Spaceflight and simulated microgravity conditions increase virulence of *Serratia marcescens* in the *Drosophila melanogaster* infection model

The study addresses how spaceflight and microgravity conditions impact host–pathogen interactions, specifically whether spaceflight alters bacterial virulence and/or the host immune response. Prior work shows astronauts exhibit immune dysregulation during and after spaceflight, and pathogens can change growth, virulence, and antibiotic resistance under spaceflight or simulated microgravity. Drosophila melanogaster is an established, cost-effective model with innate immune pathways (Imd and Toll) homologous to human pathways. Serratia marcescens, an opportunistic human pathogen detected on Mir and the ISS, provides a relevant model to assess spaceflight-induced pathogenic changes. The authors hypothesize that growth of S. marcescens in spaceflight or simulated microgravity increases virulence in D. melanogaster and seek to determine whether increased lethality arises from pathogen-intrinsic changes versus altered host immunity.

- Human immune alterations in spaceflight include changes in cytokine production, immune cell proliferation and distribution, and dysregulation across innate and adaptive systems. - Prior studies indicate microgravity/spaceflight can increase bacterial growth, virulence, antibiotic resistance, biofilm formation, and alter gene expression (including roles for global regulators like Hfq), predominantly studied in enteric pathogens (e.g., Salmonella) and other species. - Modeled microgravity using rotating wall vessel systems (low-shear modeled microgravity, LSMMG) has been used to study microbe–host interactions and can modulate virulence phenotypes. - Limited and variable human studies (small sample sizes, methodological variability) motivate the use of invertebrate models like Drosophila, which share significant conservation with human disease genes and have well-characterized innate immune pathways (Imd for Gram-negative bacteria; Toll for Gram-positive/fungi). - Previous spaceflight work focused largely on microbes in liquid culture; changes on solid/semi-solid media are less characterized, with some reports suggesting minimal changes in final cell density, though transcriptional/proteomic shifts have been observed in S. marcescens under spaceflight on semi-solid media. - The presence of S. marcescens aboard space platforms underscores its relevance as a potential opportunistic threat in space habitats.

