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Elucidation of Environmental Factors that Influence Attachment of Foodborne Pathogens to Biotic and Abiotic Surfaces

Bergholz, Teresa
North Dakota State University
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The goal of this project is to provide a comprehensive assessment of pre- and post-harvest environmental factors that influence expression of pathogen attachment mechanisms, and thereby attachment of pathogens to different surfaces. Attaining this goal is necessary for the development of targeted attachment inhibitors to complement existing pathogen control strategies.

Objective 1:Determine which environmental conditions influence attachment ofL. monocytogenes, Salmonella,and EHECto fresh produce and stainless steel

Objective 2: Determine how environmental factors influence expression of genes encoding proteins involved in attachment ofL. monocytogenes, Salmonella,and EHEC to surfaces.

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Assay conditions. In the pre- and post-harvest environment, any pathogens present on fresh produce will experience a variety of stresses related to nutrient levels, moisture, temperature and sanitizer exposure, all of which are known to impact subsequent survival and persistence in the food supply. Each stress condition will be generated in phosphate buffered saline (PBS) to increase consistency and reduce interference of the stress with components of a rich medium. These stresses include: nutrient limitation (PBS alone), low temperature (15°C, 10°C, and 5°C), sub-lethal level of chlorine (25 ppm), low pH (lactate at pH 5.5), sub-lethal level of sanitizer (18 ppm benzalkonium chloride), peroxide (5 mM H2O2), lettuce leaf exudates, and leafy green lysates (lettuce lysate and spinach lysate). The in vitro test environments will be incubated at 20°C, with the exception of the low temperature stress conditions.Attachment assays. Attachment assays will be conducted on two different biotic surfaces (baby romaine lettuce leaves and baby spinach leaves) and on an abiotic surface, stainless steel. Strains will be grown to stationary phase and washed cell pellets will be diluted to 104 CFU/mL in PBS, or PBS + stress condition, and placed into a 50mL tube. The surface to attach to will be added to the liquid (a lettuce or spinach leaf, or a stainless steel coupon), the tube sealed and placed on a rocking platform and incubated at 20°C for the length of time shown to induce or repress expression of attachment genes. Following incubation, leaves/coupons will be removed, and non-adherent cells will be removed by washing each surface three times in PBS. Attached cells will be removed by placing the washed surface into 10mL PBS with 6-mm glass beads and vortexing for 1 minute, then plating the cell suspension onto selective media using the Spiral Plater. Colonies will be enumerated with a Q-count following overnight incubation at 37°C. Attachment assays will be conducted in triplicate for each bacterial strain under each stress condition. Differences in attachment between stressed and non-stressed samples will be determined by ANOVA.Reporter fusion construction. Single copy chromosomal reporter fusions will be constructed in each strain to assay the promoter activity of selected genes involved in attachment of pathogens to biotic or abiotic surfaces. We will create reporter fusions for promoters of genes known to be involved in attachment (Table 1) and then monitor promoter activity under stress conditions representing those found in the pre- and post-harvest environment. To create the reporter fusions in L. monocytogenes, we will use pMJG2, a derivative of pPL2 with β-glucuronidase (GUS) as the reporter protein. This vector integrates into the tRNAArg - attBB' site on the chromosome, and is designed to be used in both serotype 1/2 and 4b strains. Integration into the chromosome alleviates the need for antibiotic selection to maintain the reporter fusion plasmid. For EHEC and Salmonella, we will utilize pMCI002 and RedET recombinationto generate single copy chromosomal β-galactosidase reporter fusions. As positive controls, reporter fusions will also be constructed from promoters of genes known to be induced under each environmental stress, such as SigB for L. monocytogenes and RpoS for EHEC and Salmonella. Reporter fusions will be constructed in multiple strains of the same serotype as differences in expression of attachment factors have been observed for strains of L. monocytogenes as well as EHEC O157:H7.Measuring promoter activity. Changes in expression of each attachment protein under environmental stress will be assessed by measuring either GUS (L. monocytogenes) or β-galactosidase (EHEC and Salmonella) activity of each reporter fusion strain. Prior to GUS measurements, cells will be lysed with CellLytic B reagent (Sigma-Aldrich). The enzymatic reaction will be initiated by addition of 0.4-mg/ml 4-methylumbelliferyl-β-D-glucuronide to the lysate. The reaction will be stopped after 10 min by addition of Na2CO3. Fluorescence will be measured at 460 nm using a Biotek plate reader. A standard curve of at least 5 different concentrations of 4-methylumbelliferone (MU) will be included with every plate, and the concentration of liberated MU in each sample will be calculated using the standard curve. The GUS activity for each sample will be reported as nM MU/log CFU/min. To measure β-galactosidase activity, the OD600 of each culture will be determined prior to lysis with toluene. The colorimetric substrate ortho-nitrophenyl-β-D-galactoside (ONPG) is then added to a final concentration of 1mg/ml. After development, the reaction is terminated by the addition of Na2CO3 and cell debris is removed by centrifugation. The absorbance at 420 nm (measuring the cleavage product o-nitrophenol) is used to calculate Miller units (1,000 x A420)/(reaction time in minutes x OD600). At least three independent replicates of each reporter fusion strain will be assayed, in duplicate (technical replicates), for each stress condition. Activation of a specific attachment gene by a particular stress will be determined based on a significant difference in nM MU/log CFU/min or in Miller units compared to the control condition (without environmental stress) using ANOVA. A significant difference in reporter fusion activity between the control and at least one point in time during exposure to the stress will indicate modulation of expression of the attachment gene by the stress.

Funding Source
Nat'l. Inst. of Food and Agriculture
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Bacterial Pathogens
Food Defense and Integrity