MEETING REPORT Open Access

The current state and future direction ofDoD gut microbiome research: a summaryof the first DoD gut microbiomeinformational meetingSteven Arcidiacono1, Jason W. Soares1, J. Philip Karl2, Linda Chrisey3, C. P. T. Blair C. R. Dancy4, Michael Goodson5,Fredrick Gregory6, Rasha Hammamieh4, Nancy Kelley Loughnane5, Robert Kokoska6, C. A. P. T. Mark Riddle7,Keith Whitaker8 and Kenneth Racicot1*

Abstract

The gut microbiome is increasingly recognized as integral to human health, and is emerging as a mediator ofhuman physical and cognitive performance. This has led to the recognition that US Department of Defense (DoD)research supporting a healthy and resilient gut microbiome will be critical to optimizing the health andperformance of future Warfighters. To facilitate knowledge dissemination and collaboration, identify resourcecapabilities and gaps, and maximize the positive impact of gut microbiome research on the Warfighter, DoDpartners in microbiome research participated in a 2-day informational meeting co-hosted by the Natick SoldierResearch, Engineering and Development Center (NSRDEC) and the US Army Research Institute of EnvironmentalMedicine (USARIEM) on 16–17 November 2015. Attendee presentations and discussions demonstrated that multipleDoD organizations are actively advancing gut microbiome research. Common areas of research included theinfluence of military-relevant stressors on interactions between the microbiome and Warfighter biology,manipulation of the microbiome to influence Warfighter health, and use of the microbiome as a biomarker ofWarfighter health status. Although resources and capabilities are available, they vary across laboratories and it wasdetermined that centralizing certain DoD capabilities could accelerate progress. More significantly, the meetingcreated a foundation for a coordinated gut microbiome and nutrition research program aligning key DoD partnersin the area of microbiome research. This report details the presentations and discussions presented during the 1stDoD Gut Microbiome Informational Meeting.

Keywords: Microbiome, Performance, Warfighter, Nutrition, Stressors, Biomarkers, Health, Disease, PTSD, Gut-brain axis

IntroductionThe human body hosts trillions of microorganisms, whosecollective genetic composition is collectively known as thehuman microbiome [1–3]. The human gut microbiomecomprises a diverse, dense and active microbial ecosystem,also known as a microbiota, that resides in the gastro-intestinal tract [1] and largely co-exists in a mutualistic re-lationship with the host. The composition and activity ofgut microbes are modulated by changes in host diet and

physiology while, in turn, gut microbes support immunehealth, deter pathogen invasion, regulate central andenteric nervous system activity, and generate beneficialnutrients and metabolites [4–6]. These beneficial rela-tionships, however, can be perturbed by environmentalstressors that directly or indirectly modulate the gut micro-biome, thereby resulting in a state of dysbiosis that hasbeen implicated in the development of acute health decre-ments, such as systemic inflammation [7], increased sus-ceptibility to illness and infection [8], cognitive decrements[9, 10] and the development and/or persistence of multiplechronic diseases [8].

* Correspondence: kenneth.racicot.civ@mail.mil1Natick Soldier Research Development & Engineering Center, Boston, MA,USAFull list of author information is available at the end of the article

© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Arcidiacono et al. Standards in Genomic Sciences (2018) 13:5 https://doi.org/10.1186/s40793-018-0308-0

The launch of the Human Microbiome Project in2007, supported with a $173 million investment fromthe NIH Common Fund (2007–2012) to advance humanmicrobiome research, demonstrates government recog-nition of the microbiome’s potential importance to hu-man health. The significance of microbiome-relatedresearch to global health priorities was further accentu-ated in May 2016, with the announcement of the WhiteHouse National Microbiome Initiative [11] to accelerateand coordinate efforts to explore the role of the micro-biome in the environment and in human health and dis-ease. Similarly, there is growing recognition within theUS Department of Defense (DoD) that research support-ing a healthy and resilient gut microbiome will be crit-ical for optimizing the health and performance of futureWarfighters [12]. Warfighters are commonly exposed todegrading factors that compromise physical and cogni-tive performance such as sleep loss, suboptimal nutri-tion, environmental extremes, and prolonged physicalexertion. Understanding the extent to which these fac-tors degrade Warfighter performance, determining theunderlying mechanisms, and identifying mitigation andresiliency strategies is a central theme within multiple re-cent DoD research and development directives [13–17].The gut microbiome is emerging as one factor that maymediate the effects of these stressors on Warfighter healthand performance. Since the gut microbiome is integral tohealth, but also malleable and strongly influenced bythe host diet, nutrition-based interventions could providenovel, low-cost strategies for optimizing Warfighterhealth and performance through modulation of thegut microbiome.The Natick Microbiome Interest Group (NMIG) is a

recently formed research cluster exploring interactionsbetween nutrition and gut microbiome to understand in-fluence on Warfighter health and performance. The groupis comprised of members from the US Army ResearchInstitute of Environmental Medicine (USARIEM), theCombat Feeding Directorate (CFD) and Warfighter Direct-orate (WD), US Army Natick Soldier Research, Develop-ment and Engineering Center (NSRDEC), all located atNatick Soldier Systems Center (NSSC). Upon its formationin 2014, the NMIG recognized that although multiple DoDorganizations were actively supporting gut microbiome-related research efforts, a coordinated gut microbiome andnutrition research program was absent. The group envi-sioned that enhancing coordination and information ex-change across organizations conducting microbiomeresearch could lead to more efficient use of resources andavoid duplication of efforts as DoD nutrition and gutmicrobiome research developed. To realize this goal, theNMIG hosted an informational meeting at NSSC on 16–17 November 2015 with the objective of aligning key DoDpartners in multiple areas of microbiome research. The

meeting goals included facilitating collaboration amongstscientists, identifying gaps within DoD gut microbiomeresearch, enhancing knowledge sharing and coordinationof research efforts, and identifying currently availableresources and capabilities. Attendees represented mul-tiple government organizations conducting microbiomeresearch. On Day One of the event, invited DoD scientistspresented an overview of gut microbiome research andareas of interest within their organizations (Table 1). Dur-ing Day Two, four breakout sessions allowed for deepdives into subject areas relevant to identifying DoD nutri-tion and gut microbiome research gaps and needs. Thisreport summarizes the discussion and conclusions of the1st DoD Gut Microbiome Informational Meeting.

