Antimicrobial Susceptibility and Antibacterial Mechanism of Limonene against Listeria monocytogenes

Abstract

Limonene is a monoterpenoid compound, which is founded in a lot of plants' essential oils with good antibacterial activity against food-borne pathogens, but it has an ambiguous antimicrobial susceptibility and mechanism against Listeria monocytogenes (L. monocytogenes). In this study, the antimicrobial susceptibility of Limonene to L. monocytogenes was studied, and some new sights regarding its antibacterial mechanism were further explored. Scanning electron microscopy (SEM) verified that limonene caused the destruction of the cell integrity and wall structure of L. monocytogenes. The increase in conductivity and the leakage of intracellular biomacromolecules (nucleic acids and proteins) confirmed that limonene had an obvious effect on cell membrane permeability. The results of Propidium Iodide (PI) fluorescence staining were consistent with the results of the conductivity measurements. This indicated that limonene treatment caused damage to the L. monocytogenes cell membrane. Furthermore, the decrease in ATP content, ATPase (Na⁺K⁺-ATPase, Ca²⁺-ATPase) activity and respiratory chain complex activity indicated that limonene could hinder ATP synthesis by inhibiting the activity of the respiratory complex and ATPase. Finally, differential expression of proteins in the respiratory chain confirmed that limonene affected respiration and energy metabolism by inhibiting the function of the respiratory chain complex.

Keywords: ATP; ATPase; Limonene; antibacterial mechanism; membrane damage; nucleic acid; protein; respiratory complexes.

Figure 1

Growth curves of L. monocytogenes.

Figure 2

Scanning electron microphotographs of L. monocytogenes. Cells without treatment for 6 h (a), cells without treatment for 12 h (b), cells treated with ethanol for 6 h (c), cells treated with ethanol for 12 h (d), cells treated with limonene (1 MIC) for 6 h (e), cells treated with limonene (1 MIC) for 12 h (f), cells treated with limonene (2 MIC) for 6 h (g), and cells treated with limonene (2 MIC) for 12 h (h).

Figure 3

The inhibitory effects of limonene on the membrane conductivity of L. monocytogenes.

Figure 4

Observation of L. monocytogenes by using fluorescence microscope. L. monocytogenes under optical microscope (a), L. monocytogenes under fluorescence microscope (b), L. monocytogenes treated with limonene (MIC) for 6 h (c), L. monocytogenes treated with limonene (2 MIC) for 6 h (d).

Figure 4

Observation of L. monocytogenes by using fluorescence microscope. L. monocytogenes under optical microscope (a), L. monocytogenes under fluorescence microscope (b), L. monocytogenes treated with limonene (MIC) for 6 h (c), L. monocytogenes treated with limonene (2 MIC) for 6 h (d).

Figure 5

Changes of OD 260 of L. monocytogenes.

Figure 6

Effect of limonene on protein concentration of L. monocytogenes (A). The gel electrophoresis image of intracellular protein in L. monocytogenes cutured for 6 h (B).

Figure 7

Changes in Na⁺-K⁺-ATPase (a) and Ca²⁺-ATPase (b) activities of L. monocytogenes. The effect of Limonene on ATP concentration of L. monocytogenes (c).

Figure 8

Oxidative phosphorylation system.

Figure 9

Changes in the activity of respiratory chain complex I~V of L. monocytogenes. (a) complex I, (b) complex II, (c) complex III, (d) complex IV, and (e) complex V.