Gut microbiome, stress and interventional strategies using hardy animals

Abstract

The composition of the gut microbiota has been linked to acute stressors, suggesting that modulation of the gut microbiome is a potential avenue for enhancing human health and performance. The gut microbiome exerts its effects through several metabolites, which induce epigenetic and metabolic changes. Stress is a common occurrence in humans facing challenging environments such as military personnel and astronauts, but is not limited to humans. Among various species, crocodiles are well known for their “hardiness” and ability to achieve longevity, while surviving under stressful conditions. We speculated that their microbial gut flora produces substances contributing to their ability to resist stress, “wellbeing” and “longevity”. Herein, we deliberate upon the stressors faced by individuals in testing conditions, and discuss potential avenues that can mitigate the gut microbiome compositional changes in order to augment human performance and overall health.

ARTICLE HIGHLIGHTS

• Acute and environmental stressors, including circadian disruption, infection, extreme environments and space travel, significantly influence the gut microbiome.

• Altered microbial composition and function affect immune, metabolic and neuroendocrine pathways linked to human resilience and performance.

• Integrative synthesis identifies shared microbial responses across stress domains and highlights strategies to mitigate adverse effects.

• Evidence from animal models, including resilient species such as crocodiles, reveals bioactive metabolites with translational potential.

• Understanding stress–microbiome interactions may inform new approaches to maintain health and performance in demanding environments.

PLAIN LANGUAGE SUMMARY

Stress can significantly influence the gut microbiome, leading to changes that affect human physiology, cognition and overall health. This review examines how various stressors, including circadian disruption, infection, environmental extremes and space travel, alter gut microbial composition and metabolic function. Evidence from both human and animal studies is discussed to illustrate how stress-induced microbial changes impact immune, neuroendocrine and metabolic pathways linked to performance and resilience. The review also highlights emerging strategies to mitigate these effects, including the exploration of metabolites from resilient species such as crocodiles, which may inform future microbiome-based interventions to support health under extreme conditions.

1. Introduction

The gut microbiome plays a crucial role in maintaining a balanced state with the host’s intestinal epithelial, neuronal, and immune cells [1]. This interaction between the host and microbiome is particularly important during periods of stress or illness and in maintaining optimal health [2–4]. Various factors, such as the environment, nutrition, and cognitive stress, impact the composition of the gut microbiota [5]. Several lines of evidence have highlighted the bidirectional relationship between the gut microbiome and the development of diseases, such as cancer, cardiovascular diseases, obesity, neurogenerative diseases, and accelerated aging [6–9]; however there has been limited focus on studying the effects of this interaction on acute stressors. Thus, understanding the underlying mechanisms of how various stressors may modulate both the host and the microbiome warrants further investigation. Moreover, other areas of research comprise investigating the potential for manipulating the microbiome to improve health outcomes, exploring the impact of various stressors on the interactions between the microbiome and the human body, and utilizing the microbiome as a biomarker to assess an individual’s health status [10].

Military personnel, both male and female, comprise a distinctive group of individuals who require optimal physical and mental well-being to guarantee the accomplishment of their missions [11]. A notable focus on advancing research and development geared toward enhancing human performance exists in the United States. This emphasis is evident across various branches and agencies of the Department of Defense (DoD), including the Army, Air Force, Navy, and the Defense Advanced Research Projects Agency (DARPA) [12]. Likewise, astronauts encounter challenges and various health concerns during short and extended space missions, likely due to multiple stressors like microgravity and radiation [13]. Both groups, although usually fit and healthy individuals, still face several stressors in their environments due to the nature of their work. Thus research that may alleviate these stressors via the microbiome is pertinent, considering the significance of the microbiome in maintaining human health. Stress is not limited to humans. Animals are often exposed to stressful conditions in their natural environments; hence their microbiome may offer a potential avenue of research. To this end, recent studies have explored the gut microbiome of long-lived animals, such as reptiles, to uncover their distinctive characteristics and understand how they contribute to the robust defense mechanisms observed in these species. These species regularly face stressful environments in comparison to humans; yet can thrive in these stressful conditions [14,15]. Research is underway to determine the potential benefits of animal gut microbiome for human health and the development of pre/pro/postbiotics for health and human performance [16,17].

Herein, we deliberate upon the stressors faced by individuals such as military personnel and astronauts, and discuss the potential that can mitigate the gut microbiome compositional changes to augment human performance and overall health (Figure 1). The goal is to bring together current evidence on how the gut microbiome influences human health and performance under various stress conditions. The interplay between stress, physiological function, and microbiome composition are considered, followed by focused discussions on circadian disruption, infection-related challenges such as travelers’ diarrhea, and environmental factors including temperature and hypoxia. We also examine how space travel affects astronaut health and performance through microbiome changes, and conclude with emerging strategies to beneficially modulate the gut microbiome. Relevant literature was identified through searches of PubMed, Scopus, Web of Science and Google Scholar, focusing on publications from the past decade. The search combined terms such as “gut microbiota,” “gut microbiome,” “stress,” “resilience,” “human performance,” “astronaut health,” “crocodile microbiome,” “microbial metabolites,” and “host microbiota.” Reference lists of key papers and recent reviews were also examined to identify additional studies. Only peer-reviewed English-language articles were included, with emphasis on original research and high-quality reviews addressing microbiome composition, host–microbe interactions, and their relevance to stress physiology, human performance and astronaut health.

