Benchmarking a targeted 16S ribosomal RNA gene enrichment approach to reconstruct ancient microbial communities.

The taxonomic characterization of ancient microbiomes is a key step in the rapidly growing field of paleomicrobiology. While PCR amplification of the 16S ribosomal RNA (rRNA) gene is a widely used technique in modern microbiota studies, this method has systematic biases when applied to ancient microbial DNA. Shotgun metagenomic sequencing has proven to be the most effective method in reconstructing taxonomic profiles of ancient dental calculus samples. Nevertheless, shotgun sequencing approaches come with inherent limitations that could be addressed through hybridization enrichment capture. When employed together, shotgun sequencing and hybridization capture have the potential to enhance the characterization of ancient microbial communities. Here, we develop, test, and apply a hybridization enrichment capture technique to selectively target 16S rRNA gene fragments from the libraries of ancient dental calculus samples generated with shotgun techniques. We simulated data sets generated from hybridization enrichment capture, indicating that taxonomic identification of fragmented and damaged 16S rRNA gene sequences was feasible. Applying this enrichment approach to 15 previously published ancient calculus samples, we observed a 334-fold increase of ancient 16S rRNA gene fragments in the enriched samples when compared to unenriched libraries. Our results suggest that 16S hybridization capture is less prone to the effects of background contamination than 16S rRNA amplification, yielding a higher percentage of on-target recovery. While our enrichment technique detected low abundant and rare taxa within a given sample, these assignments may not achieve the same level of specificity as those achieved by unenriched methods.

Ancient human dental calculus metadata collection and sampling strategies: Recommendations for best practices.

OBJECTIVES: Ancient human dental calculus is a unique, nonrenewable biological resource encapsulating key information about the diets, lifestyles, and health conditions of past individuals and populations. With compounding calls its destructive analysis, it is imperative to refine the ways in which the scientific community documents, samples, and analyzes dental calculus so as to maximize its utility to the public and scientific community. MATERIALS AND METHODS: Our research team conducted an IRB-approved survey of dental calculus researchers with diverse academic backgrounds, research foci, and analytical specializations. RESULTS: This survey reveals variation in how metadata is collected and utilized across different subdisciplines and highlights how these differences have profound implications for dental calculus research. Moreover, the survey suggests the need for more communication between those who excavate, curate, and analyze biomolecular data from dental calculus. DISCUSSION: Challenges in cross-disciplinary communication limit researchers' ability to effectively utilize samples in rigorous and reproducible ways. Specifically, the lack of standardized skeletal and dental metadata recording and contamination avoidance procedures hinder downstream anthropological applications, as well as the pursuit of broader paleodemographic and paleoepidemiological inquiries that rely on more complete information about the individuals sampled. To provide a path forward toward more ethical and standardized dental calculus sampling and documentation approaches, we review the current methods by which skeletal and dental metadata are recorded. We also describe trends in sampling and contamination-control approaches. Finally, we use that information to suggest new guidelines for ancient dental calculus documentation and sampling strategies that will improve research practices in the future.

Ancient dental calculus reveals oral microbiome shifts associated with lifestyle and disease in Great Britain.

The prevalence of chronic, non-communicable diseases has risen sharply in recent decades, especially in industrialized countries. While several studies implicate the microbiome in this trend, few have examined the evolutionary history of industrialized microbiomes. Here we sampled 235 ancient dental calculus samples from individuals living in Great Britain (∼2200 BCE to 1853 CE), including 127 well-contextualized London adults. We reconstructed their microbial history spanning the transition to industrialization. After controlling for oral geography and technical biases, we identified multiple oral microbial communities that coexisted in Britain for millennia, including a community associated with Methanobrevibacter, an anaerobic Archaea not commonly prevalent in the oral microbiome of modern industrialized societies. Calculus analysis suggests that oral hygiene contributed to oral microbiome composition, while microbial functions reflected past differences in diet, specifically in dairy and carbohydrate consumption. In London samples, Methanobrevibacter-associated microbial communities are linked with skeletal markers of systemic diseases (for example, periostitis and joint pathologies), and their disappearance is consistent with temporal shifts, including the arrival of the Second Plague Pandemic. This suggests pre-industrialized microbiomes were more diverse than previously recognized, enhancing our understanding of chronic, non-communicable disease origins in industrialized populations.

Microbiome ownership for Indigenous peoples.

