Degradation and inactivation of Shiga toxins by nitrogen gas plasma

Akikazu Sakudo,Yuichiro Imanishi

doi:10.1186/s13568-017-0380-7

Abstract

Shiga toxin (Stx)-producing Escherichia coli (STEC) leads to food poisoning by causing hemorrhagic colitis and hemolytic uremic syndrome. Some STEC produce Shiga toxin 1 (Stx1) and/or Shiga toxin 2 (Stx2), a relatively stable protein toxin, necessitating the development of an efficient inactivation method. Here we applied a nitrogen gas plasma apparatus to the inactivation of Stx. Samples of Stx1 and Stx2 were treated with a nitrogen gas plasma generated by a plasma device using a short high-voltage pulse applied by a static induction thyristor power supply at 1.5 kpps (kilo pulse per second). The recovered Stx samples were then analyzed for immunological and biological activities. Immunochromatography demonstrated that Stx1 and Stx2 were degraded by the gas plasma. Quantification by enzyme-linked immunosorbent assay (ELISA) showed that both toxins were efficiently degraded to less than 1/10th of their original concentration within 5 min of treatment. Western blotting further showed the gas plasma treatment degraded the A subunit, which mediates the toxicity of Stx. Moreover, an assay using HEp-2 cells as an index of cytotoxicity showed that gas plasma treatment reduced the toxic activity of Stx. Therefore, nitrogen gas plasma might be an efficient method for the inactivation of Stx.

Highlights

The virulence of Shiga toxin (Stx)-producing Escherichia coli (STEC), such as E. coli O157:H7 and other serotypes, including their most dangerous subset, enterohemorrhagic E. coli (EHEC), depends on the production of Stx (Gyles 2007; Hunt 2010)
To investigate the effect of nitrogen gas plasma on Stx, 20 μl of a 1 μg/ml solution of Shiga toxin 1 (Stx1) or Shiga toxin 2 (Stx2) was spotted onto a coverslip, which was placed on the earth electrode of the bi-polar and low-pressure plasma-triple effects sterilization (BLP-TES) device, and subjected to nitrogen gas plasma treatment (1.5 kpps, 0, 5, 15, and 30 min)
Test lines for Stx1 and Stx2, and reference lines for the internal control are indicated by arrows test lines were detected in the untreated sample (0 min) and the sample treated for 5 min, but were diminished in samples treated for 15 and 30 min (Fig. 1b)

Summary

Introduction

The virulence of Shiga toxin (Stx)-producing Escherichia coli (STEC), such as E. coli O157:H7 and other serotypes, including their most dangerous subset, enterohemorrhagic E. coli (EHEC), depends on the production of Stx (Gyles 2007; Hunt 2010). The toxins inhibit protein synthesis and are deleterious to humans, leading to life-threating complications including hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS) (Boerlin et al 1999; Scallan et al 2011). These illnesses are known as food-borne diseases, resulting from the ingestion of food contaminated with STEC and their toxins. Technologies to degrade and/or inactivate Stx are needed.

Methods

Results

Conclusion