Figure 1.

Schematic diagram showing the detrimental effects of intestinal dysbiosis (indicated by arrows) on military personnel and astronauts, and their potential reversal by transplant of crocodile intestinal microbiome.

2. The gut microbiome and impact on human health

Otherwise highly diverse, the human gut microbiome consists of the hundred trillion microorganisms, some of which are beneficial, some potentially pathogenic, and others with an unknown function [18]. This myriad of microorganisms includes bacteria, archaea, fungi, and viruses [19]. The gut microbiota is primarily comprised of bacteria, as over 100 trillion bacteria belonging to over one thousand species reside in the gut [20,21]. Yet, four bacterial phyla prevail in the gut, including Actinobacteria, Firmicutes, Proteobacteria, and Bacteroidetes [19]. Although the idea of the core microbiome, which is a collection of microbes that all humans share, is well-known, there is no exact definition of a healthy microbiome. There is significant variation in the microbiome depending on ethnicity, social context, diet, and the use of antibiotics, among other things. The gut microbiome plays an important role in host health through carrying out various metabolic processes, such as the production of short-chain fatty acids from carbohydrate fermentation, lipid metabolism, and vitamin synthesis [18]. For instance, the gut microbiome is involved in nutrient acquisition, protection from pathogens, vitamin synthesis, and even intestinal immune system maturation [22,23]. Of note, the gut microbiome can influence brain function, mood, stress, and even behavioral responses to rewards [23,24]. Yet, the importance of the gut microbiome and its metabolites to human health is still unfolding. For instance, recent evidence highlights the impact of the human gut microbiome on skeletal muscle mass through short-chain fatty acid butyrate synthesis among healthy menopausal women, its influence on host susceptibility to parasitic infection, and even preventative effects of some commensal bacterial strains against aging [25–27].

More than 90% of the taxonomic groups of human gut bacteria are thought to be made up of the Firmicutes and the Bacteroidetes [28]. Changes in the microbiome’s Firmicutes and/or Bacteroidetes composition may impact caloric intake and thus affect insulin sensitivity, diabetes risk, and cardiovascular disease risk [29]. Gut dysbiosis is a disorder of the harmony, variety, and operation of symbiotic intestine microbial populations. This can be triggered by alterations in the biological milieu and manifest as biological dysregulation, which further disrupts the body, causes the disease to develop along with appearance of various symptoms [30]. Compositional changes in gut microbiota have been linked to many diseases, such as obesity, type 2 diabetes, depression, and cardiovascular disease [19]. Furthermore, rodent studies imply that gut dysbiosis has been linked with marked behavioral anomalies, namely impaired motor performance, stress-responsiveness, memory, sociability, anxiety-like behaviors, and depression [31].

It is important to indicate that in addition to the gut, other host-associated microbiota are also important in contributing and maintaining physiological balance and influencing disease susceptibility, although are not the focus herein. The oral microbiota has been linked not only to periodontal health but also to systemic conditions such as cardiovascular and metabolic diseases through inflammation-mediated pathways [32]. Similarly, the nasal and lung microbiota modulate immune tolerance and respiratory defence, with alterations associated with asthma and chronic respiratory diseases [33]. The bladder microbiota, once thought absent, is now recognized to influence urinary health and recurrent infections, while the vaginal microbiota, typically dominated by Lactobacillus species, protects against opportunistic and sexually transmitted infections [34,35]. Together, these microbial communities across different body sites complement the gut microbiota in supporting host immunity, metabolism and overall health. Interestingly, gut dysbiosis has also been linked with acne, cancer, and potentially covid-19 [19,36]. Hence, in its eubiosis or dysbiosis, there is more to learn about the importance of the gut microbiome to human health. Logically, targeting and modulating of the microbiome is a prospective therapeutic approach that is being actively pursued to treat various diseases [37,38].

3. Stressors, human health, and performance

Stress is an omnipresent aspect of everyday human existence and is progressively acknowledged to have biological consequences, including influence on the modulation of the gut microbiota [39]. Stressors faced by military personnel may disrupt the beneficial relationships within the gut microbiome, both directly and indirectly [40]. This disruption may lead to dysbiosis, which has been linked to various acute health issues, including systemic inflammation, heightened vulnerability to illnesses and infections, cognitive decline, and the onset or persistence of multiple chronic diseases [41].

Evidence from previous in vivo studies in rodent models have highlighted the gut microbiota as a key determinant of stress vulnerability and resilience. Foundational work in germ-free mice revealed that absent or disrupted colonization exaggerated hypothalamic–pituitary–adrenal (HPA) axis reactivity to stress, whereas postnatal microbial reconstitution normalizes this response [42]. In behavioral paradigms such as chronic social defeat stress, differences in microbial composition were distinguished in susceptible and resilient rodents; enrichment of Lactobacillus was associated with resilience, and probiotic supplementation enhanced stress coping through serotonergic mechanisms, while depletion patterns accompanied vulnerability [43]. Similarly, in learned helplessness models, susceptible and resilient phenotypes exhibited distinct gut microbial profiles, further supporting a microbiota contribution to stress-related behavioral outcomes [44].

