Stress, a ubiquitous part of daily human life, has varied biological effects which are increasingly recognized as including modulation of commensal microorganisms residing in the gastrointestinal tract, the gut microbiota. In turn, the gut microbiota influences the host stress response and associated sequelae, thereby implicating the gut microbiota as an important mediator of host health. This narrative review aims to summarize evidence concerning the impact of psychological, environmental, and physical stressors on gut microbiota composition and function. The stressors reviewed include psychological stress, circadian disruption, sleep deprivation, environmental extremes (high altitude, heat, and cold), environmental pathogens, toxicants, pollutants, and noise, physical activity, and diet (nutrient composition and food restriction). Stressors were selected for their direct relevance to military personnel, a population that is commonly exposed to these stressors, often at extremes, and in combination. However, the selected stressors are also common, alone or in combination, in some civilian populations. Evidence from preclinical studies collectively indicates that the reviewed stressors alter the composition, function and metabolic activity of the gut microbiota, but that effects vary across stressors, and can include effects that may be beneficial or detrimental to host health. Translation of these findings to humans is largely lacking at present. This gap precludes concluding with certainty that transient or cumulative exposures to psychological, environmental, and physical stressors have any consistent, meaningful impact on the human gut microbiota. However, provocative preclinical evidence highlights a need for translational research aiming to elucidate the impact of stressors on the human gut microbiota, and how the gut microbiota can be manipulated, for example by using nutrition, to mitigate adverse stress responses.

Gut microbiome community dynamics are maintained by complex microbe-microbe and microbe-host interactions, which can be disturbed by stress. In vivo studies on the dynamics and manipulation of those interactions are costly and slow, but can be accelerated using in vitro fermentation. Herein, in vitro fermentation was used to determine how an acute stressor, a sudden change in diet, impacts interbacterial species competition for resistant starch-supplemented medium (RSM). Fermentation vessels were seeded with fecal samples collected from 10 individuals consuming a habitual diet or U.S. military rations for 21 days. Lactobacillus spp. growth in response to RSM was attenuated following ration consumption, whereas growth of Ruminococcus bromii was enhanced. These differences were not evident in the pre-fermentation samples. Findings demonstrate how incorporating in vitro fermentation into clinical studies can increase understanding of stress-induced changes in nutrient-microbiome dynamics, and suggest that sudden changes in diet may impact inter-species competition for substrates.

A naturally occurring antimicrobial peptide, SMAP-29, was synthesized with an n-terminal or c-terminal cysteine, termed c_SMAP and SMAP_c, respectively, for site-directed immobilization to superparamagnetic beads. Immobilized SMAP orientation-dependent activity was probed against multiple bacteria of clinical interest including Acinetobacter baumannii, Pseudomonas aeruginosa, Bacillus anthracis sterne and Staphylococcus aureus. A kinetic microplate assay was employed to reveal both concentration and time-dependent activity for elucidation of minimum bactericidal concentration (MBC) and sub-lethal effects. Immobilized SMAP activity was equivalent or reduced compared with soluble SMAP_c and c_SMAP regardless of immobilization orientation, with only one exception. A comparison of immobilized SMAP_c and c_SMAP activity revealed a bacteria-specific potency dependent on immobilization orientation, which was contrary to that seen in solution, wherein SMAP_c was more potent against all bacteria than c_SMAP. Sub-MBC kinetic studies displayed the influence of peptide exposure to the cells with multiple bacteria exhibiting increased susceptibility and efficacy at lower concentrations upon extended exposure (i.e. MBC enhancement). For instances in which complete killing was not achieved, two predominant effects were evident: retardation of growth rate and an increased lag phase. Both effects, seen independently and concomitantly, indicate some degree of induced cellular damage that can serve as a predictor toward eventual cell death. SMAP_c immobilized on glass through standard silanization chemistry was also investigated to ascertain the influence of substrate on activity against select bacteria.

