Inactivation of gram-negative bacteria by lysozyme, denatured lysozyme, and lysozyme-derived peptides under high hydrostatic pressure

We have studied the inactivation of six gram-negative bacteria (Escherichia coli, Pseudomonas fluorescens, Salmonella enterica serovar Typhimurium, Sa lmonella enteritidis, Shigella sonnei, and Shigella flexneri) by high hydro static pressure treatment in the presence of hen egg-white lysozyme, partia lly or completely denatured lysozyme, or a synthetic cationic peptide deriv ed from either hen egg white or coliphage T4 lysozyme. None of these compou nds had a bactericidal or bacteriostatic effect on any of the tested bacter ia at atmospheric pressure. Under high pressure, all bacteria except both S almonella species showed higher inactivation in the presence of 100 mug of lysozyme/ml than without this additive, indicating that pressure sensitized the bacteria to lysozyme. This extra inactivation by lysozyme was accompan ied by the formation of spheroplasts. Complete knockout of the muramidase e nzymatic activity of lysozyme by heat treatment fully eliminated its bacter icidal effect under pressure, but partially denatured lysozyme was still ac tive against some bacteria. Contrary to some recent reports, these results indicate that enzymatic activity is indispensable for the antimicrobial act ivity of lysozyme, However, partial heat denaturation extended the activity spectrum of lysozyme under pressure to serovar Typhimurium, suggesting enh anced uptake of partially denatured lysozyme through the serovar Typhimuriu m outer membrane. All test bacteria were sensitized by high pressure to a p eptide corresponding to amino acid residues 96 to 116 of hen egg white, and all except E. coli and P. fluorescens were sensitized by high pressure to a peptide corresponding to amino acid residues 143 to 155 of T4 lysozyme. S ince they are not enzymatically active, these peptides probably have a diff erent mechanism of action than all lysozyme polypeptides.