Episymbiotic Saccharibacteria TM7x modulates the susceptibility of its host bacteria to phage infection and promotes their coexistence.

Bacteriophages (phages) play critical roles in modulating microbial ecology. Within the human microbiome, the factors influencing the long-term coexistence of phages and bacteria remain poorly investigated. Saccharibacteria (formerly TM7) are ubiquitous members of the human oral microbiome. These ultrasmall bacteria form episymbiotic relationships with their host bacteria and impact their physiology. Here, we showed that during surface-associated growth, a human oral Saccharibacteria isolate (named TM7x) protects its host bacterium, a Schaalia odontolytica strain (named XH001) against lytic phage LC001 predation. RNA-Sequencing analysis identified in XH001 a gene cluster with predicted functions involved in the biogenesis of cell wall polysaccharides (CWP), whose expression is significantly down-regulated when forming a symbiosis with TM7x. Through genetic work, we experimentally demonstrated the impact of the expression of this CWP gene cluster on bacterial-phage interaction by affecting phage binding. In vitro coevolution experiments further showed that the heterogeneous populations of TM7x-associated and TM7x-free XH001, which display differential susceptibility to LC001 predation, promote bacteria and phage coexistence. Our study highlights the tripartite interaction between the bacterium, episymbiont, and phage. More importantly, we present a mechanism, i.e., episymbiont-mediated modulation of gene expression in host bacteria, which impacts their susceptibility to phage predation and contributes to the formation of "source-sink" dynamics between phage and bacteria in biofilm, promoting their long-term coexistence within the human microbiome.

Episymbiotic Saccharibacteria induce intracellular lipid droplet production in their host bacteria.

Saccharibacteria (formerly TM7) are a group of widespread and genetically diverse ultrasmall bacteria with highly reduced genomes that belong to Candidate Phyla Radiation, a large monophyletic lineage with poorly understood biology. Nanosynbacter lyticus type strain TM7x is the first Saccharibacteria member isolated from the human oral microbiome. With restrained metabolic capacities, TM7x lives on the surface of, and forms an obligate episymbiotic relationship with its bacterial host, Schaalia odontolytica strain XH001. The symbiosis allows TM7x to propagate but presents a burden to host bacteria by inducing stress response. Here, we employed super-resolution fluorescence imaging to investigate the physical association between TM7x and XH001. We showed that the binding with TM7x led to a substantial alteration in the membrane fluidity of XH001. We also revealed the formation of intracellular lipid droplets in XH001 when forming episymbiosis with TM7x, a feature that has not been reported in oral bacteria. The TM7x-induced lipid droplets accumulation in XH001 was confirmed by label-free Raman spectroscopy, which also unveiled additional phenotypical features when XH001 cells are physically associated with TM7x. Further exploration through culturing XH001 under various stress conditions showed that lipid droplets accumulation was a general response to stress. A survival assay demonstrated that the presence of lipid droplets plays a protective role in XH001, enhancing its survival under adverse conditions. In conclusion, our study sheds new light on the intricate interaction between Saccharibacteria and their host bacteria, highlighting the potential benefit conferred by TM7x to its host and further emphasizing the context-dependent nature of symbiotic relationships.

Ultrasmall epibiont Nanosynbacter lyticus strain TM7x and host bacteria transcriptional activity after initial host parasitism.

Background: Oral Saccharibacteria Nanosynbacter lyticus strain TM7× lives as an ultrasmall epibiont on the surface of its host, Schaalia odontolytica strain XH001. Establishing this interaction is a poorly understood multi-step process. The recovery phase marks a shift in the TM7×/host interaction, switching from the early killing phase, with extensive host cell death, to a stable symbiosis phase where the host and epibiont can grow together. Results: Transcriptomes of TM7× and host, XH001, were captured during the recovery phase and compared to uninfected host and the early host/epibiont interaction (initial encounter). XH001 showed increased expression for rhamnose cell wall components and for the precursor to peptidoglycan while TM7× showed increases in the peptidoglycan pathway. Transporter expression was generally increased for both organisms during recovery compared to the initial encounter, though, XH001 showed lower amino acid transporter expression. Consistent with host parasitism, XH001 showed increased expression of various stress-related genes during recovery while TM7× showed reduced stress. TM7× displayed higher expression of type IV pili, consistent with increased attachment to new hosts. Conclusion: As TM7× is a member of the broadly distributed Candidate Phyla Radiation with small genomes lacking numerous biosynthetic pathways, this study provides further insights into how these epibionts interact and modulate their host bacteria.

