RESUMO
The effects of environmental stresses on microorganisms have been well-studied, and cellular responses to stresses such as heat, cold, acids, and salts have been extensively discussed. Although high pressure processing (HPP) is becoming more popular as a preservation method in the food industry, the characteristics of the cellular damage caused by high pressure are unclear, and the microbial response to this stress has not yet been well-explored. We exposed the pathogen Listeria monocytogenes to HPP (400 MPa, 8 min, 8°C) and found that the high pressure created plasma membrane pores. Using a common staining technique involving propidium iodide (PI) combined with high-frequency fluorescence microscopy, we monitored the rate of diffusion of PI molecules into hundreds of bacterial cells through these pores on days 0, 1, 2, 3, and 4 after pressurization. We also developed a mathematical dynamic model based on mass transfer and passive diffusion laws, calibrated using our microscopy experiments, to evaluate the response of bacteria to HPP. We found that the rate of diffusion of PI into the cells decreased over the 4 consecutive days after exposure to HPP, indicating repair of the pressure-created membrane pores. The model suggested a temporal change in the size of pores until closure. To the best of our knowledge, this is the first time that pressure-created membrane pores have been quantitatively described and shown to diminish with time. In addition, we found that the membrane repair rate in response to HPP was linear, and growth was temporarily arrested at the population level during the repair period. These results support the existence of a progressive repair process in some of the cells that take up PI, which can therefore be considered as being sub-lethally injured rather than dead. Hence, we showed that a subgroup of bacteria survived HPP and actively repaired their membrane pores.
RESUMO
Over the past years, products of non-animal origin have been increasingly linked to foodborne diseases caused by the enterohemorrhagic pathogen Escherichia coli O157:H7. Contaminated fresh produce and derived ready-to-eat meals are of major concern, since no further or only minimal processing is applied. In this study, flow cytometry was evaluated as a rapid technique to detect E. coli O157:H7 by immunofluorescence, using polyclonal antibodies conjugated to R-phycoerythrin, in refrigerated ready-to-eat pasta salad containing acetic acid and benzoic acid. Signal filtering strategies were applied during sample analysis to reduce the limit of detection of the technique to 5 log CFU/g. Simultaneously with pathogen detection, physiological state was assessed by staining with the membrane integrity indicators propidium iodide and SYBR Green I. Fine tuning of dye concentrations and ratios allowed discrimination of not only cells with intact or damaged membranes, but also of cells with partially damaged membranes, which were considered injured cells. Then, changes in membrane integrity of inoculated E. coli O157:H7 cells were monitored throughout 14-day refrigerated storage. Most cells were injured at the beginning of refrigeration, but showed an intact membrane at the end. This suggests that injured E. coli O157:H7 cells underwent a membrane repair during exposure to refrigeration and acid stresses, and survived in ready-to-eat pasta salad. This highlights the importance of the implementation of control measures to limit the presence of this pathogen in non-animal origin food products. Additionally, the proposed immunodetection and membrane integrity three-color assay in food is a good tool to monitor the effect of a number of food-related treatments on E. coli O157:H7 cell membrane.
