The human oral phageome.

Oral bacteriophages (or phages), especially periodontal ones, constitute a growing area of interest, but research on oral phages is still in its infancy. Phages are bacterial viruses that may persist as intracellular parasitic deoxyribonucleic acid (DNA) or use bacterial metabolism to replicate and cause bacterial lysis. The microbiomes of saliva, oral mucosa, and dental plaque contain active phage virions, bacterial lysogens (ie, carrying dormant prophages), and bacterial strains containing short fragments of phage DNA. In excess of 2000 oral phages have been confirmed or predicted to infect species of the phyla Actinobacteria (>300 phages), Bacteroidetes (>300 phages), Firmicutes (>1000 phages), Fusobacteria (>200 phages), and Proteobacteria (>700 phages) and three additional phyla (few phages only). This article assesses the current knowledge of the diversity of the oral phage population and the mechanisms by which phages may impact the ecology of oral biofilms. The potential use of phage-based therapy to control major periodontal pathogens is also discussed.

Removable denture is a risk indicator for peri-implantitis and facilitates expansion of specific periodontopathogens: a cross-sectional study.

BACKGROUND: The prevalence of peri-implantitis ranges between 7 and 38.4% depending on risk indicators such as smoking, diabetes mellitus, lack of periodontal maintenance program, and history or presence of periodontitis. Currently, the possible effect of the type of superstructure on peri-implant health is unclear. This cross-sectional study aims to investigate the influence of the superstructure on the prevalence of peri-implant mucositis, peri-implantitis and peri-implant dysbiosis. METHODS: During a 32-month recruitment period dental implants were assessed to diagnose healthy peri-implant tissues, mucositis or peri-implantitis. The study included 1097 implants in 196 patients. Out of all peri-implantitis cases 20 randomly chosen submucosal biofilms from implants with fixed denture (FD) originating from 13 patients and 11 biofilms from implants with removable dentures (RD) originating from 3 patients were studied for microbiome analysis. Composition of transcriptionally active biofilms was revealed by RNAseq. Metatranscriptomic profiles were created for thirty-one peri-implant biofilms suffering from peri-implantitis and microbiome changes associated with superstructure types were identified. RESULTS: 16.41% of the implants were diagnosed with peri-implantitis, 25.00% of implants with RD and 12.68% of implants with FD, respectively. Multivariate analysis showed a significant positive association on patient (p = < 0.001) and implant level (p = 0.03) between the prevalence of peri-implantitis and RD. Eight bacterial species were associated either with FD or RD by linear discriminant analysis effect size method. However, significant intergroup confounders (e.g. smoking) were present. CONCLUSIONS: Within the limitations of the present work, RDs appear to be a risk indicator for peri-implantitis and seem to facilitate expansion of specific periodontopathogens. Potential ecological and pathological consequences of shift in microbiome from RDs towards higher activity of Fusobacterium nucleatum subspecies animalis and Prevotella intermedia require further investigation.

Commensal and pathogenic biofilms differently modulate peri-implant oral mucosa in an organotypic model.

The impact of oral commensal and pathogenic bacteria on peri-implant mucosa is not well understood, despite the high prevalence of peri-implant infections. Hence, we investigated responses of the peri-implant mucosa to Streptococcus oralis or Aggregatibacter actinomycetemcomitans biofilms using a novel in vitro peri-implant mucosa-biofilm model. Our 3D model combined three components, organotypic oral mucosa, implant material, and oral biofilm, with structural assembly close to native situation. S. oralis induced a protective stress response in the peri-implant mucosa through upregulation of heat shock protein (HSP70) genes. Attenuated inflammatory response was indicated by reduced cytokine levels of interleukin-6 (IL-6), interleukin-8 (CXCL8), and monocyte chemoattractant protein-1 (CCL2). The inflammatory balance was preserved through increased levels of tumor necrosis factor-alpha (TNF-α). A. actinomycetemcomitans induced downregulation of genes important for cell survival and host inflammatory response. The reduced cytokine levels of chemokine ligand 1 (CXCL1), CXCL8, and CCL2 also indicated a diminished inflammatory response. The induced immune balance by S. oralis may support oral health, whereas the reduced inflammatory response to A. actinomycetemcomitans may provide colonisation advantage and facilitate later tissue invasion. The comprehensive characterisation of peri-implant mucosa-biofilm interactions using our 3D model can provide new knowledge to improve strategies for prevention and therapy of peri-implant disease.

