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

Enteroaggregative <i>Escherichia coli</i> 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 pAA₁₀₇₀ 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 pAA₁₀₇₀ 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 <i>in vitro</i> regardless. We demonstrate the expected contribution of pAA₁₀₇₀ to intestinal cell adhesion but find that it does not contribute to biofilm formation. When assessing the impact of pAA₁₀₇₀ on motility, we discovered a region of the O104:H4 chromosome that can be excised, abolishing motility via truncation of the <i>fliR</i> gene. Ultimately, this work demonstrates the importance of mobile genetic elements to enteroaggregative <i>E. coli</i> 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.