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<p>To understand bacterial attachment and internalization, we propose a three-pronged, interdisciplinary, mechanistic approach involving engineering modeling, microfluidics and microbiology validation to study active and passive attachment and internalization at produce surfaces during exposure to contaminated water during irrigation/washing as well as passive infiltration into produce from hydro- and vacuum cooling. Applications include leafy greens and whole fruit. Our experimental approach applies microfluidics to capture processes at the bacterial scale along with microbiological and physical experiments at the real produce scale. Computational simulations include complementary approaches at various scales--particle tracking in fluid flow, microbial ecology using individual-based diffusion-reaction models, and porous media transport models at the produce scale. Synergy arising between experimentation and modeling should yield unprecedented understanding of attachment and internalization.Our quantitative understanding based on first principles complements rather than replaces current biological and experimental understanding, clarifying what happens between exposure and contamination, thereby reducing experimentation, and improving predictability.</p><p>Objectives We plan to elucidate the physical mechanisms of attachment and internalization by iteratively developing synergy between:</p><p> a) physics-based mathematical models,</p><p> b) microbiological experiments in microfluidics, and </p><p>c) microbiological experiments on real produce.</p><p> We repeat these three approaches or parts of them in three general categories:</p><p>1. Mechanisms of passive attachment or internalization at produce surface from contaminated wash water</p><p>2. Mechanisms of active attachment and internalization at fresh produce</p><p>3. Mechanisms of passive internalization in intact produce</p>
<p>We will reveal the mechanism of attachment and internalization of microorganisms into fresh produce using</p><p> i) mathematical modeling to highlight significant parameters governing these phenomena,</p><p> ii) experimental microfluidics to mimic plant tissues and verify the models under controlled conditions, and</p><p> iii) microbiological analyses for validating the proposed mechanisms. Here is a summary of our approaches:Mechanisms of passive attachment or internalization at produce surface from contaminated wash water:Develop a fluid flow-based particle-tracking model with varying fluid velocities, pore size, bacterial size and surface charge. Develop a microfluidic device with varying fluid velocities and surface conditions to observe attachment as a function of flow velocities/bacterial and solution concentrations. Obtain experimental data on attachment of bacteria over actual produce (spinach leaves) and compare against microfluidic and model results.Mechanisms of active attachment and internalization at produce surface:Develop an individual-based model (IbM) to predict the behavior of attached bacteria related to growth and biofilm formation, death, and taxis to specific locations. Develop a microfluidic device to observe the effects of chemical gradients that drive chemotaxis, growth, and biofilm formation and how temperature affects them. Obtain experimental data on internalization of bacteria over actual produce (spinach leaves) and compare against microfluidic and model results.Mechanisms of passive internalization in intact produceDevelop a porous media-based model to predict water and bacterial infiltration from hydrocooling of tomatoes and vacuum cooling of spinach. Obtain experimental data on internalization of bacteria in produce (tomatoes in hydro- cooling and spinach in vacuum cooling) and compare against model predictions.</p>