Source: CORNELL UNIVERSITY submitted to
PHYSICAL MECHANISMS OF WATER FILM-MEDIATED MICROBIAL PATHOGEN ATTACHMENT AND DETACHMENT ON FRESH VEGETABLE SURFACES
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
EXTENDED
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1005571
Grant No.
2015-67017-23075
Project No.
NYC-123563
Proposal No.
2014-05772
Multistate No.
(N/A)
Program Code
A1331
Project Start Date
Feb 1, 2015
Project End Date
Jan 31, 2019
Grant Year
2015
Project Director
Steenhuis, T.
Recipient Organization
CORNELL UNIVERSITY
(N/A)
ITHACA,NY 14853
Performing Department
Biological & Env. Engineering
Non Technical Summary
Pathogenic infections from eating contaminated fresh produce represent a growing portion of food-associated illness, with one in 15 Americans contracting illness from produce each year. While most pathogen transport is associated with water, the effect of water films on microbial pathogen attachment onto produce surfaces has surprisingly not received the attention it deserves. We envision that in most cases water films mediate initial pathogen interactions with the plant surface, and therefore should be considered as a risk reduction control point that potentially can be modified to reduce contamination potential. Drawing on our experience with micro-scale attachment processes in porous media, we will examine the mechanics of microbial association with lettuce leaves, spinach leaves and tomato skins. Subsequently, we will examine potential steps to modify conditions of the initial attachment process to weaken attachment strength and facilitate detachment with conventional washing practices.
Animal Health Component
0%
Research Effort Categories
Basic
50%
Applied
25%
Developmental
25%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
7235010202080%
1120199201020%
Knowledge Area
112 - Watershed Protection and Management; 723 - Hazards to Human Health and Safety;

Subject Of Investigation
0199 - Soil and land, general; 5010 - Food;

Field Of Science
2010 - Physics; 2020 - Engineering;
Goals / Objectives
Our overall goal is to examine the significance of water films and droplets on the initial retention and attachment of foodborne bacterial pathogens to the surfaces of fresh produce. Once this is 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. We posit the following hypotheses and objectives:Objective 1. Determine the role of water films in the retention and attachment of E. coli and Salmonella biocolloids. Under this objective, we will observe attachment extent and strength of pathogenic E. coli and Salmonella strains on lettuce, spinach and tomato surfaces under conditions that accompany water-mediated contamination events, particularly the role of sessile drop drying. Methods of inoculation via water will also emulate rainfall/irrigation impact (large droplets), fog aerosols (microdroplets) as well as immersion to link to published studies.Objective 2. Determine the effects of water film properties (ionic strength, surfactant presence) and intrinsic leaf surface properties on the extent and strength of enterobacterial attachment. This work will be primarily carried out under water impact and misting conditions encountered in the field. We will test the effects (singly and in combination) of expected primary control variables that affect the strength of water/surface interactions, including solution ionic strength, surface tension (due to surfactants), surface hydrophobicity and microstructures (a function of species/variety, leaf age, surface integrity, etc.). This will enable us to develop a general model for initial pathogen attachment.Objective 3. Test strategies to reduce the strength and extent of pathogen attachment by modifying solution characteristics (irrigation water) and surface properties (crop selection, preventive sprays) in order to enhance the safety of fresh produce. The point of initial attachment represents a potential critical control point where preventive action may substantially reduce the potential for crop contamination. In contrast, once pathogen colonization and especially internalization occurs, the options and prospects for effective decontamination rapidly dwindle.
Project Methods
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

Progress 02/01/16 to 01/31/17

Outputs
Target Audience:Our target audience is the produce industry. We have discussed our finding with colleagues. Changes/Problems:We started late and are behind on our time schedule. We will apply for a no cost extension to finish the experimentation and disseminate our results. What opportunities for training and professional development has the project provided?Two post docs were trained. Seminars were given to other undergraduate students. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?We have planned the following: Because of the limit of spatial resolution, the existence of meniscus on live bacteria was blurry with conventional confocal microscopy. We will try to apply either super resolution structured illumination microscopy (SIM) or AFM, which do not require harsh chemical treatment in sample prep, to elucidate the pinning process of E coli, the most common bacteria. This will enable us to acquire direct insight on the bacterial attachment to produce. A better understanding of the mechanism will enable us to find a solution to wash off attached bacteria in a practical manner. We will examine different surfactants for their ability to wash off the deposited material around the micro-spheres/bacteria. Once the material is removed the force required to remove the microsphere will be less. The results of the first two year will be published in refereed journals. Additional experiments will be performed to do so.

