Food Microstructure and Fat Content Affect Growth Morphology, Growth Kinetics, and Preferred Phase for Cell Growth of Listeria monocytogenes in Fish-Based Model Systems

Food microstructure significantly affects microbial growth dynamics, but knowledge concerning the exact influencing mechanisms at a microscopic scale is limited. The food microstructural influence on Listeria monocytogenes (green fluorescent protein strain) growth at 10°C in fish-based food model systems was investigated by confocal laser scanning microscopy. The model systems had different microstructures, i.e., liquid, xanthan (high-viscosity liquid), aqueous gel, and emulsion and gelled emulsion systems varying in fat content. Bacteria grew as single cells, small aggregates, and microcolonies of different sizes (based on colony radii [size I, 1.5 to 5.0 μm; size II, 5.0 to 10.0 μm; size III, 10.0 to 15.0 μm; and size IV, ≥15 μm]). In the liquid, small aggregates and size I microcolonies were predominantly present, while size II and III microcolonies were predominant in the xanthan and aqueous gel. Cells in the emulsions and gelled emulsions grew in the aqueous phase and on the fat-water interface. A microbial adhesion to solvent assay demonstrated limited bacterial nonpolar solvent affinities, implying that this behavior was probably not caused by cell surface hydrophobicity. In systems containing 1 and 5% fat, the largest cell volume was mainly represented by size I and II microcolonies, while at 10 and 20% fat a few size IV microcolonies comprised nearly the total cell volume. Microscopic results (concerning, e.g., growth morphology, microcolony size, intercolony distances, and the preferred phase for growth) were related to previously obtained macroscopic growth dynamics in the model systems for an L. monocytogenes strain cocktail, leading to more substantiated explanations for the influence of food microstructural aspects on lag phase duration and growth rate. IMPORTANCE Listeria monocytogenes is one of the most hazardous foodborne pathogens due to the high fatality rate of the disease (i.e., listeriosis). In this study, the growth behavior of L. monocytogenes was investigated at a microscopic scale in food model systems that mimic processed fish products (e.g., fish paté and fish soup), and the results were related to macroscopic growth parameters. Many studies have previously focused on the food microstructural influence on microbial growth. The novelty of this work lies in (i) the microscopic investigation of products with a complex composition and/or structure using confocal laser scanning microscopy and (ii) the direct link to the macroscopic level. Growth behavior (i.e., concerning bacterial growth morphology and preferred phase for growth) was more complex than assumed in common macroscopic studies. Consequently, the effectiveness of industrial antimicrobial food preservation technologies (e.g., thermal processing) might be overestimated for certain products, which may have critical food safety implications.acceptedVersio

Behavior of the Surviving Population of Listeria monocytogenes and Salmonella Typhimurium Biofilms Following a Direct Helium-Based Cold Atmospheric Plasma Treatment

Although the Cold Atmospheric Plasma (CAP) technology proved promising for inactivation of biofilms present on abiotic food contact surfaces, more research is required to examine the behavior of the CAP surviving biofilm-associated cells. It was therefore examined whether (i) CAP treated (Listeria monocytogenes and Salmonella Typhimurium) biofilm-associated cells were able to further colonize the already established biofilms during a subsequent incubation period and (ii) isolates of the surviving population became less susceptible toward CAP when the number of biofilm development—CAP treatment cycles increased. For this purpose, a direct treatment was applied using a helium-based Dielectric Barrier Discharge electrode configuration. Results indicated that the surviving population was able to further colonize the already established biofilms, since the cell density of the CAP treated + incubated biofilms equaled the initial density of the untreated biofilms. For the L. monocytogenes biofilms, also the total biomass proved to further increase, which might result in an even further increased resistance. The susceptibility of the biofilm-associated cells proved to be influenced by the specific number of CAP treatment cycles, which might potentially result in an overestimation of the CAP treatment efficacy and, consequently, an increased risk of food contamination

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.status: publishe

Dual-Species Model Biofilm Consisting of Listeria monocytogenes and Salmonella Typhimurium: Development and Inactivation With Cold Atmospheric Plasma (CAP)

