The Selective Antibacterial Activity of the Mixed Systems Containing Myristic Acid against Staphylococci.

Fatty acids and their derivatives are interesting cosmetic ingredients because they show the selective antibacterial activity against Staphylococcus aureus (S. aureus). However, the antibacterial activity in mixed systems containing several active ingredients is unclear because previous studies focused antibacterial systems containing one kind of fatty acid. In the present study, the minimal inhibitory concentration (MIC) and the fractional inhibitory concentration (FIC) were evaluated for myristic acid/lauric acid, myristic acid/palmitoleic acid, and myristic acid/lactic acid mixed systems to show the effect of the coexisting components on the selective antibacterial activity of myristic acid. In the myristic acid/palmitoleic acid mixed system, the antibacterial activity against S. aureus was enhanced by additive effect, whereas the antibacterial activity was not observed against S. epidermidis. On the other hand, the myristic acid/lauric acid mixed system showed antibacterial activity against S. epidermidis: Lauric acid impaired the selectivity of antibacterial activity of myristic acid. These results suggest that the selective activity of myristic acid varies with the additives. The present findings are useful for designing formulations of cosmetics and body cleansers containing myristic acid.

tivity of two-component mixed system containing myristic acid which shows the selective antibacterial activity and other antibacterial ingredients against S. aureus and S. epidermidis. The additives were lauric acid, which has the maximum antibacterial activity among saturated fatty acids, palmitoleic acid, which has characteristic selective antibacterial activity, and lactic acid, which is also used as a degradation preventing agent in foods and a topical skin inflammation treatment agent 10,18,19 . The antibacterial activity of these substances was determined using minimal inhibitory concentration MIC , which is the minimum drug concentration required to suppress bacterial growth, while the combined effect of the two-component mixture was evaluated based on the FIC index. The findings of the present study will be useful in designing skin care and cleansing products that focus on the human skin flora.

Materials
The molecular structures of the materials evaluated in the present study are shown in Fig. 1. Myristic acid C14:0 fatty acid, ≥ 99 was purchased from Sigma-Aldrich Co. LLC St. Louis, USA . Lactic acid, lauric acid C12:0 fatty acid, ≥ 99 , palmitoleic acid C16:1 fatty acid, ≥ 99 and 0.1 mol L 1 phosphate buffer solution pH 6 were obtained from Fujifilm Wako Pure Chemical Industries Ltd. Osaka, Japan . Ethanol, sodium chloride, beef extract, and polypeptone were purchased from Junsei Chemical Co. Tokyo, Japan , Kanto Chemical Co., Inc. Tokyo, Japan , Nacalai Tesque Inc. Kyoto, Japan , and Nihon Pharmaceutical Co., Ltd. Tokyo, Japan , respectively. Agar powder was purchased from Nissui Pharmaceutical Co., Ltd. Tokyo, Japan . S. aureus NBRC13276 and S. epidermidis NBRC12993 were obtained from the National Institute of Technology and Evaluation Tokyo, Japan . Water was purified using a Demi-Ace Model DX-15 demineralizer Kurita Water Industries Ltd., Tokyo, Japan .

Preparation of culture medium and bacterial disper-
sion Before the cultivation of S. aureus and S. epidermidis, a medium containing beef extract 0.15 g , polypeptone 0.30 g , sodium chloride 0.15 g , and water 30 mL was sterilized in an autoclave at 121 for 20 min. The cultures of S. aureus and S. epidermidis were prepared by shaking 30 mL of the medium containing one colony for 24 h at 37 145 rpm . For the MIC evaluation and the assays using the checkerboard method, the medium containing beef extract 1.50 g , polypeptone 3.00 g , sodium chloride 1.50 g , and pH 6 phosphate buffer 150 mL were sterilized in an autoclave at 121 for 20 min.

