Membrane Toxicity of Antimicrobial Compounds from Essential Oils ROSANGELA DI PASQUA, ²,‡ GAIL BETTS, ‡ NIKKI HOSKINS, ‡ MIKE EDWARDS, ‡ DANILO ERCOLINI, ² AND GIANLUIGI MAURIELLO* ,² Department of Food Science, Division of Microbiology, University of Naples Federico II, Via Universita ` 100, 80055 Portici, Italy, and Campden & Chorleywood Food Research Association, Chipping Campden, Gloucestershire, GL55 6LD, United Kingdom Natural antimicrobial compounds perform their action mainly against cell membranes. The aim of this work was to evaluate the interaction, meant as a mechanism of action, of essential oil antimicrobial compounds with the microbial cell envelope. The lipid profiles of Escherichia coli O157:H7, Staphylococcus aureus, Salmonella enterica serovar Typhimurium, Pseudomonas fluorescens, and Brochothrix thermosphacta cells treated with thymol, carvacrol, limonene, eugenol, and cinnamal- dehyde have been analyzed by gas chromatography. In line with the fatty acids analysis, the treated cells were also observed by scanning electron microscopy (SEM) to evaluate structural alterations. The overall results showed a strong decrease of the unsaturated fatty acids (UFAs) for the treated cells; in particular, the C18:2trans and C18:3cis underwent a notable reduction contributing to the total UFA decreases, while the saturated fatty acid C17:0 raised the highest concentration in cinnamaldehyde-treated cells. SEM images showed that the used antimicrobial compounds quickly exerted their antimicrobial activities, determining structural alterations of the cell envelope. KEYWORDS: Essential oils; natural antimicrobial compounds; cell envelope alterations; scanning electron microscopy INTRODUCTION The antimicrobial activities of some compounds known as constituents of essential oils (EOs) have already been fully discussed by many authors (1-4). The major active EO components are phenols, terpenes, and aldehydes (5), and it is also well-known that the action of these substances is principally performed against the cell cytoplasmic membrane (5-9). Among the EO constituents, the phenolic compounds were found to possess major antimicrobial activities (10). It seems that the presence of the hydroxyl group is related to the inactivation of the microbial enzymes. Most probably, this group interacts with the cell membrane causing leakage of cellular components, a change in fatty acids and phospholipids, and impairment of the energy metabolism and influencing genetic material synthesis (5). Similarly to phenolic compounds, the site of action of the terpenes is the cell membrane. They permeate through the membranes causing them to swell, thus inhibiting respiratory enzymes and causing partial dissipation of the pH gradient and electrical potential (7). Cinnamaldehyde has the highest antifungal activity among aliphatic aldehydes (5), and according to Helander et al. (2), it gains access to the periplasm and to the deeper parts of the cells but does not result in the disintegration of the outer membrane as in the case of carvacrol and thymol action. On the other hand, Gill and Holley (3) hypothesized a mechanism for cinnamaldehyde activity in which the interaction with the cell membrane causes disruption sufficient to disperse the proton motive force by leakage of small ions without leakage of larger cell components, such as ATP. In this study, we evaluated the membrane damage by analyzing the fatty acid profiles after exposure of Salmonella enterica serovar Typhimurium, Escherichia coli O157: H7, Pseudomonas fluorescence, Brochotrix thermosphacta, and Staphylococcus aureus cells to a high concentration of EO constituents. In addition, scanning electron microscope observa- tions were performed to investigate the occurrence of surface damage on the treated cultures. * To whom correspondence should be addressed. Tel: +390812539452. Fax: +390812539407. E-mail: giamauri@unina.it. ² University of Naples Federico II. ‡ Campden & Chorleywood Food Research Association. Table 1. Bacterial Strains Used in This Study a species strain E. coli O157:H7 (nontoxigenic) ATCC 43888 S. enterica serovar Typhimurium ATCC 14028 P. fluorescens NCIMB 10586 B. thermosphacta NCTC 10822 S. aureus NCTC 6571 a ATCC, American Type Culture Collection; NCIMB, National Collection of Industrial Food and Marine Bacteria; and NCTC, National Collection of Type Cultures. J. Agric. Food Chem. 2007, 55, 4863-4870 4863 10.1021/jf0636465 CCC: $37.00 © 2007 American Chemical Society Published on Web 05/12/2007