Bacterial symbionts in oral niche use type VI secretion nanomachinery for fitness increase against pathobionts.

Microbial ecosystems experience spatial and nutrient restrictions leading to the coevolution of cooperation and competition among cohabiting species. To increase their fitness for survival, bacteria exploit machinery to antagonizing rival species upon close contact. As such, the bacterial type VI secretion system (T6SS) nanomachinery, typically expressed by pathobionts, can transport proteins directly into eukaryotic or prokaryotic cells, consequently killing cohabiting competitors. Here, we demonstrate for the first time that oral symbiont Aggregatibacter aphrophilus possesses a T6SS and can eliminate its close relative oral pathobiont Aggregatibacter actinomycetemcomitans using its T6SS. These findings bring nearer the anti-bacterial prospects of symbionts against cohabiting pathobionts while introducing the presence of an active T6SS in the oral cavity.

Bacteriome and mycobiome dysbiosis in oral mucosal dysplasia and oral cancer.

It has long been considered that the oral microbiome is tightly connected to oral health and that dysbiotic changes can be detrimental to the occurrence and progression of dysplastic oral mucosal lesions or oral cancer. Improved understanding of the concepts of microbial dysbiosis together with advances in high-throughput molecular sequencing of these pathologies have charted in greater microbiological detail the nature of their clinical state. This review discusses the bacteriome and mycobiome associated with oral mucosal lesions, oral candidiasis, and oral squamous cell carcinoma, aiming to delineate the information available to date in pursuit of advancing diagnostic and prognostic utilities for oral medicine.

A Mapping Review of the Pathogenesis of Peri-Implantitis: The Biofilm-Mediated Inflammation and Bone Dysregulation (BIND) Hypothesis.

This mapping review highlights the need for a new paradigm in the understanding of peri-implantitis pathogenesis. The biofilm-mediated inflammation and bone dysregulation (BIND) hypothesis is proposed, focusing on the relationship between biofilm, inflammation, and bone biology. The close interactions between immune and bone cells are discussed, with multiple stable states likely existing between clinically observable definitions of peri-implant health and peri-implantitis. The framework presented aims to explain the transition from health to disease as a staged and incremental process, where multiple factors contribute to distinct steps towards a tipping point where disease is manifested clinically. These steps might be reached in different ways in different patients and may constitute highly individualised paths. Notably, factors affecting the underlying biology are identified in the pathogenesis of peri-implantitis, highlighting that disruptions to the host-microbe homeostasis at the implant-mucosa interface may not be the sole factor. An improved understanding of disease pathogenesis will allow for intervention on multiple levels and a personalised treatment approach. Further research areas are identified, such as the use of novel biomarkers to detect changes in macrophage polarisation and activation status, and bone turnover.

Air powder waterjet technology using erythritol or glycine powders in periodontal or peri-implant prophylaxis and therapy: A consensus report of an expert meeting.

OBJECTIVES: To attain a collective expert opinion on the use of air powder waterjet technology (APWT) with erythritol and glycine powders in the prophylaxis and therapy of periodontal and peri-implant diseases. MATERIAL AND METHODS: In the first step, a modified one-round online Delphi survey including 44 five-point Likert scale questions was conducted among a group of 10 expert clinicians and researchers with thorough knowledge and experience in this topic. In the second step, the single questions and the survey results were discussed during a meeting, and consensus statements were formulated, respectively. RESULTS: An agreement was reached on most items, especially opinions supporting glycine and erythritol powders as favorable with respect to efficiency, safety, and comfort. More scientific evidence is needed to support the improvement in clinical attachment on teeth and implants, especially when APWT with erythritol is used. In addition, APWT needs more long-term evaluation and studies in terms of microbiome/microbiological effects as well as effects on the inflammatory response on natural teeth and implants, also in light of a guided biofilm therapy concept. CONCLUSIONS: In line with the expert opinions and supported by the evidence, it was concluded that the use of APWT with erythritol and glycine powders in nonsurgical periodontal and peri-implant therapy and prophylaxis is patient compliant and efficient.

Age-Related Subgingival Colonization of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis and Parvimonas micra-A Pragmatic Microbiological Retrospective Report.

