The microbiota of the human GI tract is essential for metabolic and digestive function, development, and support of the gut-associated immune system, prevention of gut colonization by pathogenic microbial species, and support of epithelial integrity to prevent barrier translocation of microbes (Bäckhed et al., 2005; Hooper and MacPherson, 2010; Stecher and Hardt, 2011). Studies suggest that the human GI tract harbors more than 800 different individual bacterial species (Turnbaugh et al., 2010) with proportional representation, genus level distribution, and viable count of colony-forming units (CFUs) varying widely from the oral cavity to the rectum (Hayashi et al., 2005; Wang et al., 2005; Bik et al., 2006; Lazarevic et al., 2009; Hakansson and Molin, 2011) and changing with age, diet, and geographical location (Biagi et al., 2010; Claesson et al., 2011). The predominant phyla in the healthy gut are Firmicutes and Bacteroidetes, which typically represent up to 80% or more of the microbiota, with smaller contributions of Actinobacteria (∼3%), Proteobacteria (∼1%), Verrucomicrobia, and Fusobacteria (∼0.1% or less) (Arumugam et al., 2011; Hakansson and Molin, 2011; King et al., 2019).

As noted above, most studies of the effect of radiation on the GI microbiome have been conducted in the context of cancer radiotherapy, and recent reviews summarize the literature in that context (Liu et al., 2021; Tonneau et al., 2021). Indeed, therapeutic abdominopelvic radiation exposure frequently results in intestinal dysfunction and dysbiosis, with acute radiation enteritis complications observed in 50% or more of abdominally irradiated cancer patients (Touchefeu et al., 2014). Radiation enteritis is associated with high morbidity and mortality, and chronic symptoms as severe as rectal hemorrhage, strictures, and fibrosis develop 3 months to 20 years after completion of radiotherapy (Packey and Ciorba, 2010; Ding et al., 2020). However, these studies can shed light on what may happen in the event of a radiological or nuclear mass casualty incident in which victims exposed to more than 6 Gy of radiation may acutely experience nausea, vomiting, diarrhea, sepsis, and death (Wojcik, 2002).

Rapidly dividing human cells are the most sensitive to the damaging and killing effects of ionizing radiation (Donnelly et al., 2010), and in particular, the GI epithelium is very sensitive to radiation, given that the GI crypt rapidly divides to shedding villi cells every 2–4 days (Novak et al., 1979; Somosy et al., 2002; Clevers, 2013; Williams et al., 2015). Radiation-induced cell death leads to loss of GI epithelial integrity and function, leading to inflammation and penetration of the GI epithelial barrier by the luminal contents and microbiota (François et al., 2013; Shukla et al., 2016). In addition, radiation damage to endothelial cells of the blood vessels within the villi can also result in vascular damage, causing further inflammation and sepsis (Paris et al., 2001). In the context of radiotherapy, most acute symptoms generally resolve within a few weeks as mucosal crypt, and villus structures are reconstituted from surviving stem cells (Umar, 2010).

A diverse and healthy commensal intestinal microbiota plays an essential role in GI homeostasis. However, it has been found that severe postirradiation enteropathy is associated with low mucosal bacterial diversity (Ferreira et al., 2019). In rodent studies, specific findings of microbiota changes in postirradiation fecal samples include increased abundance of the phylum Proteobacteria and family Lactobacillaceae and decreased abundance of families Lachnospiraceae, Ruminococcaceae, and Clostridiaceae, with some changes observed out to 10 months (Lam et al., 2012; Goudarzi et al., 2016; Zhao et al., 2019; Li et al., 2020). In humans, one prospective study of nine gynecologic cancer patients found that Firmicutes and Fusobacterium phyla were significantly decreased in fecal samples pre- versus post-pelvic irradiation (Nam et al., 2013). While there are few prospective studies that document changes in the gut microbiota postradiation, growing research interest in this area will likely fill that gap.

