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Objectives and methodology: The long-term goal of this study is to determine and control the physiological mechanisms that trigger the increased resistance to thermal treatment of Salmonella in low water activity extruded dry cereals. <P>Our hypothesis is that Salmonella persistence and resilience to dry conditions and thermal treatments are linked by a common physiological response mechanism that relies on the iron available in the food matrices. Therefore, the modulation of iron concentration either through chelating agents (e.g., lactoferrin, EDTA, or dipirydyl), competing di-cations (e.g., Mg2+), or increase in soluble iron (e.g., Fe2+) would control the resistance of this pathogen to both thermal treatments and endurance in dry food matrices. <P>Specific objectives and approach: 1. Determine the relationship between iron available in cereal and the resistance of Salmonella to thermal inactivation and dryness. 2. Determine the molecular and physical chemical changes induced by changes in the iron availability, temperature, and water activity. 3. Determine the effective combinations of temperature, dryness, iron concentration, and iron chelators to reduce Salmonella presence in dry foods. <P>Expected Outputs: We will conduct a series of of experiments that will allow us to understand if: (1) iron plays a central role in the cross-protection induced by low aw to thermal treatments, (2) iron availability has an effect on kinetic inactivation parameters and on the survival of Salmonella to desiccation and thermal treatments , (3) there is variability among different Salmonella serovars in their response to iron concentration, (4) the effects observed are due to environmental or intracellular iron, and (5) the iron effect is mediated through oxidative stress. <P>Additional experiments will provide transcriptional information that, compared with the chemical composition data, will provide (1) the basis for a theoretical model describing the cross-resistance phenomenon, (2) the impact of the presence/absence of iron on the physiological adaptation of Salmonella to low aw environments, (3) the genes involved in this phenomenon and their physiological role. A third set of experiments will prove if our theoretical model holds true for real-world situations and identify what treatments will have the highest chances of success at decreasing the survival of Salmonella in dry foods during storage and processing.
Non-Technical Summary:<br/>
Low moisture or dry foods do not support bacterial growth, and have been considered relatively safe from contamination with pathogenic bacteria. However, recent outbreaks of salmonellosis linked to low moisture foods (e.g., chocolate, dried milk, almonds, peanut butter, peanut products, toasted oats cereal and dried spices) have demonstrated that this bacterium survives dry conditions very well. Furthermore, Salmonella cells subjected to dry conditions become more heat tolerant. This response has clear implications for food processing when heating is used to kill potential food pathogens making Salmonella an important emerging problem for this food sector. Since iron homeostasis plays a fundamental role in the survival and virulence of Salmonella, its role in bacterial tolerance to stresses has been studied. This study aims at determining and controlling the protective systems in Salmonella that trigger resistance to thermal treatments observed in dry foods. Salmonella resilience to dry conditions and thermal treatments is linked by a common physiological response relying on the iron available. Control of iron concentrations would modulate its resistance to thermal treatments.
<br/>Specific objectives: 1) Determine the relationship between iron available in cereal and the resistance of Salmonella to thermal inactivation and dryness. 2) Determine the molecular and physical chemical changes induced by changes in the iron availability, temperature, and water activity. 3) Determine the effective combinations of temperature, dryness, iron concentration, and iron chelators to reduce Salmonella presence in dry foods. Approach: Four Salmonella serovars will be inoculated in a toasted oats cereal model and subjected to very low moisture vs. thermal treatments in presence of different iron concentrations, or anaerobic conditions. The effect of similar conditions on genetic status will be determined by advanced molecular and spectroscopy to develop a theoretical model. This model will be employed to design treatments to be tested in dry cereals. Potential impact and expected outcomes: The proposed research will provide information on the factors influencing the emerging problem of thermal treatment resistance of Salmonella in dry foods.
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Approach:<br/>
Objective 1 - During this phase of the project we will determine the effect of environmental and intracellular iron on the adaptation to low water activity conditions and on the cross-protection they provide against thermal treatments. Four Salmonella serovars will be employed in a combinatorial matrix of 4 different water activity conditions and 3 different temperatures. Each serovar will be grown in complex medium, washed and re-suspended in distilled water, inoculated in cereal samples, and, for each serovar, three sets of aw vs. thermal treatment matrices will be performed. Two sets will be performed in the presence of different chelators at different concentrations to reduce the natural concentration of environmental (e.g., lactoferrin) or intracellular (e.g., dipyridyl) iron. One set will be conducted in the presence of excess reduced iron by supplementing the cereal samples with ferrous sulphate to obtain a final concentration of 500 mg iron/Kg.
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Objective 2 - This phase of the project will characterize the chemical and molecular changes that lead to the cross-protection of Salmonella. S. Typhimurium will be grown in minimal medium, washed and re-suspended in distilled water, and spotted on cellulose filters (microarray experiments) or gold-plated slides (SERS analysis). The use of vibrational spectroscopic techniques such as Raman spectroscopy has proved to be extremely useful for the biochemical characterization of microbial cells. Although Raman spectroscopy itself can provide overall and specific biochemical information about the components of bacterial cells its resolution requires a high number of cells. Recently, growing interest has been given to the surface-enhanced Raman scattering (SERS) techniques that are more sensitive and informative on the bacterial characterization. SERS is a combination technique of Raman spectroscopy and nanotechnique, where the use of nanostructure can enhance the Raman signal more than a million times. We propose here to use SERS to characterize the macromolecular changes of Salmonella enterica under different water activity. Silver dendrite nanostructures will be prepared and used to enhance the Raman signals. As in objective 1, we will test three moisture values in three sets of experiments. Two sets will be performed in the presence of lactoferrin or dipyridyl at the top concentration tested (12.5 mg/mL) and one set will be conducted in the presence of excess reduced iron (500 mg/Kg). The S. Typhimurium spotted on filter or slide placed at a aw =1 will be used as a control.
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Objective 3 - Determine the effective combinations of temperature, dryness, iron concentration, and iron chelators to reduce Salmonella presence in dry foods. This phase of the project will heavily depend on the information gathered during the previous stages. The conditions that our theoretical model identified as most conducive to cross-resistance will be tested in regular dry foods (e.g., cereals) employing wild types and mutants of four Salmonella serovars.