The aim of this study was to compare data about the prevalence and proportions of the bacterial species Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, and Parvimonas micra in periodontitis pocket samples collected from young, <35 years, and old, >35-year-old patients, YP and OP, respectively. The results from the analyses of a total of 3447 subgingival plaque samples analyzed for clinical diagnosis purposes by cultivation regarding the proportions of these species were collected from a database and elucidated. The prevalence of A. actinomycetemcomitans was found to be more than twice as high (OR = 2.96, 95% CI; 2.50-3.50) in samples from the younger (42.2%) than the older group (20.4%) (p < 0.001). The prevalence of P. micra was significantly lower in samples from the younger age group (OR = 0.43, 95%) (p < 0.001), whereas P. gingivalis was similarly distributed (OR = 0.78, 95%) in the two age groups (p = 0.006). A similar pattern was noticed for A. actinomycetemcomitans and P. gingivalis when high proportions (>50%) of the samples of these bacterial species were elucidated. In contrast, the proportion of samples containing >50% with P. micra was lower compared with the two other bacterial species. Furthermore, it was noted that the proportion of samples from old patients containing A. actinomycetemcomitans in combination with P. micra was almost three times higher than in samples when P. micra was replaced by P. gingivalis. In conclusion, A.actinomycetemcomitans showed an increased presence and proportion in samples from young patients compared with the old patients, while P. gingivalis was similarly distributed in the two age groups. P. micra showed an increased presence and proportion in samples from old patients compared with the young patients.

Gram-negative quorum sensing signalling enhances biofilm formation and virulence traits in gram-positive pathogen Enterococcus faecalis.

Acyl-homoserine lactones (AHLs) are typical quorum-sensing molecules of gram-negative bacteria. Recent evidence suggests that AHLs may also affect gram-positives, although knowledge of these interactions remains scarce. Here, we assessed the effect of AHLs on biofilm formation and transcriptional regulations in the gram-positive Enterococcus faecalis. Five E. faecalis strains were investigated herein. Crystal violet was employed to quantify the biomass formed, and confocal microscopy in combination with SYTO9/PI allowed the visualisation of biofilms' structure. The differential expression of 10 genes involved in quorum-sensing, biofilm formation and stress responses was evaluated using reverse-transcription-qPCR. The AHL exposure significantly increased biofilm production in strain ATCC 29212 and two isolates from infected dental roots, UmID4 and UmID5. In strains ATCC 29212 and UmID7, AHLs up-regulated the quorum-sensing genes (fsrC, cylA), the adhesins ace, efaA and asa1, together with the glycosyltransferase epaQ. In strain UmID7, AHL exposure additionally up-regulated two membrane-stress response genes (σV, groEL) associated with increased stress-tolerance and virulence. Altogether, our results demonstrate that AHLs promote biofilm formation and up-regulate a transcriptional network involved in virulence and stress tolerance in several E. faecalis strains. These data provide yet-unreported insights into E. faecalis biofilm responses to AHLs, a family of molecules long-considered the monopole of gram-negative signalling.

Precision periodontal care: from omics discoveries to chairside diagnostics.

The interface of molecular science and technology is guiding the transformation of personalized to precision healthcare. The application of proteomics, genomics, transcriptomics, and metabolomics is shaping the suitability of biomarkers for disease. Prior validation of such biomarkers in large and diverse patient cohorts helps verify their clinical usability. Incorporation of molecular discoveries into routine clinical practice relies on the development of customized assays and devices that enable the rapid delivery of analytical data to the clinician, while the patient is still in session. The present perspective review addresses this topic under the prism of precision periodontal care. Selected promising research attempts to innovate technological platforms for oral diagnostics are brought forward. Focus is placed on (a) the suitability of saliva as a conveniently sampled biological specimen for assessing periodontal health, (b) proteomics as a high-throughput approach for periodontal disease biomarker identification, and (c) chairside molecular diagnostic assays as a technological funnel for transitioning from the laboratory benchtop to the clinical point-of-care.

Periodontal microbiology and microbial etiology of periodontal diseases: Historical concepts and contemporary perspectives.