Day 1: Invited presentationsDr. Robert Kokoska (Army Research Office (ARO)Microbiology Program Manager) presented an overview ofextramural basic research programs sponsored by the LifeSciences Division of the US Army Research Laboratory(ARL). Dr. Kokoska described ARO programs as contribut-ing to our understanding of the characterization, inherentinteractions and downstream effects of various microbialcommunities, including the gut microbiome. The AROProgram in Microbiology, managed by Dr. Kokoska, has aprimary strategic focus on the analysis and engineer-ing of microbial communities. Single Investigator grantsand Multidisciplinary University Research Initiative (MURI)awards managed under this program address the challengesof establishing functional links between species with diver-gent roles within a microbial consortium, understandingthe role of spatial order within an interactive microbial con-sortia and the development of tools, methodologies andengineered systems that can effectively control and analyzethe physical and biochemical characteristics of both simpleand complex microbial communities. Within this program,research efforts that provide “bottom-up” approaches, i.e.,those that examine simple well-ordered communities, holdthe promise of uncovering fundamental interactive andcontrol principles within a consortium that can aid our un-derstanding of the forces that drive community structureand function.In contrast to more controllable “bottom-up” approaches,

most studies of the gut microbiome take on more data-rich“top-down” approaches toward untangling the myriad in-teractions within this highly complex natural community inorder to infer functional output from the microbial consor-tia. The downstream functional effects of the gut micro-biome are themselves highly complex and networked. Forexample, it has been theorized that there exists a bi-directional link between the gut microbiome and the brain,in which neural, hormonal, immunological and microbialsignaling pathways are networked. This link between gutmicrobes and brain function has thus drawn technical and

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programmatic interest from the Neurophysiology of Cogni-tion Program at ARO Life Sciences managed by Dr. Fred-erick Gregory. Dr. Gregory’s program is focused on themolecular, cellular and systems-level neural codes under-lying human perception and behavior. In line with both theARO Microbiology and Neurophysiology of Cognition pro-gram interests, it is thought that nutritional inputs andphysical stressors can affect the composition of the gutmicrobiome in a manner that influences these various dy-namic interactions between the gut and the brain, ultim-ately affecting cognition and behavior. The multi-layeredcomplexity of interactions between the gut microbiomeand the networked physiology, including neural and im-mune dynamics, confounds our ability to establish testablehypotheses that can point the way toward causative ratherthan correlative effects within this interactive network.Thus, a need has been identified to establish a systems-level mathematical modeling framework that can betterguide our understanding of the performance of themicrobiome-gut-brain axis. To meet this challenge, Dr.Gregory and Dr. Kokoska have teamed to establish anArmy MURI topic that looks towards the development ofa layered, cellular and system-level model that postulatescognitive and behavioral control of integrated neural,endocrine and immune interactions by commensal gutmicroorganisms in response to nutrition and physical

stress. It is envisioned that the ability to inform and refinethe model with metabolic data will generate multiscalepredictions, constrain empirical approaches, and directhypothesis-driven research toward an understanding ofthe causative linkages within the gut-brain axis.Dr. Rasha Hammamieh presented an overview of

Integrative Systems Biology (ISB) Program at US ArmyCenter for Environmental Health Research (USACEHR),where they are trying to characterize molecular eventsassociated with disease progression and identify path-ways and networks to diagnose and predict the course ofimpending illness. This is carried out using clinical andpatho-psychophysiological information with multi-omicsand phenotypic readouts. ISB has done extensive work witha multi-core Post Traumatic Stress Disorder (PTSD) groupand have used a cohort of PTSD+/− volunteers to identifyepigenomic and genomic changes unique to PTSD individ-uals. An essential project is a study to understand the regu-lation of the microbiome profile in response to traumaticstress. In a mouse model simulating features of PTSD [18],the ISB group showed that simultaneous perturbed func-tioning of multiple organ systems (e.g., brain, heart, intes-tine, liver) that can produce injuries that lead to chronicmetabolic changes associated with PTSD [19]. Themetabolomics analysis showed altered gut-derived me-tabolites in plasma at 24 h post-stressor exposure and

Table 1 DoD organizations and their general interests related to gut microbiome research

Organization Interests

Army Research Office Funding fundamental research toward understanding inherent interactions and downstreameffects of various microbial communities including the gut microbiome

Army Research Laboratory Understanding how gut microbiome influences human behavior, capability enhancementand human-system interaction, waste-to-energy conversion through metabolic pathwayengineering of gut-derived microbiota

US Army Research Institute of Environmental Medicine Conducts research providing a biomedical science basis for developing new rations,menus, policies and programs that enable Warfighter health-readiness and optimalperformance; microbiome interests include interactions between military-relevant stressors,diet, and the gut microbiome, particularly within austere environments

US Army Center for Environmental Health Research Molecular events associated with disease progression; microbiome as a tool formonitoring Warfighter exposure to environmental toxicants

Naval Medical Research Center Deployment health implications of gastrointestinal infection, the microbiome and bothacute and chronic disease

Office of Naval Research Funding programs to understand the effects of certain behavioral and environmentalstressors on a host and its gut microbiota, with an emphasis on deducing the role thegut microbiota may play in mediating psychological, cognitive and physiological effectsof such exposures

US Army Natick Soldier Research, Development andEngineering Center

In vitro studies to determine military-relevant stressor effects on the microbiome andhost to inform clinical trial design; elucidation of mechanistic knowledge of dietary inputbiotransformation by gut bacteria; optimizing combat feeding rations; performance nutrition

US Air Force Research Laboratory Influence human performance in a non-invasive manner (cognition, anxiety, stress)

Walter Reed Army Institute of Research Biomedical research that delivers lifesaving products including knowledge, technologyand medical material that sustain the combat effectiveness of the Warfighter

Uniformed Services University of the Health Sciences Nutrition, gastroenterology; Consortium for Health and Military Performance: research toimprove service member performance in the field and returning to duty; animal studies

Naval Surface Warfare Center Dahlgren Division Understanding influence of native and engineered biological threats on human health

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these remained altered up to 4 weeks after stressorwithdrawal.A recent review of the literature has demonstrated that

gut bacteria strongly influence the metabolic, immune,endocrine, peripheral and central nervous systems.These gut-brain interactions are considered as bidirec-tional with major consequences for outcome of the dis-ease. Considering the role of microbiome in psychiatricdisorders, the ISB team also utilized fecal samples fromthe longitudinal study using the established rodent modelsimulating aspects of PTSD. The ISB group’s profiling in-cluded sequencing variable regions 3 and 4 of the 16SrRNA gene from fecal samples collected every day wheresubject mice were exposed to aggressor mice for 10 days.Bacterial community analysis included looking at commu-nity composition using Quantitative Insights into Micro-bial Ecology pipelines [20]. The analyses indicated thataggressor exposure did not impact the alpha diversityamong samples at different time points. However, differ-ences in Firmicutes and Verrucomicrobia populations inaggressor-exposed mice were observed as early as 24 h.Further, Oscillospira, Lactobacillus, Akkermansia andRuminococcus were identified as the top four genera thatwere changed between control and aggressor-exposedmice. They are looking further into the functional pathwaypredictions based on bacterial composition at a particulartime point using Phylogenetic Investigation of Communi-ties by Reconstruction of Unobserved States [21], withpreliminary indications that some changes in key meta-bolic regulators can be a consequence of the microbiomedysregulation in the presence of stress. These studies willprovide new insights into how the microbiome changesupon stress exposure, although deeper analysis into thestructure of the microbiome is required to identify thespecies level classification. Further, it is important to studymucosal versus luminal microbiome to determine the rolein functional consequence of the disease given that differ-ences between responses of the mucosal and luminalmicrobiomes to stress have been reported [22].Drs. Keith Whitaker and Justin Brooks presented an

overview of the ARL Human Science Campaign, with aspecific focus on how the gut microbiome influences hu-man behavior, capability enhancement, and human-systeminteraction. Dr. Whitaker and Dr. Brooks asserted that inorder to achieve the goal of maximizing the effectivenessof Warfighters physically, perceptually, and cognitively, theArmy must invest in understanding and engineeringthe human-associated microbiome as a component ofinter- and intra-individual variability in performance. ARL’sintramural investment in the Human Sciences includes adiverse set of priorities centered on the integration of arange of technologies and concepts into multi-scale,human-centric research and advanced technology develop-ment. Within that context, there is an open question about