Multi-cohort shotgun metagenomic analysis of oral and gut microbiota overlap in healthy adults.

The multitude of barriers between the mouth and colon may eliminate swallowed oral bacteria. Ascertaining the presence of the same bacteria in the mouth and colon is methodologically challenging partly because 16S rRNA gene sequencing - the most commonly used method to characterize the human microbiota - has low confidence in taxonomic assignments deeper than genus for most bacteria. As different species of the same genus can have low-level variation across the same 16S rRNA gene region, shotgun sequencing is needed to identify a true overlap. We analyzed a curated, multi-cohort, shotgun metagenomic database with species-level taxonomy and clade-specific marker genes to fill this knowledge gap. Using 500 paired fecal/oral (4 oral sites) samples from 4 healthy adult cohorts, we found a minute overlap between the two niches. Comparing marker genes between paired oral and fecal samples with species-level overlap, the pattern of overlap in only 7 individuals was consistent with same-strain colonization. These findings argue against ectopic colonization of oral bacteria in the distal gut in healthy adults.

The oral microbiome: diversity, biogeography and human health.

The human oral microbiota is highly diverse and has a complex ecology, comprising bacteria, microeukaryotes, archaea and viruses. These communities have elaborate and highly structured biogeography that shapes metabolic exchange on a local scale and results from the diverse microenvironments present in the oral cavity. The oral microbiota also interfaces with the immune system of the human host and has an important role in not only the health of the oral cavity but also systemic health. In this Review, we highlight recent advances including novel insights into the biogeography of several oral niches at the species level, as well as the ecological role of candidate phyla radiation bacteria and non-bacterial members of the oral microbiome. In addition, we summarize the relationship between the oral microbiota and the pathology of oral diseases and systemic diseases. Together, these advances move the field towards a more holistic understanding of the oral microbiota and its role in health, which in turn opens the door to the study of novel preventive and therapeutic strategies.

Localized microbially induced inflammation influences distant healthy tissues in the human oral cavity.

Variation in human immune response to the same bacterial or viral pathogen is well established in the literature. Variation in immune response to microbial challenge has also been observed within the human oral cavity. Our recent study focused on characterizing observed variations in microbially induced gingival inflammation-resulting in three distinct clinical Inflammatory Responder Types (IRTs): High-IRT, Low-IRT, and Slow-IRT. Here, we applied a high-resolution temporal multiomic analysis during microbially induced inflammation in order to characterize the effects of localized oral inflammation on distant healthy tissues in young healthy adults. Our results highlight a nonlocalized subclinical effect with alterations in proinflammatory host mediators and an ecological shift toward dysbiosis within the subgingival microbiome in an IRT-dependent manner-despite maintained oral hygiene. Our results provide mechanistic insight into how healthy tissues within humans are influenced by distant localized inflammation and may ultimately become susceptible to disease.

Effects of a stabilized stannous fluoride dentifrice on clinical, immunomodulatory, and microbial outcomes in a human experimental gingivitis model.

