Cold Atmospheric Plasma (CAP) for inactivation of pathogenic biofilms - Case study on foodborne Listeria monocytogenes and Salmonella Typhimurium

The last decades, it has become clear that most (pathogenic) bacteria, such as the target microorganisms of this PhD research (i.e., Listeria monocytogenes and Salmonella Typhimurium), grow predominantly as biofilms on abiotic (food) contact surfaces. Biofilms are functional consortia of cells protected by a self-produced matrix of extracellular polymeric substances (EPS). The EPS matrix mainly contains polysaccharides, proteins, and extracellular DNA, and has a variety of functions, e.g., (i) retaining water and nutrients, (ii) keeping the cells attached to the surface, and (iii) limiting the diffusion of antimicrobial agents into the deeper biofilm layers. Hence, biofilm-associated cells are highly resistant towards currently applied methods for disinfection of abiotic food contact surfaces. Therefore, it needs to be examined whether novel surface disinfection methods such as Cold Atmospheric Plasma (CAP) treatment can be a suitable alternative. Plasma is the fourth state of matter and can be created by the addition of energy to a gas. As a result, the gas becomes (partially) ionized and contains a variety of (reactive) species such as ions, photons, free electrons, radicals, and excited (neutral) species. To generate CAP, a specific type of plasma, an electric discharge can be applied to a gas at room temperature and at atmospheric pressure. CAP exhibits some important advantages, i.e., (i) it is fast, (ii) it can be created at a low temperature, and (iii) most primary plasma components fade out immediately after treatment, while some secondary plasma species can remain active for a longer period of time. However, more research is still required to fully assess the efficacy of the CAP technology for inactivation of (complex) biofilms. This PhD research therefore focusses on investigating the influence of different (plasma processing) conditions on CAP's inactivation efficacy and the underlying inactivation mechanism. First, strongly adherent and mature (single-species) model biofilms were developed for two pathogenic species, i.e., L. monocytogenes (Gram positive) and S. Typhimurium (Gram negative). The adherence and the maturity of the biofilms proved to be dependent on the applied growth medium, incubation temperature, and incubation time. Moreover, optimal biofilm formation conditions differed between both species. These reference model biofilms were initially used to examine the influence of different (plasma processing) conditions on the CAP inactivation kinetics and efficacy. For this purpose, model biofilms were treated for different treatment times (0-30 min) and plate counts were used in combination with predictive models to describe the inactivation kinetics for each possible combination of plasma characteristics. Three plasma characteristics were altered, i.e., the composition of the gas flow (helium + 0.0/0.5/1.0% (v/v) oxygen), the electrode configuration (Dielectric Barrier Discharge (DBD) or Surface Barrier Discharge (SBD) electrode), and the plasma intensity (13.88/17.88/21.88 V input voltage). Results indicated that a log-linear inactivation phase was always followed by a tail phase, meaning that a resistant sub-population of cells was present within the single-species reference biofilms. Nevertheless, the inactivation kinetics and efficacy were influenced by the applied plasma characteristics. Overall, the highest log-reduction values (approximately 3.5 log10(CFU/cm²)) were obtained when (i) the feed gas only contained helium, (ii) the DBD electrode was applied, and (iii) the input voltage was set at 21.88 V. These optimal CAP treatment conditions were used to examine the influence of the biofilm maturity and complexity on the CAP inactivation kinetics and efficacy. To obtain more mature biofilms, the reference single-species biofilms (1 day old) were incubated for up to 10 days. To obtain a more complex model biofilm, a dual-species biofilm consisting of both L. monocytogenes and S. Typhimurium cells was developed. As both the kinetics and the efficacy proved to be influenced by the biofilm age and the biofilm complexity, single-species biofilm inactivation results should not be extrapolated to more complex and more mature biofilms without validation. For industrial applications, this also means that the time in between two consecutive surface disinfection cycles, using CAP treatment, needs to be minimized. As an individual optimal CAP treatment did not result in sufficiently high