This study compared four biofilm fixation protocols for scanning electron microscopy (SEM) imaging and their compatibility with Fiji-based image analysis, and introduced a complementary approach for SEM biofilm cross-section analysis to be used when assessing the antimicrobial activity of dental restorative materials. In the first phase, resin composite discs (8 × 2 mm) were characterized for degree of conversion and surface roughness, then incubated with renilla-reporter Streptococcus mutans for 1 and 24 h. Metabolic activity was assessed by luciferase assay, and biofilms were fixed using four protocols based on buffered formalin and/or glutaraldehyde, with or without graded ethanol dehydration, prior to SEM imaging. In parallel, biofilm cross-sections were analyzed using resin bars incubated under different conditions. In the second phase, the most effective fixation protocol was validated using antimicrobial-containing restorative materials, including two resin composites and a glass ionomer cement. Data were analyzed using ANOVA and Tukey's test (p < 0.05). The results demonstrated that biofilm fixation protocols significantly influence SEM image quality, biofilm architectural features, and quantitative measurements obtained through image analysis. In conclusion, standardized biofilm fixation using glutaraldehyde combined with graded ethanol dehydration and coupled with machine-learning augmented analysis of SEM images is essential for producing superior image resolution to enable clearer discrimination among bacterial cells, biofilm matrix, and material surfaces, and improved sensitivity in detecting architectural differences, particularly in biofilm cross-sections.

Background:Inflammatory oral diseases are associated with reproducible shifts from commensal-dominated microbiota toward pathobiont-enriched communities, yet the ecological mechanisms underlying the emergence of inflammophiles remain poorly understood. This study aims to investigate if host-derived inflammatory environments act as selective pressures that restructure microbial metabolism and community organization during disease progression. Methods:We performed 16S rRNA gene sequencing of patient-matched pediatric dental plaque and odontogenic abscess specimens to capture microbial community transitions across an inflammatory ecological gradient. Community ecology modeling and inferred metagenomic analyses were used to identify taxa and functional programs associated with commensal and inflammophilic states. Results:Patient-matched comparisons revealed a reproducible ecological selection gradient linking inflammatory environments to expansion of metabolically specialized inflammophiles and depletion of carbohydrate-utilizing commensals. Commensal-dominated plaque communities exhibited anabolic, carbohydrate-centered metabolic capacity, whereas abscess microbiota were enriched for catabolic metabolism, amino acid fermentation, and antimicrobial resistance, consistent with adaptation to inflammation-driven nutrient landscapes and immune pressure. Conclusions:These findings support a model in which host inflammation drives ecological restructuring of the oral microbiome toward metabolically adapted inflammophilic communities. Defining the metabolic requirements and selective pressures governing these transitions provides a framework for microbiome-directed therapeutic strategies aimed at restoring ecological stability during inflammatory dysbiosis.

The oral biofilm has been instrumental in advancing microbial research and enhancing our understanding of oral health and disease. Recent developments in next-generation sequencing have provided detailed insights into the microbial composition of the oral microbiome, enabling species-level analyses of biofilm interactions. Fluorescence in situ hybridization (FISH) has been especially valuable for studying the spatial organization of these microbes, revealing intricate arrangements such as “corncob” structures that highlight close bacterial interactions. As more genetic sequence data become available, the specificity and accuracy of existing FISH probes used in biogeographical studies require reevaluation. This study examines the performance of commonly used species-specific FISH probes, designed to differentiate oral microbes within in situ oral biofilms, when applied in vitro to an expanded set of bacterial strains. Our findings reveal that the specificity of several FISH probes is compromised, with cross-species hybridization being more common than previously assumed. Notably, we demonstrate that biogeographical associations within in situ oral biofilms, particularly involving  Streptococcus  and  Corynebacterium , may need to be reassessed to align with the latest metagenomic data.

