Genomic diversification and modular recombination of Stx2 prophages associated with chromosomal integration sites during an STEC O111 and O157 outbreak

Shiga toxin 2 (Stx2) production in Shiga toxin-producing Escherichia coli (STEC) is mediated by lambdoid Stx2-converting bacteriophages, which play a central role in the evolutionary and pathogenic potential of STEC lineages. However, the mechanisms through which Stx2 phages diversify and evolve during natural outbreaks remain unclear. Here, we analyzed a collection of 20 related Stx2 phage and prophage genomes recovered from STEC O111 and O157 isolates associated with a foodborne disease outbreak in Japan. These included 2 infectious, Stx2-converting phages isolated directly from bloody stool samples, 17 Stx2 prophages, and 1 stx -negative prophage. Genome-wide single-nucleotide polymorphism analysis identified 282 polymorphic sites, resolving two predominant lineages associated with distinct chromosomal integration sites ( argW or wrbA ), corresponding largely to the STEC O111 and O157 backgrounds, respectively. Despite this lineage structure, the genomes exhibited extensive mosaicism, including lineage-incongruent haplotype blocks and multiple recombination junctions, indicating frequent modular exchanges among co-circulating phages. The preferential association between specific haplotypes and chromosomal integration sites further suggests that the host genomic context contributes to prophage genomic variation. Additionally, the Stx2-converting phages and argW -integrated prophages encoded functionally active restriction–modification systems, indicating that prophage-encoded defense mechanisms may contribute to genome stability and diversification. Collectively, these findings highlight the genetic plasticity of Stx2 phages and prophages during a single outbreak and offer genome-based insights into the evolutionary dynamics and epidemiological significance of toxin-converting bacteriophages in STEC populations.