(C) PLOS One This story was originally published by PLOS One and is unaltered. . . . . . . . . . . Individuality and ethnicity eclipse a short-term dietary intervention in shaping microbiomes and viromes [1] ['Junhui Li', 'Vanderbilt Microbiome Innovation Center', 'Vanderbilt University', 'Nashville', 'Tennessee', 'United States Of America', 'Department Of Biological Sciences', 'Robert H. George Markowitz', 'Andrew W. Brooks', 'Vanderbilt Genetics Institute'] Date: 2022-08 Many diseases linked with ethnic health disparities associate with changes in microbial communities in the United States, but the causes and persistence of ethnicity-associated microbiome variation are not understood. For instance, microbiome studies that strictly control for diet across ethnically diverse populations are lacking. Here, we performed multiomic profiling over a 9-day period that included a 4-day controlled vegetarian diet intervention in a defined geographic location across 36 healthy Black and White females of similar age, weight, habitual diets, and health status. We demonstrate that individuality and ethnicity account for roughly 70% to 88% and 2% to 10% of taxonomic variation, respectively, eclipsing the effects a short-term diet intervention in shaping gut and oral microbiomes and gut viromes. Persistent variation between ethnicities occurs for microbial and viral taxa and various metagenomic functions, including several gut KEGG orthologs, oral carbohydrate active enzyme categories, cluster of orthologous groups of proteins, and antibiotic-resistant gene categories. In contrast to the gut and oral microbiome data, the urine and plasma metabolites tend to decouple from ethnicity and more strongly associate with diet. These longitudinal, multiomic profiles paired with a dietary intervention illuminate previously unrecognized associations of ethnicity with metagenomic and viromic features across body sites and cohorts within a single geographic location, highlighting the importance of accounting for human microbiome variation in research, health determinants, and eventual therapies. Funding: This research was supported by the Vanderbilt Microbiome Innovation Center to SRB, JFF, and HJS and funding from American Heart Association 15SDG24890015 to JFF. Core services for metagenomic sequencing at Vanderbilt were supported by scholarships from Vanderbilt University Medical Center's Digestive Disease Research Center (NIH P30DK058404 to RMP) and the Vanderbilt-Ingram Cancer Center (NIH P30CA08485 to JAP). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Data Availability: All shotgun metagenomic sequences were deposited in the NCBI under the BioProject PRJNA662107. Code for all analyses can be found at https://github.com/BordensteinLaboratory/VMI-diet-challenge (10.5281/zenodo.6643667). Metabolomic data were deposited in Metabolomics WB under the Project PR001282 (10.21228/M80Q51). Participant metadata used for analyses is available on request from the Vanderbilt University Medical Center Human Research Protections Program (615-322-2918), as public sharing of this REDCap data is restricted by the approved study IRB protocol and participant informed consent documents. Controlled studies that limit variation in intrinsic and extrinsic factors are required to associate and disentangle factors such as diet, sex, age, differential exposure to social and environmental determinants of health, and medical history in multiethnic cohorts. At the same time, combining multiomic approaches will help differentiate underlying scales of biology (e.g., microbiome, virome, metabolome) that impact ethnicity-associated variation and health outcomes. The Vanderbilt Microbiome Innovation Center (VMIC) coordinated a human clinical trial to examine whether ethnicity-associated, multiomic variation persists in gut and oral microbiomes (microbial metagenomics), gut viromes (viral metagenomics), and blood and urine metabolites (metabolomics) during a controlled, short-term, dietary intervention. This study details the results. Moreover, multiomic analyses combining metagenomics and metabolomics can provide refined insights into taxonomy, functional potential, and metabolites used or produced by microorganisms that impact health, as in the microbial generation of trimethylamine N-oxide from red meat, which contributes to cardiovascular disease [ 37 – 39 ]. Technological advances and more established reference databases also allow researchers to probe microbiomes in increasingly holistic ways by including metagenomic sequencing of purifed viral particles such as bacteriophages (viruses of bacteria; i.e., phages). Phages often outnumber gut bacteria in this environment, horizontally transfer DNA [ 40 ], and restructure microbiomes during lytic events that can occur in response to diet [ 41 – 43 ] and inflammation [ 40 , 44 ]. Ethnicity, encompassing social, environmental, geographic, and cultural variation as well as differential exposure to social and structural discrimination, is a major defining factor of health disparity incidence [ 29 – 34 ]. Diseases associated with microbiomes are also often linked with health disparities across different ethnicities in the United States, such as inflammatory bowel disease, lung cancer, and colorectal cancer [ 31 , 33 , 35 , 36 ]. A key question then is whether or not ethnicity-associated microbiome variation at the metagenomic and viromic levels is reproducible across studies and persistent in healthy participants even when diets are the same. Results may in turn lead to consideration of