The microbiota in the oral cavity has long been studied, as changes in the balance of flora in the oral cavity can lead to infections like candidiasis, also known as “thrush,” first described and attributed to a fungus in 1839 by Bernhard von Langenbeck (Hellstein and Marek, 2019). Hundreds of years of interest and easy access to the oral cavity and saliva samples have facilitated extensive research on the oral microbiome and its connection to various disease processes, including responses to radiation exposure (Anjali et al., 2020; Belstrøm, 2020). In the oral cavity, there may be from 10⁸ to 10¹⁰ CFU per gram of saliva (Lazarevic et al., 2009). It should be noted that the oral microbiota even in healthy people varies drastically across location in the oral cavity, time of day, hydration, what and when the person ate, oral hygiene, age, smoking status, and so on (Aas et al., 2005; Bik et al., 2010; Cameron et al., 2015; Leake et al., 2016; Hall et al., 2017; Belstrøm, 2020; D’Angiolella et al., 2020). In radiation exposures, oral side effects such as xerostomia (dry mouth) are seen in patients receiving external beam radiotherapy to the head and neck (Wijers et al., 2002; Dirix et al., 2006) and radioiodine therapy (Alexander et al., 1998; Solans et al., 2001; Jeong et al., 2013; Hollingsworth et al., 2016). In fact, in a follow-up Chernobyl study, 4 of 15 survivors reported experiencing xerostomia (Gottlöber et al., 2001). Salivary damage and subsequent dry mouth can lead to a variety of problems, from difficulty chewing and talking to increased dental caries, oral mucositis, osteonecrosis, and so on (Dirix et al., 2006; Gomez et al., 2011; Tolentino et al., 2011; Sroussi et al., 2017; Chen et al., 2020). While studies of the oral microbiome following a nuclear accident are limited, there are many research studies that examine the changes in the oral microbiota following head and neck radiation exposure in oncology (Anjali et al., 2020).

The oral cavity has a delicate microbiota balance that can be directly affected not only by irradiation but also from changes in saliva composition and/or volume due to radiation-induced damage of the salivary glands, which are particularly radio-sensitive organs (Kałużny et al., 2014). Since the 1970s, radiation-induced xerostomia has been known to affect the oral microbiota (Brown et al., 1975; Brown et al., 1978; Sroussi et al., 2017; Mougeot et al., 2019; Breslin and Taylor, 2020), and it has been recently discovered that Candida infections in patients who received radiotherapy are often from species that are more virulent and drug-resistant (Tarapan et al., 2019). This is particularly concerning, given that Candida is the fourth most common cause of bloodstream infections among hospital patients in the United States and can be fatal (Hajjeh et al., 2004; Lone and Ahmad, 2019). A number of studies found increased abundance of Gram-negative and Lactobacillus bacterial species, as well as Candida fungal species following radiotherapy (Vanhoecke et al., 2015). Indeed, Nishii et al. found oral candidiasis occurred in 31% of 326 oral/oropharyngeal cancer patients who underwent radiotherapy, with oral mucositis associated with a higher incidence of oral candidiasis (Nishii et al., 2020). Researchers collected buccal swabs from oral cancer patients before and after radiotherapy, and while these patients already had altered oral microbiota with high prevalence of certain species following radiotherapy such as Streptococcus pathogenic Candida albicans, Klebsiella, and Pediococcus, with elevated Candida and Pediococcus persisting out to 6 months (Anjali et al., 2020).

Another study found Streptococcus and other species were predictive of high-grade oral mucositis, while Lactobacillus and Staphylococcus were only detected in patients with low- or no-grade oral mucositis in a study of 19 patients receiving fractionated radiotherapy (Vesty et al., 2020). Patients who developed more severe oral mucositis following radiotherapy had a higher abundance of Actinobacillus (Zhu et al., 2017), and an increase in certain microbes that coincided with the onset of severe mucositis over the course of patients’ radiation treatment (Hou et al., 2018). Additionally, an in vitro study found ionizing radiation increased the adherence of Streptococcus mutans on dental restoration material and promoted the formation of biofilms (Cruz et al., 2010).

In addition to the risk of salivary and oral damage caused by prompt exposure during a radiation incident, radioactive iodine fallout can find its way into the environment and eventually into human bodies, leading to a well-documented increased risk in thyroid cancer (Robbins and Schneider, 2000; Cardis and Hatch, 2011; Thomas, 2018). Salivary glands (La Perle et al., 2013) express the sodium iodide symporter, facilitating radioiodine uptake and potential damage. Although little research on the impact of radioiodine on the oral microbiome has been conducted, given the similarities in damage and symptoms between radioiodine therapy and external beam radiotherapy, changes to the microbiota may be similar.

