| The primary objectives of this research included (1) Assessing the presence of sublethal injury by observing nonculturable E. coli O157:H7 after employing preservation methods and (2) Determining if E. coli O157:H7, that have been cultured in acidified media, show cross protection or increased resistance to certain preservation methods. These objectives were achieved by using luminometric methods to enumerate organisms that were transformed with bioluminescent markers. Three environmental strains of E. coli O157:H7 were electrotransformed using plasmid DNA (pT7-5) that codes for bioluminescence. Correlation between plate counts (CFU/mL) and bioluminescence, measured as relative light units (RLU), were established for all experimental conditions using linear regression analysis. The transformants were cultured in acidified (pH 5.0) growth media to prepare acid adapted cells or grown in neutral media to provide nonadapted cells. These conditioned E. coli were subjected to benzoate/sorbate (0.1%), freeze (–20°C) - thawing (4°C for 4 hours), and thermal (54°C) treatments and enumerated by standard plate count and luminometry. Strain-specific survival behavior was evaluated by comparing the effect of the treatments on each strain according to counts obtained. Sublethal injury was measured by obtaining a significant (α = 0.05) positive difference after subtracting plate count D-values, representing culturable bacteria, from RLU-derived D-values, which represent the total viable bacteria. Cross protection was assessed by observing (α = 0.05) significantly higher mean D-values for acid-adapted cells than mean D-values for nonadapted cells.; It was determined in this study that variation between strains existed after each treatment. Benzoate- and sorbate-treated acid-adapted E. coli O157:H7 showed a 1.5- to 3-log reduction depending on the strain. E. coli O157:H7 strains showed cell count declines ranging from 3 to 5.5-logs after freeze-thaw treatment. After thermal treatment, E. coli O157:H7 cells declined between 3.5 to 7-logs. Significant sublethal injury was apparent in all matrices, especially after freeze-thaw treatment. Significant (α = 0.05) cross protection existed after each treatment by most strains used in the study, except for freeze-thaw treated cells. This study validates the use of bioluminescence-marked E. coli O157:H7 for studying cross protection and sublethal injury. |