A plethora of stressors may affect military personnel, including psychological, environmental, and physical stressors. Examples include circadian disruption and sleep restriction, high altitudes, cold environments/heat stress, exposure to enteric pathogens, environmental toxicants and pollutants, and physical activities, as well as changes to their diet [40]. Among environmental factors, cold exposure and seasonal transitions play an important role in shaping gut microbiota and host physiology. Exposure to lower temperatures can alter microbial diversity and function, favoring taxa involved in energy harvest and thermogenic adaptation while reducing those linked to inflammatory responses. Seasonal cold influences immune regulation and gut barrier integrity, thereby affecting resilience to infection and metabolic stress. Recent work has highlighted that climatic and seasonal variations, including the timing of cold exposure, modulate gut microbiota composition and immune homeostasis, emphasizing the dynamic interplay between environmental cues and host resilience [45]. Various reports point to the connection between the gut microbiome and circadian rhythms [46,47], environmental stressors [48], exposure to enteric pathogens [49,50], as well as connection with diet as discussed in detail in the following sections [51].

4. Circadian rhythms and gut microbiome connexion

Emerging evidence indicates that circadian misalignment and sleep loss may perturb gut microbiome composition, leading to downstream effects on host mitochondrial function. Such dysbiosis may alter key metabolites, including short-chain fatty acids (SCFAs), bile acids, and reactive oxygen intermediates, which collectively regulate mitochondrial biogenesis, oxidative phosphorylation, and redox balance. Recent findings highlight that gut–mitochondria communication represents a critical interface linking metabolic and immune responses under disrupted circadian conditions. These microbiome-mediated effects on mitochondrial homeostasis may contribute to reduced resilience, impaired cognitive performance, and increased physiological vulnerability under stress.

A recent study assessed crewmembers’ sleep patterns, psychomotor vigilance performance, and work demands onboard a US Navy ship [52]. The schedule, combined with other work duties, resulted in poor sleep hygiene. Crew members experienced periodic bouts of sustained wakefulness and accrued a significant sleep debt due to extended workdays and circadian-misaligned sleep [52]. In another study examining the effect of jetlag, it was found that Firmicutes increased in abundance. Conversely, the quantity of Bacteroidetes was reduced in the gut microbiota of two human subjects one day after travel [53]. Within two weeks, the dysbiotic effects of jet lag were recovered. Interestingly, germ-free mice who received a gut microbial transplant from samples taken before jet lag, compared to the mice that received the transplant from human samples during jet lag, experienced significantly more weight gain and higher blood glucose levels after an oral glucose challenge [53].

Initial controlled laboratory studies have yielded conflicting results regarding the impact of insufficient sleep on the human gut microbiome. While some studies found no significant changes in microbial diversity after short periods of restricted sleep, such as two days with 4.25 hours of sleep opportunities [54] and five days with 4 hours of sleep opportunities [55], others observed an increase in the Firmicutes to Bacteroidetes ratio and alterations in specific microbial families. Conversely, research using animal models consistently demonstrates that sleep deprivation leads to decreased microbial diversity and shifts in microbial composition. For instance, seven days with 4 hours of sleep opportunities in rats decreased beta diversity [55], and four weeks with daily sleep fragmentation in mice altered beta diversity and increased the relative abundance of families Lachnospiraceae and Ruminococcaceae while decreasing Lactobacillaceae [56]. Moreover, transferring fecal matter from sleep-deprived animals to germ-free animals resulted in metabolic disturbances and increased inflammation markers [56], suggesting a potential link between disrupted sleep, gut microbiota, and metabolic dysfunction.

A recent interesting study investigated the effects of synbiotic ice cream supplementation on salivary IgA, gastrointestinal symptoms, wellbeing indicators, and gut microbiota in young military participants undergoing field training [57]. Sixty-five military personnel completed the study, with one group receiving synbiotic ice cream containing Lactobacillus acidophilus LA-5, Bifidobacterium animalis BB-12, and inulin, while the other received a placebo. Results showed an increased abundance of Bifidobacterium and Lactobacillus genera post-supplementation and training in both groups. Salivary IgA and gastrointestinal symptoms decreased post-training in both groups, but synbiotic supplementation did not mitigate this effect. However, the synbiotic-treated group showed decreased tenseness and sleepiness post-training compared to the placebo group. Other wellbeing indicators were not affected by supplementation. The authors concluded that synbiotic ice cream supplementation improved gut microbiota and reduced tenseness and sleepiness in healthy young military personnel during field training, thus with the potential to impact decision-making in high-stress environments [57].

SCFAs and organic acids generated by the gut microbiota through the fermentation of indigestible fiber play a crucial role in interconnecting microbiome and host tissues to regulate mammalian homeostasis. The microbiome exhibits circadian rhythmicity, contributing to the regulation of the host’s circadian clock. A recent study explored the impact of SCFAs or fiber-rich diets on adjusting the circadian phase in various peripheral tissues of mice, including the liver, kidney, and submandibular glands [58]. Initially, high concentrations of cecal SCFAs, particularly acetate and butyrate, displayed significant day-night variations during the active period, correlating with lower cecal pH levels. Through in vivo monitoring of luciferase activity associated with the clock gene Period2, oral administration of a mixture of SCFAs (acetate, butyrate, and propionate), along with lactate, or individual administration of each SCFA or lactate for three days, induced phase alterations in peripheral clocks, depending on the timing of stimulation. However, this effect was not observed in cultured fibroblasts or liver slices treated with SCFAs, suggesting an indirect modulation of circadian clocks by SCFAs in vivo [58]. Furthermore, diets containing cellobiose promoted SCFAs production and facilitated refeeding-induced adjustment of peripheral clocks. These findings suggest that the oral administration of SCFAs and prebiotic supplementation may aid in adjusting peripheral clocks, highlighting prebiotics as potential therapeutic interventions for circadian misalignment [58].

