Physiological damages caused to cells of Salmonella Enteritidis PT4 by continuous exposure to Mint (Mentha piperita L.) essential oil

Introduction: The Mentha piperita L. essential oil (MPEO) is a “green” antimicrobial. The activity of MPEO against Salmonella has been primarily associated with changes to cell membrane permeability, which may cause leakage of vital intracellular components. Little is known about the effects of continuous exposure of Salmonella cells to MPEO. Purpose: This study evaluates the effects of continuous exposure to MPEO on the physiology of a strain of Salmonella Enteritidis PT4 isolated from chicken meat associated with foodborne disease outbreaks. Methods: Salmonella Enteritidis PT4 cells (108 log CFU/ml) were exposed to 2.5 µl/ml of MPEO in brain heart infusion (BHI) broth over 252 h in 14 cycles of 18 h exposure. Cells were harvested by centrifugation (4500g×10 min, 4°C), washed twice and resuspended in PBS after every two cycles of 18 h exposure. Cells were labeled with propidium iodide for membrane integrity; bis-1,3-dibutyl barbituric acid for membrane potential and ethidium bromide for efflux activity all measured by flow cytometry. One hundred µl aliquots were also serially diluted and viable cell counts enumerated on BHI agar. The same procedures were performed with cells not exposed to MPEO. Results: After 72 h, ~95% of Salmonella Enteritidis PT4 cells exposed to MPEO presented depolarized membranes, 41% showed compromised efflux activity and 28% had damaged membranes. Cells not exposed to MPEO remained largely polarized with normal efflux pump activity and intact membranes. A decrease of approximately 0.5 log CFU/ml was observed over 252 h. Increasing exposure time reduced the population of cells with a compromised membrane or efflux activity but did not change the population of viable cells. Significance: These results show that MPEO exerts inhibitory effects through a multi-target mechanism in Salmonella Enteritidis PT4 cells, but continuous exposure appears to create a subpopulation of cells able to repair the injuries.