the relative contribution of the gut microbiome, as com-pared to other sources of performance variability, toWarfighter performance. Throughout ARL’s intramural re-search portfolio, there are research and technology devel-opment projects that can be leveraged to support theArmy’s need for altering the human gut microbiome, evenif that is not the primary objective. For example, Dr.Whitaker leads a high-risk research project on the bio-engineering of extracellular vesicles with the intention ofspecifically, temporarily and reversibly altering cellular ac-tivity to inhibit inflammation after a traumatic brain injury.Extracellular vesicles are produced by many different cellstypes, including the bacteria of the human gut, and thesecarry nucleic acids and proteins into receptive cells. Theability to manipulate the contents and characteristics ofthese nano-sized vesicles may provide new biotechnologyto influence the interaction of the human-microbiome sys-tem. On a larger scale, one of ARL’s newest programs,Continuous Multi-faceted Soldier Characterization forAdaptive Technologies (CMSCFAT), is focused on charac-terizing how the relationship between behavioral, physio-logical, environmental, and task-based factors drive andmay predict variability in task performance in real-worldenvironments. Recent advancements in our understandingof how microbial metabolism (particularly in the gut) influ-ences host factors strongly suggest that the microbiota mayhave a profound effect on human performance, state, andemotion. As part of CMSCFAT, ARL will encourage andsupport internal research proposals that examine the gutmicrobiome as a potential source of behavioral and per-formance variability within the context of other physio-logical and environmental variables. Currently, researchprojects that have collected whole blood samples may beused as part of this effort to investigate microbial metabo-lites that have entered through the enteric system, whichwill then be related to behavioral and physiological variabil-ity. While research in this area is nascent within ARL, thereis growing interest in how the gut microbiome may affecthuman performance and mechanisms that are in place tosupport the development of this line of research.Captain Blair Dancy, PhD of USACEHR, located at

Fort Detrick, Maryland, described how efforts withinUSACEHR’s Environmental Health Program (EHP) arecurrently advancing a multi-institutional initiative to ex-plore the utility of the microbiome as a tool for monitor-ing Warfighter exposure to environmental toxicants. CPTDancy then outlined the rationale underlying microbiomeresearch efforts underway or planned within the EHP. Inthe context of an environmental exposure, the micro-biome represents the very first interface between an exter-nally encountered chemical and the toxicant-manifestedclinical disease [23]. This initial interaction between atoxicant and the microbiome provides for a valuable op-portunity to develop microbiome-based biomarkers of

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exposure and susceptibility that could be used as early in-dicators of exposure or employed to provide longitudinalexposure surveillance. Furthermore, the relationship be-tween environmental toxicants and the microbiome is dy-namic. Not only is the microbiome subject to the effectsof toxicants (Toxicant Modulation of the Microbiome(TMM)), but toxicants are subject to modification by themicrobiome, potentially resulting in altered toxicity pro-files for compounds (Microbiome Modulation of Toxicity(MMT)) (Fig. 1).Initial efforts at the USACEHR to explore the role that

environmental toxicants have on the microbiome haveused fecal pellets collected from conventional rats, pre-and post-exposure to toxicants, including sodium arsenite,cadmium chloride, sodium dichromate, nickel chloride,and cobalt chloride. Fecal samples were analyzed throughacademic and industrial collaborators using 16S rRNAanalysis, small-molecule metabolomics and lipidomics.These analyses are expected to reveal panels of specificbacterial taxonomic groups and small molecules whoseperturbations are indicative of toxicant exposure. To bet-ter address the utility of the human microbiome as an ex-posure surveillance tool, the USACEHR is currently usinga humanized-gut-microbiota-mouse as a toxicant expos-ure model. These mice, which are born germ free, are pro-vided a human-gut flora through a procedure known asfecal microbiota transplantation. The USACEHR has part-nered to obtain a screened source of human fecal materialand deliver it into a commercial source of germ-free ani-mals, and has currently conducted several exposures util-izing the humanized-gut-microbiota mice. Fecal materialfrom the humanized-gut-microbiota mouse exposures willbe longitudinally collected over the course of the experi-ment’s duration. These fecal samples will then be analyzedusing next-generation sequencing, in collaboration withthe U.S. Army Medical Research Institute of Infectious

Diseases (USAMRIID), and metabolomics tools will beutilized to identify biomarkers of exposure. The USA-CEHR is also currently partnering with other organiza-tions, including the U.S. Air Force Research Laboratory(AFRL), 711th Human Performance Wing, to explore theeffect that sodium dichromate and engineered nanomater-ials have upon the microbiome. Additionally USACEHRhas partnered with the U.S. Army Public Health Centerfor completing a review on the role of the microbiome inchemical toxicity, and to host a multi-organizational meet-ing at the Defense Health Headquarters to evaluate themicrobiome role in health risk assessment. Finally, USA-CEHR also partnered with the Johns Hopkins UniversityApplied Physics Laboratory, a University Applied ResearchCenter (UARC), and the Massachusetts Institute of Tech-nology Lincoln Laboratory, a Federally Funded Researchand Development Center (FFRDC), to develop tools to ex-plore the host response to toxicant exposures and to pro-vide new sequencing and bioinformatics tools to profilebacterial communities using 16S rRNA sequencing, shot-gun metagenomic datasets, and mobile genetic elements.CAPT Mark Riddle, MD of the Naval Medical Research

Center (NMRC) presented an overview of research efforts,gaps and potential materiel solutions relative to deploy-ment health implications of gastrointestinal infection, onthe microbiome and acute/chronic disease. CAPT Riddledirects the US Military program on research relatedto vaccine development of bacterial enteric infections,as well as discovery and translational efforts in thearea of intestinal disease biomarkers and optimizedtreatment strategies. The areas explored in his overviewincluded implications on prevention and treatment ofacute travelers’ diarrhea, the chronic consequences ofthese common deployment health infections, and im-plications in vaccinology (schematic overview shownin Fig. 2).

Fig. 1 The Microbiome-Toxicant Dynamic

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Acute enteric infection during deployment is rankedas the 2nd leading infectious disease threat to militaryservice members and is a significant contribution ofmorbidity in the United States [24]. On average, 29% permonth become afflicted with a diarrheal illness duringdeployment in the developing world, and the majority ofthese infections are due to bacterial etiologies. Whilevaccines against the leading causes of bacterial diarrheas(Campylobacter jejuni, Shigella spp. and enterotoxigenicE. coli) are under development based on DoD require-ments and the Military Infectious Diseases ResearchProgram, these are long-term solutions while short- andintermediate-term solutions are needed to mitigate thisdisease burden and its effect on mission readiness. Asour understanding of the importance of the humanmicrobiome in health and disease has advanced, interestin the use of nonpathogenic bacteria and yeast (probio-tics), as well as nutrients which enhance the growth offavorable microbes (prebiotics) is an appealing concept,given the potential ease of use, and, being natural productderivatives, appear to be a safe alternative to antibiotics[25]. Mechanisms including competition for nutrients,competition for adhesion sites, direct antagonism, and im-mune stimulation have been explored and offer a strongrationale to support enhancement of the host-microbiomeas a “gut shield system” in the Warfighter. However, des-pite the attractiveness of this strategy, to date clinical stud-ies that have evaluated only a few probiotic/prebiotic

