A Longitudinal Metagenomic Comparative Analysis of Oral Microbiome Shifts in Patients Receiving Proton Radiation versus Photon Radiation for Head and Neck Cancer.

Introduction: Due to the radiation-sparing effects on salivary gland acini, changes in the composition of the oral microbiome may be a driver for improved outcomes in patients receiving proton radiation, with potentially worse outcomes in patients exposed to photon radiation therapy. To date, a head-to-head comparison of oral microbiome changes at a metagenomic level with longitudinal sampling has yet to be performed in these patient cohorts. Methods and Materials: To comparatively analyze oral microbiome shifts during head and neck radiation therapy, a prospective pilot cohort study was performed at the Maryland Proton Treatment Center and the University of Maryland Marlene and Stewart Greenebaum Comprehensive Cancer Center. A longitudinal metagenomic comparative analysis of oral microbiome shifts was performed at three time points (pre-radiation, during radiation, and immediately post-radiation). Head and neck cancer patients receiving proton radiation (n = 4) were compared to photon radiation (n = 4). Additional control groups included healthy age- and sex-matched controls (n = 5), head and neck cancer patients who never received radiation therapy (n = 8), and patients with oral inflammatory disease (n = 3). Results: Photon therapy patients presented with lower microbial alpha diversity at all timepoints, and there was a trend towards reduced species richness as compared with proton therapy. Healthy controls and proton patients exhibited overall higher and similar diversity. A more dysbiotic state was observed in patients receiving photon therapy as compared to proton therapy, in which oral microbial homeostasis was maintained. Mucositis was observed in 3/4 photon patients and was not observed in any proton patients during radiation therapy. The bacterial de novo pyrimidine biosynthesis pathway and the nitrate reduction V pathway were comparatively higher following photon exposure. These functional changes in bacterial metabolism may suggest that photon exposure produces a more permissive environment for the proliferation of pathogenic bacteria. Conclusion: Oral microbiome dysbiosis in patients receiving photon radiation may be associated with increased mucositis occurrence. Proton radiation therapy for head and neck cancer demonstrates a safer side effect profile in terms of oral complications, oral microbiome dysbiosis, and functional metabolic status.

Genomic Confirmation of Borrelia garinii, United States.

Lyme disease is a multisystem disorder primarily caused by Borrelia burgdorferi sensu lato. However, B. garinii, which has been identified on islands off the coast of Newfoundland and Labrador, Canada, is a cause of Lyme disease in Eurasia. We report isolation and whole-genome nucleotide sequencing of a B. garinii isolate from a cotton mouse (Peromyscus gossypinus) in South Carolina, USA. We identified a second B. garinii isolate from the same repository. Phylogenetic analysis does not associate these isolates with the previously described isolates of B. garinii from Canada.

Genomic hybrid capture assay to detect Borrelia burgdorferi: an application to diagnose neuroborreliosis in horses.

Antemortem diagnosis of neuroborreliosis in horses has been hindered by both the low sensitivity of PCR testing for Borrelia burgdorferi in CSF and the low specificity of serum:CSF ELISA ratios used to determine intrathecal antibody production against the bacterium. PCR testing of the CSF of an adult horse with acute neurologic disease for the B. burgdorferi flagellin gene was negative. However, we enriched B. burgdorferi DNA through nucleic acid hybrid capture, followed by next-generation sequencing, and identified B. burgdorferi in the CSF of the horse, confirming a diagnosis of neuroborreliosis.

The evolution of synaptic and cognitive capacity: Insights from the nervous system transcriptome of Aplysia.

The gastropod mollusk Aplysia is an important model for cellular and molecular neurobiological studies, particularly for investigations of molecular mechanisms of learning and memory. We developed an optimized assembly pipeline to generate an improved Aplysia nervous system transcriptome. This improved transcriptome enabled us to explore the evolution of cognitive capacity at the molecular level. Were there evolutionary expansions of neuronal genes between this relatively simple gastropod Aplysia (20,000 neurons) and Octopus (500 million neurons), the invertebrate with the most elaborate neuronal circuitry and greatest behavioral complexity? Are the tremendous advances in cognitive power in vertebrates explained by expansion of the synaptic proteome that resulted from multiple rounds of whole genome duplication in this clade? Overall, the complement of genes linked to neuronal function is similar between Octopus and Aplysia. As expected, a number of synaptic scaffold proteins have more isoforms in humans than in Aplysia or Octopus. However, several scaffold families present in mollusks and other protostomes are absent in vertebrates, including the Fifes, Lev10s, SOLs, and a NETO family. Thus, whereas vertebrates have more scaffold isoforms from select families, invertebrates have additional scaffold protein families not found in vertebrates. This analysis provides insights into the evolution of the synaptic proteome. Both synaptic proteins and synaptic plasticity evolved gradually, yet the last deuterostome-protostome common ancestor already possessed an elaborate suite of genes associated with synaptic function, and critical for synaptic plasticity.

