis stimulation. Upon detailed analysis of the TLR2 promoter CpG Island, we noted higher CpG methylation in this dysregulated cell type. When these cells were treated with DNA methyltransferase inhibitor, TLR2 mRNA and cytokine expression were significantly increased. If TLR2 expression plasmid was ectopically expressed in dysfunctional cells prior to P. gingivalis stimulation, the cytokine expression was increased, confirming the requirement of TLR2 in the P. gingivalis-mediated inflammatory response. We designed a chronic in vitro infection model to test if P. gingivalis can induce DNA methylation in normal gingival epithelial cells that express higher TLR2 upon agonist stimulation. Chronic treatment of normal epithelial cells with P. gingivalis introduced de novo DNA methylation within the cells. In addition, increased DNA methylation was observed in the gingiva of mice infected with P. gingivalis in a periodontitis oral gavage model. Moreover, tissues obtained from periodontitis patients also exhibited differential TLR2 promoter methylation, as revealed by bisulfite DNA sequencing. Taken together, DNA methylation of TLR2 can modulate host innate defense mechanisms that may confer increased disease susceptibility.