Evaluation of the combined effect of antibacterial
components based on checkerboard method The effects of the addition of lauric acid, palmitoleic acid, and lactic acid on the antibacterial activity of myristic acid were evaluated using the checkerboard method, which was a typical evaluation method for mixed antibacterial systems. In the checkerboard method, a combined effect was determined based on the FIC index, which was calculated from the MIC of a mixture containing two types of ingredients. We have reported the MICs of myristic acid and three additives in a single component against S. aureus and S. epidermidis in previously Table 1 10,17 .
Here, the antibacterial activity of the mixtures containing myristic acid and the additives in the concentrations of 1/64 to 4 times the MIC was evaluated: The concentration ranges of myristic acid and additives were as follows: 2.45 628 µg mL 1 myristic acid for S. aureus , 6.25 1600 µg mL 1 myristic acid for S. epidermidis , 0.29 75.2 µg mL 1 lauric acid for S. aureus, S. epidermidis , 0.29 75.2 µg mL 1 palmitoleic acid for S. aureus , 6.25 1600 µg mL 1 palmitoleic acid for S. epidermidis , 62.5 16000 µg mL 1 lactic acid for S. aureus , and 50.0 12800 µg mL 1 lactic acid for S. epidermidis .
To evaluate the combined effect, 20 µL of myristic acid ethanol solution, 20 µL of ethanol solution of the additive,  1000 µL of liquid medium containing beef extract 0.01 g , polypeptone 0.02 g , and sodium chloride 0.01 g , 940 µL of phosphate buffer solution, and 20 µL of a bacterial dispersion 3 10 5 2 10 6 CFU mL 1 were mixed in a test tube. When lactic acid was added, 1000 µL of the liquid medium, 940 µL of phosphate buffer solution containing lactic acid, and 20 µL of bacterial solution were added to 40 µL of myristic acid ethanol solution. Lactic acid was dispersed in a phosphate buffer solution at pH 6 because the antibacterial effect of organic acid is dependent on the pH 20 .
The prepared sample 100 µL was added to a 96-well TrueLine cell culture plate. The OD 660 of these samples was evaluated by grating microplate reader SH-1200 Lab . The conditions were as follows: wavelength 660 nm and number of flashes 10. Samples containing S. aureus or S. epidermidis were shaken at 1000 rpm by PSU-2T and were incubated at 37 for 24 and 48 h, respectively. To confirm the reproducibility, two pairs of plates containing the same composition were prepared and were evaluated twice. In order to calculate the FIC index and ΣFIC, the obtained turbidity was shown in a 9 9 board diagram. This turbidity is the difference between the turbidities at 0 h and 24 or 48 h to exclude the effect of the precipitation of components. The ΣFIC was calculated for each antibacterial combination as the sum of the individual FICs. The turbidity of the samples at MIC used for the calculation of FIC was 0.010 for all combinations. Under these conditions, no turbidity was observed in visual observation. In the board diagram, the mean turbidity values are shown in five levels, OD 660 0.000 0.099, 0.100 0.199, 0.200 0.299, 0.300 0.399, and above 0.400. In the case of myristic acid/lactic acid mixed system, 100 µL of a bacteria medium was spread on an agar medium containing 2.3 wt of agar powder to confirm the growth of S. aureus at 37 . The final concentrations of myristic acid and lactic acid were 2.45 628 and 2000 µg mL 1 , respectively.

Determination of the combination effect
The FIC index of the mixed system consisting of drugs A and B was calculated using the following equation: Based on the ΣFIC values, the interactions between drugs A and B were categorized into the following four effects: 1 Synergistic effect: When ΣFIC ≤ 0.5, the effect of the two drugs combined was much stronger than the sum of the effects of the single drug. 2 Additive effect: When 0.5 ΣFIC ≤ 1.0, two drugs combined were slightly more effective than a single drug. 3 Indifferent effect: When 1.0 ΣFIC ≤ 2.0, the combined effect is neither im-proved nor inferior to that of the single agent. 4 Antagonistic effect: When 2.0 ΣFIC, the effect of the combination was inferior to the action of the single agent 21 .