Dynamics of the infant gut microbiota in the first 18 months of life: the impact of maternal HIV infection and breastfeeding.

BACKGROUND: Access to antiretroviral therapy (ART) during pregnancy and breastfeeding for mothers with HIV has resulted in fewer children acquiring HIV peri- and postnatally, resulting in an increase in the number of children who are exposed to the virus but are not infected (HEU). HEU infants have an increased likelihood of childhood infections and adverse growth outcomes, as well as increased mortality compared to their HIV-unexposed (HUU) peers. We explored potential differences in the gut microbiota in a cohort of 272 Nigerian infants born to HIV-positive and negative mothers in this study during the first 18 months of life. RESULTS: The taxonomic composition of the maternal vaginal and gut microbiota showed no significant differences based on HIV status, and the composition of the infant gut microbiota at birth was similar between HUU and HEU. Longitudinal taxonomic composition of the infant gut microbiota and weight-for-age z-scores (WAZ) differed depending on access to breast milk. HEU infants displayed overall lower WAZ than HUU infants at all time points. We observed a significantly lower relative abundance of Bifidobacterium in HEU infants at 6 months postpartum. Breast milk composition also differed by time point and HIV infection status. The antiretroviral therapy drugs, lamivudine and nevirapine, as well as kynurenine, were significantly more abundant in the breast milk of mothers with HIV. Levels of tiglyl carnitine (C5) were significantly lower in the breast milk of mothers without HIV. ART drugs in the breast milk of mothers with HIV were associated with a lower relative abundance of Bifidobacterium longum. CONCLUSIONS: Maternal HIV infection was associated with adverse growth outcomes of HEU infants in this study, and these differences persist from birth through at least 18 months, which is a critical window for the development of the immune and central nervous systems. We observed that the relative abundance of Bifidobacterium spp. was significantly lower in the gut microbiota of all HEU infants over the first 6 months postpartum, even if HEU infants were receiving breast milk. Breastfeeding was of benefit in our HEU infant cohort in the first weeks postpartum; however, ART drug metabolites in breast milk were associated with a lower abundance of Bifidobacterium. Video abstract.

Gut microbiome and metabolome in a non-human primate model of chronic excessive alcohol drinking.

A relationship between the gut microbiome and alcohol use disorder has been suggested. Excessive alcohol use produces changes in the fecal microbiome and metabolome in both rodents and humans. Yet, these changes can be observed only in a subgroup of the studied populations, and reversal does not always occur after abstinence. We aimed to analyze fecal microbial composition and function in a translationally relevant baboon model of chronic heavy drinking that also meets binge criteria (drinking too much, too fast, and too often), i.e., alcohol ~1 g/kg and blood alcohol levels (BALs) ≥ 0.08 g/dL in a 2-hour period, daily, for years. We compared three groups of male baboons (Papio anubis): L = Long-term alcohol drinking group (12.1 years); S = Short-term alcohol drinking group (2.7 years); and C = Control group, drinking a non-alcoholic reinforcer (Tang®) (8.2 years). Fecal collection took place during 3 days of Drinking (D), followed by a short period (3 days) of Abstinence (A). Fecal microbial alpha- and beta-diversity were significantly lower in L vs. S and C (p's < 0.05). Members of the commensal families Lachnospiraceae and Prevotellaceae showed a relative decrease, whereas the opportunistic pathogen Streptococcus genus showed a relative increase in L vs. S and C (p's < 0.05). Microbiota-related metabolites of aromatic amino acids, tricarboxylic acid cycle, and pentose increased in L vs. S and C (FDR-corrected p < 0.01), with the latter two suggesting high energy metabolism and enhanced glycolysis in the gut lumen in response to alcohol. Consistent with the long-term alcohol exposure, mucosal damage and oxidative stress markers (N-acetylated amino acids, 2-hydroxybutyrate, and metabolites of the methionine cycle) increased in L vs. S and C (FDR-corrected p < 0.01). Overall, S showed few differences vs. C, possibly due to the long-term, chronic alcohol exposure needed to alter the normal gut microbiota. In the three groups, the fecal microbiome barely differed between conditions D and A, whereas the metabolome shifted in the transition from condition D to A. In conclusion, changes in the fecal microbiome and metabolome occur after significant long-term excessive drinking and are only partially affected by acute forced abstinence from alcohol. These results provide novel information on the relationship between the fecal microbiome and metabolome in a controlled experimental setting and using a unique non-human primate model of chronic excessive alcohol drinking.