Several studies have reported increased microbial diversity, or distinct microbial community compositions, in the microbiomes of Indigenous peoples around the world. However, there is a widespread failure to include Indigenous cultures and perspectives in microbiome research programmes, and ethical issues pertaining to microbiome research involving Indigenous participants have not received enough attention. We discuss the benefits and risks arising from microbiome research involving Indigenous peoples and analyse microbiome ownership as an ethical concept in this context. We argue that microbiome ownership represents an opportunity for Indigenous peoples to steward and protect their resident microbial communities at every stage of research.

A relational framework for microbiome research with Indigenous communities.

Ethical practices in human microbiome research have failed to keep pace with scientific advances in the field. Researchers seeking to 'preserve' microbial species associated with Indigenous groups, but absent from industrialized populations, have largely failed to include Indigenous people in knowledge co-production or benefit, perpetuating a legacy of intellectual and material extraction. We propose a framework centred on relationality among Indigenous peoples, researchers and microbes, to guide ethical microbiome research. Our framework centres accountability to flatten historical power imbalances that favour researcher perspectives and interests to provide space for Indigenous worldviews in pursuit of Indigenous research sovereignty. Ethical inclusion of Indigenous communities in microbiome research can provide health benefits for all populations and reinforce mutually beneficial partnerships between researchers and the public.

Community partnerships are fundamental to ethical ancient DNA research.

The ethics of the scientific study of Ancestors has long been debated by archaeologists, bioanthropologists, and, more recently, ancient DNA (aDNA) researchers. This article responds to the article "Ethics of DNA research on human remains: five globally applicable guidelines" published in 2021 in Nature by a large group of aDNA researchers and collaborators. We argue that these guidelines do not sufficiently consider the interests of community stakeholders, including descendant communities and communities with potential, but yet unestablished, ties to Ancestors. We focus on three main areas of concern with the guidelines. First is the false separation of "scientific" and "community" concerns and the consistent privileging of researcher perspectives over those of community members. Second, the commitment of the guidelines' authors to open data ignores the principles and practice of Indigenous Data Sovereignty. Further, the authors argue that involving community members in decisions about publication and data sharing is unethical. We argue that excluding community perspectives on "ethical" grounds is convenient for researchers, but it is not, in fact, ethical. Third, we stress the risks of not consulting communities that have established or potential ties to Ancestors, using two recent examples from the literature. Ancient DNA researchers cannot focus on the lowest common denominator of research practice, the bare minimum that is legally necessary. Instead, they should be leading multidisciplinary efforts to create processes to ensure communities from all regions of the globe are identified and engaged in research that affects them. This will often present challenges, but we see these challenges as part of the research, rather than a distraction from the scientific endeavor. If a research team does not have the capacity to meaningfully engage communities, questions must be asked about the value and benefit of their research.

Studying ancient human oral microbiomes could yield insights into the evolutionary history of noncommunicable diseases.

Noncommunicable diseases (NCDs) have played a critical role in shaping human evolution and societies. Despite the exceptional impact of NCDs economically and socially, little is known about the prevalence or impact of these diseases in the past as most do not leave distinguishing features on the human skeleton and are not directly associated with unique pathogens. The inability to identify NCDs in antiquity precludes researchers from investigating how changes in diet, lifestyle, and environments modulate NCD risks in specific populations and from linking evolutionary processes to modern health patterns and disparities. In this review, we highlight how recent advances in ancient DNA (aDNA) sequencing and analytical methodologies may now make it possible to reconstruct NCD-related oral microbiome traits in past populations, thereby providing the first proxies for ancient NCD risk. First, we review the direct and indirect associations between modern oral microbiomes and NCDs, specifically cardiovascular disease, diabetes mellitus, rheumatoid arthritis, and Alzheimer's disease. We then discuss how oral microbiome features associated with NCDs in modern populations may be used to identify previously unstudied sources of morbidity and mortality differences in ancient groups. Finally, we conclude with an outline of the challenges and limitations of employing this approach, as well as how they might be circumvented. While significant experimental work is needed to verify that ancient oral microbiome markers are indeed associated with quantifiable health and survivorship outcomes, this new approach is a promising path forward for evolutionary health research.

Variable preterm oral microbiome stabilizes and reflects a full-term infant profile within three months.