Quorum sensing of Streptococcus mutans is activated by Aggregatibacter actinomycetemcomitans and by the periodontal microbiome.

BACKGROUND: The oral cavity is inhabited by complex microbial communities forming biofilms that can cause caries and periodontitis. Cell-cell communication might play an important role in modulating the physiologies of individual species, but evidence so far is limited. RESULTS: Here we demonstrate that a pathogen of the oral cavity, Aggregatibacter actinomycetemcomitans (A. act.), triggers expression of the quorum sensing (QS) regulon of Streptococcus mutans, a well-studied model organism for cariogenic streptococci, in dual-species biofilms grown on artificial saliva. The gene for the synthesis of the QS signal XIP is essential for this interaction. Transcriptome sequencing of biofilms revealed that S. mutans up-regulated the complete QS regulon (transformasome and mutacins) in the presence of A. act. and down-regulated oxidative stress related genes. A.act. required the presence of S. mutans for growth. Fimbriae and toxins were its most highly expressed genes and up-regulation of anaerobic metabolism, chaperones and iron acquisition genes was observed in co-culture. Metatranscriptomes from periodontal pockets showed highly variable levels of S. mutans and low levels of A. act.. Transcripts of the alternative sigma-factor SigX, the key regulator of QS in S. mutans, were significantly enriched in periodontal pockets compared to single cultures (log2 4.159, FDR ≤0.001, and expression of mutacin related genes and transformasome components could be detected. CONCLUSION: The data show that the complete QS regulon of S. mutans can be induced by an unrelated oral pathogen and S. mutans may be competent in oral biofilms in vivo.

High-resolution taxonomic profiling of the subgingival microbiome for biomarker discovery and periodontitis diagnosis.

The oral microbiome plays a key role for caries, periodontitis, and systemic diseases. A method for rapid, high-resolution, robust taxonomic profiling of subgingival bacterial communities for early detection of periodontitis biomarkers would therefore be a useful tool for individualized medicine. Here, we used Illumina sequencing of the V1-V2 and V5-V6 hypervariable regions of the 16S rRNA gene. A sample stratification pipeline was developed in a pilot study of 19 individuals, 9 of whom had been diagnosed with chronic periodontitis. Five hundred twenty-three operational taxonomic units (OTUs) were obtained from the V1-V2 region and 432 from the V5-V6 region. Key periodontal pathogens like Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia could be identified at the species level with both primer sets. Principal coordinate analysis identified two outliers that were consistently independent of the hypervariable region and method of DNA extraction used. The linear discriminant analysis (LDA) effect size algorithm (LEfSe) identified 80 OTU-level biomarkers of periodontitis and 17 of health. Health- and periodontitis-related clusters of OTUs were identified using a connectivity analysis, and the results confirmed previous studies with several thousands of samples. A machine learning algorithm was developed which was trained on all but one sample and then predicted the diagnosis of the left-out sample (jackknife method). Using a combination of the 10 best biomarkers, 15 of 17 samples were correctly diagnosed. Training the algorithm on time-resolved community profiles might provide a highly sensitive tool to detect the onset of periodontitis.

Numerical and experimental investigation of multi-species bacterial co-aggregation.

