Vincent Meuric

Roseburia spp.: a marker of health?

The genus Roseburia consists of obligate Gram-positive anaerobic bacteria that are slightly curved, rod-shaped and motile by means of multiple subterminal flagella. It includes five species: Roseburia intestinalis, R. hominis, R. inulinivorans, R. faecis and R. cecicola. Gut Roseburia spp. metabolize dietary components that stimulate their proliferation and metabolic activities. They are part of commensal bacteria producing short-chain fatty acids, especially butyrate, affecting colonic motility, immunity maintenance and anti-inflammatory properties. Modification in Roseburia spp. representation may affect various metabolic pathways and is associated with several diseases (including irritable bowel syndrome, obesity, Type 2 diabetes, nervous system conditions and allergies). Roseburia spp. could also serve as biomarkers for symptomatic pathologies (e.g., gallstone formation) or as probiotics for restoration of beneficial flora.

Putative respiratory chain of Porphyromonas gingivalis

The electron transfer chain in Porphyromonas gingivalis, or periodontopathogens, has not yet been characterized. P. gingivalis, a strict anaerobic bacteria and the second colonizer of the oral cavity, is considered to be a major causal agent involved in periodontal diseases. Primary colonizers create a favorable environment for P. gingivalis growth by decreasing oxygen pressure. Oxygen does not appear to be the final electron acceptor of the respiratory chain. Fumarate and cytochrome b have been implicated as major components of the respiratory activity. However, the P. gingivalis genome shows many other enzymes that could be implicated in aerobic or nitrite respiration. Using bioinformatic tools and literature studies of respiratory pathways, the ATP synthesis mechanism from the sodium cycle and nutrients metabolism, the putative respirasome of P. gingivalis has been proposed.

Development of SNAP-tag-mediated live cell labeling as an alternative to GFP in Porphyromonas gingivalis

Porphyromonas gingivalis is an anaerobic periodontal pathogen that resides in the complex multispecies microbial biofilm known as dental plaque. Effective reporter tools are increasingly needed to facilitate physiological and pathogenetic studies of dental biofilm. Fluorescent proteins are ideal reporters for conveniently monitoring biofilm growth, but are restricted by several environmental factors, such as a requirement of oxygen to emit fluorescence. We developed a fluorescent reporter plasmid, known as the SNAP-tag, for labeling P. gingivalis cells, which encode an engineered version of the human DNA repair enzyme O(6)-alkylguanine-DNA alkyltransferase. Fluorescent substrates containing O(6)-benzylguanine covalently and specifically bind to the enzyme via stable thioether bonds. For the present study, we constructed a replicative plasmid carrying SNAP26b under the control of the P. gingivalis endogenous trxB promoter. The P. gingivalis-expressing SNAP26 protein was successfully labeled with specific fluorophores under anaerobic conditions. Porphyromonas gingivalis biofilm formation was investigated using flow cells and confocal laser scanning microscopy. A specific distribution of a strong fluorescence signal was demonstrated in P. gingivalis-SNAP26 monospecies and bispecies biofilms with Streptococcus gordonii-GFPmut3(*). These findings show that the SNAP-tag can be applied to studies of anaerobic bacteria in biofilm models and is a useful and advantageous alternative to existing labeling strategies.