research frameworks and interventions that are more inclusive and actively attentive to ethnic health disparities. Self-identified race and ethnicity, hereafter referred to as ethnicity, capture aspects of social, cultural, economic, geographic, and historical identity. Ethnicity is not a biological category, but rather a social construct that serves as a proxy for differences in multiple intersecting environmental and social factors and their associated structural drivers, such as racism [ 10 – 13 ]. While observational studies reproducibly link ethnicity with gut [ 14 – 18 ], oral [ 19 , 20 ], and vaginal [ 21 – 23 ] microbiome variation in populations within and between countries, the specific factors underlying these ethnicity associations are not clear. Diet is one of the most widely considered factors shaping gut microbiomes with emerging mainstream appeal [ 24 – 28 ]. While dietary differences can associate with ethnicity, the complexity of factors intertwining ethnicity and dietary regimes indicate observational studies alone cannot confidently disentangle their relative roles in shaping the microbiome, and our previous analysis of dietary metadata in 16S rRNA gene amplicon studies suggested relatively little association between diet and ethnicity-associated microbiome variation in American guts (stool samples) [ 17 ]. Notably, microbiome studies that strictly control for diet across ethnically diverse populations are lacking. Thus, we understand little about which extrinsic or intrinsic factors lead to and regulate this variation between ethnicities, and we do not yet understand how the variation within a person over time, or between different people, influences predispositions to wellness and disease. Composed of trillions of microbial cells and thousands of species, the human microbiome can substantially impact many aspects of human physiology and contribute to chronic diseases underlying health disparities [ 1 – 5 ]. The current drive toward clinical microbiome studies and personalized medicine is, however, hampered by a lack of understanding of the complex social, cultural, and economic causes that contribute to interpersonal differences in microbiome compositions and proportions of specific microbes. Many intrinsic (e.g., age, sex, ancestry) and extrinsic (e.g., lifestyle, diet) factors can associate with microbiome variation, but covariation between factors often confounds their relative importance when studying human populations. For example, investigations statistically disentangle influential factors in large observational studies that are powered to correct for multiple variables at once [ 4 , 6 – 9 ], but factors such as diet, genetics, geography, and social identities, such as race and ethnicity, often covary in complex, underlying ways that cannot be overcome by increasing sample size alone. Results Ethnicity associations are common and persistent for abundant microbial taxa and phages A total of 219 abundant taxa in cohort 1 and 182 abundant taxa in cohort 2 at or above the genus level are detected at >1% relative abundance in at least one of the gut or oral microbial metagenomes. The phylogenetic relationships of all abundant taxa are shown in Fig 4. Overall, the abundant taxa in these 2 body sites are compositionally different, as expected. Within each of the sites, the relative abundance of highly abundant taxa correlates positively with the relative abundance of that same taxa in the other site for both saliva (r = 0.63) and gut taxa (0.58, P < 0.0001, Spearman, S3 Fig), respectively. Among the 219 abundant taxa in cohort 1, a striking 49.3% (108/219) in the gut and 15.5% (34/219) in the oral microbial metagenomes vary in abundance between ethnicities (P FDR < 0.05, LinDA, Fig 4A). Similarly, in cohort 2, 49.5% (90/182) of taxa in the gut vary in abundance between ethnicities (P FDR < 0.05, LinDA, Fig 4B and S1 Data); oral taxa from cohort 2 are not differentially abundant in Black or White individuals at the same cutoff (P FDR < 0.05, LinDA, Fig 4B). While a similar number of taxa were differentially abundant between ethnicities in cohort 1 and cohort 2, only 17 taxa both replicated across cohorts and were differentially abundant in the same direction (Actinomyces oris, G_Actinomyces, Bacteroides stercoris, Bacteroides vulgatus, Eubacterium siraeum, Haemophilus parainfluenzae, Oscillibacter sp., Oscillibacter sp. CAG_241, Ruminococcaceae bacterium, Streptococcus oralis, Streptococcus parasanguinis, Streptococcus salivarius, G_Streptococcus, Veillonella atypica, Veillonella dispar, G_Veillonella, and G_Haemophilus). Of these 17 replicating taxa, 15 were more abundant in Black individuals, and 2 Bacteroides taxa were more abundant in White individuals (Fig 4). An additional 14 taxa were identified as differentially abundant in both cohorts, but the directionality of the difference was reversed in cohort 2 compared with cohort 1. Smaller proportions of species were differentially abundant between ethnicities in the assembly-free analysis for fecal samples (cohort 1: 29.7% (97/338); cohort 2: 10.6% (31/292); S7 Data). We previously showed that gut taxa also varied by ethnicity in analyses of the American Gut Project (16.2%) and the Human Microbiome Project (20.6%) [17]. PPT PowerPoint slide PNG larger image TIFF original image Download: Fig 4. Ethnicity-associated variation in abundant microbial taxa and phage. The 219 taxa in cohort 1 (A) and 182 taxa in cohort 2 (B) included in the phylogeny are present in all participants and have relative abundances of >1% in at least one of the gut or oral microbial metagenomes. The inner circle indicates differential taxa in the gut between the 2 ethnicities; the outer circle indicates differential taxa in the saliva between the 2 ethnicities. Pink or blue color gradients indicate FDR-adjusted p-value of significance (LinDA function in MicrobiomeStat package fitting linear mixed-effects models ‘~ Ethnicity + Antibiotic Use + Hormonal Contraceptive + (1|Day)’) for centered log-ratio transformed abundance between the 2 ethnicities. Data underlying this figure can be found at S1 Data. Pink indicates more abundant in Black participants, and blue indicates more abundant in White participants. Star indicates differential abundant taxa in the gut between the 2 ethnicities in both cohorts. Taxon name with a “G” before the name indicates the taxon was classified at the genus level. https://doi.org/10.1371/journal.pbio.3001758.g004 In addition to bacteria, crAss-like bacteriophages are the most abundant phages in the gut [49], and in the gut virome in cohort 1, crAssphage, which infects Bacteroides, was the most abundant viral species across both ethnicities and significantly higher in White individuals by 1.2-fold. In cohort 2, Faecalibacterium phages were collectively the most abundant phage species in all individuals but without significant abundance differences between ethnicities. Overall, 1,272 out of 4,423 (28.8%) total viral contigs are predicted to infect 85 bacterial genera in cohort 1. In cohort 2, 845 out of 2,971 (28.4%) were predicted to infect 64 bacterial genera. In cohort 1, 11 significantly different viral genera were detected between the 2 ethnicities when accounting for the day of the study (P FDR < 0.05, LinDA, S8 Data), 8 were more abundant in White participants with 1 unique phage predicted to infect Paraprevotella. In addition, 3 were more abundant in Black participants with 2 phages predicted to infect Anaerobutyricum and Coprobacter that are specific to Black participants. The largest fold-change observed in the either cohort was for phages of the Veillonella genus, which was 1,281-fold more abundant among Black individuals in cohort 1 and specific to Black individuals in cohort 2. In cohort 2, 6 differentially abundant genera were identified between ethnicities when accounting for the day of study (P FDR < 0.05, LinDA, S8 Data). Coprobacter was the only consistent finding between cohorts, being specific to Black individuals in cohort 1 and 6.4-fold more abundant in cohort 2. A single phage predicted to infect Anaerotruncus was specific to Black participants, while 2 phages predicted to infect Phascolarctobacterium and Blastocystis were specific to White individuals. The largest difference among phages was 13.4-fold for Parabacteroides phages in Black individuals in cohort 2. Ethnicity associations occur for heritable taxa Our previous analysis uncovered several gut taxa that consistently and significantly varied in abundance between ethnicities based on 16S rRNA gene amplicon sequencing from the American Gut and Human Microbiome Project [17]. Most of these recurrently varying taxa were reported to be heritable and associated with human genetic variation, which tentatively suggested human genotype may contribute to ethnicity-associated variation. Here, we identified 9 of the same bacterial taxa in the metagenomic datasets from both cohorts and tested if they also differ between ethnicities. Pairwise abundance tests validate 2 out of 9 oral taxa in cohort 1 are differentially abundant (Veillonella genus and Victivallaceae family, P FDR < 0.08, LinDA, S6 Table), and 2 out of 9 gut taxa in cohort 2 are differentially abundant (Christensenellaceae and Rikenellaceae families, P FDR < 0.05, LinDA, S6 Table). The 2 families identified in cohort 2 have higher abundances in Black participants, including the Christensenellaceae family that is the most highly heritable taxon in the gut microbiome, and higher abundances are positively associated with several health traits including obesity and inflammatory bowel disease [50–52]. The higher abundances of Rikenellaceae and Christensenellaceae families are associated with reduced visceral adipose tissue and healthier metabolic profile [53]. Pairwise abundance tests also reveal, for the first time, that 2 of the 9 heritable taxa previously identified in the gut are also differentially abundant in the oral microbiome in cohort 1 (P FDR < 0.08, LinDA, S6 Table), though they are not present in the gut of cohort 1 participants. One of these taxa, the Veillonella, is positively associated with dental calculus [54] and smoking [55]. The Veillonella genus is lower in Black participants in cohort 1, which is associated with a notably increased abundance of purified phages predicted to infect the genus Veillonella in both cohorts (P FDR < 0.05, LinDA). Thus, lytic activity of the phages may drive the reduced abundance of Veillonella. Phages predicted to infect 3 Veillonella spp. (Veillonella parvula, Veillonella sp. 3110, and Veillonella sp. AF36-20BH) are unique in Black participants. The highly heritable gut taxa Christensenellaceae is not differentially abundant in the saliva. In cohort 2, no heritable taxa were identified as differentially abundant in the oral microbiome (S6 Table). [END] --- [1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3001758 Published and (C) by PLOS One Content appears here under this condition or license: Creative Commons - Attribution BY 4.0. via Magical.Fish Gopher News Feeds: gopher://magical.fish/1/feeds/news/plosone/