Recent research has recognized humans as metaorganisms— multispecies consortia consisting of the human host and its associated microbial inhabitants commonly referred to as the microbiome. The subsequent flood of research in this field has led to a dramatic improvement of our understanding into how the host, its microbiome, and the environment interact to determine human health and disease. On Earth, probiotics have been shown to interact with the host and its GI microbiome to improve the immune response, protect against pathogens, and improve gut barrier function.
Long-duration missions in space will expose astronauts to chronic stresses imposed by microgravity, ionizing radiation, and confinement. These aspects of space flight have been shown to alter astronaut physiology, affecting nearly every system including musculoskeletal, neurological, endocrine, cardiovascular, respiratory, excretory, cognitive, and immune systems. Development of effective countermeasures to maintain astronaut health and performance under the extreme conditions of deep space exploration is an actively ongoing NASA endeavor.
Research into changes to astronaut microbiomes has revealed evidence of both compositional and functional changes during long-duration space flight. Dysregulation of the astronaut immune system in space has been identified as a major contributor to numerous spaceflight syndromes. Further, The Design Reference Mission (DRM) for a Mars mission states that all nutrients sufficient for three plus years will be preserved and stored onboard, and no resupply activity or cultivation of fresh food is planned. This necessitates research into the stability and shelf-life of food items, supplements, and pharmaceuticals in space over the course of a Mars DRM. Probiotics stored as freeze-dried powders in capsules are a promising option for long-term stability. However, probiotics consisting of bacterial spores may serve as a better, albeit unexplored, option for long-duration missions due to their extreme longevity and documented resistance to ionizing radiation.
The hypothesis of this experiment is exposure of probiotic bacteria to simulated space radiation will result in a decrease in survival and potency at a rate which can be empirically measured. Probiotics containing Bacillus spores will demonstrate enhanced long-term stability and potency compared to traditional Lactobacillus- or Bifidobacterium-containing probiotics.
This experiment has three specific aims.
(1) Select probiotic candidates to test.
(2) Expose probiotic samples to simulated Galactic Cosmic Radiation (GCRSim) and Solar Particle Events (SPESim) at Mars DRM-relevant doses.
(3) Characterize probiotic viability of pre- and post-irradiation probiotic samples and potency of these samples in simulated gastric and intestinal juices in vitro.
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For Aim 1, the researchers applied a set of criteria to select three candidate probiotic formulations. These criteria included (1) the probiotics contain only a single species, (2) the selected species should be readily cultured on standard laboratory media, (3) the candidate probiotics are commercially available in a form amenable to long-duration space flight, and (4) the candidate probiotics should have a proven record of safety and efficacy documented in the peer-reviewed literature. The selected probiotics included Lactobacillus acidophilus DDS-1, Bifidobacterium longum BB536, and Bacillus subtilis HU58.
Following the selection of the probiotic strains, 16S taxonomy will be used to ensure the species identity of selected commercial probiotic cultures. The universal bacterial primers B27F (5'-GAGTTTGATCMTGGCTCAG-3') and B1512R (5'-AAGGAGGTGATCCANCCRCA-3') will be used to amplify the selected 16S sequence, which will subsequently be identified to the species level using the Ribosomal Database Project server. Further, the researchers will characterize the proportion of viable cells in each preparation using live/dead fluorescent staining. This measure will provide a baseline for comparison to samples exposed to radiation during Aim 2.
In Aim 2, the samples will be exposed to Mars DM-relevant doses of GCRSim or SPESim beams at the NASA Space Radiation Laboratory (NSRL). GCRSim consists of a mixture of atomic nuclei (H, He, C, O, Si, Ti, and Fe) delivered in series at a variety of energies and dosages to simulate the flux of GCR expected to be encountered during a three-year Mars DRM. SPESim consists of protons delivered at a variety of different energies simulating a typical SPE. Each probiotic will be loaded into capsules and arranged on 28-place blister cards according to a random number generator. Four blister cards total will be irradiated, each containing nine samples of each of the three selected probiotics, resulting in a total 36 treated samples per probiotic strain.
In Aim 3, the 36 treated samples and 36 control samples of each strain will be compared across four assays: (1) viability assessment by plating, (2) live/dead fluorescent staining, and survival to simulated (3) gastric and (4) intestinal juices in vitro. Working suspensions of each sample will be prepared from probiotic lyophilized powder within the capsules for all four assays. Aliquots of serial tenfold dilutions will be plated in duplicate on either LB (Bacillus subtilis) or MRSC (Lactobacillus acidophilus, Bifidobacterium longum) agar plates and incubated at 37 °C for up to 72 hours. Plates yielding 20-200 well-separated colonies will be counted and the number of Colony Forming Units (CFUs) will be extrapolated. The same dilution series used for plating will be used for live/dead fluorescent staining in which the nucleic acids will be stained green in live cells and be stained red in dead cells. For in vitro experiments, aliquots of the working suspension will be transferred to commercial preparations of simulated gastric and intestinal juices and incubated in a 37 °C shaking water bath for 0, 0.5, 1, 2, and 3 hours (gastric) or 0, 2, 4, 6, 12, and 24 hours (intestinal). The potency of cells exposed to simulated gastric or intestinal fluids will be evaluated using plating and live/dead fluorescent staining.
This study aims to assess the viability and potency of three common probiotics, Lactobacillus acidophilus, Bifidobacterium longum, and Bacillus subtilis, after exposure to ionizing radiation simulating a three-year Mars DRM. The researchers hypothesize that all three bacteria will be inactivated to some extent by radiation exposure, but that Bacillus subtilis spores will retain a higher degree of viability and potency than the other two strains due to the well-documented enhanced radiation resistance of spores. The results of this experiment will provide valuable baseline information for future planning decisions regarding the inclusion of probiotics on future Mars missions.