Corynebacteria are abundant members of the oral microbiome and increasingly recognized as key structural organizers of supragingival biofilms. Despite their prevalence, the ecological roles and phenotypic traits of many oral corynebacterial species remain poorly defined. Here, we isolated and characterized two new strains, Corynebacterium durum JJ2 and Corynebacterium argentoratense MB1, and compared them with previously characterized and published Corynebacterium durum JJ1 and reference strain Corynebacterium matruchotii ATCC 14266. Phenotypic assays revealed that C. durum strains displayed robust aggregation, thick biofilm formation, and extensive extracellular polymeric substance (EPS) networks, whereas C. argentoratense MB1 and C. matruchotii ATCC 14266 formed thinner biofilms with minimal EPS production. All four strains secreted extracellular membrane vesicles capable of inducing chain elongation in Streptococcus sanguinis, underscoring a conserved interspecies signaling function. Genomic analysis demonstrated close relatedness between C. durum and C. matruchotii, while C. argentoratense MB1 was more distantly related, with a reduced genome, fewer metabolic pathways, and the absence of nitrate reductase genes, consistent with its inability to grow under anaerobic conditions. These findings suggest that C. argentoratense MB1 may represent a less specialized or transient inhabitant of the oral cavity, whereas C. durum and C. matruchotii are well adapted to the oral niche. Together, this study expands our understanding of phenotypic diversity, metabolic capacity, and interspecies interactions among selected oral corynebacteria, highlighting their potential importance as biofilm organizers and contributors to oral microbial ecology.IMPORTANCEOral corynebacteria contribute to the structural and ecological stability of supragingival communities. Yet, their species-level functions remain poorly defined. By isolating and characterizing new strains of Corynebacterium durum and Corynebacterium argentoratense, and comparing them with reference strains including Corynebacterium matruchotii, we provide new insight into their phenotypic diversity, metabolic capacity, and ecological roles. Our results demonstrate that C. durum strains form robust biofilms enriched in extracellular polymeric substances, while C. argentoratense produces thinner biofilms and lacks the genomic features required for anaerobic growth, suggesting a less specialized or transient role in the oral cavity. Importantly, we show that extracellular membrane vesicles secreted by all tested strains promote chain elongation in Streptococcus sanguinis, highlighting a conserved mechanism of interspecies communication. These findings advance our understanding of how oral corynebacteria contribute to biofilm organization and microbial homeostasis and position them as critical but understudied players in oral microbial ecology.

ABSTRACT The commensal Streptococcus sanguinis is highly prevalent in the oral cavity and characterized for its ability to inhibit growth of oral pathogens. Like many other cell types, streptococci produce extracellular membrane vesicles (EMVs), which contain specific molecular cargo and facilitate interactions with host cells. We previously demonstrated that EMVs from S. sanguinis are internalized by gingival epithelial cells (GECs) without causing cell death. Our aim is to characterize the effects of vesicles on eukaryotic cells. Microscopy studies of gingival epithelial cells inoculated with EMVs from wild-type and specific deletion mutants show differential uptake, with decreased uptake of ΔSSA1099 EMVs and increased uptake of ΔSSA1882 EMVs relative to SK36 EMVs. However, EMVs from wild-type and deletion mutants showed similar patterns in cytokine and chemokine secretion. Transcriptomic analysis of gingival epithelial cells inoculated with SK36 EMVs showed a downregulation of genes implicated in apoptosis as well as interferon signaling, while showing an upregulation of genes involved in cytokine production. Gelatin zymography results show that SK36 EMVs have a contrasting result on production of MMP2/9; MMP2 production is decreased while MMP9 is increased by 48 hours post-inoculation (hpi). Dual-inoculation studies demonstrate that prior internalization of S. sanguinis EMVs protects gingival epithelial cells from exposure to pathobiont Porphyromonas gingivalis outer membrane vesicles (OMVs), preventing dissociation and cell death. Our overall findings suggest that S. sanguinis EMVs trigger an immune response on gingival epithelial cells; however, this response suggests inhibition of some immune signaling pathways. Our results highlight an important role in commensalism, in which a microbe induces an immune response but avoids damage to host cells, thus discouraging infection by pathobionts.

