Increased Adhesion of Listeria monocytogenes Strains to Abiotic Surfaces under Cold Stress

Abstract

Food contamination by Listeria monocytogenes remains a major concern for some food processing chains, particularly for ready-to-eat foods, including processed foods. Bacterial adhesion on both biotic and abiotic surfaces is a source of contamination by pathogens that have become more tolerant or even persistent in food processing environments, including in the presence of adverse conditions such as cold and dehydration. The most distinct challenge that bacteria confront upon entry into food processing environments is the sudden downshift in temperature, and the resulting phenotypic effects are of interest. Crystal violet staining and the BioFilm Ring Test^® were applied to assess the adhesion and biofilm formation of 22 listerial strains from different serogroups and origins under cold-stressed and cold-adapted conditions. The physicochemical properties of the bacterial surface were studied using the microbial adhesion to solvent technique. Scanning electron microscopy was performed to visualize cell morphology and biofilm structure. The results showed that adhesion to stainless-steel and polystyrene was increased by cold stress, whereas cold-adapted cells remained primarily in planktonic form. Bacterial cell surfaces exhibited electron-donating properties regardless of incubation temperature and became more hydrophilic as temperature decreased from 37 to 4°C. Moreover, the adhesion of cells grown at 4°C correlated with affinity for ethyl acetate, indicating the role of cell surface properties in adhesion.

Keywords: BRT®; Listeria monocytogenes; MATS; SEM; adhesion; biofilm; cold stress; crystal violet staining.

FIGURE 1

Experimental scheme. Time frame for incubation at 37 or 4°C is shown, with arrows indicating cells in stationary phase used for experiments. Each box (A–C) contains the temperatures and names used for sample conditions, with a list of experiments in parentheses.

FIGURE 2

Increased adhesion of cold-stressed cells compared to cold-adapted cells, measured by BRT^®. Sudden exposure to cold for the first time was denoted as cold-stressed cells (A,B), and exposure for a second time was denoted as cold-stressed cells² (C,D); initial inocula were at an OD₆₀₀ of 0.5 (A,C) and 0.17 (B,D). Strains and serogroups are indicated on the X-axis, and data are presented as the mean ± standard deviation of the BFI. A BFI of 0 represents full blockage of the magnetic beads. ^∗p < 0.05.

FIGURE 3

Cold-stressed cells form more biomass than cold-adapted cells (A), while cold-adapted cells exceed total cell densities (B) as assessed using microtiter plate assay (MPA). Adherent cells are quantified by CV staining (A), and total cell densities combining planktonic and sessile cells are measured based on the turbidity of wells (B). Strains and serogroups are indicated on the X-axis, and data are presented as the mean ± standard deviation. ^∗p < 0.05.

FIGURE 4

Solvent affinities (%) of all 22 Listeria monocytogenes strains in stationary phase at 37 and 4°C. Box plot whiskers indicate minimum and maximum values, and the line in the middle of the box is plotted at the median. The affinity for each solvent under two temperatures differs significantly (p < 0.01).

FIGURE 5

Correlation (r² = 0.3055, p < 0.01) between affinity for ethyl acetate and quantification of adherent cells, measured by CV staining of cold-adapted cells.

FIGURE 6

Comparison of L. monocytogenes biofilm formation under different conditions. Positive control cells demonstrated great variance in terms of interstrain biofilm formation (left column), while cold-stressed cells formed a single biofilm layer with cell aggregates (arrowhead) (middle column), and cold-adapted cells adhered sparsely on the surface (right column). Scale bars: 10 μm.

FIGURE 7

Observation of L. monocytogenes adhesion patterns and cellular morphology. (A–F) Cold-stressed cells; (G–K) cold-adapted cells; (L) positive control cells. EPS are marked in red circles, arrows indicate elongated cells and arrowheads indicate cells in crevices. A scale bar (length in μm) is indicated in yellow in each figure.