BACKGROUND: Stannous fluoride dentifrice is well established for its beneficial clinical effects. In this study, we evaluated the effects of stannous fluoride on inflammation and oral microbiome. METHODS: In this randomized, parallel-arm, double-blind, controlled clinical trial, we compared clinical resolution of experimental gingivitis by evaluating bleeding on probing, gingival index, and plaque index between stannous fluoride stabilized with zinc phosphate (test) and sodium fluoride (control) dentifrices. Further, these groups were compared for oral neutrophil counts, systemic priming of neutrophils, gingival crevicular fluid (GCF) expression of inflammatory markers, and the oral microbiome. RESULTS: We found significant reduction in bleeding on probing in the test group compared to the control group in experimental gingivitis when participants used the test dentifrice prior to induction of experimental gingivitis. The test group also showed significant reductions in GCF levels of inflammatory markers (matrix metalloproteinase 8 [MMP8], receptor activator of nuclear factor kappa-Β ligand [RANKL]), oral polymorphonuclear neutrophil (PMN) counts, and systemic neutrophil priming (CD11b expression) during experimental gingivitis. Further, significant reductions in the gram-negative genera Porphyromonas, Tannerella, and Treponema were noted in the test group. CONCLUSION: The stannous fluoride stabilized with zinc phosphate dentifrice formulation demonstrated clinical reduction in gingival inflammation and a beneficial effect on microbiome and immune markers. This intervention should be explored as a preventive aid in the progression of plaque-induced gingivitis to periodontitis.

Episymbiotic bacterium induces intracellular lipid droplet production in its host bacteria.

Saccharibacteria (formerly TM7) Nanosynbacter lyticus type strain TM7x exhibits a remarkably compact genome and an extraordinarily small cell size. This obligate epibiotic parasite forms a symbiotic relationship with its bacterial host, Schaalia odontolytica, strain XH001 (formerly Actinomyces odontolyticus strain XH001). Due to its limited genome size, TM7x possesses restrained metabolic capacities, predominantly living on the surface of its bacterial host to sustain this symbiotic lifestyle. To comprehend this intriguing, yet understudied interspecies interaction, a thorough understanding of the physical interaction between TM7x and XH001 is imperative. In this study, we employed super-resolution fluorescence imaging to investigate the physical association between TM7x and XH001. We found that the binding with TM7x led to a substantial alteration in the membrane fluidity of the host bacterium XH001. Unexpectedly, we revealed the formation of intracellular lipid droplets in XH001 when forming episymbiosis with TM7x, a feature not commonly observed in oral bacteria cells. The TM7x-induced LD accumulation in XH001 was further confirmed by label-free non-invasive Raman spectroscopy, which also unveiled additional phenotypical features when XH001 cells are physically associated with TM7x. Further exploration through culturing host bacterium XH001 alone under various stress conditions showed that LD accumulation was a general response to stress. Intriguingly, a survival assay demonstrated that the presence of LDs likely plays a protective role in XH001, enhancing its overall survival under adverse conditions. In conclusion, our study sheds new light on the intricate interaction between Saccharibacteria and its host bacterium, highlighting the potential benefit conferred by TM7x to its host, and further emphasizing the context-dependent nature of symbiotic relationships.

Genetic manipulation of Patescibacteria provides mechanistic insights into microbial dark matter and the epibiotic lifestyle.

Patescibacteria, also known as the candidate phyla radiation (CPR), are a diverse group of bacteria that constitute a disproportionately large fraction of microbial dark matter. Its few cultivated members, belonging mostly to Saccharibacteria, grow as epibionts on host Actinobacteria. Due to a lack of suitable tools, the genetic basis of this lifestyle and other unique features of Patescibacteira remain unexplored. Here, we show that Saccharibacteria exhibit natural competence, and we exploit this property for their genetic manipulation. Imaging of fluorescent protein-labeled Saccharibacteria provides high spatiotemporal resolution of phenomena accompanying epibiotic growth, and a transposon-insertion sequencing (Tn-seq) genome-wide screen reveals the contribution of enigmatic Saccharibacterial genes to growth on their hosts. Finally, we leverage metagenomic data to provide cutting-edge protein structure-based bioinformatic resources that support the strain Southlakia epibionticum and its corresponding host, Actinomyces israelii, as a model system for unlocking the molecular underpinnings of the epibiotic lifestyle.

