Mechanisms of bactericidal action of cinnamaldehyde against Listeria monocytogenes and of eugenol against L. monocytogenes and Lactobacillus sakei

Abstract

The spice oil components eugenol and cinnamaldehyde possess activity against both gram-positive and gram-negative bacteria, but the mechanisms of action remain obscure. In broth media at 20 degrees C, 5 mM eugenol or 30 mM cinnamaldehyde was bactericidal (>1-log reduction in the number of CFU per milliliter in 1 h) to Listeria monocytogenes. At a concentration of 6 mM eugenol was bactericidal to Lactobacillus sakei, but treatment with 0.5 M cinnamaldehyde had no significant effect. To investigate the role of interference with energy generation in the mechanism of action, the cellular and extracellular ATP levels of cells in HEPES buffer at 20 degrees C were measured. Treatment of nonenergized L. monocytogenes with 5 mM eugenol, 40 mM cinnamaldehyde, or 10 microM carbonyl cyanide m-chlorophenylhydrazone (CCCP) for 5 min prevented an increase in the cellular ATP concentration upon addition of glucose. Treatment of energized L. monocytogenes with 40 mM cinnamaldehyde or 10 microM CCCP caused a rapid decline in cellular ATP levels, but 5 mM eugenol had no effect on cellular ATP. Treatment of L. sakei with 10 mM eugenol prevented ATP generation by nonenergized cells and had no effect on the cellular ATP of energized cells. CCCP at a concentration of 100 microM had no significant effect on the cellular ATP of L. sakei. No significant changes in extracellular ATP were observed. Due to their rapidity, effects on energy generation clearly play a major role in the activity of eugenol and cinnamaldehyde at bactericidal concentrations. The possible mechanisms of inhibition of energy generation are inhibition of glucose uptake or utilization of glucose and effects on membrane permeability.

FIG. 1.

Structures of the substituted aromatic compounds (from left to right) eugenol, trans-cinnamaldehdye, carvacrol, and thymol.

FIG. 2.

Effects of eugenol (A) and cinnamaldehyde (B) on L. monocytogenes in TSB+YE at 20°C and pH 7.0. The data are averages for three experiments, and the error bars indicate one standard deviation.

FIG. 3.

Effect of eugenol on L. sakei in TSB+YE at 20°C and pH 7.0. The data are averages for three experiments, and the error bars indicate one standard deviation.

FIG. 4.

Effects of antimicrobial agents on L. monocytogenes (A) and L. sakei (B) in 25 mM HEPES (pH 7.0) at 20°C with 0.25% dextrose. The data are averages of results from two experiments, and the error bars indicate one standard deviation.

FIG. 5.

Cellular ATP concentrations expressed as femtomoles per microgram of protein from L. monocytogenes in 25 mM HEPES buffer (pH 7.0) at 20°C. (A) Cells were exposed to antimicrobial agents at zero time, and all treatments except the buffer treatment were energized with 0.25% glucose at 5 min. (B) All treatments except the buffer treatment were energized with 0.25% glucose at zero time, and antimicrobial agents were added at 5 min. The treatments included buffer, glucose, eugenol (5 mM), cinnamaldehyde (40 mM), and CCCP (10 μM). The data are averages for four experiments, and values that are significantly different as determined by a Student t test (α = 0.05) are indicated by different letters.

FIG. 6.

Cellular ATP concentrations expressed as femtomoles per microgram of protein from L. sakei in 25 mM HEPES buffer (pH 7.0) at 20°C. (A) Cells were exposed to antimicrobial agents at zero time, and all treatments except the buffer treatment were energized with 0.25% glucose at 5 min. (B) All treatments except the buffer treatment were energized with 0.25% glucose at zero time, and antimicrobial agents were added at 5 min. The treatments included buffer, glucose, eugenol (10 mM), and CCCP (100 μM). The data are averages for four experiments, and values that are significantly different as determined by a Student t test (α = 0.10) are indicated by different letters.