Bifidobacterium animalis subsp. lactis BB-12 Protects against Antibiotic-Induced Functional and Compositional Changes in Human Fecal Microbiome.

The administration of broad-spectrum antibiotics is often associated with antibiotic-associated diarrhea (AAD), and impacts gastrointestinal tract homeostasis, as evidenced by the following: (a) an overall reduction in both the numbers and diversity of the gut microbiota, and (b) decreased short-chain fatty acid (SCFA) production. Evidence in humans that probiotics may enhance the recovery of microbiota populations after antibiotic treatment is equivocal, and few studies have addressed if probiotics improve the recovery of microbial metabolic function. Our aim was to determine if Bifidobacterium animalis subsp. lactis BB-12 (BB-12)-containing yogurt could protect against antibiotic-induced fecal SCFA and microbiota composition disruptions. We conducted a randomized, allocation-concealed, controlled trial of amoxicillin/clavulanate administration (days 1-7), in conjunction with either BB-12-containing or control yogurt (days 1-14). We measured the fecal levels of SCFAs and bacterial composition at baseline and days 7, 14, 21, and 30. Forty-two participants were randomly assigned to the BB-12 group, and 20 participants to the control group. Antibiotic treatment suppressed the fecal acetate levels in both the control and probiotic groups. Following the cessation of antibiotics, the fecal acetate levels in the probiotic group increased over the remainder of the study and returned to the baseline levels on day 30 (-1.6% baseline), whereas, in the control group, the acetate levels remained suppressed. Further, antibiotic treatment reduced the Shannon diversity of the gut microbiota, for all the study participants at day 7. The magnitude of this change was larger and more sustained in the control group compared to the probiotic group, which is consistent with the hypothesis that BB-12 enhanced microbiota recovery. There were no significant baseline clinical differences between the two groups. Concurrent administration of amoxicillin/clavulanate and BB-12 yogurt, to healthy subjects, was associated with a significantly smaller decrease in the fecal SCFA levels and a more stable taxonomic profile of the microbiota over time than the control group.

Differences in the Platelet mRNA Landscape Portend Racial Disparities in Platelet Function and Suggest Novel Therapeutic Targets.

The African American (AA) population displays a 1.6 to 3-fold higher incidence of thrombosis and stroke mortality compared with European Americans (EAs). Current antiplatelet therapies target the ADP-mediated signaling pathway, which displays significant pharmacogenetic variation for platelet reactivity. The focus of this study was to define underlying population differences in platelet function in an effort to identify novel molecular targets for future antiplatelet therapy. We performed deep coverage RNA-Seq to compare gene expression levels in platelets derived from a cohort of healthy volunteers defined by ancestry determination. We identified > 13,000 expressed platelet genes of which 480 were significantly differentially expressed genes (DEGs) between AAs and EAs. DEGs encoding proteins known or predicted to modulate platelet aggregation, morphology, or platelet count were upregulated in AA platelets. Numerous G-protein coupled receptors, ion channels, and pro-inflammatory cytokines not previously associated with platelet function were likewise differentially expressed. Many of the signaling proteins represent potential pharmacologic targets of intervention. Notably, we confirmed the differential expression of cytokines IL32 and PROK2 in an independent cohort by quantitative real-time polymerase chain reaction, and provide functional validation of the opposing actions of these two cytokines on collagen-induced AA platelet aggregation. Using Genotype-Tissue Expression whole blood data, we identified 516 expression quantitative trait locuses with Fst values > 0.25, suggesting that population-differentiated alleles may contribute to differences in gene expression. This study identifies gene expression differences at the population level that may affect platelet function and serve as potential biomarkers to identify cardiovascular disease risk. Additionally, our analysis uncovers candidate novel druggable targets for future antiplatelet therapies.

Changes in the Gut Microbiota Following Bariatric Surgery Are Associated with Increased Alcohol Intake in a Female Rat Model.

