The prevalence and genomic context of Shiga toxin 2a genes in E. coli found in cattle

Shiga toxin-producing Escherichia coli (STEC) that cause severe disease predominantly carry the toxin gene variant stx 2a . However, the role of Shiga toxin in the ruminant reservoirs of this zoonotic pathogen is poorly understood and strains that cause severe disease in humans (HUSEC) likely constitute a small and atypical subset of the overall STEC flora. The aim of this study was to investigate the presence of stx 2a in samples from cattle and to isolate and characterize stx 2a -positive E. coli . In nationwide surveys in Sweden and Norway samples were collected from individual cattle or from cattle herds, respectively. Samples were tested for Shiga toxin genes by real-time PCR and amplicon sequencing and stx 2a -positive isolates were whole genome sequenced. Among faecal samples from Sweden, stx 1 was detected in 37%, stx 2 in 53% and stx 2a in 5% and in skin samples in 64%, 79% and 2% respectively. In Norway, 79% of the herds were positive for stx 1 , 93% for stx 2 and 17% for stx 2a . Based on amplicon sequencing the most common stx 2 types in samples from Swedish cattle were stx 2a and stx 2d . Multilocus sequence typing (MLST) of 39 stx 2a -positive isolates collected from both countries revealed substantial diversity with 19 different sequence types. Only a few classical LEE-positive HUSEC were found among the stx 2a -positive isolates, notably a single O121:H19 and an O26:H11. Known LEE-negative HUSEC lineages were also recovered including O113:H21 (ST-223), O130:H11 (ST-297), and O101:H33 (ST-330). We conclude that E. coli encoding stx 2a in cattle are ranging from well-known HUSEC to unknown STEC variants. Comparison of isolates from human HUS cases to related STEC from the ruminant reservoirs can help identify combinations of virulence attributes necessary to cause HUS, as well as provide a better understanding of the routes of infection for rare and emerging pathogenic STEC.