In Vitro Virulence Potential, Surface Attachment, and Transcriptional Response of Sublethally Injured Listeria monocytogenes following Exposure to Peracetic Acid

Abstract

The disinfectant peracetic acid (PAA) can cause high levels of sublethal injury to Listeria monocytogenes. This study aims to evaluate phenotypic and transcriptional characteristics concerning the surface attachment and virulence potential of sublethally injured L. monocytogenes ScottA and EGDe after exposure to 0.75 ppm PAA for 90 min at 4°C and subsequent incubation in tryptic soy broth supplemented with yeast extract (TSBY) at 4°C. The results showed that injured L. monocytogenes cells (99% of the total population) were able to attach (after 2 and 24 h) to stainless steel coupons at 4°C and 20°C. In vitro virulence assays using human intestinal epithelial Caco-2 cells showed that injured L. monocytogenes could invade host cells but could not proliferate intracellularly. The in vitro virulence response was strain dependent; injured ScottA was more invasive than EGDe. Assessment of PAA injury at the transcriptional level showed the upregulation of genes (motB and flaA) involved in flagellum motility and surface attachment. The transcriptional responses of L. monocytogenes EGDe and ScottA were different: only injured ScottA demonstrated upregulation of the virulence genes inlA and plcA. Downregulation of the stress-related genes fri and kat and upregulation of lmo0669 were observed in injured ScottA. The obtained results indicate that sublethally injured L. monocytogenes cells may retain part of their virulence properties as well as their ability to adhere to food-processing surfaces. Transmission to food products and the introduction of these cells into the food chain are therefore plausible scenarios that are worth taking into consideration in terms of risk assessment. IMPORTANCE L. monocytogenes is the causative agent of listeriosis, a serious foodborne illness. Antimicrobial practices such as disinfectants used for the elimination of this pathogen in the food industry can produce a sublethally injured population fraction. Injured cells of this pathogen that may survive antimicrobial treatment may pose a food safety risk. Nevertheless, knowledge regarding how sublethal injury may impact important cellular traits and phenotypic responses of this pathogen is limited. This work suggests that sublethally injured L. monocytogenes cells maintain virulence and surface attachment potential and highlights the importance of the occurrence of sublethally injured cells regarding food safety.

Keywords: Listeria monocytogenes; cell invasion; disinfectant; intracellular growth; oxidative stress genes; pathogenicity; sublethal damage; surface adhesion; virulence genes.

FIG 1

Populations of L. monocytogenes ScottA (A) and EGDe (B) in PAA for 90 min followed by TSBY for 30 min at 4°C. Gray bars represent counts on TSAY (primary axis). Triangles (secondary axis) demonstrate the percentage of sublethal injury.

FIG 2

In vitro virulence potential of L. monocytogenes. The invasion efficiency (A) and intracellular growth (B) of L. monocytogenes ScottA and EGDe were evaluated for unstressed cells that were exposed in MW (black bars) or MW-TSBY (light gray bars) and injured cells that were exposed in PAA (dark gray bars) or PAA-TSBY (white bars). Virulence assays were performed using Caco-2 cells that were infected for 1 h with L. monocytogenes and incubated for 45 min (invasion) or 4 h (intracellular proliferation) in the presence of gentamicin. Invasion data are expressed as percentages of bacteria recovered after 45 min of incubation in the presence of gentamicin compared to the initial inoculum. The IGC was calculated as the number of intracellular bacteria after 4 h minus the number of bacteria recovered after 45 min divided by the number of bacteria recovered after 45 min. Data presented are mean values ± standard errors from three biological replicates performed in triplicate. * indicates statistically significant differences between ScottA and EGDe under each experimental condition. Different letters indicate statistically significant differences among experimental conditions for each strain.

FIG 3

Adhesion capacity of L. monocytogenes ScottA (A and C) and EGDe (B and D) 2-h-attached and 24-h-attached populations (log CFU per square centimeter) on stainless steel coupons at 20°C (A and B) and 4°C (C and D). The adhesion assay was evaluated for (i) unstressed cells that were exposed in MW (light gray bars) (control) and (ii) stressed cells exposed to PAA (dark gray bars). Population data are represented by bars corresponding to the primary axis. Triangles (secondary axis) demonstrate the percentage of sublethal injury of the attached population due to previous exposure to PAA (0.75 ppm at 4°C for 90 min). Data presented are mean values ± standard deviations from three biological replicates performed in duplicate. * indicates statistically significant differences of PAA from the control (MW).

FIG 4

Relative transcription of genes involved in virulence and motility in ScottA (dark gray bars) and EGDe (light gray bars) exposed to PAA and PAA-TSBY at 4°C. Data were normalized to the reference gene (tpi), and relative gene transcription was estimated in comparison to the respective controls (MW and MW-TSBY at 4°C). Error bars represent standard deviations of the means for three biological and two technical replicates. Stars indicate statistically significant up- or downregulation (greater than 1 or less than −1) of genes in PAA-exposed L. monocytogenes cells compared to nonexposed L. monocytogenes cells. Letters indicate significant changes in the regulation of genes after TSBY incubation. Lowercase letters correspond to ScottA genes, and uppercase letters correspond to EGDe genes. Different letters for the same strain indicate a significant difference.

FIG 5

Relative transcription of genes involved in the general and oxidative stress responses in ScottA (dark gray bars) and EGDe (light gray bars) exposed to PAA and PAA-TSBY at 4°C. Data were normalized to the reference gene (tpi), and relative gene transcription was estimated in comparison to the respective controls (MW and MW-TSBY at 4°C). Error bars represent standard deviations of the means for three biological and two technical replicates. Stars indicate statistically significant up- or downregulation (greater than 1 or less than −1) of genes in PAA-exposed L. monocytogenes cells compared to nonexposed L. monocytogenes. Letters indicate a significant change in the regulation of genes after TSBY incubation. Lowercase letters correspond to ScottA genes, and uppercase letters correspond to EGDe genes. Different letters for the same strain indicate a significant difference.