The long-term response of microbial communities to the microgravity environment of space is not yet fully understood. Of special interest is the possibility that members of these communities may acquire antibiotic resistance. In this study, Escherichia coli cells were grown under low-shear modeled microgravity (LSMMG) conditions for over 1,000 generations (1000G) using chloramphenicol treatment between cycles to prevent contamination. The results were compared with data from an earlier control study done under identical conditions using steam sterilization between cycles rather than chloramphenicol. The sensitivity of the final 1000G-adapted strain to a variety of antibiotics was determined using Vitek analysis. In addition to resistance to chloramphenicol, the adapted strain acquired resistance to cefalotin, cefuroxime, cefuroxime axetil, cefoxitin, and tetracycline. In fact, the resistance to chloramphenicol and cefalotin persisted for over 110 generations despite the removal of both LSMMG conditions and trace antibiotic exposure. Genome sequencing of the adapted strain revealed 22 major changes, including 3 transposon-mediated rearrangements (TMRs). Two TMRs disrupted coding genes (involved in bacterial adhesion), while the third resulted in the deletion of an entire segment (14,314 bp) of the genome, which includes 14 genes involved with motility and chemotaxis. These results are in stark contrast with data from our earlier control study in which cells grown under the identical conditions without antibiotic exposure never acquired antibiotic resistance. Overall, LSMMG does not appear to alter the antibiotic stress resistance seen in microbial ecosystems not exposed to microgravity. IMPORTANCE Stress factors experienced during space include microgravity, sleep deprivation, radiation, isolation, and microbial contamination, all of which can promote immune suppression (1, 2). Under these conditions, the risk of infection from opportunistic pathogens increases significantly, particularly during long-term missions (3). If infection occurs, it is important that the infectious agent should not be antibiotic resistant. Minimizing the occurrence of antibiotic resistance is, therefore, highly desirable. To facilitate this, it is important to better understand the long-term response of bacteria to the microgravity environment. This study demonstrated that the use of antibiotics as a preventive measure could be counterproductive and would likely result in persistent resistance to that antibiotic. In addition, unintended resistance to other antimicrobials might also occur as well as permanent genome changes that might have other unanticipated and undesirable consequences.

https://journals.asm.org/doi/pdf/10.1128/mBio.02637-18

Evaluation of Acquired Antibiotic Resistance in Escherichia coli Exposed to Long-Term Low-Shear Modeled Microgravity and Background Antibiotic Exposure

BioSentinel is the first biological CubeSat designed and developed for deep space. The main objectives of this NASA mission are to assess the effects of deep space radiation on biological systems and to engineer a CubeSat platform that can autonomously support and gather data from model organisms hundreds of thousands of kilometers from Earth. The articles in this special collection describe the extensive optimization of the biological payload system performed in preparation for this long-duration deep space mission. In this study, we briefly introduce BioSentinel and provide a glimpse into its technical and conceptual heritage by detailing the evolution of the science, subsystems, and capabilities of NASA's previous biological CubeSats. This introduction is not intended as an exhaustive review of CubeSat missions, but rather provides insight into the unique optimization parameters, science, and technology of those few that employ biological model systems.

BioSentinel: A Biological CubeSat for Deep Space Exploration.

We report here complete 6-month results from the orbiting Space Environment Survivability of Living Organisms (SESLO) experiment. The world’s first and only long-duration live-biology cubesat experiment, SESLO was executed by one of two 10-cm cube-format payloads aboard the 5.5-kg O/OREOS (Organism/Organic Exposure to Orbital Stresses) free-flying nanosatellite, which launched to a 72°-inclination, 650-km Earth orbit in 2010. The SESLO experiment measured the long-term survival, germination, metabolic, and growth responses of Bacillus subtilis spores exposed to microgravity and ionizing radiation including heavy-ion bombardment. A pair of radiation dosimeters (RadFETs, i.e., radiation-sensitive field-effect transistors) within the SESLO payload provided an in-situ dose rate estimate of 6–7.6 mGy/day throughout the mission. Microwells containing samples of dried spores of a wild-type B. subtilis strain and a radiation-sensitive mutant deficient in Non-Homologoous End Joining (NHEJ) were rehydrated after 14, 91, and 181 days in space with nutrient medium containing with the redox dye alamarBlue (aB), which changes color upon reaction with cellular metabolites. Three-color transmitted light intensity measurements of all microwells were telemetered to Earth within days of each 24-hour growth experiment. At 14 and 91 days, spaceflight samples germinated, grew, and metabolized significantly more slowly than matching ground-control samples, as measured both by aB reduction and optical density changes; these rate differences notwithstanding, the final optical density attained was the same in both flight and ground samples. After 181 days in space, spore germination and growth appeared hindered and abnormal. We attribute the differences not to an effect of the space environment per se, as both spaceflight and ground-control samples exhibited the same behavior, but to a pair of ~15-day thermal excursions, after the 91-day measurement and before the 181-day experiment, that peaked above 46 °C in the SESLO payload. Because the payload hardware operated nominally at 181 days, the growth issues point to heat damage, most likely to component(s) of the growth medium (RPMI 1640 containing aB) or to biocompatibility issues caused by heat-accelerated outgassing or leaching of harmful compounds from components of the SESLO hardware and electronics.

