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. 2020 Jan 28;8(2):184.
doi: 10.3390/microorganisms8020184.

Benzalkonium Chloride Induces a VBNC State in Listeria monocytogenes

Matthias Noll  1 , Katharina Trunzer  1 , Antje Vondran  1 , Szilvia Vincze  2 , Ralf Dieckmann  2 , Sascha Al Dahouk  2 , Carolin Gold  1
Affiliations

Benzalkonium Chloride Induces a VBNC State in Listeria monocytogenes

Matthias Noll et al. Microorganisms. .

Abstract

The objective of our study was to investigate the effects of benzalkonium chloride (BC) adaptation of L. monocytogenes on the susceptibility to antimicrobial agents and on the viable but non culturable (VBNC) state of the bacterial cells. We adapted L. monocytogenes SLCC2540 to BC by applying BC below minimum inhibitory concentration (MIC) to above minimum bactericidal concentration (MBC). The culturable fractions and the susceptibility of adapted and parental cells to BC were assessed. In addition, cell membrane permeability and glucose uptake were analyzed by multi parametric flow cytometry using the fluorescent agents SYTO9, propidium iodide, and 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose (2-NBDG). Adapted cells displayed a two-fold MIC increase of BC and reduced antibiotic susceptibility. At high BC concentrations, the decrease in the number of colony forming units was significantly lower in the population of adapted cells compared to parental cells. At the same time, the number of metabolically active cells with intact membranes was significantly higher than the number of culturable cells. Growth-independent viability assays revealed an adapted subpopulation after BC application that was not culturable, indicating increased abundance of viable but nonculturable (VBNC) cells. Moreover, adapted cells can outcompete non-adapted cells under sublethal concentrations of disinfectants, which may lead to novel public health risks.

Keywords: 2-NBDG; Listeria monocytogenes; VBNC; antibiotic susceptibility; benzalkonium chloride; colony forming units; flow cytometry; metabolic activity; viable but nonculturable.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Colony forming units (A) and SYTO9-positive cells (B) of parent (black bars) and adapted (white bars) Listeria monocytogenes SLCC2540 following cultivation in brain heart infusion broth (BHI) containing benzalkonium chloride (BC) in various concentrations. Error bars indicate standard deviation of nine replicates. Different letters above bars within panels indicate significant differences (p < 0.05) according to two-way analysis of variance (ANOVA).
Figure 2
Figure 2
Mean ratio of PI-negative to SYTO9-positive parent (black bars) and adapted (white bars) Listeria monocytogenes SLCC2540 cells following cultivation in BHI containing benzalkonium chloride (BC) in various concentrations. SYTO9-positive cells are indicative of total cell counts, while PI-negative cells are a surrogate for intact cell membranes. Error bars indicate the standard deviation of three replicates. Different letters above bars within panels represent significant differences (p < 0.05) according to two-way analysis of variance (ANOVA).
Figure 3
Figure 3
Mean ratio of 2-NBDG-positive to SYTO9-positive parent (black bars) and adapted Listeria monocytogenes SLCC2540 cells (white bars) following cultivation in brain heart infusion broth containing benzalkonium chloride (BC) in various concentrations. 2-NBDG-positive cells are indicative of metabolically active cells, while SYTO9-positive cells represent the total number of cells. Error bars show the standard deviation of three replicates. Different letters above bars within panels indicate significant differences (p < 0.05) according to two-way analysis of variance (ANOVA).
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