SBIR-STTR Award

One of the most severe foodborne outbreaks in Europe, and the second-largest “Shiga toxin-producing” E. coli outbreak worldwide, was traced to E. coli O104: H4 in fresh fenugreek sprouts from a farm in Germany. It affected more than 4,075 individuals in 16 countries and included 908 cases complicated by hemolytic uremic syndrome (HUS) and 50 deaths. In the U.S., sprouts were implicated as the cause of 58 foodborne outbreaks between 1996 and 2017, encompassing at least 1,953 illnesses, 212 hospitalizations, and 5 deaths. Raw and lightly- cooked edible sprouts pose high risk for outbreaks of foodborne illness which typically originate in the seeds (vs. processing condition genesis of typical plant foodborne illnesses). While seeds contaminated with small amounts of human enteric pathogens, such as Salmonella enterica, Escherichia coli and Listeria monocytogenes have little to no impact on final non-sprout field grown crops, the distinct warm, humid growing conditions of sprouts serve to dramatically amplify growth of these pathogens to dangerous levels.2-5 As the consumption of edible sprouts, including alfalfa, mung bean and clover continues to increase worldwide, largely due to their short growing period, easy availability and high nutritive value,5 so too does the risk of sprout?related foodborne illness outbreak. The FDA has expressed serious concern over the increasing number of outbreaks. While the FDA recommends soaking the seeds in 20,000 ppm solution of calcium hypochlorite, as well as applying a least one approved antimicrobial treatment immediately before sprouting,6 high concentrations of antimicrobials fail to completely eliminate bacteria from seed, particularly those that have infiltrated seed tissue. According to the agency, “There is no single treatment so far that has been shown to completely eliminate pathogens on seeds or sprouts that cause foodborne illness without affecting germination or yield.” To prevent outbreaks of foodborne pathogenic bacteria in edible sprouts, Ascribe Bioscience is developing novel resistance-conferring seed treatments based on a natural, microbiome-derived molecule that has been shown to activate a plant's natural defenses to provide protection against a broad range of pathogens in a variety of crops, both pre- and post-emergence. To establish the feasibility of the proposed approach as a means to effectively confer protection to sprouts against human enteric pathogens, Phase I development will target four specific aims: 1) Develop an efficacious formulation for sprout seed treatment based on the microbiome-derived molecule, 2) Test the efficacy of the formulation against human enteric pathogens, 3) Test the efficacy of the seed treatment against human enteric pathogens during seed production and after harvest, 4) Test the efficacy of additional related molecules on growth inhibition of human enteric pathogens. In Phase II, we will finalize the sprout seed treatment formulation and conduct broad spread challenges on other types of spouts. Once fully developed, this intervention has the potential to reduce sprout- related infections and outbreaks.

Public Health Relevance Statement:
Narrative Raw and lightly-cooked edible sprouts pose high risk for outbreaks of foodborne illness that typically originate in the seeds, due to trace amounts of human enteric pathogens which are then amplified under warm, humid sprouting conditions to high microbial loads that are not easily addressed by application of antimicrobials. Ascribe Bioscience is developing a novel sprout seed treatment based on a natural molecule that activates plant defenses, potentially offering reliable protection against a broad range of bacterial pathogens regardless of seed type or degree of microbial sorption. Successful development of this innovative approach has the potential to dramatically improve the microbial safety of edible sprouts and reduce incidence of sprout-related foodborne outbreak, by uniquely conferring protection that begins at the seed stage and persists through growth, harvest, and up to the point of consumption.

