Effects of origanun vulgare on physiological functions of salmonella enteritidis sessile cells in mature biofilms

Introduction: The survival of Salmonella Enteritidis in the food chain is related to its ability to form biofilms on surfaces. Origanum vulgare L essential oil (OVEO) is suggested as a sustainable disinfectant to replace chloride derived agents because of its strong antimicrobial activity. However, little is known about the action mechanism of OVEO on Salmonella Enteritidis sessile cells in mature biofilms. Purpose: This study assesses the effects of OVEO on physiological functions and counts of Salmonella Enteritidis sessile cells of mature biofilms formed on food-contact surfaces. Methods: Food-contact stainless steel surfaces (2 by 2 by 0.2 cm) were immersed in brain heart infusion broth (BHI) inoculated with Salmonella Enteritidis (final count six log CFU/mL) and incubated at 37°C. After 72 h, surfaces were washed twice with distilled water and exposed to OVEO (2.4 μl/ml), NaCLO (250 mg/ml) or distilled water for 15 min. Afterward, coupons were again washed and submitted to ultrasound (40 kHz, five min). Cells were resuspended in PBS and labeled with the fluorochromes: propidium iodide for membrane integrity; bis- (1,3-dibutyl barbituric acid) trimethine oxonol for membrane potential and ethidium bromide for efflux activity, all measured by flow cytometry (FC). Resuspended cells were also serially diluted and enumerated on BHI agar. Results: No viable sessile cells were recovered from surfaces exposed to OVEO or NaClO (less than one log CFU/ cm ). Among the non-cultivable sessile cells exposed to OVEO or NaClO, ~40% and 80% showed compromised membranes and efflux activity, respectively. Around 20% of the non-cultivable sessile cells exposed to OVEO showed depolarized membrane, while 51.6% of those exposed to NaClO were depolarized. No physiological damages related to membrane or efflux activity was observed in >90% of sessile cells exposed to distilled water. Significance: OVEO compromised the viability of Salmonella Enteritidis sessile cells in mature biofilms formed on stainless steel surfaces by affecting the membrane function and the efflux activity.