Elucidating the mechanisms and factors influencing biofilm formation in foods

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This submission requests continued support for the study of biofilm antimicrobial resistance. Bacterial biofilms develop on surfaces where they become more resistant to antimicrobial agents and other stressors than their counterpart bacteria grown in liquid suspension. Biofilms thus tend to survive antimicrobial treatment and sanitation regimes in food processing plants, where they rapidly regrow and contribute to downstream losses in food quality, reduced product shelf life, or food borne disease. The mechanisms responsible for biofilm survival following antimicrobial treatment are not entirely clear; consequently, effective biofilm control strategies remain a goal of social and economic importance. The core of my research program focuses on elucidating structural and metabolic response pathways of biofilms that contribute to enhanced resistance to antimicrobial agents such as benzalkonium chloride, chlorhexidine and triclosan, using wild-type and mutant strains of Salmonella enterica, Escherichia coli O157, and defined co-culture systems. It is hypothesized that prior stress events (growth at low temperature) and formation of protective microniches in co-culture biofilms, both induce stress response pathways that lead to enhanced resistance. Comparative studies of pre-stressed (adapted) and unadapted biofilms will be performed using confocal laser microscopy (CLM) and whole cell protein expression patterns to document effects. Fluorescent probes and CLM will provide in situ physical and metabolic information on biofilm cell condition in treated and untreated biofilms, whereas protein analyses will reveal how adaptation to antimicrobial agents, as well as location within the biofilm, affect biofilm metabolic processes. The overall effect of 'prior biofilm stress' will be evaluated by subjecting biofilms and resuspended biofilm bacteria to a panel of imposed stresses (e.g., antimicrobial agents and heat) and determining their respective survival. This research will also employ correlative microscopy approaches (synchrotron soft x-ray transmission microscopy, atomic force microscopy, confocal microscopy) to examine bacterial-antimicrobial interactions on surfaces, as well as the effects of food-grade nanomaterials on bacteria.