With a surface area of approximately 2 m², the skin is the largest organ and is highly complex, with structures such as hair follicles and sweat ducts increasing its true surface area to about 25 m² (Gallo, 2017). The variable surface of the skin supports a vast ecosystem of distinct microorganisms, where more exposed areas tend to be drier and less populated by resident bacteria (Roth and James, 1988). However, the overall number of microorganisms present on the skin is held relatively constant under normal conditions (Davis, 1996). The commensal relationship between cutaneous tissue and the diverse community of microorganisms plays a critical role in barrier protection from invading pathogenic microorganisms, homeostasis, and the adaptive immune response (Dréno et al., 2016; Sfriso et al., 2020).

Much is still to be learned of the interplay between the skin microbiome and ionizing radiation-induced cutaneous injury. Most clinical studies focus on posttreatment inflammation, particularly dermatitis in breast cancer patients after radiotherapy (Eslami et al., 2020). As it is very likely that many individuals will have cutaneous and combined injuries following a radiation mass-casualty incident, mediating changes in the skin microbiota with preventative or mitigative treatments is of particular importance for chronic and acute wound healing outcomes and to prevent systemic complications. Combined injury, consisting of total body irradiation (TBI) followed by punch wounding resulted in early detection of bacteria in the blood, heart, and liver, although detection of bacteria was delayed in mice that received radiation alone. Only transient bacteremia occurred in mice that underwent wounding alone. Results suggest that increased levels of iNOS, cytokines, and bacterial infection triggered by combined injury may contribute to mortality in this model (Kiang et al., 2010).

Thermal and radiation burns are also likely during a radiation incident. However, standard medical management for thermal burns such as medications, wound dressings, therapy, and surgery may not be appropriate for radiation burns, which have a different damage profile with cyclic waves of inflammation and progressive lesion formation over time (DiCarlo et al., 2020). Adding to this complex scenario is the possibility of bacterial infection. Researchers have demonstrated extremophilic bacteria such as Aeribacillus, likely introduced during debridement of flame or scald wounds, correlated with patient comorbidities, such as pneumonia, infection, and sepsis (Plichta et al., 2017). Germ-free mice have been shown to have accelerated wound closure and scar reduction with elevated levels of anti-inflammatory cytokine IL-10, angiogenic growth factor VEGF, and angiogenesis in the germ-free wound tissue, suggesting the influence of an inflammatory component in wound healing (Canesso et al., 2014). A few case reports of mesenchymal stem cell treatment of patients with severe radiation burns also showed a resolution of inflammation (Bey et al., 2007; Bey et al., 2010). Although these studies suggest bacteria delay skin injury healing, certain bacterial species, such as Lactobacillus plantarum, can inhibit biofilm growth of harmful bacterial (e.g., Pseudomonas aeruginosa), subsequently improving tissue repair (Valdéz et al., 2005). These studies suggest it is possible to harness the beneficial power of the skin microbiome, expanding therapeutic options.

Although different from radiation injury, the microbiome research conducted for other skin injuries, such as those involving ultraviolet irradiation (Wolf et al., 2016; Patra et al., 2019; Patra et al., 2020), diabetic ulcers, and other chronic skin diseases (Wolcott et al., 2016; Johnson et al., 2018), may shed light and help guide future skin microbiome research in the context of radiation injury. Additionally, clinical strategies currently used to treat these complicated skin wounds may provide insight into identifying effective therapeutics and improving patient outcomes. While a wealth of information can be found in the literature on processes governing wound healing, the role of the skin microbiome is less clear. Research shows that differences exist between normal and pathological microbial responses after a skin injury (Singer and Clark, 1999; Schultz et al., 2011; Johnson et al., 2018); therefore, a better understanding of the skin microbiome and its influence on the immune response has great medicinal potential with regard to radiation injuries.

