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<p>1. To define conditions to assure a 5 log reduction of acid tolerant pathogens in refrigerated or bulk stored acidified vegetables.</p>
<p>2. To determine how the metabolism of Escherichia coli O157:H7 (internal pH, membrane potential, ion concentrations, and cell metabolites) are affected as cells are exposed to organic acid and salt conditions typical of acidified foods.</p>
<p>3. To determine the survival of E. coli O157:H7 in commercial fermentation brines, with and without competing microflora, and under a variety of extrinsic and intrinsic conditions.</p>
Approach:
A cocktail of five or more pathogenic Escherichia coli O157:H7 strains from the USDA/ARS Food Science Research Unit culture collection will be used for these studies. While our previous work has focused on E. coli O157:H7 (from human, food, animal, and environmental sources) additional serotypes, including O145 strains obtained from ARS sources will also be used in this research. Previous research on acidified vegetable brines has shown that E. coli O157:H7 is the most acid resistant vegetative pathogen of concern for acidified vegetable products. E. coli O157:H7 and related serotypes can't grow in most acidified vegetable products, the objective is to prevent bacterial pathogens from surviving long enough in non-heat treated acid and acidified foods to cause disease. Bacterial strains will be grown statically for 15 h at 37°C in non-selective broth (Luria broth) supplemented with 1 g/L glucose to induce acid resistance. Cell viability before, during and after acid treatments will be determined by plating on non-selective media to allow enumeration of injured cells with a spiral plater and an automated plate reader (Spiral Biotech). Samples from acid treatment of bacterial cells will be diluted in MOPS buffer at neutral pH prior to plating. The lower limit for detection is 10^2 to 10^3 CFU/mL for this method. In addition to standard plating, an MPN method done with microtiter plates, custom MatlabTM software, and a microtiter plate reader has been developed in our laboratory. This method can be used to determine log number for a range of cell concentrations from 10^8 to <30 CFU/mL, and will supplement spiral or standard plating techniques when cell numbers are lower than 10^3 CFU/mL. Most acid solutions will be prepared based on the protonated acid concentration. The acid concentration required to achieve specific protonated concentration for a given pH and ionic strength will be determined using a Matlab computer program (pHTools ) developed in our laboratory, or custom Matlab functions. Sodium gluconate will be used as a non-inhibitory buffer in acid solutions to allow comparisons of the effects of organic acids with the effect of pH alone. Cucumber juice medium or brined cucumbers will be used for these studies as representative of brined vegetable products, because these media do not contain inhibitors of microbial survival or growth, but do contain amino acids and other compounds that may aid in survival of the pathogens. Acid concentrations will be confirmed by HPLC using a Thermo Separation Products HPLC system with a Bio-Rad HPX-87H column and UV detector. For acid challenge experiments requiring anaerobic conditions, a Coy anaerobic chamber will be used and media or acid solutions allowed to equilibrate in the chamber for 24 h to remove dissolved oxygen.