In vitro fermentation systems offer significant opportunity for deconvoluting complex metabolic dynamics within polymicrobial communities, particularly those associated with the human gut microbiome. In vitro gut models have broad experimental capacity allowing rapid evaluation of multiple parameters, generating knowledge to inform design of subsequent in vivo studies. Here, our method describes an in vitro fermentation test bed to provide a physiologically-relevant assessment of engineered probiotics circuit design functions. Typically, engineered probiotics are evaluated under pristine, monoor co-culture conditions and transitioned directly into animal or human studies, commonly resulting in a loss of desired function when introduced to complex gut communities. Our method encompasses a systematic workflow entailing fermentation, molecular and functional characterization, and statistical analyses to validate an engineered probiotic’s persistence, plasmid stability and reporter response. To demonstrate the workflow, simplified polymicrobial communities of human gut microbial commensals were utilized to investigate the probiotic Escherichia coli Nissle 1917 engineered to produce a fluorescent reporter protein. Commensals were assembled with increasing complexity to produce a mock community based on nutrient utilization. The method assesses engineered probiotic persistence in a competitive growth environment, reporter production and function, effect of engineering on organism growth and influence on commensal composition. The in vitro test bed represents a new element within the Design-Build-Test-Learn paradigm, providing physiologically-relevant feedback for circuit re-design and experimental validation for transition of engineered probiotics to higher fidelity animal or human studies.

The Tri-Service Microbiome Consortium (TSMC) was founded to enhance collaboration, coordination, and communication of microbiome research among U.S. Department of Defense (DoD) organizations and to facilitate resource, material and information sharing among consortium members. The 2019 annual symposium was held 22-24 October 2019 at Wright-Patterson Air Force Base in Dayton, OH. Presentations and discussions centered on microbiome-related topics within five broad thematic areas: 1) human microbiomes; 2) transitioning products into Warfighter solutions; 3) environmental microbiomes; 4) engineering microbiomes; and 5) microbiome simulation and characterization. Collectively, the symposium provided an update on the scope of current DoD microbiome research efforts, highlighted innovative research being done in academia and industry that can be leveraged by the DoD, and fostered collaborative opportunities. This report summarizes the presentations and outcomes of the 3rd annual TSMC symposium.

Background Interactions between diet, stress and the gut microbiome are of interest as a means to modulate health and performance. Here, in vitro fermentation was used to explore the effects of a sudden change in diet, 21 days sole sustenance on the Meal, Ready-to-Eat (MRE) U.S. military combat ration, on inter-species competition and functional potential of the human gut microbiota. Human fecal samples collected before and after MRE intervention or consuming a habitual diet (HAB) were introduced to nutrient-rich media supplemented with starch for in vitro fermentation under ascending colon conditions. 16S rRNA amplicon and Whole-metagenome sequencing (WMS) were used to measure community composition and functional potential. Specific statistical analyses were implemented to detect changes in relative abundance from taxa, genes and pathways. Results Differential changes in relative abundance of 11 taxa, Dorea, Lachnospira, Bacteroides fragilis, Akkermansia muciniphila, Bifidobacterium adolescentis, Betaproteobacteria, Enterobacteriaceae, Bacteroides egerthii, Ruminococcus bromii, Prevotella, and Slackia, and nine Carbohydrate-Active Enzymes, specifically GH13_14, over the 24 h fermentation were observed as a function of the diet intervention and correlated to specific taxa of interest. Conclusions These findings suggest that consuming MRE for 21 days acutely effects changes in gut microbiota structure in response to carbohydrate but may induce alterations in metabolic capacity. Additionally, these findings demonstrate the potential of starch as a candidate supplemental strategy to functionally modulate specific gut commensals during stress-induced states.

