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

Using colony blot hybridization withstx 2 andeae probes and agglutination in anti-O157 lipopolysaccharide serum, we isolatedstx 2 -positive andeae -positive sorbitol-fermenting (SF) enterohemorrhagicEscherichia coli (EHEC) O157:NM (nonmotile) strains from initial stool specimens andstx -negative andeae -positive SFE. coli O157:NM strains from follow-up specimens (collected 3 to 8 days later) from three children. Thestx -negative isolates from each patient shared with the correspondingstx 2 -positive isolatesfliC H7 , non-stx virulence traits, and multilocus sequence types, which indicates that they arose from thestx 2 -positive strains by loss ofstx 2 during infection. Analysis of the integrity of theyecE gene, a possiblestx phage integration site in EHEC O157, in the consecutivestx 2 -positive andstx -negative isolates demonstrated thatyecE was occupied instx 2 -positive but intact instx -negative strains. It was possible to infect and lysogenize thestx -negativeE. coli O157 strains in vitro using anstx 2 -harboring bacteriophage from one of the SF EHEC O157:NM isolates. The acquisition of thestx 2 -containing phage resulted in the occupation ofyecE and production of biologically active Shiga toxin 2. We conclude that theyecE gene in SFE. coli O157:NM is a hot spot for excision and integration of Shiga toxin 2-encoding bacteriophages. SF EHEC O157:NM strains and theirstx -negative derivatives thus represent a highly dynamic system that can convert in both directions by the loss and gain ofstx 2 -harboring phages. The ability to recyclestx 2 , a critical virulence trait, makes SFE. coli O157:NM strains ephemeral EHEC that can exist asstx -negative variants during certain phases of their life cycle.