Optimization of Liquid Resuscitation for Repair of Heat and Acid-injured Foodborne Pathogens

Abstract

Because of the presence of injured cells, the inactivation efficacy of various interventions may be overestimated. Therefore, verifying the exact populations of not only healthy but also injured cells is becoming increasingly important. However, recovery through commonly used method, overlay and SPRAB have several limitations. As there is a need to overcome limitations, liquid resuscitation method was developed and optimized. The purpose of this research was to determine optimum conditions for the liquid resuscitation of heat/acid-injured Salmonella Typhimurium and Escherichia coli O157:H7. Preliminary experiments showed that exposure to 55°C in PW for 15 and 25 min respectively as heat-treatment, and to 2% lactic acid for both 60 s as acid-treatment induced cellular injury of S. Typhimurium and E. coli O157:H7. BHI, TSB, and TSBYE were selected for recovery broth through optical density measurements at 600 nm. After microorganisms were heat and acid-treated at appropriate conditions, microbes were ten-fold diluted with BHI, TSB, and TSBYE and incubated at 37°C for up to 3 h. At hourly intervals, diluents were plated onto XLD or SMAC for the enumeration of healthy cells. Simultaneously, diluents were plated onto TSA for total viable counts (including recovered injured cells) followed by further incubation at 37°C. Comparison was made with the commonly used overlay method using XLD for recovering injured cells of S. Typhimurium. For E. coli O157:H7, SPRAB was used instead of the overlay method. One hour resuscitation were significantly recover heat or acid-injured S. Typhimurium and E. coli O157:H7, in any of the following broth media: BHI, TSB, and TSBYE. No growth of non-injured cells was observed during incubation. When liquid resuscitation applied to injured cells in food samples (low fat milk, apple juice), also 1 h incubation were enough to recover Thus, the liquid resuscitation method is a convenient alternative to the conventional overlay method.