Historically, lungs have been considered sterile. When it was first reported in 2010 that the microbiome in the lower airways was comparable to the upper bowel, the phenomenon was attributed to possible contamination during the bronchoalveolar lavage (BAL) procedure (Hilty et al., 2010). Since then, the existence of a microbiome in healthy lung has been widely accepted (Kiley and Caler, 2014; Mathieu et al., 2018). The lung microbiome is situated in the lower airways of healthy lung and houses a large number of microbes, including phyla Bacteroidetes and Firmicutes (Charlson et al., 2011; Dickson et al., 2013; Liu et al., 2020). The microbiome landscape changes dramatically under disease conditions affecting the lung, such as asthma and chronic obstructive pulmonary disease (Segal et al., 2014; Evsyutina et al., 2017), through processes involving immigration, elimination, and local growth conditions (Evsyutina et al., 2017).

Microbial migration occurs via air inhalation, micro-aspiration, and direct dispersion through the respiratory tract mucosa, while microbiome elimination occurs by mucociliary clearance, cough, and immune mechanisms. Microbiome growth conditions can be influenced by pO₂, pH, blood perfusion, alveolar ventilation, temperature, lung epithelium, mucociliary clearance, and inflammatory cell activity. Furthermore, microbiome expansion is affected by bacteriostatic activity from surfactant produced in the distal alveoli. Finally, under disease conditions, the lung microbiome can be entirely destroyed and replaced with a single pathogen, as can occur during pneumonia (Araghi, 2020). Interestingly, the gut microbiota can affect general pulmonary health through a vital cross-talk between the gut microbiota and the lungs, referred to as the “gut–lung axis” (Keely et al., 2012). The gut–lung axis is bidirectional, denoting that the endotoxins and microbial metabolites released into systemic circulation by the gut can affect the lung, and if inflammation occurs in the pulmonary tissue, the gut microbiota is also affected (Dumas et al., 2018).

Though progress has been made, lung microbiome research is complicated by the difficulty in collecting biospecimens specific to the lung and lower airways. Clinically, sputum is used as a surrogate for lower airway samples; however, this process leads to contamination from microbes inhabiting the upper airways and oral cavity. Unfortunately, other than sputum, there are few reliable approaches to lower airway sampling, which is an obstacle to large-scale investigations of lung disease for studies requiring frequent sampling. Similarly, lung microbiome analysis using BAL fluid can also be contaminated by contributions from upper airway microbiota. Several studies analyzing lung tissue acquired via sterile surgical explant demonstrated that the lower respiratory tract contains a microbiome that is distinct from but related to that of the upper airways (Dickson et al., 2013).

While there are some publications related to radiotherapy and lung microbiome, there are no publications specific to the role of lung microbiome in radiation-induced lung injury at the writing of this review. One study described the prophylactic (pre-irradiation) use of heat-inactivated Salmonella typhimurium in ameliorating thoracic radiation-induced lung injury in mice by reducing apoptosis, inflammation, and endothelial mesenchymal remodeling of lung tissue (Kun et al., 2019). Some recent publications indicate that low-dose radiation therapy can be used in treating SARS-CoV-2–induced pneumopathy (Prasanna et al., 2020; Salomaa et al., 2020; Wilson et al., 2020); however, the relationship to the normal lung microbiome and the potential for a mitigation or biodosimetry strategy from these few studies is relatively unclear. Researchers in the radiation community can draw upon publications on the microbiome of the lung to better understand the significance of the microbiome in radiation-induced lung injury and how the microbiota are implicated in intervention strategies. These could include determining (1) whether an altered lung microbiome initiates radiation-induced disease pathogenesis, promotes chronic inflammation, or is merely a marker of injury and inflammation; (2) whether the lung microbiome can be manipulated therapeutically to change radiation-induced lung disease progression; and (3) what molecules (metabolites) generated during an inflammatory response can serve as biomarkers for pulmonary injury diagnosis and prognosis of the therapeutic interventions.

The following microbiome niches are of lesser interest to the radiation emergency mission space. Radiation damage to these systems has low to no impact on lethality and no well-established animal models of injury. However, radiation exposure can still greatly damage these tissues and their resident microbiota and have been included here for completeness.

