Evaluating the antimicrobial mechanism of neutral electrochemically activated water on foodborne pathogens and their biofilms

Abstract

Sanitizing is a key step in ensuring food safety. Neutral electrochemically activated water (NECAW) not only has antimicrobial effects but also is relatively friendly to handlers and foods, and it complies with organic food practices. However, its antimicrobial effects on different pathogens and strains have not been examined and its antimicrobial mechanisms are not fully understood. The goal of this project was to investigate the use of NECAW as a sanitizer in inactivating foodborne pathogens. The first objective of this study was to determine the effectiveness and broadspectrum activity of NECAW against foodborne pathogens. The sanitizing efficacy of NECAW against 40 different strains of E. coli O157:H7, L. monocytogenes, and Salmonella as liquid cultures, dried cells on stainless steel (SS) surfaces, and biofilms on SS was examined. It was found that NECAW with 100 mg/l free available chlorine (FAC) caused more than 5 log CFU/ml reductions for all strains in liquid culture, more than 3 log CFU/coupon reductions for 92.5% of the strains dried on stainless steel (SS) surfaces and for 27.5% of biofilms. Among all the strains, S. Newport B4442CDC was the most resistant strain to NECAW on surfaces while E. coli O157:H7 ATCC 43895 was the most resistant strain in biofilms and liquid pure cultures. Overall, NECAW was effective and had a broad-spectrum activity against foodborne pathogens. The second objective was to evaluate the sanitizing effect of NECAW and other commercial ‘active water’ technologies on foodborne pathogens. Five individual strains of each foodborne pathogen including E. coli O157:H7, L. monocytogenes, and Salmonella as liquid cultures and dried cells on stainless steel surfaces were studied by iv comparing the sanitizing effect of NECAW and available commercial technologies on these pathogens. The results showed that 100 mg/l ECAW had sanitizing effects of at least 5 log CFU/ml reductions on liquid culture and more than 4 log CFU/coupon reductions for pathogens dried on SS surfaces. In contrast, the other commercial technologies tested were not effective in sanitizing. They, however, washed the bacteria off the surface into rinse water, which would lead to a significant safety concern of cross contamination. The third objective of this study was to examine the three dimensional morphology of foodborne pathogen biofilms using atomic force microscopy (AFM). AFM experiments were conducted by directly imaging the three-dimensional morphology of the foodborne pathogens biofilms (five strains for each pathogen), and imaging the biofilms pre- and post-NECAW treatment. Images of AFM showed tree-like structures as well as individual cells. L. monocytogenes biofilms had a higher percentage of tree-like structures than E. coli O157:H7 and Salmonella. The biofilm structures and microbial cells on SS were destroyed by the treatment with NECAW according to the AFM study, providing morphological evidence that NECAW was effective in controlling surface contamination of pathogenic bacteria and biofilm growth. The fourth objective of this study was to assess the role of sigB and inlA genes in L. monocytogenes biofilm formation and the antimicrobial efficacy of NECAW treatment on L. monocytogenes. The expression levels of sigB and inlA genes were investigated by using qPCR, and sanitizing effects of NECAW on wild-type (WT) and sigB/inlA mutant L. monocytogenes strains were determined by the direct plating method. After NECAW v treatment, expression of both genes increased for the WT. While sigB gene expression of the ΔinlA strain increased to a level comparable to that of the WT, inlA gene expression of the ΔsigB strain did not significantly increase. Both genes were expressed more in biofilms than in liquid cultures. The level of inlA gene expression in WT increased by 4.28 and 5.51-fold by treatment with 4 mg/l NECAW for 10 min in liquid cultures and biofilms, respectively, while the corresponding values were 5.91 and 10.05-fold for the sigB gene. Mutant strains were more sensitive to NECAW treatment than the WT strain. The sigB gene was more important than inlA for the pathogen’s survival under NECAW treatment. Surviving L. monocytogenes cells post-sublethal NECAW treatment might become resistant to further sanitizer treatment.