Purpose: Previous work in our laboratory suggested that the genome region encoding two tandemly integrated prophage, designated Sp11 and Sp12, undergoes spontaneous deletion(s). In this study we chose to further characterize these deletions to determine whether they may impact molecular subtyping results.
Methods: The counter-selectable marker sacB was integrated into Sp11 to quantify the frequency of prophage curing. The limits of deleted regions in each mutant were defined by PCR.
Results: Sucrose-resistant colonies (indicating loss of sacB) were observed at a frequency between 6.23x10-4 and 1.30x10-3. Out of 20 colonies screened, only 1 had a precise excision of Sp11. While we were unable to define the exact limits of the deletions in other colonies, we did observe 4 other deletion events that included both Sp11 and Sp12, of which the minimal deletion sizes ranged from 35 to 45 kb. Sequencing of the phage released from another clinical isolate of E. coli O157:H7 suggested that these regions become incorporated into mature phage particles.
Significance: This study identified a region of instability within the genome of E. coli O157:H7. Characterization of additional regions and determining the effects deletions have on PFGE patterns will help improve the accuracy of this molecular subtyping method.