Antimicrobial activity of resveratrol-derived monomers and dimers against foodborne pathogens

Abstract

Plant polyphenolic compounds are considered a promising source for new antibacterial agents. In this study, we evaluated the antimicrobial activity of a collection of resveratrol-derived monomers and dimers screened as single molecules against a panel of nine foodborne pathogens. The results demonstrated that two monomers (i.e., pterostilbene 2 and (E)-3-hydroxy-4',5-dimethoxystilbene 9) and three dimers (i.e., δ-viniferin 10, viniferifuran 14 and dehydro-δ-viniferin 15) were endowed with significant antibacterial activity against gram-positive bacteria. The exposure of gram-positive foodborne pathogens to 100 µg/mL of 2, 9 and 15 induced severe cell membrane damage, resulting in the disruption of the phospholipid bilayer. The most promising dimeric compound, dehydro-δ-viniferin 15, was tested against Listeria monocytogenes, resulting in a loss of cultivability, viability and cell membrane potential. TEM analysis revealed grave morphological modifications on the cell membrane and leakage of intracellular content, confirming that the cell membrane was the principal biological target of the tested derivative.

Figure 1

Structures of stilbenoid monomers (1–9) and dimers (10–15).

Figure 2

Synthesis of resveratrol-derived monomers.

Figure 3

Synthesis of resveratrol-derived dimers.

Figure 4

Leakage of cFSE fluorescence outside the Gram-positive cells due to membrane damages after exposure to 100 µg/mL of each compounds for 30 min at 30‒37 °C. (A) B. cereus DSM 9378. (B) S. aureus ATCC 25923. (C) L. monocytogenes Scott A. (D) E. faecium DSM 20477. (E) E. faecalis DSM 20478. Data are means of three replicates; standard deviation is shown as error bars. In the same chart, different letters indicate statistically significant differences between groups (p < 0.001).

Figure 5

(a) Dot plot of cell viability determined by flow cytometry. Cell suspensions were stained with SYTO™ 24 and PI just after exposure to either 15 or chlorhexidine, for 30 min at 37 °C. Green gate: AFU (considered as live cells); dark gate: damaged cells; red gate: non-AFU (considered as dead cells). Inoculum was 9.0 ± 0.20 log₁₀ AFU L. monocytogenes cells. Control cells were incubated in PBS; cells exposed to DMSO served as treatment control. (b) Effect of 15 and chlorhexidine exposure on membrane potential of L. monocytogenes ScottA cells. The membrane potential is reported as normalized green/red fluorescence (GF/RF). Fluorescence was measured by flow cytometry and GF/RF ratio considers the fluorescence of 30000 events. Gramicidin A used as standard molecule inducing membrane depolarization. Data are means of three replicates; standard deviation is shown as error bars. In the same chart, different letters indicate statistically significant differences between groups (p < 0.001).

Figure 6

TEM micrographs of L. monocytogenes Scott A suspended in either PBS (a) or exposed to DMSO (b), 15 (c,d) or chlorhexidine (e,f). Intact cell wall (CW) and cell membrane (CM) were present in control samples (a,b). Cell wall or cell membrane interruptions (arrows) were visible in 15 exposed cells (c). Bar is 500 nm.