Microbial metabolism and trophic interactions between microbes give rise to complex multispecies communities in microbe-host systems. Bacteroides thetaiotaomicron (B. theta) is a human gut symbiont thought to play an important role in maintaining host health. Untargeted nuclear magnetic resonance metabolomics revealed B. theta secretes specific organic acids and amino acids in defined minimal medium. Physiological concentrations of acetate and formate found in the human intestinal tract were shown to cause dose-dependent changes in secretion of metabolites known to play roles in host nutrition and pathogenesis. While secretion fluxes varied, biomass yield was unchanged, suggesting feedback inhibition does not affect metabolic bioenergetics but instead redirects carbon and energy to CO2 and H2 Flux balance analysis modeling showed increased flux through CO2-producing reactions under glucose-limiting growth conditions. The metabolic dynamics observed for B. theta, a keystone symbiont organism, underscores the need for metabolic modeling to complement genomic predictions of microbial metabolism to infer mechanisms of microbe-microbe and microbe-host interactions.IMPORTANCE Bacteroides is a highly abundant taxon in the human gut, and Bacteroides thetaiotaomicron (B. theta) is a ubiquitous human symbiont that colonizes the host early in development and persists throughout its life span. The phenotypic plasticity of keystone organisms such as B. theta is important to understand in order to predict phenotype(s) and metabolic interactions under changing nutrient conditions such as those that occur in complex gut communities. Our study shows B. theta prioritizes energy conservation and suppresses secretion of "overflow metabolites" such as organic acids and amino acids when concentrations of acetate are high. Secreted metabolites, especially amino acids, can be a source of nutrients or signals for the host or other microbes in the community. Our study suggests that when metabolically stressed by acetate, B. theta stops sharing with its ecological partners.

Thiamine (vitamin B1) is an essential cofactor for all organisms. Humans primarily acquire thiamine through their diet, and thiamine deficiencies have adverse neurological effects. However, the role gut microbes play in modulating thiamine availability is poorly understood, and little is known about how thiamine impacts the stability of microbial gut communities. To investigate thiamine's role in the gut, we utilized the model gut microbe Bacteroides thetaiotaomicron. Transcriptome sequencing (RNA-seq) revealed a global downregulation of thiamine and amino acid biosynthesis, glycolysis, and purine metabolism when thiamine was present. Using genetic mutants with thiamine biosynthesis and transport locus mutations, we determined both systems were critical for growth in thiamine-deficient medium. The defect in the double transport mutant suggests an uncharacterized feedback mechanism between thiamine transport and biosynthesis in B. thetaiotaomicron. Mutant phenotypes were recapitulated during pairwise competitions, reinforcing the importance of encoding versatile thiamine acquisition mechanisms when thiamine concentrations are variable. In addition, liquid chromatography-mass spectrometry (LC-MS) analyses corroborate that exogenous thiamine levels affect the internal thiamine pool of B. thetaiotaomicron. Furthermore, we computationally examined the ability of other gut microbes to acquire thiamine and identified lineage-specific differences in thiamine acquisition strategies. Among the Bacteroidetes, the capacities for both thiamine transport and biosynthesis are common. Together, these data show that thiamine acquisition mechanisms used by B. thetaiotaomicron not only are critical for its physiology and fitness but also provide the opportunity to model how other gut microbes may respond to the shifting availability of thiamine in the gut. IMPORTANCE Variation in the ability of gut microbes to transport, synthesize, and compete for vitamin B1 (thiamine) is expected to impact the structure and stability of the microbiota, and ultimately this variation may have both direct and indirect effects on human health. Our study identifies the diverse strategies employed by gut Bacteroidetes to acquire thiamine. We demonstrate how the presence or absence of thiamine biosynthesis or transport dramatically affects the abundance of B. thetaiotaomicron in a competitive environment. This study adds further evidence that altering the presence or concentrations of water-soluble vitamins such as thiamine may be an effective method for manipulating gut community composition. In turn, targeted thiamine delivery could be used therapeutically to alter dysbiotic communities linked to disease. Author Video: An author video summary of this article is available.

Functional metagenomics is a powerful method that allows the isolation of genes whose role may not have been predicted from DNA sequence. In this approach, first, environmental DNA is cloned to generate metagenomic libraries that are maintained in Escherichia coli, and second, the cloned DNA is screened for activities of interest. Typically, functional screens are carried out using E. coli as a surrogate host, although there likely exist barriers to gene expression, such as lack of recognition of native promoters. Here, we describe efforts to develop Bacteroides thetaiotaomicron as a surrogate host for screening metagenomic DNA from the human gut. We construct a B. thetaiotaomicron-compatible fosmid cloning vector, generate a fosmid clone library using DNA from the human gut, and show successful functional complementation of a B. thetaiotaomicron glycan utilization mutant. Though we were unable to retrieve the physical fosmid after complementation, we used genome sequencing to identify the complementing genes derived from the human gut microbiome. Our results demonstrate that the use of B. thetaiotaomicron to express metagenomic DNA is promising, but they also exemplify the challenges that can be encountered in the development of new surrogate hosts for functional screening. IMPORTANCE Human gut microbiome research has been supported by advances in DNA sequencing that make it possible to obtain gigabases of sequence data from metagenomes but is limited by a lack of knowledge of gene function that leads to incomplete annotation of these data sets. There is a need for the development of methods that can provide experimental data regarding microbial gene function. Functional metagenomics is one such method, but functional screens are often carried out using hosts that may not be able to express the bulk of the environmental DNA being screened. We expand the range of current screening hosts and demonstrate that human gut-derived metagenomic libraries can be introduced into the gut microbe Bacteroides thetaiotaomicron to identify genes based on activity screening. Our results support the continuing development of genetically tractable systems to obtain information about gene function.

