Tracing back the evolutionary route of the Enteroinvasive Escherichia coli (EIEC) and Shigella through the example of the highly pathogenic O96:H19 EIEC clone

Enteroinvasive Escherichia coli (EIEC) cause intestinal illness mainly in developing countries. The pathogenic mechanism of EIEC is indistinguishable from that caused by Shigella strains, which can be typed through genomic and phenotypic methods used for E. coli and have been proposed for reclassification in E. coli species.Recently an emerging highly pathogenic O96:H19 EIEC clone was described in Europe as causative agent of two large outbreaks occurring in Italy and in the United Kingdom. This serotype had not previously been identified as belonging to the EIEC pathotype. In contrast to Shigella spp and to the majority of EIEC strains, O96:H19 EIEC are lactose-fermenting, lack pathoadaptive mutations and show a good fitness in extracellular environment, similarly to non-pathogenic E. coli, suggesting they have emerged following recent acquisition of the invasion plasmid by a non-pathogenic E. coli.Here we describe the whole genome comparison of two O96:H19 EIEC strains isolated from severe cases of diarrhea occurred in Uruguay in 2014 with reference EIEC and Shigella strains and with available sequences of O96:H19 EIEC. A phylogenetic comparison performed through cgMLST analysis grouped all the O96:H19 strains in a central cluster, with reference EIEC and Shigella strains branching in different clusters. Interestingly the ipaH-positive O96:H19 strain from which all the clusters branched lacked the “entry region” harboring the main virulence genes of EIEC and Shigella in the plasmid, thus resembling a non-pathogenic strain which could be considered as a missing link in the phylogenetic studies on EIEC and Shigella and confirming the current evolutionary hypothesis.Moreover, the comparison of the virulence plasmids highlighted the presence of a complete conjugation region in some O96:H19 EIEC. Reverse Transcriptase Real Time PCR experiments confirmed the expression of the pilin-encoding gene in the strain isolated in Italy in 2012 and conjugation experiments showed its ability to mobilize an accessory plasmid in a recipient strain.Altogether, these results support the hypothesis of a common origin of EIEC and Shigella from a non-pathogenic E. coli through acquisition of the virulence plasmid via conjugation. Noteworthy, this study highlights the ability of circulating EIEC strains to perform conjugation, possibly leading to the emergence of novel EIEC clones.