1 INTRODUCTION

The field of human microbiome research has undergone a revolution in its approach toward understanding how microorganisms influence the physiology of their host.1 Development of culture-independent methods has resulted in increased detection and classification of microbial species within microbial communities.2 technologies, biomarker sequencing, and shotgun metagenomics have become standard tools used to determine the composition and genetic makeup of the human microbiome.3 Other “-omics” technologies, such as proteomics and metabolomics, support mechanistic hypotheses involved in causal microbial pathways that are related to states of health and disease.4, 5 Since Antonie van Leeuwenhoek first discovered the existence of microbes in the 1700s while analyzing dental plaque under a microscope, the composition of oral microbial communities has been extensively studied.6 Over 250 species from the oral cavity have been isolated in culture and characterized, including several key pathogens, such as Streptococcus mutans, Porphyromonas gingivalis, Tannerella forsythia, and Aggregatibacter actinomycetemcomitans, involved in the etiology of dental caries and periodontal disease.7-9 An integrated approach toward understanding states of oral disease from the polymicrobial perspective has emerged over time, attributing disease pathology not only to key pathogens but rather to networks of co-occurring microbes, the collective activities of which contribute to pathogenesis.9-12 As such, the importance of understanding the divergences, between oral health and disease, in the microbes comprising the system as well as their relative abundance and functional activity, in addition to genetic factors and ecological pressures that drive such changes, is a primary focus of research within the field of oral health research.9, 13-17 In recent decades, genetic approaches have shed light on the functional capacity of members of oral microbiomes, the mechanistic underpinnings of caries and periodontal disease pathogenesis, and the complex dynamics and fitness factors of key organisms in oral microbiomes.3, 18

The oral microbial ecosystem is constantly exposed to exogenous foreign substances.17 Such circumstances are defining factors for founding microbes and their ability to persist in this environment, and make for distinct relationships between microbe and host that rely on selective pressures. Pioneer microbial colonizers of the oral cavity, such as Streptococcus mitis, Streptococcus sanguinis, Streptococcus gordonii, and Streptococcus salivarius, display core characteristics that make them well suited to this specific niche as they are able to bind selectively to tongue and cheek cells before the teeth emerge and can outcompete other microbial species.17, 19 Emerging teeth acquire a protective glycoprotein coat, which sets in motion successional microbial colonization, resulting in the development of complex polymicrobial biofilm communities, namely dental plaque.20 These complex dental plaque matrices create unique microenvironments that harbor acidic and anaerobic microenvironments, and thus select for organisms distinct from those growing directly on the tooth surface.21

Diet provides nutritional resources for the oral microbiota and also serves as a selective pressure by enriching for organisms best adapted to utilize specific host-derived dietary resources.22 Major historical dietary shifts throughout evolution are accompanied by significant changes in the oral microbiota.4, 23 Lifestyle changes taking place at the Neolithic Revolution, and the later Industrial Revolution, resulted in development of the Westernized Diet, characterized by dietary staples such as farmed animal meats, dairy products, refined vegetable oils, and processed cereal grains that substantially diverged from pre-agricultural diets. Today, such dietary constituents are staples of the American diet, as well as in many developed and developing countries. Such changes in diet were paralleled by pathologic changes to the oral microbiota, including greater representation of acid-producing and acid-tolerant organisms and periodontal pathogens.17, 22, 24, 25 While diet influences the oral microbiome,22, 24, 26 recent data indicate that the oral microbiome influences the dietary preferences of its host. Certain bacteria, such as some Clostridia and Prevotella species, have been associated with taste thresholds, such as sweet, sour, salty, and bitter, plausibly representing a mechanism by which the oral microbiota influences dietary preferences to sustain its membership and persistence in the oral cavity.27, 28 Oral hygiene habits are another consistent source of influence on the oral microflora.29-31 Toothbrushing and flossing can be powerful means to disrupt plaque, the microbial inhabitants of which can cause tooth demineralization and gingival inflammation long-term.32 Toothbrushes themselves, however, can also serve as reservoirs for pathogenic bacteria that can then inoculate the oral cavity, bringing into view the importance of properly sanitizing and storing personal dental hygiene equipment.28, 33 Novel toothpastes have also entered the market to intentionally to shape the oral microbiota via proteins designed to foster species associated with healthy oral bacterial communities, while other products have more general antimicrobial properties.33, 34 Mouthwashes are also designed to have the same effect as toothpaste in that they reduce microbial load via antimicrobial and bactericidal mechanisms.35-37

