Antibacterial interactions of pulegone and 1,8‐cineole with monolaurin ornisin against Staphylococcus aureus

Abstract The aim of this study was to investigate the antibacterial interactions of pulegone and 1,8‐cineole with monolaurin ornisin against Staphylococcus aureus. The individual and combined antibacterial activities of the compounds were evaluated using minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), fractional inhibitory concentration index (FICi), and time‐kill methods. Furthermore, the mechanism of the antibacterial action of the compounds was tested by measuring the release of cell constituents. The MIC values of pulegone, 1,8‐cineole, nisin, and monolaurin were 5.85 µl/ml, 23.43 µl/ml, 6.25 µg/ml, and 0.031 mg/ml, respectively. A synergistic antibacterial activity (FICi = 0.5) was found between 1,8‐cineole and nisin. The time‐kill assay showed that the populations of S. aureus exposed to 1,8‐cineole, nisin, and their combination were decreased by 5.9, 5.3, and 7.1 log CFU (colony‐forming units)/mL, respectively. The combination of 1,8‐cineole and nisin also induced the highest release of cell constituents. It was concluded that the combination of 1,8‐cineole and nisin could be considered as a novel and promising combination which may reduce the required dose of each antibacterial compound.

as headaches, palpitations, allergies, stomach cancer, asthma, skin rashes, and contact dermatitis (Sharma, 2015). In fact, due to the increasing awareness of consumers about the adverse effects of chemical antimicrobials, the tendency to use natural compounds to protect food and control food-borne pathogens has increased significantly (Baygar, 2019).
Pulegone is a monoterpene ketone found in the leaves and flowers of several members of the mint family (Mkaddem et al., 2009). Terpenes are capable of penetrating into the bacterial cell wall, leading to denaturation of proteins and disintegration of the cell membrane, leading to cytoplasmic leakage, cell lysis, and eventually cell death (Oussalah et al., 2007). Based on the published reports, pulegone can effectively destroy S. aureus, S. typhimurium, and Escherichia coli (Mkaddem et al., 2009).
Nisin is a natural preservative used in the food industry to prevent the growth of microorganisms, especially Gram-positive bacteria, such as S. aureus . However, it has been reported that some bacterial species, including S. aureus, may acquire resistance to nisin (Zhou et al., 2014).
Monolaurin, an ester of lauric acid, is used as an antimicrobial and emulsifier agent in the food industry. It possesses the strongest antimicrobial effect among fatty acid esters (Raeisi et al., 2016). Generally, this monoester is very effective against Gram-positive bacteria such as Staphylococcus and Streptococcus, as well as effectively inhibits toxin production by Staphylococci (Lieberman et al., 2006). Higher concentrations of monolaurin may result in undesirable soapy aroma and taste (Bell & De Lacy, 1987). Antibacterial interaction between nisin and monolaurin has been previously reported (Mansour & Millière, 2001;Zhang et al., 2009). Some previous studies have reported the individual antibacterial effect of pulegone, 1,8-cineole, monolaurin, and nisin and their combination with other antimicrobial agents. However, there is no report on the antibacterial interactions of two essential oil components (pulegone and 1,8-cineole) with monolaurin or nisin. Then, the objective of the current study was to evaluate the combined antibacterial activity of pulegone with monolaurin/nisin and 1,8-cineole with monolaurin/nisin against Staphylococcus aureus.

| Preparation of the bacterial suspension
Staphylococcus aureus (PTCC1431) was procured from the microbial collection of the Department of Food Hygiene and Quality Control, Urmia University. The colonies of the bacterium grown on the PCA were transferred to BHI broth (10 ml) and incubated at 37°C for 24 h. Then, the broth containing bacteria was centrifuged at 9342 g for 10 min and washed with peptone water (0.1%) thrice. After that, 10 ml of peptone water (0.1%) was added to the bacterial pellet and resuspended. Finally, the turbidity of the bacterial suspension was adjusted to 0.1 at 600 nm (≈10 8 CFU/ml) using a spectrophotometer

| Preparation of the antimicrobial solutions
First, 600 μl pulegone or 1,8-cineole was mixed with 1 ml dimethyl sulfoxide (10%) to prepare their stock solutions (375 μl/ml) and 1 ml of this solution was added to 9 ml BHI broth. Then, different twofold concentrations (187.5-0.73 μl/ml) of the compounds were prepared in BHI broth from the stock solutions. To prepare the stock solution of monolaurin, 500 mg of monolaurin was dissolved in 5 ml of ethanol (96%) and 0.1 ml of this solution was added to 9.9 ml BHI broth.
Then, the solution was diluted to reach concentrations ranging from 1 to 0.0156 mg/ml (Razavi-Rohani & Griffiths, 1994). The amount of 10 mg of nisin was also dissolved in 1 ml of HCl (0.02 M, pH = 1.6) to prepare its stock solution. Different concentrations from this stock solution (200 to 3.125 µg/ml) were subsequently prepared (Dufour et al., 2003). The stock solutions of all the antimicrobial compounds were initially sterilized using a syringe filter (0.45 μm).

