In vitro fermentation test bed for evaluation of engineered probiotics in polymicrobial communities.

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, mono- or 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.

Intestinal in vitro and ex vivo Models to Study Host-Microbiome Interactions and Acute Stressors.

The gut microbiome is extremely important for maintaining homeostasis with host intestinal epithelial, neuronal, and immune cells and this host-microbe interaction is critical during times of stress or disease. Environmental, nutritional, and cognitive stress are just a few factors known to influence the gut microbiota and are thought to induce microbial dysbiosis. Research on this bidirectional relationship as it pertains to health and disease is extensive and rapidly expanding in both in vivo and in vitro/ex vivo models. However, far less work has been devoted to studying effects of host-microbe interactions on acute stressors and performance, the underlying mechanisms, and the modulatory effects of different stressors on both the host and the microbiome. Additionally, the use of in vitro/ex vivo models to study the gut microbiome and human performance has not been researched extensively nor reviewed. Therefore, this review aims to examine current evidence concerning the current status of in vitro and ex vivo host models, the impact of acute stressors on gut physiology/microbiota as well as potential impacts on human performance and how we can parlay this information for DoD relevance as well as the broader scientific community. Models reviewed include widely utilized intestinal cell models from human and animal models that have been applied in the past for stress or microbiology research as well as ex vivo organ/tissue culture models and new innovative models including organ-on-a-chip and co-culture models.

Associations between the gut microbiota and host responses to high altitude.

Hypobaric hypoxia and dietary protein and fat intakes have been independently associated with an altered gastrointestinal (GI) environment and gut microbiota, but little is known regarding host-gut microbiota interactions at high altitude (HA) and the impact of diet macronutrient composition. This study aimed to determine the effect of dietary protein:fat ratio manipulation on the gut microbiota and GI barrier function during weight loss at high altitude (HA) and to identify associations between the gut microbiota and host responses to HA. Following sea-level (SL) testing, 17 healthy males were transported to HA (4,300 m) and randomly assigned to consume provided standard protein (SP; 1.1 g·kg-1·day-1, 39% fat) or higher protein (HP; 2.1 g·kg-1·day-1, 23% fat) carbohydrate-matched hypocaloric diets for 22 days. Fecal microbiota composition and metabolites, GI barrier function, GI symptoms, and acute mountain sickness (AMS) severity were measured. Macronutrient intake did not impact fecal microbiota composition, had only transient effects on microbiota metabolites, and had no effect on increases in small intestinal permeability, GI symptoms, and inflammation observed at HA. AMS severity was also unaffected by diet but in exploratory analyses was associated with higher SL-relative abundance of Prevotella, a known driver of interindividual variability in human gut microbiota composition, and greater microbiota diversity after AMS onset. Findings suggest that the gut microbiota may contribute to variability in host responses to HA independent of the dietary protein:fat ratio but should be considered preliminary and hypothesis generating due to the small sample size and exploratory nature of analyses associating the fecal microbiota and host responses to HA. NEW & NOTEWORTHY This study is the first to examine interactions among diet, the gut microbiota, and host responses to weight loss at high altitude (HA). Observed associations among the gut microbiota, weight loss at HA, and acute mountain sickness provide evidence that the microbiota may contribute to variability in host responses to HA. In contrast, dietary protein:fat ratio had only minimal, transient effects on gut microbiota composition and bacterial metabolites which were likely not of clinical consequence.

Effects of Psychological, Environmental and Physical Stressors on the Gut Microbiota.

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.

Comparison of the fecal microbiota in feral and domestic goats.

Animals have co-evolved with mutualistic microbial communities, known as the microbiota, which are essential for organ development and function. We hypothesize that modern animal husbandry practices exert an impact on the intestinal microbiota. In this study, we compared the structure of the fecal microbiota between feral and domestic goats using the G2 PhyloChip and assessed the presence of five tetracycline resistance genes [tet(M), tet(S), tet(O), tet(Q) and tet(W)] by PCR. Feces were collected from 10 goats: 5 domestic from a farm in the main island of Puerto Rico and 5 feral from the remote dry island of Mona. There were 42 bacterial phyla from 153 families detected in the goats' feces. A total of 84 PhyloChip-OTUs were different in the fecal microbiota of feral and domestic goat. Both feral and domestic goats carried antibiotic resistance genes tet(O) and tet(W), but domestic goats additionally carried tet(Q). Diet, host genetics and antibiotic exposure are likely determinant factors in shaping the intestinal microbiota and may explain the differences observed between feral and domestic goats fecal microbiota.