log-reduction values (i.e., the detection limit of approximately 1.0 log10(CFU/cm²) was not reached), a combined treatment was applied in order to possibly obtain complete biofilm inactivation. The single-species reference biofilms were therefore treated with H2O2 (10 min - 0.05 or 0.20% (v/v)) and CAP (10 min - DBD - helium - 21.88 V). H2O2 was selected since this antimicrobial agent is often used for disinfection of abiotic food contact surfaces, either on its own or in combination with other chemicals. In addition, it is generally recognized as safe (GRAS) and previous studies indicated that H2O2 is able to cause damage to DNA, proteins, lipids, and cell membranes. It was determined whether there was an optimal combined treatment sequence, i.e., whether significantly higher log-reduction values were obtained if (i) first CAP, then H2O2, (ii) first H2O2, then CAP, or (iii) a simultaneous treatment was applied. In addition, it was examined whether the (lack of an) increased combined treatment efficacy was (partially) the result of (i) biofilm removal, (ii) the presence of catalase within the model biofilms, and/or (iii) the induction of sub-lethal injury. Especially the latter is of high importance since these sub-lethally injured cells can either become more susceptible or resistant towards a subsequent treatment. Results indicated that some of the examined combined treatments resulted in an increased combined treatment efficacy, while others resulted in a decreased combined treatment efficacy. The increased combined treatment efficacy was deemed to be a consequence of the induction of sub-lethal injury, partial removal of the biofilm, and/or an increased porosity of the biofilm matrix following the first individual treatment. The decreased combined efficacy, especially observed for the simultaneous treatments, was most likely the result of a limited diffusion of the plasma species into the H2O2 solution and the presence of catalase within the model biofilms. When the optimal CAP treatment was followed by the chemical treatment (0.20% (v/v)), the detection limit was reached. Therefore, it was concluded that the implementation of the optimal CAP treatment in an entire surface cleaning schedule can result in an (almost) complete biofilm inactivation. Even though the optimal CAP treatment conditions resulted in promising log-reduction values, either alone or in combination with H2O2, it was examined whether similar log-reduction values can be obtained with an air-based plasma system. This is of high importance since the use of air as feed gas is less expensive. The model biofilms were therefore treated with an air-based SBD electrode and the inactivation kinetics and efficacy were compared with those observed using the helium-operated DBD and SBD set-up. The results of this study proved that the efficacy of the treatment was more influenced by the electrode configuration than by the composition of the feed gas, i.e., (i) similar log-reduction values were obtained using the air-based and helium-operated SBD system and (ii) the helium-operated DBD proved to be more effective than the air-based SBD. Therefore, it was concluded that air can be a suitable substitute for helium, although it would be advised to apply a DBD electrode. Finally, it was examined how the (plasma processing) conditions had an influence on the biofilm inactivation mechanism of CAP. For this purpose, it was assessed for each of the combinations whether the biofilm-associated cells were inactivated due to (i) the generation of UV photons, (ii) the presence of reactive oxygen and nitrogen species (ROS and RNS), and/or (iii) a drop in pH. Moreover, it was investigated whether the inactivation was attributed to membrane damage and/or damage to the intracellular DNA. In order to comment on the influence of the Gram type on the CAP inactivation mechanism for biofilms, both single-species model biofilms (i.e., developed by L. monocytogenes (Gram positive) and S. Typhimurium (Gram negative)) were CAP treated. Results indicated that the generation of CAP species was indeed influenced by the applied plasma conditions and that the lethal effect of CAP was the result of both damage to the membrane and the DNA of the cells. Nevertheless, membrane damage was deemed to be more important. In addition, the Gram type of the biofilm forming species had no major effect on the CAP inactivation mechanism. This increased knowledge concerning the biofilm inactivation mechanism of CAP is essential for the further optimization of this technology for surface disinfection.

Jaar van publicatie:2020

Toegankelijkheid:Open