Antibacterial Properties of Nootkatone Against Gram-Positive Bacteria

Abstract

Nootkatone is one of the sesquiterpenes contained in citrus peels, especially in grapefruits. It is known that nootkatone has various physiological activities such as antioxidative and antifibrotic actions. This study showed that nootkatone, a natural sesquiterpene, exhibited antibacterial activities against Gram-positive bacteria such as Staphylococcus aureus, Enterococcus faecalis, Listeria monocytogenes, Corynebacterium diphtheriae, and Bacillus cereus, with the antibacterial effect against C. diphtheriae being most pronounced. However, no growth-inhibitory effects or bactericidal activity was observed against Gram-negative bacteria. In addition, the bactericidal activity of nootkatone at a high concentration was observed against Gram-positive bacilli. These results suggested that nootkatone may exert an antibacterial effect by targeting cell wall structures or a particular metabolite. Moreover, even at a low concentration (0.25 mM), nootkatone was capable of inhibiting biofilm formation by Staphylococcus aureus. Thus, this study demonstrated antibacterial efficacy for nootkatone against Gram-positive bacteria, indicating that nootkatone could be a potential candidate for the development of new antibacterial agents.

Terpenoids are a wide variety of compounds that possess an isoprene unit as the basic structure, and are classified as monoterpenes, sesquiterpenes, diterpenes, and others based on the number of carbon atoms. Plants synthesize numerous terpenoids that play important roles as temporary metabolites, photosynthetic pigments, and growth hormones. ^1,2 In addition, terpenoids provide protection against pathogenic microorganisms. ^3,4 Citrus terpenoids are present in oils called essential oils and are widely utilized commercially as aroma-providing components in foods and cosmetics. Furthermore, terpenoids and other components of plant essential oils exhibit a wide range of properties such as antioxidant activity, ⁵ antitumor effect, ^6,7 and osteogenic action. ^8,9 Currently, clinical studies are being conducted on the anticancer action of terpenoids, ⁶ suggesting the possibility that these compounds may be effective medicines against several diseases.

It is known that some essential oils containing terpenoids inhibit bacterial growth. ¹⁰ Adukwu et al ¹¹ reported that essential oil components derived from lemongrass inhibited the growth of Acinetobacter baumannii and Staphylococcus aureus. ¹¹ Further, Lahmar et al ¹² reported synergistic antibacterial activity by combining various essential oil components and antibiotics. ¹² Moreover, other studies have reported that eugenol and limonene inhibit Staphylococcus aureus biofilm formation. ^3,13 Carvacrol and thymol decreased the resistance of Bacillus spores to heat and high pressure. ^14,15 Ansari et al ¹⁶ reported that perillyl alcohol inhibited the growth of Candida albicans under physiological stresses, such as alkaline, ionic, and high-temperature conditions. ¹⁶ Thus, these reports suggest that essential oils and terpenoids may be used as therapeutic agents for infectious diseases, or drug-resistant bacteria.

Nootkatone is one of the sesquiterpenes contained in grapefruit and is synthesized by the oxidation of the precursor, valencene. ^17,18 It is known that nootkatone has various physiological activities such as antioxidation activities against diesel exhaust particles in the lung and antifibrotic actions against carbon tetrachloride-induced tumor necrosis of the liver. ^{19 –22} However, there are very few reports about the antibacterial activity of nootkatone. It was hypothesized that nootkatone could serve as a candidate for the development of a new therapeutic agent against infectious diseases. Therefore, the antibacterial activity was investigated in terms of growth suppression and the bactericidal action of nootkatone against various bacterial species.

First, the effects of nootkatone on the growth of each bacterial strain were investigated. When bacterial strains were cultured in a medium supplemented with 0.25-2.0 mM nootkatone, growth suppression was observed only in Gram-positive bacteria, such as Listeria monocytogenes, Corynebacterium diphtheriae, Staphylococcus aureus, and Enterococcus faecalis (Figure 1a,b,c,d). Complete growth suppression occurred in L. monocytogenes and C. diphtheriae at 1 and 0.5 mM, respectively (Figure 1a,b), while growth suppression was not observed in the Gram-negative bacteria, Salmonella enteritidis and Pseudomonas aeruginosa (Figure 1e,f). This suggests that nootkatone exhibited an antibacterial effect by targeting a structure or metabolite specific to Gram-positive bacteria such as peptidoglycan. Kim et al ^23,24 reported that synthetic retinoids contained an isoprene unit that killed methicillin-resistant Staphylococcus aureus (MRSA) by penetrating and disrupting lipid bilayers. ²⁴ Therefore, there is a possibility that nootkatone may inhibit proliferation by acting on the synthetic pathway of peptidoglycan.