| Determination of the MIC and MBC of the antimicrobials
The microdilution method was used to determine the minimal in-

| Time-kill method
To investigate the lethal effects of pulegone and 1,8-cineole combined with nisin or monolaurin, 50 μl of bacterial suspension (≈10 8 CFU/ml) was inoculated into tubes containing 4450 μl of BHI broth. Then, 500 µl from each antimicrobial solution was added to four tubes at the final concentration of MIC. In combined treatments, an amount of 250 µl of each antimicrobial at MIC concentration was also used as follows: 250 µl pulegone + 250 µl monolaurin, 250 µl pulegone + 250 µl nisin, 250 µl 1,8-cineole + 250 µl monolaurin, and 250 µl 1,8-cineole + 250 µl nisin. One tube was also considered as growth control, which contained BHI broth and bacterial culture. Subsequently, all of the tubes were incubated at 37°C. Serial dilutions from the tubes were prepared at 0, 2, 4, 8, and 24 h and cultured on the BHI agar. Finally, the colonies were enumerated after 24 h incubation at 37°C, and then the time-kill curve was plotted (Avila et al., 1999;Lavigne et al., 2020).

| Estimation of the cell constituents' release
The effect of the antimicrobial compounds on the cell membrane integrity of S. aureus was evaluated by determining the release of cell constituents to the supernatant. The overnight culture of S. aureus in the BHI broth was centrifuged at 4000g for 15 min, and the supernatant was discarded. The bacterial cells were resuspended in phosphate-buffered saline (PBS) and centrifuged three times again at 4000g for 15 min. Then, each individual antimicrobial compound (at 2 × MIC concentration) and its combination (at MIC concentration of each antimicrobial) were added to the bacterial suspension in PBS. A tube containing bacteria suspended in PBS was considered as a control. All tubes were incubated for 1 h in a shaker incubator at 250 rpm and 37°C. Then, the tubes were centrifuged at 13,400g for 20 min. Eventually, the concentration of cell constituents in the supernatants was measured using a spectrophotometer at 260 nm.
The PBS buffer was considered as a blank to zero the spectrophotometer (Mutlu-Ingok & Karbancioglu-Guler, 2017). To calculate cell constituent release, the optical density (OD) of the tube containing antimicrobial solution and the bacterial suspension was subtracted from that of the antimicrobial solution.

| Statistical analysis
The experiments of MIC, MBC, and FIC were carried out three times,

| MIC and MBC of the antimicrobial compounds
The results of the MIC and MBC values of pulegone, 1,8-cineole, monolaurin, and nisin against S. aureus are presented in Table 1.
According to the reported results, the MIC values could be varied.
The antimicrobial performance of essential oils in vitro depends on various factors such as antimicrobial components, type of microorganism, culture medium, amount of inoculum, pH, temperature, and food composition (Tajkarimi et al., 2010;H. Zhou et al., 2014).
The MIC value of monolaurin was 0.031 mg/ml. This finding is similar to the results reported by other researchers (Raeisi et al., 2016). Evidence exists that monolaurin destroys the cell membrane by its lipophilic characteristics and inhibits the growth of Gram-positive bacteria, while it has no effect on Gram-negative bacteria (Delamare et al., 2007;Tajik et al., 2014). In one study, the MIC of monolaurin against S. aureus was 0.0625 mg/ml (Preuss et al., 2005), while Tangwatcharin and Khopaibool (2012) reported that the MIC value of monolaurin against S. aureus ATCC 25923 was 0.1 mg/ml. The other study demonstrated that monolaurin alone could inhibit S. aureus at a level of 128 μg/ml (pH = 7) and 16 μg/ ml (pH = 5) but had no effect on E. coli. Therefore, the most potent antibacterial activity against S. aureus was observed at the lower pH (Aminzare et al., 2014). The MIC values of monolaurin against S. aureus ATCC 25923 and ATCC 1885 were 100 and 250 μg/ml, respectively (Sadiq et al., 2016;Tajik et al., 2014).
In this study, the MIC value of nisin was determined to be 6.25 µg/ ml. In agreement with our finding, other researchers showed that the MIC value of nisin against S. aureus ranged from 2 to 32 µg/ml (Dosler et al., 2012). It was shown that the MIC value of nisin against S. aureus was 8 µg/ml (Zhao et al., 2014). However, another study showed that the MIC value of nisin against S. aureus strains was in the range of 16-32 µg/ml (Shi et al., 2017).