Inflammatory dysbiotic diseases present an intriguing biological paradox. Like most other infectious disease processes, the alarm bells of the host are potently activated by tissue-destructive pathobionts, triggering a cascade of physiological responses that ultimately mobilize immune cells like neutrophils to sites of active infection. Typically, these inflammatory host responses are critical to inhibit and/or eradicate infecting microbes. However, for many inflammatory dysbiotic diseases, inflammophilic pathobiont-enriched communities not only survive the inflammatory response, but they actually obtain a growth advantage when challenged with an inflammatory environment. This is especially true for those organisms that have evolved various strategies to resist and/or manipulate components of innate immunity. In contrast, members of the commensal microbiome typically experience a competitive growth disadvantage under inflammatory selective pressure, hindering their critical ability to restrict pathobiont proliferation. Here, we examine examples of bacteria-neutrophil interactions from both conventional pathogens and inflammophiles. We discuss some of the strategies utilized by them to illustrate how inflammophilic microbes can play a central role in the positive feedback cycle that exemplifies dysbiotic chronic inflammatory diseases.

Glucan-dependent biofilm formation is a crucial process in the establishment of Streptococcus mutans as a cariogenic oral microbe. The process of glucan formation has been investigated in great detail, with glycosyltransferases GtfB, GtfC, and GtfD shown to be indispensable for the synthesis of glucans from sucrose. Glucan production can be visualized during biofilm formation through fluorescent labeling, and its abundance, as well as the effect of glucans on general biofilm architecture, is a common phenotype to study S. mutans virulence regulation. Here, we describe an entirely new phenotype associated with glucan production, caused by a mutation in the open reading frame SMU_848, which is located in an operon encoding ribosome-associated proteins. This mutation led to the excess production and accumulation of glucan-containing droplets on the surface of biofilms formed on agar plates after prolonged incubation. While not characterized in S. mutans, SMU_848 shows homology to the phage-related ribosomal protease Prp, essential in cleaving off the N-terminal extension of ribosomal protein L27 for functional ribosome assembly in Staphylococcus aureus. We present a further characterization of SMU_848/Prp, demonstrating that the deletion of this gene leads to significant changes in S. mutans gtfBC expression. Surprisingly, it also profoundly impacts the interkingdom interaction between S. mutans and Candida albicans, a relevant dual-species interaction implicated in severe early childhood caries. The presented data support a potential broader role for SMU_848/Prp, possibly extending its functionality beyond the ribosomal network to influence important ecological processes.

MecA is a highly conserved adaptor protein encoded by prokaryotes from the Bacillota phylum. MecA mutants exhibit similar pleiotropic defects in a variety of organisms, although most of these phenotypes currently lack a mechanistic basis. MecA mediates ClpCP-dependent proteolysis of its substrates, but only several such substrates have been reported in the literature and there are suggestions that proteolysis-independent regulatory mechanisms may also exist. Here, we provide the first comprehensive characterization of the MecA interactome and further assess its regulatory role in Clp-dependent proteolysis. Untargeted coimmunoprecipitation assays coupled with mass spectrometry revealed that the MecA ortholog from the oral pathobiont Streptococcus mutans likely serves as a major protein interaction network hub by potentially complexing with >100 distinct protein substrates, most of which function in highly conserved metabolic pathways. The interactome results were independently verified using a newly developed prokaryotic split luciferase complementation assay (SLCA) to detect MecA protein-protein interactions in vivo. In addition, we further develop a new application of SLCA to support in vivo measurements of MecA relative protein binding affinities. SLCA results were independently verified using targeted coimmunoprecipitation assays, suggesting the general utility of this approach for prokaryotic protein-protein interaction studies. Our results indicate that MecA indeed regulates its interactome through both Clp-dependent proteolysis as well as through an as-yet undefined proteolysis-independent mechanism that may affect more than half of its protein interactome. This suggests a significant aspect of the MecA regulatory function still has yet to be discovered.IMPORTANCEDespite multiple decades of study, the regulatory mechanism and function of MecA have remained largely a mystery. The current study provides the first detailed roadmap to investigate these functions in other medically significant bacteria. Furthermore, this study developed new genetic approaches to assay prokaryotic protein-protein interactions via the split luciferase complementation assay (SLCA). SLCA technology is commonly employed in eukaryotic genetic research but has not yet been established for studies of bacterial protein-protein interactions. The SLCA protein binding affinity assay described here is a new technological advance exclusive to the current study and has not been reported elsewhere.