Recent evidence enhances our understanding of the gut microbiome’s role in metabolic traits. However, there’s a pressing need for rigorous studies using advanced methodologies in both metabolic and sleep-circadian research. These studies are crucial for uncovering how the microbiome influences responses to insufficient sleep and circadian disruptions. Certain microbial taxa and their metabolites may contribute to metabolic dysregulation linked to sleep deprivation and circadian misalignment. Greater microbial diversity appears to be associated with improved sleep quality. Additionally, preliminary evidence suggests prebiotic supplementation could positively impact the microbiome and improve sleep [59].

5. Travelers’ diarrhea and enteric pathogens

During military deployments, acute enteric infections are considered the second most prevalent infectious disease risk for service members and contribute significantly to illness rates in the United States; approximately 29% of individuals experience diarrheal illnesses per month while deployed in developing countries [10,41]. A previous review of studies on diarrhea in long-term travelers, mainly focusing on populations such as the US military, presented several key findings. First, diarrhea is a common occurrence, often without healthcare intervention, prompting questions regarding its severity and efficacy of self-treatment. Second, globally significant pathogens such as Enterotoxigenic Escherichia coli (ETEC), Campylobacter, and Shigella, mainly the latter two, are associated with more severe symptoms and longer durations of illness. Third, various other bacterial, viral, and parasitic pathogens, including Enteroaggregative Escherichia coli (EAEC), Salmonella, norovirus, and rotavirus, continue to be significant contributors to disease burden in these populations. the combined effect of both treated and untreated diseases, along with the resulting incapacitation, poses a significant health risk. Therefore, it is imperative to conduct further research into developing timely and effective management strategies and exploring vaccination options for prevention.

A recent pilot study investigated the impact of deployment on the gut microbiome and susceptibility to traveler’s diarrhea [60,61]. The investigation focused on the microbiome of military personnel before and after their deployment abroad, aiming to pinpoint specific microbial markers. Comprehensive full-length 16S rRNA gene sequencing was employed in identifying predictive microbial markers. Analysis of 16S rRNA data from fecal samples collected before and after deployment revealed several marker taxa that exhibited significant differences in abundance among individuals who reported experiencing diarrhea. These taxa included Weissella, Butyrivibrio, Corynebacterium, uncultivated Erysipelotrichaceae, Jeotgallibaca, unclassified Ktedonobacteriaceae, Leptolinea, and uncultivated Ruminiococcaceae. The ability to identify the risk of developing traveler’s diarrhea before travel holds the potential for informing preventive measures and interventions to mitigate susceptibility to diarrhea while traveling [61]. Although identifying particular pathogens is undoubtedly essential, particularly in military or traveler populations, it may be more beneficial to understand whether specific microorganisms or microbiome populations can predict susceptibility or resilience to diarrhea. This knowledge could help prevent the issue from becoming a hindrance to performance. A prior study conducted within the US Air Force (USAF) at an air base in Honduras identified microbial taxa that potentially predicted the development of traveler’s diarrhea within the study population [62]. The study was conducted for two nine-month deployment cycles in Honduras from 2015 to 2016. The findings revealed that 56.7% reported reduced performance, and 21.1% reported lost duty days. Passive surveillance of diarrhea cases presented to the medical unit revealed 152 total cases, primarily caused by EAEC, ETEC, and enteropathogenic E. coli (EPEC). Active longitudinal surveillance of 67 subjects identified diarrheagenic E. coli as the primary cause [62]. A subset of 11 subjects participated in a nested longitudinal study, revealing differences in baseline microbiota between those who developed traveler’s diarrhea and those who did not, along with the detection of taxa associated with gastrointestinal distress [62]. Qualitative observations showed disrupted microbiota in the weeks surrounding diarrhea incidents. These findings highlight the multifaceted nature of diarrhea among deployed military personnel and its impact on job performance [62]. Identifying factors influencing resistance or susceptibility can inform future clinical trials for prevention and treatment strategies.

6. Environmental stressors and gut microbiome

Environmental stressors, such as heat stress on gut barrier function have been extensively reviewed [63] and investigated in various human and animal models. Even brief exposure, ranging from 4 to 6 hours, can inflict significant damage on the intestinal epithelium [64]. Moreover, hyperthermia within the intestinal wall can damage gut barrier, resulting in increased permeability of tight junctions [65], potentially leading to inflammation and sepsis [66]. Animal studies have demonstrated alterations in gut microbiota composition due to environmental heat stress. These changes include decreased diversity [67] and reduced levels of Lactobacillus and Bifidobacterium in chickens [67,68]. These findings suggest that environmental heat stress could negatively impact gut microbiota. Heat-related illnesses pose significant health risks, particularly with the projected increase in global temperatures and extreme weather events. While heat acclimation training (HAT) shows promise in preventing such illnesses, the precise mechanisms through which it promotes beneficial changes in organ function, immunity, and gut microbiota remain unclear.