Genetic manipulation of candidate phyla radiation bacteria provides functional insights into microbial dark matter.

The study of bacteria has yielded fundamental insights into cellular biology and physiology, biotechnological advances and many therapeutics. Yet due to a lack of suitable tools, the significant portion of bacterial diversity held within the candidate phyla radiation (CPR) remains inaccessible to such pursuits. Here we show that CPR bacteria belonging to the phylum Saccharibacteria exhibit natural competence. We exploit this property to develop methods for their genetic manipulation, including the insertion of heterologous sequences and the construction of targeted gene deletions. Imaging of fluorescent protein-labeled Saccharibacteria provides high spatiotemporal resolution of phenomena accompanying epibiotic growth and a transposon insertion sequencing genome-wide screen reveals the contribution of enigmatic Saccharibacterial genes to growth on their Actinobacteria hosts. Finally, we leverage metagenomic data to provide cutting-edge protein structure-based bioinformatic resources that support the strain Southlakia epibionticum and its corresponding host, Actinomyces israelii , as a model system for unlocking the molecular underpinnings of the epibiotic lifestyle.

Specific host metabolite and gut microbiome alterations are associated with bone loss during spaceflight.

Understanding the axis of the human microbiome and physiological homeostasis is an essential task in managing deep-space-travel-associated health risks. The NASA-led Rodent Research 5 mission enabled an ancillary investigation of the gut microbiome, varying exposure to microgravity (flight) relative to ground controls in the context of previously shown bone mineral density (BMD) loss that was observed in these flight groups. We demonstrate elevated abundance of Lactobacillus murinus and Dorea sp. during microgravity exposure relative to ground control through whole-genome sequencing and 16S rRNA analyses. Specific functionally assigned gene clusters of L. murinus and Dorea sp. capable of producing metabolites, lactic acid, leucine/isoleucine, and glutathione are enriched. These metabolites are elevated in the microgravity-exposed host serum as shown by liquid chromatography-tandem mass spectrometry (LC-MS/MS) metabolomic analysis. Along with BMD loss, ELISA reveals increases in osteocalcin and reductions in tartrate-resistant acid phosphatase 5b signifying additional loss of bone homeostasis in flight.

Colonization resistance is dispensable for segregation of oral and gut microbiota.

BACKGROUND: The oral and colonic microbiota are distinct in healthy individuals. However, this distinction is diminished in common diseases such as colon cancer and inflammatory bowel disease, suggesting a potential pathogenic role for oral bacteria when ectopically colonized in the gut. A key mechanism for the segregation of oral and colonic microbiota niches is thought to be microbiota-mediated colonization resistance whereby the commensal gut microbiota outcompete and eliminate the ingested oral bacteria. METHODS: We tested this theory by analyzing exact amplicon sequence variants generated from concurrent fecal and oral samples from healthy volunteers exposed to a brief course of a single antibiotic (cohort 1), acute leukemia patients (cohort 2), and stem cell transplant recipients (cohort 3). Cohorts 2 and 3 represent extreme clinical scenarios with respect to antibiotic pressure and severity of gut microbiota injury. RESULTS: While mild antibiotic exposure in cohort 1 was not sufficient for colonization of any oral bacteria in the gut, even with extreme antibiotic pressure and severe gut microbiota disruptions in cohorts 2 and 3, only one oral species in each cohort colonized the gut. CONCLUSIONS: Colonization resistance is dispensable for segregation of oral and colonic microbiota in humans. This finding implies that the presence of oral bacteria in the distal gut in diseases such as colon cancer and inflammatory bowel disease is not driven by impaired colonization resistance.

Exploitation of a Bacterium-Encoded Lytic Transglycosylase by a Human Oral Lytic Phage To Facilitate Infection.