This narrative review summarizes the collective knowledge on periodontal microbiology, through a historical timeline that highlights the European contribution in the global field. The etiological concepts on periodontal disease culminate to the ecological plaque hypothesis and its dysbiosis-centered interpretation. Reference is made to anerobic microbiology and to the discovery of select periodontal pathogens and their virulence factors, as well as to biofilms. The evolution of contemporary molecular methods and high-throughput platforms is highlighted in appreciating the breadth and depth of the periodontal microbiome. Finally clinical microbiology is brought into perspective with the contribution of different microbial species in periodontal diagnosis, the combination of microbial and host biomarkers for this purpose, and the use of antimicrobials in the treatment of the disease.

Profiling Antibiotic Susceptibility among Distinct Enterococcus faecalis Isolates from Dental Root Canals.

Enterococcus faecalis, a leading multi-resistant nosocomial pathogen, is also the most frequently retrieved species from persistently infected dental root canals, suggesting that the oral cavity is a possible reservoir for resistant strains. However, antimicrobial susceptibility testing (AST) for oral enterococci remains scarce. Here, we examined the AST profiles of 37 E. faecalis strains, including thirty-four endodontic isolates, two vanA-type vancomycin-resistant isolates, and the reference strain ATCC-29212. Using Etest gradient strips and established EUCAST standards, we determined minimum inhibitory concentrations (MICs) for amoxicillin, vancomycin, clindamycin, tigecycline, linezolid, and daptomycin. Results revealed that most endodontic isolates were susceptible to amoxicillin and vancomycin, with varying levels of intrinsic resistance to clindamycin. Isolates exceeding the clindamycin MIC of the ATCC-29212 strain were further tested against last-resort antibiotics, with 7/27 exhibiting MICs matching the susceptibility breakpoint for tigecycline, and 1/27 reaching that of linezolid. Both vanA isolates confirmed vancomycin resistance and demonstrated resistance to tigecycline. In conclusion, while most endodontic isolates remained susceptible to first-line antibiotics, several displayed marked intrinsic clindamycin resistance, and MICs matched tigecycline's breakpoint. The discovery of tigecycline resistance in vanA isolates highlights the propensity of clinical clone clusters to acquire multidrug resistance. Our results emphasize the importance of implementing AST strategies in dental practices for continued resistance surveillance.

Clinical laboratory diagnostics in dentistry: Application of microbiological methods.

Diagnosis and treatment in dentistry are based on clinical examination of the patients. Given that the major oral diseases are of microbial biofilm etiology, it can be expected that performing microbiological analysis on samples collected from the patient could deliver supportive evidence to facilitate the decision-making process by the clinician. Applicable microbiological methods range from microscopy, to culture, to molecular techniques, which can be performed easily within dedicated laboratories proximal to the clinics, such as ones in academic dental institutions. Periodontal and endodontic infections, along with odontogenic abscesses, have been identified as conditions in which applied clinical microbiology may be beneficial for the patient. Administration of antimicrobial agents, backed by microbiological analysis, can yield more predictable treatment outcomes in refractory or early-occurring forms of periodontitis. Confirming a sterile root canal using a culture-negative sample during endodontic treatment may ensure the longevity of its outcome and prevent secondary infections. Susceptibility testing of samples obtained from odontogenic abscesses may facilitate the selection of the appropriate antimicrobial treatment to prevent further spread of the infection.

Extracellular Vesicle Subproteome Differences among Filifactor alocis Clinical Isolates.

Filifactor alocis is a Gram-positive asaccharolytic, obligate anaerobic rod of the Firmicutes phylum, which has recently been implicated in oral infections. Extracellular vesicles (EVs) are crucial conveyors of microbial virulence in bacteria and archaea. Previously, in highly purified EVs from the F. alocis reference strain ATCC 35896 (CCUG 47790), 28 proteins were identified. The present study aimed to use label-free quantification proteomics in order to chart these EV proteins, in the reference strain, and in nine less-well-characterized clinical F. alocis isolates. In total, 25 of the EV proteins were identified and 24 were quantified. Sixteen of those were differentially expressed between the ten strains and the novel FtxA RTX toxin and one lipoprotein were among them. Consistent expression was observed among ribosomal proteins and proteins involved in L-arginine biosynthesis and type IV pilin, demonstrating a degree of EV protein expression preservation among strains. In terms of protein-protein interaction analysis, 21 functional associations were revealed between 19 EV proteins. Interestingly, FtxA did not display predicted interactions with any other EV protein. In conclusion, the present study charted 25 EV proteins in ten F. alocis strains. While most EV proteins were consistently identified among the strains, several of them were also differentially expressed, which justifies that there may be potential variations in the virulence potential among EVs of different F. alocis strains.

