Progress 01/01/05 to 12/31/08
Outputs
Progress Report Objectives (from AD-416) The objective of this cooperative research project is to develop and optimize a product for use against Listeria monocytogenes biofilm in meat and poultry processing equipment and to develop standard methodologies for biofilm removal for registration of EPA biofilm claims for products used in food processing. The additional time is required to assure completion of the original approved project scope and objectives. The initial two years of the project focused upon methods development and mechanistic studies. In the extended time, we will grow Listeria monocytogenes biofilms to test the effect of Sterilex experimental samples on biofilm, and perform additional antimicrobial efficacy studies. Approach (from AD-416) Develop methods to produce biofilms for further study and for quantification of biofilms on surfaces including batch culture within a reactor. Establish baseline data of Sterilex technology against L. monocytogenes biofilms and mixed species biofilms containing L. monocytogenes under food processing plant conditions with AOAC use- dilution testing. Determine mechanism of action of current Sterilex technology in conjunction with chemical screening by total organic carbon analysis. Optimize product against multi-species biofilms in meat and poultry processing equipment and test final product with use-dilution test. Significant Activities that Support Special Target Populations This research supports the parent project through the development of approaches for processing biofilms to provide for quantitative measurement of bacterial populations in situ. Objective 3 of the parent project is to �Develop approaches for processing biofilm samples to allow quantitative measurement of bacterial populations in situ.� Our focus for this year has been completion of all project experiments. Methods that were developed during the course of the project to produce Listeria monocytogenes biofilms and test Sterilex technology were used to complete chemical screening of potential product components for biofilm removal activity. We have implemented new methods to screen efficacy against L. monocytogenes biofilms, with concomitant testing of biofilm sustainability and bacterial cell culturability from the biofilms. Methods which were recommended by experts in the area of biofilm biology and our previous experiments with Campylobacter were not effective with Listeria. We have analyzed chemical screening data to optimize product formulations for selection of test components. After testing the chemical components in the formulations, we completed measurement of extractions of L. monocytogenes biofilm. The presence of biofilm mass, before and after treatment, was confirmed by specialized microscopy. We have determined the mechanism of action of current Sterilex technology in conjunction with chemical screening. We have optimized products against biofilms and tested multiple products. The results of these experiments yielded an optimized formulation of a disinfectant for meat and poultry food processing and production equipment that is more effective in reducing L. monocytogenes biofilm growth than currently used disinfectants, thus reducing the risks to food safety. Protocol development and validation is critical to the establishment of standards for registration of biofilm claims for used in food processing. Such claims will educate end users about biofilms and the important of utilizing products that can address the problem of biofilms in food processing environments. During the past year, monitoring activities included weekly to bi-weekly conference calls and/or emails to review progress, data, and exchange technical advice. Review of project plans, program goals, and accomplishments were conducted off-site at a stakeholders meeting. The Cooperator�s annual progress report was presented to the Authorized Departmental Officer in writing, on schedule. A cumulative, detailed research report was submitted to the Research Leader, Area Director, and granting agency. A final comprehensive report for the entire project will be submitted within 90 days of termination as required.
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Progress 10/01/06 to 09/30/07
Outputs
Progress Report Objectives (from AD-416) The objective of this cooperative research project is to develop and optimize a product for use against Listeria monocytogenes biofilm in meat and poultry processing equipment and to develop standard methodologies for biofilm removal for registration of EPA biofilm claims for products used in food processing. Approach (from AD-416) Develop methods to produce biofilms for further study and for quantification of biofilms on surfaces including batch culture within a reactor. Establish baseline data of Sterilex technology against L. monocytogenes biofilms and mixed species biofilms containing L. monocytogenes under food processing plant conditions with AOAC use- dilution testing. Determine mechanism of action of current Sterilex technology in conjunction with chemical screening by total organic carbon analysis. Optimize product against multi-species biofilms in meat and poultry processing equipment and test final product with use-dilution test. Significant Activities that Support Special Target Populations This report documents research conducted under a trust agreement between ARS and Sterilex. Additional details of research can be found in the report for the in-house associated project 6612-32000-055-00D, Molecular Characterization and Gastrointestinal Tract Ecology of Commensal Human Foodborne Bacterial Pathogens in the Chicken. The focus for this year has been completion of baseline data established during the first year. Methods developed during the first year to produce Listeria monocytogenes biofilms and test Sterilex technology were used to complete chemical screening of potential product components for biofilm removal activity. New methods were implemented to screen for detection efficacy against L. monocytogenes biofilms, with concomitant testing of biofilm sustainability and bacterial cell culturability from the biofilms. Methods recommended by experts in the area of biofilm biology and our previous experiments with Campylobacter were not effective with Listeria. We have analyzed chemical screening data to optimize product formulations for selection of test components. After variance of the calcium sequestrants and nonionic surfactants in the formulations, we completed colorimetric measurement of dye staining and extractions of L. monocytogenes biofilm as the biofilm screening test agent. Field strains continued to be more susceptible to the chemistry than laboratory strains. We are ready to test the final product to meet USDA �zero tolerance� regulations for L. monocytogenes biofilms, and its activity will be confirmed by the AOAC use-dilution test. Protocol development and validation is critical to the establishment of standards for registration of EPA biofilm claims for used in food processing. Such claims will educate end users about biofilms and the important of utilizing products that can address the problem of biofilms in food processing environments. Understanding the mechanism of action underlying the effectiveness of the current Sterilex technology is critical for the future development of effective products for use against biofilms containing Listeria monocytogenes and other harmful pathogens. The results of these experiments will yield an optimized formulation of a disinfectant for meat and poultry RTE food processing and production equipment that is more effective in reducing Listeria monocytogenes biofilm growth than currently used disinfectants, thus minimizing the risks to food safety from pathogenic contamination. During the past year, monitoring activities included weekly to bi-weekly conference calls and/or emails to review progress, data, and exchange technical advice. Review of project plans, program goals, and accomplishments were conducted off-site at a stakeholders meeting in July, 2007. The Cooperator�s annual progress report was presented in person to the granting agency at a celebration for NRI awardees, October, 2006; and to the ADO in writing, on schedule, as required, in December, 2006. A cumulative, detailed research report was submitted to the RL, Area Director, and granting agency, June 2007.