BACKGROUND: Preterm infants suffer higher morbidity and mortality rates compared to full-term infants, but little is known about how changes to oral and respiratory tract microbiota may impact disease development. METHODS: Here, very preterm neonates (n = 50) were selected to study oral and respiratory microbiota development during the first few months post-birth, where 26 individuals were diagnosed with BPD and/or sepsis. These infants were compared to 14 healthy full-term infants and 16 adults. Microbiota diversity, composition, and species abundances were calculated from 16S ribosomal RNA gene sequences in buccal swabs and tracheal aspirates at two time points (within a week and 1-3 months post-birth). RESULTS: Collection time point was the biggest factor to significantly influence the preterm oral microbial diversity and composition. In addition, BPD and sepsis were linked to distinct preterm oral microbiota diversity and composition, and opportunistic pathogens previously associated with these diseases were identified in the initial sample for both healthy preterm neonates and those with the disease. Compared to the full-term infant and adult dataset, preterm infant diversity and composition was initially significantly different, but resembled full-term infant diversity and composition over time. CONCLUSION: Overall, consequences of microbiota development need further examination in preterm infant infections and later development. IMPACT: Non-gut microbiota research on preterm infants is limited. At one week post-birth, preterm infants harbor distinct oral microbiota that are not shared with full-term children or adults, eventually becoming similar to full-term infants at 36 weeks postmenstrual age. DNA from potential opportunistic pathogens was observed in the mouth and lungs of preterm infants within a week of birth, and microbes associated with BPD were identified in the lungs. Oral microbiota in preterm infants over the first 2-3 months is unique and may be connected to short- and long-term health outcomes in these children.

Type 1 diabetes, periodontal health, and a familial history of hyperlipidaemia is associated with oral microbiota in children: a cross-sectional study.

BACKGROUND: Hyperlipidaemia may play a significant role in the interrelationship between type 1 diabetes (T1D) and periodontal disease. A potential mechanism that links these three aspects together is the oral microbiota. We wanted to determine if there is an association between hyperlipidaemia, periodontal disease, and the oral microbiota of children with T1D, as this has not yet been explored. METHODS: In a post-hoc, cross-sectional study using 16S rRNA gene sequencing, we explored links between oral bacterial diversity and composition of gingival swab samples from 72 children with T1D to periodontal risk factors and hyperlipidaemia status of first-degree relatives. While multiple periodontal risk factors were assessed, we used periodontal pocket depth of 3 mm to characterise periodontal risk. As periodontal pocket depth confounded the analysis of familial history of hyperlipidaemia, a multivariate analyses were performed (i.e., no periodontal risk markers in children with or without a family history of hyperlipidaemia were compared to counterparts who did not have periodontal risk markers) to examine linkages between these factors and diversity and composition of the microbiome. RESULTS: In participants with no periodontitis risk, children with a family history of dyslipidemia had different bacterial diversity and composition compared to those without a familar hisitory. In contrast, such differences did not exist in the children with periodontal risk, whether or not they had a family history of hyperlipidaemia. Co-occurrence networks showed that these differences in children with no periodontists risk were linked to the presence of fewer oral microbial networks, but more microbes linked to mature plaque structures. In contrast, children with periodontal risk markers, regardless of family history of hyperlipidaemia, contained co-occurrence networks that were associated with microbes linked to periodontal disease. CONCLUSIONS: In children diagnosed with T1D, our findings support an association between oral microbiota and two different exposure variables: familial history of hyperlipidaemia and periodontal risk factors.

Schoolyard Biodiversity Determines Short-Term Recovery of Disturbed Skin Microbiota in Children.

Creating biodiverse urban habitat has been proposed, with growing empirical support, as an intervention for increasing human microbial diversity and reducing associated diseases. However, ecological understanding of urban biodiversity interventions on human skin microbiota remains limited. Here, we experimentally test the hypotheses that disturbed skin microbiota recover better in outdoor schoolyard environments and that greater biodiversity provides a greater response. Repeating the experiment three times, we disturbed skin microbiota of fifty-seven healthy 10-to-11-year-old students with a skin swab (i.e., cleaning), then exposed them to one school environment-either a 'classroom' (n = 20), 'sports field' (n = 14), or biodiverse 'forest' (n = 23)-for 45 min. Another skin swab followed the exposure to compare 'before' and 'after' microbial communities. After 45 min, the disturbance immediately followed by outdoor exposure, especially the 'forest', had an enriching and diversifying effect on skin microbiota, while 'classroom' exposure homogenised inter-personal variability. Each effect compounded over consecutive days indicating longer-term exposure outcomes. The experimental disturbance also reduced the core skin microbiota, and only outdoor environments were able to replenish lost species richness to core membership (n species > 50% prevalent). Overall, we find that environmental setting, especially including biodiversity, is important in human microbiota recovery periods and that the outdoors provide resilience to skin communities. This work also has implications for the inclusion of short periods of outside or forest exposure in school scheduling. Future investigations of the health impacts of permanent urban biodiversity interventions are needed.