This paper deals with the mathematical modeling of bacterial co-aggregation and its numerical implementation in a FEM framework. Since the concept of co-aggregation refers to the physical binding between cells of different microbial species, a system composed of two species is considered in the modeling framework. The extension of the model to an arbitrary number of species is straightforward. In addition to two-species (multi-species growth) dynamics, the transport of a nutritional substance and the extent of co-aggregation are introduced into the model as the third and fourth primary variables. A phase-field modeling approach is employed to describe the co-aggregation between the two species. The mathematical model is three-dimensional and fully based on the continuum description of the problem without any need for discrete agents which are the key elements of the individual-based modeling approach. It is shown that the use of a phase-field-based model is equivalent to a particular form of classical diffusion-reaction systems. Unlike the so-called mixture models, the evolution of each component of the multi-species system is captured thanks to the inherent capability of phase-field modeling in treating systems consisting of distinct multi-phases. The details of numerical implementation in a FEM framework are also presented. Indeed, a new multi-field user element is developed and implemented in ANSYS for this multiphysics problem. Predictions of the model are compared with the experimental observations. By that, the versatility and applicability of the model and the numerical tool are well established.

Evidence for inoculum size and gas interfaces as critical factors in bacterial biofilm formation on magnesium implants in an animal model.

Infections of medical implants caused by bacterial biofilms are a major clinical problem. Bacterial colonization is predicted to be prevented by alkaline magnesium surfaces. However, in experimental animal studies, magnesium implants prolonged infections. The reason for this peculiarity likely lies within the ‒still largely hypothetical‒ mechanism by which infection arises. Investigating subcutaneous magnesium implants infected with bioluminescent Pseudomonas aeruginosa via in vivo imaging, we found that the rate of implant infections was critically dependent on a surprisingly high quantity of injected bacteria. At high inocula, bacteria were antibiotic-refractory immediately after infection. High cell densities are known to limit nutrient availability, restricting proliferation and trigger quorum sensing which could both contribute to the rapid initial resistance. We propose that gas bubbles such as those formed during magnesium corrosion, can then act as interfaces that support biofilm formation and permit long-term survival. This model could provide an explanation for the apparent ineffectiveness of innovative contact-dependent bactericidal implant surfaces in patients. In addition, the model points toward air bubbles in tissue, either by inclusion during surgery or by spontaneous gas bubble formation later on, could constitute a key risk factor for clinical implant infections.

Non-Invasive Luciferase Imaging of Type I Interferon Induction in a Transgenic Mouse Model of Biomaterial Associated Bacterial Infections: Microbial Specificity and Inter-Bacterial Species Interactions.

The performance of biomaterials is often compromised by bacterial infections and subsequent inflammation. So far, the conventional analysis of inflammatory processes in vivo involves time-consuming histology and biochemical assays. The present study employed a mouse model where interferon beta (IFN-ß) is monitored as a marker for non-invasive rapid detection of inflammation in implant-related infections. The mouse model comprises subcutaneous implantation of morphologically modified titanium, followed by experimental infections with four taxonomically diverse oral bacteria: Streptococcus oralis, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis and Treponema denticola (as mono culture or selected mixed-culture). IFN-ß expression increased upon infections depending on the type of pathogen and was prolonged by the presence of the implant. IFN-ß expression kinetics reduced with two mixed species infections when compared with the single species. Histological and confocal microscopy confirmed pathogen-specific infiltration of inflammatory cells at the implant-tissue interface. This was observed mainly in the vicinity of infected implants and was, in contrast to interferon expression, higher in infections with dual species. In summary, this non-invasive mouse model can be used to quantify longitudinally host inflammation in real time and suggests that the polymicrobial character of infection, highly relevant to clinical situations, has complex effects on host immunity.

Diversity patterns of bacteriophages infecting Aggregatibacter and Haemophilus species across clades and niches.

Aggregatibacter and Haemophilus species are relevant human commensals and opportunistic pathogens. Consequently, their bacteriophages may have significant impact on human microbial ecology and pathologies. Our aim was to reveal the prevalence and diversity of bacteriophages infecting Aggregatibacter and Haemophilus species that colonize the human body. Genome mining with comparative genomics, screening of clinical isolates, and profiling of metagenomes allowed characterization of 346 phages grouped in 52 clusters and 18 superclusters. Less than 10% of the identified phage clusters were represented by previously characterized phages. Prophage diversity patterns varied significantly for different phage types, host clades, and environmental niches. A more diverse phage community lysogenizes Haemophilus influenzae and Haemophilus parainfluenzae strains than Aggregatibacter actinomycetemcomitans and "Haemophilus ducreyi". Co-infections occurred more often in "H. ducreyi". Phages from Aggregatibacter actinomycetemcomitans preferably lysogenized strains of specific serotype. Prophage patterns shared by subspecies clades of different bacterial species suggest similar ecoevolutionary drivers. Changes in frequencies of DNA uptake signal sequences and guanine-cytosine content reflect phage-host long-term coevolution. Aggregatibacter and Haemophilus phages were prevalent at multiple oral sites. Together, these findings should help exploring the ecoevolutionary forces shaping virus-host interactions in the human microbiome. Putative lytic phages, especially phiKZ-like, may provide new therapeutic options.