Project Terms:
Address; Affect; Alfalfa; antimicrobial; Appearance; Bacteria; Bacterial Counts; base; Biological Sciences; Calcium; Cessation of life; Clover; commercialization; Consumption; cooking; Country; Dangerousness; defense response; Development; Disease; Disease Outbreaks; Disease Resistance; efficacy testing; enteric pathogen; Enterobacteriaceae; Escherichia coli; Europe; Face; Farming environment; Food Safety; foodborne illness; foodborne outbreak; Formulation; Germany; Germination; Goals; Growth; Harvest; hazard; Hemolytic-Uremic Syndrome; high risk; Hospitalization; Human; human pathogen; Hypochlorite; improved; Incidence; Individual; Industry; Infection; innovation; Intervention; Listeria monocytogenes; microbial; microbiome; mung bean; nanomolar; novel; Nutritive Value; Onions; Pantoea; pathogen; pathogenic bacteria; Pathogenicity; Phase; Plants; prevent; Production; Pseudomonas syringae; Recommendation; Resistance; Rice; Risk; Safety; Sales; Salmonella enterica; Seeds; Shiga Toxin; Smell Perception; Soil; Source; Soybeans; Taste Perception; Technology; Time; Tissues; Trigonella foenum-graecum; Xanthomonas

Ascribe Bioscience is developing a novel technology to prevent foodborne outbreaks caused by the consumption of edible sprouts. Sprouts are highly vulnerable to infection with human enteric bacterialpathogens at the seed stage. These pathogens then grow exponentially in the warm and humid conditions usedfor sprouting. Even low initial bacterial counts on seeds can result in unsafe microbial loads by the end of sproutproduction and processing. Moreover, the internalization of contaminating bacteria into seed tissuescontaminates the sprout tissues from inside, rendering the application of antimicrobials largely ineffective.Sprouts have emerged as a significant source of foodborne illness and have become a public health hazard.Between 1996 and 2017, the U.S. experienced 58 foodborne illness outbreaks associated with sprouts,encompassing at least 1,953 illnesses, 212 hospitalizations, and 5 deaths. According to the FDA, no singletreatment so far has been shown to eliminate pathogens on seeds or sprouts that cause foodborneillness. Ascribe Bioscience will use a natural, microbiome-derived molecule that activates and/or prime plantdefenses at the seed stage, thereby conferring to sprouts an enhanced resistance against contamination bydisease-causing bacterial pathogens. Ascribe's proposed treatment technology has the potential todramatically improve the safety of edible sprouts by addressing both external and internal bacterialcontamination. In Phase I, Ascribe has established the feasibility of the technology for the prevention of outbreaksrelated to human consumption of sprouts by demonstrating that treatment with our molecule can preventSalmonella growth during the sprouting process in alfalfa. In Phase II, we will finalize the sprout seed treatmentformulation and expand testing to a broader spectrum of sprout-pathogen systems. To supportcommercialization, we will perform toxicity and residue testing for regulatory approval, and refine synthesismethodologies to enable large-scale production of the active ingredient. The specific aims of this Phase II projectare 1) Refine treatment formulation, including optimized formulation development, determination of the minimumeffective dose and treatment duration, comparison of the effectiveness of the formulation versus the sproutindustry-standard treatments, and measuring the effects of the formulation on germination, growth and sproutquality; 2) Test the efficacy of treatments against other illness-causing pathogens on a variety of sprout types,and evaluate it's role in altering bacterial abilities to form biofilm; 3) Demonstrate safety via toxicology studiesand residue testing to support EPA approval; 4) Develop methods to scale the synthesis of active ingredient topilot scale, and 5) Investigate the ability of the molecule to provide post-harvest protection to leafy greens. Theproposed research is expected to yield commercial products that will prevent or dramatically reduce theincidence of sprout-related foodborne outbreaks.

Public Health Relevance Statement:
Narrative Edible sprouts pose a high risk for outbreaks of foodborne illnesses that typically originate in the seeds due to trace amounts of human enteric pathogens, which thrive under the warm, humid conditions needed for sprout production. Ascribe Bioscience is developing a novel sprout treatment product based on a natural molecule that activates plant defenses, offering reliable long-term protection against a broad range of bacterial pathogens. Successful development of this innovation has the potential to dramatically improve the safety of edible sprouts and reduce the incidence of sprout-related foodborne illness outbreaks, by uniquely conferring protection that begins at the seed stage and persists through growth, harvest, and up to the point of consumption.

Project Terms: