Induction of lysogenic bacteriophages of Shiga toxin-producing Escherichia coli by antimicrobial growth promoters used in food- producing animals in South Africa

Abstract

Shiga toxin-producing Escherichia coli (STEC) O157:H7 is a foodborne disease characterized by diarrhoea and complications such as haemorrhagic colitis and the haemolytic uremic syndrome, a complication which can lead to kidney failure in 5-10% of humans showing STEC disease. The major virulence factors of STEC are two toxins (Stx1 and Stx2) encoded on bacteriophages, commonly termed Shiga toxin-converting bacteriophages (stx-converting bacteriophages). Previous studies have shown that a number of antimicrobials which are used for livestock growth promotion can induce stx-converting bacteriophages at subinhibitory concentrations. Induced stx-converting bacteriophages are considered the main “drivers” of STEC emergence and evolution as they transfer bacteriophage-encoded Shiga toxin-encoding genes from STEC to naïve E. coli by transduction. This phenomenon is considered to be behind the formation of novel STEC strains. Although a European Union-wide ban on the use of antimicrobial growth promoters in animal agriculture exists since 2006, this controversial practice remains in effect in a number of countries around the world including South Africa. Therefore, in this study, four antimicrobials which are approved for livestock growth-promotion in South Africa were tested at sub-inhibitory concentrations for their capacity to induce Shiga toxin-converting bacteriophages from 47 STEC O157:H7 isolates using the double-layer agar-plaque assay. The antimicrobials tested included josamycin, virginiamycin, flavophospholipol and poly 2-propenal 2-propenoic acid. These antimicrobial growth promoters had never been tested for their capacity to induce bacteriophages. In addition, induced bacteriophages were characterized for the presence of genes encoding different Shiga toxin subtypes (stx1, stx2, stx2c and stx2d), restriction fragment length polymorphisms and morphology by electron microscopy. The following bacteriophage induction rates were obtained for each antimicrobial growth promoter tested: poly 2-propenal 2-propenoic acid, 42.6% (20/47); virginiamycin, 34.0% (16/47); josamycin, 34.0% (16/47); flavophospholipol, 29.8% (14/47). A small number of STEC O157:H7 isolates induced bacteriophages spontaneously (14.9%; 7/47). Most of the induced bacteriophages carried the stx2 and stx2c-encoding genes, independent of the induction method while only a few bacteriophages carried stx2d except for josamycin and spontaneously-induced bacteriophages which stx2d at higher rates of 87.5% (14/16) and 100% (7/7). Electron microscopy revealed only four representative groups of virion particle morphologies: three groups of bacteriophages with either a long hexagonal, oval/circular, and elongated head which all had long tails and one group of bacteriophages with an icosahedral/hexagonal head and a thick contractile tail. These results showed that josamycin, virginiamycin, flavophospholipol and poly 2-propenal 2-propenoic acid induce stx-converting bacteriophages from STEC O157:H7. Induced bacteriophages were largely stx2 and stx2c positive, but stx2d positive to a lesser extent. Induction of stx-converting bacteriophages by antimicrobial growth promoters may be contributing to the conversion of naïve E. coli into virulent STEC strains as a result of bacteriophage transduction and horizontal transfer of stx-encoding genes from STEC to naive E. coli. This phenomenon has been identified as the driving force behind STEC emergence, expansion and evolution. The formulation of policies and implementation of strategies which promote the prudent use of antimicrobials growth promoters in animal agriculture in South Africa and elsewhere where these compounds are still in use are recommended.