Abstract
In the food industry, the increasing antimicrobial resistance of food-borne pathogens to conventional sanitizers poses the risk of food contamination and a decrease in product quality and safety. Therefore, we explored alternative antimicrobials N-Acetyl-l-cysteine (NAC), rhamnolipids (RLs), and usnic acid (UA) as a novel approach to prevent biofilm formation and reduce existing biofilms formed by important food-borne pathogens (three strains of Salmonella enterica and two strains of Escherichia coli, Listeria monocytogenes, Staphylococcus aureus). Their effectiveness was evaluated by determining minimum inhibitory concentrations needed for inhibition of bacterial growth, biofilm formation, metabolic activity, and biofilm reduction. Transmission electron microscopy and confocal scanning laser microscopy followed by image analysis were used to visualize and quantify the impact of tested substances on both planktonic and biofilm-associated cells. The in vitro cytotoxicity of the substances was determined as a half-maximal inhibitory concentration in five different cell lines. The results indicate relatively low cytotoxic effects of NAC in comparison to RLs and UA. In addition, NAC inhibited bacterial growth for all strains, while RLs showed overall lower inhibition and UA inhibited only the growth of Gram-positive bacteria. Even though tested substances did not remove the biofilms, NAC represents a promising tool in biofilm prevention.
Highlights
Food-borne pathogens are responsible for infections and intoxications with significant effects on human health and adverse economic impacts for the food industry worldwide [1,2,3].Contamination may occur at any stage during food processing as a result of insufficient cooking during food preparation, improper food storage, unhygienic food handling, inadequate refrigeration, or cross-contamination from surfaces in direct contact with food or directly from infected people [1,4]
Clusters of microorganisms that usually stick to surfaces, are dynamic, highly organized communities of organisms embedded in extracellular polymeric substances (EPS), such as exopolysaccharides, extracellular DNA, proteins, and lipids, and are highly resistant to conventional eradication methods [8,9,10,11]
MIC was defined as the lowest substance concentration able to inhibit at least 80% of microbial growth (MICPC80 for planktonic cells, MICBC80 for further growth of biofilm cells), inhibit 80% of metabolic activity (MICBM80 for biofilm metabolic activity, MICMPB80 for metabolic activity of preformed biofilm), prevent biofilm formation by at least 80% (MICBF80 for biofilm formation), or reduce a preformed biofilm by at least 80%
Summary
Food-borne pathogens are responsible for infections and intoxications with significant effects on human health and adverse economic impacts for the food industry worldwide [1,2,3].Contamination may occur at any stage during food processing as a result of insufficient cooking during food preparation, improper food storage, unhygienic food handling, inadequate refrigeration, or cross-contamination from surfaces in direct contact with food or directly from infected people [1,4]. Due to slow penetration through the biofilm, cells attached in the deeper layers have a longer time to adapt to adverse conditions Their physical proximity and the presence of eDNA result in horizontal gene transfer and further spread of antimicrobial resistance [12,13,14]. One possible strategy is to employ natural substances, such as N-Acetyl- L -cysteine (NAC), rhamnolipids (RLs; R90), and (+)-usnic acid (UA), all of which previously showed promising antimicrobial efficacy against important clinical pathogens [15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33] Other benefits of these substances are their solubility, affordability, and chemical stability. Little is known about their cytotoxicity and antimicrobial efficacy against food-borne pathogens frequently occurring in the food industry
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