# External factors influence intrinsic differences in Stx2e production by Porcine Shiga Toxin-producing Escherichia coli strains

## TL;DR

This study shows how genetic and environmental factors influence toxin production in Shiga toxin-producing E. coli strains that affect pigs.

## Contribution

The study identifies a role for phage holin genes in regulating Stx2e secretion in response to antibiotics and bile acids.

## Key findings

- Porcine STEC strains show significant variation in extracellular Stx2e levels.
- Bile acids and antibiotics like ciprofloxacin and enrofloxacin increase toxin production in specific strains.
- A holin gene downstream of the Stx2e operon is crucial for antibiotic-induced toxin secretion.

## Abstract

Porcine Shiga toxin-producing Escherichia coli (STEC) strains pose significant challenges to the pig industry. The toxins produced by these strains, particularly Shiga toxin subtype 2e (Stx2e), are associated with a range of clinical symptoms such as diarrhoea and oedema disease, which in severe cases result in death. Understanding the factors that influence the production and secretion of Stx2e is crucial to elucidate porcine STEC pathogenesis and to develop effective therapeutic strategies. Therefore, this study aimed to characterize the variability in Stx2e production among different porcine STEC strains and assess the effect of several external factors, including bile acids and antibiotics. Our results highlighted a substantial variation in extracellular Stx2e levels by porcine STEC strains. In addition, bile acids, especially the bile acid deoxycholate, exerted strain-specific effects on these extracellular Stx2e levels. Antibiotics also affected extracellular Stx2e levels with ciprofloxacin and enrofloxacin inducing a substantial increase in toxin production in certain strains. Genome analysis revealed that these strains encode a holin gene downstream of the Stx2e operon. Deleting this holin gene abolished the antibiotic-induced increase in extracellular Stx2e levels, while introducing holin expression in unresponsive strains increased the presence of Stx2e in the extracellular environment. These findings unravel a role for phage holins in Stx2e secretion and highlight the intricate interplay between genetic and environmental factors in regulating Stx2e production in porcine STEC strains. Together, our results offer insights into STEC pathogenesis.

Shiga toxin-producing Escherichia coli (STEC) can cause serious disease in pigs, including diarrhea and oedema disease, sometimes leading to death. These symptoms are mainly caused by the Shiga toxin Stx2e. However, the factors influencing the production and secretion of this toxin by STEC strains are not fully understood. In this study, we examined various porcine STEC strains and tested how external factors like bile acids and antibiotics affect extracellular toxin levels. We found that both strain differences and environmental conditions strongly influence these extracellular Stx2e levels. The bile acid deoxycholate and the antibiotics ciprofloxacin and enrofloxacin, increased toxin production and release in specific STEC strains. This increased toxin release in response to antibiotics was driven by the presence of a gene encoding a holin, a protein produced by viruses that infect bacteria. Removing this holin gene stopped the antibiotic-induced increase in extracellular toxin, while adding it to unresponsive strains increased toxin release. These findings reveal how bacterial genetics and external factors interact to control toxin production, improving our understanding of STEC infections in pigs.