AIMS: We aimed to investigate if differences in gut microbiota diversity and composition are associated with post-operative alcohol intake following bariatric surgery in a rat model. METHODS: Twenty-four female rats were randomized to three treatment groups: sham surgery, vertical sleeve gastrectomy (VSG) or Roux-en-Y gastric bypass (RYGB). Stool was collected pre- and post-operatively and 16S rRNA gene amplification and sequencing was performed. Analysis focused on correlating microbial diversity, type of surgery and alcohol (EtOH) intake. RESULTS: Pre-operative stools samples on regular diet showed similar taxonomic composition and Shannon diversity among the three treatment groups. There was a significant decrease in Shannon diversity and a change in taxonomic composition of the gut microbiota after rats was fed high fat diet. Post-operatively, the RYGB group showed significantly lower taxonomic diversity than the VSG and sham groups, while the VSG and sham groups diversity were not significantly different. Taxonomic composition and function prediction based on PICRUSt analysis showed the RYGB group to be distinct from the VSG and sham groups. Shannon diversity was found to be negatively associated with EtOH intake. CONCLUSIONS: Changes in the taxonomic profile of the gut microbiota following bariatric surgery, particularly RYGB, are associated with increased EtOH intake and may contribute to increased alcohol use disorder risk through the gut-brain-microbiome axis.

Nasal Microbiota and Infectious Complications After Elective Surgical Procedures.

Importance: The association of the nasal microbiome with outcomes in surgical patients is poorly understood. Objective: To characterize the composition of nasal microbiota in patients undergoing clean elective surgical procedures and to examine the association between characteristics of preoperative nasal microbiota and occurrence of postoperative infection. Design, Setting, and Participants: Using a nested matched case-control design, 53 individuals who developed postoperative infection were matched (approximately 3:1 by age, sex, and surgical procedure) with 144 individuals who were not infected (ie, the control group). The 2 groups were selected from a prospective cohort of patients undergoing surgical procedures at 2 tertiary care university hospitals in Baltimore, Maryland, who were at high risk for postoperative infectious complications. Included individuals were aged 40 years or older; had no history of autoimmune disease, immunocompromised state, immune-modulating medication, or active infection; and were scheduled to undergo elective cardiac, vascular, spinal, or intracranial surgical procedure. Data were analyzed from October 2015 through September 2020. Exposures: Nasal microbiome cluster class served as the main exposure. An unsupervised clustering method (ie, grades of membership modeling) was used to classify nasal microbial samples into 2 groups based on features derived from 16S ribosomal RNA gene sequencing. The microbiome cluster groups were derived independently and agnostic of baseline clinical characteristics and infection status. Main Outcomes and Measures: Composite of surgical site infection, bacteremia, and pneumonia occurring within 6 months after surgical procedure. Results: Among 197 participants (mean [SD] age, 64.1 [10.6] years; 63 [37.7%] women), 553 bacterial taxa were identified from preoperative nasal swab samples. A 2-cluster model (with 167 patients in cluster 1 and 30 patients in cluster 2) accounted for the largest proportion of variance in microbial profiles using grades of membership modeling and was most parsimonious. After adjusting for potential confounders, the probability of assignment to cluster 2 was associated with 6-fold higher odds of infection after surgical procedure (odds ratio [OR], 6.18; 95% CI, 3.33-11.7; P < .001) independent of baseline clinical characteristics, including nasal carriage of Staphylococcus aureus. Intrasample (ie, α) diversity was inversely associated with infectious outcome in both clusters (OR, 0.57; 95% CI, 0.42-0.75; P < .001); however, probability of assignment to cluster 2 was associated with higher odds of infection independent of α diversity (OR, 4.61; 95% CI, 2.78-7.86; P < .001). Conclusions and Relevance: These findings suggest that the nasal microbiome was an independent risk factor associated with infectious outcomes among individuals who underwent elective surgical procedures and may serve as a biomarker associated with infection susceptibility in this population.

FADU: a Quantification Tool for Prokaryotic Transcriptomic Analyses.

