Effect and Mechanism of Eliminating Staphylococcus aureus by Electron Beam Irradiation and Reducing the Toxicity of Its Metabolites

Abstract

The purpose of this study was to evaluate the mechanism of sterilization of Staphylococcus aureus by electron beam irradiation (0.5-, 1-, 2-, 4-, and 6-kGy treatments) and whether it reduces the toxicity of its fermentation supernatant. In this study, we investigated the mechanism of sterilization of S. aureus by electron beam irradiation using colony count, membrane potential, intracellular ATP, and UV absorbance measurements; we used hemolytic, cytotoxic, and suckling mouse wound models to verify that electron beam irradiation reduced the toxicity of the S. aureus fermentation supernatant. The results showed that 2 kGy of electron beam irradiation treatment completely inactivated S. aureus in suspension culture, and 4 kGy inactivated cells in S. aureus biofilms. This study suggests that the bactericidal effect of electron beam irradiation on S. aureus may be attributed to reversible damage to the cytoplasmic membrane, resulting in its leakage and the significant degradation of genomic DNA. The combined results of hemolytic, cytotoxic, and suckling mouse wound models demonstrated that the toxicity of S. aureus metabolites was significantly reduced when the electron beam irradiation dose was 4 kGy. In summary, electron beam irradiation has the potential to control S. aureus and reduce its toxic metabolites in food. IMPORTANCE Electron beam irradiation of >1 kGy damaged the cytoplasmic membrane, and reactive oxygen species (ROS) penetrated the cells. Electron beam irradiation of >4 kGy reduces the combined toxicity of virulent proteins produced by Staphylococcus aureus. Electron beam irradiation of >4 kGy can be used to inactivate Staphylococcus aureus and biofilms on milk.

Keywords: Staphylococcus aureus; electron beam irradiation; inactivation; metabolites; toxicity.

FIG 1

Colony counts of EBI (0-, 0.5-, 1-, 2-, 4-, and 6-kGy)-treated S. aureus suspensions (A) and milk with biofilm (B). ****, P < 0.0001 for highly significant differences compared to the 0-kGy-treated group.

FIG 2

Effects of different doses of EBI on the intracellular ATP concentration (A), cell membrane potential (B), ROS in cells (C), cell leakage determined by the UV absorbance (D), and genomic DNA (E) of strain ATCC 29213. Significance was determined compared with the lethal heat treatment group in panel A (**, P < 0.0005 [significant difference]; ****, P < 0.0001 [highly significant difference]) and compared with the 0-kGy-treated group in panel B (***, P = 0.0010 [significant difference]; ****, P < 0.0001 [highly significant difference]), panel C (***, P = 0.0005 [significant difference]; ****, P < 0.0001 [highly significant difference]), and panel D (****, P < 0.0001 [highly significant difference]). In panel E, agarose gel electrophoresis (left) and band grayscale analysis (right) are shown. IntDen, integrated density.

FIG 3

Effect of EBI on the cell membrane integrity of strain ATCC 29213 as observed by confocal laser scanning microscopy (A), analysis of quantitative fluorescence (B), and scanning electron microscopy (C). *, P = 0.0166 (significant difference); ****, P < 0.0001 (highly significant difference) (compared with the 0-kGy-treated group) (B).

FIG 4

Effect of EBI on strain ATCC 29213 FS proteins (A), hemolysis (B and C), and cytotoxicity (D and E). (A) Effects of EBI on protein primary structures in the FS by SDS-PAGE (top) and on SEA and Hla in the FS by Western blotting (bottom). β-Actin served as a loading control. M, molecular marker. (B) Hemolysis rate for the FS at 5%. NC, negative control; PC, positive control; BC, blank control. (C) Hemolysis rate. **, P = 0.0067 compared to the 0-kGy-treated group (significant difference); ****, P < 0.0001 (very significant difference). (D) Caco-2 cells were viewed under an electron microscope at a magnification of ×200. (E) Cell viability. ***, P = 0.0005 (significant difference); ****, P < 0.0001 (highly significant difference) (compared with the 0-kGy-treated group).

FIG 5

Dorsal wound recordings of suckling mice 0 and 8 h after receiving FSs by subcutaneous injection (A) and staining of subcutaneous tissue sections (HE) at 8 h (B).