Cronobacter sakazakii is an important pathogen associated with the development of necrotizing enterocolitis (NEC), infant sepsis, and meningitis. Several randomized prospective clinical trials demonstrated that oral probiotics could decrease the incidence of NEC. Previously, we isolated and characterized a novel probiotic, Bacteroides fragilis strain ZY-312. However, it remains unclear how ZY-312 protects the host from the effects of C. sakazakii infection. To understand the underlying mechanisms triggering the probiotic effects, we tested the hypothesis that there was cross talk between probiotics/probiotics-modulated microbiota and the local immune system, governed by the permeability of the intestinal mucosa, using in vitro and in vivo models for the intestinal permeability. The probiotic effects of ZY-312 on intestinal epithelial cells were first examined, and the results revealed that ZY-312 inhibited C. sakazakii invasion, C. sakazakii-induced dual cell death (pyroptosis and apoptosis), and epithelial barrier dysfunction in vitro and in vivo The presence of ZY-312 also resulted in decreased expression of an inflammasome (NOD-like receptor family member pyrin domain-containing protein 3 [NLRP3]), caspase-3, and serine protease caspase-1 in a neonatal rat model. Furthermore, ZY-312 significantly modulated the compositions of the intestinal bacterial communities and decreased the relative abundances of Proteobacteria and Gammaproteobacteria but increased the relative abundances of Bacteroides and Bacillus in neonatal rats. In conclusion, our findings have shown for the first time that the probiotic B. fragilis ZY-312 suppresses C. sakazakii-induced NEC by modulating the proinflammatory response and dual cell death (apoptosis and pyroptosis).IMPORTANCE Cronobacter sakazakii is an opportunistic pathogenic bacterium that can cause necrotizing enterocolitis (NEC). However, the mechanism of pathogenicity of C. sakazakii is largely unknown. Here we have now demonstrated that apoptotic and pyroptotic stimuli are effectors of C. sakazakii-induced NEC. Previously, we isolated a novel probiotic strain candidate from fecal samples from healthy infants and characterized it as Bacteroides fragilis strain ZY-312. Functional characterization reveals that ZY-312 inhibited C. sakazakii invasion, restoring epithelial barrier dysfunction, decreasing the expression of inflammatory cytokines, and reducing dual cell death (pyroptosis and apoptosis). Furthermore, the presence of ZY-132 was sufficient to hinder the adverse reaction seen with C. sakazakii in a C. sakazakii-induced NEC model. Taking the results together, our study demonstrated the utility of ZY-312 as a promising probiotic agent for the prevention of NEC.

Human gut dysbiosis is associated with type 2 diabetes mellitus (T2DM); however, the gut microbiome in pregnant women with pregestational type 2 diabetes mellitus (PGDM) remains unexplored. We investigated the alterations in the gut microbiota composition in pregnant women with or without PGDM. The gut microbiota was examined using 16S rRNA sequencing data of 234 maternal fecal samples that were collected during the first (T1), second (T2), and third (T3) trimesters. The PGDM group presented a reduction in the number of gut bacteria taxonomies as the pregnancies progressed. Linear discriminant analyses revealed that Megamonas, Bacteroides, and Roseburia intestinalis were enriched in the PGDM group, whereas Bacteroides vulgatus, Faecalibacterium prausnitzii, Eubacterium rectale, Bacteroides uniformis, Eubacterium eligens, Subdoligranulum, Bacteroides fragilis, Dialister, Lachnospiraceae, Christensenellaceae R-7, Roseburia inulinivorans, Streptococcus oralis, Prevotella melaninogenica, Neisseria perflava, Bacteroides ovatus, Bacteroides caccae, Veillonella dispar, and Haemophilus parainfluenzae were overrepresented in the control group. Correlation analyses showed that the PGDM-enriched taxa were correlated with higher blood glucose levels during pregnancy, whereas the taxonomic biomarkers of normoglycemic pregnancies exhibited negative correlations with glycemic traits. The microbial networks in the PGDM group comprised weaker microbial interactions than those in the control group. Our study reveals the distinct characteristics of the gut microbiota composition based on gestational ages between normoglycemic and PGDM pregnancies. Further longitudinal research involving women with T2DM at preconception stages and investigations using shotgun metagenomic sequencing should be performed to elucidate the relationships between specific bacterial functions and PGDM metabolic statuses during pregnancy and to identify potential therapeutic targets. IMPORTANCE The incidence of pregestational type 2 diabetes mellitus (PGDM) is increasing, with high rates of serious adverse maternal and neonatal outcomes that are strongly correlated with hyperglycemia. Recent studies have shown that type 2 diabetes mellitus is associated with gut microbial dysbiosis; however, the gut microbiome composition and its associations with the metabolic features of patients with PGDM remain largely unknown. In this study, we investigated the changes in the gut microbiota composition in pregnant women with and without PGDM. We identified differential taxa that may be correlated with maternal metabolic statuses during pregnancy. Additionally, we observed that the number of taxonomic and microbial networks of gut bacteria were distinctly reduced in women with hyperglycemia as their pregnancies progressed. These results extend our understanding of the associations between the gut microbial composition, PGDM-related metabolic changes, and pregnancy outcomes.

