Comparison of Shiga toxin-encoding bacteriophages in highly pathogenic strains of Shiga toxin-producing Escherichia coli O157:H7 in the UK

Abstract

Over the last 35 years in the UK, the burden of Shiga toxin-producing Escherichia coli (STEC) O157:H7 infection has, during different periods of time, been associated with five different sub-lineages (1983-1995, Ia, I/IIa and I/IIb; 1996-2014, Ic; and 2015-2018, IIb). The acquisition of a stx2a-encoding bacteriophage by these five sub-lineages appears to have coincided with their respective emergences. The Oxford Nanopore Technologies (ONT) system was used to sequence, characterize and compare the stx-encoding prophages harboured by each sub-lineage to investigate the integration of this key virulence factor. The stx2a-encoding prophages from each of the lineages causing clinical disease in the UK were all different, including the two UK sub-lineages (Ia and I/IIa) circulating concurrently and causing severe disease in the early 1980s. Comparisons between the stx2a-encoding prophage in sub-lineages I/IIb and IIb revealed similarity to the prophage commonly found to encode stx2c, and the same site of bacteriophage integration (sbcB) as stx2c-encoding prophage. These data suggest independent acquisition of previously unobserved stx2a-encoding phage is more likely to have contributed to the emergence of STEC O157:H7 sub-lineages in the UK than intra-UK lineage to lineage phage transmission. In contrast, the stx2c-encoding prophage showed a high level of similarity across lineages and time, consistent with the model of stx2c being present in the common ancestor to extant STEC O157:H7 and maintained by vertical inheritance in the majority of the population. Studying the nature of the stx-encoding bacteriophage contributes to our understanding of the emergence of highly pathogenic strains of STEC O157:H7.

Keywords: Escherichia coli O157:H7; Shiga toxin; bacteriophage; whole-genome sequencing.

Fig. 1.

Maximum-likelihood phylogenetic tree of 105 genomes including the 17 (labelled) publicly available genomes and nanopore sequenced genomes produced during this study. Sub-lineages are coloured as follows: I/II, grey; Ia, yellow; Ib, orange; Ic, red; IIa, purple; IIb, blue; IIc, green. Scale bar indicates kbp.

Fig. 2.

Easyfig diagram representing the chromosome and prophage content within the samples sequenced in this study (in descending order PT4, PT2, PT49, PT21/28, PT8 and PT34). stx2a-encoding, stx2c-encoding and stx1-encoding prophages are highlighted in red, orange and yellow, respectively. Non-stx-encoding prophages are coloured black. Prophage-like elements are coloured blue and the locus of enterocyte effacement is shown in green.

Fig. 3.

Mid-rooted tree of stx-encoding prophages based on Jaccard distance produced from Mash. Strains are annotated with strain ID, length (bp), stx profile and SBI. Strains sequenced during this study have prophages that are hown preceded by an *. Strains are coloured by sub-lineage: green, Ia; red, Ic; blue, I/IIa; grey, I/IIb; orange, IIa; black, IIb; purple, IIc. Scale bar indicates Jaccard distance.

Fig. 6.

Two Easyfig plots comparing the stx2a-encoding prophages from E45000 with E116508 (above) and 155 and 267849 (below), in descending order. Arrows indicate gene directions. stx genes are shown in red; recombination/replication genes shown in light blue; regulation-associated genes are shown in dark blue; effector genes are shown in pink; structure- and lysis-associated genes are shown in light and dark green, respectively; tRNAs are shown as purple lines; finally, hypothetical genes are shown in grey.

Fig. 4.

Easyfig plot comparing the stx1a-encoding prophages from 644 (×2), 180 (×2), 350, E30288, EDL933 and Sakai. Arrows indicate gene directions. stx genes are shown in red; recombination/replication genes are shown in light blue; regulation-associated genes are shown in dark blue; effector genes are shown in pink; structure- and lysis-associated genes are shown in light and dark green, respectively; tRNAs are shown as purple lines; finally, hypothetical genes are shown in grey.

Fig. 5.

Easyfig plot comparing the stx2c-encoding prophages from all samples in the study, including two stx2a prophages that are in a stx2c-associated prophage structure (315126 and E45000). Arrows indicate gene directions. stx genes are shown in red; recombination/replication genes are shown in light blue; regulation-associated genes are shown in dark blue; effector genes are shown in pink; structure- and lysis-associated genes are shown in light and dark green, respectively; tRNAs are shown as purple lines; finally, hypothetical genes are shown in grey.