Figure 1

Growth curve assessment. Effect of increasing concentrations of nootkatone on the growth of Listeria monocytogenes (a), Corynebacterium diphtheriae (b), Staphylococcus aureus (c), Enterococcus faecalis (d), Pseudomonas aeruginosa (e), and Salmonella enteritidis (f). Bacterial strains were precultured overnight at 37°C in appropriate media. Strains were diluted and cultured at 37°C in medium supplemented with nootkatone. Dimethyl sulfoxide (•), 0.25 mM (▴), 0.5 mM (♦), 1.0 mM (▪), 2.0 mM (×) nootkatone. OD: optical density.

Next, the bactericidal effect of nootkatone was investigated against the four strains of Gram-positive bacteria in which growth suppression was observed. Bactericidal action was evident 30 minutes after the addition of 2 mM nootkatone in L. monocytogenes and C. diphtheriae (Figure 2a,b). Further, 60 minutes following the addition of 2 and 4 mM nootkatone, the bactericidal activity was found to be stronger for C. diphtheriae than L. monocytogenes. Interestingly, bactericidal action was not observed for Staphylococcus aureus or E. faecalis (Figure 2c,d). Ansari et al ¹⁶ reported that perillyl alcohol damaged the cell wall of C. albicans and resulted in morphological changes that caused wrinkling and corrugation. However, changes in morphology were not observed in cells treated with nootkatone (data not shown).

Figure 2

Killing curve of Gram-positive bacteria by nootkatone. Nootkatone was added to a stationary phase culture of Listeria monocytogenes (a), Corynebacterium diphtheriae (b), Staphylococcus aureus (c), and Enterococcus faecalis (d) in appropriate media, which were then incubated at 37°C. Samples were taken at 15, 30, and 60 minutes after adding nootkatone, serially diluted with phosphate-buffered saline, and then plated onto Luria-Bertani or brain heart infusion agar plates. Dimethyl sulfoxide (•), 1.0 mM (▴), 2.0 mM (♦), 4.0 mM (▪) nootkatone.

Since the bactericidal effect of nootkatone was found to be confined to Gram-positive bacillus, the effect of nootkatone on the growth and spores of the Gram-positive spore-forming Bacillus cereus was investigated. Growth suppression was observed when the cells were exposed to more than 0.5 mM nootkatone, and the bacterial cell number was decreased after treatment with 2 and 4 mM nootkatone (Figure 3a,b). However, nootkatone was ineffective in inhibiting spore formation in B. cereus (Figure 3c).

Figure 3

Effect of nootkatone on Bacillus cereus. Growth of B. cereus in the presence of nootkatone (a). DMSO (•), 0.25 mM (▴), 0.5 mM (♦), 1.0 mM (▪) 2.0 mM (×) nootkatone. Killing curve of B. cereus (b) and spore of B. cereus (c) by nootkatone. Growth curve and cell viability were measured by the same method as for other bacteria. DMSO (•), 1.0 mM (▴), 2.0 mM (♦), 4.0 mM (▪) nootkatone. DMSO: dimethyl sulfoxide.

In this study, bactericidal action was not observed for the Gram-positive cocci, Staphylococcus aureus and E. faecalis, although Gram-positive bacilli such as L. monocytogenes, C. diphtheriae, and B. cereus were killed in medium containing more than 2 mM nootkatone. No substantial differences were noted in the cell wall structures of the Gram-positive cocci and the Gram-positive bacilli. Therefore, these data suggested that the bactericidal activity of nootkatone against Gram-positive bacteria was not the result of a mechanism targeting cell wall structure. It is also known that microorganisms oxidize valencene through cytochrome P450, and nootkatone is synthesized during this process. ¹⁷ Further, microbial P450 enzymes further oxidized the synthesized nootkatone into various metabolites such as nootkatone-11 and 12-epoxide. ¹⁸ Thus, differences found in the antimicrobial action of nootkatone among bacterial strains could be attributed to the metabolic capacity of the bacteria.

A previous study showed that eugenol inhibits biofilm formation in MRSA and methicillin-sensitive Staphylococcus aureus strains. ³ Four Staphylococcus aureus strains (GTC1772, GTC1881, GTC1886, and 04SNISCCII) were incubated in tryptic soy broth (TSB) with 0.5% glucose on 24-well plates and treated with a different concentration of nootkatone to elucidate antibiofilm activity. The results showed that at lower concentrations (0.125 and 0.25 mM), nootkatone did not inhibit growth of Staphylococcus aureus (Figure 4). It is clear that the growth of Staphylococcus aureus was suppressed by nootkatone at higher concentrations of 1 and 2 mM (Figure 1c). These experiments showed that nootkatone repressed the growth and biofilm formation of Staphylococcus aureus, suggesting that it could be used as a countermeasure against drug-resistant Staphylococcus aureus infections. Subramenium et al ¹³ reported that limonene changed the gene expression associated with biofilm formation and the virulence factors of Streptococcus mutans and Streptococcus pyogenes. It has been reported that treatment of Staphylococcus aureus with eugenol inhibited colony formation in the ears of mice. ³ These results suggest that treatment with nootkatone might reduce infection rates for Gram-positive bacteria.

Figure 4

Effect of nootkatone on biofilm formation by Staphylococcus aureus. Four Staphylococcus aureus strains (GTC1181, 1186, 1770, and 97LSAP3) were incubated at 37°C for 2 days in a 24-well plate. Decreases in biofilm biomass were detected using the crystal violet assay. Bars represent the mean ± SD of 3 independent measurements. * P < 0.05. DMSO: dimethyl sulfoxide.

Different types of essential oils contained various types and concentrations of terpenoids. ^25,26 Several studies have reported on the antibacterial activity of essential oil components. Although the concentration of terpenoids in essential oils is low, it is known that essential oils containing monoterpenes suppressed the growth of pathogenic bacteria. ^10,16 It has been reported that an essential oil component derived from Cymbopogon flexuosus exerts bactericidal action against A. baumannii and Staphylococcus aureus. ¹¹ Based on our results, a combination of terpenoid or essential oil components derived from nootkatone-containing pericarp should be investigated as a new antimicrobial agent. While further investigation is required, it is suggested that combinations of these antibacterial ingredients could potentially be utilized as therapeutic drugs customized to each infection.

Experimental

Bacterial Strain and Media

Pseudomonas aeruginosa, Salmonella enteritidis (NHp18SE), B. cereus (NHp7), E. faecalis (NHp6), and Staphylococcus aureus (04SNISCCII) were obtained from Ryukyus University, Okinawa, Japan. Staphylococcus aureus (GTC01181, GTC01186, and GTC01770), L. monocytogenes (GTC00149T), and C. diphtheriae (GTC00263T) were obtained from Gifu University, Gifu, Japan. P. aeruginosa, Salmonella enteritidis, B. cereus, and Staphylococcus aureus were grown at 37°C in Luria-Bertani (LB) broth (Lennox) (Sigma-Aldrich, St Louis, MO, USA) or agar (1.5% agar) aerobically. E. faecalis, L. monocytogenes, and C. diphtheriae were grown at 37°C in brain heart infusion (BHI) broth (Nissui, Tokyo, Japan) or agar (1.5% agar) aerobically.

Antibacterial Activity Analysis

Cultures of bacteria grown in LB broth or BHI broth were prepared overnight at 37°C with shaking and diluted 100-fold with fresh LB broth. Various concentrations of nootkatone were added (0-2 mM). Flasks were incubated at 37°C, and the cell mass was measured spectrophotometrically by assessing turbidity at the 600 nm wavelength.

For the killing assay, nootkatone (1-4 mM) was added to a stationary phase culture in LB broth or BHI broth, and then the bacteria were cultured at 37°C. Samples were obtained at intervals and cell numbers determined by plating the cultures and counting the colony-forming units per milliliter (CFU/mL).

Anti-Spore Activity Assay

B. cereus was incubated on LB-agar for 3 days at 37°C. For all microorganisms, once 95% sporulation was achieved (determined by microscopy), the spores on the LB-agar plate were transferred into sterile distilled water. Following harvesting, the spores were washed three times by centrifugation at 3 000 × g for 15 minutes at 4°C. The number of spores in suspension was checked by the plate count method and stored at 4°C until use.

Antibiofilm Activity

The biofilm assay was performed using 24-well polystyrene flat-bottom microtiter plates, according to a previously described procedure. ¹¹ Overnight cultures of Staphylococcus aureus in BHI broth were diluted 1:200 with fresh sterile TSB containing 0.5% glucose. For the screening of test compounds, 500 µL aliquots of the diluted cultures were transferred into the wells with nootkatone (0.0625-0.25 mM) or DMSO controls; this was performed in triplicate. Nootkatone was added to the cell suspension, and these suspensions were inoculated into a 24-well microtiter plate and incubated for 24 hours at 37°C. Following incubation, the medium was discarded, and the biofilms associated with the surface of the wells were fixed using 4% paraformaldehyde in phosphate-buffered saline (PBS) for 15 minutes. Then the paraformaldehyde was discarded, and the plates were washed 3 times with sterile PBS. Subsequently, the plates were stained with 500 µL of crystal violet (0.1%) for 15 minutes. Excess stain was removed by washing with water. The crystal violet (CV) attached to the biofilm samples was dissolved with 1 mL of 0.1 M sodium citrate in 50% ethanol. The absorbance at 570 nm was measured using a spectrophotometer to quantify biofilm formation. Inhibition of biofilm formation by the drug treatment as measured by CV staining was calculated as a percentage of the mean of the DMSO-treated control samples.