Recycling of Shiga Toxin 2 Genes in Sorbitol-Fermenting Enterohemorrhagic Escherichia coli O157:NM

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Abstract
Using colony blot hybridization with stx 2 and eae probes and agglutination in anti-O157 lipopolysaccharide serum, we isolated stx 2 -positive and eae -positive sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:NM (nonmotile) strains from initial stool specimens and stx -negative and eae -positive SF E. coli O157:NM strains from follow-up specimens (collected 3 to 8 days later) from three children. The stx -negative isolates from each patient shared with the corresponding stx 2 -positive isolates fliC H7 , non- stx virulence traits, and multilocus sequence types, which indicates that they arose from the stx 2 -positive strains by loss of stx 2 during infection. Analysis of the integrity of the yecE gene, a possible stx phage integration site in EHEC O157, in the consecutive stx 2 -positive and stx -negative isolates demonstrated that yecE was occupied in stx 2 -positive but intact in stx -negative strains. It was possible to infect and lysogenize the stx -negative E. coli O157 strains in vitro using an stx 2 -harboring bacteriophage from one of the SF EHEC O157:NM isolates. The acquisition of the stx 2 -containing phage resulted in the occupation of yecE and production of biologically active Shiga toxin 2. We conclude that the yecE gene in SF E. coli O157:NM is a hot spot for excision and integration of Shiga toxin 2-encoding bacteriophages. SF EHEC O157:NM strains and their stx -negative derivatives thus represent a highly dynamic system that can convert in both directions by the loss and gain of stx 2 -harboring phages. The ability to recycle stx 2 , a critical virulence trait, makes SF E. coli O157:NM strains ephemeral EHEC that can exist as stx -negative variants during certain phases of their life cycle.