Bacteriophages (phages) are an integral part of the human oral microbiome. Their roles in modulating bacterial physiology and shaping microbial communities have been discussed but remain understudied due to limited isolation and characterization of oral phage. Here, we report the isolation of LC001, a lytic phage targeting human oral Schaalia odontolytica (formerly known as Actinomyces odontolyticus) strain XH001. We showed that LC001 attached to and infected surface-grown, but not planktonic, XH001 cells, and it displayed remarkable host specificity at the strain level. Whole-genome sequencing of spontaneous LC001-resistant, surface-grown XH001 mutants revealed that the majority of the mutants carry nonsense or frameshift mutations in XH001 gene APY09_05145 (renamed ltg-1), which encodes a putative lytic transglycosylase (LT). The mutants are defective in LC001 binding, as revealed by direct visualization of the significantly reduced attachment of phage particles to the XH001 spontaneous mutants compared that to the wild type. Meanwhile, targeted deletion of ltg-1 produced a mutant that is defective in LC001 binding and resistant to LC001 infection even as surface-grown cells, while complementation of ltg-1 in the mutant background restored the LC001-sensitive phenotype. Intriguingly, similar expression levels of ltg-1 were observed in surface-grown and planktonic XH001, which displayed LC001-binding and nonbinding phenotypes, respectively. Furthermore, the overexpression of ltg-1 failed to confer an LC001-binding and -sensitive phenotype to planktonic XH001. Thus, our data suggested that rather than directly serving as a phage receptor, ltg-1-encoded LT may increase the accessibility of phage receptor, possibly via its enzymatic activity, by cleaving the peptidoglycan structure for better receptor exposure during peptidoglycan remodeling, a function that can be exploited by LC001 to facilitate infection. IMPORTANCE The evidence for the presence of a diverse and abundant phage population in the host-associated oral microbiome came largely from metagenomic analysis or the observation of virus-like particles within saliva/plaque samples, while the isolation of oral phage and investigation of their interaction with bacterial hosts are limited. Here, we report the isolation of LC001, the first lytic phage targeting oral Schaalia odontolytica. Our study suggested that LC001 may exploit the host bacterium-encoded lytic transglycosylase function to gain access to the receptor, thus facilitating its infection.

Transcriptome of Epibiont Saccharibacteria Nanosynbacter lyticus Strain TM7x During the Establishment of Symbiosis.

Saccharibacteria Nanosynbacter lyticus strain TM7x is a member of the broadly distributed candidate phylum radiation. These bacteria have ultrasmall cell sizes, have reduced genomes, and live as epibionts on the surfaces of other bacteria. The mechanisms by which they establish and maintain this relationship are not yet fully understood. The transcriptomes of the epibiont TM7x and its host bacteria Schaalia odontolytica strain XH001 were captured across the establishment of symbiosis during both the initial interaction and stable symbiosis. The results showed a dynamic interaction with large shifts in gene expression for both species between the initial encounter and stable symbiosis, notably in transporter genes. During stable symbiosis, the host XH001 showed higher gene expression for peptidoglycan biosynthesis, mannosylation, cell cycle and stress-related genes, whereas it showed lower expression of chromosomal partitioning genes. This was consistent with the elongated cell shape seen in XH001 infected with TM7x and our discovery that infection resulted in thickened cell walls. Within TM7x, increased pili, type IV effector genes, and arginine catabolism/biosynthesis gene expression during stable symbiosis implied a key role for these functions in the interaction. Consistent with its survival and persistence in the human microbiome as an obligate epibiont with reduced de novo biosynthetic capacities, TM7x also showed higher levels of energy production and peptidoglycan biosynthesis, but lower expression of stress-related genes, during stable symbiosis. These results imply that TM7x and its host bacteria keep a delicate balance in order to sustain an episymbiotic lifestyle. IMPORTANCE Nanosynbacter lyticus type strain TM7x is the first cultivated member of the Saccharibacteria and the candidate phyla radiation (CPR). It was discovered to be ultrasmall in cell size with a highly reduced genome that establishes an obligate epibiotic relationship with its host bacterium. The CPR is a large, monophyletic radiation of bacteria with reduced genomes that includes Saccharibacteria. The vast majority of the CPR have yet to be cultivated, and our insights into these unique organisms to date have been derived from only a few Saccharibacteria species. Being obligate parasites, it is unknown how these ultrasmall Saccharibacteria, which are missing many de novo biosynthetic pathways, are maintained at a high prevalence within the human microbiome as well as in the environment.

