Spaceflight Modifies Escherichia coli Gene Expression in Response to Antibiotic Exposure and Reveals Role of Oxidative Stress Response

Abstract

Bacteria grown in space experiments under microgravity conditions have been found to undergo unique physiological responses, ranging from modified cell morphology and growth dynamics to a putative increased tolerance to antibiotics. A common theory for this behavior is the loss of gravity-driven convection processes in the orbital environment, resulting in both reduction of extracellular nutrient availability and the accumulation of bacterial byproducts near the cell. To further characterize the responses, this study investigated the transcriptomic response of Escherichia coli to both microgravity and antibiotic concentration. E. coli was grown aboard International Space Station in the presence of increasing concentrations of the antibiotic gentamicin with identical ground controls conducted on Earth. Here we show that within 49 h of being cultured, E. coli adapted to grow at higher antibiotic concentrations in space compared to Earth, and demonstrated consistent changes in expression of 63 genes in response to an increase in drug concentration in both environments, including specific responses related to oxidative stress and starvation response. Additionally, we find 50 stress-response genes upregulated in response to the microgravity when compared directly to the equivalent concentration in the ground control. We conclude that the increased antibiotic tolerance in microgravity may be attributed not only to diminished transport processes, but also to a resultant antibiotic cross-resistance response conferred by an overlapping effect of stress response genes. Our data suggest that direct stresses of nutrient starvation and acid-shock conveyed by the microgravity environment can incidentally upregulate stress response pathways related to antibiotic stress and in doing so contribute to the increased antibiotic stress tolerance observed for bacteria in space experiments. These results provide insights into the ability of bacteria to adapt under extreme stress conditions and potential strategies to prevent antimicrobial-resistance in space and on Earth.

Keywords: Escherichia coli; RNA-sequencing; antibiotic; bioastronautics; microgravity; oxidative stress; spaceflight; tolerance.

FIGURE 1

Microgravity experimental design and response of E. coli to antibiotic exposure in space and Earth. (A) The bacterial samples were launched aboard the Orbital CRS-1 mission and were carried up to the International Space Station, where they underwent a 49-h experiment consisting of a growth phase (19 h) and an antibiotic challenge phase (30 h), before a fixative was introduced. (B) By using rubber septa, the BioServe Fluid Processing Apparatus (FPA) was divided in four chambers. From left to right: growth medium with two Teflon balls to ensure mixing in microgravity (astronauts were requested to shake the hardware after each mixing), inoculum, antibiotic, fixative. Astronaut operations pushed all chambers from right to left, to enable the mixing of two consecutive chambers via the bypass (in this figure, observed over the growth medium chamber). (C) Cells/mL of surviving E. coli for each experimental condition upon introduction of antibiotic. Cells did not grow on Earth at higher than 75 μg/mL gentamicin concentration.

FIGURE 2

Overall gene response to gentamicin and the trp and thi operons. (A) Heat map of the 63 genes that show differential expression consistently with respect to increasing gentamicin concentration (complete heat map with genes in Supporting Information). Emphasis on genes with differing response on Earth versus in space. (B) Representative expression patterns in two representative genes from the trp and thi operons. Expression change is measured in log2 fold change (^∗P-value < 0.05, ^∗∗∗P-value < 0.001).

FIGURE 3

Oxidative stress response versus antibiotic concentration. Comparison of soxR and soxS (A), shows conflicting regulation patterns relative to the 25 μg/mL experiments, as soxR is known to control transcription of soxS. Expression patterns of rpoS (B), oxyR (C), and marA (D) relative to the 25 μg/mL experiments on Earth and in space show consistent upregulation of oxidative stress response in space independent of the soxRS operon. Expression change is measured in log2 fold change (^∗P-value < 0.05, ^∗∗∗P-value < 0.001).

FIGURE 4

Oxidative stress response genes in matched drug concentration experiments. Expression levels of rpoS (A), oxyR (B), and marA (C) relative to respective Earth 25 μg/mL experiment shows a consistent expression pattern for all three genes. The 25 μg/mL experiment comparison is the only one to show significant differential expression. Expression change is measured in log2 fold change (^∗P-value < 0.05, ^∗∗∗P-value < 0.001).

FIGURE 5

Analysis of the microgravity stress response and differential variability. (A) A pie chart distribution of stress response genes that are differentially expressed between space and Earth in matched experiments shows a broad assortment of stressors. Every differentially expressed stress response gene in this analysis is upregulated in space. (B) Box plot distributions for the coefficient of variation of expression of genes pooled across space or Earth in comparisons where one environment showed significantly different variability. The plot shows an overwhelming tendency for gene expression to be more variable in space. (C) Plots for the pooled coefficients of variation for each gene in operons that were less variable in space.