Similar to H-ARS, chemotherapy can induce myelosuppression in cancer patients, resulting in increased risk of infection. Thus, the IDSA has published guidelines recommending neutropenic cancer patients be given fluroquinolone antibiotics (Freifeld et al., 2011). As it is likely that antibiotics will be first-line therapeutics in the event of a mass casualty radiation emergency (Coleman et al., 2015), this IDSA recommendation was initially put forward as a recommendation of the Strategic National Stockpile Radiation Working Group, convened in 2002 (Waselenko et al., 2004). This guidance is supported by studies carried out in mice at various institutions. For example, in developing a model of H-ARS, investigators tested several antibiotic regimens in mice given various doses of TBI—finding MST was increased in antibiotic-treated mice, although levofloxacin did not provide a better outcome than ciprofloxacin (Plett et al., 2012). They also found that the use of different combinations of antibiotics (e.g., doxycycline + neomycin) increased survival (Plett et al., 2012).

Additionally, iliac bacteria counts in mice exposed to 10 Gy of TBI were found to be reduced, and anaerobe repopulation was delayed (Brook et al., 1988). Anaerobic bacteria appear to be protective, as treatment with metronidazole caused a further decrease in the anaerobic population and quicker onset of mortality. A subsequent review (Brook et al., 2004) noted that administration of quinolones to mice reduced levels of Gram-negative aerobes while sparing the anaerobic population, which is in alignment with IDSA guidelines and is the preferred choice.

Researchers have long known that administration of antibiotics to irradiated animals can affect their survival, as noted above. This modification has generally been attributed to the ability of these molecules to reduce the likelihood of opportunistic infections in animals that are immunosuppressed—but what if the efficacy could also involve a more direct modification of the natural flora of the animal? Fluoroquinolones, such as enrofloxacin and tetracycline, have been shown to reduce radiation damage to hematopoietic progenitor cells grown in culture. Thus, the radiation dose-modifying effect of some antibiotics may allow them to serve as radiation mitigators in addition to their ability to slow the growth of microbes (Epperly et al., 2010). These findings were further explored in another model of GI-ARS that demonstrated that oral fluoroquinolones also led to higher survival rates in irradiated mice (Booth et al., 2012). In a mouse model of radiation combined injury, ciprofloxacin provided similar protection (Kiang et al., 2014), and in a TAI model, where radiation exposure was used to reduce the number of GI microbes, a cocktail of antibiotics given prior to radiation exposure improved bacterial regrowth in the gut (Zhao et al., 2020).

In addition, the use of acidified water, which is frequently employed in animal colonies, could mask the impact of radiation-induced GI injury. Acid water (pH 2.5–3.0) is used to prevent bacterial infections from spreading within an animal colony. ¹ It is often accomplished using hydrochloric or sulfuric acid or tetracycline (Hermann et al., 1982). Its use provides protection not only primarily against Pseudomonas aeruginosa but also against other Gram-negative organisms (Small and Deitrich, 2007), and in mouse models, water acidification has been shown to reduce the diversity of the gut microbiome (Sofi et al., 2014). Therefore, researchers considering the use of radiation injury models to study microbiome traits should be aware of these kinds of husbandry details in their animal facilities.

The idea of altering the host microbiome was first introduced by Russian embryologist Elie Metchnikoff in the early 1900s (Podolsky, 2012). In the 1990s, a resurgence of probiotic research occurred and only in 2001 was the term “microbiome” used in the literature to describe the collective genome in a host. In late 2001, the Food and Agriculture Organization of the United Nations and the World Health Organization held an expert consultation in Cordoba, Argentina, to evaluate the health and nutritional properties of probiotics in food, which led to a joint report to provide assessment and safety guidelines for research in the field (Food and Agriculture Organization of the United Nations World Health Organization, 2006). Since then, many studies have demonstrated the beneficial effect that live, naturally occurring microorganisms can have on the immune system (Hardy et al., 2013; Peters et al., 2019), gut (Gourbeyre et al., 2011; Quigley, 2012), food allergies (Di Costanzo et al., 2020), colon (Pujo et al., 2020; Wang et al., 2020), skin (Friedrich et al., 2017; Patra et al., 2020), and central nervous system (Kim et al., 2020; Loniewski et al., 2020). Of particular importance for this review are the therapeutic effects of probiotics that are seen when these systems are exposed to ionizing radiation.

