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

Abstract

Shiga toxin-producing Escherichia coli (STEC) that cause severe disease predominantly carry the toxin gene variant stx2a. 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 stx2a in samples from cattle and to isolate and characterize stx2a-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 stx2a-positive isolates were whole genome sequenced. Among faecal samples from Sweden, stx1 was detected in 37%, stx2 in 53% and stx2a in 5% and in skin (ear) samples in 64%, 79% and 2% respectively. In Norway, 79% of the herds were positive for stx1, 93% for stx2 and 17% for stx2a. Based on amplicon sequencing the most common stx2 types in samples from Swedish cattle were stx2a and stx2d. Multilocus sequence typing (MLST) of 39 stx2a-positive isolates collected from both countries revealed substantial diversity with 19 different sequence types. Only a few classical LEE-positive strains similar to HUSEC were found among the stx2a-positive isolates, notably a single O121:H19 and an O26:H11. Lineages known to include LEE-negative HUSEC were also recovered including, such as O113:H21 (sequence type ST-223), O130:H11 (ST-297), and O101:H33 (ST-330). We conclude that E. coli encoding stx2a in cattle are ranging from strains similar to 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.

Fig 1. Prevalence of Shiga toxin 2 variants in 31 stx₂-positive skin samples from Swedish cattle evaluated by amplicon sequencing.

The figure shows (A) the prevalence, with 95% confidence intervals, of each stx₂ variant and (B) combinations of different toxin variants found in individual samples.

Fig 2. Virulence gene profiles, sequence types, phylogroups and serotypes of 39 stx_2a-positive E. coli isolates from cattle in Sweden and Norway.

Two outbreak strains of EHEC (O157:H7 TW14359 and O121:H19 16–9255) are included as references. U; undetermined phylogroup or sequence type. ONT; antigen non-typable. O2 not distinguishable from O50. O101 not distinguishable from O162. O153 not distinguishable from O178.

Fig 3. Minimum spanning tree of sequence types among stx_2a-positive E. coli from cattle.

All three major clonal complexes comprise isolates of both Swedish (blue) and Norwegian (red) origin. A difference in only one SNP is displayed by a thick line whereas a difference in two SNP’s is displayed by a thin line.

Fig 4. Multiple correspondence analysis biplots of the virulence genes of stx_2a-positive E. coli from Norwegian and Swedish cattle.

Association between virulence genes is estimated based on co-occurence in isolates (A), and association between isolates is estimated based on the genes they possess or lack (B).