Metatranscriptome and Resistome of the Endodontic Microbiome.

INTRODUCTION: In this study, we used metatranscriptomics for the first time to investigate microbial composition, functional signatures, and antimicrobial resistance gene expression in endodontic infections. METHODS: Root canal samples were collected from ten teeth, including five primary and five persistent/secondary endodontic infections. RNA from endodontic samples was extracted, and RNA sequencing was performed on a NovaSeq6000 system (Illumina). Taxonomic analysis was performed using the Kraken2 bacterial database. Then, sequences with a taxonomic classification were annotated against the Universal Protein Knowledgebase for functional annotation and the Comprehensive Antibiotic Resistance Database for AR-like gene identification. RESULTS: Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria represented the dominant phyla, whereas Fusobacteria, Spirochetes, and Synergistetes were among the nondominant phyla. The top ten species were mainly represented by obligate (or quasiobligate) anaerobes, including Gram-negative (eg, Capnocytophaga sp. oral taxon 323, Fusobacterium nucleatum, Prevotella intermedia, Prevotella oris, Tannerella forsythia, and Tannerella sp. oral taxon HOT-286) and Gram-positive species (eg, Olsenella uli and Parvimonas micra). Transcripts encoding moonlighting proteins (eg, glycolytic proteins, translational elongation factors, chaperonin, and heat shock proteins) were highly expressed, potentially affecting bacterial adhesion, biofilm formation, host defense evasion, and inflammation induction. Endodontic bacteria expressed genes conferring resistance to antibiotic classes commonly used in dentistry, with a high prevalence and expression of tetracycline and lincosamide resistance genes. Antibiotic efflux and antibiotic target alteration/protection were the main resistance mechanisms. CONCLUSIONS: Metatranscriptomics revealed the activity of potential endodontic pathogens, which expressed putative virulence factors and a wide diversity of genes potentially involved in AR.

The alterations of oral, airway and intestine microbiota in chronic obstructive pulmonary disease: a systematic review and meta-analysis.

Background: Increasing evidence indicates the microbial ecology of chronic obstructive pulmonary disease (COPD) is intricately associated with the disease's status and severity, and distinct microbial ecological variations exist between COPD and healthy control (HC). This systematic review and meta-analysis aimed to summarize microbial diversity indices and taxa relative abundance of oral, airway, and intestine microbiota of different stages of COPD and HC to make comparisons. Methods: A comprehensive systematic literature search was conducted in PubMed, Embase, the Web of Science, and the Cochrane Library databases to identify relevant English articles on the oral, airway, and intestine microbiota in COPD published between 2003 and 8 May 2023. Information on microbial diversity indices and taxa relative abundance of oral, airway, and intestine microbiota was collected for comparison between different stages of COPD and HC. Results: A total of 20 studies were included in this review, involving a total of 337 HC participants, 511 COPD patients, and 154 AECOPD patients. We observed that no significant differences in alpha diversity between the participant groups, but beta diversity was significantly different in half of the included studies. Compared to HC, Prevotella, Streptococcus, Actinomyces, and Veillonella of oral microbiota in SCOPD were reduced at the genus level. Most studies supported that Haemophilus, Lactobacillus, and Pseudomonas were increased, but Veillonella, Prevotella, Actinomyces, Porphyromonas, and Atopobium were decreased at the genus level in the airway microbiota of SCOPD. However, the abundance of Haemophilus, Lactobacillus and Pseudomonas genera exhibited an increase, whereas Actinomyces and Porphyromonas showed a decrease in the airway microbiota of AECOPD compared to HC. And Lachnospira of intestine microbiota in SCOPD was reduced at the genus level. Conclusion: The majority of published research findings supported that COPD exhibited decreased alpha diversity compared to HC. However, our meta-analysis does not confirm it. In order to further investigate the characteristics and mechanisms of microbiome in the oral-airway- intestine axis of COPD patients, larger-scale and more rigorous studies are needed. Systematic review registration: PROSPERO (https://www.crd.york.ac.uk/prospero/), identifier CRD42023418726.

Overexpression of proinflammatory cytokines in dental pulp tissue and distinct bacterial microbiota in carious teeth of Mexican Individuals.