Gut microbial β-glucuronidase (GUS) enzymes play important roles in drug efficacy and toxicity, intestinal carcinogenesis, and mammalian-microbial symbiosis. Recently, the first catalog of human gut GUS proteins was provided for the Human Microbiome Project stool sample database and revealed 279 unique GUS enzymes organized into six categories based on active-site structural features. Because mice represent a model biomedical research organism, here we provide an analogous catalog of mouse intestinal microbial GUS proteins-a mouse gut GUSome. Using metagenome analysis guided by protein structure, we examined 2.5 million unique proteins from a comprehensive mouse gut metagenome created from several mouse strains, providers, housing conditions, and diets. We identified 444 unique GUS proteins and organized them into six categories based on active-site features, similarly to the human GUSome analysis. GUS enzymes were encoded by the major gut microbial phyla, including Firmicutes (60%) and Bacteroidetes (21%), and there were nearly 20% for which taxonomy could not be assigned. No differences in gut microbial gus gene composition were observed for mice based on sex. However, mice exhibited gus differences based on active-site features associated with provider, location, strain, and diet. Furthermore, diet yielded the largest differences in gus composition. Biochemical analysis of two low-fat-associated GUS enzymes revealed that they are variable with respect to their efficacy of processing both sulfated and nonsulfated heparan nonasaccharides containing terminal glucuronides.IMPORTANCE Mice are commonly employed as model organisms of mammalian disease; as such, our understanding of the compositions of their gut microbiomes is critical to appreciating how the mouse and human gastrointestinal tracts mirror one another. GUS enzymes, with importance in normal physiology and disease, are an attractive set of proteins to use for such analyses. Here we show that while the specific GUS enzymes differ at the sequence level, a core GUSome functionality appears conserved between mouse and human gastrointestinal bacteria. Mouse strain, provider, housing location, and diet exhibit distinct GUSomes and gus gene compositions, but sex seems not to affect the GUSome. These data provide a basis for understanding the gut microbial GUS enzymes present in commonly used laboratory mice. Further, they demonstrate the utility of metagenome analysis guided by protein structure to provide specific sets of functionally related proteins from whole-genome metagenome sequencing data.

Emerging evidence shows that modulation of the microbiome can suppress intra-abdominal hypertension (IAH)-induced intestinal barrier damage through the regulation of amino acid (AA) biosynthesis. Here, we investigated the protective effects of orally gavaged Lactobacillus acidophilus L-92 (L92) and a mixture of AA in rats with induced IAH. The results showed that both L92 and AA pretreatments effectively mitigated IAH-induced intestinal damage. Interestingly, L92 but not AA prevented metagenomic changes induced by IAH. Bacteroides fragilis, Bacteroides eggerthii, Bacteroides ovatus, Faecalibacterium prausnitzii, Prevotella, and extensively altered functional pathways were associated with L92-mediated host protection. Metabolomic profiling revealed that tryptophan metabolism was involved in both L92- and AA-mediated gut protection. The tryptophan metabolite 5-hydroxyindoleacetic acid (5-HIAA) is a sensitive biomarker for IAH in rats and patients with either gut-derived sepsis (n = 41) or all-source sepsis (n = 293). In conclusion, we show that microbiome and metabolic modulations can effectively prevent IAH-induced intestinal damage and that 5-HIAA is a potential metabolic marker for IAH and sepsis. IMPORTANCE Gut protection through modulation of the microbiome for critically ill patients has been gaining much attention recently. Intra-abdominal hypertension (IAH) is a prevailing clinical feature of acute gastrointestinal injuries in critically ill patients, characterized by nonspecific intestinal barrier damage. Prolonged IAH can induce or aggravate the development of sepsis and multiorgan dysfunctions. Therefore, the prevention of IAH-induced damage in rats through microbiome and metabolic interventions by commercially available L92 and AA treatments and the identification of 5-HIAA as an important marker for IAH/sepsis have important clinical implications for the treatment and early diagnosis of critically ill patients.