products provide results that are too variable to supportcurrent use [26, 27]. Findings from relevant studies havealso been difficult to interpret due to differences in thespecies, formulations and dosages of probiotics studies,and also due to methodological problems within the stud-ies themselves (i.e., poor compliance, recall bias). Compli-cating this matter is the fact that these products, whichhave been evaluated for the indication of acute enteric ill-ness prevention, have not undergone the necessary FDAregulated trials required for a product to be used for a pre-ventative indication under Force Health Protection regula-tions. However, despite limitations in the current evidencebase, a commercially available prebiotic has demonstratedsome evidence of efficacy [28], and researchers at theNaval Medical Research Center are actively pursuing anInvestigational New Drug clinical trial under industrysponsorship. In addition to the acute symptoms andmorbidity associated with diarrhea during deployment, agrowing body of literature from both military and non-military populations is defining a significant burden ofchronic gastrointestinal disorders following acute entericinfections [29, 30]. Further, the recognition of multi-drugresistant organism (MDRO) acquisition among travelerswith diarrhea independent of antibiotics being used,calls for further efforts to consider how to enhanceeither colonization resistance against such MDRO ordisplacement of these colonizers during or post travel ordeployment [31]. While gaps remain in our complete

Fig. 2 Schematic representation of NMRC microbiome research and key questions currently under consideration. Horizontal bars show scientifictasks associated with the research domains vaccinology (blue), chronic consequences (purple), and TD treatment and prevention (turquoise) andwhat technology readiness levels they fit. 6.1, 6.2 etc. are DoD designations for the various technology levels

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understanding of these complex disorders, gut barrier in-tegrity, inflammation, and dysbiosis are hallmarks of dis-ease, which can result in life-long disability. Researchersfrom the NMRC have begun discovery efforts in collabor-ation with professional society and industry support totry and understand basic pathobiology underpinningindividual susceptibility and diagnostic markers thatmay eventually lead to precision medicine directedstrategies for disease prevention, interception or treatment[32, 33]. Interesting observations were discussed related tothe host-microbiome and vaccinology. For example, re-cent studies have shown how the host microbiome is animportant component in the host response to seasonal in-fluenza vaccination [34] and how interference of inductionof functional antibody responses to an experimental hu-man HIV-1 vaccine was associated with cross-reactingmicrobiota antibodies [35]. These early observations high-light the importance of understanding how the micro-biome may impact the variation in host responses,specifically to enteric vaccines under development and theimpact of enteric vaccines on the host-microbiome. Fi-nally, CAPT Riddle led a discussion on the challenges re-lated to lack of specified research priorities and fundingprograms to address these gaps, but noted the incredibleopportunity to uniquely study these important questionsthough active field site studies, biorepositories, and rele-vant small animal models.Dr. Linda Chrisey (Program Officer, Office of Naval

Research (ONR) Gut Microbiology for Warfighter Resili-ence Program) provided a background and objectives ofONR programs. The fairly young basic research pro-gram, is primarily funded through ONR’s Basic ResearchChallenge program (FY14 start) and an FY15 Multidis-ciplinary University Research Initiative topic (late FY15start). Dr. Chrisey provided background for her pro-grams by explaining that the commensal bacteria, fungiand viruses that comprise the gut microbiome, have tre-mendous influence on the development of the brain,central and enteric nervous systems; mucosal immunesystem and protection against pathogens, perception ofpain; extraction of nutrients from food and distributionof fat; as well as the microbiome’s direct role in severaldiseases. Thus, it is critically important to enhance ourunderstanding of this ‘microbial organ’ with a focus on itsrole in response to external stressors that can affect humanresilience. She elaborated by noting that specific roles forthe gut microbiome in emotional behavior, anxiety, mem-ory and cognitive function, and neuromodulator expres-sion have been documented, as well as supporting a rolefor the gut microbiome in bi-directional signaling via thegut-brain-axis, the hippocampal-pituitary axis, and foroverall homeostasis. Of particular interest to ONR is thestudy of behavioral stressors (e.g., fear, anxiety, socialcrowding) and environmental stressors (e.g., environmental

shifts in altitude, temperature, time zone, round-the-clockwork shifts, altered circadian rhythms, fatigue, sporadic nu-trition, pain, anticipatory anxiety, fear), to begin to func-tionally link specific microbes to observed host responses.Dr. Chrisey defined the objective of the ONR program asaiming to understand the effects of certain behavioral andenvironmental stressors on a host and its gut microbiota,with an emphasis on deducing the role the gut microbiotamay play in mediating psychological, cognitive and physio-logical effects of such exposures. More specifically “Doesthe gut microbiome play a role in host response to physicaland psychosocial stressors, and if so, can we envision waysto manipulate the microbiome to minimize negative re-sponses?” The potential impact of the program is to de-velop the capability to increase Warfighter resilience tostressors via manipulation of the gut microbiota, such asusing pre−/pro- or antibiotic prophylaxis or treatments.Such manipulation of the host-gut microbiota may allowus to enhance Warfighter fitness and resilience to thesestressors. The program will attempt to study analogs ofthese stressors for which solid animal models and usefulparadigms for human research exist and determine the role(if any) of the gut microbiota in the host response. Theprogram will: a) characterize the psychological, physio-logical and cognitive responses of the host and gut micro-biome following exposure to various stressors; b) ideallycapture system response from metabolites/small signalmolecules to whole organism(s); c) ascertain if specific gutmicrobial community members, metabolites or functionsplay a role in the observed stress response; d) utilize estab-lished animal models, assays, and standardized tests tomeasure psychological state, cognitive performance, andphysiological condition to link functions to microbes; e)utilize human studies to measure psychological state, cog-nitive performance, physiological condition after stress(sleep deprivation), measurement of microbial metabolites,and treatments to test hypotheses (pre−/pro−/antibiotics;membrane vesicles, microbial metabolites); and f) supportthe development of novel tools for real-time analysis of mi-crobes, the surfaces they adhere to, and microbial productsin the GI tract to more closely map heterogeneity andidentify links to function.Dr. J. Philip Karl presented an overview of the collab-

orative gut microbiome research efforts and related cap-abilities at USARIEM [36] and NSRDEC [37] co-locatedat the NSSC, Natick MA. Collectively, this team has initi-ated a multi-disciplinary research program that aims toidentify nutrition-based strategies for optimizing resiliencyin the gut microbiome to military-relevant stressors. Dr.Karl began by providing a high-level overview of theUSARIEM-NSRDEC team’s joint research program. Theinitial phase of the program was described as a nascent ef-fort aiming to characterize the effects of military-relevantoperational stressors on gut microbiota composition,