Signature of Microbial Dysbiosis in Periodontitis

ABSTRACT Periodontitis is driven by disproportionate host inflammatory immune responses induced by an imbalance in the composition of oral bacteria; this instigates microbial dysbiosis, along with failed resolution of the chronic destructive inflammation. The objectives of this study were to identify microbial signatures for health and chronic periodontitis at the genus level and to propose a model of dysbiosis, including the calculation of bacterial ratios. Published sequencing data obtained from several different studies (196 subgingival samples from patients with chronic periodontitis and 422 subgingival samples from healthy subjects) were pooled and subjected to a new microbiota analysis using the same Visualization and Analysis of Microbial Population Structures (VAMPS) pipeline, to identify microbiota specific to health and disease. Microbiota were visualized using CoNet and Cytoscape. Dysbiosis ratios, defined as the percentage of genera associated with disease relative to the percentage of genera associated with health, were calculated to distinguish disease from health. Correlations between the proposed dysbiosis ratio and the periodontal pocket depth were tested with a different set of data obtained from a recent study, to confirm the relevance of the ratio as a potential indicator of dysbiosis. Beta diversity showed significant clustering of periodontitis-associated microbiota, at the genus level, according to the clinical status and independent of the methods used. Specific genera (Veillonella, Neisseria, Rothia, Corynebacterium, and Actinomyces) were highly prevalent (>95%) in health, while other genera (Eubacterium, Campylobacter, Treponema, and Tannerella) were associated with chronic periodontitis. The calculation of dysbiosis ratios based on the relative abundance of the genera found in health versus periodontitis was tested. Nonperiodontitis samples were significantly identifiable by low ratios, compared to chronic periodontitis samples. When applied to a subgingival sample set with well-defined clinical data, the method showed a strong correlation between the dysbiosis ratio, as well as a simplified ratio (Porphyromonas, Treponema, and Tannerella to Rothia and Corynebacterium), and pocket depth. Microbial analysis of chronic periodontitis can be correlated with the pocket depth through specific signatures for microbial dysbiosis. IMPORTANCE Defining microbiota typical of oral health or chronic periodontitis is difficult. The evaluation of periodontal disease is currently based on probing of the periodontal pocket. However, the status of pockets “on the mend” or sulci at risk of periodontitis cannot be addressed solely through pocket depth measurements or current microbiological tests available for practitioners. Thus, a more specific microbiological measure of dysbiosis could help in future diagnoses of periodontitis. In this work, data from different studies were pooled, to improve the accuracy of the results. However, analysis of multiple species from different studies intensified the bacterial network and complicated the search for reproducible microbial signatures. Despite the use of different methods in each study, investigation of the microbiota at the genus level showed that some genera were prevalent (up to 95% of the samples) in health or disease, allowing the calculation of bacterial ratios (i.e., dysbiosis ratios). The correlation between the proposed ratios and the periodontal pocket depth was tested, which confirmed the link between dysbiosis ratios and the severity of the disease. The results of this work are promising, but longitudinal studies will be required to improve the ratios and to define the microbial signatures of the disease, which will allow monitoring of periodontal pocket recovery and, conceivably, determination of the potential risk of periodontitis among healthy patients.

A new mathematical model of bacterial interactions in two-species oral biofilms

Periodontitis are bacterial inflammatory diseases, where the bacterial biofilms present on the tooth-supporting tissues switch from a healthy state towards a pathogenic state. Among bacterial species involved in the disease, Porphyromonas gingivalis has been shown to induce dysbiosis, and to induce virulence of otherwise healthy bacteria like Streptococcus gordonii. During biofilm development, primary colonizers such as S. gordonii first attach to the surface and allow the subsequent adhesion of periodontal pathogens such as P. gingivalis. Interactions between those two bacteria have been extensively studied during the adhesion step of the biofilm. The aim of the study was to understand interactions of both species during the growing phase of the biofilm, for which little knowledge is available, using a mathematical model. This two-species biofilm model was based on a substrate-dependent growth, implemented with damage parameters, and validated thanks to data obtained on experimental biofilms. Three different hypothesis of interactions were proposed and assayed using this model: independence, competition between both bacteria species, or induction of toxicity by one species for the other species. Adequacy between experimental and simulated biofilms were found with the last hypothetic mathematical model. This new mathematical model of two species bacteria biofilms, dependent on different substrates for growing, can be applied to any bacteria species, environmental conditions, or steps of biofilm development. It will be of great interest for exploring bacterial interactions in biofilm conditions.

The Cytochrome bd Oxidase of Porphyromonas gingivalis Contributes to Oxidative Stress Resistance and Dioxygen Tolerance

Porphyromonas gingivalis is an etiologic agent of periodontal disease in humans. The disease is associated with the formation of a mixed oral biofilm which is exposed to oxygen and environmental stress, such as oxidative stress. To investigate possible roles for cytochrome bd oxidase in the growth and persistence of this anaerobic bacterium inside the oral biofilm, mutant strains deficient in cytochrome bd oxidase activity were characterized. This study demonstrated that the cytochrome bd oxidase of Porphyromonas gingivalis, encoded by cydAB, was able to catalyse O2 consumption and was involved in peroxide and superoxide resistance, and dioxygen tolerance.