Aggregatibacter actinomycetemcomitans and Filifactor alocis: Two exotoxin-producing oral pathogens.

Periodontitis is a dysbiotic disease caused by the interplay between the microbial ecosystem present in the disease with the dysregulated host immune response. The disease-associated microbial community is formed by the presence of established oral pathogens like Aggregatibacter actinomycetemcomitans as well as by newly dominant species like Filifactor alocis. These two oral pathogens prevail and grow within the periodontal pocket which highlights their ability to evade the host immune response. This review focuses on the virulence factors and potential pathogenicity of both oral pathogens in periodontitis, accentuating the recent description of F. alocis virulence factors, including the presence of an exotoxin, and comparing them with the defined factors associated with A. actinomycetemcomitans. In the disease setting, possible synergistic and/or mutualistic interactions among both oral pathogens might contribute to disease progression.

Photo-oxidative stress response and virulence traits are co-regulated in E. faecalis after antimicrobial photodynamic therapy.

Knowledge of photo-oxidative stress responses in bacteria that survive antimicrobial photodynamic therapy (aPDT) is scarce. Whereas aPDT is attracting growing clinical interest, subsequent stress responses are crucial to evaluate as they may lead to the up-regulation of pathogenic traits. Here, we aimed to assess transcriptional responses to sublethal aPDT-stress and identify potential connections with virulence-related genes. Six Enterococcus faecalis strains were investigated; ATCC 29212, three dental root-canal isolates labelled UmID1, UmID2 and UmID3 and two vancomycin-resistant isolates labelled A1 and A2. TMPyP was employed as a photosensitiser. A viability dose-response curve to increasing concentrations of TMPyP was determined by culture plating. Differential expression of genes involved in oxidative stress responses (dps and hypR), general stress responses (dnaK, sigma-factorV and relA), virulence-related genes (ace, fsrC and gelE) and vancomycin-resistance (vanA) was assessed by reverse-transcription qPCR. TMPyP-mediated aPDT inactivated all strains with comparable efficiencies. TMPyP at 0.015 µM was selected to induce sublethal photo-oxidative stress. Despite heterogeneities in gene expression between strains, transcriptional profiles revealed up-regulations of transcripts dps, hypR as well as dnaK and sigma factorV after exposure to TMPyP alone and to light-irradiated TMPyP. Specifically, the alternative sigma factorV reached up to 39 ± 113-fold (median ± IQR) (p = 0.0369) in strain A2. Up-regulation of the quorum sensing operon, fsr, and its downstream virulence-related gelatinase gelE were also observed in strains ATCC-29212, A1, A2 and UmID3. Finally, photo-oxidative stress induced vanA-type vancomycin-resistance gene in both carrier isolates, reaching up to 3.3 ± 17-fold in strain A2 (p = 0.015). These findings indicate that, while aPDT successfully inactivates vancomycin-resistant and naïve strains of E. faecalis, subpopulations of surviving cells respond by co-ordinately up-regulating a network of genes involved in stress survival and virulence. This includes the induction of vancomycin-resistance genes in carrier isolates. These data may provide the mechanistic basis to circumvent bacterial responses and improve future clinical protocols.

Proteomic Characterization of the Oral Pathogen Filifactor alocis Reveals Key Inter-Protein Interactions of Its RTX Toxin: FtxA.