The Institut des Maladies de l’Appareil Digestif conducted a systematic review of six preclinical and seven clinical studies (Touchefeu et al., 2014), which found that decreases in Bifidobacterium, Clostridium cluster XIVa, Faecalibacterium prausnitzii, and increases in Enterobacteriaceae and Bacteroides after radiotherapy contributed to GI mucositis, leading to increased diarrhea and bacteremia. Many probiotic strains were investigated as preventative therapeutics, most of which led to a reduction in diarrhea or bacteremia incidence. Another systematic review considered 15 clinical trials studying varied GI pathologies (Picó-Monllor and Mingot-Ascencao, 2019). They concluded that a combination of probiotics could reduce the incidence of mucositis in chemo- or radiotherapy-treated patients. Likewise, a meta-analysis of randomized controlled trials showed that supplementation with Lactobacillus acidophilus plus Bifidobacterium bifidum had a modest effect at preventing radiation-induced diarrhea after abdominal or pelvic radiotherapy (Liu et al., 2017). Clearly, probiotics within Lactobacillus and Bifidobacterium genera were found effective in many of the trials.

Nonpathogenic bacterial species in genera such as Lactobacillus and Bifidobacterium are commonly used and have demonstrated a wide range of health benefits (Hardy et al., 2013). Understanding the role these bacteria play in the processing and biotransformation of xenobiotics or foreign compounds (e.g., drugs and antibiotics) in the host gut can lead to personalized therapeutics to avoid or circumvent antibiotic resistance (Maurice et al., 2013). In the case of a mass casualty radiation emergency, antibiotics will likely be used as first-line therapeutics (Coleman et al., 2015). Therefore, understanding this interplay will be essential to selecting the proper antibiotics. It may also be possible to co-administer a probiotic that can manage the microbial variability of the human gut.

Research on the potential for probiotics to serve as radiation MCMs is limited; however, the prophylactic use of probiotics has been explored extensively. The knowledge gained about underlying mechanisms in these kinds of studies could lead to druggable pathways and aid in the development of MCMs, specifically to address GI-ARS. For example, death was delayed for mice fed Lactobacillus rhamnosus GG (LGG) prior to exposure to 14 Gy of TBI (Dong et al., 1987). LGG, the first bacterial strain to be patented in 1989, has since demonstrated benefit against GI issues (Dong et al., 1987; Ciorba et al., 2012; Capurso, 2019; Riehl et al., 2019), perhaps by altering the immune system (Capurso, 2019), and protecting intestinal epithelium (Riehl et al., 2019). In another study, LGG protected the intestinal epithelium in mice that were administered the probiotic or LGG-conditioned media by oral gavage, 3 days prior to 12-Gy TBI (Ciorba et al., 2012). Researchers showed that LGG administration prior to irradiation increased the number of regenerative crypt cells and reduced epithelial cell apoptosis. This effect was observed both for mice administered LGG and mice administered LGG-conditioned media. Moreover, a head-to-head comparison of commercially available probiotics demonstrated that Culturelle offered a similar level of radioprotection to that produced by live, cultured LGG; however, protection was not provided by another non-Lactobacillus, commercially available probiotic (B infantis 35624; Align) (Ciorba et al., 2012). Administration of probiotics (LGG and Bifidobacterium longum) has also been shown to improve survival in pediatric mice after the onset of sepsis resulting from a cecal ligation and puncture (Khailova et al., 2013). In addition, several probiotic species were shown to be effective at displacing dangerous enteropathogens (Candela et al., 2008). Together, these studies suggest that Lactobacillus may be the probiotic genus of choice for ameliorating radiation-induced GI injury.

Lactobacillus is a member of the Firmicutes phylum, and another recent study found elevated Firmicutes bacteria levels in irradiated mice were associated with a survival benefit. Mice exposed to 9.2-Gy TBI that had an abundance of bacteria in the Lachnospiraceae, and Enterococcaceae families present in their gut had a significant survival advantage or were considered “elite-survivors” (Guo et al., 2020). Upon exposing germ-free mice to “elite-survivor” dirty cages or FMT via oral administration of feces, specific pathogen-free mice had significantly higher rates of survival than non-FMT controls. To substantiate these findings in humans, researchers also looked at fecal samples from 21 leukemia patients undergoing TBI as a pre-hematopoietic stem cell transplant conditioning. Patients with higher levels of Lachnospiraceae and Enterococcaceae generally had shorter bouts of diarrhea, as well as increased levels of propionate and tryptophan metabolites (Guo et al., 2020).