The influence of the oral microbiota is not confined to this location.38 Oral cavity-associated microbes have been detected in many distant organ sites, including the small intestines, lungs, heart, placenta, and brain.39 Many associations between oral microbes, specifically those implicated in periodontal disease, and other common chronic conditions, such as cardiovascular disease and high blood pressure, have been established.40 Data on the mechanistic connections involved in the development of disease at sites distant from the oral cavity remain sparse, but early research demonstrates that oral cavity-associated microbes can influence immune responses and disease pathogenesis outside the oral cavity, and that their ability to colonize ectopic sites depends on the current state of health of that site.39, 41 These data suggest that the oral microbiome may serve as a reservoir for pathobionts that can either contribute to or exacerbate disease at remote body niches or organ systems. The oral microbiome has become an increasingly important component of recommendations and practices in dental medicine.42 New approaches to modulate oral microbiomes are being presented. For example, some oral probiotics are being designed to increase the alkalinity of the oral cavity and plaque and others are developed to target pathogenic species, such as S mutans.43-45 Administration of supplements, such as arginine, can also substantially affect the composition and metabolic output of an oral microbial community and represents another modular handle.43

2 RESEARCH TECHNIQUES 2.1 Sequence-based culture-independent approaches to assess the microbiome

The field of human microbiome research has revolutionized our view of the role of microbes on mammalian development and health. Traditional approaches revolved around culturing clinical samples in vitro, prior to testing their roles in pathogenesis using in vitro or in vivo assays. A major advancement in this domain has been the development of germ-free mice—animals bred, fed, and raised under sterile conditions—which offer a useful tool for studying the microbiome.46 First conceptualized by Louis Pasteur in 1885 but with uses only fully appreciated in recent decades, this approach allows experimentation in mammals with either no pre-existing microbiome or a highly defined microbial background.46 Seminal studies using germ-free mice confirmed the role of the gut microbiome in a number of diseases, including obesity,47 Kwashiorkor,48 and autism-spectrum disorder,49 by inducing disease features in recipient animals following transfer of a patient-associated microbiome. Studies using germ-free mice have also been employed in the context of the oral microbiome, demonstrating the role of diabetes in disrupting the equilibrium of the oral microbiota50 and confirming the role of the oral microbiota in periodontal disease pathogenesis.51, 52 Studies in germ-free mice have also recently identified a novel role of masticatory forces in eliciting immune surveillance responses in the gingiva in a microbiota-independent manner, consistent with the role of gingival tissues as a physiological barrier in the face of ongoing masticatory challenges.53, 54

In vitro biofilm culture systems using human saliva or defined media have served as a useful surrogate for oral biofilm research and have shed light on the mechanisms involved in microbial adherence, species interactions, antibiofilm treatments, and organization of the microbial community.55-59 Biofilms, with anaerobic centers and aerobic peripheries, exhibit gradients of oxygenation.60, 61 As oral bacteria can be fastidious, slow growing, or require specialized growth media, and many are strict anaerobes, samples should be collected and cultivated appropriately.62, 63 Thus, in vitro models need to be designed to take into account selective pressures in the oral cavity, including salivary flow, species-mediated biofilm succession, and inflammatory substrates, in the development of subgingival biofilm communities.56, 64, 65 In vivo and in vitro biofilm models have been combined in an ex vivo oral biofilm growth model to facilitate the complex interactions taking place in the oral cavity during microbial biofilm succession.66 While powerful insights into oral biofilms have been obtained from in vitro biofilm studies, the difficulty of recapitulating the complexity of the oral cavity in vitro complicates the use of such approaches to investigate microbial communities and understand their holistic functional qualities in their entirety.

