Adaptive radioresistance of Enterohemorrhagic Escherichia coli O157:H7 results in genomic loss of shiga toxin-encoding prophages

Abstract

Enterohemorragic Escherichia coli (EHEC) O157:H7 is a foodborne pathogen producing shiga toxins (Stx1 and Stx2), can cause hemorrhagic diarrhea, and life-threatening infections. O157:H7 strain EDL933 carries prophages CP-933V and BP-933W that encode shiga toxin genes (stx1 and stx2 respectively). The aim of this work was to investigate the mechanisms of adaptive resistance of EHEC strain EDL933 to a typically "lethal" dose of {gamma}-irradiation (1.5 kGy). Adaptive selection through six passages of exposure to 1.5 kGy resulted in the loss of CP-933V and BP-933W prophages from the genome and mutations within three genes: wrbA, rpoA, and Wt_02639 (molY). Three selected EHEC clones that became irradiation-adapted to the 1.5 kGy dose (C1, C2 and C3) demonstrated increased resistance to oxidative stress, sensitivity to acid pH, and decreased cytotoxicity to Vero cells. To confirm that loss of prophages plays a role in increased radioresistance, C1 and C2 clones were exposed to bacteriophage containing lysates. Although, phage BP-933W could lysogenize C1, C2, and E.coli K-12 strain MG1655, it was not found to have integrated into the bacterial chromosome in C1-{Phi} and C2-{Phi} lysogens. Interestingly, for the E. coli K-12 lysogen (K12-{Phi}), BP-933W DNA had integrated at the wrbA gene (K12-{Phi}). Both C1-{Phi} and C2-{Phi} lysogens regained sensitivity to oxidative stress, were more effectively killed by a 1.5 kGy {gamma}-irradiation dose and had regained cytotoxicity and acid resistance phenotypes. Further, the K12-{Phi} lysogen became cytotoxic, more sensitive to {gamma}-irradiation and oxidative stress and slightly more acid resistant. ImportanceGamma ({gamma})-irradiation of food products can provide an effective means of eliminating bacterial pathogens such as enterohemorrhagic Escherichia coli (EHEC) O157:H7, a significant foodborne pathogen that can cause severe disease due to the production of Shiga toxins. To decipher the mechanisms of adaptive resistance of the O157:H7 strain EDL933, we evolved clones of this bacterium resistant to a lethal dose of {gamma}-irradiation by repeatedly exposing bacterial cells to irradiation following a growth restoration over six successive passages. Our findings provide evidence that adaptive selection involved modifications in the bacterial genome including deletion of the CP-933V and BP-933W prophages. These mutations in EHAC O157:H7 resulted in loss of stx1, stx2, loss of cytotoxicity to epithelial cells and decreased resistance to acidity, critical virulence determinants of EHEC, concomitant with increased resistance to lethal irradiation and oxidative stress. These findings demonstrate that the potential adaptation of EHEC to high doses of radiation would involve elimination of the Stx encoding phages and likely lead to a substantial attenuation of virulence.