This study investigated the in vitro mechanism of action of a commercial citrus EO, Brazilian orange terpenes (BOT), on an enterotoxigenic Escherichia coli (ETEC) isolated from pig gut and on Lactobacillus rhamnosus. Firstly, bacteria were exposed sequentially to BOT every 3 h (three times) at sub-minimal inhibitory concentrations and results showed that sequential exposure to BOT provoked a higher reduction of bacteria viability than a single exposure and the reduction of ETEC viability was higher compared to that of L. rhamnosus. Then, evaluation of the BOT effects on the cell membrane permeability and integrity, indicated that BOT increased the membrane permeability and caused disruptive effects on the integrity of bacterial cells as reflected by an increase of the relative electric conductivity and the release of essential cell constituents. Interestingly, BOT effects were more pronounced on the ETEC than on L. rhamnosus. This was ratified by scanning electron microscopy, which showed more noticeable morphological damages and disturbances on ETEC cells than on the L. rhamnosus cells. Limonene was detected as the major compound in BOT by polar/nonpolar GC-MS (78·65%/79·38%). Results revealed that the probable mechanism of the selective antibacterial action of the citrus EO, BOT, can be described as altering more remarkable the permeability and integrity of the cytoplasmic membrane as well as the external structure of ETEC cells than L. rhamnosus cells. This study provides information about the mechanism of antibacterial action displayed by a citrus EO, a by-product of the citrus processing industry, as a natural alternative to antibiotics used in pig production sector to combat pathogens such as ETECs.
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