Sequencing of enterohemorrhagic Escherichia coli virulence phages reveals variants promoting increased Shiga-toxin mobilization

Each year in the US alone approximately 75,000 people are infected with enterohemorrhagic E. coli (EHEC) of the O157:H7 serotype. Symptoms can be life threating and include hemolytic uremic syndrome and hemorrhagic colitis. Pathogenesis is linked to the production of Shiga toxin during infection of the gastrointestinal tract. Although shotgun sequences are publically available for a large number of E. coli O157:H7 strains, the high sequence similarity with other prophages makes it difficult to accurately assemble the genomes of stx-producing phages. This study presented Stx phage plasticity in EHEC through complete phage sequencing as prerequisite to correlate differences in phage inventory and architecture to variation in Stx phage mobilization and production as direct mediator of disease. A total number of 9 strains were selected for this comparative study. Phage DNA was isolated after induction with ciprofloxacin and sequenced on the Illumina MiSeq. Phages were assembled with both Spades and Velvet in order to extract complete stx2 -producing phage DNA. Comparison with deposited Stx phages was accomplished using Geneious. Strains were phenotyped for variation in phage mobilization and Stx expression by qPCR. The phage mobilization assay allowed classifying strains into low, moderate and high phage/toxin inducers. This study characterized variation in Stx production and mobilization among the different clades, and identified different types of Stx-producing phages. Sequencing and assembly of bacteriophage DNA provided a refined picture of the extant Stx phage plasticity that will allow to correlate catalogued phage genotypes and architectures to Stx mobilization and production.