Broad and efficient control of major foodborne pathogenic strains of Escherichia coli by mixtures of plant-produced colicins

Significance Enterohemorrhagic Escherichia coli-contaminated food products are among the leading causes of bacterial enteric infections in the United States and worldwide. Currently, other than thermal inactivation, there are no effective methods to control pathogenic bacteria in food. We investigated colicins, nonantibiotic antimicrobial proteins produced by certain E. coli strains and active against other strains of the species, as potential pathogen control agents. We demonstrate that most colicins can be expressed at high yields in plants and are fully functional. We show that mixtures of colicins applied at low concentrations are highly and broadly active against all major pathogenic E. coli strains of concern for foodborne illness. We propose plant-produced colicins as an inexpensive food treatment for the broad control of pathogenic E. coli strains. Enterohemorrhagic Escherichia coli (EHEC) is one of the leading causes of bacterial enteric infections worldwide, causing ∼100,000 illnesses, 3,000 hospitalizations, and 90 deaths annually in the United States alone. These illnesses have been linked to consumption of contaminated animal products and vegetables. Currently, other than thermal inactivation, there are no effective methods to eliminate pathogenic bacteria in food. Colicins are nonantibiotic antimicrobial proteins, produced by E. coli strains that kill or inhibit the growth of other E. coli strains. Several colicins are highly effective against key EHEC strains. Here we demonstrate very high levels of colicin expression (up to 3 g/kg of fresh biomass) in tobacco and edible plants (spinach and leafy beets) at costs that will allow commercialization. Among the colicins examined, plant-expressed colicin M had the broadest antimicrobial activity against EHEC and complemented the potency of other colicins. A mixture of colicin M and colicin E7 showed very high activity against all major EHEC strains, as defined by the US Department of Agriculture/Food and Drug Administration. Treatments with low (less than 10 mg colicins per L) concentrations reduced the pathogenic bacterial load in broth culture by 2 to over 6 logs depending on the strain. In experiments using meats spiked with E. coli O157:H7, colicins efficiently reduced the population of the pathogen by at least 2 logs. Plant-produced colicins could be effectively used for the broad control of pathogenic E. coli in both plant- and animal-based food products and, in the United States, colicins could be approved using the generally recognized as safe (GRAS) regulatory approval pathway.