Spaceflight growth of S. marcescens DB11 on solid media: - Strain and media: S. marcescens DB11 obtained from the University of Minnesota C. elegans stock center. Pre-culture: 20 h in LB with 100 µg/mL streptomycin at 37 °C; diluted to OD600 0.100 in PBS. 100 µL spread onto trays with 2 mL semi-defined solid fly food (composition: 48% brewer’s yeast, 6% sucrose, 3% potato extract, 2% gelatin, 1% agar, trace sulfate, 0.05% CaCl2·2H2O, 0.5% blue food dye, 0.001% 6-hydroxybenzothiazole, 0.05% propionic acid). - Sample sets: Two trays designated Space 1 and Space 2 were flown; two trays Ground 1 and Ground 2 were prepared identically and grown as ground controls in matching hardware and matched environmental profiles (gas composition, temperature, humidity). - Flight logistics: Trays stored at 4 °C pre-launch. Spaceflight on SpaceX CRS-5; growth on ISS per recorded conditions; ground controls matched to ISS environmental profiles. Post-flight, bacteria scraped, suspended in 50% glycerol, and stored at −80 °C (same for ground controls). In-flight fly infections were not performed; infections were conducted post-flight on Earth. Fly infection and survival assays (spaceflight vs ground bacteria): - Bacterial preparation: Frozen stocks in 50% glycerol quantified by colony counts on LB; diluted to 4 × 10^4 CFU/mL in sterile PBS to deliver <10 CFU per injection. Sham control: 12.5% glycerol in PBS. - Injections: 2–3-day-old anesthetized female D. melanogaster injected in ventral abdomen (fine injector; volume setting noted at 32 µL; adjusted to achieve <10 CFU per fly). After a 1 h recovery at ~30 °C, flies were maintained with food and water ad libitum; survival was monitored. In vivo bacterial load quantification: - Time points: 0, 9, 12, 15 h post-infection. Individual flies homogenized in 200 µL PBS; serial dilutions plated on LB agar; incubated 24 h at 37 °C; CFUs counted. Each experiment independently repeated three times with three replicates per time point. Ground subculture reversibility test: - To assess persistence of virulence changes, Space and Ground samples were subcultured on Earth: loop-inoculated into LB + streptomycin (100 µg/mL), 37 °C, 225 rpm, 24 h (~23 generations), then diluted to 4 × 10^4 CFU/mL in PBS and injected as above. Survival and in vivo growth assessed. Simulated microgravity (LSMMG) using rotating wall vessel: - Non-flight DB11 revived on LB-strep agar (100 µg/mL), 18–24 h, stored at 4 °C up to 2 weeks. Liquid cultures grown 24 h in LB + streptomycin at 37 °C, 25 rpm. Cultures diluted and loaded into sterile disposable 10 mL rotating wall vessels (RWW). Air bubbles removed and sealed. - Orientation: Horizontal rotation (LSMMG) vs vertical (normal gravity control), 37 °C, 25 rpm, for 24 h to stationary phase. - Post-culture: Cultures diluted to ~6 × 10^7 CFU in PBS; ~0.5 µL injected into 2–3-day-old flies. Survival and in vivo growth measured as above. Host genotype assays and RNA-seq: - Survival assays included wild-type flies (e.g., w1118, yw) and immune mutants of the Imd pathway (PGPR-CLAS, imd1, relE20) and Toll pathway mutants to assess host pathway contributions to susceptibility. - RNA-seq: Wild-type flies infected with spaceflight-exposed vs ground bacteria; RNA extracted 18 h post-infection (Trizol, Qiagen RNeasy, on-column DNase). Sequencing on Illumina HiSeq (paired-end 100 bp). Transcript abundance by kallisto v0.42.0; differential expression by sleuth v0.300 (Wald test), significance at q < 0.05. Statistical analyses: - Software: GraphPad Prism 7.0, JMP Pro 13. - Survival: Cox Proportional Hazards model for multi-group comparisons; log-rank (Mantel–Cox) for mutant/roll comparisons. - In vivo growth: One-way ANOVA with Tukey–Kramer post hoc. Significance threshold generally p < 0.05. Selected detailed outputs reported in Tables/Figures (e.g., hazard ratios, ANOVA F and p values).

- Spaceflight exposure increased bacterial virulence in flies: • Fly survival was significantly lower after infection with spaceflight-exposed S. marcescens (Space 1 and Space 2) compared with ground controls (all comparisons P < 0.0001 in Cox PH; Table 1). - Altered in vivo growth kinetics at later time points: • No significant differences at early time points (e.g., p = 0.489 overall; 6 h F = 0.5316, p = 0.6772; 9 h F = 1.169, p = 0.9364). • At 12 h post-injection: Space 1 > Ground (p = 0.0002) and Space 1 > Space 2 (p = 0.04); Space 2 > Ground (p = 0.0006). Spaceflight samples showed higher in vivo CFUs particularly 12 h and later (Fig. 1b). • In vitro growth of space-flown bacteria also increased at later time points versus ground controls (Supplementary Fig. 1). - Increased virulence was reversible after ground subculture: • After one overnight subculture on Earth, survival of hosts injected with Space 1 was not significantly different from Ground 1 (p = 0.995) or Ground 2 (p = 0.337); similar lack of differences for Space 2 (Table 2 hazard ratios ~1; non-significant between space and ground after subculture). - Simulated microgravity (LSMMG) recapitulated increased virulence and in vivo growth: • LSMMG-treated bacteria caused reduced survival compared to RWV-control and sham (Fig. 3a; statistical details not fully enumerated but survival differences evident). • In vivo CFUs: No difference at 0 h (F = 3.551, p = 0.128) or 6 h (F = 7.493, p = 0.0544); significant increases for LSMMG vs RWV-control at 9 h (F = 20.248, p = 0.008), 12 h (F = 12.265, p = 0.01), and 15 h (F = 18.427, p = 0.009) (Fig. 3b). - Host immune pathway genotype did not eliminate the relative increase in lethality from spaceflight bacteria: • Imd pathway mutants (PGPR-CLAS, imd1, relE20) were more susceptible overall to Serratia as expected; Toll mutants behaved more similarly to wild-type for Gram-negative infections. • Space 1 bacteria caused greater lethality than Ground 1 across wild-type and mutant genotypes, indicating pathogen-intrinsic changes drive increased virulence (Fig. 4; Supplementary Tables 2–6). - Host transcriptional responses showed few differences between infections with spaceflight vs ground bacteria: • RNA-seq at 18 h post-infection found only 11 significantly differentially expressed transcripts (q < 0.05); only Attacin-A (AttA) directly immune-related and upregulated. • PCA showed substantial overlap between groups, indicating minimal global differences in host gene expression (Fig. 4e–f).

