Assessment of sanitation efficacy against Escherichia coli O157:H7 by rapid measurement of intracellular oxidative stress,membrane damage or glucose active uptake

To reduce cross contamination with foodborne pathogens during washing procedures, sodium hypochlorite (NaOCl) and hydrogen peroxide (H₂O₂) are widely used in the fresh produce industry. To ensure food safety and minimize excessive use of sanitizers and water during sanitation, it is critical to develop a rapid method to assess sanitation efficacy. This study examines the potential for employing oxidative stress, membrane damage and glucose uptake measurements to assess the antimicrobial efficacy of NaOCl (0–70.4 ppm) and H₂O₂ (0–1.6% v/v) toward Escherichia coli O157:H7. The relative amount of intracellular reactive oxygen species (ROS) was measured using Aminophenyl fluorescein (APF) and 2′,7′- dichlorodihydrofluorescein diacetate (H₂DCFDA) for cells treated with NaOCl and H₂O₂ respectively. Results from these ROS sensitive probes revealed a limited correlation between these oxidative stress measurements and inactivation of bacteria measured using the plate counting method. Sanitation experiments, conducted with contaminated pre-cut lettuce leaves in water, were also carried out. Measurement of the bacterial membrane integrity was assessed using the membrane permeable probe propidium iodide (PI) and by evaluating effects on active transport by the glucose transport system using 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose (2-NBDG). The results demonstrate that the relative increase in membrane permeability using PI correlated with NaOCl, but did not correlate with H₂O₂ induced reduction of bacterial survival quantified by the plate counting method. In contrast to the other approaches, monitoring the loss of glucose transport system function by measuring the uptake of 2-NBDG displayed strong correlation with the reduction of bacterial survival for both NaOCl and H₂O₂ treatment. Overall, the study demonstrated potential of glucose uptake measurements with 2-NBDG to serve as surrogate method for the traditional plate counting, which is the current gold standard used by the Food and Drugs Administration, to provide a faster analysis of sanitation processes.