Results
The antibacterial activity was evaluated for mixed systems containing myristic acid and three additives including lauric acid, palmitoleic acid, and lactic acid against S. aureus and S. epidermidis. Culture medium containing S. aureus or S. epidermidis was added to a mixture of a Myristic acid/lauric acid mixed system. Board diagrams of myristic acid/lauric acid mixed systems against S. aureus and S. epidermidis are shown in Figs. 2 a and 2 b , respectively. The myristic acid/lauric acid mixture exhibited an antibacterial ability against S. aureus as the increase of the turbidity was low OD 660 0.010 when concentrations of myristic acid and lauric acid were above 39.3 and 18.8 µg mL 1 , respectively. The ΣFIC for this mixed system was 0.56 0.12, which suggests that the addition of lauric acid to myristic acid induced an additive effect on the antibacterial activity against S. aureus Table 2 . On the other hand, turbidity did not increase against S. epidermidis when the concentrations of lauric acid was ≥ 18.8 µg mL 1 regardless of the mixing ratio of myristic acid and exhibited the antibacterial activity. This concentration was consistent with the MIC of lauric acid. This suggests that lauric acid impaired the selectivity of antibacterial activity of myristic acid.
b Myristic acid/palmitoleic acid mixed system.  a and 3 b show the board diagrams of the antibacterial activity of myristic acid/palmitoleic acid mixed systems against S. aureus and S. epidermidis, respectively. The myristic acid/palmitoleic acid mixtures exhibited an antibacterial activity against S. aureus as the increase of the turbidity was low OD 660 0.010 when concentrations of myristic acid and palmitoleic acid were above 78.5 and 9.4 µg mL 1 , respectively. The MICs of myristic acid and palmitoleic acid are 157 and 18.8 µg mL 1 , respectively. When the two-components were mixed, a smaller amount of the components showed antibacterial activity against S. aureus. These results suggest that the addition of palmitoleic acid to myristic acid results in additive effect on the antibacterial behavior against S. aureus as ΣFIC was 0.57 0.20 Table 2 . This mixed system did not show significant antibacterial activity against S. epidermidis. As shown in Fig. 3 b , the turbidity of the liquid medium increased in all compositions. Not only did neither myristic acid nor palmitoleic acid exhibit antimicrobial activity, but also no antimicrobial activity was observed under conditions where the two components were mixed.
c Myristic acid/lactic acid mixed system. Figs. 4 a and 4 b show the board diagrams of the antibacterial activity of myristic acid/lactic acid mixed systems against S. aureus and S. epidermidis, respectively. The myristic acid/lactic acid mixture exhibited an antibacterial activity against S. aureus as the increase of the turbidity was low OD 660 0.010 when the concentrations of myristic acid and lactic acid were above 78.5 and 4000 µg mL 1 , respectively. However, the turbidity increased specifically under all myristic acid concentrations when 2000 µg mL 1 of lactic acid was added. We evaluated the system on eight plates to check for reproducibility and obtained the same results. Here, the liquid medium in which the turbidity specifically increased was spread on agar plates and incubated at 37 for 24 h to confirm the growth of bacteria. When the liquid medium containing myristic acid and 2000 µg mL 1 lactic acid was spread, S. aureus grew under all conditions: The increases in turbidity are caused by an increase in bacteria. The ΣFIC was 1.57 0.12, which suggested that the antibacterial activities of myristic acid and lactic acid against S. aureus were indifferent Table 2 . On the other hand, the antibacterial activity against S. epidermidis was similar to that of myristic acid/lauric acid mixed system. The myristic acid/lactic acid mixture exhibited the antibacterial activity against S. epidermidis when lactic acid was added above 3200 µg mL 1 regardless of the mixing ratio of myristic acid. This concentration was consistent with the MIC of lactic acid individually. When the concentrations of myristic acid and lactic acid added were 1600 and 800 µg mL 1 , respectively, the change in turbidity at 48 h was as small as 0.025. This is because insoluble myristic acid dispersed in the medium and was the turbidity increased at 0 h. These results suggest that lactic acid impaired the selectivity of antibacterial activity of myristic acid.