The prevalence of dental caries in the Mexican adult population aged 20 to 85 years is around 93.3%, and 50% in Mexican children and adolescents. Worldwide, it is the most common non-communicable disease. One of the main etiological factors for dental caries is the oral microbiome and changes in its structure and function, with an expansion of pathogenic bacteria like Streptococcus mutans. The exposed dental pulp tissue triggers an innate immune response to counteract this bacterial invasion. The relation between oral dysbiosis and innate immune responses remains unclear. We aimed to understand the relationship between innate immune response and the oral microbiota by quantifying the expression of Toll-like receptors (TLRs) and proinflammatory markers (cytokines and a chemokine) in dental pulp tissue, either exposed or not to carious dentin, and to correlate this information with the oral microbiome found in healthy teeth and those with moderate caries. RNA was purified from pulp tissue, subjected to RT-qPCR and analysed with the ΔΔCt method. Supragingival dental plaque of non-carious teeth and dentin of carious teeth were subjected to 16S targeted sequencing. Principal coordinate analysis, permutational multivariate ANOVA, and linear discriminant analysis were used to assess differences between non-carious and carious teeth. Correlations were assessed with Spearman´s test and corrected for multiple comparisons using the FDR method. The relative abundance (RA) of Lactobacillus, Actinomyces, Prevotella, and Mitsuokella was increased in carious teeth; while the RA of Haemophilus and Porphyromonas decreased. Olsenella and Parascardovia were only detected in carious teeth. Significant overexpression of interleukin 1 beta (IL1 ß), IL6, and CXCL8 was detected in pulp tissue exposed to carious dentin. IL1ß correlated positively with TLR2 and Actinomyces; yet negatively with Porphyromonas. These findings suggest that immune response of pulp tissue chronically exposed to cariogenic microbiome is triggered by proinflammatory cytokines IL1ß and IL6 and the chemokine CXCL8.

Impact of different oral treatments on the composition of the supragingival plaque microbiome.

Background: Dental plaque consists of a diverse microbial community embedded in a complex structure of exopolysaccharides. Dental biofilms form a natural barrier against pathogens but lead to oral diseases in a dysbiotic state. Objective: Using a metaproteome approach combined with a standard plaque-regrowth study, this pilot study examined the impact of different concentrations of lactoperoxidase (LPO) on early plaque formation, and active biological processes. Design: Sixteen orally healthy subjects received four local treatments as a randomized single-blind study based on a cross-over design. Two lozenges containing components of the LPO-system in different concentrations were compared to a placebo and Listerine®. The newly formed dental plaque was analyzed by mass spectrometry (nLC-MS/MS). Results: On average 1,916 metaproteins per sample were identified, which could be assigned to 116 genera and 1,316 protein functions. Listerine® reduced the number of metaproteins and their relative abundance, confirming the plaque inhibiting effect. The LPO-lozenges triggered mainly higher metaprotein abundances of early and secondary colonizers as well as bacteria associated with dental health but also periodontitis. Functional information indicated plaque biofilm growth. Conclusion: In conclusion, the mechanisms on plaque biofilm formation of Listerine® and the LPO-system containing lozenges are different. In contrast to Listerine®, the lozenges led to a higher bacterial diversity.

Microbioma oral: variabilidad entre regiones y poblaciones / Oral Microbiome: Variability Between Regions and Populations

Resumen De acuerdo con la Organización Mundial de la Salud (OMS), 3.58 billones de personas son afectadas por desórdenes orales, donde la caries, seguida de la enfermedad periodontal son las más frecuentes y las principales causas de daño al tejido pulpar y pérdida de órganos dentales. En México, el Sistema de Vigilancia Epidemiológica de Patologías Bucales (SIVEPAB) reportó que el 53% de la población se ve afectada por algún grado de enfermedad periodontal, mientras que en promedio la caries afecta al 93.3% de la población de entre 20 a 85 años y más, así como a alrededor del 50.0% de niños y adolescentes, por lo que ambos padecimientos son considerados un problema de salud pública importante en este país. Adicionalmente, se sabe que el microbioma oral humano está asociado con la salud y la enfermedad bucodental. Entre los géneros bacterianos que comúnmente habitan la cavidad oral humana destacan Streptococcus spp., Lactobacillus spp. y Porphyromonas spp. que, a través del desequilibrio del microbioma oral (disbiosis), se asocian con la caries o la enfermedad periodontal. En vista de que estamos constantemente expuestos a este tipo de infecciones crónicas inflamatorias, se sabe que las bacterias orales se trasladan a otras partes del cuerpo contribuyendo al desarrollo y exacerbación de la inflamación sistémica y otras enfermedades. Ya que existen factores como la ubicación geográfica, además de la disbiosis, la edad, la dieta y la genética, que influyen en la variabilidad del microbioma humano. Es importante analizar la diversidad del microbioma oral desde esta perspectiva, ya que el conocimiento que se tiene hasta el momento aún es escaso; por lo anterior se realizó una búsqueda de artículos publicados entre 2010 y 2020 en poblaciones de Asia, África, América y Europa, con el fin de responder la siguiente pregunta: ¿el factor geográfico tiene un impacto en la composición de la variabilidad del microbioma oral humano?