The host–microbe interaction is critical for intestinal homeostasis. By‐products from microbial metabolism of unabsorbed dietary components have been studied increasingly as potential contributors to health and disease. In vitro fermentation systems provide a way to simulate microbial activity and by‐product production of the colon using human fecal samples. Objectives of the study were to determine how clarified supernatants from two different fermentation conditions affect markers of cell proliferation, differentiation, barrier function, and immune function in a human‐induced pluripotent (iPSC) colon organoid model. SCFA and BCFA's of the supernatants were analyzed and were similar to known in vivo concentrations. Molecular results showed 25% of the clarified supernatant from batch fermentation led to a more physiological intestinal phenotype including increased markers of differentiation, including alkaline phosphatase, chromogranin A, SCFA transport monocarboxylate transporter‐1, (6.2‐fold, 2.1‐fold, and 1.8‐fold, respectively; p < 0.05). Mucin production (mucin‐2, mucin‐4) was increased in cells treated with 25% supernatant, as observed by confocal microscopy. In addition, increased tight junction expression (claudin‐3) was noted by immunofluorescence in 25% supernatant‐ treated cells. A dose–response increase in barrier function was observed over the 72‐h time course, with a twofold increase in transepithelial electrical resistance (TER) in the 25% group compared to the control group (p < 0.05). To further investigate host effects, clarified supernatants from a continuous multistage fermentation representing the ascending (AC), transverse (TC), and descending (DC) colonic domains were utilized and some regional differences were observed including increased markers of inflammation (IL‐1β, 6.15 pg/ml; IL‐6, 27.58 pg/ml; TNFα, 4.49 pg/ml; p < 0.05) in DC‐treated samples only. Overall, clarified supernatants represent a valuable model to examine effects of microbial by‐products on host intestinal development and function and future efforts will be designed to further understand microbial communities and metabolites, along with additional host response measures.

Objectives Vitamin K (VK) is a family of structurally-related quinones, phylloquinone (PK) and menaquinones (MKn, n = prenyl units in side chain), that share a common napthoquinone ring (menadione, MD). VK quinones function as an essential dietary nutrient for humans. MD is considered a pro-vitamin form of VK. Plants and bacteria that produce VK quinones (PK and MKn, respectively) use them as an electron carrier in energy production. Little is known about the interaction of dietary VK quinones with gut bacteria, which may be bi-directional. The objective of this study was to investigate the influence of VK quinones and MD on human gut bacteria composition and MKn production. Methods Stool from 5 healthy male donors was pooled and inoculated in bioreactors under conditions mimicking the colon (anaerobic, pH 6.8, 37°C) for 48 h. Bioreactors were treated with deuterium (2H)-labeled quinones (2H-PK, 2H-MK4, 2H-MK9 or 2H-MD); no quinones (cell controls); or 2H-quinone treatment with no stool (cell-free controls). Culture aliquots were collected at 0, 5, 10, 24, and 48 h, and separated into pellet and supernatant fractions. Experiments were conducted in triplicate. All fractions were analyzed for VK quinone content using LC-MS. DNA from 0 and 24 h pellet fractions was extracted and amplified for paired-end 16S sequencing on an Illumina MiSeq 2500. Differences in bacterial composition were assessed using PERMANOVA. Results Supplemented 2H-quinones accumulated in the pellet fraction over time. This was not observed in cell-free controls and was thus not a function of culture media solubility. Endogenous (unlabeled) production of MKn was unaffected by supplementation of 2H-quinones. Generated 2H-MKn (2H-MK4, 2H-MK9, 2H-MK10, and 2H-MK11) were only detected in 2H-MD supplemented vessels. Community-wide bacterial composition significantly differed between 0 h and 24 h (r2 = 0.85, P = 0.001), but not by quinone treatment. Conclusions PK and MKn, dietary viamin K quinones, were not transformed by gut microbes to MKn in vitro, whereas the pro-vitamin quinone MD was transformed to MKn of multiple side chain lengths. Although no quinone induced community-wide changes in bacteria composition, additional analyses are needed to assess species-specific growth promotion. Funding Sources USDA ARS and DOD Health Program.