Genomic analysis of shiga toxin-containing Escherichia coli O157:H7 isolated from Argentinean cattle

Abstract

Cattle are the main reservoir of Enterohemorrhagic Escherichia coli (EHEC), with O157:H7 the distinctive serotype. EHEC is the main causative agent of a severe systemic disease, Hemolytic Uremic Syndrome (HUS). Argentina has the highest pediatric HUS incidence worldwide with 12-14 cases per 100,000 children. Herein, we assessed the genomes of EHEC O157:H7 isolates recovered from cattle in the humid Pampas of Argentina. According to phylogenetic studies, EHEC O157 can be divided into clades. Clade 8 strains that were classified as hypervirulent. Most of the strains of this clade have a Shiga toxin stx2a-stx2c genotype. To better understand the molecular bases related to virulence, pathogenicity and evolution of EHEC O157:H7, we performed a comparative genomic analysis of these isolates through whole genome sequencing. The isolates classified as clade 8 (four strains) and clade 6 (four strains) contained 13 to 16 lambdoid prophages per genome, and the observed variability of prophages was analysed. An inter strain comparison show that while some prophages are highly related and can be grouped into families, other are unique. Prophages encoding for stx2a were highly diverse, while those encoding for stx2c were conserved. A cluster of genes exclusively found in clade 8 contained 13 genes that mostly encoded for DNA binding proteins. In the studied strains, polymorphisms in Q antiterminator, the Q-stx2A intergenic region and the O and P γ alleles of prophage replication proteins are associated with different levels of Stx2a production. As expected, all strains had the pO157 plasmid that was highly conserved, although one strain displayed a transposon interruption in the protease EspP gene. This genomic analysis may contribute to the understanding of the genetic basis of the hypervirulence of EHEC O157:H7 strains circulating in Argentine cattle. This work aligns with other studies of O157 strain variation in other populations that shows key differences in Stx2a-encoding prophages.

Fig 1. Schematic lineal representation of aligned EHEC O157 genomes.

The origin of replication is placed at the 5´and 3’end. Lambdoid prophages are represented as boxes of blue color, except for those carrying stx genes. In those cases, stx1, stx2_a and stx2_c prophages were indicated in yellow, red and light blue boxes, respectively. Artemis comparison tool was used to indicate regions of similarity (red), inversions (blue) and In/dels (clear).

Fig 2. Phylogenetic tree constructed with the core genome from the Argentinian strains and previously published strains using RAxML.

Bootstrap values are located at each branch point.

Fig 3. Alignment of pO157 plasmids from EHEC O157 isolates.

Plasmid are represented in lineal forms with the ORF colored blue. The tagA gene was oriented at the 5´end to allow for comparison of plasmid architecture. Artemis comparison tool was used to indicate regions of similarity (red), inversions (blue) and In/dels (clear).

Fig 4. Alignment of the p55 plasmid of strains 7.1Anguil and 146N4 with a related pMCR1plasmid.

Plasmid are represented in lineal forms with blue and red arrows indicating genes. Artemis comparison tool was used to indicate regions of similarity (red), inversions (blue) and In/dels (clear).

Fig 5. Alignment of prophages carrying stx2_a, the red arrows represent genes of the stx2_a operon; the grey arrows indicate hypothetical proteins, and the blue arrows refer to genes encoding for protein with assigned function.

Some relevant genes of lambda phage are marked. Artemis comparison tool was used to indicate regions of similarity (red), inversions (blue) and In/dels (clear) or no homology (no lines). Columns at the right show cytotoxicity in Vero cells, by ELISA with or without mitomicin and O and P protein subtype.

Fig 6

(A). amino acid substitutions (shaded in grey) in the Q antiterminator gene, (B) SNPs in Q-stx2A intergenic region. EDL933 sequence is used as reference. Dots represent nucleotide that are identical to the reference. SNPs are represented by the substituting nucleotide. A tRNA present in the intergenic region is shaded in grey.