Culture-independent methods have improved our understanding of microbial diversity in the oral cavity. Advances in sequencing and mass spectrometry technologies have permitted assessment of microbial community membership, their functional activities, and molecular products.9, 14, 67-70 Sequence-based approaches to assess microbial composition include biomarker approaches that focus on a single kingdom-specific ubiquitous microbial gene or region that exhibits sequence hypervariability (eg, the 16S ribosomal RNA gene in bacteria or the interspacer region in fungi).71 Such hypervariable genes or regions permit identification of microbial community members but can be limited in their capacity to resolve phylogenetically related species or strains and do not provide information on functional gene content of the microbial members present.72 As such, they are useful for cataloging differences in microbiota composition, particularly in large studies, because the approach is relatively inexpensive compared with other, more highly resolving, sequence-based approaches.72, 73 Newer approaches have developed strategies to assess the presence of bacteria in environments with a low-burden microbial signal.74, 75 One such application is depletion of abundant sequences by hybridization, in which the nonmicrobial DNA burden is depleted via CRISPR-associated endonuclease Cas9 targeting to enhance bacterial signals in samples with a low bacterial burden.74, 76

Unlike 16S ribosomal RNA sequencing, shotgun metagenomics sequences permit a parallel assessment of all microbial kingdoms (bacterial, fungal, viral) in a given sample.77, 78 This approach employs random fragmentation and adapter ligation sequence in an unbiased manner all extracted DNA, enabling more in-depth analyses of the pan-genomic gene content in microbiomes.3 Whole genomes of organisms, in addition to strain tracking, can be extracted from the data, allowing for evolutionary analysis of specific organisms associated with a particular disease or environment.79-81 The usefulness of these techniques has been greatly accelerated by the advent of next-generation sequencing technologies, which provide increased read-depth, improved accuracy, and are higher throughput than older methods, such as Sanger sequencing. Three prominent companies have dominated this field so far, with Illumina MiSeq offering shorter-read sequencing, and PacBio and Oxford Nanopore technologies providing longer read lengths.77 There are pros and cons of both short- and long-read lengths: short-read technology provides abundant sequencing data that is less error prone than long-read technology; however, it can be difficult to assemble complete genomes using short read-lengths due to limitations in the technology to distinguish repetitive elements. By contrast, long-read technology provides more read-length but at the cost of higher error rates.82 The generation of high-molecular-weight DNA was a limitation that prevented long-read technology from demonstrating its full potential in advancing shotgun metagenomic methodologies; however, recent improvements in sample preparation have resulted in increased interest for use of this technology in metagenomic studies.83-85 Using shotgun metagenomic sequencing, it is now possible to study members of the microbiome other than bacteria, including the oral virome, in the context of oral diseases, together with periodontal disease.86 Results obtained from the small number of studies performed using shotgun metagenomic sequencing suggest that the oral virome may be as significant in disease pathogenesis as the oral bacteriome.87, 88 Metatranscriptomics, or sequencing of mRNA in a sample, provides a snapshot of transcriptionally active microbes.89 RNA has low stability and thus a short half-life. Hence, the ability of transcriptomic approaches to detect RNA from functionally active and viable bacteria overcomes the limitations of metagenomic approaches. Advances in RNA sequencing, such as random hexamer priming, permit assessment of microbial and host transcriptomes in parallel and are now being explored to assess the interactome.90 Single-cell sequencing was developed initially for immune-profiling purposes but has been adapted to permit assessments of single microbial cells.91 Assessing individual cells from environmental samples increases the detection rate of unculturable organisms while providing the opportunity to ask more novel questions related to the functional capacities and significant roles of single organisms within complex microbial communities.92, 93 Application of transcriptomic approaches has proved extremely significant for delineating both microbial and host gene expression in the context of oral health and pathology.14, 68, 94

The field faces some key hurdles, namely the challenge of separating out the highly complex mixtures that are typical of clinical samples while simultaneously visualizing many molecules of a diverse chemical nature. In periodontal disease, there is a characteristic shift in the composition of oral bacteria that is in part mediated by bacterial metabolites.95 Despite its drawbacks, use of metabolomics could provide valuable mechanistic insights into how and why this shift occurs, and may offer clues to critical time points at which therapeutic or lifestyle interventions may be beneficial. Ultimately, longitudinal, integrated multimodal analyses, involving a range of high-resolution profiling techniques, represent, together with clinical data, the next frontier to understanding microbial host interactions from species level to the molecular level and the implications of these on oral health.

