On-line fluorescence determination of pressure mediated outer membrane damage in Escherichia coli

The outer membrane (OM) of Gram-negative bacteria provides a protective barrier for natural occurring inhibitors. Pressure mediated OM permeabilisation therefore contributes to the elimination of Escherichia coli and Salmonella by pressure preservation processes. Pressure mediated inactivation, sublethal injury, and membrane permeabilisation of E. coli were determined using two strains differing in their barotolerance. Pressure treatment of E. coli TMW 2.427 at 300, 500 and 600 MPa for 40 min resulted in a 0, 1, and greater 6 log decrease of viable cell counts, respectively. The kinetics of OM and cytoplasmic membrane permeabilisation after pressure treatment were determined by staining of pressure treated cells with the fluorescent dyes propidium iodide (PI) and 1-N-phenylnaphtylamine (NPN), respectively. A slight increase of PI fluorescence was observed at conditions resulting in a greater 6 log decrease of viable cell counts only. In contrast, increased NPN fluorescence indicating OM permeabilisation was observed prior to cell death and sublethal injury. An on-line assay for determination of pressure mediated OM damage based on NPN fluorescence was established to distinguish between reversible and irreversible OM damage. Generally, the same degree of outer membrane damage was observed by either on line or off line determinations. However, whereas reversible membrane damage occurred fast and in thermodynamic equilibrium with pressure conditions, irreversible outer membrane damage was a time dependent process.