In a recent study, 32 healthy young soldiers were recruited and randomly assigned to four teams to undergo HAT for 10 days [69]. These teams included the equipment-assisted training team in high temperature (HE), the equipment-assisted training team under typical hot weather (NE), the high-intensity interval training team in high temperature (HIIT), and a control group without training. A standard heat tolerance test (HTT) was conducted before (HTT-1st) and after (HTT-2nd) the training to assess heat acclimation (HA) criteria. The results showed that participants in both the HE and NE teams exhibited significantly higher acclimation rates than those in the HIIT team. Moreover, the HE team demonstrated superior effects of HAT compared to the NE team. In the HA group, physiological indicators and plasma organ damage biomarkers notably decreased after HTT-2nd compared to HTT-1st, while immune factors increased. Additionally, significant changes were observed in the composition, metabolism, and pathogenicity of gut microbes, with a decrease in potentially pathogenic bacteria and an increase in probiotics [69]. Overall, these findings indicate that HA leads to a reduction in pathogenic gut bacteria and an increase in probiotics, accompanied by elevated immune factors and reduced organ damage during heat stress. This suggests an improved heat adaptation of the body following HAT, particularly with prolonged training in high temperature and humidity conditions [69].

Another stressor is high altitudes (≥2500 m); individuals often experience gastrointestinal symptoms like loss of appetite, indigestion, nausea, vomiting, gas, and abdominal pain due to hypobaric hypoxia, a condition characterized by reduced oxygen levels [70]. This hypoxia lowers arterial oxygen saturation, impacting oxygen delivery to tissues and potentially affecting GI motility, oxidative stress, and inflammation [71]. Studies on the effects of hypobaric hypoxia on gut microbiota are scarce. Research in rats suggests that exposure to hypobaric hypoxia can lead to intestinal changes, inflammation, increased serum endotoxin levels, bacterial translocation, and alterations in gut microbiota composition [72,73]. However, distinguishing hypobaric hypoxia effects from other factors like underfeeding and weight loss is challenging. Observational studies in humans at high altitudes face similar challenges in isolating hypobaric hypoxia effects from dehydration, foodborne pathogens, undernutrition, and increased physical activity. Nonetheless, some studies suggest increased inflammation and changes in anaerobic bacterial counts during mountaineering expeditions [74].

Recent studies investigating the effects of hypoxia on gut microbiota under controlled conditions suggest alterations in bacteria and bacterial genes related to metabolism and virulence [75,76]. However, the relevance of these findings to high-altitude environments is uncertain due to differences between normobaric and hypobaric hypoxia. Overall, existing studies suggest an association between high altitude exposure and increases in pro-inflammatory gut bacteria, but evidence on potentially beneficial bacteria is limited. Further randomized controlled trials are needed to understand the independent effects of hypobaric hypoxia on gut microbiota and its implications for health and performance.

7. Space travel and astronaut health and performance

Numerous studies have suggested that spaceflight has a significant impact on the composition of the gut microbiome in astronauts [77,78]. As already stated, the gut microbiome plays a crucial role in maintaining the body’s physiological balance and immune system. Extended space travel and the microgravity environment can influence vascular physiology and the composition of the gut microbiome. To better understand these effects, the “Astronaut Microbiome project” is currently underway, aiming to study microbiome changes during space travel [79]. In addition, radiation exposure during space travel can notably alter the composition of the gut microbiome and disrupt gut homeostasis [80]. Researchers hope that by studying the impact of space travel on the gut microbiome, they can determine its effects on astronaut health and wellbeing. Initial findings from these studies indicate that the gut microbiome of astronauts becomes less diverse. There is an increase in Faecalibacterium, which is beneficial as it produces butyrate. However, there is also an increase in Parasutterella, which has been associated with chronic intestinal inflammation. Moreover, there is a decrease in the levels of Akkermansia, which is known for its anti-inflammatory properties. As a result, prebiotics containing Akkermansia have been suggested to reduce the risk of diseases associated with chronic inflammation [80].

In addition to studies conducted on astronauts, analog mission projects on Earth have provided further evidence of the gut microbiome’s importance. For example, the “MARS500 study” involved six astronauts confined within a Mars-surface habitat for 520 days. This study observed changes in the gut microbiota throughout the mission, including an increased abundance of Bacteroides spp. and a decreased abundance of Faecalibacterium prausnitzii [81]. Similar findings were also observed in the “Skylab Medical Experiments Altitude Test” conducted in the 1970s during a 56-day confinement period [80]. These studies demonstrate the adaptability of the gut microbiome in response to prolonged space-like conditions. Recent advancements in sequencing technology have allowed for a more detailed analysis of the gut microbiome during space missions. One study reanalyzed data from the MARS500 project using improved bioinformatics technology and identified significant changes in the gut microbiome composition over time [82]. Certain species associated with inflammation and glucose homeostasis were found to be altered during the study period. Other studies have utilized shotgun metagenomic sequencing and microarray approaches to investigate the microbiome of astronauts before, during, and after spaceflight, as well as environmental samples from the International Space Station (ISS). These studies have revealed changes in the composition and diversity of the astronaut’s microbiome during spaceflight, which generally returned to normal upon their return to Earth [83].

Furthermore, in vitro and in vivo studies conducted in microgravity or simulated microgravity environments have demonstrated the impact of these conditions on gut microbiome diversity and the effects on beneficial microbes. For instance, a study using a mouse model of hindlimb unloading (HU), which mimics microgravity, showed reduced intestinal immunity and disturbance of the gut microbiome [21].

