Improved Methods to Control Biofilms Containing Listeria in Meat and Poultry Processing Environments

NON-TECHNICAL SUMMARY: The reduction of microbial pathogens in food products is the most pressing food safety problem today. A major source of food-borne illnesses in the United States is contamination of RTE meat and poultry products by L. monocytogenes pathogens when disinfection is inadequate. While there are multiple causes of disinfection failure in meat and poultry processing facilities, the generation of protective biofilms by microorganisms that aggregate on food processing equipment surfaces is a major contributing factor to contamination. Traditional disinfectants, such as bleach and halogenated organics, do not effectively penetrate and remove the biofilm matrix and are therefore, unable to adequately control pathogens that are embedded within it. This project will establish baseline efficacy of Sterilex technology that has shown excellent results in preliminary studies against L. monocytogenes, elucidate the mechanism of action for penetration into biofilms, and optimize formulations for use in meat and poultry food processing environments against biofilms containing L. monocytogenes. The overall goal of the research is an optimized formulation of a disinfectant that is effective in controlling L. monocytogenes biofilm, thus minimizing the risks to food safety from pathogenic contamination.

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APPROACH: Currently, there are no products that both disinfect effectively and remove the biofilm matrix in food processing equipment. This is due in large part to the presence of the bacterial-produced polymer layer called biofilm that provides a physical barrier to the penetration of chemical disinfectants and leads to the expression of resistance mechanisms in microorganisms. Biocides used in meat and poultry processing equipment typically include chlorine-based formulations and quaternary ammonium salts as primary ingredients, which have been shown to be ineffective against L. monocytogenes biofilm. The Sterilex anti-biofilm technology is based on the discovery that combining peroxides with a phase transfer (PT) catalyst creates products that have powerful antimicrobial activity and unique ability to breakdown and dislodge biofilm. Therefore, the first objective of this study is to conduct baseline experimental tests to demonstrate product efficacy. Experiments in Objective #2 are designed to study the structure-activity relationships of quaternary ammonium compounds (QACs), calcium sequestrants and nonionic surfactants, and pH of the product solutions to elucidate the mechanisms of action. Results of Objectives 1 and 2 will be applied to the optimization of a commercial product that is acceptable to the EPA for claims of L. monocytogenes biofilm disinfection in RTE meat and poultry processing equipment. The product attributes will include: antibiofilm activity meeting USDA 'zero tolerance' regulations for L. monocytogenes, low residue after rinsing, minimal corrosion of materials used in processing equipment, ease of use, and shelf-stability. The stability of the formulations will be tested over periods of three months to one year by measuring hydrogen peroxide and QACs by the following methods that Sterilex has adopted for excellence in quality control. The optimized biofilm disinfectant will be field tested against mixed species biofilms on actual poultry and meat processing equipment. Total bacterial enumeration and detection of L. monocytogenes will be performed before and after cleaning and after sanitizing. The optimized product will be applied at varying concentrations and contact times to the processing equipment and its activity against biofilms will be measured. The data obtained will be used to determine the lowest-cost and most efficient protocol that achieves elimination of L. monocytogenes on surfaces and equipment. The optimized protocol will be used to develop directions for use for the commercial product.

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PROGRESS: 2004/12 TO 2008/12<BR>
OUTPUTS: 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. <BR>
APPROACH: 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. <BR>
OBJECTIVE COMPLETED The above-stated objective was successfully completed. The grant review panel stated that: This project could allow optimizing and improving methods and could have a practical impact to control biofilms in food facilities. This research has generated technique-sensitive methods that provide quantitative data for comparison and analysis of Listeria biofilm production and tests for disinfectant activity against L. monocytogenes. Methods were developed to produce biofilms under flow conditions that mimic poultry environments. Fluorescent labels were tested for specificity for Listeria monocytogenes biofilms. Analytical procedures and assays were developed to quantitatively measure pathogens within biofilms produced under flow conditions. Assays were developed and tested to measure the efficacy of products for the killing and removal of L. monocytogenes biofilms. This was the first use of the assay using epifluorescence for quantification of the foodborne pathogen, L. monocytogenes, within bacterial biofilms on stainless steel. The test methods are applicable to other bacteria and substrata. The assay methods compared multiple disinfectants for killing and biofilm removal of L. monocytogenes biofilm. The results showed which test compounds were effective, with total kill and more than 90 percent biofilm removal for successful compounds.We have established a Listeria test method for the Official Methods of Analysis of the AOAC International. In addition, we have established a test method protocol acceptable to the EPA against L. monocytogenes biofilm growth under flow conditions. Sterilex has developed a cost-effective cleaner/disinfectant product for use in poultry and meat production and processing environments with instructions for use that will achieve USDA zero tolerance regulations for L. monocytogenes. <BR>
TARGET AUDIENCES: This project is relevant to those involved in meat and poultry food processing, particularly as it relates to adequate disinfection procedures, infection control, and zero tolerance for Listeria. <BR>
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IMPACT: 2004/12 TO 2008/12<BR>