Abstract According to the World Health Organization (WHO), 3.58 billion people were affected by oral disorders, where caries, followed by periodontal disease are the most frequent and the main causes of damage to pulp tissue and loss of dental organs. In Mexico, the Epidemiological Surveillance System for Oral Pathologies (SIVEPAB) reported that 53% of the population is affected by some degree of periodontal disease, while on average caries affects 93.3% of the population between 20 and 85 years old and older, as well as about 50.0% of children and adolescents, so both conditions are considered an important public health problem in this country. Additionally, the human oral microbiome is known to be associated with oral health and disease. An imbalance in the oral microbiome (dysbiosis) can result in the proliferation of Streptococcus mutans and Porphyromonas gingivalis, linked to caries and periodontal disease. The latter two conditions, the most prevalent oral diseases worldwide, are the main causes of damage to pulp tissue and loss of dental organs. In the presence of these pathologies, constant exposure to the corresponding inflammatory chronic infection could lead to the translocation of oral bacteria to other parts of the body, where they may contribute to the development and/or exacerbation of systemic inflammation and trigger disease. Since age, diet, genetics, and geographical location are known to influence the variability of the human microbiome, it is important to analyze differences in the oral microbiome between distinct populations. Up to now, little attention has been given to this task. The current review carried out for articles published between 2010 and 2020 and describes the human oral microbiome in populations of Asia, Africa, America and Europa, to explore whether geographical differences have an impact on the variability of the human oral microbiome.

Over 50,000 Metagenomically Assembled Draft Genomes for the Human Oral Microbiome Reveal New Taxa.

The oral cavity of each person is home to hundreds of bacterial species. While taxa for oral diseases have been studied using culture-based characterization as well as amplicon sequencing, metagenomic and genomic information remains scarce compared to the fecal microbiome. Here, using metagenomic shotgun data for 3346 oral metagenomic samples together with 808 published samples, we obtain 56,213 metagenome-assembled genomes (MAGs), and more than 64% of the 3589 species-level genome bins (SGBs) contain no publicly available genomes. The resulting genome collection is representative of samples around the world and contains many genomes from candidate phyla radiation (CPR) that lack monoculture. Also, it enables the discovery of new taxa such as a genus Candidatus Bgiplasma within the family Acholeplasmataceae. Large-scale metagenomic data from massive samples also allow the assembly of strains from important oral taxa such as Porphyromonas and Neisseria. The oral microbes encode genes that could potentially metabolize drugs. Apart from these findings, a strongly male-enriched Campylobacter species was identified. Oral samples would be more user-friendly collected than fecal samples and have the potential for disease diagnosis. Thus, these data lay down a genomic framework for future inquiries of the human oral microbiome.

Episymbiotic Saccharibacteria suppresses gingival inflammation and bone loss in mice through host bacterial modulation.

Saccharibacteria (TM7) are obligate epibionts living on the surface of their host bacteria and are strongly correlated with dysbiotic microbiomes during periodontitis and other inflammatory diseases, suggesting they are putative pathogens. However, due to the recalcitrance of TM7 cultivation, causal research to investigate their role in inflammatory diseases is lacking. Here, we isolated multiple TM7 species on their host bacteria from periodontitis patients. These TM7 species reduce inflammation and consequential bone loss by modulating host bacterial pathogenicity in a mouse ligature-induced periodontitis model. Two host bacterial functions involved in collagen binding and utilization of eukaryotic sialic acid are required for inducing bone loss and are altered by TM7 association. This TM7-mediated downregulation of host bacterial pathogenicity is shown for multiple TM7/host bacteria pairs, suggesting that, in contrast to their suspected pathogenic role, TM7 could protect mammalian hosts from inflammatory damage induced by their host bacteria.