The HU model was developed in the 1970s by National Aeronautics and Space Agency (NASA)-Ames Research Center, this method employs rats and mice to simulate conditions resembling space flight, bed rest, and acute stress experienced by humans [84]. It offers an alternative approach for studying the mechanisms underlying skeletal muscle mass loss and interventions to mitigate atrophy resulting from hindlimb unloading. Additionally, this model enables the examination of bone quality and alterations in various physiological parameters, including blood pressure, heart rate, lipid composition in plasma or tissues, and blood sugar levels, as well as an examination of acute stress markers such as oxidative stress [85].

A recent study investigated the potential benefits of a novel bacterial conditioned media derived from two strains found in the crocodile gut microbiome in HU mice, aiming to counteract the effects of unloading in this mouse model and elucidating the potential of these strains as potential probiotic strains [86]. The study examined the impact of this bacterial-conditioned media on the diversity and quantity of intestinal microbes in HU mice, as well as its systemic effects on the microarchitecture of retinas and kidneys. Male C57/Bl6 mice aged four months were divided into groups: a ground-based control group, HU mice given a placebo (HU-placebo group), and HU mice given bacterial conditioned media (HU-CP group), maintained under controlled conditions for three weeks. Gut dissections were conducted, and metagenomic analysis of bacterial species was performed through DNA extraction and 16S rRNA analysis. The results indicated that HU reduced intestinal microbial diversity, accompanied by an increase in pathogenic bacteria, primarily Firmicutes (45%). However, supplementation with bacterial-conditioned media for three weeks significantly increased gut microbial diversity and caused noticeable changes in the abundance of operational taxonomic units (OTUs) in HU mice. Furthermore, HU-induced muscle weakness and structural abnormalities in the retina and kidney were partially mitigated by bacterial-conditioned media. Additionally, HU-CP mice exhibited a greater diversity of various bacteria, including Bacteriodota, Firmicutes, Proteobacteria, Actionobacteriota, Verrucomicorbiota, Cyanobacteria, Gemmatimonadota, Acidobacteriota, Chloroflexi, Myxococcota, among others. These findings underscore the potential of bacterial metabolite-conditioned media to modulate gut microbiota diversity and alleviate some systemic effects of chronic stress in experimental animal models. However, further research, particularly molecular mechanistic studies, is warranted to fully understand the systemic effects of bacterial metabolite-conditioned media in such models [86]. The study revealed future research should focus on identifying unique bacterial species that may be developed as pro/prebiotics based on the specific needs of astronauts. Further work investigating functional aspects of the microbiome, such as virulence genes, metabolic genes, and antibiotic resistance genes, should also be accomplished. An integrated overview of microbiome alterations, metabolic implications, and intervention strategies related to acute and environmental stressors is presented in Table 1. Additionally, understanding the long-term response of such unique bacteria to microgravity environments is crucial for further insights.

Table 1.

A comparative synthesis of acute and environmental stressors, associated gut microbiome alterations, and functional implications. It integrates evidence across physiological, occupational and extreme environments to highlight cross-domain resilience mechanisms and translational opportunities.

Stressor category	Representative context	Microbiome alterations (comparative synthesis)	Functional and metabolic implications	Potential resilience or mitigation strategies
Circadian disruption and sleep loss	Human and rodent models of jetlag or sleep restriction	Reduced microbial diversity and beneficial taxa (Lactobacillaceae); altered Firmicutes/Bacteroidetes ratio; disrupted rhythmicity of bacterial metabolism	Perturbation of short-chain fatty acid (SCFA) and bile acid pathways; mitochondrial and metabolic dysregulation	Restoration of SCFA-producing taxa through prebiotic or timed feeding interventions improves circadian alignment and host resilience
Psychological and physical stress	Military and occupational training, high workload	Loss of beneficial genera such as Bifidobacterium and enrichment of proinflammatory taxa	HPA axis overactivation, reduced microbial metabolites linked to stress buffering	Synbiotic formulations combining Lactobacillus acidophilus, Bifidobacterium animalis and inulin improved physiological and cognitive outcomes
Thermal and environmental stress	Heat acclimation and cold exposure in animal and human studies	Selective expansion of probiotic species (Lactobacillus, Bifidobacterium); reduction of opportunistic pathogens	Enhanced thermotolerance and immune stability under temperature variation	Thermal acclimation and nutritional support promote microbiome-mediated adaptation
Hypobaric hypoxia (high altitude)	Mountaineering and hypoxia chamber exposure	Increase in proinflammatory bacteria; depletion of anaerobic commensals	Inflammation, oxidative stress and impaired gastrointestinal integrity	Glutamine and antioxidant supplementation support microbial balance and barrier function
Infectious stressors (travelers’ diarrhea)	Enterotoxigenic Escherichia coli, Shigella and Campylobacter exposure	Transient dysbiosis, reduction of protective taxa and overgrowth of pathogens	Gut barrier disruption, systemic inflammation and reduced nutrient absorption	Galacto-oligosaccharide supplementation and probiotic therapy shown to reduce diarrhea incidence and restore microbiota
Spaceflight and microgravity	Astronauts (International Space Station, MARS500) and hindlimb unloading models	Reduced microbial diversity, altered abundance of Parasutterella and Akkermansia, increased Firmicutes	Dysregulated immunity and glucose metabolism, enhanced oxidative stress	Probiotic supplementation and crocodile gut-derived bacterial metabolites shown to mitigate dysbiosis in simulated microgravity
Hardy species and adaptive microbiomes	Crocodylus and Varanus species	Presence of stress-tolerant microbes and producers of bioactive metabolites such as leucrocin, cathelicidin and hepcidin	Potential for antimicrobial, anti-inflammatory and epigenetic modulation via secondary metabolites	Isolation and characterization of microbial metabolites for translational probiotic applications
Dietary and nutritional modulation	High-fat or low-fiber diets, malnutrition	Reduced SCFA-producing taxa and overall microbial diversity	Disturbed nutrient metabolism, immune dysregulation and inflammation	Fiber-rich and prebiotic diets restore microbial homeostasis and support physiological resilience

