Payload hardware and experimental protocol for testing the effect of 1 space microgravity on the resistance to gentamicin of stationary-phase 2 uropathogenic Escherichia coli and its σ s-deficient mutant 3 4

26 Human immune response is compromised and bacteria can become more antibiotic resistant in 27 space microgravity (MG). We report that under low-shear modeled microgravity (LSMMG) 28 stationary-phase uropathogenic Escherichia coli (UPEC) become more resistant to gentamicin 29 (Gm). UPEC causes urinary tract infections (UTIs), reported to afflict astronauts; Gm is a 30 standard treatment, so these findings could impact astronaut health. Because LSMMG has been 31 shown to differ from MG, we report here preparations to examine UPEC’s Gm sensitivity during 32 spaceflight using the E. coli Anti-Microbial Satellite (EcAMSat) on a free-flying “nanosatellite” 33 in low Earth orbit. Within EcAMSat’s payload, a 48-microwell fluidic card contains and supports 34 study of bacterial cultures at constant temperature; optical absorbance changes in cell 35 suspensions are made at three wavelengths for each microwell and a fluid-delivery system 36 provides growth medium and predefined Gm concentrations. Performance characterization is 37 reported for spaceflight prototypes of this payload system. Using conventional microtiter plates, 38 we show that Alamar Blue (AB) absorbance changes due to cellular metabolism accurately 39 reflect E. coli viability changes: measuring AB absorbance onboard EcAMSat will enable 40 telemetry of spaceflight data to Earth. Laboratory results using payload prototypes are consistent 41 with wellplate and flask findings of differential sensitivity of UPEC and its rpoS strain to Gm. 42 Space MG studies using EcAMSat should clarify inconsistencies from previous space 43 experiments on bacterial antibiotic sensitivity. Further, if σ plays the same role in space MG as 44 in LSMMG and Earth gravity, EcAMSat results would facilitate utilizing our previously45 developed terrestrial UTI countermeasures in astronauts. 46 47