Analysis of Sanitizer Rotation on the Susceptibility, Biofilm Forming Ability and Caco-2 Cell Adhesion and Invasion of Listeria

Abstract

The purpose of this study was to determine the effect of sanitizer use conditions on the susceptibility, biofilm forming ability and pathogenicity of Listeria monocytogenes. Two different strains of L. monocytogenes and a non-pathogenic L. innocua were exposed to sodium hypochlorite, benzalkonium chloride and peroxyacetic acid at different concentrations (4 to 512 ppm) and treatment times (30 s to 5 min), respectively. Under the tested conditions, no significant difference (p > 0.05) in reduction was observed among the three tested sanitizers. A reduction of 1 to 8 log CFU/mL was observed depending upon the sanitizer concentration and treatment times. The survived cells at the highest sublethal concentration and treatment time of a particular sanitizer upon re-exposure to the same or different sanitizer showed either no change or increased susceptibility when compared to parent strains. Upon repeated exposure to sanitizers at progressively increasing concentrations from 1 to 128 ppm, L. innocua was able to survive concentrations of up to 32 ppm benzalkonium chloride and 64 ppm peroxyacetic acid treatments, respectively. At the tested sub-lethal concentrations, no significant difference (p > 0.05) in biofilm formation was observed among the tested strains. Caco-2 interaction with L. innocua showed a reduction in invasion ability with sublethal concentrations of sanitizers.

Keywords: Listeria monocytogenes; biofilm; pathogenicity; repeated exposure; resistance; sanitizers.

Figure 1

Effect of type of strain, type of sanitizer and its concentration, and treatment time on the log reduction. (A–C) Log reduction of L. innocua subjected to different concentrations of sodium hypochlorite (SHC), peroxyacetic acid (PAA), and benzalkonium chloride (BAC), respectively. (D–F) The same sanitizer treatments against L. monocytogenes (101M, Lm-1), respectively. (G–I) The same sanitizer treatments against L. monocytogenes (F8385, Lm-2), respectively. represent treatment times of 30 s, 1, 2.5, and 5 min, respectively.

Figure 2

Measurement of biofilm production ability of different Listeria strains (which were either pre-exposed or not exposed to different sanitizers) in 16 ppm of (a) sodium hypochlorite (SHC), (b) benzalkonium chloride (BAC), and (c) peroxyacetic acid (PAA) for 72 h.

Figure 3

CaCO-2 cell adhesion and invasion of repeatedly sanitizer-exposed L. innocua compared to parent strain. BAC 4, 16, 32 and PAA4, 16, 32 refers to L. innocua cells that were previously subjected to repeated treatment with peroxyacetic acid and benzalkonium chloride at 4, 16 and 32 ppm, respectively. The adhesion and invasion of L. innocua to Caco-2 cells were expressed as a fold change relative to the untreated parent strain. The error bars represent the standard error of the mean. * p < 0.05 and ** p < 0.01.

Figure 4

Preexposure of L. innocua to PAA or BAC decreased its invasiveness into Caco-2 cells. Representative confocal image showing SYOT-9-stained L. innocua bacterial invasion of Caco-2 cells in the parent (A) after exposure to PAA (4 ppm) (B) and BAC (4 ppm) (C). The bottom images in each panel are magnified versions of the square areas depicted in the top images. Scale bar = 30 µm. Green channel: SYTO-9-stained nuclei of L. innocua (excitation 485 nm, emission 501 nm). Blue channel: DAPI-stained nuclei of Caco-2 cells (excitation 359 nm, emission 457 nm). Gray channel: phase contrast, merged: the image of SYTO-9 and DAPI staining and phase contrast were overlayed. Experimental details are described in the Materials and Methods section.