Plasma-Treated Water Affects Listeria monocytogenes Vitality and Biofilm Structure

Abstract

Background: Plasma-generated compounds (PGCs) such as plasma-processed air (PPA) or plasma-treated water (PTW) offer an increasingly important alternative for the control of microorganisms in hard-to-reach areas found in several industrial applications including the food industry. To this end, we studied the antimicrobial capacity of PTW on the vitality and biofilm formation of Listeria monocytogenes, a common foodborne pathogen. Results: Using a microwave plasma (MidiPLexc), 10 ml of deionized water was treated for 100, 300, and 900 s (pre-treatment time), after which the bacterial biofilm was exposed to the PTW for 1, 3, and 5 min (post-treatment time) for each pre-treatment time, separately. Colony-forming units (CFU) were significantly reduced by 4.7 log₁₀ ± 0.29 log₁₀, as well as the metabolic activity decreased by 47.9 ± 9.47% and the cell vitality by 69.5 ± 2.1%, compared to the control biofilms. LIVE/DEAD staining and fluorescence microscopy showed a positive correlation between treatment and incubation times, as well as reduction in vitality. Atomic force microscopy (AFM) indicated changes in the structure quality of the bacterial biofilm. Conclusion: These results indicate a promising antimicrobial impact of plasma-treated water on Listeria monocytogenes, which may lead to more targeted applications of plasma decontamination in the food industry in the future.

Keywords: MidiPLexc; PTW; antimicrobial; cold plasma; food production industry; sustainability; viability.

FIGURE 1

Schematic illustration of the MidiPLexc. The image shows the MidiPLexc with a 1 l glass bottle connected to the bottle adapter below the plasma source as a 3D model. Image is modified according to Handorf et al. (2020a).

FIGURE 2

Colony-formingun its of Listeria monocytogenes biofilms after PTW treatment. The graph shows the colony forming units of Listeria monocytogenes biofilms before and after plasma treatment. The data are presented in the form of a grouped bar chart where the values on the x-axis represent the different pre-treatment times and the different bars represent the various post-treatment times. The experiment was performed in four independent experiments with three technical replicates each. *p ≤ 0.05 and **p ≤0.005, tested with ANOVA. All treated groups were testes in relation to the control group.

FIGURE 3

LIVE/DEAD assay of Listeria monocytogenes biofilms after PTW treatment. The figure shows the LIVE/DEAD assay of Listeria monocytogenes biofilms after treatment with plasma-treated water. The different bars show the control biofilms and the different post-treatment times and the x-axis shows the different pre-treatment times of the water. The experiment was performed in four independent experiments with three technical replicates each. *p ≤ 0.05 and ***p ≤ 0.0005, tested with ANOVA. All treated groups were testes in relation to the control group.

FIGURE 4

XTT assay of Listeria monocytogenes biofilms after PTW treatment. The influence on the metabolic activity of the biofilms after treatment with plasma treated water is shown in this image. The grouped bar chart shows the different post-treatment times and the x-axis shows the different pre-treatment times of the biofilms. The experiment was performed in four independent experiments with three technical replicates each. *p ≤ 0.05 and ***p ≤ 0.0005, tested with ANOVA. All treated groups were testes in relation to the control group.

FIGURE 5

Comparison of the effects of PTW, ClO₂, 10 M HCl, and 70% Alcohol against L. monocytogenes biofilms investigating CFU, Fluorescence and XTT assay and influence of the pH value concerning these effects. (A) CFU assay of L. monocytogenes biofilms after treatment with different chemicals frequently used in the food industry with their own pH value after preparation and compared with the results of the chemicals adapted to the pH value of the PTW of 1.271. (B) Fluorescence assay of L. monocytogenes biofilms (C) XTT assay of L. monocytogenes biofilms. *p ≤ 0.05, **p ≤ 0.005, and ***p ≤ 0.0005, tested with ANOVA. All treated groups were testes in relation to the control group.

FIGURE 6

Fluorescence microscopy of Listeria monocytogenes biofilms after PTW treatment. The images show inverse footage of the Listeria monocytogenes biofilms. The pre-treatment time defines the time period in which the water came into contact with the plasma gas. The post-treatment time is the period of time where the PTW came into contact with the biofilm. The biofilms are stained with SYTO9 (green) for living cells and propidium iodide (PI) (red) for dead cells. (A) Control biofilm (B) 100 s pre-treatment, 1 min post-treatment (C) 100 s pre-treatment time, 3 min post-treatment time (D) 100 s pre-treatment, 5 min post-treatment (E) 300 s pre-treatment, 1 min post-treatment (F) 300 s pre-treatment, 3 min post-treatment (G) 300 s pre-treatment, 5 min post-treatment (H) 900 s pre-treatment, 1 min post-treatment (I) 900 s pre-treatment, 3 min post-treatment (J) 900 s pre-treatment, 5 min post-treatment.

FIGURE 7

Confocal laser scanning microscopy (CLSM) of Listeria monocytogenes biofilms after PTW treatment. The picture shows the untreated control biofilm and the Listeria monocytogenes biofilms processed with plasma-treated water in confocal laser scanning microscopy (CLSM). Left panels show a topographical view of the biofilm layer (height view of the biofilms in μm). Central and right panels show 3D images with a top and a bottom view of the biofilms, respectively. The pre-treatment time is the processing time of the water by the plasma source (MidiPLexc) and the post-treatment time is the contact time of the PTW with the biofilm. For each biofilm, an area of 100 × 100 μm is visualized.

FIGURE 8

Atomic-force microscopy (AFM) of Listeria monocytogenes biofilms after PTW treatment. AFM images of Listeria monocytogenes biofilms. Left) topographical images right) error images. The pre-treatment time defines the period in which the water were exposed to the plasma gas. The post-treatment time represents the period, where the PTW were exposed to the biofilm. (A) Control biofilm (B) 100 s pre-treatment, 5 min post-treatment (C) 300 s pre-treatment, 5 min post-treatment (D) 900 s pre-treatment, 5 min post-treatment.

FIGURE 9

Scanning-electron microscopy (SEM) of Listeria monocytogenes biofilms after PTW treatment. SEM images of Listeria monocytogenes biofilms. (A) Control biofilm (B) 100 s pre-treatment, 5 min post-treatment (C) 300 s pre-treatment, 5 min post-treatment (D) 900 s pre-treatment, 5 min post-treatment. The red arrows indicated cells with membrane damages.