Draft Genome Sequences of Nine "Candidatus Nanosynbacter sp. HMT-352" Strains Cultured from the Human Oral Cavity.

Here, we report draft genome sequences for nine strains of "Candidatus Nanosynbacter sp. HMT-352." These strains and their sequences were used to interrogate strain-level variations in host range, gene content, and growth dynamics among the phylum "Candidatus Saccharibacteria."

Acquisition of the arginine deiminase system benefits epiparasitic Saccharibacteria and their host bacteria in a mammalian niche environment.

Saccharibacteria are a group of widespread and genetically diverse ultrasmall bacteria with highly reduced genomes that belong to the Candidate Phyla Radiation. Comparative genomic analyses suggest convergent evolution of key functions enabling the adaptation of environmental Saccharibacteria to mammalian microbiomes. Currently, our understanding of this environment-to-mammal niche transition within Saccharibacteria and their obligate episymbiotic association with host bacteria is limited. Here, we identified a complete arginine deiminase system (ADS), found in further genome streamlined mammal-associated Saccharibacteria but missing in their environmental counterparts, suggesting acquisition during environment-to-mammal niche transition. Using TM7x, the first cultured Saccharibacteria strain from the human oral microbiome and its host bacterium Actinomyces odontolyticus, we experimentally tested the function and impact of the ADS. We demonstrated that by catabolizing arginine and generating adenosine triphosphate, the ADS allows metabolically restrained TM7x to maintain higher viability and infectivity when disassociated from the host bacterium. Furthermore, the ADS protects TM7x and its host bacterium from acid stress, a condition frequently encountered within the human oral cavity due to bacterial metabolism of dietary carbohydrates. Intriguingly, with a restricted host range, TM7x forms obligate associations with Actinomyces spp. lacking the ADS but not those carrying the ADS, suggesting the acquired ADS may also contribute to partner selection for cooperative episymbiosis within a mammalian microbiome. These data present experimental characterization of a mutualistic interaction between TM7x and their host bacteria, and illustrate the benefits of acquiring a novel pathway in the transition of Saccharibacteria to mammalian microbiomes.

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.

Human variation in gingival inflammation.

Oral commensal bacteria actively participate with gingival tissue to maintain healthy neutrophil surveillance and normal tissue and bone turnover processes. Disruption of this homeostatic host-bacteria relationship occurs during experimental gingivitis studies where it has been clearly established that increases in the bacterial burden increase gingival inflammation. Here, we show that experimental gingivitis resulted in three unique clinical inflammatory phenotypes (high, low, and slow) and reveal that interleukin-1ß, a reported major gingivitis-associated inflammatory mediator, was not associated with clinical gingival inflammation in the slow response group. In addition, significantly higher levels of Streptococcus spp. were also unique to this group. The low clinical response group was characterized by low concentrations of host mediators, despite similar bacterial accumulation and compositional characteristics as the high clinical response group. Neutrophil and bone activation modulators were down-regulated in all response groups, revealing novel tissue and bone protective responses during gingival inflammation. These alterations in chemokine and microbial composition responses during experimental gingivitis reveal a previously uncharacterized variation in the human host response to a disruption in gingival homeostasis. Understanding this human variation in gingival inflammation may facilitate the identification of periodontitis-susceptible individuals. Overall, this study underscores the variability in host responses in the human population arising from variations in host immune profiles (low responders) and microbial community maturation (slow responders) that may impact clinical outcomes in terms of destructive inflammation.

