Shiga toxin-producing Escherichia coli strains negative for locus of enterocyte effacement

Abstract

Most Shiga toxin-producing Escherichia coli (STEC) infections that are associated with severe sequelae such as hemolytic uremic syndrome (HUS) are caused by attaching and effacing pathogens that carry the locus of enterocyte effacement (LEE). However, a proportion of STEC isolates that do not carry LEE have been associated with HUS. To clarify the emergence of LEE-negative STEC, we compared the genetic composition of the virulence plasmids pO113 and pO157 from LEE-negative and LEE-positive STEC, respectively. The complete nucleotide sequence of pO113 showed that several plasmid genes were shared by STEC O157:H7. In addition, allelic profiling of the ehxA gene demonstrated that pO113 belongs to a different evolutionary lineage than pO157 and that the virulence plasmids of LEE-negative STEC strains were highly related. In contrast, multilocus sequence typing of 17 LEE-negative STEC isolates showed several clonal groups, suggesting that pathogenic LEE-negative STEC has emerged several times throughout its evolution.

Figure 1

Circular map of virulence plasmid pO113 generated by using circular_diagram.pl (Sanger Institute, Cambridge, UK) and Inkscape software (www.inkscape.org). The locations of proteins encoded on the leading and lagging strand are shown on the outer 2 rings. The colors indicate the assigned GenoList functional category (Institut Pasteur, Paris, France). The black ring indicates GC content, with high GC content outermost. The innermost ring shows GC skew.

Figure 2

Graphic overview of sequences related to virulence plasmid pO113 in the plasmids ColIb-P9 and pO157. The overview was generated by ACT (www.sanger.ac.uk); related sequences are indicated as boxes between the horizontal bars representing each of the plasmid sequences. Similarity between sequences was established by using TBLASTX with the pO113 sequence as the subject and either ColIb-P9 or pO157 as the query sequence. Blue indicates that open reading frames occur in the same order; red indicates a DNA inversion.

Figure 3

Neighbor-joining tree of ehxA (A) and repA (B) as implemented in ClustalW (www.ebi.ac.uk/Tools/clustalw2). This rectangular cladogram demonstrates the distinct clades (shown by boxes) for ehxA that delineate locus of enterocyte effacement (LEE)–negative and LEE-positive Shiga toxin–producing Escherichia coli strains. Exceptions to this pattern are shown in boldface, strain names are shown in parentheses, and + or – indicates the presence or absence of LEE. Significant nodes were identified by bootstrapping (Monte Carlo randomization); nodes were present in >70% of the 1,000 bootstrap trees highlighted and identified as significant.

Figure 4

Phylogenetic relationships of 17 locus of enterocyte effacement (LEE)–negative and 13 LEE-positive Shiga toxin–producing Escherichia coli (STEC) strains (highlighted in gray) compared with a cohort of reference E. strains. Phylogeny was demonstrated by a neighbor-joining algorithm from 7 housekeeping gene sequences. Each isolate has been assigned a sequence type (ST) (in boldface), and assigned clonal groups (CGs) are displayed. The scale bar demonstrates the branch length that corresponds to 2 nucleotide substitutions per 1,000 nucleotide sites. Significant nodes were identified by bootstrapping (Monte Carlo randomization); nodes were present in >70% of the 1,000 bootstrap trees highlighted and identified as significant.