Weakening of Salmonella with selected microbial metabolites of berry-derived phenolic compounds and organic acids

Gram-negative bacteria are important food spoilage and pathogenic bacteria. Their unique outer membrane (OM) provides them with a hydrophilic surface structure, which makes them inherently resistant to many antimicrobial agents, thus hindering their control. However, with permeabilizers, compounds that disintegrate and weaken the OM, Gram-negative cells can be sensitized to several external agents. Although antimicrobial activity of plant-derived phenolic compounds has been widely reported, their mechanisms of action have not yet been well demonstrated. The aim of our study was to elucidate the role of selected colonic microbial metabolites of berry-derived phenolic compounds in the weakening of the Gram-negative OM. The effect of the agents on the OM permeability of Salmonella was studied utilizing a fluorescence probe uptake assay, sensitization to hydrophobic antibiotics, and lipopolysaccharide (LPS) release. Our results show that 3,4-dihydroxyphenylacetic acid, 3-hydroxyphenylacetic acid, 3-(3,4-dihydroxyphenyl)propionic acid (3,4-diHPP), 3-(4-hydroxyphenyl)propionic acid, 3-phenylpropionic acid, and 3-(3-hydroxyphenyl)propionic acid efficiently destabilized the OM of Salmonella enterica subsp. enterica serovar Typhimurium and S. enterica subsp. enterica serovar Infantis as indicated by an increase in the uptake of the fluorescent probe 1-N-phenylnaphthylamine (NPN). The OM-destabilizing activity of the compounds was partially abolished by MgCl2 addition, indicating that part of their activity is based on removal of OM-stabilizing divalent cations. Furthermore, 3,4-dihydroxyphenylacetic acid, 3-hydroxyphenylacetic acid, and 3,4-diHPP increased the susceptibility of S. enterica subsp. enterica serovar Typhimurium strains for novobiocin. In addition, organic acids present in berries, such as malic acid, sorbic acid, and benzoic acid, were shown to be efficient permeabilizers of Salmonella as shown by an increase in the NPN uptake assay and by LPS release.