Comparative Genomics and stx Phage Characterization of LEE-Negative Shiga Toxin-Producing Escherichia coli

Abstract

Infection by Escherichia coli and Shigella species are among the leading causes of death due to diarrheal disease in the world. Shiga toxin-producing E. coli (STEC) that do not encode the locus of enterocyte effacement (LEE-negative STEC) often possess Shiga toxin gene variants and have been isolated from humans and a variety of animal sources. In this study, we compare the genomes of nine LEE-negative STEC harboring various stx alleles with four complete reference LEE-positive STEC isolates. Compared to a representative collection of prototype E. coli and Shigella isolates representing each of the pathotypes, the whole genome phylogeny demonstrated that these isolates are diverse. Whole genome comparative analysis of the 13 genomes revealed that in addition to the absence of the LEE pathogenicity island, phage-encoded genes including non-LEE encoded effectors, were absent from all nine LEE-negative STEC genomes. Several plasmid-encoded virulence factors reportedly identified in LEE-negative STEC isolates were identified in only a subset of the nine LEE-negative isolates further confirming the diversity of this group. In combination with whole genome analysis, we characterized the lambdoid phages harboring the various stx alleles and determined their genomic insertion sites. Although the integrase gene sequence corresponded with genomic location, it was not correlated with stx variant, further highlighting the mosaic nature of these phages. The transcription of these phages in different genomic backgrounds was examined. Expression of the Shiga toxin genes, stx1 and/or stx2, as well as the Q genes, were examined with quantitative reverse transcriptase polymerase chain reaction assays. A wide range of basal and induced toxin induction was observed. Overall, this is a first significant foray into the genome space of this unexplored group of emerging and divergent pathogens.

Shiga toxins and stx phages: highly diverse entities

“…Table 1 lists stx phages whose complete sequences are available in GenBank. Recent comparisons of whole genomes have confirmed that stx phages share a general genomic organization, but with a significant degree of sequence diversity, reinforcing the concept of their mosaic nature (Ahmed et al, 2012;Smith et al 2012;Steyert et al, 2012;Cooper et al, 2014;Tozzoli et al, 2014a). Furthermore, some studies identified different types of stx phages harbouring even the same stx 2 subtype (Ahmed et al, 2012;Tozzoli et al, 2014b).…”

“…argW, potC, prfC, serU, ssrA, wrbA, yciD, yecD, yecE, yjbM, ynfH and Z2577 (Recktenwald & Schmidt, 2002;Koch et al, 2003;Ahmed et al, 2012;Steyert et al, 2012;Cooper et al, 2014).…”

“…In an interesting study evaluating chromosomal site specificity, Serra-Moreno et al (2007) detected that phages preferentially use one insertion site depending on the host strain; however, if the preferred locus is unavailable, the A. Krü ger and P. M. A. Lucchesi phage integrates into a secondary insertion site. In addition, Steyert et al (2012) observed heterogeneity in integrase genes amongst stx phages in LEE (locus of enterocyte effacement)-negative strains. Such variants could be associated with phage insertion at specific genomic locations.…”

“…Moreover, a relationship between the Q allele and the level of stx expression has been suggested (LeJeune et al, 2004;Ahmad & Zurek, 2006;Zhang et al, 2010;Mellor et al, 2012). Recently, Steyert et al (2012) observed phylogenetic diversity of stx phages in an analysis of Q sequences, identifying seven clusters amongst 15 selected strains. The Q sequences associated with the highest level of stx expression were found to be clustered together.…”

Shiga toxin-producing Escherichia coli in human, cattle and foods. Strategies for detection and control

“…Strains laking eae are named as LEE-negative STEC. Steyert et al (2012) demonstrate that the overall genome content, phage location, and combination of potential virulence factors are variable in this strains group.…”