2.2 Metabolomics and proteomics

Recent developments in high-resolution profiling techniques have additionally focused on the profile of small molecules, such as metabolites and proteins, that are detected via liquid or gas chromatography-mass spectrometry or nuclear magnetic resonance spectroscopy. Metabolomics represents the study of molecules in biological samples, which, in the case of human samples, may be produced by the host or its microbiome.5 Metabolomics provides insight into the metabolic and functional activities of the host and its microbiome and intricate interspecies interactions encoded within this pangenome.89 Metabolite production is influenced by the availability of energy sources, environmental stressors, and competition among microbes within a system.5 Metabolomics provides important information related to changes in functional and metabolic pathways via analysis of divergent metabolite profiles presented in the context of health or disease.96, 97 In periodontal disease, there is a characteristic shift in the profile of oral bacteria that is in part mediated by bacterial metabolites, which comprise a chemical communication network. Despite its drawbacks, metabolomics could provide valuable mechanistic insights into how and why this shift occurs, and offer clues to potential therapeutic or lifestyle interventions. Longitudinal, integrated multimodal analyses are ideal for investigating the species that are present, active, and that interact with host cells over time. By contrast, proteomics seeks to analyze the proteome, namely, the profile of all proteins within an organism, tissue, cell, or biological fluid, or subcomponent of any of these.98 Such applications provide insight related to expression and modulation of proteins under specific conditions, such as in health or disease.99 These analyses present the opportunity to identify proteins present within a sample, as well as the abundance, post-translational modifications, isoforms, and molecular interactions of proteins.100 Such technologies use 1- or 2-dimensional gel electrophoresis/mass spectrometry or liquid chromatography/mass spectrometry.100 Proteomic applications have been applied to understand changes in the proteome that diverge states of periodontal health from disease and to further characterize various periodontal disease states, including gingivitis, mild, moderate, chronic, and aggressive periodontitis.70, 101-104

3 COMMUNITY ASSEMBLY OF THE ORAL MICROBIOME 3.1 Oral colonization in early life

The oral cavity is a site of first encounters. As the gatekeeper of the alimentary canal, the oral cavity is the first organ to encounter ingested food and drink, exogenous microbes, allergens, and antigens before they pass further into the gastrointestinal and/or respiratory tracts.54 These direct environmental exposures in the absence of keratinized epithelium pose the oral cavity as a highly susceptible site for infection.105 Regardless, specialized immune-cell networks in the oral cavity respond to the challenges of this fluctuating environment via tissue-specific cues and exclusive immunologic responses that are tailored to the oral cavity.53, 106-108 In line with the role of oral mucosa as a physiological barrier, the functions of immune networks within this mucosa reflect the site-specific challenges faced within the oral cavity. For example, they contribute to homeostasis in response to masticatory forces and trigger immune responses to the development of pathologic microbial communities.52, 53

Oral immune ontogeny, as with the gut, develops by 11 weeks of gestation, at which point cellular components related to the prenatal secretary immune system demonstrate organization of tissue into Peyer's patches.109 Current research findings suggest that the prenatal oral cavity is sterile until birth after which colonization with microorganisms occurs upon exposure to the external environment.110 However, oral bacteria have been detected at various sites within the uterus.110, 111 Interestingly, in a study of 12 mother-neonate pairs, it was found that the microbiota of the neonatal oral cavity displayed clear associations with that of the placenta and was not significantly altered by the birth canal or maternal microbiotas, suggesting that the neonatal microbiota may have a prenatal origin.112 As such, studies pertaining to this question warrant future investigation. Pathogenic bacteria from the oral cavity found at various sites within the uterus are associated with adverse pregnancy outcomes, such as preterm delivery and preeclampsia.113-115 Studies investigating the placental microbiome to understand its role in preterm pregnancies have identified bacteria associated with periodontal disease, suggesting a relationship between the oral and placental microbiomes.113-116 Oral microbes from genera including Streptococcus, Fusobacterium, Neisseria, Prevotella, and Porphyromonas have been recovered from placenta.111 Interestingly, the placental microbiota more closely resembles that of the maternal oral microbiome than that of the gut.116 Animal studies have helped to confirm the direct role of the oral microbiota, and more specifically periodontal disease-related pathogens, in adverse pregnancy outcomes.117 Similarly, bacteria of oral origin, namely Fusobacterium nucleatum, were identified in samples of amniotic fluid and cord blood from women with pregnancy complications, suggesting oral translocation via hematogenous mechanisms.115 Fusobacterium nucleatum has also been associated with stillbirth.118

