Location: Food Systems Research Unit
Project Number: 8090-10700-001-009-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Aug 3, 2026
End Date: Aug 2, 2028
Objective:
This project will focus on enhancing microbial food safety of grass- or grain-finished beef-on-dairy calves and cattle. Beef cattle operations harbor a wide variety of microorganisms, including foodborne pathogens, that persist and interact in multispecies biofilms. The environmental microbial communities could affect pathogen survival via mixed biofilm formation and provide an ecological niche for pathogens to better colonize and gain tolerance against sanitization. Understanding the diverse microbial community in biofilms and their interactions with biotic and abiotic factors is a critical step in the development of microbial interventions to combat foodborne pathogens in the pre-harvest environment. To address this problem, this project has two objectives.
1. Characterize bacterial stress responses and multispecies biofilm ecology in beef-on-dairy farm environments.
2. Identify and evaluate sustainable preharvest interventions to disrupt biofilms and reduce zoonotic foodborne pathogen occurrence in beef on dairy production environments.
Objective 1 will define how environmental niches in beef-on-dairy systems, including pen flooring, soil, drains, water trough sediments, feces, feed troughs, and cattle contact surfaces, shape microbial community composition, biofilm formation, and pathogen persistence. Emphasis will be placed on multispecies environmental biofilms that confer sanitizer tolerance, protect foodborne pathogens, and facilitate the persistence and dissemination of Shiga toxin producing Escherichia coli (STEC), Salmonella, and other zoonotic bacteria. The work will further characterize stress response traits, antimicrobial resistance determinants, and biofilm associated survival mechanisms that may allow these pathogens to persist on or near cattle before entry into the food chain.
Objective 2 will assess practical, sustainable strategies to disrupt environmental biofilms and reduce pathogen loads on cattle entering the food chain in the New England and New York beef-on-dairy systems. Candidate interventions will include conventional sanitizers, plant-based antimicrobials, organic acids, and biological controls, including beneficial bacteria, bacteriophages, phage-derived enzymes, and biofilm degrading enzymes. Each intervention will be evaluated for efficacy against multispecies biofilms and associated foodborne pathogens, feasibility for implementation on small and midsized farms, compatibility with resource conserving production practices, and potential contribution to improved preharvest food safety and system sustainability.
Approach:
To pursue this research, the USDA, ARS, Food Systems Research Unit (Agency) and the Cooperator will take a five-part approach.
1. Environmental sampling across farm niches: The Agency will collect samples monthly from at least two Vermont beef-on-dairy farms during spring, summer, and fall, targeting niches linked to bacterial persistence and pathogen exposure: cattle drinking water, lagoon water, sediments, pen-floor and floor-drain swabs, fecal slurry, and surface swabs (water containers, feed bins). Temperature will be recorded at collection; samples transported on ice and held at 4°C. Each sample will be aseptically split, with one portion retained at the Agency for enumeration, pathogen screening, and downstream analyses and the matched portion shipped under cold chain to the Cooperator for biofilm, sequencing, microscopy, and synthetic-consortium work.
2. Enumeration and pathogen detection: The Agency will enumerate total mesophiles, Enterobacteriaceae, coliforms, and E. coli, and screen for STEC and Salmonella using established culture, biochemical, serological, and molecular methods, defining pathogen occurrence and high-relevance environmental reservoirs.
3. Biofilm and pathogen-protection assays: The Agency will assess biofilm-forming potential by crystal violet assay. The Cooperator will (i) test whether mixed-species biofilms from farm samples protect STEC and Salmonella under environmental stress and sanitizer exposure; (ii) quantify pathogen persistence by selective culture and shotgun metagenomic sequencing, characterizing community composition, species richness, core microbiome, resistance determinants, and functional genes; and (iii) isolate biofilm-associated species into defined synthetic communities to probe structure- and species-specific protection mechanisms.
4. Microscopy-based biofilm structure analysis: The Cooperator will use scanning electron and three-dimensional confocal microscopy to visualize biofilm architecture and locate STEC and Salmonella within protected structures, surface matrices, or multispecies aggregates, supporting the quantitative and sequencing findings.
5. In vitro evaluation of mitigation strategies: The Agency and Cooperator, both, will evaluate candidate treatments in parallel under farm-relevant in vitro conditions to test consistency across labs. Treatments will include chlorine, peracetic acid, plant-derived antimicrobials, organic acids, and biological controls (lactic acid bacteria, bacteriophages, phage depolymerases, biofilm-degrading enzymes), applied to farm-derived consortia and pathogen-containing biofilms on coupons (stainless steel, HDPE, PVC, galvanized steel, concrete). Effects will be measured by viability assays, selective culture, and confocal microscopy. Pathogens will be expressed as log10 CFU/mL or cm² and phage as PFU/mL or cm²; data analyzed by ANOVA and generalized/general linear mixed models at P<0.05 to identify the strongest candidates.