Bottom-Up Community Proteome Analysis of Saliva Samples and Tongue Swabs by Data-Dependent Acquisition Nano LC-MS/MS Mass Spectrometry.

Analysis using mass spectrometry enables the characterization of metaproteomes in their native environments and overcomes the limitation of proteomics of pure cultures. Metaproteomics is a promising approach to link functions of currently actively expressed genes to the phylogenetic composition of the microbiome in their habitat. In this chapter, we describe the preparation of saliva samples and tongue swabs for nLC-MS/MS measurements and their bioinformatic analysis based on the Trans-Proteomic Pipeline and Prophane to study the oral microbiome .

Taxonomic and Gene Category Analyses of Subgingival Plaques from a Group of Japanese Individuals with and without Periodontitis.

Periodontitis is an inflammation of tooth-supporting tissues, which is caused by bacteria in the subgingival plaque (biofilm) and the host immune response. Traditionally, subgingival pathogens have been investigated using methods such as culturing, DNA probes, or PCR. The development of next-generation sequencing made it possible to investigate the whole microbiome in the subgingival plaque. Previous studies have implicated dysbiosis of the subgingival microbiome in the etiology of periodontitis. However, details are still lacking. In this study, we conducted a metagenomic analysis of subgingival plaque samples from a group of Japanese individuals with and without periodontitis. In the taxonomic composition analysis, genus Bacteroides and Mycobacterium demonstrated significantly different compositions between healthy sites and sites with periodontal pockets. The results from the relative abundance of functional gene categories, carbohydrate metabolism, glycan biosynthesis and metabolism, amino acid metabolism, replication and repair showed significant differences between healthy sites and sites with periodontal pockets. These results provide important insights into the shift in the taxonomic and functional gene category abundance caused by dysbiosis, which occurs during the progression of periodontal disease.

The relationship between oral-origin bacteria in the fecal microbiome and albumin-bilirubin grade in patients with hepatitis C.

BACKGROUND: Bacteria of oral origin (BO) in the gut are associated with prognosis in patients with cirrhosis. The Greengenes database (gg_13_8) is widely used in microbiome analysis, but the expanded Human Oral Microbiome Database (eHOMD), a specialized database for BO, can add more detailed information. We used each database to evaluate the relationship between the albumin-bilirubin grade (ALBI) and the microbiome in patients with hepatitis C. METHODS: Eighty patients were classified into the low ALBI group (LA; n = 34) or high ALBI group (HA; n = 46). Isolated DNA from stool was amplified to target the V3-4 regions of 16S rRNA. The microbiomes of the two groups were compared using gg_13_8 or eHOMD. We evaluated the associations between microbiomes and prognoses using Cox proportional hazards models. RESULTS: At the genus level, the two groups differed significantly regarding 6 (gg_13_8) and 7 (eHOMD) types of bacteria. All types except Akkermansia are classified as BO. Both databases showed an increase in Streptococcus and Veillonella. eHOMD showed a decrease in Fusobacterium and an increase in Fretibacterium; both produce various types of short-chain fatty acids. At the species level, the two groups demonstrated significant differences in 2 (gg_13_8) and 6 (eHOMD) bacterial types. Selenomonas noxia and Streptococcus salivarius were related to poor prognosis in univariate analysis. CONCLUSION: The HA group demonstrated increased BO, most of which produce lactic acid or acetic acid. The correlation between the microbiome and metabolism might be related to prognosis. eHOMD was a useful database for analyzing BO.

Metaproteomics analysis of microbial diversity of human saliva and tongue dorsum in young healthy individuals.

Background: The human oral microbiome influences initiation or progression of diseases like caries or periodontitis. Metaproteomics approaches enable the simultaneous investigation of microbial and host proteins and their interactions to improve understanding of oral diseases. Objective: In this study, we provide a detailed metaproteomics perspective of the composition of salivary and tongue microbial communities of young healthy subjects. Design: Stimulated saliva and tongue samples were collected from 24 healthy volunteers, subjected to shotgun nLC-MS/MS and analyzed by the Trans-Proteomic Pipeline and the Prophane tool. Results: 3,969 bacterial and 1,857 human proteins could be identified from saliva and tongue, respectively. In total, 1,971 bacterial metaproteins and 1,154 human proteins were shared in both sample types. Twice the amount of bacterial metaproteins were uniquely identified for the tongue dorsum compared to saliva. Overall, 107 bacterial genera of seven phyla formed the microbiome. Comparative analysis identified significant functional differences between the microbial biofilm on the tongue and the microbiome of saliva. Conclusion: Even if the microbial communities of saliva and tongue dorsum showed a strong similarity based on identified protein functions and deduced bacterial composition, certain specific characteristics were observed. Both microbiomes exhibit a great diversity with seven genera being most abundant.

Oral microbiome: Unveiling the fundamentals.

The oral cavity has the second largest and diverse microbiota after the gut harboring over 700 species of bacteria. It nurtures numerous microorganisms which include bacteria, fungi, viruses and protozoa. The mouth with its various niches is an exceptionally complex habitat where microbes colonize the hard surfaces of the teeth and the soft tissues of the oral mucosa. In addition to being the initiation point of digestion, the oral microbiome is crucial in maintaining oral as well as systemic health. Because of the ease of sample collection, it has become the most well-studied microbiome till date. Previously, studying the microbiome was limited to the conventional culture-dependent techniques, but the abundant microflora present in the oral cavity could not be cultured. Hence, studying the microbiome was difficult. The emergence of new genomic technologies including next-generation sequencing and bioinformatics has revealed the complexities of the oral microbiome. It has provided a powerful means of studying the microbiome. Understanding the oral microbiome in health and disease will give further directions to explore the functional and metabolic alterations associated with the diseased states and to identify molecular signatures for drug development and targeted therapies which will ultimately help in rendering personalized and precision medicine. This review article is an attempt to explain the different aspects of the oral microbiome in health.

Oral Dysbiotic Communities and Their Implications in Systemic Diseases.

The human body supports the growth of a wide array of microbial communities in various niches such as the oral cavity, gastro-intestinal and urogenital tracts, and on the surface of the skin. These host associated microbial communities include yet-un-cultivable bacteria and are influenced by various factors. Together, these communities of bacteria are referred to as the human microbiome. Human oral microbiome consists of both symbionts and pathobionts. Deviation from symbiosis among the bacterial community leads to “dysbiosis”, a state of community disturbance. Dysbiosis occurs due to many confounding factors that predispose a shift in the composition and relative abundance of microbial communities. Dysbiotic communities have been a major cause for many microbiome related systemic infections. Such dysbiosis is directed by certain important pathogens called the “keystone pathogens”, which can modulate community microbiome variations. One such persistent infection is oral infection, mainly periodontitis, where a wide array of causal organisms have been implied to systemic infections such as cardio vascular disease, diabetes mellitus, rheumatoid arthritis, and Alzheimer’s disease. The keystone pathogens co-occur with many yet-cultivable bacteria and their interactions lead to dysbiosis. This has been the focus of recent research. While immune evasion is one of the major modes that leads to dysbiosis, new processes and new virulence factors of bacteria have been shown to be involved in this important process that determines a disease or health state. This review focuses on such dysbiotic communities, their interactions, and their virulence factors that predispose the host to other systemic implications.

Comparative analysis of Salivette® and paraffin gum preparations for establishment of a metaproteomics analysis pipeline for stimulated human saliva.

The value of saliva as a diagnostic tool can be increased by taxonomic and functional analyses of the microbiota as recently demonstrated. In this proof-of-principle study, we compare two collection methods (Salivette® (SV) and paraffin gum (PG)) for stimulated saliva from five healthy participants and present a workflow including PG preparation which is suitable for metaproteomics.

A Massively Parallel Sequence Similarity Search for Metagenomic Sequencing Data.

Sequence similarity searches have been widely used in the analyses of metagenomic sequencing data. Finding homologous sequences in a reference database enables the estimation of taxonomic and functional characteristics of each query sequence. Because current metagenomic sequencing data consist of a large number of nucleotide sequences, the time required for sequence similarity searches account for a large proportion of the total time. This time-consuming step makes it difficult to perform large-scale analyses. To analyze large-scale metagenomic data, such as those found in the human oral microbiome, we developed GHOST-MP (Genome-wide HOmology Search Tool on Massively Parallel system), a parallel sequence similarity search tool for massively parallel computing systems. This tool uses a fast search algorithm based on suffix arrays of query and database sequences and a hierarchical parallel search to accelerate the large-scale sequence similarity search of metagenomic sequencing data. The parallel computing efficiency and the search speed of this tool were evaluated. GHOST-MP was shown to be scalable over 10,000 CPU (Central Processing Unit) cores, and achieved over 80-fold acceleration compared with mpiBLAST using the same computational resources. We applied this tool to human oral metagenomic data, and the results indicate that the oral cavity, the oral vestibule, and plaque have different characteristics based on the functional gene category.

The Salivary Microbiome in Polycystic Ovary Syndrome (PCOS) and Its Association with Disease-Related Parameters: A Pilot Study.

BACKGROUND: Polycystic ovary syndrome (PCOS) is a common female endocrine condition of unclear etiology characterized by hyperandrogenism, oligo/amenorrhoea, and polycystic ovarian morphology. PCOS is often complicated by infertility, overweight/obesity, insulin resistance, and low-grade inflammation. The gut microbiome is known to contribute to several of these conditions. Recently, an association between stool and saliva microbiome community profiles was shown, making saliva a possible convenient, non-invasive sample type for detecting gut microbiome changes in systemic disease. In this study, we describe the saliva microbiome of PCOS patients and the association of microbiome features with PCOS-related parameters. METHODS: 16S rRNA gene amplicon sequencing was performed on saliva samples from 24 PCOS patients and 20 healthy controls. Data processing and microbiome analyses were conducted in mothur and QIIME. All study subjects were characterized regarding reproductive, metabolic, and inflammatory parameters. RESULTS: PCOS patients showed a decrease in bacteria from the phylum Actinobacteria and a borderline significant shift in bacterial community composition in unweighted UniFrac analysis. No differences between patients and controls were found in alpha diversity, weighted UniFrac analysis, or on other taxonomic levels. We found no association of saliva alpha diversity, beta diversity, or taxonomic composition with serum testosterone, oligo/amenorrhoea, overweight, insulin resistance, inflammatory markers, age, or diet. CONCLUSIONS: In this pilot study, patients with PCOS showed a reduced salivary relative abundance of Actinobacteria. Reproductive and metabolic components of the syndrome were not associated with saliva microbiome parameters, indicating that the majority of between-subject variation in saliva microbiome profiles remains to be explained.

Gene Sequence Analyses of the Healthy Oral Microbiome in Humans and Companion Animals.

It has long been accepted that certain oral bacterial species are responsible for the development of periodontal disease. However, the focus of microbial and immunological research is shifting from studying the organisms associated with disease to examining the indigenous microbial inhabitants that are present in health. Microbiome refers to the aggregate genetic material of all microorganisms living in, or on, a defined habitat. Recent developments in gene sequence analysis have enabled detection and identification of bacteria from polymicrobial samples, including subgingival plaque. Diversity surveys utilizing this technology have demonstrated that bacterial culture techniques have vastly underestimated the richness and diversity of microorganisms in vivo, since only certain bacteria grow in vitro. Surveys using gene sequence analysis have demonstrated that the healthy oral microbiome is composed of an unexpectedly high number of diverse species, including putative pathogens. These findings support the view that coevolution microorganisms and macroscopic hosts has occurred in which certain microorganisms have adapted to survive in the oral cavity and host immune tolerance has allowed the establishment of a symbiotic relationship in which both parties receive benefits (mutualism). This review describes gene sequence analysis as an increasingly common, culture-independent tool for detecting bacteria in vivo and describes the results of recent oral microbiome diversity surveys of clinically healthy humans, dogs, and cats. Six bacterial phyla consistently dominated the healthy oral microbiome of all 3 host species. Previous hypotheses on etiology of periodontitis are reviewed in light of new scientific findings. Finally, the consideration that clinically relevant periodontal disease occurs when immune tolerance of the symbiotic oral microbiome is altered to a proinflammatory response will be discussed.