Bactériémies d'origine buccale.

Transient bacteremia from oral cavity related to oral anaerobic bacteria may occur as a result of dental healthcare procedures but also as a result of daily gestures involving the gums (chewing and oral hygiene). The risk of presenting a transient bacteremia is related to oral cavity bacterial load and to the severity of inflammation in the oral cavity. Although bacteremia is transient, in patients with immunodeficiency or comorbidity, this bacteremia may cause extra-oral infections. The bacteremia rate and the identified bacteria vary from one study to the next, depending on the method used to isolate and identify bacteria. Nevertheless, the risk for bacteremia is determined by the infectious and inflammatory conditions of each patient.

Increased transferrin saturation is associated with subgingival microbiota dysbiosis and severe periodontitis in genetic haemochromatosis

Genetic haemochromatosis (GH) is responsible for iron overload. Increased transferrin saturation (TSAT) has been associated with severe periodontitis, which is a chronic inflammatory disease affecting tissues surrounding the teeth and is related to dysbiosis of the subgingival microbiota. Because iron is essential for bacterial pathogens, alterations in iron homeostasis can drive dysbiosis. To unravel the relationships between serum iron biomarkers and the subgingival microbiota, we analysed samples from 66 GH patients. The co-occurrence analysis of the microbiota showed very different patterns according to TSAT. Healthy and periopathogenic bacterial clusters were found to compete in patients with normal TSAT (≤45%). However, significant correlations were found between TSAT and the proportions of Porphyromonas and Treponema, which are two genera that contain well-known periopathogenic species. In patients with high TSAT, the bacterial clusters exhibited no mutual exclusion. Increased iron bioavailability worsened periodontitis and promoted periopathogenic bacteria, such as Treponema. The radical changes in host-bacteria relationships and bacterial co-occurrence patterns according to the TSAT level also suggested a shift in the bacterial iron supply from transferrin to NTBI when TSAT exceeded 45%. Taken together, these results indicate that iron bioavailability in biological fluids is part of the equilibrium between the host and its microbiota.

New growth media for oral bacteria

New growth media have been designed for the iron-controlled co-cultures of three oral bacteria. These media share a common core composition enabling the switch from mono- to co-cultures, and efficiently promote both planktonic and biofilm cultures of Porphyromonas gingivalis, Treponema denticola and Streptococcus gordonii.

The Cytochrome bd Oxidase ofPorphyromonas gingivalis Contributes toOxidative Stress Resistance and DioxygenTolerance

Porphyromonas gingivalis is an etiologic agent of periodontal disease in humans. The disease is associated with the formation of a mixed oral biofilm which is exposed to oxygen and environmental stress, such as oxidative stress. To investigate possible roles for cytochrome bd oxidase in the growth and persistence of this anaerobic bacterium inside the oral biofilm, mutant strains deficient in cytochrome bd oxidase activity were characterized. This study demonstrated that the cytochrome bd oxidase of Porphyromonas gingivalis, encoded by cydAB, was able to catalyse O2 consumption and was involved in peroxide and superoxide resistance, and dioxygen tolerance. Introduction Porphyromonas gingivalis is a gram negative anaerobe populating the oral cavity. P. gingivalis resides in the dental plaque and it is a main contributor to periodontal diseases. The oral biofilm is a model of microbial multicellularity and multicellular behaviour ranging from commensal microbiome to virulent infection. The accumulated body of studies of bacterial pathogens in periodontal acute and chronic infections have designated P. gingivalis, Tannerella forsythia and Treponema denticola (also called “the red complex”) as the tripartite cornerstone of the community in its pathogenic state, as recently confirmed by metagenomic microbiome analysis [1]. Not only do they produce proteases, toxins and inflammatory compounds that attack host tissue, but they can shape the whole behaviour of the community [2]. Nevertheless, the physiological properties of each individual of the red complex are not fully understood. In this respect, P. gingivalis remains a puzzling organism. Although it is non-motile, and possesses a rather undersized armament for adaptive responses compared to most ubiquitous organisms, P. gingivalis sustains the infection disease by surviving in the unfavourable environment of the oral cavity and periodontal pockets, and by invading host tissues. For example, the P. gingivalis PLOS ONE