title: Evolution of a zoonotic pathogen: investigating prophage diversity in enterohaemorrhagicEscherichia coliO157 by long-read sequencing.
abstract: EnterohaemorrhagicEscherichia 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 asstxand 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.
authors.affiliation: 1​Division of Infection and Immunity, The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush EH25 9RG, UK.
authors.forename: Sharif
authors.initials: S
authors.lastname: Shaaban
authors.affiliation: 2​Gastrointestinal Bacterial Reference Unit, 61 Colindale Avenue, Public Health England, London NW9 5EQ, UK.
authors.forename: Lauren A
authors.initials: LA
authors.lastname: Cowley
authors.affiliation: 1​Division of Infection and Immunity, The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush EH25 9RG, UK.
authors.forename: Sean P
authors.initials: SP
authors.lastname: McAteer
authors.affiliation: 2​Gastrointestinal Bacterial Reference Unit, 61 Colindale Avenue, Public Health England, London NW9 5EQ, UK.
authors.forename: Claire
authors.initials: C
authors.lastname: Jenkins
authors.affiliation: 2​Gastrointestinal Bacterial Reference Unit, 61 Colindale Avenue, Public Health England, London NW9 5EQ, UK.
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Dallman
authors.affiliation: 3​U.S. Meat Animal Research Center, Agricultural Research Service, U.S. Department of Agriculture, Clay Center, NE 68933-0166, USA.
authors.forename: James L
authors.initials: JL
authors.lastname: Bono
authors.affiliation: 1​Division of Infection and Immunity, The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush EH25 9RG, UK.
authors.forename: David L
authors.initials: DL
authors.lastname: Gally
mesh_terms: D000818:Animals; D002417:Cattle; D019453:Escherichia coli O157 / Q000235:genetics / Q000821:virology*; D019143:Evolution, Molecular; D014644:Genetic Variation; D016680:Genome, Bacterial / Q000235:genetics; D006801:Humans; D039002:Prophages / Q000145:classification* / Q000235:genetics*
keywords: Eschrichia coli 0157; Shiga toxin; bacteriophage; prophage