The ubiquitous inflammophilic oral pathobiont Fusobacterium nucleatum (Fn) is widely recognized for its strong association with inflammatory dysbiotic diseases and cancer. Fn is subdivided into four subspecies, which are historically considered functionally interchangeable in the oral cavity. To test this assumption, we analyzed patient-matched dental plaque and odontogenic abscess clinical specimens and examined whether an inflammatory environment selects for/against particular Fn subspecies. Dental plaque harbored a greater diversity of fusobacteria, with Fn. polymorphum dominating, whereas odontogenic abscesses were exceptionally biased for the largely uncharacterized organism Fn. animalis. Comparative genomic analyses revealed significant genotypic distinctions among Fn subspecies that correlate with their preferred ecological niches and support a taxonomic reassignment of each as a distinct Fusobacterium species. Despite originating as a low-abundance organism in dental plaque, Fn. animalis typically outcompetes other oral fusobacteria within the inflammatory abscess environment, which may explain its prevalence in other oral and extraoral diseases.

Parvimonas micra is a pathobiont of humans that is often found in abundance at sites of mucosal inflammation as well as within malignant tumors. Here, we report the complete genome sequence of P. micra strain JM503A, which is a genetically tractable clinical isolate derived from a human odontogenic abscess specimen.

The cell wall plays an important structural role for bacteria and is intimately tied to a variety of critical processes ranging from growth and differentiation to pathogenesis. Our understanding of cell wall biogenesis is primarily derived from a relatively small number of heavily studied model organisms. Consequently, these processes can only be inferred for the vast majority of prokaryotes, especially among groups of uncharacterized and/or genetically intractable organisms. Recently, we developed the first tractable genetic system for Parvimonas micra, which is a ubiquitous Gram-positive pathobiont of the human microbiome involved in numerous types of inflammatory infections as well as a variety of malignant tumors. P. micra is also the first, and currently only, member of the entire Tissierellia class of the Bacillota phylum in which targeted genetic manipulation has been demonstrated. Thus, it is now possible to study cell wall biogenesis mechanisms within a member of the Tissierellia, which may also reveal novel aspects of P. micra pathobiology. Herein, we describe a procedure for cloning-independent genetic manipulation of P. micra, including allelic replacement mutagenesis and genetic complementation. The described techniques are also similarly applicable for the study of other aspects of P. micra pathobiology and physiology.

Polymicrobial diseases such as periodontal disease and caries pose significant treatment challenges due to their resistance to common approaches like antibiotic therapy. These infections exhibit increased resilience, due to microbial interactions that also disrupt host immune responses. Current research focuses on virulence and disease-promoting interactions, but less is known about interactions that could inhibit or prevent disease development. Normally human-associated microbiomes maintain homeostasis, preventing pathobionts from becoming dominant. In conditions like chronic disseminated candidiasis or severe early childhood caries (s-ECC), an overgrowth of microbes such as Candida albicans disrupts this balance. Typically, C. albicans coexists benignly within the microbial community but can become pathogenic, forming biofilms and interacting with other microbes such as cariogenic Streptococcus mutans. This interaction is particularly significant in s-ECC, where it exacerbates the disease's progression and severity. Here, we present that Corynebacterium durum, itself and through its extracellular membrane vesicles disrupts interkingdom assemblages between C. albicans and S. mutans. Mechanistically the interaction interference occurs at the genetic level with downregulated HWP1 expression, a surface protein specifically induced in the presence of S. mutans promoting the interkingdom interaction. Additionally, we show that C. durum can impede C. albicans systemic virulence in the Galleria mellonella infection model. This suggests that oral corynebacteria may act as a beneficial commensal species, exerting antifungal effects within polymicrobial communities and opening new avenues for managing polymicrobial diseases.IMPORTANCEPolymicrobial diseases such as severe early childhood caries (s-ECC) lack effective treatment options. Prevention, requiring a deeper understanding of ecological processes before the onset of disease symptoms, could be a potential strategy. In this context, we investigated how relatively abundant oral biofilm Corynebacterium species, which are associated with oral health, can interfere with the interkingdom partnership of Streptococcus mutans and Candida albicans. This partnership is a significant driver of tooth decay in s-ECC due to synergistic activities that increase cariogenicity. Our study reveals that oral corynebacteria, through the production of extracellular membrane vesicles, can disrupt the S. mutans and C. albicans partnership by inhibiting fungal hyphae formation. Additionally, the fatty acid cargo within these vesicles exhibits antifungal properties, suggesting that corynebacteria play a role in shaping microbial dynamics within the oral biofilm.

