Carvacrol, (+)-carvone, thymol, and trans-cinnamaldehyde were tested for their inhibitory activity against Escherichia coli O157:H7 and Salmonella typhimurium. In addition, their toxicity to Photobacterium leiognathi was determined, utilizing a bioluminescence assay. Their effects on the cell surface were investigated by measuring the uptake of 1-N-phenylnaphthylamine (NPN), by measuring their sensitization of bacterial suspensions toward detergents and lysozyme, and by analyzing material released from cells upon treatment by these agents. Carvacrol, thymol, and trans-cinnamaldehyde inhibited E. coli and S. typhimurium at 1-3 mM, whereas (+)-carvone was less inhibitory. trans-Cinnamaldehyde was the most inhibitory component toward P. leiognathi. Carvacrol and thymol disintegrated the outer membrane and released outer membrane-associated material from the cells to the external medium; such release by (+)-carvone or trans-cinnamaldehyde was negligible. Of the tested components, carvacrol and thymol decreased the intracellular ATP pool of E. coli and also inreased extracellular ATP, indicating disruptive action on the cytoplasmic membrane.

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The effect of lactic acid on the outer membrane permeability of Escherichia coli O157:H7, Pseudomonas aeruginosa, and Salmonella enterica serovar Typhimurium was studied utilizing a fluorescent-probe uptake assay and sensitization to bacteriolysis. For control purposes, similar assays were performed with EDTA (a permeabilizer acting by chelation) and with hydrochloric acid, the latter at pH values corresponding to those yielded by lactic acid, and also in the presence of KCN. Already 5 mM (pH 4.0) lactic acid caused prominent permeabilization in each species, the effect in the fluorescence assay being stronger than that of EDTA or HCl. Similar results were obtained in the presence of KCN, except for P. aeruginosa, for which an increase in the effect of HCl was observed in the presence of KCN. The permeabilization by lactic and hydrochloric acid was partly abolished by MgCl 2 . Lactic acid sensitized E. coli and serovar Typhimurium to the lytic action of sodium dodecyl sulfate (SDS) more efficiently than did HCl, whereas both acids sensitized P. aeruginosa to SDS and to Triton X-100. P. aeruginosa was effectively sensitized to lysozyme by lactic acid and by HCl. Considerable proportions of lipopolysaccharide were liberated from serovar Typhimurium by these acids; analysis of liberated material by electrophoresis and by fatty acid analysis showed that lactic acid was more active than EDTA or HCl in liberating lipopolysaccharide from the outer membrane. Thus, lactic acid, in addition to its antimicrobial property due to the lowering of the pH, also functions as a permeabilizer of the gram-negative bacterial outer membrane and may act as a potentiator of the effects of other antimicrobial substances.Lactic acid, as produced by lactic acid starter culture bacteria or as an additive to foods, functions as a natural antimicrobial having a generally recognized as safe status. As reviewed by Doores (8), lactic acid is able to inhibit the growth of many types of food spoilage bacteria, including gram-negative species of the families Enterobacteriaceae and Pseudomonadaceae. Among other organic acids, lactic acid is recognized as a biopreservative in naturally fermented products (25), and numerous applications for decontamination of meat by lactic acid have been described (7,10,22,29,32,33). The antibacterial action of lactic acid is largely, but not totally, assigned to its ability in the undissociated form to penetrate the cytoplasmic membrane, resulting in reduced intracellular pH and disruption of the transmembrane proton motive force (25).The relative efficacy of lactic acid against gram-negative bacteria is not unexpected considering that as a small watersoluble molecule lactic acid gains access to the periplasm through the water-filled porin proteins of the outer membrane (OM), as reviewed by Nikaido (18). The OM, however, functions as an efficient permeability barrier that is able to exclude macromolecules (such as bacteriocins or enzymes) and hydrophobic substances (i.e., hydrophobic antibiotics). The permeability ...

Uptake of the fluorescent probe 1-N-phenylnaphthylamine (NPN), as adapted to an automated spectrofluorometer enabling multiwell reading of microtitre plates, was applied to determine permeability changes in Gram-negative bacteria. An intact outer membrane is a permeability barrier, and excludes hydrophobic substances such as NPN but, once damaged, it can allow the entry of NPN to the phospholipid layer, resulting in prominent fluorescence. With Escherichia coli O157, Pseudomonas aeruginosa, and Salmonella typhimurium as test organisms and ethylenediaminetetraacetic acid and sodium hexametaphosphate as the model permeabilizers, quantitative and highly reproducible NPN uptake levels were obtained that differed characteristically between the test bacteria. Furthermore, citric acid was shown to be a potent permeabilizer at millimolar concentrations, its effect being partly (Ps. aeruginosa, Salm. typhimurium) or almost totally (E. coli O157) abolished by MgCl2, suggesting that part of the action occurs by chelation. Sodium citrate induced weak NPN uptake, which was totally abolished by MgCl2. In conclusion, the NPN uptake assay with the automated spectrofluorometer serves as a convenient method in analysing and quantifying the effects of external agents, including potential food preservatives, on Gram-negative bacteria.

Antimicrobial activity and mechanisms of phenolic extracts of 12 Nordic berries were studied against selected human pathogenic microbes. The most sensitive bacteria on berry phenolics were Helicobacter pylori and Bacillus cereus. Campylobacter jejuni and Candida albicans were inhibited only with phenolic extracts of cloudberry, raspberry, and strawberry, which all were rich in ellagitannins. Cloudberry extract gave strong microbicidic effects on the basis of plate count with all studied strains. However, fluorescence staining of liquid cultures of virulent Salmonella showed viable cells not detectable by plate count adhering to cloudberry extract, whereas Staphylococcus aureus cells adhered to berry extracts were dead on the basis of their fluorescence and plate count. Phenolic extracts of cloudberry and raspberry disintegrated the outer membrane of examined Salmonella strains as indicated by 1-N-phenylnaphthylamine (NPN) uptake increase and analysis of liberation of [14C]galactose- lipopolysaccharide. Gallic acid effectively permeabilized the tested Salmonella strains, and significant increase in the NPN uptake was recorded. The stability of berry phenolics and their antimicrobial activity in berries stored frozen for a year were examined using Escherichia coli and nonvirulent Salmonella enterica sv. Typhimurium. The amount of phenolic compounds decreased in all berries, but their antimicrobial activity was not influenced accordingly. Cloudberry, in particular, showed constantly strong antimicrobial activity during the storage.