Validation of Antimicrobial Interventions for Non-O157 and O157 Shiga-Toxigenic Escherichia coli (STEC) on Beef

<P>NON-TECHNICAL SUMMARY: Antimicrobial treatments designed for use on beef surfaces will be evaluated for effectiveness in reducing the risk of contamination with eight different serotypes of shiga-toxin producing E. coli. The research will assist in developing Standard Operating Procedures (SOPs) for application parameters to be used with the treatments. In addition,parallel reduction data for antimicrobial treatments using pathogenic bacterial strains and non-pathogenic surrogate bacteria will be evaluated for effectiveness in validation of beef carcass decontamination procedures. The validation procedures will be tested using the surrogates within commercial plant environments to determine ability to validate process effectiveness. This will generate scientific-based data to assist in developing best practices for control of enteric bacterial pathogens in beef products and validation of antimicrobial treatments. </P>
<P>APPROACH: Commercially available antimicrobial treatments, including lactic acid, peracetic acid, cetyl piridinium chloride, and lauric arginate, will be evaluated for reduction of eight strains of STEC (O26, O45, O103, O111, O121, O145, O157:H7, and O104:H4) on pre-rigor and refrigerated beef carcass surface regions (outside round, brisket, and clod) and on subprimal beef cuts, beef trimmings, and similar veal products. The surface of carcass regions, subprimals cuts and beef trimmings will be inoculated with a cocktail of the eight STEC strains. Each STEC strain will be inoculated in tryptic soy broth at 37°C for 18 h and after incubation, the bacterial cocktail will be prepared by mixing 1 ml of each culture with 92 ml of 0.1% Peptone Water (PW). The final concentration of each strain in the cocktail will be approximately 7 log CFU/ml. A 400-cm2 area (20 cm X 20 cm) of carcass surface regions, subprimal cuts, and beef trimmings will be inoculated with the bacterial cocktail under a biosafety hood. After inoculation, the carcass surfaces will be kept under the hood to allow for bacterial attachment. Subprimal cuts will be vacuum packaged and stored under refrigeration conditions (4°C) for up to 28 days to simulate commercial practices and handling, and further unpacked before antimicrobial treatments are applied. Antimicrobial treatments, including lactic acid, peracetic acid, cetyl piridinium chloride, and lauric arginate will be sprayed on inoculated carcass surface regions, subprimal cuts, and beef trimmings in a model spray cabinet. Standard Operating Procedures (SOPs) for application parameters will be designed to be used in data collection. The SOPs will include a detailed description of each solution to be used (concentration, temperature, pH, etc.) and application parameters including type of spray nozzle, pressure, distance from the meat surface, volume sprayed, and contact time. Composite samples will be collected from treated and untreated carcass surfaces, subprimal cuts, and beef trimmings. Each composite sample will consist of two 10-cm2 pieces excised with a sterile surgical blade. The composite samples will be placed into stomacher bags and pummeled with 99 ml of 0.1% PW in a stomacher for 1 min. Appropriate decimal dilutions will be plated on Tryptic Soy Agar (TSA), allowed to resuscitate for 2 h, and then covered with an overlay of selective agar for STEC enumeration. All plates will be incubated at 37ºC and colonies will be enumerated after 24 h. Log reductions (CFU/cm2) for STEC will be calculated by subtracting the log counts (CFU/cm2) obtained after antimicrobial treatments from the initial log count (CFU/cm2) obtained before each treatment was applied. Statistical procedures will be conducted to determine significant differences for mean log reductions among treatments. All experiments will be performed in triplicate. Parallel reduction data for antimicrobial treatments will also be collected for non-pathogenic E. coli biotype I strains previously proposed as surrogates for validation of beef carcass decontamination procedures. Carcass surface regions, subprimal cuts, beef trimmings, and similar veal products will be collected as previously described. The surface of carcass regions, subprimal cuts, and beef trimmings will be inoculated with a cocktail of surrogates consisting of three non-pathogenic E. coli ATCC cultures BAA-1427, BAA-1428 and BAA-1430. Each surrogate strain will be inoculated in tryptic soy broth at 37°C for 18 h, and after incubation the bacterial cocktail will be prepared by mixing 1 ml of each culture with 97 ml of 0.1% PW. The final concentration of each surrogate in the cocktail will be approximately 7 log CFU/ml. A 400-cm2 area (20 cm X 20 cm) of each carcass region, subprimal cut, and beef trimming will be inoculated with the bacterial cocktail under a biosafety hood. After inoculation, the carcass surfaces will be kept under the hood to allow for bacterial attachment. Subprimal cuts will be vacuum packaged to simulate commercial practices and handling, stored under refrigeration conditions (4°C) for a minimum of 28 days, and opened before antimicrobial treatments are applied. Similar antimicrobial treatments as described in the previous section will be applied according to the Standard Operating Procedures (SOPs) designed. From each treatment, composite samples will be collected from treated and untreated carcass surfaces, subprimal cuts, and beef trimmings as previously described. The surrogates will be enumerated on TSA, allowed to resuscitate for 2 h, and then covered with an overlay of selective agar for enumeration. Plates will be incubated at 37ºC for 24 h. Log reductions (CFU/cm2) for surrogates will be calculated by subtracting the log counts (CFU/cm2) obtained after antimicrobial treatments from the initial log count (CFU/cm2) obtained before each treatment was applied. Statistical procedures will be conducted to determine significant differences for mean log reductions among treatments and also between STEC and surrogates. Three independent replications will be performed for each of the experiments. Validation of commercial beef decontamination treatments will be accomplished by inoculating the surface of beef carcasses, subprimal cuts, and beef trimming with the surrogate strains evaluated in Objective 2, subjecting the cuts to commercial antimicrobial treatments, and then validating effectiveness of the treatments by measuring the reduction of bacterial inoculum levels. Data collected in these experiments will provide validation of antimicrobial treatment effectiveness in commercial production environments and will assist in developing best practices for control of STEC in beef products. </P>