Individuals with Inflammatory Bowel Disease Have an Altered Gut Microbiome Composition of Fungi and Protozoa.

It is known that the bacterial gut microbiome is altered in inflammatory bowel disease (IBD), but far less is known about the role of eukaryotic microorganisms in IBD. While eukaryotes are rarer than bacteria within the gastrointestinal environment, the current literature suggests that they may also be implicated in IBD. In our study, we characterized these often-neglected eukaryotic microbial communities by identifying fungi and protozoa in published shotgun stool metagenomes from 355 people with IBD (206 with Crohn's disease, 126 with ulcerative colitis, and 23 with IBD-unclassified) and 471 unaffected healthy individuals. The individuals with IBD had a higher prevalence of fungi, particularly Saccharomyces cerevisiae, and a lower prevalence of protozoa, particularly Blastocystis species (subtypes 1, 2, 3, and 4). Regression analysis showed that disease state, age, and BMI were associated with the prevalence and abundance of these two genera. We also characterized the eukaryotic gut microbiome in a shotgun stool metagenomic dataset from people with IBD who received fecal transplants, with samples pre- and post-transplantation, and from their donors. We found that in some FMT recipients, a single eukaryotic species remained stable over time, while in other recipients, the eukaryotic composition varied. We conclude that the eukaryotic gut microbiome is altered and varies over time in IBD, and future studies should aim to include these microbes when characterizing the gut microbiome in IBD.

Effect of Periodontal Interventions on Characteristics of the Periodontal Microbial Profile: A Systematic Review and Meta-Analysis.

Our systematic review aimed to evaluate the effect of periodontal interventions on the diversity and composition of periodontal microbiota assessed by high throughput sequencing (HTS) metagenomics analysis. An electronic search was conducted from database inception to November 2021. All clinical trials that evaluated the effect of periodontal interventions on the gingival microbiota through HTS were selected. The measures of alpha diversity, richness, Shannon diversity index, and the Chao1 index, were used as the primary outcome, whereas relative abundances of bacterial genera were considered as the secondary outcome. Overall, 24 studies were eligible for the systematic review, of which 13 studies were included in the meta-analysis. Periodontal intervention for the test group decreased Shannon diversity, richness, and Chao1 index (alpha diversity), as observed from baseline to post-treatment. The most common genera that increased after periodontal therapy were Rothia, Actinomyces, Streptococcus, Veillonella, and Hemophilus, whilst Porphyromonas, Tannerella, Fusobacterium, and Treponema decreased after periodontal therapy. Periodontal interventions may decrease the bacterial diversity and richness and alter the composition of oral microbiota in the short term. Periodontal microbiota signatures could potentially be used for the assessment of periodontal disease development, progression, and success of the intervention.

A Case Study for the Recovery of Authentic Microbial Ancient DNA from Soil Samples.

High Throughput DNA Sequencing (HTS) revolutionized the field of paleomicrobiology, leading to an explosive growth of microbial ancient DNA (aDNA) studies, especially from environmental samples. However, aDNA studies that examine environmental microbes routinely fail to authenticate aDNA, examine laboratory and environmental contamination, and control for biases introduced during sample processing. Here, we surveyed the available literature for environmental aDNA projects­from sample collection to data analysis­and assessed previous methodologies and approaches used in the published microbial aDNA studies. We then integrated these concepts into a case study, using shotgun metagenomics to examine methodological, technical, and analytical biases during an environmental aDNA study of soil microbes. Specifically, we compared the impact of five DNA extraction methods and eight bioinformatic pipelines on the recovery of microbial aDNA information in soil cores from extreme environments. Our results show that silica-based methods optimized for aDNA research recovered significantly more damaged and shorter reads (<100 bp) than a commercial kit or a phenol−chloroform method. Additionally, we described a stringent pipeline for data preprocessing, efficiently decreasing the representation of low-complexity and duplicated reads in our datasets and downstream analyses, reducing analytical biases in taxonomic classification.

Heritage-specific oral microbiota in Indigenous Australian dental calculus.

