Bacterial radiosensitization by using radiation processing in combination with essential oil: Mechanism of action

Abstract

Spice extracts under the form of essential oils were tested for their efficiency to increase the relative radiosensitivity of Listeria monocytogenes and Escherichia coli O157H7 in culture media. The two pathogens were treated by gamma-irradiation alone or in combination with oregano essential oil to evaluate their mechanism of action. The membrane murein composition, and the intracellular and extracellular concentration of ATP was determined. The bacterial strains were treated with two irradiation doses: 1.2 kGy to induce cell damage and 3.5 kGy to cause cell death for L. monocytogenes. A dose of 0.4 kGy to induce cell damages, 1.1 kGy to obtain viable but nonculturable (VBNC) state and 1.3 kGy to obtain a lethal dose was also applied on E. coli O157H7. Oregano essential oil was used at 0.020% and 0.025% (w/v), which is the minimum inhibitory concentration (MIC) for L. monocytogenes. For E. coli O157H7, a concentration of 0.006% and 0.025% (w/v) which is the minimum inhibitory concentration was applied. The use of essential oils in combination with irradiation has permitted an increase of the bacterial radiosensitization by more than 3.1 times. All treatments had also a significant effect (p⩽0.05) on the murein composition, although some muropeptides did not seem to be affected by the treatment. Each treatment influenced differently the relative percentage and number of muropeptides. There was a significant (p⩽0.05) correlation between the reduction of intracellular ATP and increase in extracellular ATP following treatment of the cells with oregano oil. The reduction of intracellular ATP was even more important when essential oil was combined with irradiation, but irradiation of L. monocytogenes alone induced a significant decrease (p⩽0.05) of the internal ATP without affecting the external ATP.

Introduction

New trends in food irradiation technology consist to develop combined treatments to reduce the irradiation doses required to kill pathogenic bacteria and/or reduce overall microbial load (Caillet et al., 2006a, Caillet et al., 2006b; Lafortune et al., 2005). It was demonstrated that the combination of natural products and irradiation had a positive effect in increasing the radiosensitization of bacteria (Caillet et al., 2006a, Caillet et al., 2006b; Chiasson et al., 2004; Mahrour et al., 2003). The use of essential oils from spices as antibacterial compounds seem to be an interesting way to control the presence of pathogenic bacteria and/or to extend the shelf life of processed foods (Oussalah et al., 2004, Oussalah et al., 2007). Among these essential oils, oregano oil has been shown to possess antimicrobial activities and could serve as a source of antimicrobial agent against L. monocytogenes (Oussalah et al., 2007). Essential oil can produce protein denaturation and membrane structure disruption, leading to release of intracellular ATP and cell death (Rhayour et al., 2003). Although the actions of essential oils on the cell membrane and the release of cell components were already studied (Oussalah et al., 2007; Rhayour et al., 2003), their actions and the radiation effects on bacterial murein (peptidoglycan) are less well known. Most bacteria are enveloped with a rigid exoskeleton, called murein sacculus, allowing them to maintain their typical shape in spite of a high osmotic pressure inside the cell. Because the peptidoglycan sacculus is unique to bacteria and important for their survival, it is important to determine the effect of antimicrobial agents or treatments on murein walls and cell shape. The objective of this study was to evaluate the effect of three treatments, oregano essential oil alone, irradiation alone and a combination of oregano essential oil and irradiation, on the peptidoglycans of Escherichia coli 0157:H7 and L. monocytogenes murein. The antimicrobial mechanism was also examined by monitoring the release of ATP.