Investigation into the Physiological State of Heat Stressed Escherichia coli Used in the Evaluation Testing of an Intrinsic Fluorescence-Based RMM

Rapid microbiological methods (RMMs) have been used as novel quality control technologies in industry. The ability of RMMs to detect stressed bacteria, in particular, is of continued interest due to the limitations of the conventional method in stressed bacteria detection. Accordingly, there is a need to better characterize an RMM’s ability to detect stressed microorganisms. Previously we reported on the detection ability of an intrinsic fluorescence-based RMM using a 50% injured (determined based on colony-forming ability) bacterial cell group after heat treatment at 55°C for 8 min. In this study, we added further information about the physiological state of the heat treated Escherichia coli, besides proliferation ability, by investigating respiratory activity using CTC fluorescent staining and expression of DnaK, a heat shock protein. It was found that 89% of cells (control 96%) retained respiratory activity, but only 20% (control 41%) retained proliferation ability after heat treatment. The difference between the percentage of cells with respiratory activity versus that of cells still capable of proliferation further supports the existence of viable but non-culturable stressed cells in the test sample. Also, we suggest such analysis would be one approach to confirming the use of stressed as opposed to dead cells when evaluating an RMM’s ability to detect stressed microorganisms.