Historically, the study of microbe-associated diseases has focused primarily on pathogens, guided by Koch's postulates. This pathogen-centric view has provided a mechanistic understanding of disease etiology and microbial pathogenesis. However, next-generation sequencing approaches have revealed a far more nuanced view of the roles various microbes play in disease, highlighting the importance of microbial diversity beyond individual pathogens. This broader perspective acknowledges the roles of host and microbial communities in disease development and resistance. In particular, the concept of dysbiosis, especially within the oral cavity, has gained attention for explaining the emergence of complex polymicrobial diseases. Such diseases often stem from resident microbes rather than foreign pathogens, complicating their treatment and even clouding our understanding of disease etiology. Oral health is maintained through a delicate balance between commensal microbes and the host, with diseases like caries and periodontal disease arising from pathogenic perturbations of this balance. Commensal microbes, such as certain streptococci and Corynebacterium spp., play crucial roles in maintaining oral health through mechanisms involving hydrogen peroxide production and membrane vesicle secretion, which can inhibit pathogenic species and modulate host immune responses. Recent research focused upon the mechanisms of molecular commensalism has expanded our understanding of these key functions of the commensal microbiome, demonstrating their central role in promoting oral health and preventing disease. These abilities represent a largely untapped reservoir of potential innovative strategies for disease prevention and management, emphasizing the need to bolster a symbiotic microbiome that inherently suppresses pathogenesis.

It is widely acknowledged that the human-associated microbial community influences host physiology, systemic health, disease progression, and even behavior. There is currently an increased interest in the oral microbiome, which occupies the entryway to much of what the human initially encounters from the environment. In addition to the dental pathology that results from a dysbiotic microbiome, microbial activity within the oral cavity exerts significant systemic effects. The composition and activity of the oral microbiome is influenced by (1) host-microbial interactions, (2) the emergence of niche-specific microbial "ecotypes," and (3) numerous microbe-microbe interactions, shaping the underlying microbial metabolic landscape. The oral streptococci are central players in the microbial activity ongoing in the oral cavity, due to their abundance and prevalence in the oral environment and the many interspecies interactions in which they participate. Streptococci are major determinants of a healthy homeostatic oral environment. The metabolic activities of oral Streptococci, particularly the metabolism involved in energy generation and regeneration of oxidative resources vary among the species and are important factors in niche-specific adaptations and intra-microbiome interactions. Here we summarize key differences among streptococcal central metabolic networks and species-specific differences in how the key glycolytic intermediates are utilized.

During oral biofilm development, interspecies interactions drive species distribution and biofilm architecture. To understand what molecular mechanisms determine these interactions, we used information gained from recent biogeographical investigations demonstrating an association of corynebacteria with streptococci. We previously reported that Streptococcus sanguinis and Corynebacterium durum have a close relationship through the production of membrane vesicle and fatty acids leading to S. sanguinis chain elongation and overall increased fitness supporting their commensal state. Here we present the molecular mechanisms of this interspecies interaction. Coculture experiments for transcriptomic analysis identified several differentially expressed genes in S. sanguinis. Due to its connection to fatty acid synthesis, we focused on the glycerol-operon. We further explored the differentially expressed type IV pili genes due to their connection to motility and biofilm adhesion. Gene inactivation of the glycerol kinase glpK had a profound impact on the ability of S. sanguinis to metabolize C. durum secreted glycerol and impaired chain elongation important for their interaction. Investigations on the effect of type IV pili revealed a reduction of S. sanguinis twitching motility in the presence of C. durum, which was caused by a decrease in type IV pili abundance on the surface of S. sanguinis as determined by SEM. In conclusion, we identified that the ability to metabolize C. durum produced glycerol is crucial for the interaction of C. durum and S. sanguinis. Reduced twitching motility could lead to a closer interaction of both species, supporting niche development in the oral cavity and potentially shaping symbiotic health-associated biofilm communities.

