Control of Salmonella in mung bean sprouts by antagonistic spore-forming Bacilli

Abstract

Sprouting seeds are considered part of a healthy and nutritious diet, and accordingly, their consumption increases globally. However, edible sprouts are occasionally linked to outbreaks of foodborne diseases, with Salmonella enterica being a frequent contaminant. Biocontrol was recently proposed as a possible approach to combat sprout contamination during production. The purpose of this study was to identify sprout-derived Salmonella-antagonists as a basis for the development of new biocontrol agents. To this end, spore-forming bacteria were isolated from sprouted mung bean seeds, based on either in vitro anti-Salmonella activity or rough colony morphology and tested in planta for inhibition of mung bean sprout colonization. Artificially contaminated seeds (2 log CFU/g) treated with selected Bacillus strains (7 log CFU/ml) demonstrated extensive inhibition of Salmonella growth after 4-days of sprouting, resulting in 3.3–5.4 log CFU/g reduction compared to control (untreated). The potent antagonistic activity of the selected Bacillus isolates was confirmed for different S. enterica serovars. Interestingly, some of the antagonistic strains that demonstrated activity in vitro were not active in planta and vice versa, indicating that in vitro testing may not be the best strategy for screening potential biocontrol strains in sprouts. Confocal microscopy imaging demonstrated that in the absence of Bacillus, Salmonella readily proliferates and colonizes the entire sprouting seed. Treatment with an antagonistic Bacillus isolate markedly inhibited the colonization of Salmonella. Scanning electron microscopy of Bacillus-treated sprouts demonstrated the colonization of the sprout with biofilm-like structures, indicating the activation of the biofilm formation mechanism during sprout colonization. The identified Bacillus isolates provide a basis for the future development of novel biocontrol agents against Salmonella and perhaps other foodborne pathogens in edible sprouts.