(177m) Unravelling the Mechanisms of Resistance of Escherichia coli, Salmonella, and Listeria biofilms to Cold Atmospheric Plasma, As Affected By Age and Surface Physico-Chemistry

Introduction:

There is an increasing demand for effective decontamination of food with minimal processing techniques such as the use of cold-atmospheric plasma (CAP), to ensure high product quality¹. CAP is a partly ionized non-thermal plasma generated at atmospheric pressure which creates reactive neutral species that can damage and inactivate bacterial cells². Bacteria can grow on food surfaces as biofilms were the cells secrete exopolysaccharide (EPS) matrix that acts as a protective layer. The susceptibility of biofilms to antimicrobials is known to depend on the stage of biofilm maturity³. So far, there is no systematic study that investigates the impact of surface physicochemical properties of food systems on the biofilm’s response to CAP. Recently, we have developed biphasic gel systems that mimic the structure of real food and it was found that there was a phase preferential localization and growth of bacteria on their surface⁴.

The aim of this study is to investigate the effect of CAP on Gram-positive Escherichia coli and Salmonella, and Gram-negative Listeria monocytogenes biofilms of variable age and to investigate how the different surface physicochemical properties may affect the efficacy of CAP.

Methodology:

E. coli, Salmonella and L. monocytogenes (10³-10⁴CFU/mL) were used to contaminate the surface of tryptic soy agar (TSA) or our previously developed biphasic Xanthan gum/Whey protein-based viscoelastic systems⁴. Thereafter, the samples were incubated at 37^oC for 2, 4, 6 and 24-hours to form biofilms of different maturity stages. The biofilms were exposed to CAP (120 seconds) and viability was assessed by plate counts. Furthermore, flow cytometric analysis was employed to quantify live/injured cells and reactive oxygen species (ROS). Confocal fluorescence microscopy was used to measure the thickness of the biofilms and to study the localization/ spatial distribution of dead and/or injured cells within the biofilm and at different locations of the viscoelastic models while Scanning Electron Microscopy (SEM) was used to observe morphological changes and membrane damage at single cell level.

Results:

The antibacterial effects of CAP on biofilms was shown to be species, age and surface dependent. Generally, more EPS was secreted in the mixed biphasic gel as compared to the monophasic gel. Early stage biofilms were the most susceptible to CAP showing an increase in percentage of dead and injured cells while no antibacterial effects were observed in 6-hour(E. coli and Salmonella) and 24-hour (L. monocytogenes) biofilms. The results indicate that E. coli and Salmonella biofilms were more resistant to CAP in comparison to L. monocytogenes biofilm. Moreover, the survival of biofilms was correlated with an increase of the EPS thickness. However, re-suspended cells from un-treated late stage biofilms were completely inactivated after CAP treatment. In comparison, planktonic cells of a similar number of viable cells compared to late stage un-treated biofilms at each stage of maturity were completely inactivated after CAP treatment further confirming the protective role of EPS. The mechanisms of antibacterial activity involved elevated levels of intracellular ROS and damage to cellular membranes was observed using SEM.

Conclusions and relevance:

Our findings show that the susceptibility of biofilms to CAP was species, age and surface type dependent. Furthermore, the biofilm resistance to plasma depended on the EPS layer thickness and not on the physiological status of the cells which composed the biofilm. Our results present the first systematic study of the response of biofilms of various maturity levels to CAP as affected by surface structure. These results contribute to a better understanding of intrinsic (species, age, EPS secretion) and extrinsic (surface physicochemistry) factors which affect the response of biofilms to CAP. Therefore, our findings are of significance to the food industry for the effective decontamination of food products using CAP.

Acknowledgements: This work was supported by the National Biofilm Innovation Centre, the Royal Society, the EPSRC and the Department of Chemical and Process Engineering of the University of Surrey.

References:

(1) Baka, M. et al, 2015. Food.Res.Int. 70, 94-100.

(2) Modic, M. el at, 2017. Int.J.Antimicrobial.Agents. 49, 375-378.

(3) Pandit et al., 2015. Oral.Disease. 21, 565-571

(4) Costello, K.M. et al, 2018. Int.J.Food.Microbiol. 286,15-30.