Clinically Healthy Human Gingival Tissues Show Significant Inter-individual Variability in GCF Chemokine Expression and Subgingival Plaque Microbial Composition.

Aim: Clinically healthy gingival tissue is maintained through controlled regulation of host defense mechanisms against plaque biofilm overgrowth. One key component is the transit of neutrophils from the vasculature into gingival tissue where the expression of different neutrophil chemokines are tightly regulated. This cross-sectional study examines the inter-individual variability in chemokine profiles within gingival crevicular fluid (GCF) in relation to the subgingival bacterial community in a state of gingival health. Methods: Gingival crevicular fluid and subgingival plaque samples were collected from mesiobuccal surfaces of all six Ramfjord teeth of 20 systemically healthy individuals (14.55 ± 1.67 years). A multiplex immunoassay was carried out to quantify the expression of 40 different chemokines in the healthy gingival tissue. Neutrophils were assessed indirectly by myeloperoxidase (MPO) in GCF using traditional ELISA. Characterization of healthy subgingival plaque was conducted with the Illumina Miseq targeting the 16S rRNA gene. Results: In health, there are distinct variations within individual gingival crevicular fluid chemokine expression profiles, as well as in the concentration of neutrophils, that divided the participants into high or low chemokine expressing groups. Specifically, key differences were identified within MIF (2683.54 ± 985.82 pg per 30-s sample), IL-8/CXCL8 (170.98 ± 176.96 pg per 30-s sample), Gro-α/CXCL1 (160.42 ± 94.21 pg per 30-s sample), ENA-78/CXCL5 (137.76 ± 76.02 pg per 30-s sample), IL-1ß (51.39 ± 37.23 pg per 30-s sample), TNF-α (1.76 ± 1.79 pg per 30-s sample), and IFN-γ (0.92 ± 0.54 pg per 30-s sample). Of these identified chemokines, the highest correlation was associated between IL-8/CXCL8 and neutrophils (r = 0.54, p = 0.014). Furthermore, species characterization of healthy subgingival plaque revealed significant inter-individual variability that identified two unique groups unrelated to the previously identified chemokine groups. Conclusion: The lack of concordance between the microbial composition and chemokine profile during health may be a reflection of the unique microbial composition of each individual coupled with variations within their host response, emphasizing the vast complexity of the defense mechanisms in place to maintain gingival health.

Modified SHI medium supports growth of a disease-state subgingival polymicrobial community in vitro.

Developing a laboratory model of oral polymicrobial communities is essential for in vitro studies of the transition from healthy to diseased oral plaque. SHI medium is an enriched growth medium capable of supporting in vitro biofilms with similar diversity to healthy supragingival inocula; however, this medium does not maintain the diversity of gram-negative bacteria more associated with subgingival plaque. Here, we systematically modified SHI medium components to investigate the impacts of varying nutrients and develop a medium capable of supporting a specific disease-state subgingival community. A diseased subgingival plaque sample was inoculated in SHI medium with increasing concentrations of sucrose (0%, 0.1%, 0.5%), fetal bovine serum (FBS) (0%, 10%, 20%, 30%, 50%), and mucin (0.1, 2.5, 8.0 g/L) and grown for 48 hrs, then the 16S rRNA profiles of the resulting biofilms were examined. In total, these conditions were able to capture 89 of the 119 species and 43 of the 51 genera found in the subgingival inoculum. Interestingly, biofilms grown in high sucrose media, although dominated by acidogenic Firmicutes with a low final pH, contained several uncultured taxa from the genus Treponema, information that may aid culturing these periodontitis-associated fastidious organisms. Biofilms grown in a modified medium (here named subSHI-v1 medium) with 0.1% sucrose and 10% FBS had a high diversity closest to the inoculum and maintained greater proportions of many gram-negative species of interest from the subgingival periodontal pocket (including members of the genera Prevotella and Treponema, and the Candidate Phyla Radiation phylum Saccharibacteria), and therefore best represented the disease community.