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function and activity, and to elucidate the consequences onWarfighter health and performance. Relevant operationalstressors were described as including dietary intake, under-nutrition, sleep deprivation, psychological stress, prolongedphysical activity, dietary supplements and medications, andenvironmental extremes (e.g., heat, altitude). Performancewas defined as primarily acute effects on physical and cog-nitive function. Dr. Karl then described the objective of theprogram’s second phase, which is to identify pre-, intra- orpost-exposure nutritional interventions for mitigating ad-verse effects of operational stressors on gut microbiomecomposition and activity. The latter portion of the presen-tation highlighted how the unique capabilities of theUSARIEM and NSRDEC partners will be integrated. Theresearch mission of the Military Nutrition Division, USAR-IEM is to conduct research that provides a biomedical sci-ence basis for developing new rations, menus, policies andprograms that enable Warfighter health-readiness and op-timal performance. Dr. Karl highlighted several ongoinghuman subject trials in which dietary intake (e.g., militaryration consumption) and environmental conditions (e.g.,military combat operations training) [38] were being ma-nipulated to elucidate effects on gut microbiota compos-ition and activity, and gut barrier function. Dr. Karl brieflydescribed the importance of the gut barrier in maintaininga selective boundary between the host and exogenouscompounds [39, 40], and cited evidence suggesting thatmilitary-relevant stressors could compromise Warfighterhealth and performance by altering gut barrier permeability[41, 42]. Further, the gut microbiome was identified as amalleable target for building resiliency to such stressors,and dietary intake was identified as the tool that would beutilized by the USARIEM-NSRDEC team to manipulatethe gut microbiome in future studies. Dr. Karl then transi-tioned to noting one of the WD, NSRDEC research mis-sions, which is to develop physiologically-relevant in vitrofermentation capabilities to provide models for under-standing nutrient-gut microbiome interactions. Dr. Karlhighlighted recent work by that team in which the gutmicrobiomes of human donors were maintained in an invitro batch fermentation system to study microbe-mediated metabolism of cranberry polyphenols [43]. Dr.Karl continued by describing how NSRDEC in vitro fer-mentation capabilities will expand into a continuous fer-mentation system capable of replicating the environmentwithin the ascending, transverse and descending regions ofthe human colon [44]. Physiologic relevance of the modelwill be enhanced through collaboration with the CombatFeeding Directorate, NSRDEC, wherein cell culture cap-abilities provide a means of challenging human cell lineswith fermentate generated during in vitro fermentation ex-periments. Coupling in vitro mammalian intestinal cell cul-ture models with in vitro fermentation experiments willallow basic mechanistic studies of pathways relevant to

inflammation, innate immunity, and gut permeability. Thisplatform will be integrated into the USARIEM-NSRDECresearch program as a cost-effective, high-throughputmeans for screening candidate nutrients and nutrient com-binations for their ability to restore gut microbiomehomeostasis following stressor-induced perturbations andfor their potential to enhance resiliency of the gut micro-biome to such stressors. Dr. Karl concluded by elaboratingon the mission of the CFD, NSRDEC, which includesidentifying and developing feeding solutions for all militaryservice branches. This critical position will provide oppor-tunities for transitioning the knowledge gleaned fromindependent and joint USARIEM-NSRDEC human and invitro studies into nutritional interventions that will subse-quently be evaluated in future human trials.Dr. Nancy Kelley-Loughnane, of the 711 Human Per-

formance Wing (HPW), AFRL, presented an overview ofhow the microbiome and synthetic biology fits into theAir Force’s strategic science and technology vision of a‘layered information’ concept of personalizing Airmanhealth and performance. In order to address the AirForce’s future needs as set forth in Air Force Future Op-erating Concept (September 2015) and Global Vigilance,Global Reach, Global Power for America, 711 HPW hasdeveloped cross cutting research primary mission areasin order to synergize its research and development andmedical efforts. The four primary mission areas (PMAs)include Airman Machine Teaming, Education-Training,Force Protection, and Airman Health and Performance.Understanding the microbiome and manipulating it viasynthetic biology approaches will play an important rolein the PMAs, especially Airman Health and Performance,which is defined as “the convergence of science, medicine,and engineering to enhance, optimize, and sustain thephysical, psychological, cognitive, and behavioral statesacross the Airman’s lifecycle to achieve airpower domin-ance.” The 711 HPW expertise in biological sciences andperformance measurement places it in a unique positionto revolutionize personalized performance optimizationfor the Airman.Ongoing microbiome-related efforts within the 711

HPW were highlighted that explore how microbiomesare modified by environmental conditions and how theymight be modified to augment human performance. TheOffice of Naval Research-funded study, led by Dr. VictorChan, is investigating how sleep-deprivation affects host in-testinal function and gut microbiota composition and me-tabolism. This will allow development of a host-microbemetabolic interaction model for sleep deprivation, enablingin silico testing of potential strategies to modulate gutmicrobiota to counter the effects of sleep deprivation.Studies led by Dr. Camilla Mauzy at 711 HPW are investi-gating the effect of toxicants on the microbiome of the gutand the lung, and how this affects host epidermal

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permeability. These data will be used to determine if spe-cific microbiome signatures can predict exposure andhealth. Finally, Dr. Michael Goodson, also from 711 HPW,highlighted an Air Force School of Aerospace Medicine-funded project that is investigating the role of the gutmicrobiome in deployment-associated diarrhea. The broadgoal of this research is to identify components of the gutmicrobiome that are protective against diarrheal infectionso that they can be used as a basis for prophylactic treat-ment in deployed personnel to reduce the incidence ofdiarrheal disease during and after deployment.Overall, the 711 HPW team along with its service and

academic partners are addressing the need to understandand the possibility of controlling the microbiome inorder to improve Airman Health and Performance.

Day two: Group breakout sessionsDay two of the information meeting featured workinggroup breakout sessions to identify the following withinthe DoD: 1) current capabilities; 2) research areas ofinterest; 3) rationale for gut microbiome research andpotential impact on the Warfighter; 4) fundamental andapplied knowledge gaps; 5) resource gaps; 6) future re-search directions. Two parallel sessions were held in themorning that focused on programmatic issues, followedby two parallel afternoon sessions that focused on tech-nical aspects of microbiome research. The programmaticsessions described what is needed to create an environ-ment for success in DoD gut microbiome research. Thetechnical sessions discussed the resources needed to exe-cute these research programs.

Programmatic perspectives for DoD gutmicrobiome researchDiscussion on programmatic perspectives was structuredaround the knowledge that could be gleaned frommicrobiome research, the impact and implications of theknowledge to the Warfighter, and how that knowledgecould be translated into strategies and products for im-proving Warfighter performance. Open discussions cen-tered on how Warfighters are exposed to multipleunique stressors potentially impacting the microbiome,often in combination, and that gut microbiome researchshould largely focus on interactions between suchstressors, the gut microbiome, and Warfighter healthand performance. This focus should complement andbuild upon, rather than duplicate, the gut microbiomeefforts being funded by the NIH and similar organiza-tions, which largely emphasizes disease outcomes andchronic health. Avoiding duplication of efforts withinthe DoD was also discussed as necessary to maximize re-sources, knowledge generation and technology transi-tion. To that end, it was suggested each service branchshould consider establishing specific expertise within

their research programs, making that expertise availableto other service agencies, and communicating their re-search goals, directions and outcomes. This effort couldinclude establishing a centralized infrastructure for someresearch activities, such as high-performance computing.However, it was recognized that some unique resourcesare not likely to be replicated within DoD laboratories.As such, partnering with academic institutions and in-dustry to meet gut microbiome-related research goalswill continue to be necessary.Research towards “purposeful manipulation” of the gut