Second-generation probiotics are also being developed to take advantage of the natural properties of these bacteria, using microbial-mediated delivery of drugs to target the gut. Researchers have engineered probiotics that produce IL-22 (Zhang et al., 2020), a cytokine with anti-inflammatory properties known to stabilize both intestinal Paneth cells and Lgr5+ intestinal stem cells (Zha et al., 2019). In this study, C57BL/6 mice were exposed to 9.25-Gy TBI and then treated with Lactobacillus reuteri–producing IL-22 strains postirradiation via oral gavage. A 30% improvement in survival was noted, as compared to animals dosed only with the IL-22 protein. Time of administration of the bacteria was also examined, and a survival advantage could be seen even when dosed at 72-h postirradiation, with the highest benefit seen at 24 h (85%) and 48 h (70%) postirradiation administration (Zhang et al., 2020).

Probiotics may be therapeutic in systems beyond the GI. Oral probiotics have been found to affect microbial communities and local inflammation within these axes as well as the vaginal microbiota (Petricevic et al., 2008), skin (Eslami et al., 2020), and more. Additionally, the emerging information in the area of microbiome/gut–brain axis opens up new opportunities for the development of effective treatments for CNS disorders. Changes in the gut microbiota postirradiation have been associated with psychoneurological symptoms in cancer patients (Bai et al., 2020). Psychobiotics (bacterially mediated biotherapeutics, which include probiotics, prebiotics, and synbiotics—a combination of probiotics and prebiotics) are currently being investigated for their potential in treating neurologic disorders. Psychobiotics can be delivered through supplements, functional foods, and dietary changes (Long-Smith et al., 2020).

As the field of probiotics has continued to mature, researchers have found that synbiotics may provide a superior outcome than either one alone, by providing an optimal GI environment to allow the probiotics to survive and colonize the gut (Markowiak and Śliżewska, 2017). Another important consideration is the risk associated with certain strains of probiotics such as the Enterococcus genus, which can acquire antibiotic resistance and become pathogenic. To date, no enterococcal probiotics have been approved for human use, leading the European Food Safety Authority to conclude that “Enterococci do not meet the standard for Qualified Presumption of Safety” (Wang et al., 2020). Given these data, along with studies showing their systemic effects (Valdéz et al., 2005; Petricevic et al., 2008; Keely et al., 2012), probiotics are a promising potential treatment for GI-ARS and other radiation injuries.

In considering the GI microbiome, dietary supplementation can play a major role in the composition of gut bacteria and impact of radiation exposure. For example, normal tissue injuries from administration of abdominal radiotherapy to treat gynecologic malignancies can sometimes evolve into chronic radiation enteritis. Therefore, a clinical trial (NCT01549782) was carried out to study the effect of consumption of certain prebiotics, in this case fiber and plant sugars, on stool consistency in postirradiation patients (Garcia-Peris et al., 2016). Some improvement was noted in the group that consumed the prebiotic diet (reduction in days of diarrhea), suggesting that these dietary changes could lead to improved quality of life for these patients. Although the causal role of modulating microbiome by supplements to improve radiation injury resulting from accidental exposure to large doses is not as widely published, supplements are reported to protect gamma-irradiated mice (Shimoi et al., 1994) and improve survival (Satyamitra et al., 2011; Obrador et al., 2020). However, there are conflicting reports that underscore the need for caution in the use of all supplements without supporting data. For instance, investigators reported that high-protein diet such as methionine-supplemented diet (MSD) is used to build muscle mass in patients undergoing chemo- and/or radiotherapy; however, when this diet was fed to CBA/CaJ mice exposed to 3–8.5 Gy of TBI, the mice developed acute radiation toxicity, even at sublethal doses of 3 Gy, and demonstrated higher mortality (Miousse et al., 2020). Another study reported that MSD increased GI toxicity in abdominal irradiated CBA/CaJ mice, with a concomitant shift in gut microbiome, reduction in microbiome diversity, and significant increase in pro-inflammatory genus Bacteroides (Ewing et al., 2021). In addition, omega-3 polyunsaturated fatty acids were shown to reduce intestinal inflammation following radiotherapy (Zhang et al., 2019), a finding that was attributed to its ability to reduce oxidative stress in the GI tract. Similarly, consideration of the diet of astronauts has been a major source of concern, since space flight involves exposure to cosmic radiation (Turner et al., 2002). By providing extra antioxidants to the diet, in the form of vitamins such as E and C, as well as flavonoids, polyphenols, and folic acid, it may be possible to modify the composition of gut bacteria and reduce the risks associated with radiation exposure. This could be applicable to a wide range of scenarios involving radiation exposure including during space missions.