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Progress 10/01/05 to 09/30/06
Outputs
Progress Report 4d Progress report. This report serves to document research conducted under a trust agreement between ARS and Sterilex Corporation through and NRI grant. Our focus for the second year of this three-year project has been to further study with baseline data established during the first year. Methods that were developed during the first year to produce Listeria monocytogenes biofilms and test Sterilex technology were used to complete chemical screening of potential product components for biofilm removal activity. We will use the results of the chemical screening data to optimize product formulations for efficacy against L. monocytogenes biofilms. Selection of test components varied the calcium sequestrants and nonionic surfactants in the formulations. Colorimetric measurement of dye staining and extractions of L. monocytogenes biofilm were used as the biofilm screening test agent. Four strains of Listeria biofilms were grown and tested: two laboratory
strains and two field strains. Field strains appeared to be more susceptible to the chemistry than laboratory strains. Six concentrations of 18 product components have been screened against the four strains of Listeria for three time periods. Each variable was tested against L. monocytogenes biofilms in two trials, and duplicate samples were measured within each trial. Data from the experiments are currently undergoing analysis. The data will allow us to quantify the percent reduction in biofilm between treated and untreated samples, as well as to determine the standard deviation of the efficacy measure. The analysis will determine the role of phase-transfer in antibiofilm activity and if high pH is a prerequisite for efficacy. Finally, the data will be used to determine the mechanism of action of current technology in conjunction with the chemical screening to formulate two compounds to be further tested for efficacy. The final product will be developed in the third year of research
to meet USDA 'zero tolerance' regulations for L. monocytogenes biofilms, and its activity will be confirmed by the AOAC use-dilution test. Expected Impact Protocol development and validation is critical to the establishment of standards for registration of EPA biofilm claims for used in food processing. Such claims will educate end users about biofilms and the important of utilizing products that can address the problem of biofilms in food processing environments. Understanding the mechanism of action underlying the effectiveness of the current Sterilex technology is critical for the future development of effective products for use against biofilms containing Listeria monocytogenes and other harmful pathogens. The results of these experiments will yield an optimized formulation of a disinfectant for meat and poultry RTE food processing and production equipment that is more effective in reducing Listeria monocytogenes biofilm growth than currently used disinfectants, thus minimizing
the risks to food safety from pathogenic contamination. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Arnold, J.W., Zambelli-Weiner, A., deLaubenfels, E., and Kramer, S. 2006. Quantitative assessment of hard surface disinfectant activity against the food-borne pathogen, Listeria monocytogenes. J AOAC Int.
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Progress 10/01/04 to 09/30/05
Outputs
4d Progress report. This report serves to document research conducted under a three-year Trust Agreement between ARS and Sterilex Corporation. This is the first year (four months) of the agreement. Additional details of research can be found in the report for the parent project 6612-41420-006-00D Reduction and control of pathogens associated with food processing surfaces. The objective of this cooperative research project is to develop and optimize a product for use against Listeria monocytogenes biofilm in meat and poultry processing equipment and to develop standard methodologies for biofilm removal. We have initiated development of methods to produce biofilms for further study and for quantification of biofilms on surfaces including batch culture within a reactor. We have evaluated a primary test compound with the AOAC use-dilution test against three standard bacteria: Pseudomonas aeruginosa, Staphylococcus aureus, and Salmonella enteritidis. We developed a
modification of the test to measure activity against Listeria monocytogenes. The compound was tested against each organism in three or more trials, and each sample was replicated within each trial. The minimum inhibitory concentration for the compound was determined for six strains of Listeria monocytogenes taken from contaminated meat and poultry sources by the Food Safety and Inspection Service.
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