Alterations in the gut microbiome are associated with neurocognition and related disorders, including in the context of human immunodeficiency virus (HIV) and hepatitis C virus (HCV) infection. However, the connection between the gut microbiome and cognitive decline, gauged by increased dependence in instrumental activities of daily living (IADL), remains largely unexplored in the context of these diseases. Here we characterized the gut microbiome using 16S rRNA amplicon sequencing and untargeted metabolomics with liquid chromatography-mass spectrometry from 347 people with HIV, HIV and HCV, or neither, all of whom underwent a comprehensive neuropsychiatric assessment. We observed that IADL-dependent and -independent HIV-monoinfected (HIV-positive [HIV+]/HCV-negative [HCV-]) and coinfected (HIV+/HCV+) individuals have distinct gut microbiomes. Moreover, we found that dependent individuals with HIV or HIV and HCV were enriched in Bacteroides These results may have implications for the characterization of cognitive decline, as well as the development of potential prevention and treatment strategies for individuals infected with HIV and/or HCV. Of particular interest is the possibility that dietary interventions that are known to modify the microbiome could be used to shift the microbiome toward more favorable states for preserving independence.IMPORTANCE The microbes in the gut and the chemicals they produce by metabolism have been linked to brain function. In earlier work, we showed that infection with two viruses, HIV and HCV, changed the gut microbes and metabolism in ways that were associated with a lifetime history of major depressive disorder. Here, we extend this analysis looking at a measurement of independence in daily living. We find that in individuals with HIV, whether or not they also have HCV, those who reported reduced independence were enriched in a genus of bacteria called Bacteroides This result is interesting because Bacteroides is strongly associated with diets low in carbohydrates and high in animal protein, suggesting that diet changes may help preserve independent living in people living long-term with HIV (although clinical intervention trials would be needed in order to confirm this).

Bovine digital dermatitis (DD) is a skin disorder that is a significant cause of infectious lameness in cattle around the world. However, very little is known about the etiopathogenesis of the disease and the microbiota associated with DD in beef cattle. In this study, we provide a comprehensive characterization of DD and healthy skin microbiota of feedlot beef cattle. We also developed and validated a novel multiplex quantitative PCR (qPCR) assay to quantify the distribution of DD-associated bacterial species across DD lesion stages. We determined the DD-associated microbiota with deep amplicon sequencing of the V3-V4 hypervariable region of the 16S rRNA gene, followed by the application of novel and existing qPCR assays to quantify species distributions of Treponema, Porphyromonas, Fusobacterium, and Bacteroides across lesion stages. Deep amplicon sequencing revealed that Treponema, Mycoplasma, Porphyromonas, and Fusobacterium were associated with DD lesions. Culturing of DD biopsy specimens identified Porphyromonas levii, Bacteroides pyogenes, and two Fusobacterium spp. within DD lesions. Using species-specific qPCR on DD lesion DNA, we identified P. levii in 100% of active lesion stages. Early-stage lesions were particularly associated with Treponema medium, T. phagedenis, and P. levii. This study suggests a core DD microbial group consisting of species of Treponema, Fusobacterium, Porphyromonas, and Bacteroides, which may be closely tied with the etiopathogenesis of DD. Further characterizations of these species and Mycoplasma spp. are necessary to understand the microbial factors involved in DD pathogenesis, which will help elucidate DD etiology and facilitate more targeted and effective mitigation and treatment strategies. IMPORTANCE Previous work, primarily in dairy cattle, has identified various taxa associated with digital dermatitis (DD) lesions. However, there is a significant gap in our knowledge of DD microbiology in beef cattle. In addition, characterization of bacteria at the species level in DD lesions is limited. In this study, we provide a framework for the accurate and reproducible quantification of major DD-associated bacterial species from DNA samples. Our findings support DD as a polymicrobial infection, and we identified a variety of bacterial species spanning multiple genera that are consistently associated with DD lesions. The DD-associated microbiota identified in this study may be capable of inducing the formation and progression of DD lesions and thus should be primary targets in future DD pathogenesis studies.

Vancomycin-resistant Enterococcus faecium (VREfm) is an emerging antibiotic-resistant pathogen. Strain-level investigations are beginning to reveal the molecular mechanisms used by VREfm to colonize regions of the human bowel. However, the role of commensal bacteria during VREfm colonization, in particular following antibiotic treatment, remains largely unknown. We employed amplicon 16S rRNA gene sequencing and metabolomics in a murine model system to try and investigate functional roles of the gut microbiome during VREfm colonization. First-order taxonomic shifts between Bacteroidetes and Tenericutes within the gut microbial community composition were detected both in response to pretreatment using ceftriaxone and to subsequent VREfm challenge. Using neural networking approaches to find cooccurrence profiles of bacteria and metabolites, we detected key metabolome features associated with butyric acid during and after VREfm colonization. These metabolite features were associated with Bacteroides, indicative of a transition toward a preantibiotic naive microbiome. This study shows the impacts of antibiotics on the gut ecosystem and the progression of the microbiome in response to colonization with VREfm. Our results offer insights toward identifying potential nonantibiotic alternatives to eliminate VREfm through metabolic reengineering to preferentially select for Bacteroides IMPORTANCE This study demonstrates the importance and power of linking bacterial composition profiling with metabolomics to find the interactions between commensal gut bacteria and a specific pathogen. Knowledge from this research will inform gut microbiome engineering strategies, with the aim of translating observations from animal models to human-relevant therapeutic applications.