Quantification tools for RNA sequencing (RNA-Seq) analyses are often designed and tested using human transcriptomics data sets, in which full-length transcript sequences are well annotated. For prokaryotic transcriptomics experiments, full-length transcript sequences are seldom known, and coding sequences must instead be used for quantification steps in RNA-Seq analyses. However, operons confound accurate quantification of coding sequences since a single transcript does not necessarily equate to a single gene. Here, we introduce FADU (Feature Aggregate Depth Utility), a quantification tool designed specifically for prokaryotic RNA-Seq analyses. FADU assigns partial count values proportional to the length of the fragment overlapping the target feature. To assess the ability of FADU to quantify genes in prokaryotic transcriptomics analyses, we compared its performance to those of eXpress, featureCounts, HTSeq, kallisto, and Salmon across three paired-end read data sets of (i) Ehrlichia chaffeensis, (ii) Escherichia coli, and (iii) the Wolbachia endosymbiont wBm. Across each of the three data sets, we find that FADU can more accurately quantify operonic genes by deriving proportional counts for multigene fragments within operons. FADU is available at https://github.com/IGS/FADUIMPORTANCE Most currently available quantification tools for transcriptomics analyses have been designed for human data sets, in which full-length transcript sequences, including the untranslated regions, are well annotated. In most prokaryotic systems, full-length transcript sequences have yet to be characterized, leading to prokaryotic transcriptomics analyses being performed based on only the coding sequences. In contrast to eukaryotes, prokaryotes contain polycistronic transcripts, and when genes are quantified based on coding sequences instead of transcript sequences, this leads to an increased abundance of improperly assigned ambiguous multigene fragments, specifically those mapping to multiple genes in operons. Here, we describe FADU, a quantification tool for prokaryotic RNA-Seq analyses designed to assign proportional counts with the purpose of better quantifying operonic genes while minimizing the pitfalls associated with improperly assigning fragment counts from ambiguous transcripts.

Molecular mechanisms of probiotic prevention of antibiotic-associated diarrhea.

Antibiotic-associated diarrhea (AAD) is a common and unintended adverse effect of antibiotic treatment. It is characterized by the disruption of the gut microbiota, decreased intestinal short chain fatty acid (SCFA) concentrations, accumulation of luminal carbohydrates and colonic bile acids, altered water absorption, and ultimately diarrhea. Probiotics were shown to prevent AAD in numerous clinical trials. This review examines what is currently known about how probiotics reduce the risk for AAD via modulating the gut microbiota, altering nutrient and bile acid metabolism, inducing epithelial solute transporter activity, supporting intestinal barrier function, and influencing the immune system. Although probiotics are frequently prescribed with antibiotic use, mechanistic evidence verifying how they confer protection against AAD is extremely limited. This information is urgently needed for improving recommendations for sustaining probiotic development and for implementing probiotics in clinical settings.

Transcriptional dysregulation in developing trigeminal sensory neurons in the LgDel mouse model of DiGeorge 22q11.2 deletion syndrome.

LgDel mice, which model the heterozygous deletion of genes at human chromosome 22q11.2 associated with DiGeorge/22q11.2 deletion syndrome (22q11DS), have cranial nerve and craniofacial dysfunction as well as disrupted suckling, feeding and swallowing, similar to key 22q11DS phenotypes. Divergent trigeminal nerve (CN V) differentiation and altered trigeminal ganglion (CNgV) cellular composition prefigure these disruptions in LgDel embryos. We therefore asked whether a distinct transcriptional state in a specific population of early differentiating LgDel cranial sensory neurons, those in CNgV, a major source of innervation for appropriate oropharyngeal function, underlies this departure from typical development. LgDel versus wild-type (WT) CNgV transcriptomes differ significantly at E10.5 just after the ganglion has coalesced. Some changes parallel altered proportions of cranial placode versus cranial neural crest-derived CNgV cells. Others are consistent with a shift in anterior-posterior patterning associated with divergent LgDel cranial nerve differentiation. The most robust quantitative distinction, however, is statistically verifiable increased variability of expression levels for most of the over 17 000 genes expressed in common in LgDel versus WT CNgV. Thus, quantitative expression changes of functionally relevant genes and increased stochastic variation across the entire CNgV transcriptome at the onset of CN V differentiation prefigure subsequent disruption of cranial nerve differentiation and oropharyngeal function in LgDel mice.

Strains used in whole organism Plasmodium falciparum vaccine trials differ in genome structure, sequence, and immunogenic potential.

