Adaptive Radioresistance of Enterohemorrhagic Escherichia coli O157:H7 Results in Genomic Loss of Shiga Toxin-Encoding Prophages.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a foodborne pathogen producing Shiga toxins (Stx1 and Stx2), which can cause hemorrhagic diarrhea and life-threatening infections. O157:H7 strain EDL933 carries prophages CP-933V and BP-933W, which 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, clones C1 and C2 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-Φ and C2-Φ lysogens. Interestingly, for the E. coli K-12 lysogen (K-12-Φ), BP-933W DNA had integrated at the wrbA gene (K-12-Φ). Both C1-Φ and C2-Φ 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 K-12-Φ lysogen became cytotoxic, more sensitive to gamma irradiation and oxidative stress, and slightly more acid resistant. IMPORTANCE 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 Stx. 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 EHEC O157:H7 resulted in loss of stx1 and 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.

Identification and frequency of the associated genes with virulence and antibiotic resistance of Escherichia coli isolated from cow's milk presenting mastitis pathology.

Antimicrobial resistance, genotype, and virulence gene content of Escherichia coli isolated from bovine clinical mastitis in Tehran Province, Iran, were studied. Two hundred seven (207) milk samples from individual cows presenting mastitis symptoms collected from different dairy farms were used to determine the presence of specific genes of E. coli responsible for this pathology. Multiplex PCR was used to differentiate E. coli isolates into different phylogenetic groups/subgroups and to detect their virulence and involved resistance genes. All the isolated strains were tested for the susceptibility to 21 antimicrobial agents. The results showed that E. coli was detected in 42 (20.3%) samples and 69% of them belonged to the phylogenetic groups A and B1. The phylogenetic subgroup A1 (31%) and subgroup B1 (28.6%) demonstrated the highest prevalence of virulence genes (f17c-A, and eae (n = 6), f17b-A, and f17d-A (n = 5), afaD-8, afaE-8, aucD, and bfpA (n = 4), clpG and VT (n = 2), and LT and ST genes (n = 1)). The highest antimicrobial resistance was observed for tetracycline (45.2%) followed by streptomycin (26.2%). The antimicrobial resistance genes tetB (31%), tetA (28.6%), and aadA (26.2%) were the most prevalent. Moreover, integron class 1 and 2 were found in 24 (57.1%) and 8 (19%) of the E. coli isolates.