The findings indicate that growth of S. marcescens under spaceflight or modeled microgravity conditions increases virulence in a D. melanogaster infection model, with pronounced effects on bacterial growth kinetics at later stages of infection (≥12 h). The reversion of increased virulence after brief regrowth on Earth suggests the phenotype is primarily physiological and reversible rather than due to stable genetic mutations from spaceflight (e.g., radiation). LSMMG experiments reproduced key phenotypes, implicating microgravity-associated low-shear conditions as a primary driver. Survival outcomes across wild-type and immune pathway mutant flies, coupled with minimal changes in host immune gene expression, support that the increased lethality is largely attributable to pathogen-intrinsic changes rather than altered host immunity. Contrary to expectations that solid media growth might be less affected in microgravity than liquid cultures, S. marcescens exhibited altered phenotypes consistent with prior reports of transcriptional and proteomic changes under spaceflight on semi-solid media. Increased in vivo and in vitro growth at later time points suggests that microgravity exposure may confer advantages under nutrient-limiting or low-shear conditions, potentially via changes in metabolism, cell polarity, motility (e.g., swarming behavior), or biofilm propensity. These observations underscore the need to dissect the molecular mechanisms underlying altered growth and virulence of opportunistic pathogens in space-relevant environments.

This study demonstrates that S. marcescens grown during spaceflight exhibits increased virulence and accelerated growth kinetics at later infection stages in D. melanogaster, and that similar phenotypes arise under simulated microgravity (LSMMG). The virulence increase is reversible after brief regrowth under Earth conditions, indicating a predominantly physiological adaptation. Results suggest pathogen-intrinsic changes, rather than major shifts in host immune responses, primarily drive the observed lethality. The work provides the first evidence that spaceflight can influence growth and virulence of bacteria grown on a solid substrate. Future research should elucidate the molecular mechanisms (transcriptional, proteomic, metabolic) underlying these phenotypes, assess the roles of media composition and nutrient limitation, and compare liquid versus solid culture systems to define how microgravity-related physical forces modulate pathogen behavior and host outcomes.

- Limited bacterial material recovered from the spaceflight experiment precluded transcriptomic/proteomic analyses of the bacteria themselves in this study. - In-flight infection of flies was not performed due to logistical constraints; infections were conducted post-flight on Earth, potentially missing in situ interactions. - Not all samples (Space 1/2, Ground 1/2) were included in every experiment due to limited sample availability and the labor-intensive nature of injections, which may reduce statistical power for some comparisons. - RNA-seq sampled flies alive at 18 h post-infection, potentially biasing toward less advanced infections and underestimating transcriptional differences. - Some methodological parameters (e.g., precise injection volumes, exact RWV model nomenclature) had minor inconsistencies/typographical issues in reporting, though overall procedures were clear. - Ground environmental matching relied on recorded ISS conditions; unmeasured variables could contribute to differences.