Impacts
What was accomplished under these goals? As the main source of food-borne contamination, the bacterial pathogen attachment to the fresh produce surface is poorly understood. In this project, we attempt to show that a "passive" physical force can pin bacterial pathogens to the surface of fresh produce during drying of a water film. In our hypothesis, capillary forces associated with the water meniscus can overcome the repulsive electrostatic DLVO force and push bacteria close enough to the produce surface so that the attractive van der Waals forces can bind the bacterial pathogen strongly to the produce. In the second year, we used several imaging techniques (laser scanning confocal, spinning disk confocal, and bright field microscopy) and atomic force microscope (AFM), to investigate the passive pinning process by the capillary forces along with the drying process for model systems of a hydrophobic slide representing the hydrophobic leaf surface and microsphere representing the pathogenic bacteria. We accomplished the following: Using the laser scanning microscope, we studied the colloid pinning in an evaporating droplet containing micro-spheres on a hydrophobic slide. Evaporation makes the droplet smaller. Both the wetter area on the slide and the contact angle of the meniscus with the slide decrease. Initially, colloids located near the intersection of the meniscus of the droplet and slide were pushed inward by the retracting meniscus. The colloids became pinned after the contact angle decreased sufficiently. We were able to show that when the colloids stopped moving, the distance between the colloid and the slide surface decreased. Consistent with our hypothesis, we confirmed that capillary force resulting from a deformation of the meniscus is the dominant force to pin the micro-spheres. The confocal images clearly showed meniscus was stretched over the colloid and was pushed outwards. The vertical component of the capillary force (normal to surface) increases with decreasing contact angle when the droplet evaporates, explaining that the pining occurred when the contact angle was sufficient small vertical force is above threshold to overcome the repulsive energy barrier. Our observations confirmed the theoretical calculations. Using the atomic force microscope (AFM), we measured the force required to detach colloidal particles from a hydrophobic surface after the water in droplet with suspended colloids had evaporated. The force exhorted by a sharpened tip of the AFM to a colloid pinned by a drying meniscus was many times greater than for then colloids placed on the surface without water The force required was independent of the surfactant concentration but dependent on the height of the deposited material around the colloids. More experiments are required to confirm this.

Publications


    Progress 02/01/15 to 01/31/16

    Outputs
    Target Audience: Nothing Reported Changes/Problems:Post doc were hired relatively late so we are behind with spending the funds and with the time table. No need to make changes for the next project period. We are following the research plan. What opportunities for training and professional development has the project provided?Two post-doctoral associated are performing the research How have the results been disseminated to communities of interest?We have been discussing our results with other professionals What do you plan to do during the next reporting period to accomplish the goals?In preliminary work, we have found that surfactants can alter the layout of beads on the substrate surface during drying process. We proposed that this may be the result of the change of both surface tension and contact angle associated with the fluid dynamic mass transport during the drying process. We further hypothesize that extracellular biomolecules may act similarly and minimize the repulsion between bacteria and target substrate, so that bacteria will initially be closer to the substrate and have a smaller water cushion between them and the surface. As with beads, capillary forces are expected to pin bacteria during the drying process and we will investigate how surface biomolecules affect the pinning process. We will use curli as a model surface biomolecule to characterize this process on the pinning of E. coli in upcoming experiments. We will also test the effects of dissolved solutes on both colloids and pathogen in upcoming studies. The upcoming experiments will provide additional insight that will enable us to qualitatively and quantitatively characterize this physical mechanism.

    Impacts
    What was accomplished under these goals? We are studying a "passive" mechanism through which bacterial pathogens may be pinned to the surface of fresh produce by capillary force during drying of the surrounding water. Capillary forces are strong enough to push bacterial close to the surface and overcome the repulsive electrical double layer force. According to the DLVO model, a strong attachment occurs between bacteria and the surface when bacteria is being pushed to the closest proximity (Morales, Parlange et al. 2013). We also explore the role of surfactants in weakening the effect of capillary pinning by altering the surface tension of the surrounding water. In addition, this capillary pinning process may play a dominant role in bacterial attachment to the surfaces. In order to characterize this physical mechanism, we designed a series of studies that include analysis of pinning using a simplified model system of inert colloids, and in vitro experiments with pathogenic bacteria. We apply Bright Field, Confocal, Spinning-disk and Atomic Force Microscopy to quantify the attachment force and possible factors that affect the attachment strength. We are making steady and consistent progress toward our research aims. As per this reporting period, 1. In order to mimic fresh produce surface, we have adapted a hydrophobic coating protocol for preparation of our glass microscope slides. Specifically, we have treated regular hydrophilic microscope slides with linear alkyl-silane to form chemically a stable hydrophobic coating, which has similar hydrophobicity to fresh produce surfaces. Imaging results indicate that carboxylate colloids form similar patterns on the coated glass slides, on acrylic surfaces, and on fresh produce, in solutions with similar surfactant concentrations. 2. We captured direct imaging evidence of bulging water films around colloids before and after they were pinned to the substrate. This enables us to calculate the actual contact angle, the capillary force, and the corresponding variation during this process. 3. We developed a new imaging analysis method to quantitatively analyze confocal 3D images. By analyzing fluorescence intensity variation in different channels, we were able to calculate the dimension of the water film on colloids as the capillary forces moved them on the slide. Our measured film thickness (0.2 µm) is in excellent agreement with theoretical predictions (Renard and Ortoleva 1997). 4. Furthermore, using the same analysis method, we were able to calculate the thickness of water cushion between beads and substrate. Experimental results quantitatively reveal the shrinking of the distance during the drying process. When water film disappeared caused by evaporation, all colloids are very close to the surface(<< 0.1µm). This result strongly supports the initial hypothesis that colloids/pathogen are being pressed by the capillary force through the surrounding water and thereby making strong and close associations with the surface. Morales, V. L., J. Y. Parlange, et al. (2013). "Surfactant-mediated control of colloid pattern assembly and attachment strength in evaporating droplets." Langmuir 29(6): 1831-1840. Renard, F. and P. Ortoleva (1997). "Water films at grain-grain contacts: Debye-Huckel, osmotic model of stress, salinity, and mineralogy dependence." Geochimica Et Cosmochimica Acta 61(10): 1963-1970.

    Publications