Induction of Shiga Toxin-Encoding Prophage by Abiotic Environmental Stress in Food

Abstract

The prophage-encoded Shiga toxin is a major virulence factor in Stxproducing Escherichia coli (STEC). Toxin production and phage production are linked and occur after induction of the RecA-dependent SOS response. However, foodrelated stress and Stx-prophage induction have not been studied at the single-cell level. This study investigated the effects of abiotic environmental stress on stx expression by single-cell quantification of gene expression in STEC O104:H4 Δstx2::gfp:: amp r . In addition, the effect of stress on production of phage particles was determined. The lethality of stressors, including heat, HCl, lactic acid, hydrogen peroxide, and high hydrostatic pressure, was selected to reduce cell counts by 1 to 2 log CFU/ml. The integrity of the bacterial membrane after exposure to stress was measured by propidium iodide (PI). The fluorescent signals of green fluorescent protein (GFP) and PI were quantified by flow cytometry. The mechanism of prophage induction by stress was evaluated by relative gene expression of recA and cell morphology. Acid (pH Ͻ 3.5) and H 2 O 2 (2.5 mM) induced the expression of stx 2 in about 18% and 3% of the population, respectively. The mechanism of prophage induction by acid differs from that of induction by H 2 O 2 . H 2 O 2 induction but not acid induction corresponded to production of infectious phage particles, upregulation of recA, and cell filamentation. Pressure (200 MPa) or heat did not induce the Stx2-encoding prophage (Stx2-prophage). Overall, the quantification method developed in this study allowed investigation of prophage induction and physiological properties at the single-cell level. H 2 O 2 and acids mediate different pathways to induce Stx2-prophage.IMPORTANCE Induction of the Stx-prophage in STEC results in production of phage particles and Stx and thus relates to virulence as well as the transduction of virulence genes. This study developed a method for a detection of the induction of Stxprophages at the single-cell level; membrane permeability and an indication of SOS response to environmental stress were additionally assessed. H 2 O 2 and mitomycin C induced expression of the prophage and activated a SOS response. In contrast, HCl and lactic acid induced the Stx-prophage but not the SOS response. The lifestyle of STEC exposes the organism to intestinal and extraintestinal environments that impose oxidative and acid stress. A more thorough understanding of the influence of food processing-related stressors on Stx-prophage expression thus facilitates control of STEC in food systems by minimizing prophage induction during food production and storage.KEYWORDS single-cell detection, E. coli O104:H4, prophage induction, environmental stress, RecA-independent SOS response, membrane permeability, STEC, Shiga toxin, Shiga toxin-producing E. coli S higa toxin-producing Escherichia coli (STEC) causes the life-threatening hemolyticuremic syndrome (HUS), which is associated with acute renal failure and significant mortality (1, 2). Shiga toxins (Stx) include St...

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Prediction, Diversity, and Genomic Analysis of Temperate Phages Induced From Shiga Toxin-Producing Escherichia coli Strains

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