Pulsed electric fields cause sublethal injuries in the outer membrane of Enterobacter sakazakii facilitating the antimicrobial activity of citral

Aims: The objective was to evaluate the relation of sublethal injury in the outer membrane of Enterobacter sakazakii to the inactivating effect of the combination of pulsed electric fields (PEF) treatments and citral. Methods and Results: The occurrence of sublethal injury in the outer membrane was measured using selective recovery media containing bile salts. Loss of membrane integrity was measured by the increased uptake of the fluorescent dye propidium iodide (PI). PEF caused nonpermanent and permanent envelope permeabilization of Ent. sakazakii at pH 4 center dot 0. After PEF, most surviving cells showed transient cell permeabilization and sublethal injury in their outer membranes. The simultaneous application of a mild PEF treatment (100 pulses, 25 kV cm-1) and 200 mu l l-1 of citral to cells suspended in pH 4 center dot 0 buffer at a final concentration of 107 cells per ml showed an outstanding synergistic lethal effect, causing the inactivation of more than two extra log(10) cycles. Conclusions: Our results confirm that the detection of sublethal injury in the outer membrane after PEF may contribute to the identification of the treatment conditions under which PEF may act synergistically with hydrophobic compounds such as citral. Significance and Impact of the Study: Knowledge about the mechanism of microbial inactivation by PEF will aid the establishment of successful combined preservation treatments.