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

Authors: SHAABAN, SHARIF - Roslin Institute; COWLEY, LAUREN - Public Health England (PHE); MCATEER, SEAN - Roslin Institute; JENKINS, CLAIRE - Public Health England (PHE); DALLMAN, TIMOTHY - Public Health England (PHE); Bono, James - Jim; GALLY, DAVID - Roslin Institute

Submitted to: Microbial Genomics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/2/2016
Publication Date: 12/1/2016
Publication URL: https://handle.nal.usda.gov/10113/5934977
Citation: Shaaban, S., Cowley, L., McAteer, S., Jenkins, C., Dallman, T., Bono, J.L., Gally, D. 2016. Evolution of a zoonotic pathogen: investigating prophage diversity in enterohaemorrhagic Escherichia coli O157 by long-read sequencing. Microbial Genomics. doi:10.1099/mgen.0.000096.

Interpretive Summary: Pathogenic Escherichia coli O157:H7 strains pose a threat to human health and are usually acquired from ruminants, the environment or fresh produce. Recent whole genome sequencing based on short-read technologies has helped trace outbreaks and provided significant insights into the evolution of this pathogen, but these methods don’t capture the complete genomic variation that underpins differences that determines a strains potential to cause disease. This variation is, in part, driven by the acquisition of virus that infect bacteria called bacteriophages (phages). Phages contain many similar sequences that short-read sequencing isn’t able to resolve. This study used long-read read sequencing to define the composition and position in the genome of phages from a strain set selected to represent the wide diversity of pathogenic E. coli O157. We demonstrate that the most recent genome modification correlates with acquisition of phages encoding specific types of toxin, which are responsible for damage to kidneys in humans and may have life-threatening consequences. Profiling of phages by composition and position in the genome will aid identification of virulent strains from short read sequencing currently being adopted in diagnostic laboratories.

Technical Abstract: Enterohaemorrhagic E. coli 0157 is a zoonotic pathogen for which colonisation of cattle and virulence in humans is associated with the expression of multiple horizontally acquired genes, the majority present in active or cryptic prophages. Our understanding of the evolution and phylogeny of E. coli 0157 continues to develop primarily based on core genome analyses from short-read sequences, including a recent study of over one thousand isolates from humans and cattle in the United Kingdom (ref). However, due to limitations of short-read sequencing it has been difficult to compare prophage content, location, and sequences between the main clonal clusters. Our current study has applied SMRT sequencing, using the Pacific Bioscience long read sequencing platform, to a subset of representative strains from these clusters and then extracted sequences for the prophage regions as well as from reference strains for comparison. The genome position and diversity of the prophages was analysed along with their genetic content. Prophages could be assigned into different clusters with smaller prophages generally exhibiting less diversity with preferential loss of structural genes. Prophages encoding Stx2a and Stx1a showed were the most diverse and were more variable than prophages encoding Stx2c,further evidence that Stx2c prophage integration is more ancestral within the lineages. Specifically, the concept that PT21/28 (stx2a+ stx2c+) evolved from PT32 (stx2c+) strains was supported by Stx- prophage deletion. IS elements were over-represented In prophage sequences compared to the rest of the genome with integration in key genes such as stx and excisionases, the latter potentially capturing the bacteriophage in the genome. Prophage profiling should allow more accurate prediction of the pathogenic potential of isolates.