Evolution of a zoonotic pathogen: investigating prophage diversity in enterohaemorrhagic Escherichia coli O157 by long-read sequencing.

Sharif Shaaban,David L Gally,Sean P Mcateer,Timothy J Dallman,Lauren A Cowley,Claire Jenkins,James L Bono

doi:10.1099/mgen.0.000096

Abstract

Enterohaemorrhagic Escherichia coli (EHEC) O157 is a zoonotic pathogen for which colonization of cattle and virulence in humans is associated with multiple horizontally acquired genes, the majority present in active or cryptic prophages. Our understanding of the evolution and phylogeny of EHEC O157 continues to develop primarily based on core genome analyses; however, such short-read sequences have limited value for the analysis of prophage content and its chromosomal location. In this study, we applied Single Molecule Real Time (SMRT) sequencing, using the Pacific Biosciences long-read sequencing platform, to isolates selected from the main sub-clusters of this clonal group. Prophage regions were extracted from these sequences and from published reference strains. Genome position and prophage diversity were analysed along with genetic content. Prophages could be assigned to clusters, with smaller prophages generally exhibiting less diversity and preferential loss of structural genes. Prophages encoding Shiga toxin (Stx) 2a and Stx1a were the most diverse, and more variable compared to prophages encoding Stx2c, further supporting the hypothesis that Stx2c-prophage integration was ancestral to acquisition of other Stx types. The concept that phage type (PT) 21/28 (Stx2a+, Stx2c+) strains evolved from PT32 (Stx2c+) was supported by analysis of strains with excised Stx-encoding prophages. Insertion sequence elements were over-represented in prophage sequences compared to the rest of the genome, showing integration in key genes such as stx and an excisionase, the latter potentially acting to capture the bacteriophage into the genome. Prophage profiling should allow more accurate prediction of the pathogenic potential of isolates.

Highlights

The availability of additional sequences to compare with the first sequenced Escherichia coli genome, E. coli K12 MG1655, has highlighted how the evolution of this species is intimately associated with the integration of bacteriophages into the bacterial genome, and their subsequent entrapment, recombination and degradation as prophage regions (Ohnishi et al, 2001; Shaikh & Tarr, 2003)
All Shiga toxin (Stx)-encoding prophages were found in previously documented insert sites (Shaikh & Tarr, 2003): prophages encoding Stx2c were found only in sbcB; while those encoding Stx2a were found in multiple insert sites – wrbA, argW, yecE; and Stx1aencoding prophages were detected in yehV and argW
This study aimed to analyse prophage regions extracted from E. coli O157 : H7 strains representative of the main clusters found in the extensive phylogeny presented by Dallman et al (2015)

Summary

Introduction

The availability of additional sequences to compare with the first sequenced Escherichia coli genome, E. coli K12 MG1655, has highlighted how the evolution of this species is intimately associated with the integration of bacteriophages into the bacterial genome, and their subsequent entrapment, recombination and degradation as prophage regions (Ohnishi et al, 2001; Shaikh & Tarr, 2003). The importance of specific prophages and their longer-term legacy are evident when considering the emergence of enterohaemorrhagic E. coli (EHEC) as a serious zoonotic pathogen (Hayashi et al, 2001; Ogura et al, 2009; Perna et al, 2001). Phylogenetic studies have established that E. coli O157 can be delineated into three main lineages, as well as nine clades (Eppinger et al, 2011; Zhang et al, 2007; Manning et al, 2008). In the USA, clade 8 strains of lineage I/II are associated with more severe human disease (Manning et al, 2008); while in the UK, the main human isolates reside in lineage I and clade 4/5 (Dallman et al, 2015)

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Conclusion