Validation of Methods Used To Recover Escherichia coli O157:H7 and Salmonella spp. Subjected to Stress Conditions†

Abstract

Escherichia coli O157:H7, Salmonella spp., and Salmonella Typhimurium DT104 were stressed with lactic acid and cell-free supernatants from lactic acid bacteria and plated on three different media to determine if injured cells were recovered. A comparison of the susceptibility and recovery of antibiotic-resistant strains of the pathogens and nonresistant strains was also made. Acid stress conditions were created by adjusting the pH of a cocktail mixture (two to four strains) of the pathogen to 3.50 with lactic acid and holding for 18 h. The pathogen cocktail was also stressed with a cell-free supernatant of Lactobacillus lactis (pH 3.90) in a 4:6 ratio. Both nonstressed and stressed cocktail cultures were plated on Trypticase soy agar (TSA) and violet red bile agar (VRBA) for E. coli and xylose lysine tergitol4 (XLT4) for Salmonella. Repair of injured cells was evaluated by pour plating the stressed cells on a 5-ml thin layer of TSA and allowing a 2-h room temperature incubation followed by overlaying with VRBA or XLT4. There were significant reductions in the populations of both pathogens under both stress conditions when plating was done on nonselective media. Injured E. coli O157:H7 was not recovered on recovery or selective media compared with TSA. Numbers of cells of supernatant-stressed Salmonella spp. plated on selective and recovery media were similar to those on TSA. Acid-stressed cells for all Salmonella spp. were not recovered on TSA, selective, or recovery media at levels comparable to recovery on TSA. Antibiotic-resistant strains showed similar recovery patterns on all media evaluated. However, the antibiotic-resistant strains were less sensitive to both stress conditions. The use of antibiotic-resistant strains resulted in a greater recovery of stressed pathogens than the use of recovery media.