Following birth, overt colonization of the oral cavity with microbes occurs within 8-16 hours as a result of transmission of microbes vertically (through exposure to maternal skin and vaginal microbiomes), from the diet via oral fixation by the infant, and horizontally (from human interactions additional to those already mentioned).119, 120 The infant mouth becomes colonized by early oral colonizers associated with the infant's mode of delivery,121 demonstrated by finding distinct differences in the bacterial phyla predominant in the oral cavities of babies delivered vaginally compared with those delivered by Cesarean section.121 Firmicutes, Bacteroides, and Actinobacteria were found to be most abundant, respectively, in babies delivered vaginally, while Bacteroides, Proteobacteria, and Firmicutes were most abundant in babies delivered by Cesarean section. Regarding mode of delivery, vast differences in relative abundance at the genus level have been observed for most phyla, with the most marked increases being observed for Lactobacillus species in children delivered vaginally and for Petrimonas species in children delivered by Cesarean section. Lactobacillus species are common constituents of the vaginal microbiome, with strong consistency found between lactobacilli in the microbiota of the vagina and those in the oral cavity of infants delivered vaginally after a natural labor and birth.

The composition of the oral microbiome is shaped throughout life by factors including host genetics and maternal transmission, as well as by environmental factors, such as dietary habits, oral hygiene practice, medications, stress levels, and systemic factors (Figure 1).24, 129 Rather than being fixed, the composition of the oral microbiota changes throughout life, consistent with the oral cavity being a dynamic microbial environment. Eruption of primary deciduous dentition introduces new substrata for microbial colonization, thus introducing ecological shifts within the oral microbiome.130, 131 Additional changes throughout one's life, including to dietary habits, age, hygiene regimens, and behaviors (such as tobacco and alcohol use), also influence changes to the oral microbiome.132 A 2020 study of crowd-sourced oral microbial swabs, taken from a large sample representative of the general population, showed that bacterial diversity of oral microbiomes seems to decline with age,133 an observation that has also been made in lower gastrointestinal microbiomes in relation to diet and health status.134 As children age, their oral microbiomes tend to stabilize, a feature attributed to the establishment of independent oral-hygiene maintenance habits, acquisition of permanent dentition, and consumption of an adult diet with more-or-less defined dietary patterns.135

The Oral Microbiome: From First Encounters to Lifelong Encounters. Prenatal. The prenatal oral cavity is thought to be sterile until birth, with colonization occurring soon after delivery. The composition of the oral microbiota in infants has been shown to correlate with mode of delivery. However, infants share an oral microbiota similar to that of their mothers, suggesting that the infant oral microbiota may derive from hematogenous or intrauterine transmission from the mother. Detection of oral microbes of maternal origin among several intrauterine locations, as well as associations with adverse pregnancy outcomes, demonstrate the role of the maternal oral microbiome in prenatal health and suggests in utero colonization. Early life. Microbial colonization begins shortly following birth through vertical transmission from the mother, transmission from the diet, and transmission from infant-to-human interactions. Microbial diversity increases upon eruption of primary teeth as this process permits the expansion of microbial niches in the oral cavity. Eruption of primary teeth also results in deviation from the maternal oral microbiota. As children age, their oral microbiotas begin to stabilize. Adult life. The oral microbiota continues to be shaped throughout life by genetic and environmental factors. Environmental factors that influence the composition and function of the oral microbiome include diet, stress, oral hygiene practices, drinking alcohol, and smoking. Genetic factors are linked to conserved phylogenetic and functional microbial signatures related to development of dental caries and heritable predisposition to periodontal disease. Aging and systemic disease. Oral microbiome diversity has been shown to decrease with age. The phylogeny and functional signatures of the oral microbiome are linked to states of dental and periodontal diseases, as well as being implicated in various systemic diseases, including cardiovascular disease, cancers, and Alzheimer's disease. Systemic conditions, such as stress and diabetes, can additionally affect the oral microbiome. Figure courtesy of Dr Ryutaro Kuraji, Assistant Professor, Department of Life Science Dentistry, The Nippon Dental University, Tokyo, Japan; Department of Periodontology, The Nippon Dental University School of Life Dentistry at Tokyo, Tokyo, Japan; Visiting Assistant Professor, Department of Orofacial Sciences, School of Dentistry, University of California San Francisco, San Francisco, CA, USA