AbstractThe ubiquitous inflammophilic pathobiontFusobacterium nucleatumis widely recognized for its strong association with a variety of human dysbiotic diseases such as periodontitis and oral/extraoral abscesses, as well as multiple types of cancer. F. nucleatumis currently subdivided into four subspecies:F. nucleatumsubspeciesnucleatum(Fn. nucleatum), animalis(Fn. animalis), polymorphum(Fn. polymorphum), andvincentii/fusiforme(Fn. vincentii). Although these subspecies have been historically considered as functionally interchangeable in the oral cavity, direct clinical evidence is largely lacking for this assertion. Consequently, we assembled a collection of oral clinical specimens to determine whetherF. nucleatumsubspecies prevalence in the oral cavity stratifies by local oral health status. Patient-matched clinical specimens of both disease-free dental plaque and odontogenic abscess were analyzed with newly developed culture-dependent and culture-independent approaches using 44 and 60 oral biofilm/tooth abscess paired specimens, respectively. Most oral cavities were found to simultaneously harbor multipleF. nucleatumsubspecies, with a greater diversity present within dental plaque compared to abscesses. In dental plaque,Fn. polymorphumis clearly the dominant organism, but this changes dramatically within odontogenic abscesses whereFn. animalisis heavily favored over all other fusobacteria. Surprisingly, the most commonly studiedF. nucleatumsubspecies,Fn. nucleatum,is only a minor constituent in the oral cavity. To gain further insights into the genetic basis for these phenotypes, we subsequently performed pangenome, phylogenetic, and functional enrichment analyses of oral fusobacterial genomes using the Anvi’o platform, which revealed significant genotypic distinctions amongF. nucleatumsubspecies. Accordingly, our results strongly support a taxonomic reassignment of eachF. nucleatumsubspecies into distinctFusobacteriumspecies. Of these,Fn. animalisshould be considered as the most clinically relevant at sites of active inflammation, despite being among the least characterized oral fusobacteria.

Advancements in DNA sequencing technologies within the last decade have stimulated an unprecedented interest in the human microbiome, largely due the broad diversity of human diseases found to correlate with microbiome dysbiosis. As a direct consequence of these studies, a vast number of understudied and uncharacterized microbes have been identified as potential drivers of mucosal health and disease. The looming challenge in the field is to transition these observations into defined molecular mechanistic studies of symbiosis and dysbiosis. In order to meet this challenge, many of these newly identified microbes will need to be adapted for use in experimental models. Consequently, this review presents a comprehensive overview of the molecular microbiology tools and techniques that have played crucial roles in genetic studies of the bacteria found within the human oral microbiota. Here, we will use specific examples from the oral microbiome literature to illustrate the biology supporting these techniques, why they are needed in the field, and how such technologies have been implemented. It is hoped that this information can serve as a useful reference guide to help catalyze molecular microbiology studies of the many new understudied and uncharacterized species identified at different mucosal sites in the body.

Membrane vesicles are produced by Gram-negative and Gram-positive bacteria. While membrane vesicles are potent elicitors of eukaryotic cells and involved in cell-cell communication, information is scarce about their general biology in the context of community members and the environment. Streptococcus sanguinis, a Gram-positive oral commensal, is prevalent in the oral cavity and well-characterized for its ability to antagonize oral pathobionts. We have found that production and dissemination of membrane vesicles by S. sanguinis is dependent on environmental and community factors. Co-culture with interacting commensal Corynebacterium durum, as well as with the periodontal pathobiont Filifactor alocis had no effect on S. sanguinis vesicle number and size, whereas the periodontal pathobiont Porphyromonas gingivalis abolished S. sanguinis vesicle production. Using both correlation and differential expression analyses to examine the transcriptomic changes underlying vesicle production, we found that differential expression of genes encoding proteins related to the cytoplasmic membrane and peptidoglycan correlate with the abundance of membrane vesicles. Proteomic characterizations of the vesicle cargo identified a variety of proteins, including those predicted to influence host interactions or host immune responses. Cell culture studies of gingival epithelial cells demonstrated that both crude and highly purified membrane vesicles could induce the expression of IL-8, TNF-α, IL-1β, and Gro-α within 6 hours of inoculation at levels comparable to whole cells. Our findings suggest that production of membrane vesicles by S. sanguinis is heavily influenced by community and environmental factors and plays an important role in communication with host cells.