Escherichia coli Cells Exposed to Lethal Doses of Electron Beam Irradiation Retain Their Ability to Propagate Bacteriophages and Are Metabolically Active

Abstract

Reports in the literature suggest that bacteria exposed to lethal doses of ionizing radiation, i.e., electron beams, are unable to replicate yet they remain metabolically active. To investigate this phenomenon further, we electron beam irradiated Escherichia coli cells to a lethal dose and measured their membrane integrity, metabolic activity, ATP levels and overall cellular functionality via bacteriophage infection. We also visualized the DNA double-strand breaks in the cells. We used non-irradiated (live) and heat-killed cells as positive and negative controls, respectively. Our results show that the membrane integrity of E. coli cells is maintained and that the cells remain metabolically active up to 9 days post-irradiation when stored at 4°C. The ATP levels in lethally irradiated cells are similar to non-irradiated control cells. We also visualized extensive DNA damage within the cells and confirmed their cellular functionality based on their ability to propagate bacteriophages for up to 9 days post-irradiation. Overall, our findings indicate that lethally irradiated E. coli cells resemble live non-irradiated cells more closely than heat-killed (dead) cells.

Keywords: DNA damage; bacteria; bacteriophages; electron beam; ionizing radiation.

FIGURE 1

Representative images depicting membrane integrity in eBeam inactivated, heat-killed, and live Escherichia coli cells. Cultures were incubated at 4°C in LB broth post-treatment and images were taken at 0, 4, 24 h, and 9 days.

FIGURE 2

Representative images showing the detection of DNA double-strand breaks in E. coli cells using the neutral comet assay. Cells were exposed to either a lethal eBeam irradiation dose (absorbed dose: 7.04 kGy), a lethal heat treatment (70°C for 60 min) or no treatment. Arrows indicate DNA tails (control), putative DNA fragments (eBeam) or both (heat-killed).

FIGURE 3

(A) Metabolic activity of eBeam inactivated, heat-killed, and live E. coli cells. Heat-killed cells did not have measurable levels. Bacterial cultures were incubated at 4°C in LB broth post-treatment and measurements were taken at 0, 4, 24 h, and 9 days. Two independent experiments were performed, with standard deviations shown. C^∗ denotes statistical significance (p < 0.0001). (B) ATP levels of eBeam inactivated, heat-killed, and non-treated E. coli cells. Samples were incubated at 4°C in LB broth post-treatment and measurements were taken at 0, 4, 24 h, and 9 days. Two independent experiments were performed, with standard deviations shown. ^∗∗Denotes statistical significance (p < 0.01); ^∗∗∗denotes statistical significance (p < 0.001).

FIGURE 4

Bacteriophage λ numbers after incubation (at 37°C for 24 h) with eBeam inactivated host cells (EB), heat-killed host cells (HK), non-treated live host cells (PC – positive control), and no host cells (NC – negative control). The 0, 4, 24 h, and day 9 time points represent the time after host cell treatment. Two independent experiments were performed, with standard deviations shown. ^∗Denotes statistical significance (p < 0.05); ^∗∗denotes statistical significance (p < 0.01); ^∗∗∗∗denotes statistical significance (p < 0.0001).

FIGURE 5

T4 bacteriophage numbers after incubation (at 37°C for 24 h) with eBeam inactivated host cells (EB), heat-killed host cells (HK), non-treated live host cells (PC – positive control), and no host cells (NC – negative control). The 0, 4, 24 h, and day 9 time points represent the time after host cell treatment. Two independent experiments were performed, with standard deviations shown. ^∗∗∗Denotes statistical significance (p < 0.001); ^∗∗∗∗denotes statistical significance (p < 0.0001).

FIGURE 6

T7 bacteriophage numbers after incubation (at 37°C for 24 h) with eBeam inactivated host cells (EB), heat-killed host cells (HK), non-treated live host cells (PC – positive control), and no host cells (NC – negative control). The 0, 4, 24 h, and day 9 time points represent the time after host cell treatment. Two independent experiments were performed, with standard deviations shown. ^∗∗∗∗Denotes statistical significance (p < 0.0001).