Symbiotic bacteria are responsible for the majority of complex carbohydrate digestion in the human colon. Since the identities and amounts of dietary polysaccharides directly impact the gut microbiota, determining which microorganisms consume specific nutrients is central for defining the relationship between diet and gut microbial ecology. Using a custom phenotyping array, we determined carbohydrate utilization profiles for 354 members of the Bacteroidetes, a dominant saccharolytic phylum. There was wide variation in the numbers and types of substrates degraded by individual bacteria, but phenotype-based clustering grouped members of the same species indicating that each species performs characteristic roles. The ability to utilize dietary polysaccharides and endogenous mucin glycans was negatively correlated, suggesting exclusion between these niches. By analyzing related Bacteroides ovatus/Bacteroides xylanisolvens strains that vary in their ability to utilize mucin glycans, we addressed whether gene clusters that confer this complex, multilocus trait are being gained or lost in individual strains. Pangenome reconstruction of these strains revealed a remarkably mosaic architecture in which genes involved in polysaccharide metabolism are highly variable and bioinformatics data provide evidence of interspecies gene transfer that might explain this genomic heterogeneity. Global transcriptomic analyses suggest that the ability to utilize mucin has been lost in some lineages of B. ovatus and B. xylanisolvens, which harbor residual gene clusters that are involved in mucin utilization by strains that still actively express this phenotype. Our data provide insight into the breadth and complexity of carbohydrate metabolism in the microbiome and the underlying genomic events that shape these behaviors. IMPORTANCE Nonharmful bacteria are the primary microbial symbionts that inhabit the human gastrointestinal tract. These bacteria play many beneficial roles and in some cases can modify disease states, making it important to understand which nutrients sustain specific lineages. This knowledge will in turn lead to strategies to intentionally manipulate the gut microbial ecosystem. We designed a scalable, high-throughput platform for measuring the ability of gut bacteria to utilize polysaccharides, of which many are derived from dietary fiber sources that can be manipulated easily. Our results provide paths to expand phenotypic surveys of more diverse gut bacteria to understand their functions and also to leverage dietary fibers to alter the physiology of the gut microbial community.

Eczema is a major allergic disease in children, which is particularly prevalent in Chinese children during their first year of life. In this study, we showed that alterations in the infant gut microbiota precede the development of eczema in a prospective Chinese cohort. In particular, we discovered enrichments of the genera Clostridium sensu stricto 1 and Finegoldia in the cases at 3 and 1 month of age, respectively, which may represent potential targets for intervention to prevent eczema. Besides, we identified a depletion of Bacteroides from 1 to 6 months of age and an enrichment of Clostridium sensu stricto 1 at 3 months in the eczema cases, patterns also observed in C-section-born infants within the same time frames, providing first evidence to support a role of the gut microbiota in previously reported associations between C-section and increased risk of eczema in infancy.

Dietary composition and antibiotic use have major impacts on the structure and function of the gut microbiome, often resulting in dysbiosis. Despite this, little research has been done to explore the role of host diet as a determinant of antibiotic-induced microbiome disruption. Here, we utilize a multi-omic approach to characterize the impact of Western-style diet consumption on ciprofloxacin-induced changes to gut microbiome structure and transcriptional activity. We found that Western diet consumption dramatically increased Bacteroides abundances and shifted the community toward the metabolism of simple sugars and mucus glycoproteins. Mice consuming a Western-style diet experienced a greater expansion of Firmicutes following ciprofloxacin treatment than those eating a control diet. Transcriptionally, we found that ciprofloxacin reduced the abundance of tricarboxylic acid (TCA) cycle transcripts on both diets, suggesting that carbon metabolism plays a key role in the response of the gut microbiome to this antibiotic. Despite this, we observed extensive diet-dependent differences in the impact of ciprofloxacin on microbiota function. In particular, at the whole-community level we detected an increase in starch degradation, glycolysis, and pyruvate fermentation following antibiotic treatment in mice on the Western diet, which we did not observe in mice on the control diet. Similarly, we observed diet-specific changes in the transcriptional activity of two important commensal bacteria, Akkermansia muciniphila and Bacteroides thetaiotaomicron, involving diverse cellular processes such as nutrient acquisition, stress responses, and capsular polysaccharide (CPS) biosynthesis. These findings demonstrate that host diet plays a role in determining the impacts of ciprofloxacin on microbiome composition and microbiome function.IMPORTANCE Due to the growing incidence of disorders related to antibiotic-induced dysbiosis, it is essential to determine how our "Western"-style diet impacts the response of the microbiome to antibiotics. While diet and antibiotics have profound impacts on gut microbiome composition, little work has been done to examine their combined effects. Previous work has shown that nutrient availability, influenced by diet, plays an important role in determining the extent of antibiotic-induced disruption to the gut microbiome. Thus, we hypothesize that the Western diet will shift microbiota metabolism toward simple sugar and mucus degradation and away from polysaccharide utilization. Because of bacterial metabolism's critical role in antibiotic susceptibility, this change in baseline metabolism will impact how the structure and function of the microbiome are impacted by ciprofloxacin exposure. Understanding how diet modulates antibiotic-induced microbiome disruption will allow for the development of dietary interventions that can alleviate many of the microbiome-dependent complications of antibiotic treatment.