8. Novel strategies to modulate the gut microbiome

A recent report introduced a methodology that utilizes an in vitro fermentation test bed for evaluating engineered probiotics in a physiologically relevant manner. The approach involves a systematic workflow comprising fermentation, molecular and functional characterization, and statistical analyses to validate the persistence, plasmid stability, and reporter response of the engineered probiotic. The methodology evaluates the engineered probiotic’s ability to persist in a competitive growth environment, assesses reporter production and functionality, examines the impact of engineering on organism growth and commensal composition, and provides experimental validation for the transition of engineered probiotics to more comprehensive animal or human studies [41].

Understanding the precise biological mechanisms of the gut microbiome in human health and performance is crucial, especially in the military context. Exploring the mechanisms, processes, and genes that contribute to enhanced human performance and protection against environmental stressors may be facilitated by studying long-lived animals. Reptiles, particularly crocodiles, are remarkable examples of such species and are often referred to as “living fossils” [87]. With an origin dating back 310–320 million years, crocodiles have successfully survived multiple mass extinction events, including the Cretaceous-Tertiary extinction event. In contrast, humans are relatively recent additions to the planet, making it worthwhile to investigate the defense mechanisms of long-lived animals, such as reptiles, which have evolved, adapted, and thrived in harsh environments containing heavy metals and intense radiation. Recent research suggests that the gut microbiome of reptiles plays a crucial role in their robust immune system, their capability to resist infectious diseases and cancer, and, in some cases, negligible or minimal cellular senescence [13,14].

The presence of a unique gut microbiome in reptiles, particularly crocodiles, is now evident, and it is believed to play a significant role in their ability to produce antimicrobial peptides, resist infections and cancer, and exhibit longevity [15]. Recent research has revealed that specific gut bacterial metabolites derived from crocodiles possess potent activity versus various cancer cell lines, highlighting their potential for investigating the effects of these molecules on human performance, aging, and longevity [14]. Although further mechanistic studies need to be performed, the ability of bacteria isolated from crocodile gut to induce nitric oxide (NO) production in response to cellular stress, along with the levels of pro-inflammatory cytokines (IL-1β, TNF-α, PGE2) was recently investigated [17]. Senolytic compounds such as fisetin and quercetin were used for comparison, as they are known to selectively clear senescent cells and hold promise for preventing or treating various diseases and age-related conditions in humans. The findings revealed that conditioned media containing metabolites CP27 and CP36 from crocodile gut microflora significantly inhibited NO production compared to the positive control taxol, which induced 100% NO production under stress. Furthermore, CP27 and CP36 conditioned media, along with senolytic compounds, notably reduced taxol-mediated NO production in cerebrovascular endothelial cells. Interestingly, these effects were not observed when using conditioned media from nonpathogenic E. coli K-12. Importantly, crocodile gut bacterial conditioned media (CP27 and CP36) exhibited even greater potency in reducing NO production than senolytic compounds, even in the presence of taxol, underscoring their protective properties against cellular stress. Comparative insights into hardy species and their microbiome-derived metabolites have been described and reviewed recently, highlighting potential adaptive mechanisms relevant to human resilience and performance [88].

In order to utilize these novel bacterial metabolites for human health, a multi-step process is suggested: an exploration of the effects of these reptile gut microbial metabolites/strains in animal models or germ-free in vivo models and an investigation of host-gut microbial interactions and stressors via in vitro and ex vivo intestinal models, in the military context [89], as this may provide an alternative approach for developing novel interventions to modulate the gut microbiome. The HU model described earlier could be utilized to evaluate these probiotic strains and explore any epigenetic mechanisms associated with acute stress, using DNA methylation studies and analyzing various biomarkers associated with stress. Nonetheless, there is a need to explore potential risks that may also arise from these gut microbial constituents. For example, in reptiles such as lizards, Salmonella species have been detected as part of the normal gut flora and may act as opportunistic pathogens under favorable conditions [90]. Similar concerns may apply to crocodilians, emphasizing the necessity of thorough biosafety evaluation prior to any translational application. An alternative and more practical approach may be to identify and utilize bioactive metabolites or postbiotic components derived from these microbes, which may retain beneficial antimicrobial or anti-inflammatory properties while minimizing pathogen transmission risks [91]. Future work should therefore focus on systematic screening of crocodilian gut microbiota to distinguish beneficial metabolites from potential pathogens, ensuring safety and efficacy in downstream applications.