BACKGROUND: Plasmodium falciparum (Pf) whole-organism sporozoite vaccines have been shown to provide significant protection against controlled human malaria infection (CHMI) in clinical trials. Initial CHMI studies showed significantly higher durable protection against homologous than heterologous strains, suggesting the presence of strain-specific vaccine-induced protection. However, interpretation of these results and understanding of their relevance to vaccine efficacy have been hampered by the lack of knowledge on genetic differences between vaccine and CHMI strains, and how these strains are related to parasites in malaria endemic regions. METHODS: Whole genome sequencing using long-read (Pacific Biosciences) and short-read (Illumina) sequencing platforms was conducted to generate de novo genome assemblies for the vaccine strain, NF54, and for strains used in heterologous CHMI (7G8 from Brazil, NF166.C8 from Guinea, and NF135.C10 from Cambodia). The assemblies were used to characterize sequences in each strain relative to the reference 3D7 (a clone of NF54) genome. Strains were compared to each other and to a collection of clinical isolates (sequenced as part of this study or from public repositories) from South America, sub-Saharan Africa, and Southeast Asia. RESULTS: While few variants were detected between 3D7 and NF54, we identified tens of thousands of variants between NF54 and the three heterologous strains. These variants include SNPs, indels, and small structural variants that fall in regulatory and immunologically important regions, including transcription factors (such as PfAP2-L and PfAP2-G) and pre-erythrocytic antigens that may be key for sporozoite vaccine-induced protection. Additionally, these variants directly contributed to diversity in immunologically important regions of the genomes as detected through in silico CD8+ T cell epitope predictions. Of all heterologous strains, NF135.C10 had the highest number of unique predicted epitope sequences when compared to NF54. Comparison to global clinical isolates revealed that these four strains are representative of their geographic origin despite long-term culture adaptation; of note, NF135.C10 is from an admixed population, and not part of recently formed subpopulations resistant to artemisinin-based therapies present in the Greater Mekong Sub-region. CONCLUSIONS: These results will assist in the interpretation of vaccine efficacy of whole-organism vaccines against homologous and heterologous CHMI.

Cost effective, experimentally robust differential-expression analysis for human/mammalian, pathogen and dual-species transcriptomics.

As sequencing read length has increased, researchers have quickly adopted longer reads for their experiments. Here, we examine 14 pathogen or host-pathogen differential gene expression data sets to assess whether using longer reads is warranted. A variety of data sets was used to assess what genomic attributes might affect the outcome of differential gene expression analysis including: gene density, operons, gene length, number of introns/exons and intron length. No genome attribute was found to influence the data in principal components analysis, hierarchical clustering with bootstrap support, or regression analyses of pairwise comparisons that were undertaken on the same reads, looking at all combinations of paired and unpaired reads trimmed to 36, 54, 72 and 101 bp. Read pairing had the greatest effect when there was little variation in the samples from different conditions or in their replicates (e.g. little differential gene expression). But overall, 54 and 72 bp reads were typically most similar. Given differences in costs and mapping percentages, we recommend 54 bp reads for organisms with no or few introns and 72 bp reads for all others. In a third of the data sets, read pairing had absolutely no effect, despite paired reads having twice as much data. Therefore, single-end reads seem robust for differential-expression analyses, but in eukaryotes paired-end reads are likely desired to analyse splice variants and should be preferred for data sets that are acquired with the intent to be community resources that might be used in secondary data analyses.

Establishing What Constitutes a Healthy Human Gut Microbiome: State of the Science, Regulatory Considerations, and Future Directions.

On December 17, 2018, the North American branch of the International Life Sciences Institute (ILSI North America) convened a workshop "Can We Begin to Define a Healthy Gut Microbiome Through Quantifiable Characteristics?" with >40 invited academic, government, and industry experts in Washington, DC. The workshop objectives were to 1) develop a collective expert assessment of the state of the evidence on the human gut microbiome and associated human health benefits, 2) see if there was sufficient evidence to establish measurable gut microbiome characteristics that could serve as indicators of "health," 3) identify short- and long-term research needs to fully characterize healthy gut microbiome-host relationships, and 4) publish the findings. Conclusions were as follows: 1) mechanistic links of specific changes in gut microbiome structure with function or markers of human health are not yet established; 2) it is not established if dysbiosis is a cause, consequence, or both of changes in human gut epithelial function and disease; 3) microbiome communities are highly individualized, show a high degree of interindividual variation to perturbation, and tend to be stable over years; 4) the complexity of microbiome-host interactions requires a comprehensive, multidisciplinary research agenda to elucidate relationships between gut microbiome and host health; 5) biomarkers and/or surrogate indicators of host function and pathogenic processes based on the microbiome need to be determined and validated, along with normal ranges, using approaches similar to those used to establish biomarkers and/or surrogate indicators based on host metabolic phenotypes; 6) future studies measuring responses to an exposure or intervention need to combine validated microbiome-related biomarkers and/or surrogate indicators with multiomics characterization of the microbiome; and 7) because static genetic sampling misses important short- and long-term microbiome-related dynamic changes to host health, future studies must be powered to account for inter- and intraindividual variation and should use repeated measures within individuals.