Background and objectives: Aboriginal Australians and Torres Strait Islanders (hereafter respectfully referred to as Indigenous Australians) experience a high burden of chronic non-communicable diseases (NCDs). Increased NCD risk is linked to oral diseases mediated by the oral microbiota, a microbial community influenced by both vertical transmission and lifestyle factors. As an initial step towards understanding the oral microbiota as a factor in Indigenous health, we present the first investigation of oral microbiota in Indigenous Australian adults. Methodology: Dental calculus samples from Indigenous Australians with periodontal disease (PD; n = 13) and non-Indigenous individuals both with (n = 19) and without PD (n = 20) were characterized using 16S ribosomal RNA gene amplicon sequencing. Alpha and beta diversity, differentially abundant microbial taxa and taxa unique to different participant groups were analysed using QIIME2. Results: Samples from Indigenous Australians were more phylogenetically diverse (Kruskal-Wallis H = 19.86, P = 8.3 × 10-6), differed significantly in composition from non-Indigenous samples (PERMANOVA pseudo-F = 10.42, P = 0.001) and contained a relatively high proportion of unique taxa not previously reported in the human oral microbiota (e.g. Endomicrobia). These patterns were robust to stratification by PD status. Oral microbiota diversity and composition also differed between Indigenous individuals living in different geographic regions. Conclusions and implications: Indigenous Australians may harbour unique oral microbiota shaped by their long relationships with Country (ancestral homelands). Our findings have implications for understanding the origins of oral and systemic NCDs and for the inclusion of Indigenous peoples in microbiota research, highlighting the microbiota as a novel field of enquiry to improve Indigenous health.

The Oral-Microbiome-Brain Axis and Neuropsychiatric Disorders: An Anthropological Perspective.

In the 21st century, neuropsychiatric disorders (NPDs) are on the rise, yet the causal mechanisms behind this global epidemic remain poorly understood. A key to these unknowns may lie within the vast communities of bacteria, fungi, and viruses in the body (microbiota), which are intimately linked with health and disease. NPDs were recently shown to be connected to gut microbiota, which can communicate with and influence the brain through the Gut-Brain-Axis (GBA). Parallel studies examining oral microbiota and their connections to the brain also suggest that microbes in the mouth can similarly influence NPD outcomes. However, the mechanisms and pathways that illuminate how oral microbiota and brain communicate in NPDs remain unknown. Here, we review identified mechanisms and pathways that oral microbiota use to engage the brain, and we lay the theoretical foundation for an oral-microbiota-brain axis (OMBA). Specifically, we examine established neuroinflammatory and immune system activation responses that underpin interactions between the oral microbiota and the central nervous system (CNS), detailing four specific mechanisms: (1) microbial and metabolite escape, (2) neuroinflammation, (3) CNS signaling, and (4) response to neurohormones. We then scrutinize why including the OMBA, in addition to the GBA, is critically needed to elucidate specific causal relationships between microbial dysbiosis and observed NPD development and progression. Furthermore, we argue for comprehensive, interdisciplinary approaches that integrate lab-based microbiome research and population-level studies that examine the OMBA to improve NPDs. We specifically identify key anthropological perspectives that integrate sociocultural, epidemiological, genetic, and environmental factors that shape the oral microbiome and its interactions with NPDs. Together, future studies of the OMBA in conjunction with interdisciplinary approaches can be used to identify NPD risks and improve outcomes, as well as develop novel intervention and treatment strategies.

Rare genera differentiate urban green space soil bacterial communities in three cities across the world.

Vegetation complexity is potentially important for urban green space designs aimed at fostering microbial biodiversity to benefit human health. Exposure to urban microbial biodiversity may influence human health outcomes via immune training and regulation. In this context, improving human exposure to microbiota via biodiversity-centric urban green space designs is an underused opportunity. There is currently little knowledge on the association between vegetation complexity (i.e. diversity and structure) and soil microbiota of urban green spaces. Here, we investigated the association between vegetation complexity and soil bacteria in urban green spaces in Bournemouth, UK; Haikou, China; and the City of Playford, Australia by sequencing the 16S rRNA V4 gene region of soil samples and assessing bacterial diversity. We characterized these green spaces as having 'low' or 'high' vegetation complexity and explored whether these two broad categories contained similar bacterial community compositions and diversity around the world. Within cities, we observed significantly different alpha and beta diversities between vegetation complexities; however, these results varied between cities. Rare genera (<1% relative abundance individually, on average 35% relative abundance when pooled) were most likely to be significantly different in sequence abundance between vegetation complexities and therefore explained much of the differences in microbial communities observed. Overall, general associations exist between soil bacterial communities and vegetation complexity, although these are not consistent between cities. Therefore, more in-depth work is required to be done locally to derive practical actions to assist the conservation and restoration of microbial communities in urban areas.