Alzheimer's disease (AD) is a heterogeneous disorder that spans a continuum with multiple phases, including preclinical, mild cognitive impairment, and dementia. Unlike for most other chronic diseases, human studies reporting on AD gut microbiota in the literature are very limited. With the scarcity of approved drugs for AD therapies, the rational and precise modulation of gut microbiota composition using diet and other tools is a promising approach to the management of AD. Such an approach could be personalized if an AD continuum can first be deconstructed into multiple strata based on specific microbiota features by using single or multiomics techniques. However, stratification of AD gut microbiota has not been systematically investigated before, leaving an important research gap for gut microbiota-based therapeutic approaches. Here, we analyze 16S rRNA amplicon sequencing of stool samples from 27 patients with mild cognitive impairment, 47 patients with AD, and 51 nondemented control subjects by using tools compatible with the compositional nature of microbiota. To stratify the AD gut microbiota community, we applied four machine learning techniques, including partitioning around the medoid clustering and fitting a probabilistic Dirichlet mixture model, the latent Dirichlet allocation model, and we performed topological data analysis for population-scale microbiome stratification based on the Mapper algorithm. These four distinct techniques all converge on Prevotella and Bacteroides stratification of the gut microbiota across the AD continuum, while some methods provided fine-scale resolution in stratifying the community landscape. Finally, we demonstrate that the signature taxa and neuropsychometric parameters together robustly classify the groups. Our results provide a framework for precision nutrition approaches aiming to modulate the AD gut microbiota. IMPORTANCE The prevalence of AD worldwide is estimated to reach 131 million by 2050. Most disease-modifying treatments and drug trials have failed, due partly to the heterogeneous and complex nature of the disease. Recent studies demonstrated that gut dybiosis can influence normal brain function through the so-called "gut-brain axis." Modulation of the gut microbiota, therefore, has drawn strong interest in the clinic in the management of the disease. However, there is unmet need for microbiota-informed stratification of AD clinical cohorts for intervention studies aiming to modulate the gut microbiota. Our study fills in this gap and draws attention to the need for microbiota stratification as the first step for microbiota-based therapy. We demonstrate that while Prevotella and Bacteroides clusters are the consensus partitions, the newly developed probabilistic methods can provide fine-scale resolution in partitioning the AD gut microbiome landscape.

Previous study found that appropriate high-fiber diet (containing 19.10% total dietary fiber [TDF], treatment II) did not reduce apparent fiber digestibility of Chinese Suhuai finishing pigs and increased the yield of short-chain fatty acids (SCFAs), but too high-fiber diet (containing 24.11% TDF, treatment IV) significantly reduced apparent fiber digestibility compared with normal diet (containing 16.70% TDF, control group). However, characteristics of microbiota at the species level and histological structure in pigs with the ability to digest appropriate high-fiber diets were still unknown. This study conducted comparative analysis of cecal physiology and microbial populations colonizing cecal mucosa. The results showed intestinal development indexes including cecum length, densities of cecal goblet cells, and renewal of cecal epithelial cells in treatment II and IV had better performance than those in the control. Paludibacter jiangxiensis, Coprobacter fastidiosus, Bacteroides coprocola CAG:162, Bacteroides barnesiae, and Parabacteroides merdae enriched in treatment II expressed large number of glycoside hydrolase (GH)-encoding genes and had the largest number of GH families. In addition, pathogenic bacteria (Shigella sonnei, Mannheimia haemolytica, and Helicobacter felis) were enriched in treatment IV. Correlation analysis revealed that the intestinal development index positively correlated with the relative abundance of cecal mucosal microbiota and the amount of digested fiber. These results indicated that increased proportions of fiber-degrading microbes and enhanced intestinal development jointly promote the host to digest an appropriate high-fiber diet. However, although too-high fiber levels in diet could maintain the adaptive development of cecal epithelium, the proportions of pathogenic bacteria increased, which might lead to a decrease of fiber digestion in pigs. IMPORTANCE Although studies about the effects of dietary fiber on fiber digestion and intestinal microbiota of pigs were widely in progress, few studies have been conducted on the dynamic response of intestinal microbiota to dietary fiber levels, and the characteristics of intestinal microbiota and intestinal epithelial development adapted to high-fiber diet s were still unclear. Appropriate high fiber promoted the thickness of large intestine wall, increased the density of cecal goblet cells, and promoted the renewal of cecal epithelial cells. In addition, appropriate high fiber improves the microbial abundance with fiber-digesting potential. However, excessive dietary fiber caused an increase in the abundance of pathogenic bacteria. These results indicated that an increased proportion of fiber-degrading microbes and enhanced intestinal development jointly promote host to digest appropriate high-fiber diets. However, although too-high fiber levels in diet could maintain the adaptive development of cecal epithelium, the proportions of pathogenic bacteria increased, which might lead to a decrease of fiber digestion in pigs. Our data provided a theoretical basis for rational and efficient utilization of unconventional feed resources in pig production.