The relative importance, on the oral microbiota, of host genetics versus environmental factors has been debated.123, 135-139 In a large study of many sets of young and middle-aged Swedish twins, the effect of host genetic factors on the salivary microbiota, predicted metabolic functions, and immune responses to oral bacteria were explored.108 Here, the use of young and middle-aged twins granted an understanding of genetics versus environment, as the young twins in the study were living together and exposed to similar environmental backgrounds, whereas the middle-aged twins shared genetic factors but had lived apart for many years. The presence and relative abundance of all species identified were influenced by environmental factors, and thus differed according to the environmental factors to which they had been exposed. Conversely, the influence of host genetic factors on these parameters were variable at the species level, mainly demonstrating strong effects on the presence and relative abundance of 27 bacterial species. The bacteria associated with host genetic factors were caries-associated species, including S mutans, Scardovia wiggisae, and Stomatobaculum longum. Genetic factors were also associated with predicted metabolic pathways among the salivary microbiota, most specifically in relation to carbohydrate metabolism. In support of this, a different study found that heritability of microbial functions related to acid production was nearly 76%.122 Moreover, heritability of caries is nearly 50% among Swedish twins.140 In terms of immune responses, genetic factors were more strongly associated with serum antibody responses to the putative periodontal pathogen P gingivalis. Notably, strong host genetic factors existed for several taxa and microbial metabolic functions relevant to caries development in the younger cohort, while strong host genetic factors on serum antibody levels against known periodontal pathogens existed in the older age group.108

3.2 Biofilm colonization

The mucosal surfaces serve as the chief substrata available for microbial colonization in the infant oral cavity.141 The most frequently detected early colonizers of the predental oral cavity include Streptococcus, Staphylococcus, and Fusobacterium species.142, 143 Streptococci are capable of adhering to epithelial cells and are a dominant bacterial group in breast milk.144 As such, streptococcal species constitute the majority of the infant oral microbiota. Streptococcus salivarius demonstrates the highest relative abundance among newborns and shows a steady decrease after 3 months of age.119 Additional early colonizers, such as Gemella, Rothia, Granulicatella, and Haemophilus, present at 3-6 months of age and increase in abundance with time.141, 142 In a cross-sectional study on acquisition of the oral microbiota by infants, Mason and colleagues found that in 85% of infants, the composition of the oral microbial community was similar to that of their mothers, suggesting a significant role of the mother in introducing oral microbial communities to their children. Moreover, maternal smoking was associated with increased levels of F nucleatum and Campylobacter conscisus among infants. The eruption of deciduous teeth creates additional niches, such as nonshedding enamel surfaces, dentogingival borders, and a subgingival environment, for colonization of microbes.143, 145 The eruption of teeth leads to divergence of the infant oral microbiota from the maternal microbiota, and such changes persist among mixed and permanent dentition states.

The human oral microbiota is markedly diverse among individuals.29 However, despite dissimilar phylogeny, functional signatures among the oral microbiota are often conserved from one individual to another.13, 14 Among infants, phylogenetic divergences across individuals are observed prior to eruption of deciduous teeth143; however, functional signatures remain conserved.143 Marked expansion of oral microbial phylogenetic and functional diversity is observed with the eruption of deciduous teeth, suggesting the significance of related microbial ecosystems and parallel changes in dietary habits (solid foods) to oral microbiome diversity.143 Interestingly, salivary microbial communities from oral cavities of primary teeth-only cohorts demonstrate greater microbial diversity than do pre-dentate, mixed dentition, and permanent teeth cohorts. Tooth eruption introduces the relative abundance of Streptococcus, Gemella, Granulicatella, and Veillonella species.143 Expansion of microbial functions at the time of tooth eruption reflect changes in the oral ecosystem, including increased expression of genes related to adhesion, biofilm formation, membrane transport, cell mobility, secretion systems, chemotaxis, flagella assembly, and oxidative phosphorylation.143 Exfoliation of primary dentition, the presence of mixed dentition, and the emergence of permanent teeth continue to alter the oral microbiome in early life and childhood.143

Although the oral microbiome in infants evolves with advancing age, initial colonizers of the oral cavity remain as permanent colonizers that influence this colonization trajectory into adulthood.146 The significance of primary colonizers suggests that such pioneer organisms play a key role in determining development of the oral microbiome and thus the long-term oral health status of individuals.146 Not only does the composition of one's oral microbiome locally impact oral health, it may also affect systemic health throughout life.38 This is demonstrated by the association of various disease states with the oral microbiome, including not only caries and periodontal disease but also oral, esophag