Impact of simulated microgravity on the growth and proteomic profile of <i>Enterobacter cloacae</i>

Previous studies have shown decreased microbial diversity in astronaut gut microbiomes during spaceflight, raising potential health concerns. <i>Enterobacter cloacae</i>, a commensal member of the human gastrointestinal tract microbiota, has the potential to cause opportunistic infections in immunocompromised patients. However, little is known about how <i>E. cloacae</i> cells adapt to extreme environments such as microgravity, and whether this bacterium is of medical concern for astronauts during long-term spaceflight missions, as astronaut immune systems are compromised by microgravity. To this end, <i>E. cloacae</i> growth and proteomic profiles were obtained from high aspect ratio vessel (HARV) cultures grown in the rotary cell culture system (RCCS) in three orientations: low-shear modeled microgravity (LSMMG), normal gravity (NG; oxygenation membrane on bottom), and inverted normal gravity (INV; oxygenation membrane on top). Both NG and INV controls were included to determine if <i>E. cloacae</i> LSMMG and INV HARV cultures overlapped in their growth properties and/or proteomics profiles, as was previously observed for <i>Staphylococcus aureus</i>. Our results revealed significant proteomic changes in <i>E. cloacae</i> that overlapped in LSMMG and INV conditions compared to NG, suggesting that adaptive responses may be driven by both simulated microgravity and orientation of the oxygenation membrane. These findings offer insight into the molecular mechanisms underlying <i>E. cloacae</i>'s adaptation to microgravity and its potential health risks for astronauts. This study also underlines the importance of multiple control orientations in ground-based microgravity simulations using the RCCS, guiding future research on microbial behavior in space conditions.IMPORTANCE<i>Enterobacter cloacae</i> can transition from a gut commensal to an opportunistic pathogen in immunocompromised hosts and in closed environments such as hospitals. This danger can be exacerbated by the emergence of multidrug-resistant <i>E. cloacae</i> strains. Astronauts undergo changes in their immune systems during spaceflight that could predispose them to infection and spend extended time in the International Space Station and other closed environments. Therefore, elucidating the impacts of actual spaceflight and simulated microgravity on the biology of <i>E. cloacae</i> and other commensal organisms is vital due to the challenges of antibiotic treatment (such as limited shelf life) during extended spaceflight missions. The findings in this study highlight the importance of using multiple control conditions in ground-based microgravity simulations and lay groundwork for future research into microbial adaptation to space and other extreme environments.