Oxford Nanopore sequencing elucidates a novel stx2f carrying prophage in a Shiga toxin producing Escherichia coli(STEC) O63:H6 associated with a case of haemolytic uremic syndrome (HUS)

The aim of this study was to use Oxford Nanopore sequencing to characterise a Shiga-toxin producing Escherichia coli(STEC) O63:H6 responsible for a recent case of haemolytic uremic syndrome (HUS).

STEC isolate 377323 and a comparator stxnegative E. coli isolate of the same lineage and serotype (382634) were sequenced on an Oxford Nanopore minION R9.4 flow cell and an Illumina HiSeq 2500. Nanopore FAST5 data were basecalled and demultiplexed by Albacore. Assemblies were generated using Unicycler and polished using Nanopolish, Pilon and Racon before screening for prophages using PHASTER.

The Shiga toxin subtype was confirmed to be stx2f, which is rarely associated with the onset of HUS in STEC. Comparing the two genomes, the stxnegative isolate harboured five prophages compared to eleven prophages in the STEC isolate, with only one in common. Both samples contained an intact locus of enterocyte effacement pathogenicity island. The Shiga toxin encoding prophage was 42.5 kb in size and predicted to contain 71 coding sequences. 377323 also encoded an 85kbp IncFIB plasmid, not present in 382634. This plasmid encodes a traconjugation cassette and the virulence bfpcassette encoding for a type IV bundle forming pili.

Long read sequencing enabled the characterisation of a rare STEC and related E. coli IN relation to both prophage content and plasmid content and revealed significant differences in the pan-genome in addition to the acquisition of the novel Stx2f phage. This data contributes to the understanding of non-O157 STEC associated with HUS.