Nanosatellites for Biology in Space: In Situ Measurement of Bacillus subtilis Spore Germination and Growth after 6 Months in Low Earth Orbit on the O/OREOS Mission

BioSentinel is the first biological CubeSat designed and developed for deep space. The main objectives of this NASA mission are to assess the effects of deep space radiation on biological systems and to engineer a CubeSat platform that can autonomously support and gather data from model organisms hundreds of thousands of kilometers from Earth. The articles in this special collection describe the extensive optimization of the biological payload system performed in preparation for this long-duration deep space mission. In this study, we briefly introduce BioSentinel and provide a glimpse into its technical and conceptual heritage by detailing the evolution of the science, subsystems, and capabilities of NASA's previous biological CubeSats. This introduction is not intended as an exhaustive review of CubeSat missions, but rather provides insight into the unique optimization parameters, science, and technology of those few that employ biological model systems. 

/pdf/biosentinel-a-biological-cubesat-for-deep-space-exploration-1bciaphp.pdf

BioSentinel: A Biological CubeSat for Deep Space Exploration

Space microbiology research contributes to human safety and performance during space exploration by advancing understanding of the effects of reduced gravity and/or radiation on cells and organisms. Astrobiology, on the other hand, seeks a more fundamental understanding of life and the universe; its objectives are also furthered by spaceflight experiments. Experimental progress in both fields is limited partly by access to space aboard human-tended spacecraft and space stations. CubeSats are helping address such limitations by proving to be effective biological experiment spaceflight platforms. NASA's Ames Research Center has developed and operated five CubeSats in low Earth orbit with microbiology or astrobiology missions; technical advances and lessons learned from each “nanosatellite” served as the starting point for the next. Here, we present technical and performance overviews of these biological CubeSats and their scientific results. A preview of the next biological CubeSat mission, bound for interplanetary space, along with potential future applications, is included.

CubeSats for microbiology and astrobiology research

Human immune response is compromised and bacteria can become more antibiotic resistant in space microgravity (MG). We report that under low-shear modeled microgravity (LSMMG), stationary-phase uropathogenic Escherichia coli (UPEC) become more resistant to gentamicin (Gm), and that this increase is dependent on the presence of σs (a transcription regulator encoded by the rpoS gene). UPEC causes urinary tract infections (UTIs), reported to afflict astronauts; Gm is a standard treatment, so these findings could impact astronaut health. Because LSMMG findings can differ from MG, we report preparations to examine UPEC's Gm sensitivity during spaceflight using the E. coli Anti-Microbial Satellite (EcAMSat) as a free-flying "nanosatellite" in low Earth orbit. Within EcAMSat's payload, a 48-microwell fluidic card contains and supports study of bacterial cultures at constant temperature; optical absorbance changes in cell suspensions are made at three wavelengths for each microwell and a fluid-delivery system provides growth medium and predefined Gm concentrations. Performance characterization is reported here for spaceflight prototypes of this payload system. Using conventional microtiter plates, we show that Alamar Blue (AB) absorbance changes can assess the Gm effect on E. coli viability, permitting telemetric transfer of the spaceflight data to Earth. Laboratory results using payload prototypes are consistent with wellplate and flask findings of differential sensitivity of UPEC and its ∆rpoS strain to Gm. if σs plays the same role in space MG as in LSMMG and Earth gravity, countermeasures discovered in recent Earth studies (aimed at weakening the UPEC antioxidant defense) to control UPEC infections would prove useful also in space flights. Further, EcAMSat results should clarify inconsistencies from previous space experiments on bacterial antibiotic sensitivity and other issues.

Payload hardware and experimental protocol development to enable future testing of the effect of space microgravity on the resistance to gentamicin of uropathogenic Escherichia coli and its σ s -deficient mutant

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Payload hardware and experimental protocol development to enable future testing of the effect of space microgravity on the resistance to gentamicin of uropathogenic Escherichia coli and its σ s -deficient mutant