Preparation for and performance of a Pseudomonas aeruginosa biofilm experiment on board the International Space Station

Biofilms are problematic on Earth due to their ability to both degrade the materials upon which they grow and \npromote infections. Remarkably, 65% of infections and 80% of chronic diseases on Earth are associated with \nbiofilms. The impact of biofilms is even greater in space, as the crew’s lives and mission success depend on \nnominal operation of mechanical systems which can be interrupted by material damage associated with biofilm \ngrowth. Furthermore, the isolated confined environment nature of spaceflight may increase the rates of disease \ntransmission. In the case of the International Space Station (ISS), biofilms are an identified problem on the \nEnvironmental Control and Life Support System (ECLSS), namely on the water processor assembly (WPA). In late \n2019, the bacterial component of the Space Biofilms experiment launched to ISS to (i) characterize the mass, \nthickness, morphology, and gene expression of biofilms formed in space compared to matched Earth controls, (ii) \ninterrogate the expression of antimicrobial resistance genes, and (iii) test novel materials as potential biofilm \ncontrol strategies for future ECLSS components. For this, 288 bacterial samples were prepared prior to the launch \nof the Northrop Grumman CRS-12 mission from NASA’s Wallops Flight Facility. The samples were integrated \ninto the spaceflight hardware, BioServe’s Fluid Processing Apparatus (FPA), packed in sets of eight in Group \nActivation Packs (GAP). Half of these samples were activated and terminated on orbit by NASA astronauts Jessica \nMeir and Christina Koch, while the remaining half were processed equivalently on Earth. The spaceflight bacterial samples of Space Biofilms returned on board the SpaceX CRS-19 Dragon spacecraft in early 2020. We here \ndescribe the test campaign implemented to verify the experiment design and confirm it would enable us to \nachieve the project’s scientific goals. This campaign ended with the Experiment Verification Test (EVT), from \nwhich we present example morphology and transcriptomic results. We describe in detail the sample preparation \nprior to flight, including cleaning and sterilization of the coupons of six materials (SS316, passivated-SS316, \nlubricant impregnated surface, catheter-grade silicone with and without a microtopography, and cellulose \nmembrane), loading and integration of growth media, bacterial inoculum, and the fixative and preservative to \nenable experiment termination on orbit. Additionally, we describe the performance of the experiment on board the ISS, including crew activities, use of assets, temperature profile, and experiment timeline; all leading to a \nsuccessful spaceflight experiment. Hence, this manuscript focuses on the steps implemented to ensure the \nexperiment would be ready for spaceflight, as well as ISS and ground operations, with results presented elsewhere. The processes discussed here may serve as a guideline to teams planning their own gravitational \nmicrobiology experiments. This material is based upon work supported by the National Aeronautics and Space \nAdministration under Grant No. 80NSSC17K0036.