Tracing Back the Evolutionary Route of Enteroinvasive Escherichia coli (EIEC) and Shigella Through the Example of the Highly Pathogenic O96:H19 EIEC Clone

Abstract

Enteroinvasive Escherichia coli (EIEC) cause intestinal illness through the same pathogenic mechanism used by Shigella spp. The latter species can be typed through genomic and phenotypic methods used for E. coli and have been proposed for reclassification within E. coli species. Recently the first appearance of a highly pathogenic EIEC O96:H19 was described in Europe as the causative agent of two large outbreaks that occurred in Italy and in the United Kingdom. In contrast to Shigella spp and to the majority of EIEC strains, EIEC O96:H19 fermented lactose, lacked pathoadaptive mutations, and showed good fitness in extracellular environment, similarly to non-pathogenic E. coli, suggesting they have emerged following acquisition of the invasion plasmid by a non-pathogenic E. coli. Here we describe the whole genome comparison of two EIEC O96:H19 strains isolated from severe cases of diarrhea in Uruguay in 2014 with the sequences of EIEC O96:H19 available in the public domain. The phylogenetic comparison grouped all the O96:H19 strains in a single cluster, while reference EIEC strains branched into different clades with Shigella strains occupying apical positions. The comparison of the virulence plasmids showed the presence of a complete conjugation region in at least one O96:H19 EIEC. Reverse Transcriptase Real Time PCR experiments confirmed in this strain the expression of the pilin-encoding gene and conjugation experiments suggested its ability to mobilize an accessory plasmid in a recipient strain. Noteworthy, the tra region was comprised between two reversely oriented IS600 elements, which were also found as remnants in another EIEC O96:H19 plasmid lacking the tra locus. We hypothesize that an IS-mediated recombination mechanism may have caused the loss of the conjugation region commonly observed in EIEC and Shigella virulence plasmids. The results of this study support the hypothesis of EIEC originating from non-pathogenic E. coli through the acquisition of the virulence plasmid via conjugation. Remarkably, this study showed the ability of a circulating EIEC strain to mobilize plasmids through conjugation, suggesting a mechanism for the emergence of novel EIEC clones.

Keywords: EIEC; conjugation; emerging clones; evolution; phylogenomics.

Figure 1

Comparison of invasion plasmids of O96:H19 EIEC strains. The completely assembled pINV from EF432 strain (inner circle, red color) responsible of the outbreak occurred in Italy in 2012 was used as reference for alignment and gene annotation.

Figure 2

Genetic map of the pINV virulence plasmid of EF432 strain obtained through OriT Finder. The annotation includes gene prediction obtained through Prokka (Seemann, 2014) or progressive numbers for the coding sequences identified for which no gene could be called.

Figure 3

Genetic map of the pINV virulence plasmid of 152661 strain obtained through OriT Finder. The annotation includes gene prediction obtained through Prokka (Seemann, 2014) or progressive numbers for the coding sequences identified for which no gene could be called.

Figure 4

Alignment of tra regions in pINV of EF432 strain, used as reference, and pINV of 152661, produced with MAUVE tool (Rissman et al., 2009). Conserved segments that appear to be internally free from big genome rearrangements are visualized as colored blocks. A decrease in percentage of sequence identity is represented as a decrease in coloring. The genetic organization is represented under each sequence, with tra genes indicated with the corresponding letters (traMJYAVCNQSDI) and T4CP and Relaxase-encoding genes highlighted for their encoded function. The red boxes indicate the two identical and inverted copies of an IS600 element in the reference, while the orange box highlights the IS600 remnant in the corresponding region of pINV in 152661 strain.

Figure 5

Phylogenomic analysis of EIEC and Shigella strains through cgMLST. For EIEC isolates, the strains identifiers include the strain name and the O and H antigens, each separated by underscores. For Shigella isolates, the strains identifiers comprise the species and the strain name. The different colors categorize the Sequence Types derived for the corresponding strains, as detailed in the legend. The numbers in red indicate the number of allelic differences identified for each link.