microbiome will likely result in the development of novelstrategies for improving Warfighter health and perform-ance. The panel envisioned research aimed at engineeringthe gut microbiome by introduction of bacteria into theecosystem (e.g., probiotics), targeting bacteria currently inthe ecosystem through nutritional modulation, or viagenetically engineered novel microorganisms. The goalsidentified for gut microbiome manipulation were myriadand included stimulating production of metabolites to en-hance cognition and emotional state, modulating immuneresponsiveness to vaccines, promoting resistance to chem-ical attack, use of personalized medicine to inform inter-vention strategies, and reducing chronic disease risk.The microbiome was also seen as a predictive tool for per-formance and chronic health that could garner informationfor proactive decision-making to assist in determining themicrobiome response for certain tasks or resistance tostressors. Proposed 10 year research goals included devel-opment of foods or identification of supplements targetingthe gut microbiome that could be incorporated into mili-tary feeding scenarios. Additionally, introducing geneticallyengineered microbes into the human gut microbiome wasconsidered a 20 year and beyond research objective.Discussion also centered on the types of studies and

outcomes needed to elucidate the role of the gut micro-biome in Warfighter health, and to identify effective ap-proaches for leveraging the gut microbiome to benefitthe Warfighter. One current limitation is a lack of bio-logical sample repositories for fecal, urine and bloodsamples from new recruits and Warfighters throughouttheir military careers. Extensive discussion centered onthe utility of establishing policies for collecting and stor-ing biological samples, recording Warfighter health in-formation, being in compliance with guidelines for humanuse research, and creating protocols for retrieval of healthinformation by various organizations. These facilities andrepositories would facilitate prospective cohort studies,the lack of which was identified as a programmatic gap.Such studies would enable investigations into the long-term impact of military-relevant environments on the gutmicrobiome (e.g., pre- and post-deployment) and the rela-tion to Warfighter health. It was suggested that program-matic initiatives could also focus on improving current

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methods of obtaining and recording health and other in-formation from Warfighters. Current in-theatre informa-tion records were noted as incomplete, thereby creatinggaps in Warfighter health timelines. Few widely acceptedstandards exist for conducting gut microbiome research.Therefore, establishing standard protocols for sample col-lection and analysis is a prerequisite to establishing bios-pecimen repositories and large databases. This need iscorroborated by a recent study investigating the influenceof sample collection and handling on detection of bacter-ial taxa via qPCR, which correlated multiple parameters toDNA degradation [45]. Another standardization aspect toconsider is next-generation sequencing, typically through16S rRNA sequencing. Although a recent study hasdemonstrated that multiple bioinformatics pipelinescan identify similar bacterial composition within hu-man fecal samples from 16S rRNA sequencing data [46],the influence of the multitude of variables within the 16SrRNA sequencing technique, including extraction tech-niques, on the data sets generated for bioinformatics ana-lysis is unclear. As such, a programmatic focus on protocolstandardization was suggested. Subsequent to this informa-tion meeting, the National Institutes of Standards andTechnology hosted, from 9 to 10 August 2016, a Standardsfor Microbiome Measurements Workshop to discussstandardizing methods for gut microbiome research [47].A separate, but related programmatic issue identified

was the need to validate, define and standardize modelsand the outcomes used to measure the effects of inter-ventions. In order to translate research, establishing ap-propriate animal and in vitro models to complementhuman studies is critical. Currently there is a lack of val-idated robust models within DoD laboratories, althoughseveral groups (e.g. rat models at AFRL, humanized-gutmicrobiota mouse models at USACEHR, and in vitrofermentation models at NSRDEC and AFRL) are activelydeveloping promising models that can be leveraged bythe DoD research community. Research outcomes wouldbe significantly enhanced via use of standardized models.An additional barrier to translating gut microbiomeresearch is the fact that challenges posed by inter-individual differences and environmental factors can im-pede detection of intra-individual variances over time inresponse to stress (e.g., sleep circadian disruptions),highlighting the importance of longitudinal studies forthe duration of stressor exposure. In addressing theseissues, it was agreed that systems-level approachesthat integrate and correlate multiple biological samplematrices (e.g., blood, urine, fecal, tissue, saliva, etc.)would facilitate knowledge discovery and translation.The panel agreed that collaboration in using these modelsand incorporating standardized techniques and measure-ments will ultimately enhance DoD gut microbiomeresearch.

The panel recommended establishing a DoD Tri-ServiceMicrobiome Consortium (TSMC) to promote coordin-ation and collaboration of gut microbiome research andto provide a forum for sharing ideas, research activities,resources and data. In order to improve collaboration andresearch progress, it was recognized that capabilities needto be identified across various organizations. Increasingawareness of existing capabilities within organizations andthe development of novel capabilities was recognized ashaving a clear benefit to DoD gut microbiome research.Finally, it was recognized that microbiome research is re-source intensive, requires significant funding, and that oneof the primary challenges to existing research includes thecurrent availability and stability of funding. Sharing of re-sources and leveraging programs such as MURIs, SmallBusiness Technology Transfer (STTR)/Small BusinessInnovative Research (SBIR), Laboratory University Collab-oration Initiative (LUCI), and Defense University ResearchInstrumentation Program (DURIP) will be necessary. Sev-eral participants noted that requirements stated in DoDcapability documents are particularly influential in settingresearch priorities and funding, and that funding is oftendetermined by threat; however, at present, there is noestablished direct threat related to the human micro-biome, although microbiome-related interventions arepossible solutions. A focus of the TSMC would be cham-pioning the need for and importance of gut microbiomeresearch within the DoD, and identifying and securingfunding where appropriate.

Technical aspects of current and future DoD gutmicrobiome researchDiscussion of technical issues centered on the mechanicsfor achieving near- and long-term research objectives inrelation to existing technical resources, gaps, barriers, andprojected research efforts. Overall, there are sufficienttechnological resources for developing tangible gut micro-biome targeted solutions in the near-term. However, thisresearch, and related capabilities, should also advance toinform future research and develop capabilities that facili-tate achievement of longer term research objectives.Several established DoD research and development

capabilities offering potential for near-term knowledgetransition were discussed. These proficiencies includedfood product development, access to Warfighter popula-tions in austere environments and controlled settings, anability to collect, store and transport biospecimens, ani-mal and in vitro models for studying mechanisms ofhost-microbiome interactions, 16S rRNA and metage-nomic sequencing capabilities, and bioinformatics ex-pertise. These capabilities collectively enabled potentialmanipulation of the microbiome using pre-, pro- andsynbiotics, case-control studies for identifying signaturesof health and dysbiosis within the gut microbiome,

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longitudinal studies of gut microbiome responses tomilitary-relevant stressors for both acute (sleep, under-nutrition, fatigue, diet, etc.) and chronic (PTSD, anxiety,depression, prolonged sleep deprivation, etc.) scenarios,the persistence of these effects, and the ability to buildresistance to infection. However, it was noted that funda-mental knowledge gaps impede interpretation of resultsfrom these studies and results are constrained. To addressthese gaps, new and improved methodologies are needed.The most immediate identified need was an improvedunderstanding of mechanisms of host-microbiome andmicrobe-microbe interactions, including characterizationof the role of fungi and viruses within the gut microbiome.One approach involved model systems (e.g., designedbacterial communities), rather than targeting the wholehost-microbiome system, to generate more targeted andinterpretable data. In parallel, integrated multi-omic (e.g.,metagenome, metatranscriptome, metabolome, proteome,lipidome) approaches to studying dynamic environment-host-microbiome interactions were also suggested. Refin-ing and standardizing current protocols for fecal samplecollection [48, 49] and analysis [50–52] should be ex-tended to approaches that may be more amenable to theaustere environments in which Warfighters operate. Inaddition, technologies are also needed for real-time andnon-invasive spatiotemporal sampling throughout thegastrointestinal tract to provide more relevant sam-ples for both human and animal studies. Several panelistsexpressed concern over the translatability of animal models,particularly murine models in gut microbiome research.However, although animal models may not always translateto human outcomes, especially regarding human perform-ance and cognition, they are, nonetheless, integral to devel-oping mechanistic insights.Interpretation of human and animal studies and the de-