A novel investigative treatment is the use of FMT. Briefly, fecal material is obtained from a screened, healthy donor (or in the case of radiation exposure, an unirradiated host) followed by a dilution, homogenization, and filtration processing. The resulting preparation is then administered to the colon of the recipient, either through oral ingestion of a capsule, or via colonoscopy or enema. In preclinical studies, animals are typically fed donor feces. Initially conceived as a means of correcting the microbiome imbalance in individuals suffering from chronic GI infections, the therapy has completed a randomized, controlled clinical trial for treatment of antibiotic-resistant Clostridium difficile infection (Kelly et al., 2021). The therapy is believed to work by “out-competing” growth of C. difficile with other more protective species. Studies have shown that this treatment can mitigate infections in 80–90% of patients (van Nood et al., 2013). FMT procedures have also been studied to address a number of different disease states, such as multiple sclerosis (NCT03975413), diabetes (NCT04124211), autism (NCT03408886), AIDS (NCT02256592), and liver diseases (NCT03152188) (Lo, 2019). These findings of efficacy across multiple organ systems and disease states are not surprising, given the acknowledged role of the GI microbiome in the “gut–brain–skin axis” (Vojvodic et al., 2019) and the “gut–lung axis” (Dumas et al., 2018; Nie et al., 2020), which involve a close interplay between the systems and regulation by signaling molecules. Therefore, balance of microbes in the GI tract is important for maintenance of many conditions outside the gut.

Novel therapeutics are being developed in search of effective MCMs against ARS, including radiation mitigators that have a common 4-nitro-phenyl-piperazine pharmacophore (NPSP) (Micewicz et al., 2019). In this study, C3H mice were exposed to an LD_70/30 dose of radiation and then treated with an NPSP mitigator. To track long-term changes in the mice microbiota, fecal samples were collected from both irradiated and control mice on days 162, 214, and 442. The colonic microbiota was analyzed by 16S rDNA enrichment and sequencing, showing a consistent level of Firmicutes-to-Bacteroidetes composition in both treated and control mice until day 214. At this point, mice treated with NPSP 5355512 exhibited a decreased amount of Bacteroidetes, while the level of Firmicutes increased as compared to control mice (Micewicz et al., 2019). The Firmicutes-to-Bacteroidetes ratio is often analyzed as a marker for gut health but can fluctuate often and change with age (Mariat et al., 2009). While the significance of the change still needs to be elucidated, it is interesting to note that composition of the microbiome differed between the treated and non-treated groups.

Other therapeutics such as phycocyanin (PC), an active protein found in the genus Arthrospira, have been examined for efficacy against radiation-induced GI injury after radiotherapy. PC has been shown to have anti-inflammatory (Remirez et al., 2002) and antioxidant (Villegas et al., 2014) properties. In one study, C57BL/6 mice were administered PC daily for a month prior to an exposure of 12-Gy TAI (Lu et al., 2019). PC treatment provided protection against radiation-induced GI injury and maintained a healthier level of diversity in the microbiota, which is usually reduced after irradiation. In general, the levels of beneficial bacteria were increased, harmful bacteria were decreased, and inflammatory cytokines such as TNF-α and IL-6 were downregulated (Lu et al., 2019). Another drug simvastatin, commonly used to treat high cholesterol, has also been shown to alter the gut microbiota to provide a therapeutic advantage against radiation-induced injury in mice (Cui et al., 2019). Maintenance of a healthy gut microbiome appears to be essential in overcoming radiation-induced injury, as supported by studies that highlight the importance of this balance. It may be possible to repurpose existing products to modify the microbiome.