Genetic and phenotypic analysis of the virulence plasmid of a non-Shigatoxigenic enteroaggregative O104:H4 outbreak strain.

Enteroaggregative O104:H4 is best known for causing a worldwide outbreak in 2011 due to the acquisition of a Shiga-like toxin alongside traditional enteroaggregative virulence traits; however, whilst the 2011 outbreak strain has been well studied, the virulence plasmid of O104:H4 has been subjected to far less experimental analysis. In this paper, we analyse the genetic and phenotypic contribution of the pAA virulence plasmid to a non-Shigatoxigenic O104:H4 strain (1070/13) that was nonetheless implicated in a substantial UK outbreak in 2013. We find that pAA is 99.95% identical across 88% of the plasmid sequence to pTY2 from the 2011 outbreak strain and has a copy number of ~2-3 plasmid molecules per chromosome. We demonstrate that pAA carries a functional CcdAB plasmid addiction system that only marginally impacts its stability under the conditions tested. None of the other toxin-antitoxin systems encoded by the plasmid appear to be functional, though we note a surprisingly high stability of the plasmid regardless. We demonstrate the expected contribution of pAA to intestinal cell adhesion but find that it does not contribute to biofilm formation. When assessing the impact of pAA on motility, we discovered a region of the O104:H4 chromosome that can be excised, abolishing motility via truncation of the gene. Ultimately, this work demonstrates the importance of mobile genetic elements to enteroaggregative as a pathovar in its own right and highlights the complexity but necessity of experimentally characterizing genuine outbreak strains rather than laboratory strains in order to understand virulence phenotypes.