sign of subsequent studies would be facilitated and en-hanced by next generation computational tools/platformsfor complex consortia including predictive modeling util-izing multi-omic readouts from organisms exposed to dif-ferent military-relevant stressors and interventions. Suchmodels would enable prediction of testable strategies forbuilding gut microbiome resiliency and deemed develop-ment of such models as likely feasible within 10–15 years.Within the same time frame, the panel posited that bettercorrelation of in vitro and animal models to human stud-ies will be established. The state of gut microbiome re-search in 20 years and beyond was briefly discussed and itwas thought that a systems biology approach would pre-dominate in this area [53]. Moreover, the interaction ofthe gut microbiome with other microbial communities onthe human body (e.g., lung, oral, vaginal, skin) is likely toemerge as relevant to Warfighter health and performance.Further, designer bacteria that were engineered to producemetabolites of interest or were able to sense and respond

to the environment in order to manipulate and modulatethe microbiome would also be desirable.Technological discussions considered relevant physio-

logical outcomes for DoD nutrition and gut microbiomeresearch and how to measure them. Relevant outcomesfell into three broad categories: 1) maintaining gut resili-ency; 2) optimizing function to improve performance; 3)restoring function after dysbiosis. Measuring resistance toinfection should be a top priority for future research asbuilding gut microbiome resiliency to pathogens woulddirectly reduce lost duty time and improve Warfighterreadiness. Increased energy and reduced fatigue were shortterm objectives that may be potentially relevant to gutmicrobiome homeostasis. During such efforts, multiplebiological sample matrices, including real-time tissue/intes-tinal lining sampling, if possible, will need to be collectedand cohesively analyzed to identify mechanisms underlyinghost-microbe communication. Understanding differenceswithin individual gut microbiomes of responders and non-responders to a stressor or intervention is also critical.Studies should be conducted and outcomes measured insettings closely mimicking mission and/or training envi-ronments with detailed cohort studies as a vehicle to bothgain knowledge and facilitate translation of data for tar-geted solutions. Such studies would be aided by the devel-opment of novel methods for real-time data assessments.

Conclusions and recommendationsThe 1st DoD Gut Microbiome Informational Meetingon nutrition and gut microbiome research included pre-sentations and discussion among DoD scientists workingin the field. Attendees agreed that the microbiome ishighly relevant to Warfighter health and performance, andcontinued research is critical to mission readiness. Severalcommon themes emerged during the meeting: 1) a needfor increased collaboration, coordination, and communi-cation, possibly via a central portal 2) a need to establishlongitudinal studies of incoming recruits prior to initialmilitary training, as well as pre- and post-deployment 3)although bioinformatics resources exists, the area wasidentified as a bottleneck that could be remedied by in-creasing bioinformatics expertise within the DoD 4) gutmicrobiome research is a relatively new field and rapidlyevolving; thus research results must be interpreted withinthe context of the limitations to the methods used to col-lect the data, and staying abreast of technological ad-vances is critical 5) many capabilities and resourcesrequired for gut microbiome research currently existwithin the DoD; 6) collaborative use of these capabilitiesand resources in addition to method standardization willfacilitate the translation/interpretation of results fromdifferent studies across the DoD, and ultimately researchimpact. Also identified were the potential DoD-specificbarriers to gut microbiome research, such as funding,

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security clearances when working with non-DoD partners,contracting requirements that may impede collaboration,and meeting attendance and cybersecurity restrictions.Since 2015, progress has been made in a number of theseareas: 1) steps to improve communication, collaborationand coordination have occurred as evidenced by establish-ment of the DoD Tri-Service Microbiome Consortium(TSMC) and its inaugural workshop held in May 2017; 2)interagency efforts have been undertaken to coordinateand streamline handling of large data sets from bioinfor-matics analyses (e.g. DoD cloud); and 3) the NIST/NIHsponsored Standards for Microbiome MeasurementsWorkshop held in August 2016 met to prioritize needs fordeveloping standards for microbiome measurements thatwill enable federal, academic, and industry labs to reliablyreproduce and advance research.Over the course of the meeting, common research inter-

ests were evident, including understanding the influence ofmilitary-relevant stressors on interactions between themicrobiome and Warfighter biology, manipulating themicrobiome to influence Warfighter health, and using themicrobiome as a biomarker of Warfighter health status.Current DoD research efforts appear complimentary ratherthan duplicative. Toward addressing common research in-terests, several key focus areas emerged across the tech-nical and programmatic break-out sessions (Fig. 3).

Four research thrust areas – Engineering the Micro-biome, Bioinformatics, Relevant Data Sets, and Predict-ive Modeling – were identified as research domains thatare essential for both near- and long-term Warfightersolutions. Each of the areas can be broken down intomultiple layers of sub-focus areas that entail specific ap-proaches to addressing key challenges within the mainresearch thrust. For example, two key approaches wereidentified as a means to engineer the microbiome - syn-thetic biology and nutrition. The synthetic biology ap-proach can further be distilled into two main researchconcepts – designer organisms for targeted persistenceand/or for sense/respond functions. Both concepts fallwithin the research interest areas of manipulating themicrobiome and using the microbiome as a biomarkerfor health status. The additional research thrust areassimilarly break down into sub-focus areas and link tothe common areas of research interest. The identifica-tion of key research thrust areas and concepts to addresstechnical/programmatic aspects of those areas was a keyoutcome of the meeting and serves as a starting point forestablishing DoD gut microbiome research directions.An objective of the meeting was to gain an under-

standing of the current state-of-the art at that time inDoD gut microbiome research and identify researchgaps, as they pertain to the common research interest

Fig. 3 Several research thrust areas (large, dark ovals) emerged across the break-out sessions that can be further refined into sub-focus areas(medium ovals) and research concepts (smaller, outer ovals) to address the challenges in working toward common research areas of interestidentified during the informational meeting

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across the Services. Toward this goal, information wascompiled to summarize the active DoD gut microbiomeresearch, which areas of active research need expansion,and where the current gaps exist within the collectiveServices, as can be seen in Fig. 4. While work inthese areas and gaps have been ongoing, they remainrelevant today.Active research across the Services center on develop-

ing tools and capabilities to gain baseline knowledge onthe role of the gut microbiome in Warfighter health andperformance. Current active areas in need of expansionrepresent program aspects that are at sufficient levelsnow to derive baseline knowledge. However, attendeesrecognized the need for enhancement in tools, capabil-ities, and, in some areas, research focus in order to gen-erate more in-depth knowledge. Lastly, research gapswere identified as areas determined as essential but inwhich very limited or where an absence of current inves-tigation was evident. The information gathered is not allinclusive, as the gut microbiome area is dynamic andnew gaps or new research efforts can readily emerge;however the information can serve as a starting pointfor a collaborative, joint roadmap as DoD gut micro-biome research programs are developed.Although consensus could not be generated on a num-

ber of technical aspects, it was agreed that translatingsolutions to the Warfighter is the ultimate outcome ofDoD gut microbiome research. Relevant research out-comes were identified as being extremely important toarticulate to stakeholders and decision makers. Towardthis end, the 2-day meeting information was compiled togenerate near- (0–10 year), mid- (10–20 year) and far-term

