Physical Mechanisms Of Water Film-Mediated Microbial Pathogen Attachment And Detachment On Fresh Vegetable Surfaces

<p>Our overall experimental approach will be to examine the mechanics of initial retention and attachment of foodborne bacterial pathogens to the surfaces of fresh produce (vegetable leaves, fruit skins), particularly the role of water-mediated events. Once this is better understood, we can examine potential steps to modify conditions of the initial attachment process so that pathogens can be washed off more easily, thereby contributing to enhanced safety and quality of commercial fresh vegetables. This section will first describe materials and general experimental methods, and then will describe specific experiments by the objective they seek to help define.General Materials & MethodsVegetable substrates Yellow tomatoes (which enable better visualization than red) will be purchased for use within 1 week. Spinach and romaine lettuce grown hydroponically at a Cornell greenhouse for 2 weeks will be used. Immediately prior to experimentation, 0.5 x 1 cm sections of the leaves or tomato skin will be cut and mounted on microscope slides with double-sided tape. Leaf segments will also be used as templates for polymethyldisiloxane (PDMS) polymer (http://naldc.nal.usda.gov/download/8916/PDF) molds at the Cornell Nanobiotechnology center so that reproducible surrogates of leaf surfaces can be obtained and systematically modified with respect to surface hydrophobicity and topography. Streaming potentials of vegetable surfaces will be measured with a Brookhaven EKA Electro-kinetic analyzer (Holtsville, NY) so that zeta potential can be calculated.Microbes We will select two strains each of E. coli (non-shigatoxin variants of O157:H7 and O104:H4) as well as S. enterica (e.g. Saintpaul, Newport). These are readily available and all express (or will be modified to express) fluorescent protein labels for cell visualization in this study. Particle zeta potential will be measured with a Malvern Zetasizer Nano ZS (Worcesterhsire, UK).Model pathogen suspensions Proposed experiments will often be conducted first with pathogen surrogates (polystyrene microspheres) in order to optimize the protocols and understand the abiotic factors affecting the system. For these reasons, suspensions of synthetic red microspheres (5 μm diameter, surfactant free) of carboxyl modified polystyrene (Magsphere, Pasadena, California) have been selected as pathogen surrogates for their similarity in size and surface charge.Other solution components Stock solution of non-ionic and non-volatile surfactant (Surfynol® 485; Air Products, Allentown, PA) solutions will be prepared at concentrations of 0, 0.125, 0.250, and 5 µL/mL, to which colloids and/or microbes will be added as appropriate. Surface tension for the same concentrations has been measured with a semiautomatic tensiometer (Model 21 Tensiomat, Fisher ScientificPathogen attachment on vegetable surfaces will follow the drop evaporating method developed by Morales et al (2013)Surface attachment forces We propose to integrate two complimentary experimental methods to improve our understanding of bacterial adhesion in terms of kinetics and force. First, flow displacement experiments are proposed to obtain information about desorption kinetics under flow (mimicking vegetable washing processes). Second, to directly quantify changes in the strength of attachment between various colloid/pathogen and surface combinations, samples will be examined with atomic force microscopy (AFM).Visual data collection A digital bright field microscope (Hirox, model KH-7700 equipped with lenses capable of magnification up to 2500x) and a macro lens camera (d-VID plugged to a computer interface) will be used to concurrently record changes in diameter and contact angle of water drops (colloidal suspension) on the vegetable surfaces (Morales et al., 2013).Image processing to quantify the detachment of cells from flow displacement experiments includes segmentation and thresholding to permit quantification of the number of colloids or cells on the vegetable surface before and after going through the washing process, thus, quantitatively evaluating the effectiveness of the washing treatment and/or the effectiveness of surface retention. Having defined our standard procedures, we next describe the treatments and protocols relevant to each set of experiments.Objective 1 Experiments. Determine the role of water films in the retention and attachment of E. coli and Salmonella biocolloids Using a primary treatment array of three vegetables (spinach leaves, romaine lettuce leaves, and tomato skin) and four pathogen candidates (two each of E. coli and S. enterica) in addition to synthetic microsphere pathogen surrogates, we will examine and compare the extent and strength of pathogen retention and attachment in water-mediated attachment events. In addition, we will also examine the effects of two additional variables that may affect the water film dynamics, namely application mode and drying conditions.Objective 2 Experiments. Determine the effects of droplet solution composition properties (ionic strength, surfactant presence) and intrinsic leaf surface properties on the extent and strength of enterobacterial attachment. These experiments will examine the effects of varied suspension liquid properties - specifically a realistic range of ionic strengths and the presence of surfactants - known from our prior work to alter water film/colloid/surface mechanics, as well as the effects of several leaf properties including leaf age and the presence of epidermal damage. For simplicity, we will select a subset of experimental conditions related to application method and drying conditions, most likely the baseline application and drying methods, and run these experiments in Year 2.We strongly suspect (as per Morales et al. 2013) that the effect of higher surfactant concentration leading to easier detachment is due to the formation of hemi-micelles on the surfaces of both colloid and substrate. Using a scanning electron microscope (Leica 440 SEM) we will be able to test this. If capillary forces and friction are used to explain observed patterns for the above listed effects, we need to know the surface roughness before and after deposition of surfactant or other suspension components.Objective 3 Experiments. Test strategies to reduce the strength and extent of pathogen attachment by modifying solution characteristics (irrigation water or wash water) and surface properties (crop selection, preventive sprays) in order to enhance the safety of fresh produce.The goal of this objective is to apply results from prior experiments in order to test and optimize various mitigation approaches to reduce surface pathogen contamination that may occur prior to, during, and after the processing of fresh produce. We will use those pathogens identified in Objective 1 testing as possessing the strongest attachment traits, and will again use the same array of plant tissue as used in the prior objectives. Given the applied nature of this objective's testing, we will use current industry-standard washing and/or sanitization conditions as starting points, and alter conditions iteratively to attempt to improve observed impacts on pathogen exclusion or removal.ReferencesMarcotte, L.; Tabrizian, M. 2008. Sensing surfaces: Challenges in studying the cell adhesion process and the cell adhesion forces on biomaterials. IRBM 29:2-3, 77-88.Morales, V.L., Parlange, J.-Y., Wu, M., Pérez-Reche, F.J., Zhang, W., Sang, W., Steenhuis, T.S. 2013. Surfactant-mediated control of colloid pattern assembly and attachment strength in evaporating droplets. Langmuir DOI: 10.1021/la304685b</p>