Filifactor alocis is a Gram-positive asaccharolytic, obligate anaerobic rod that has been isolated from a variety of oral infections including periodontitis, peri-implantitis, and odontogenic abscesses. As a newly emerging pathogen, its type strain has been investigated for pathogenic properties, yet little is known about its virulence variations among strains. We previously screened the whole genome of nine clinical oral isolates and a reference strain of F. alocis, and they expressed a novel RTX toxin, FtxA. In the present study, we aimed to use label-free quantification proteomics to characterize the full proteome of those ten F. alocis strains. A total of 872 proteins were quantified, and 97 among them were differentially expressed in FtxA-positive strains compared with the negative strains. In addition, 44 of these differentially expressed proteins formed 66 pairs of associations based on their predicted functions, which included clusters of proteins with DNA repair/mediated transformation and catalytic activity-related function, indicating different biosynthetic activities among strains. FtxA displayed specific interactions with another six intracellular proteins, forming a functional cluster that could discriminate between FtxA-producing and non-producing strains. Among them were FtxB and FtxD, predicted to be encoded by the same operon as FtxA. While revealing the broader qualitative and quantitative proteomic landscape of F. alocis, this study also sheds light on the deeper functional inter-relationships of FtxA, thus placing this RTX family member into context as a major virulence factor of this species.

Cytokine profiles and the dynamic of gingivitis development in humans.

AIM: To investigate the relationship between cytokine profiles and "fast" and "slow" patterns of gingival inflammation development. MATERIALS AND METHODS: Forty-two adults participated in an experimental gingivitis study, comprising a 2-week hygiene phase (clinical examination and professional cleaning); a 3-week induction phase (absence of oral hygiene); and a 2-week resolution phase (re-establishment of oral hygiene). Plaque and gingival inflammation scores were assessed. Interferon-gamma (IFN-γ), interleukin (IL)-1ß, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, and tumour necrosis factor-alpha (TNF-α) from gingival crevicular fluid were collected and measured by multiplex ELISA. Group-based-trajectory-modelling (GBTM) was used to model cytokine profiles over the induction phase. The effect of gingival inflammation on cytokine levels over time was estimated with mixed-effects modelling. RESULTS: GBTM analysis revealed two cytokine profiles, "non-organized response" (IL-4, IL-6, IL-8, IL-12, and IL-13) and "organized response" (IL-2, IL-10, and TNF-α). Among the "slow" responders, neither cytokine profile was associated with gingivitis. In contrast, a "fast" response was associated with a higher "non-organized response" factor (coef. 0.14) and a lower "organized response" factor (coef. -0.03). CONCLUSION: A "fast" gingivitis development was associated with a higher "non-organized response" and a lower "organized response", which may elucidate the role of individual variability in gingivitis susceptibility.

Antibiofilm activity of nanosilver coatings against Staphylococcus aureus.

Implant infections due to bacterial biofilms constitute a major healthcare challenge today. One way to address this clinical need is to modify the implant surface with an antimicrobial nanomaterial. Among such nanomaterials, nanosilver is arguably the most powerful one, due to its strong and broad antimicrobial activity. However, there is still a lack of understanding on how physicochemical characteristics of nanosilver coatings affect their antibiofilm activity. More specifically, the contributions of silver (Ag)+ ion-mediated vs. contact-based mechanisms to the observed antimicrobial activity are yet to be elucidated. To address this knowledge gap, we produce here nanosilver coatings on substrates by flame aerosol direct deposition that allows for facile control of the coating composition and Ag particle size. We systematically study the effect of (i) nanosilver content in composite Ag silica (SiO2) coatings from 0 (pure SiO2) up to 50 wt%, (ii) the Ag particle size and (iii) the coating thickness on the antibiofilm activity against Staphylococcus aureus (S. aureus), a clinically-relevant pathogen often present on the surface of surgically-installed implants. We show that the Ag+ ion concentration in solution largely drives the observed antibiofilm effect independently of Ag size and coating thickness. Furthermore, co-incubation of both pure SiO2 and nanosilver coatings in the same well also reveals that the antibiofilm effect stems predominantly from the released Ag+ ions, which is especially pronounced for coatings featuring the smallest Ag particle sizes, rather than direct bacterial contact inhibition. We also examine the biocompatibility of the developed nanosilver coatings in terms of pre-osteoblastic cell viability and proliferation, comparing it to that of pure SiO2. This study lays the foundation for the rational design of nanosilver-based antibiofilm implant coatings.

A critical analysis of research methods to study clinical molecular biomarkers in Endodontic research.