(20 year +) possible relevant outcomes for gut microbiomeresearch (Fig. 5).Near-term outcomes center on two primary aspects: 1)

avoiding duplicity in both capability and tool developmentas well as research focus, and, when necessary, avenues toeffectively communicate and leverage complementarystudies across all Services and 2) generation of novel plat-forms and standardization of methods and analyses thatwill facilitate the translation/interpretation of results fromdifferent studies across the DoD. Mid-term outcomesfocus on those research areas that have been identified asnecessary to expand to advance understanding of thecomplex interactions within the gut microbiome, includ-ing computational tool advancements and real-time data/sample acquisition. It is also feasible to extend focus alongspecific axes in which the gut plays a pivotal role. Ultim-ately, in the mid-term, scientific direction should centeron translating solutions to the Warfighter to realize thebenefits of using the gut microbiome to modulate healthand performance. Far-term outcomes are more difficult toarticulate due to the nascent nature of gut microbiome re-search in the DoD. However, relevant outcomes may cen-ter on looking at a more holistic level and focus primarilyon engineering the microbiome for tailored resiliency onan individualized basis that can extend to mission-specificnutrition, utilizing models to predict health status and po-tential impact on performance. To realize the relevantoutcomes, DoD gut microbiome research must be con-ducted through a coordinated, collaborative research en-vironment that not only reaches throughout the Servicesbut also includes academic, industrial and other relevantpartners. Since 2015, several near-term outcomes have

Fig. 4 Current state of the science derived from the informational meeting. The current state includes active areas of research across theattendees, areas that are ongoing but need expansion through additional resources, capability development and novel techniques, and alsoresearch gaps that emerged as critical to providing translatable solutions to the Warfighter for improving health and performance. While effortshave been made to address these research areas and gaps, they remain relevant today

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been advanced: 1) the chartering of the TSMC and itsinaugural workshop sought to improve integration andcoordination of research efforts, and 2) leveraging oppor-tunities and collaborative efforts will be advanced by es-tablishment of the Applied Research for the Advancementof S&T Priorities (ARAP) Program in Synthetic Biologyfor Military Environments (SBME) that will result in bybuilding DoD capabilities in synthetic biology.Although it was hoped that explicit technical recom-

mendations would be realized during the meeting,breakout panels did not come to a consensus on alltechnical needs and gaps or approaches to address thosetopics. This is not uncommon to the field, and indicatesa need for continued communication within the DoDand the broader scientific community. The immediateand foremost outcome of this meeting was the identifi-cation of DoD organizations active in gut microbiomeresearch and complementary interests and capabilities.Furthermore, inter-institute and inter-agency coordin-ation and communication was improved and/or estab-lished, which will allow collaboration within the DoD toleverage capabilities and enhance productivity, whichcollectively will facilitate working toward the desiredrelevant outcomes within gut microbiome research. TheDecember 2016 establishment of the TSMC clearly servesas a powerful example of the motivation and momentumof the DoD microbiome community to coordinate and le-verage the microbiome as a tool for improving the healthand performance of the Warfighter. The outcome of theTSMC workshop in May 2017 determined that methodsand analysis standardization, big data sharing and syn-thetic biology are priority areas that apply to all micro-biome research. Similar meetings were deemed necessary

to be conducted on a regular basis to continue communi-cation, further identify collaborative opportunities, andcontinue to refine/update the scientific direction for DoDgut microbiome research. There was overwhelming sup-port for holding future gut microbiome informationalmeetings, and to integrate academic and industry partnerswithin these discussions.

AbbreviationsAFRL: US Air Force Research Laboratory; ARL: US Army Research Laboratory;ARO: US Army Research Office; CFD: Combat Feeding Directorate;CMSCFAT: Continuous Multifaceted Soldier Characterization for AdaptiveTechnologies; DoD: Department of Defense; DURIP: Defense UniversityResearch Instrumentation Program; FFRDC: Federally Funded Research andDevelopment Center; GM: Gut Microbiota; HPW: Human Performance Wing;ISB: Integrative Systems Biology Program; LUCI: Laboratory UniversityCollaboration Initiative; MDRO: Multi-Drug Resistant Organisms;MMT: Microbiome Modulation of Toxicity; MURI: Multidisciplinary UniversityResearch Initiative; NIH: National Institutes of Health; NMIG: NatickMicrobiome Interest Group; NMRC: Naval Medical Research Center;NSRDEC: US Army Natick Soldier Research Engineering and DevelopmentCenter; NSSC: Natick Soldier Systems Center; OASD(R&D): Office of theAssistant Secretary of Defense for Research and Engineering; ONR: Office ofNaval Research; PTSD: Post-Traumatic Stress Disorder; SBIR: Small BusinessInnovative Research; STTR: Small Business Technology Transfer;TMM: Toxicant Modulation of the Microbiome; TSMC: Tri-Service MicrobiomeConsortium; UARC: University Applied Research Center; USACEHR: US ArmyCenter for Environmental Health Research; USAMRIID: US Army MedicalResearch Institute of Infectious Diseases; USARIEM: US Army ResearchInstitute of Environmental Medicine; WD: Warfighter Directorate

AcknowledgementsThe authors would like thank Mary Olmstead, Donna Glowka, Joanne Gordonand Oliver Bradford for logistical support in setting up and conducting themeeting. The authors also thank Scott Montain, Betty Davis, John Player andDanielle Anderson for support and guidance.

FundingCongressionally Directed Medical Research Program, Defense MedicalResearch and Development Program.

Fig. 5 A compilation of the information across the 2-day meeting identified possible relevant outcomes of DoD gut microbiome research for thenear-, mid-, and far-term. Identification of the possible outcomes assists in developing scientific direction for the area, however, additional assessmentis still necessary

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Availability of data and materialsNot applicable.

DeclarationsThe opinions or assertions contained herein are the private views of the authorsand are not to be construed as official or as reflecting the views of the Army orthe Department of Defense. Citation of commercial organizations or tradenames in this report does not constitute an official Department of the Armyendorsement or approval of the products or services of these organizations.Opinions, interpretations, conclusions, and recommendations are those of theauthors and are not necessarily endorsed by the US Army.

Authors’ contributionsKR, SA, JPK and JWS compiled the information and wrote the report. All otherauthors contributed to the report, read, edited and approved the final manuscript.

Ethics approval and consent to participateNot applicable.

Competing interestsThe authors declare that they have no competing interests.

Publisher’s NoteSpringer Nature remains neutral with regard to jurisdictional claims in publishedmaps and institutional affiliations.

Author details1Natick Soldier Research Development & Engineering Center, Boston, MA,USA. 2US Army Research Institute of Environmental Medicine, Boston, MA,USA. 3Office of Naval Research, Arlington, VA, USA. 4US Army Center forEnvironmental Health Research, Annapolis, MD, USA. 5Air Force ResearchLaboratory, Columbus, OH, USA. 6US Army Research Office, Annapolis, MD,USA. 7Naval Medical Research Center, Annapolis, MD, USA. 8US ArmyResearch Laboratory, Annapolis, MD, USA.

Received: 7 October 2017 Accepted: 26 February 2018

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