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There are three Specific Aims to this project: <br/>1. Composition and chemical structure of L. monocytogenes EPS. Many types of beneficial and pathogenic bacteria adhere to the surfaces of plants via exopolysaccharides (EPS). Currently the structure and function of EPS in L. monocytogenes is unknown. Our labs have identified L. monocytogenes genes involved in the synthesis of EPS and have developed mutant strains that either lack or over-express the polymer. Under this Aim we will isolate L. monocytogenes EPS from the over-expression strain, and determine its composition and structure. These studies will for the first time reveal the composition and structure of EPS synthesized by L. monocytogenes. <P>2. Function of EPS in the attachment and formation of L. monocytogenes biofilms on produce. Under this Aim, we will investigate how listerial EPS is involved in attachment of L. monocytogenes to cantaloupe and other produce, such as lettuce and red peppers, which have been identified as sources of L. monocytogenes contamination in previous listeriosis outbreaks. Experiments will be performed with EPS-negative, wild type, and EPS-over-producing strains, as well as with strains isolated from contaminated produce. EPS production will be quantified in these strains, and we will determine how attachment is correlated with EPS level. The studies will provide a thorough evaluation of the role of EPS in L. monocytogenes attachment to produce. <P>3. Function of L. monocytogenes EPS in resistance to environmental and food preservation stressors. In these experiments we will explore the function of EPS in the resistance of L. monocytogenes to environmental stress agents, including disinfectants and preservatives used in food decontamination and preservation. <P>Experiments will be performed with produce-grown biofilms using our set of laboratory strains and selected environmental isolates that synthesize low or high levels of EPS. Several chemical and physical stressors will be tested in these experiments, including desiccation and bleach. These experiments will show for the first time the relationship between EPS production and resistance of the bacterium to control agents.
Non-Technical Summary:<br/>
Listeria monocytogenes is a pervasive food-borne pathogen that causes hundreds of cases of the severe disease, listeriosis, in the USA every year. Although other food-borne bacteria such as Salmonella and E. coli cause more illnesses, L. monocytogenes is the deadliest of the common food-borne bacteria, having a mortality rate of approximately 20%. Infants, the elderly, immunocompromised individuals such as cancer patients, and pregnant women have the greatest risk of being infected. Traditionally, listeriosis cases have been associated with the consumption of contaminated meats, cheeses, smoked fish, and raw milk. However, over the last two decades the number of diseases caused by the consumption of contaminated fresh produce has significantly increased. Last year's multi-state outbreak of listeriosis caused by contamination of cantaloupe has been the deadliest (>30 deaths) outbreak caused by a food-borne pathogen in the USA in the last 80 years. L. monocytogenes is able to adhere to and grow on the surface of fresh produce such as cantaloupe in biofilms, i.e. multicellular communities embedded in extracellular substances secreted by bacteria. These substances facilitate bacterial attachment to surfaces and protect bacteria from environmental stressors. Among extracellular components of biofilms, exopolysaccharides are most commonly used by bacteria to attach to and grow on plant surfaces. The compositions of biofilms formed by L. monocytogenes on plant surfaces have not been characterized at the molecular level. An exopolysaccharide component of L. monocytogenes biofilms has recently been discovered by the PIs. The objectives of this study are to characterize the composition and structure of this novel exopolysaccharide, and to determine its role in L. monocytogenes attachment to produce and resistance to disinfectants and other control agents. This research will lead to a better understanding of the factors underlying the adherence and persistence of L. monocytogenes on produce and will guide efforts to prevent colonization and inhibit the growth of L. monocytogenes on produce.
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Approach:<br/>
Specific Aim 1. Composition and chemical structure of L. monocytogenes EPS. EPS will be obtained for chemical composition and structure analysis from our L. monocytogenes EGD-e mutant strain that overproduces EPS. To obtain EPS, cells will be grown in liquid medium and EPS removed from the cell surface by stirring. After pelleting cells by centrifugation, residual proteins will be removed by trichloroacetic acid precipitation, EPS will be precipitated from the culture medium using ethanol, precipitated EPS will be taken up in water and dialyzed against water to remove small molecules, and dialyzed EPS will be freeze-dried to obtain a crude EPS fraction. The next steps of EPS structural analysis will be to determine the monomer composition of the polymer, identify the types of linkages between monomer units, and determine its 3D structure by NMR spectroscopy.
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Specific Aim 2. Function of EPS in the attachment and formation of L. monocytogenes biofilms on produce. The analysis of the role of EPS in produce attachment and biofilm formation will be performed using a wild type lab strain (EGD-e), and EPS-negative and EPS-over-producing derivatives of EGD-e constructed in the lab. Experiments also will be performed with strains associated with outbreaks of produce origin. All strains will be assayed to determine the level of EPS they synthesize. The produce items that will be studied include cantaloupes, lettuce, and peppers. Data will be evaluated to determine if the number of cells recovered from the produce items is correlated with the EPS synthesis levels of the strains. For selected strains, we will examine the structure of the biofilms formed on produce surfaces by scanning electron microscopy.
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Specific Aim 3. Function of L. monocytogenes EPS in resistance to environmental and food preservation stressors. In these experiments we will expose biofilm cells grown on plastic microtiter plates and cantaloupe rinds to stress agents, and then determine survival of cells in the biofilm. Experiments will be conducted with our three lab strains that produce high, intermediate, and no EPS, and selected strains isolated from produce-associated listeriosis outbreaks. As stressors, we will evaluate nine agents and conditions that are known to impact L. monocytogenes viability. For disinfectants/sanitizers we will evaluate benzalkonium chloride and hypochlorous acid (bleach), which are used for control of L. monocytogenes growth in food processing plants. The food preservation agents that will be evaluated include heat, cold, pH variation, high salt concentration, sodium lactate, and the bacteriocin nisin. We also will evaluate the resistance of the strains to desiccation. Strains will be compared to one another to determine if the level of EPS in the biofilm affects cell survival.