A different behavior was observed in three gram-positive bacteria exposed to hen egg white lysozyme by plate counts and phase-contrast microscopy. The inactivation of Lactobacillus johnsonii was accompanied by spheroplast formation, which is an indication of peptidoglycan hydrolysis. Staphylococcus aureus was resistant to lysozyme and showed no signs of peptidoglycan hydrolysis, and Listeria innocua was inactivated and showed indications of cell leakage but not of peptidoglycan hydrolysis. Under high hydrostatic pressure, S. aureus also became sensitive to lysozyme but did not form spheroplasts and was not lysed. These results suggested the existence of a nonlytic mechanism of bactericidal action of lysozyme on the latter two bacteria, and this mechanism was further studied in L. innocua. Elimination of the enzymic activity of lysozyme by heat denaturation or reduction with β-mercaptoethanol eliminated this bactericidal mechanism. By means of a LIVE/DEAD viability stain based on a membrane-impermeant fluorescent dye, the nonlytic mechanism was shown to involve membrane perturbation. In the absence of lysozyme, high-pressure treatment was shown to induce autolytic activity in S. aureus and L. innocua.

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