The Neglected Gut Microbiome: Fungi, Protozoa, and Bacteriophages in Inflammatory Bowel Disease.

The gut microbiome has been implicated in the pathogenesis of inflammatory bowel disease (IBD). Studies suggest that the IBD gut microbiome is less diverse than that of the unaffected population, a phenomenon often referred to as dysbiosis. However, these studies have heavily focused on bacteria, while other intestinal microorganisms-fungi, protozoa, and bacteriophages-have been neglected. Of the nonbacterial microbes that have been studied in relation to IBD, most are thought to be pathogens, although there is evidence that some of these species may instead be harmless commensals. In this review, we discuss the nonbacterial gut microbiome of IBD, highlighting the current biases, limitations, and outstanding questions that can be addressed with high-throughput DNA sequencing methods. Further, we highlight the importance of studying nonbacterial microorganisms alongside bacteria for a comprehensive view of the whole IBD biome and to provide a more precise definition of dysbiosis in patients. With the rise in popularity of microbiome-altering therapies for the treatment of IBD, such as fecal microbiota transplantation, it is important that we address these knowledge gaps to ensure safe and effective treatment of patients.

Fungi, protozoa, and bacteriophages are often neglected in gut microbiome research of inflammatory bowel disease. Here, we review what is currently known of these microbes in inflammatory bowel disease and how they can be studied using high-throughput DNA sequencing methods.

Late Holocene climate anomaly concurrent with fire activity and ecosystem shifts in the eastern Australian Highlands.

The alpine area of the Australian mainland is highly sensitive to climate and environmental change, and potentially vulnerable to ecosystem tipping points. Over the next two decades the Australian alpine region is predicted to experience temperature increases of at least 1 °C, coupled with a substantial decrease in snow cover. Extending the short instrumental record in these regions is imperative to put future change into context, and potentially provide analogues of warming. We reconstructed past temperatures, using a lipid biomarker palaeothermometer technique and mercury flux changes for the past 3500 years from the sediments of Club Lake, a high-altitude alpine tarn in the Snowy Mountains, southeastern Australia. Using a multi-proxy framework, including pollen and charcoal analyses, high-resolution geochemistry, and ancient microbial community composition, supported by high-resolution 210Pb and AMS 14C dating, we investigated local and regional ecological and environmental changes occurring in response to changes in temperature. We find the region experienced a general warming trend over the last 3500 years, with a pronounced climate anomaly occurring between 1000 and 1600 cal yrs. BP. Shifts in vegetation took place during this warm period, characterised by a decline in alpine species and an increase in open woodland taxa which co-occurred with an increase in regional fire activity. Given the narrow altitudinal band of Australian alpine vegetation, any future warming has the potential to result in the extinction of alpine species, including several endemic to the area, as treelines are driven to higher elevations. These findings suggest ongoing conservation efforts will be needed to protect the vulnerable alpine environments from the combined threats of climate changes, fire and invasive species.

Development and characterization of an oral microbiome transplant among Australians for the treatment of dental caries and periodontal disease: A study protocol.

BACKGROUND: Oral microbiome transplantation (OMT) is a novel concept of introducing health-associated oral microbiota into the oral cavity of a diseased patient. The premise is to reverse the state of oral dysbiosis, and restore the ecological balance to maintain a stable homeostasis with the host immune system. This study will assess the effectiveness, feasibility, and safety of OMT using an interdisciplinary approach. METHODS/DESIGN: To find donors suitable for microbial transplantation, supragingival plaque samples will be collected from 600 healthy participants. Each sample (200µL) will subsequently be examined in two ways: 1) 100µL of the sample will undergo high-throughput 16S rRNA gene amplicon sequencing and shotgun sequencing to identify the composition and characterisation of a healthy supragingival microbiome, 2) the remaining 100µL of the plaque sample will be mixed with 25% artificial saliva medium and inoculated into a specialised in-vitro flow cell model containing a hydroxyapatite disk. To obtain sufficient donor plaque, the samples would be grown for 14 days and further analysed microscopically and sequenced to examine and confirm the growth and survival of the microbiota. Samples with the healthiest microbiota would then be incorporated in a hydrogel delivery vehicle to enable transplantation of the donor oral microbiota. The third step would be to test the effectiveness of OMT in caries and periodontitis animal models for efficacy and safety for the treatment of oral diseases. DISCUSSION: If OMTs are found to be successful, it can form a new treatment method for common oral diseases such as dental caries and periodontitis. OMTs may have the potential to modulate the oral microbiota and shift the ecological imbalances to a healthier state.