Most environmental biofilms contain a variety of species. These species can establish cooperative and competitive interactions, possibly resulting in an increase or a decrease in antimicrobial resistance. Therefore, results obtained following inactivation of single-species biofilms by means of different technologies (e.g., Cold Atmospheric Plasma, CAP) should be validated for multi-species biofilms. First, a strongly adherent and mature Listeria monocytogenes and S. Typhimurium dual-species biofilm was developed by altering different incubation conditions, i.e., growth medium, incubation temperature, inoculum ratio of L. monocytogenes and S. Typhimurium cells, and incubation time. Adherence and maturity were quantified by means of optical density measurements and viable plate counts, respectively. Secondly, both the (1 day old) reference biofilm and a more mature 7 days old biofilm were treated for different CAP treatment times (0–30min). Viable plate counts were again used to determine the (remaining) cell density. For both the biofilm development and inactivation, predictive models were applied to describe the growth/inactivation kinetics. Finally, the kinetics of the [1 and 7 day(s) old] dual-species biofilms were compared with those obtained for the corresponding single-species biofilms. Results implied that a strongly adherent and mature reference dual-species biofilm was obtained following 24 h of incubation at 25◦C using 20-fold diluted TSB and an inoculum ratio of 1:1. Main observations regarding CAP inactivation were: (i) the dual-species biofilm age had no influence on the CAP efficacy, although a longer treatment time was required for the oldest biofilm, (ii) for the 1 day old biofilms, CAP treatment became less efficient for S. Typhimurium inactivation when this species was part of the dual-species biofilm, while L. monocytogenes inactivation was not influenced by the biofilm type, and (iii) for the 7 days old biofilms, CAP inactivation of both species became more efficient when they were part of the dual-species biofilms. It can be concluded that the efficacy of the CAP treatment is altered when cells become part of a dual-species biofilm, which is quite important with respect to a possible application of CAP for biofilm inactivation within the food industry.status: Published onlin

The Complex Effect of Food Matrix Fat Content on Thermal Inactivation of Listeria monocytogenes: Case Study in Emulsion and Gelled Emulsion Model Systems

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Combined Effect of Cold Atmospheric Plasma and Hydrogen Peroxide Treatment on Mature Listeria monocytogenes and Salmonella Typhimurium Biofilms

Cold Atmospheric Plasma (CAP) is a promising novel method for biofilm inactivation as log-reduction values up to 4.0 log10 (CFU/cm2) have been reported. Nevertheless, as the efficacy of CAP itself is not sufficient for complete inactivation of mature biofilms, the hurdle technology could be applied in order to obtain higher combined efficacies. In this study, CAP treatment was combined with a mild hydrogen peroxide (H2O2) treatment for disinfection of 1 and 7 day(s) old Listeria monocytogenes and Salmonella Typhimurium biofilms. Three different treatment sequences were investigated in order to determine the most effective treatment sequence, i.e., (i) first CAP, then H2O2, (ii) first H2O2, then CAP, and (iii) a simultaneous treatment of CAP and H2O2. Removal of the biofilm, induction of sub-lethal injury, and H2O2 breakdown due to the presence of catalase within the biofilms were investigated in order to comment on their possible contribution to the combined inactivation efficacy. Results indicated that the preferred treatment sequence was dependent on the biofilm forming species, biofilm age, and applied H2O2 concentration [0.05 or 0.20% (v/v)]. At the lowest H2O2 concentration, the highest log-reductions were generally observed if the CAP treatment was preceded by the H2O2 treatment, while at the highest H2O2 concentration, the opposite sequence (first CAP, then H2O2) proved to be more effective. Induction of sub-lethal injury contributed to the combined bactericidal effect, while the presence of catalase within the biofilms resulted in an increased resistance. In addition, high log-reductions were partially the result of biofilm removal. The highest overall log-reductions [i.e., up to 5.42 ± 0.33 log10 (CFU/cm2)] were obtained at the highest H2O2 concentration if CAP treatment was followed by H2O2 treatment. As this resulted in almost complete inactivation of the L. monocytogenes and S. Typhimurium biofilms, the combined treatment of CAP and H2O2 proved to be a promising method for disinfection of abiotic surfaces.status: Published onlin