SMRT sequencing reveals differential patterns of methylation in two O111:H- Shiga toxigenic Escherichia coli isolates from a historic hemolytic uremic syndrome outbreak in Australia

Abstract

Shiga toxigenic Escherichia coli (STEC) are important food-borne pathogens and a major cause of haemorrhagic colitis and haemolytic-uremic syndrome (HUS) worldwide. In 1995 a severe HUS outbreak in Adelaide occurred. A recent genomic analysis of STEC O111:H-strains 95JB1 and 95NR1 from this outbreak found that the more virulent isolate, 95NR1, harboured two additional copies of the Shiga toxin 2 (Stx2) genes although the structure of the Stx2-converting prophages could not be fully resolved due to the fragmented assembly. In this study we have used Pacific Biosciences (PacBio) single molecule real-time (SMRT) long read sequencing to characterise the complete epigenome (genome and methylome) of 95JB1 and 95NR1. Using long reads we completely resolved the structure of two, tandemly inserted, stx2-converting phage in 95NR1. Our analysis of the methylome of 95NR1 and 95JB1 identified hemi-methylation of a novel motif (5-CTGCm6AG-3) in more than 4000 sites in the 95NR1 genome. These sites were entirely unmethalyted in the 95JB1, including at least 180 potential promoter regions that could explain regulatory differences between the strains. We identified a Type IIG methyltransferase encoded in both genomes in association with three additional genes in an operon-like arrangement. IS1203 mediated disruption of this operon in 95JB1 is the likely cause of the observed differential patterns of methylation between 95NR1 and 95JB1. This study demonstrates the enormous potential of PacBio SMRT sequencing to resolve complex prophage regions and reveal the genetic and epigenetic heterogeneity within a clonal population of bacteria.