Space exploration necessitates understanding the effects of spaceflight conditions—microgravity, radiation, isolation, and hypergravity—on human physiology, particularly the immune system. Evidence suggests that spaceflight not only alters the human immune system but also impacts the virulence of pathogens. Opportunistic pathogens like *Serratia marcescens* show increased growth and virulence, and antibiotic resistance in spaceflight conditions. Studying these effects directly in humans is challenging due to limitations in sample size and experimental design. *Drosophila melanogaster* serves as a valuable model organism due to its well-characterized immune system, which shares significant homology with humans (28% of human disease-causing genes have functional homologs in *Drosophila*). *S. marcescens*, a human opportunistic bacterium found on spacecraft, provides an ideal pathogen model for studying spaceflight-induced virulence changes. The specific *S. marcescens* strains used in this study are not pathogenic to *D. melanogaster*, allowing for safe study of spaceflight effects.

Previous research has demonstrated the impact of spaceflight on the human immune system, including altered cytokine production, immune cell proliferation and distribution changes, and overall immune system alteration. Studies also show significant effects of microgravity and spaceflight on pathogens, including increased growth, virulence, and antibiotic resistance. *Drosophila melanogaster* has proven useful in studying the effects of spaceflight on immunity and pathogenesis, exhibiting dramatic shifts in immune gene expression after spaceflight. While invertebrates lack an adaptive immune system, their inducible immune response is highly similar to that of humans. *S. marcescens* has recently become a model for rapid virulence and antibiotic resistance shifts due to microbial host responses, and its presence on spacecraft makes it a crucial organism for study. Existing research on the effects of spaceflight on humans is varied, likely due to low sample sizes and methodological inconsistencies. Time-accelerated studies are also limited by factors such as cost, launch variability, and the constraints of spaceflight experiments.

The study used *Serratia marcescens* Db11 grown on the ISS during the SpaceX-Fox 5 mission and compared it to ground controls. Solid semi-defined fly food media were used for bacterial growth on the ISS and in a ground-based incubator programmed to simulate spaceflight conditions. *D. melanogaster* flies were injected with spaceflight and ground samples of *S. marcescens*, and their survival was monitored. In vivo bacterial growth was assessed at various time points post-injection. To test reversibility, spaceflight samples were subcultured on the ground, and their virulence was reassessed. Simulated microgravity (SMG) conditions were created using a rotating wall vessel (RWV), and the virulence of bacteria grown under SMG was also tested. Immune-deficient *Drosophila* mutants (imd and Toll pathways) were used to examine the role of host immune response. RNA sequencing (RNA-seq) was performed on wild-type flies infected with spaceflight and ground bacteria to assess host gene expression changes. Statistical analyses included Cox Proportional Hazards models and one-way ANOVA with post-hoc analysis.

Flies infected with spaceflight-exposed *S. marcescens* showed significantly lower survival rates compared to those infected with ground controls. In vivo growth of spaceflight bacteria was significantly higher at 12 h post-injection. The increased virulence of spaceflight bacteria was reversible after subculturing on the ground. Bacteria grown under simulated microgravity conditions also exhibited increased virulence. Increased virulence was observed in both wild-type and immune-deficient flies, suggesting that changes in host immunity were not the major contributing factor. RNA-seq analysis showed few significant differences in host gene expression between spaceflight and ground bacteria infections, with only one gene (AtfA, an antimicrobial peptide) directly related to the immune response showing differential expression. In vitro growth of spaceflight bacteria was also significantly higher than ground controls at later time points.

This study demonstrates that spaceflight and simulated microgravity can significantly increase the virulence of *S. marcescens*. The reversibility of the increased virulence suggests that the changes are physiological rather than genetic. The lack of significant differences in host gene expression between spaceflight and ground bacteria infections implies that the increased virulence is primarily due to changes in the pathogen itself, rather than altered host immunity. The increased in vitro and in vivo growth rates of spaceflight bacteria support this conclusion. The results highlight the need to understand the underlying mechanisms that mediate these changes and have implications for the health risks posed by pathogens in space environments.

This study provides the first demonstration of spaceflight influencing the growth and virulence of bacteria grown on a solid substrate. The increased virulence of *S. marcescens* after spaceflight and simulated microgravity is likely due to physiological changes in the bacteria rather than alterations in host immunity. Future research should focus on identifying the molecular mechanisms responsible for these changes, which could have important implications for astronaut health during long-duration space missions.

The study used a single bacterial strain and a single *Drosophila* model. While the use of immune-deficient flies provided insights into the host immune response, additional experiments with different immune pathways would strengthen the conclusions. The limited sample size from the spaceflight mission restricted the scope of some analyses, particularly transcriptomic and proteomic studies. Future studies could expand upon these limitations by including multiple strains and fly models and conducting more comprehensive molecular analyses.