Influence of sublethal concentrations of common disinfectants on expression of virulence genes in Listeria monocytogenes

Abstract

Listeria monocytogenes is a food-borne human pathogen that causes listeriosis, a relatively rare infection with a high fatality rate. The regulation of virulence gene expression is influenced by several environmental factors, and the aim of the present study was to determine how disinfectants used routinely in the food industry affect the expression of different virulence genes in L. monocytogenes when added at sublethal concentrations. An agar-based assay was developed to screen the effect of disinfectants on virulence gene promoter expression and was validated at the transcriptional level by Northern blot analysis. Eleven disinfectants representing four different groups of active components were evaluated in this study. Disinfectants with the same active ingredients had a similar effect on gene expression. Peroxy and chlorine compounds reduced the expression of the virulence genes, and quaternary ammonium compounds (QAC) induced the expression of the virulence genes. In general, a disinfectant had similar effects on the expression of all four virulence genes examined. Northern blot analyses confirmed the downregulation of prfA and inlA expression by Incimaxx DES (a peroxy compound) and their upregulation by Triquart Super (a QAC) in L. monocytogenes EGD. Hence, sublethal concentrations of disinfectants routinely used in the food industry affect virulence gene expression in the human pathogen L. monocytogenes, and the effect depends on the active components of the disinfectant. From a practical perspective, the study underlines that disinfectants should be used at the lethal concentrations recommended by the manufacturers. Further studies are needed to elucidate whether the changes in virulence gene expression induced by the disinfectants have impact on virulence or other biological properties, such as antibiotic resistance.

FIG. 1.

Listeria monocytogenes with lacZ fusions with pprfA, pplcA, pinlA, and phly promoters (from left to right, respectively) diluted 1,000-fold and cast in BHI (upper row) and BHI-AC (lower row). Overfladedesinfektion was added at decreasing concentrations in wells 1 to 6 from right to left. Water was added as control (K) and did not alter the color of the agar. The results presented are representative of two independent experiments.

FIG. 2.

(A) Listeria monocytogenes with a lacZ fusion with the phly promoter diluted 1,000-fold and cast in BHI-AC. Incimaxx DES was added at decreasing concentrations in wells 1 to 6. (B) L. monocytogenes with a lacZ fusion with the pprfA promoter diluted 1,000-fold and cast in BHI. Triquart Super was added at decreasing concentrations in well 1 to 6. The results are representative of two independent experiments.

FIG. 3.

(A) Growth of Listeria monocytogenes EGD in BHI-AC (0.2% charcoal) broth with water (control) (⧫) or 0.25% (▪) or 0.125% (▴) Incimaxx DES. (B) Growth of Listeria monocytogenes EGD in BHI broth with water (control) (⧫) or 0.0031% (▪) or 0.0016% (▴) Triquart Super. The results are representative of two independent experiments.

FIG. 4.

Listeria monocytogenes prfA (A) and inlA (B) transcription measured by Northern blotting using RNA isolated from L. monocytogenes EGD grown in BHI-AC (0.2% charcoal) for 15 min (lanes A, B, and C), 30 min (lanes D, E, and F), 60 min (lanes G, H, and I), or 180 min (lanes J, K, and L) with water (control) (lanes A, D, G, and J) or 0.125% (lanes B, E, H, and K) or 0.250% (lanes C, F, I, and L) Incimaxx DES. The arrows show the plcA-prfA (2.1-kb), prfA (0.8- and 0.9-kb), inlAB (5-kb), and inlA (2.9-kb) transcripts. The results are representative of two independent experiments.

FIG. 5.

prfA transcription measured by Northern blotting using RNA isolated from Listeria monocytogenes EGD grown for 15 min (lanes A, B, and C), 30 min (lanes D, E, and F), 60 min (lanes G, H, and I), or 180 min (lanes J, K, and L) with water (control) (lanes A, D, G, and J) or 0.0031% (lanes B, E, H, and K) or 0.0016% (lanes C, F, I, and L) Triquart Super. The arrows show the sizes of the plcA-prfA (2.1-kb) and prfA (0.8- and 0.9-kb) transcripts. The results are representative of two independent experiments.