Liquid-Infused Structured Titanium Surfaces: Antiadhesive Mechanism to Repel Streptococcus oralis Biofilms.

To combat implant-associated infections, there is a need for novel materials which effectively inhibit bacterial biofilm formation. In the present study, the antiadhesive properties of titanium surface functionalization based on the "slippery liquid-infused porous surfaces" (SLIPS) principle were demonstrated and the underlying mechanism was analyzed. The immobilized liquid layer was stable over 13 days of continuous flow in an oral flow chamber system. With increasing flow rates, the surface exhibited a significant reduction in attached biofilm of both the oral initial colonizer  Streptococcus oralis and an oral multispecies biofilm composed of S. oralis, Actinomyces naeslundii, Veillonella dispar, and Porphyromonas gingivalis. Using single cell force spectroscopy, reduced S. oralis adhesion forces on the lubricant layer could be measured. Gene expression patterns in biofilms on SLIPS, on control surfaces, and expression patterns of planktonic cultures were also compared. For this purpose, the genome of S. oralis strain ATCC 9811 was sequenced using PacBio Sequel technology. Even though biofilm cells showed clear changes in gene expression compared to planktonic cells, no differences could be detected between bacteria on SLIPS and on control surfaces. Therefore, it can be concluded that the ability of liquid-infused titanium to repel S. oralis biofilms is mainly due to weakened bacterial adhesion to the underlying liquid interface.

The use of bacteriophages to biocontrol oral biofilms.

Infections induced by oral biofilms include caries, as well as periodontal, and peri-implant disease, and may influence quality of life, systemic health, and expenditure. As bacterial biofilms are highly resistant and resilient to conventional antibacterial therapy, it has been difficult to combat these infections. An innovative alternative to the biocontrol of oral biofilms could be to use bacteriophages or phages, the viruses of bacteria, which are specific, non-toxic, self-proliferating, and can penetrate into biofilms. Phages for Actinomyces naeslundii, Aggregatibacter actinomycetemcomitans, Enterococcus faecalis, Fusobacterium nucleatum, Lactobacillus spp., Neisseria spp., Streptococcus spp., and Veillonella spp. have been isolated and characterised. Recombinant phage enzymes (lysins) have been shown to lyse A. naeslundii and Streptococcus spp. However, only a tiny fraction of available phages and their lysins have been explored so far. The unique properties of phages and their lysins make them promising but challenging antimicrobials. The genetics and biology of phages have to be further explored in order to determine the most effective way of applying them. Studying the effect of phages and lysins on multispecies biofilms should pave the way for microbiota engineering and microbiota-based therapy.

Dysbiosis in chronic periodontitis: Key microbial players and interactions with the human host.

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.

Screening of Compounds against Gardnerella vaginalis Biofilms.

Bacterial vaginosis (BV) is a common infection in reproductive age woman and is characterized by dysbiosis of the healthy vaginal flora which is dominated by Lactobacilli, followed by growth of bacteria like Gardnerella vaginalis. The ability of G. vaginalis to form biofilms contributes to the high rates of recurrence that are typical for BV and which unfortunately make repeated antibiotic therapy inevitable. Here we developed a biofilm model for G. vaginalis and screened a large spectrum of compounds for their ability to prevent biofilm formation and to resolve an existing G. vaginalis biofilm. The antibiotics metronidazole and tobramycin were highly effective in preventing biofilm formation, but had no effect on an established biofilm. The application of the amphoteric tenside sodium cocoamphoacetate (SCAA) led to disintegration of existing biofilms, reducing biomass by 51% and viability by 61% and it was able to increase the effect of metronidazole by 40% (biomass) and 61% (viability). Our data show that attacking the biofilm and the bacterial cells by the combination of an amphoteric tenside with the antibiotic metronidazole might be a useful strategy against BV.