| FIC
The results of FIC indices for the antimicrobial compounds (combination of pulegone with monolaurin/ nisin and 1,8-cineole with monolaurin/nisin) are presented in Table 2. It was found that the com-  (Dufour et al., 2003). Furthermore, a synergistic effect between nisin and coenzyme Q has been reported against S. aureus isolates (Zhao et al., 2014). Another similar study showed a synergistic effect between nisin and cinnamaldehyde against S. aureus strains with the FIC value of 0.3 (Shi et al., 2017). It has been reported that the combined use of 1,8-cineole and carvacrol could synergistically inhibit Listeria monocytogenes, Aeromonas hydrophila, and Pseudomonas fluorescens (De Sousa et al., 2012). In another study, synergy was found between 1,8-cineole and aromadendrene against S. aureus (Mulyaningsih et al., 2010). Meanwhile, 1,8-cineole in combination with limonene showed a synergistic effect against S. aureus (van Vuuren & Viljoen, 2007). However, it has been reported that the antibacterial effects of 1,8-cineole in combination with α-terpineol or linalool were additive (FICi = 1) against S. aureus (Zengin & Baysal, 2014).

| Time-kill assay
The antibacterial interaction between pulegone and monolaurin against S. aureus during 24 h incubation is shown in Figure 1. Within the first 8 h, monolaurin showed stronger antibacterial activity than pulegone against S. aureus. However, at the end of 24 h incubation, there was no significant difference between their effects.
Within the first 2 h, no significant difference was detected between the antibacterial performance of monolaurin and the combination of pulegone and monolaurin (P+ML). Nevertheless, at subsequent incubation times, monolaurin had surprisingly stronger antibacterial activity than the combination of pulegone and monolaurin. The combination of pulegone and monolaurin had a significantly stronger antibacterial effect than pulegone alone at the first 8 h. After 24 h incubation, however, there was no significant difference between their effects. Furthermore, all treatments displayed significant effects on S. aureus at the end of incubation, and approximately a 6-log reduction in bacteria counts was induced by the treatments compared to control. Figure 2 shows the antibacterial performance of pulegone, nisin, and their combination. Within the first 8 h, the antibacterial activity of nisin and the combination of pulegone and nisin (P + N) were similar, and their effects were significantly stronger than pulegone alone. After 24 h, a weak regrowth was observed in nisin treatment.
The antibacterial interaction between 1,8-cineol and monolaurin is illustrated in Figure 3. Many studies have shown that some antimicrobials, such as nisin and monolaurin alone, are more effective against Grampositive bacteria but have no effect on Gram-negative ones (Raeisi et al., 2016;Wang et al., 2020). However, it has been proven that the combined use of the antimicrobials can increase not only the antibacterial effect but also reduce the required dose of each antimicrobial . It has been reported that the combination of nisin and cell-free supernatant of Bacillus licheniformis showed synergistic bactericidal activity against S. aureus after 4 h (He & Chen, 2006). The results of the time-kill test in broth and pasteurized milk showed that nisin in combination with cinnamaldehyde had a synergistic antibacterial effect against S. aureus (Shi et al., 2017).

| Cell constituents' release
The cell membrane integrity was evaluated by measuring the release of cell constituents such as nucleic acids and proteins into the supernatant at 260 nm. The internal cell constituents were released into the supernatant following cell membrane disruption, and then the absorbance of the supernatant at 260 nm was increased.  (Zengin & Baysal, 2014). The results of another study showed that the combination of nisin and cinnamaldehyde induced more damage on the cell membrane of S. aureus compared to nisin or cinnamaldehyde alone (Shi et al., 2017).
The combination of nisin and Zataria multiflora essential oil significantly increased the cell constituents' release from S. aureus (Moosavy et al., 2008).

| CON CLUS IONS
The present study investigated the antibacterial effects of pule-

ACK N OWLED G M ENT
The authors would like to thank for financial support of Urmia University.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author.

R E FE R E N C E S
OD b = optical density of PBS + antimicrobial.

TA B L E 3
The cell constituents' release in Staphylococcus aureus induced by individual (at 2 × MIC (minimum inhibitory concentration)) and combined antimicrobials (at MIC of each antimicrobial)