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

Abstract

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.

Keywords: Listeria monocytogenes; Salmonella Typhimurium; biofilms; cold atmospheric plasma; regrowth; resistance and susceptibility; surviving population.

Figure 1

Experimental design used within the presented study. (A) Ability of surviving Cold Atmospheric Plasma (CAP) treated biofilm-associated cells to continue multiplying and/or forming Extracellular Polymeric Substances (EPS). (B) Susceptibility of biofilm-associated cells toward consecutive CAP treatments.

Figure 2

Viable cell density [log (CFU/cm²)] of the untreated, CAP treated, and CAP treated + incubated model biofilms determined on non-selective and selective medium (n = 3). Moreover, the corresponding sub-lethal injury values (average ± stdev) have been presented at the top of the bars. (A) Listeria monocytogenes model biofilms and (B) Salmonella Typhimurium model biofilms.

Figure 3

Optical density (OD; -) following crystal violet staining of the untreated, CAP treated, and CAP treated + incubated model biofilms (n = 5). (A) Listeria monocytogenes model biofilms and (B) Salmonella Typhimurium model biofilms.

Figure 4

(A) Cell density [log₁₀ (CFU/cm²)] and (B) percentage (%) of sub-lethally injured (SI) cells, both as function of the CAP treatment time for the Listeria monocytogenes model biofilms (n = 2). Five consecutive CAP treatment cycles were performed as: isolates from cycle × survivors were used to re-develop mature biofilms, which were again CAP treated (up to 30 min) in cycle x + 1. For the cell density, both the experimental data (symbols) and the global fit (line) of the Geeraerd et al. (2000) model are represented for each CAP treatment cycle: total viable population on non-selective medium (o, solid line) and uninjured viable population on selective medium (x, dashed line). For both the cell density and the percentage of SI, different CAP treatment cycles are indicated in different colors, i.e., black, red, blue, green, and light blue are used to illustrate the results obtained for the 1st, 2nd, 3rd, 4th, and 5th CAP treatment cycle, respectively.

Figure 5

(A) Cell density [log₁₀ (CFU/cm²)] and (B) percentage (%) of SI cells, both as function of the CAP treatment time for the Salmonella Typhimurium model biofilms (n = 2). Five consecutive CAP treatment cycles were performed as: isolates from cycle × survivors were used to re-develop mature biofilms, which were again CAP treated (up to 30 min) in cycle x + 1. For the cell density, both the experimental data (symbols) and the global fit (line) of the Geeraerd et al. (2000) model are represented for each CAP treatment cycle: total viable population on non-selective medium (o, solid line) and uninjured viable population on selective medium (x, dashed line). For both the cell density and the percentage of SI, different CAP treatment cycles are indicated in different colors, i.e., black, red, blue, green, and light blue are used to illustrate the results obtained for the 1st, 2nd, 3rd, 4th, and 5th CAP treatment cycle, respectively.