Differential Expression of E. coli O157:H7 Virulence Genes in Model Ready-to-Eat Produce Microenvironment during Temperature Drop and Refrigeration

Introduction: The contamination and persistence of pathogens in ready-to-eat produce have emerged as a significant food safety and public health concern.  Viable produce-borne pathogens cope with several stresses during production and storage of the commodities.  During such stressful exposures (such as temperature fluctuations, low-temperature storage), pathogens must change physiologically to adapt to the new environment. Understanding the implication of this stress-induced adaptation on pathogenicity of the organisms is important.

Purpose: This study aims to evaluate the differential virulence gene expressions of E. coli O157:H7 at 25°C and 4°C by real-time PCR and in vitro bioassay in bacteriological medium and simulated produce microenvironment.

Methods: Rifampin-resistant E. coli O157:H7, strain EDL933 grown at 37°C was exposed to 25°C for 1 h, 4°C for 1 h, and 4°C for 10 min in lettuce leaf lysates or Luria-Bertani medium. QRT PCR utilizing comparative critical threshold method was performed to evaluate the expression of selected genes including virulence, heat- and cold-shock protein, etc., with reference to selected housekeeping genes. Adhesion of treated bacterial cells to mammalian cells (MAC-T) was quantified by plating-based method and Giemsa staining.

Results: Bacterial cold-shock protein (cspA) gene was found to be up-regulated at both the temperatures (25°C and 4°C) as compared to expression at 37°C (P < 0.05).  The results also revealed that two key virulence genes stx1A and stx1B were up-regulated significantly (P < 0.05) upon cold-shock treatments; but another virulence gene eae was down-regulated.  MAC-T cell adhesion assay revealed a temperature-dependent reduction in attachment of cold shocked E. coli cells. 

Significance: Cold-shock resulted in the reduction in attachment of E. coli to epithelial cells while promoting higher levels of Shiga Toxin gene expressions at molecular level. The present study reveals the role of cold-shock (refrigeration) on the potential severity of sublethally injured E. coli O157:H7 infections associated with ready-to-eat produce.