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.

To unravel the biological function of the widely used probiotic bacterium Lactobacillus rhamnosus GG, we compared its 3.0-Mbp genome sequence with the similarly sized genome of L. rhamnosus LC705, an adjunct starter culture exhibiting reduced binding to mucus. Both genomes demonstrated high sequence identity and synteny. However, for both strains, genomic islands, 5 in GG and 4 in LC705, punctuated the colinearity. A significant number of strain-specific genes were predicted in these islands (80 in GG and 72 in LC705). The GG-specific islands included genes coding for bacteriophage components, sugar metabolism and transport, and exopolysaccharide biosynthesis. One island only found in L. rhamnosus GG contained genes for 3 secreted LPXTG-like pilins (spaCBA) and a pilin-dedicated sortase. Using anti-SpaC antibodies, the physical presence of cell wall-bound pili was confirmed by immunoblotting. Immunogold electron microscopy showed that the SpaC pilin is located at the pilus tip but also sporadically throughout the structure. Moreover, the adherence of strain GG to human intestinal mucus was blocked by SpaC antiserum and abolished in a mutant carrying an inactivated spaC gene. Similarly, binding to mucus was demonstrated for the purified SpaC protein. We conclude that the presence of SpaC is essential for the mucus interaction of L. rhamnosus GG and likely explains its ability to persist in the human intestinal tract longer than LC705 during an intervention trial. The presence of mucus-binding pili on the surface of a nonpathogenic Gram-positive bacterial strain reveals a previously undescribed mechanism for the interaction of selected probiotic lactobacilli with host tissues.genome ͉ probiotics ͉ adhesion ͉ pilus ͉ lactic acid bacteria

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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 ...

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