Functional biomarkers for chronic periodontitis and insights into the roles of Prevotella nigrescens and Fusobacterium nucleatum; a metatranscriptome analysis.

BACKGROUND/OBJECTIVES: Periodontitis is the most prevalent inflammatory disease worldwide and is caused by a dysbiotic subgingival biofilm. Here we used metatranscriptomics to determine the functional shift from health to periodontitis, the response of individual species to dysbiosis and to discover biomarkers. METHODS: Sixteen individuals were studied, from which six were diagnosed with chronic periodontitis. Illumina sequencing of the total messenger RNA (mRNA) yielded ~42 million reads per sample. A total of 324 human oral taxon phylotypes and 366,055 open reading frames from the HOMD database reference genomes were detected. RESULTS: The transcriptionally active community shifted from Bacilli and Actinobacteria in health to Bacteroidia, Deltaproteobacteria, Spirochaetes and Synergistetes in periodontitis. Clusters of orthologous groups (COGs) related to carbohydrate transport and catabolism dominated in health, whereas protein degradation and amino acid catabolism dominated in disease. The LEfSe, random forest and support vector machine methods were applied to the 2,000 most highly expressed genes and discovered the three best functional biomarkers, namely haem binding protein HmuY from Porphyromonas gingivalis, flagellar filament core protein FlaB3 from Treponema denticola, and repeat protein of unknown function from Filifactor alocis. They predicted the diagnosis correctly for 14 from 16 individuals, and when applied to an independent study misclassified one out of six subjects only. Prevotella nigrescens shifted from commensalism to virulence by upregulating the expression of metalloproteases and the haem transporter. Expression of genes for the synthesis of the cytotoxic short-chain fatty acid butyrate was observed by Fusobacterium nucleatum under all conditions. Four additional species contributed to butyrate synthesis in periodontitis and they used an additional pathway. CONCLUSION: Gene biomarkers of periodontitis are highly predictive. The pro-inflammatory role of F. nucelatum is not related to butyrate synthesis.

Cross-feeding and interkingdom communication in dual-species biofilms of Streptococcus mutans and Candida albicans.

Polymicrobial biofilms are of large medical importance, but relatively little is known about the role of interspecies interactions for their physiology and virulence. Here, we studied two human pathogens co-occuring in the oral cavity, the opportunistic fungus Candida albicans and the caries-promoting bacterium Streptococcus mutans. Dual-species biofilms reached higher biomass and cell numbers than mono-species biofilms, and the production of extracellular polymeric substances (EPSs) by S. mutans was strongly suppressed, which was confirmed by scanning electron microscopy, gas chromatography-mass spectrometry and transcriptome analysis. To detect interkingdom communication, C. albicans was co-cultivated with a strain of S. mutans carrying a transcriptional fusion between a green fluorescent protein-encoding gene and the promoter for sigX, the alternative sigma factor of S. mutans, which is induced by quorum sensing signals. Strong induction of sigX was observed in dual-species biofilms, but not in single-species biofilms. Conditioned media from mixed biofilms but not from C. albicans or S. mutans cultivated alone activated sigX in the reporter strain. Deletion of comS encoding the synthesis of the sigX-inducing peptide precursor abolished this activity, whereas deletion of comC encoding the competence-stimulating peptide precursor had no effect. Transcriptome analysis of S. mutans confirmed induction of comS, sigX, bacteriocins and the downstream late competence genes, including fratricins, in dual-species biofilms. We show here for the first time the stimulation of the complete quorum sensing system of S. mutans by a species from another kingdom, namely the fungus C. albicans, resulting in fundamentally changed virulence properties of the caries pathogen.