The impact of gut fungi and (1→3)-β-d-glucan (BG), a major fungal cell wall component, on uremia was explored by Candida albicans oral administration in bilateral nephrectomy (BiNx) mice because of the prominence of C. albicans in the human intestine but not in mice. As such, BiNx with Candida administration (BiNx-Candida) enhanced intestinal injury (colon cytokines and apoptosis), gut leakage (fluorescein isothiocyanate [FITC]-dextran assay, endotoxemia, serum BG, and bacteremia), systemic inflammation, and liver injury at 48 h postsurgery compared with non-Candida BiNx mice. Interestingly, uremia-induced enterocyte apoptosis was severe enough for gut translocation of viable bacteria, as indicated by culture positivity for bacteria in blood, mesenteric lymph nodes (MLNs), and other organs, which was more severe in BiNx-Candida than in non-Candida BiNx mice. Candida induced alterations in the gut microbiota of BiNx mice as indicated by (i) the higher fungal burdens in the feces of BiNx-Candida mice than in sham-Candida mice by culture methods and (ii) increased Bacteroides with decreased Firmicutes and reduced bacterial diversity in the feces of BiNx-Candida mice compared with non-Candida BiNx mice by fecal microbiome analysis. In addition, lipopolysaccharide plus BG (LPS+BG), compared with each molecule alone, induced high supernatant cytokine levels, which were enhanced by uremic mouse serum in both hepatocytes (HepG2 cells) and macrophages (RAW264.7 cells). Moreover, LPS+BG, but not each molecule alone, reduced the glycolysis capacity and mitochondrial function in HepG2 cells as determined by extracellular flux analysis. Additionally, a probiotic, Lactobacillus rhamnosus L34 (L34), attenuated disease severity only in BiNx-Candida mice but not in non-Candida BiNx mice, as indicated by liver injury and serum cytokines through the attenuation of gut leakage, the fecal abundance of fungi, and fecal bacterial diversity but not fecal Gram-negative bacteria. In conclusion, Candida enhanced BiNx severity through the worsening of gut leakage and microbiota alterations that resulted in bacteremia, endotoxemia, and glucanemia.IMPORTANCE The impact of fungi in the intestine on acute uremia was demonstrated by the oral administration of Candida albicans in mice with the removal of both kidneys. Because fungi in the mouse intestine are less abundant than in humans, a Candida-administered mouse model has more resemblance to patient conditions. Accordingly, acute uremia, without Candida, induced intestinal mucosal injury, which resulted in the translocation of endotoxin, a major molecule of gut bacteria, from the intestine into blood circulation. In acute uremia with Candida, intestinal injury was more severe due to fungi and the alteration in intestinal bacteria (increased Bacteroides with decreased Firmicutes), leading to the gut translocation of both endotoxin from gut bacteria and (1→3)-β-d-glucan from Candida, which synergistically enhanced systemic inflammation in acute uremia. Both pathogen-associated molecules were delivered to the liver and induced hepatocyte inflammatory responses with a reduced energy production capacity, resulting in acute uremia-induced liver injury. In addition, Lactobacillus rhamnosus attenuated intestinal injury through reduced gut Candida and improved intestinal bacterial conditions.

The role of dietary amino acids or intact proteins in the progression of colitis remains controversial, and the mechanism involving gut microbes is unclear. Here, we investigated the effects of an elemental diet (ED) enriched in amino acids and a polymeric diet enriched in intact protein on the pathogenesis of dextran sulfate sodium (DSS)-induced colitis in mice. Our results showed that the ED induced remission of colitis in mice. Notably, ED treatment reduced the abundance of the mucolytic bacteria Akkermansia and Bacteroides, which was attributed to decreased colonic protein fermentation. Consistently, the activities of mucolytic enzymes were decreased, leading to protection against mucus layer degradation and microbial invasion. Fecal microbiota transplantation from ED-fed mice reshaped microbial ecology and alleviated intestinal inflammation in recipient mice. The ED failed to induce remission of colitis in pseudogermfree mice. Together, our results demonstrate the critical role of the gut microbiota in the prevention of colitis by an ED. IMPORTANCE The prevalence of inflammatory bowel disease is rapidly increasing and has become a global burden. Several specific amino acids have been shown to benefit mucosal healing and colitis remission. However, the role of amino acids or intact proteins in diets and enteral nutrition formulas is controversial, and the mechanisms involving gut microbes remain unclear. In this study, we investigated the effects of an elemental diet (ED) enriched in amino acids and a polymeric diet enriched in intact protein on the pathogenesis of colitis in mice. The underlying mechanisms were explored by utilizing fecal microbiota transplantation and pseudogermfree mice. ED treatment reduced the abundance of mucolytic bacteria, thereby protecting the mucus layer from microbial invasion and degradation. For the first time, we convincingly demonstrated the critical role of gut microbiota in the effects of the ED. This study may provide new insights into the gut microbiota-diet interaction and its role in human health.