Protocol development and validation is critical to the establishment of standards for biofilm claims for use of disinfectants in food processing. Such claims will educate end users about biofilms and the importance 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 L. monocytogenes and other harmful pathogens. The results of these experiments yielded an optimized formulation of a disinfectant for meat and poultry food processing and production equipment that is effective in reducing L. monocytogenes biofilm growth, thus minimizing the risks to food safety from pathogenic contamination. Effective products will be of great use for sanitation in food and health areas to help reduce cases of listeriosis across the nation.
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PROGRESS: 2004/12/15 TO 2005/12/14<BR>

The goals for the first year were: to establish baseline data of Sterilex technology against L. monocytogenes biofilms by AOAC use-dilution testing, to develop methods to produce biofilms for further study and for quantification of biofilms on surfaces including batch culture within a reactor, and to perform chemical screening of potential products for biofilm removal activity. Preliminary efficacy of Sterilex technology (a quaternary ammonium compound with hydroperoxide ion, QAC) was determined by measuring the minimum inhibitory concentrations (MICs) against 26 bacterial species. To do so, 15 field isolates from meat and poultry were obtained from the Food Safety and Inspection Service, as well as controls from the American Type Culture Collection, including Listeria monocytogenes, Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, and Salmonella choleraesius. All of the strains grew well on TSB. The test compounds were diluted with water in series from 1:20 to 1:1000. For 26 bacterial species, the zones of inhibition ranged from 7.0 to 12.5 mm, while the minimum inhibitory concentration (MIC) ranged from 5 to 250 parts per million. At least two trials have been performed for all species. The Sterilex product was most effective on L. monocytogenes and was more effective on field isolates than on laboratory control strains. Of the Listeria species, the compound was least effective on two nonpathogenic species, ivanovii and grayi. The MIC results were predictive of the results for the use-dilution method (below). For final efficacy, methodology for the use-dilution test (modification of the AOAC Official method 955.11) for L. monocytogenes (ATCC19114) was developed to measure the disinfectant activity of the QAC. Three broth media were tested, and brain heart infusion broth was selected for liquid culture media. Aliquots of the test compound were placed into a series of test tubes. Stainless steel penicylinders were contaminated with L. monocytogenes and added separately to the tubes for five, ten, and 15 minutes. Growth on the penicylinders was about 105 colony-forming units. After exposure, the carriers were removed, placed into two series of D/E neutralization broth, incubated and observed for growth. The potency of the disinfectant was compared with that of phenol (phenol coefficient, PC): a PC greater than 1 means that the disinfectant is more effective than phenol. For three replications with duplicate samples, the PC was 3.3 (250/75). Colorimetric measurement of dye staining and extractions was selected as the biofilm screening mechanism. The peak absorbance for crystal violet and Congo red were determined by spectrophotometry. Multiple concentrations of the dyes were scanned, and optimum concentrations with bacterial mixtures were determined. Crystal violet was selected for optimal extraction of L. monocytogenes biofilm. MICs have been performed concomitantly with the extractions for comparison of bacterial killing with biofilm removal. Three products have been tested, and data is currently undergoing analysis.
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IMPACT: 2004/12/15 TO 2005/12/14<BR>

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 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.