Salt and nitrite at concentrations relevant to meat processing affects Shiga toxin II production of Escherichia coli O157:H7

Escherichia coli (E. coli) O157:H7 is a major food safety threat. It has the ability to produce several virulence factors. Shiga toxins (Stxs) are the key virulence factors of E. coli O157:H7 that are responsible for hemorrhagic colitis and serious renal failure. Despite the extensive study of E. coli O157:H7 survival during meat processing, the production of Stxs by E. coli O157:H7during these processes has not been studied. This becomes a very important question since Stx2 is resistant to heat treatment. The objective of this study is to elucidate the effect of essential additives to meat products, salt and sodium nitrite, on Stx2 production by E. coli O157:H7. E. coli O157:H7 (EDL933 and 86-24) was treated with different concentrations of salt (1%, 2%, 3%, W/V) or sodium nitrite (0, 100, 200, and 300 ppm) solutions for 6 h. The Stx2 production and CFU were analyzed for both strains in both salt and sodium nitrite solutions. RecA expression, PFU, and Stx2 carrying bacteriophages were analyzed for strain EDL933 in salt solutions. Solutions supplemented with 2% and 3% salt decreased (P < 0.05) E. coli O157:H7 population, but supplementing with 2% salt increased (P < 0.05) Stx2 production per CFU compared to that of the control (1% salt), while including 3% salt decreased (P < 0.05) Stx2 production as analyzed through western blotting for both EDL933 and 86-24. Relative expression of Stx2 mRNA expression for strain 86-24 showed a tendency for increased ( P < 0.1) Stx2 expression in 2% salt while there was a tendency for less (P < 0.1) Stx2 expression in 3% salt expression compared with the control. Relative expression of Stx2 and RecA mRNA expression for strain EDL933 showed an increase (P < 0.05) in 2% and 3% solutions compared with the control. Strain EDL933 showed greatest bacteriophage activation in 2% salt (P < 0.05). 3% salt suppressed bacteriophage expression (P < 0.05). Bacteriophages were isolated and qRT-PCR was performed to measure the amount of Stx2A gene in the bacteriophages. Again, 2% salt showed a greater expression ( P < 0.05). When E. coli O157:H7 was grown in sodium nitrite solutions, there was no difference seen in growth for solutions containing 100, 200 and 300 ppm sodium nitrite compared with the control (0 ppm) for both strains. Stx2 concentration in solutions containing 100 and 200 ppm sodium nitrite did not differ from the control while 300 ppm sodium nitrite had significantly less (P < 0.05) Stx2 protein concentration for both strains. Since 2% salt concentration is commonly used in processed meats, the current study suggests that this concentration promotes Stx2 production. Therefore, not only the survival of E. coli O157:H7 during meat processing is important. Stx production by E. coli O157:H7 is another critical control point for producing safe processed meats, such as fermented sausage where E. coli O157:H7 could grow and accumulate Stx2 during fermentation.