The authors of this narrative review aimed to address various experimental methods and make recommendations for how research should move forward in the context of studying biomarkers in clinical Endodontic research. The approach adopted is exemplified using two prominent clinical problems, namely (a) the 'reversible' versus 'irreversible' pulpitis conundrum and (b) persistent idiopathic dentoalveolar pain (PIDAP). Pulpitis under deep caries or dentinal cracks is understood from a histological perspective, but clinical assessment tools to indicate irreversibly inflamed aspects of the dental pulp are elusive. PIDAP, on the other hand, is a diagnosis of exclusion; its pathophysiology is complex and not understood sufficiently to avoid unnecessary dental treatments. This review addresses how diagnostic biomarkers could further our understanding of those and other clinical problems, and how issues can be tackled from a methodological point of view. Hence, different methodological approaches to identify suitable diagnostic biomarker(s) or use known biomarkers are presented. The importance of asking a relevant research question, collecting the most suitable fluid and using the ideal collection vehicle for the research question under investigation is discussed based on the defined clinical problems.

OralDisk: A Chair-Side Compatible Molecular Platform Using Whole Saliva for Monitoring Oral Health at the Dental Practice.

Periodontitis and dental caries are two major bacterially induced, non-communicable diseases that cause the deterioration of oral health, with implications in patients' general health. Early, precise diagnosis and personalized monitoring are essential for the efficient prevention and management of these diseases. Here, we present a disk-shaped microfluidic platform (OralDisk) compatible with chair-side use that enables analysis of non-invasively collected whole saliva samples and molecular-based detection of ten bacteria: seven periodontitis-associated (Aggregatibacter actinomycetemcomitans, Campylobacter rectus, Fusobacterium nucleatum, Prevotella intermedia, Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola) and three caries-associated (oral Lactobacilli, Streptococcus mutans, Streptococcus sobrinus). Each OralDisk test required 400 µL of homogenized whole saliva. The automated workflow included bacterial DNA extraction, purification and hydrolysis probe real-time PCR detection of the target pathogens. All reagents were pre-stored within the disk and sample-to-answer processing took < 3 h using a compact, customized processing device. A technical feasibility study (25 OralDisks) was conducted using samples from healthy, periodontitis and caries patients. The comparison of the OralDisk with a lab-based reference method revealed a ~90% agreement amongst targets detected as positive and negative. This shows the OralDisk's potential and suitability for inclusion in larger prospective implementation studies in dental care settings.

Dysbiosis of the Human Oral Microbiome During the Menstrual Cycle and Vulnerability to the External Exposures of Smoking and Dietary Sugar.

Physiological hormonal fluctuations exert endogenous pressures on the structure and function of the human microbiome. As such, the menstrual cycle may selectively disrupt the homeostasis of the resident oral microbiome, thus compromising oral health. Hence, the aim of the present study was to structurally and functionally profile the salivary microbiome of 103 women in reproductive age with regular menstrual cycle, while evaluating the modifying influences of hormonal contraceptives, sex hormones, diet, and smoking. Whole saliva was sampled during the menstrual, follicular, and luteal phases (n = 309) of the cycle, and the participants reported questionnaire-based data concerning their life habits and oral or systemic health. No significant differences in alpha-diversity or phase-specific clustering of the overall microbiome were observed. Nevertheless, the salivary abundances of genera Campylobacter, Haemophilus, Prevotella, and Oribacterium varied throughout the cycle, and a higher species-richness was observed during the luteal phase. While the overall community structure maintained relatively intact, its functional properties were drastically affected. In particular, 11 functional modules were differentially abundant throughout the menstrual cycle, including pentose phosphate metabolism, and biosynthesis of cobalamin and neurotransmitter gamma-aminobutyric acid. The menstrual cycle phase, but not oral contraceptive usage, was accountable for greater variations in the metabolic pathways of the salivary microbiome. Further co-risk factor analysis demonstrated that Prevotella and Veillonella were increased in current smokers, whereas high dietary sugar consumption modified the richness and diversity of the microbiome during the cycle. This is the first large study to systematically address dysbiotic variations of the oral microbiome during the course of menstrual cycle, and document the additive effect of smoking and sugar consumption as environmental risk factors. It reveals the structural resilience and functional adaptability of the oral microbiome to the endogenous hormonal pressures of the menstrual cycle, while revealing its vulnerability to the exogenous exposures of diet and smoking.