Many commensal bacteria antagonize each other or their host by producing syringe-like secretion systems called contractile injection systems (CIS). Members of the Bacteroidales family have been shown to produce only one type of CIS-a contact-dependent type 6 secretion system that mediates bacterium-bacterium interactions. Here, we show that a second distinct cluster of genes from Bacteroidales bacteria from the human microbiome may encode yet-uncharacterized injection systems that we term Bacteroidales injection systems (BIS). We found that BIS genes are present in the gut microbiomes of 99% of individuals from the United States and Europe and that BIS genes are more prevalent in the gut microbiomes of healthy individuals than in those individuals suffering from inflammatory bowel disease. Gene clusters similar to that of the BIS mediate interactions between bacteria and diverse eukaryotes, like amoeba, insects, and tubeworms. Our findings highlight the ubiquity of the BIS gene cluster in the human gut and emphasize the relevance of the gut microbiome to the human host. These results warrant investigations into the structure and function of the BIS and how they might mediate interactions between Bacteroidales bacteria and the human host or microbiome.IMPORTANCE To engage with host cells, diverse pathogenic bacteria produce syringe-like structures called contractile injection systems (CIS). CIS are evolutionarily related to the contractile tails of bacteriophages and are specialized to puncture membranes, often delivering effectors to target cells. Although CIS are key for pathogens to cause disease, paradoxically, similar injection systems have been identified within healthy human microbiome bacteria. Here, we show that gene clusters encoding a predicted CIS, which we term Bacteroidales injection systems (BIS), are present in the microbiomes of nearly all adult humans tested from Western countries. BIS genes are enriched within human gut microbiomes and are expressed both in vitro and in vivo Further, a greater abundance of BIS genes is present within healthy gut microbiomes than in those humans with with inflammatory bowel disease (IBD). Our discovery provides a potentially distinct means by which our microbiome interacts with the human host or its microbiome.

The complex interactions between the gut microbiome and host or pathogen colonization resistance cannot be understood solely from community composition. Missing are causal relationships, such as metabolic interactions among species, to better understand what shapes the microbiome. Here, we focused on metabolic niches generated and occupied by the Oligo-Mouse-Microbiota (OMM) consortium, a synthetic community composed of 12 members that is increasingly used as a model for the mouse gut microbiome. Combining monocultures and spent medium experiments with untargeted metabolomics revealed broad metabolic diversity in the consortium, constituting a dense cross-feeding network with more than 100 pairwise interactions. Quantitative analysis of the cross-feeding network revealed distinct C and N food webs, highlighting the two Bacteroidetes members Bacteroides caecimuris and Muribaculum intestinale as primary suppliers of carbon and a more diverse group as nitrogen providers. Cross-fed metabolites were mainly carboxylic acids, amino acids, and the so far not reported nucleobases. In particular, the dicarboxylic acids malate and fumarate provided a strong physiological benefit to consumers, presumably used in anaerobic respiration. Isotopic tracer experiments validated the fate of a subset of cross-fed metabolites, such as the conversion of the most abundant cross-fed compound succinate to butyrate. Thus, we show that this consortium is tailored to produce the anti-inflammatory metabolite butyrate. Overall, we provide evidence for metabolic niches generated and occupied by OMM members that lays a metabolic foundation to facilitate an understanding of the more complex in vivo behavior of this consortium in the mouse gut. IMPORTANCE This article maps out the cross-feeding network among 10 members of a synthetic consortium that is increasingly used as the model mouse gut microbiota. Combining metabolomics with in vitro cultivations, two dense networks of carbon and nitrogen exchange are described. The vast majority of the ∼100 interactions are synergistic in nature, in several cases providing distinct physiological benefits to the recipient species. These networks lay the groundwork toward understanding gut community dynamics and host-gut microbe interactions.

Recently, dysbiosis in the human gut microbiome and shifts in the relative abundances of several bacterial species have been recognized as important factors in colorectal cancer (CRC). However, these studies have been carried out mainly in developed countries where CRC has a high incidence, and it is unclear whether the host-microbiome relationships deduced from these studies can be generalized to the global population. To test if the documented associations between the microbiome and CRC are conserved in a distinct context, we performed metagenomic and metabolomic association studies on fecal samples from 30 CRC patients and 30 healthy controls from two different locations in India, followed by a comparison of CRC data available from other populations. We confirmed the association of Bacteroides and other bacterial taxa with CRC that have been previously reported in other studies. However, the association of CRC with Flavonifractor plautii in Indian patients emerged as a novel finding. The plausible role of F. plautii appears to be linked with the degradation of beneficial anticarcinogenic flavonoids, which was also found to be significantly correlated with the enzymes and modules involved in flavonoid degradation within Indian CRC samples. Thus, we hypothesize that the degradation of beneficial flavonoids might be playing a role in cancer progression within this Indian cohort. We also identified 20 potential microbial taxonomic markers and 33 potential microbial gene markers that discriminate the Indian CRC from healthy microbiomes with high accuracy based on machine learning approaches.IMPORTANCE This study provides novel insights on the CRC-associated microbiome of a unique cohort in India, reveals the potential role of a new bacterium in CRC, and identifies cohort-specific biomarkers, which can potentially be used in noninvasive diagnosis of CRC. The study gains additional significance, as India is among the countries with a very low incidence of CRC, and the diet and lifestyle in India have been associated with a distinct gut microbiome in healthy Indians compared to other global populations. Thus, in this study, we hypothesize a unique relationship between CRC and the gut microbiome in an Indian population.