An intriguing example from 1917 involved a German corporal who displayed immunity to dysentery during an epidemic. It was hypothesized that his gut flora contained a strain of E. coli that exhibited antagonistic properties against various pathogens. This specific E. coli strain was isolated, cultivated, and patented as the therapeutic product “Mutaflor©,” which continues to be available in Germany and other countries today [92]. This case highlights the potential of studying and utilizing gut microbial species or metabolites from animals, such as crocodiles and other unique species, in a similar manner. This could be followed by the synthesis of bioactive molecules derived from the gut microbiome of these unique organisms, target identification, screening, testing of purified molecules or bacterial strains, lead optimization, and augmentation/modification of identified molecules to enhance their specificity to target sites. Subsequently, extensive evaluation in pre-clinical/clinical settings, product analysis, and regulatory approvals should be accomplished.

Another interesting approach is using technologies such as Organ-on-a-Chip (OOC) to test interesting and novel molecules. These OOC aim to mimic native tissue complexity in vitro, particularly in studying the gut with and without microbiota [93]. These models offer alternatives to animal testing for understanding physiology, pathology, and pharmacology, surpassing traditional 2D cell cultures. However, they still lack accurate representation of endocrine and immunological functions, and constructing complex models remains challenging. Despite limitations, gut-on-a-chip models hold promise for advancing our understanding of gut interactions and have implications for studying diseases, drug development, and personalized medicine [94].

A recent study investigated the potential of synbiotics, a promising new category of live therapeutics that utilize engineered genetic circuits [95]. This study aimed to develop a simplified gut-chip model using common Caco2 and HT-29 cell lines, providing a dynamic human screening platform for evaluating a synbiotic designed to sustain cognitive performance by sensing cortisol and producing tryptamine. The synbiotic the authors focused on, named SYN, was engineered from the well-known probiotic E. coli Nissle 1917 strain. It was engineered to detect cortisol at physiological levels, triggering a genetic circuit to produce tryptophan decarboxylase, an enzyme that converts available tryptophan into tryptamine. Within the gut-chip environment, SYN exhibited growth patterns similar to the wild-type strain during the log phase. Upon exposure to 5 μM cortisol, the rapid metabolism of tryptophan was observed, leading to complete conversion into tryptamine within the gut compartment. The gut-chip model provided a stable platform for assessing the sensitivity and dynamic range of the cortisol sensor by manipulating cortisol and tryptophan concentrations. Additionally, it enabled evaluation of tryptophan and tryptamine metabolism, production, and transport while allowing analyses of cellular viability and secretion of pro-inflammatory cytokines. Overall, these findings highlighted the efficacy of the human gut-chip in providing relevant insights into synbiotic function. This study shows that organ-on-a-chip technology holds great promise in accelerating the screening of potential therapeutics, thereby reducing failure rates and expediting the development of clinical interventions [95].

9. Concluding remarks

The composition of the gut microbiota has been linked to acute stressors faced by military personnel and astronauts, suggesting the modulation of the gut microbiome as a potential avenue for enhancing human health and performance. Dietary interventions, including prebiotics, probiotics, synbiotics, and microbiota transplantation, may offer promising methods for modulating the gut microbiota. Optimal strategies involve controlling diets and prebiotics to promote beneficial microbes associated with health and performance. Microbiota transplantation and/or supplementation could potentially shift the gut microbiota composition of individuals facing acute stressors toward that of healthier counterparts. Parallel advances in other biological systems, including aquaculture and microecological medicine, highlight the expanding translational potential of microbiome research and provide comparative perspectives relevant to stress adaptation and host resilience [96,97]. In framing future directions, it will be important to place acute stress responses within the broader context of long-term environmental change and its effects on food security, dietary access, and public health. Environmental fluctuations such as temperature variation, seasonal transitions, and extreme weather events can influence gut microbiome composition and immune balance, thereby shaping vulnerability to acute stressors [45].

Developing safe and effective delivery methods for these interventions is paramount. Research should focus on well-defined probiotic strains and novel bacterial strains, considering combinations, dosage, timing, and formulation. These strains can be evaluated through leveraging technologies such as Organ-on-a-Chip (OOC). OOC platforms aim to replicate native tissue complexity in vitro, providing insights into gut physiology and microbiota interactions. Another valuable way to test these bacterial strains/metabolites of interest will be to utilize the HU model, a rodent model which mimic conditions akin to space flight, bed rest, and acute stress experienced by humans. It provides a viable method for investigating skeletal muscle mass loss and interventions to counteract hindlimb unloading-induced atrophy. Moreover, this model facilitates the assessment of various physiological parameters, including blood pressure, heart rate, lipid composition, and blood sugar levels, as well as acute stress markers like oxidative stress and cortisol. Finally, research should utilize studies investigating epigenetic alterations in these in vivo models/organ-on-a-chip under acute stress compared to normal conditions, and after treatment with novel metabolites/probiotic strains. Future work conducting mechanistic studies, such as DNA methylation studies, to identify distinct epigenetic changes and assess potential adverse effects is necessary.

Funding Statement

This work was funded by the Air Force Office of Scientific Research (AFOSR), grant number: FA9550-23-1-0711.

Author contribution statement

NAK and RS envisioned the concept for the review. RS and RQ reviewed literature and prepared the first draft of the manuscript under the supervision of NAK and SM. All authors reviewed and approved the final manuscript.

Author’s note on AI-assisted editing

An AI-based language editing tool was used to improve the grammar and readability of this manuscript under the supervision of the authors. All scientific content, analysis, and interpretation were performed by the authors, who take full responsibility for the final manuscript.

Disclosure statement

The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

European Office of Aerospace Research and Development;