Sensitization of Outer-Membrane Mutants of Salmonella Typhimurium and Pseudomonas aeruginosa to Antimicrobial Peptides under High Pressure

Abstract

High pressure can sensitize gram-negative bacteria to antimicrobial peptides or proteins through the permeabilization of their outer membranes; however, the range of compounds to which sensitivity is induced is species and strain dependent. We studied the role of outer-membrane properties in this sensitization by making use of a series of rough and deep rough mutants of Salmonella enterica serovar Typhimurium that show an increased degree of lipopolysaccharide(LPS) truncation, along with Pseudomonas aeruginosa PhoP and PhoQ mutants with altered outer-membrane properties. The outer-membrane properties of P. aeruginosa were also modulated through the use of different Mg2+ concentrations in the growth medium. Each of these strains was challenged under high pressure (15 min at 270 MPa for Salmonella Typhimurium and 15 min at 100 MPa for P. aeruginosa) in phosphate buffer with lysozyme (100 μg/ml), nisin (100 IU/ml), lactoferricin (20 μg/ml), and HEL96-116 (100 μg/ml), a synthetic lysozyme-derived peptide, and sensitization levels were compared. The results obtained indicated that outer-membrane properties affected high-pressure sensitization differently for different compounds. LPS truncation in Salmonella Typhimurium was correlated with increased sensitization to lysozyme (up to 1.5 log10 units) and nisin (up to 1.2 log10 units) but with decreased sensitization to lactoferricin under pressure. For P. aeruginosa, the pattern of sensitization to lactoferricin and nisin resembled that of polymyxin B at atmospheric pressure, suggesting that pressure induces the self-promoted uptake of both peptides. Sensitization to HEL96-116 was not affected by outer-membrane properties for either organism. Hence, outer-membrane permeabilization by high pressure cannot be explained by a single unifying mechanism and is dependent on the organism, the outer-membrane properties, and the nature of the antimicrobial compound. On the basis of these findings, the use of antimicrobial cocktails targeting different bacteria and fractions of bacterial populations may enhance the efficacy of high pressure as a preservation treatment.