Discussion
In the mixed systems containing myristic acid, some ingredients showed additive or indifferent effects on the antimicrobial effect against S. aureus Table 2 . On the other hand, some of these mixed systems did not show antibacterial activity against S. epidermis. These differences in behavior reflect the antibacterial mechanism of each component.
First, we discuss the antibacterial behavior against S. aureus. The antibacterial properties against S. aureus were enhanced in the myristic acid/lauric acid and myristic acid/palmitoleic acid mixed systems. This effect suggests that the mixture of the two types fatty acids potentiate their action on the cell membrane. Desbois et al. reported that fatty acids affect the membrane function through the solubilization of cell membranes, disruption of electron transport chains, inhibition of oxidative phosphorylation, impaired enzyme activity, and formation of autoxidative degradation products 22 24 . These two ingredients act on cell membranes by the same mechanism and thus have an additive antibacterial effect.
On the other hand, synergistic antibacterial effect was not observed in the myristic acid/lactic acid mixed system. In particular, the increase of the turbidity was low when the concentration of lactic acid was above 4000 µg mL 1 , which is the MIC against S. aureus. We speculate that this result is due to the fact that the antibacterial mechanism of lactic acid is different from that of fatty acids. Undissociated acid molecules can penetrate the cell membrane and dissociate in the intracellular region, causing a pH decrease in the bacterial cell 25 27 . The pH was 3.3 4.4 for myristic acid/lactic acid mixed system when the concentration of lactic acid was above 4000 µg mL 1 . The pH of these mixed systems was out of the buffer region of the phosphate buffer pH6 , because they contained high concentration of lactic acid. Since the pKa value of lactic acid is 3.83, many lactic acid molecules exist in an undissociated state in a system under these conditions 28 . Furthermore, the turbidity specifically increased for all myristic acid concentrations when 2000 µg mL 1 of lactic acid was added Fig. 4 a . When these samples were spread on agar medium, the growth of S. aureus was confirmed. The exact mechanism of this phenomenon is not clear now. Here, we suggest a possible mechanism: Significant antibacterial effect was not observed, because the bacteria may have altered metabolic pathways and gene expression to maintain homeostasis when they were exposed to lactic acid 29 31 . Rode et al. reported that in the environment acidified by lactic acid, S. aureus produced larger amounts of ammonium, 2,3-butanedione, and pyrazine than in the environment exposed to acetic acid or hydrochloric acid. Furthermore, the expression of genes involved in energy production was enhanced 31 .
Finally, the antibacterial behavior against S. epidermidis is discussed. The myristic acid/lauric acid and myristic acid/lactic acid mixed systems confirmed the antibacterial activity as the increase of the turbidity was low when the concentration of lauric acid and lactic acid were 18.8 and 3200 µg mL 1 , respectively, which were the MICs against S. epidermidis. We found that myristic acid does not show antibacterial activity against S. epidermidis 10 . Therefore, these results suggest that myristic acid did not involve the antibacterial activity, while lauric acid and lactic acid showed antibacterial activity against S. epidermidis independently: Lauric acid and lactic acid inhibited the selective antibacterial properties of myristic acid against S. aureus.
In the case of myristic acid/palmitoleic acid mixed system, the turbidity increased under all mixed conditions: The antibacterial behavior was not observed against S. epidermidis. Palmitoleic acid also does not exhibit antibacterial activity against S. epidermidis as a single component as well as myristic acid 10 . This result suggests that the selective antibacterial activity against S. aureus is maintained even when the two components, which do not exhibit antibacterial activity against S. epidermidis, are mixed. The exact mechanism of the selective antibacterial behavior is unclear. We introduce a hypothesis here. It may be influenced by differences in the composition of lipid fatty acids in the cell membranes of S. aureus and S. epidermidis. Tiwai et al. reported that the composition of branched fatty acids in the cell membrane of S. epidermidis is higher than that of S. aureus and fluidity of the cell membrane in S. epidermidis is higher than that in S. epidermidis 32 . Branched fatty acids can disrupt the order of cell membranes more than straight-chain fatty acids and increase membrane permeability and fluidity of the cell membrane 33 . Furthermore, the cell membrane of S. aureus contains staphyloxanthin, which is a carotenoid that contributes to membrane stabilization, while this component is absent in S. epidermidis 34,35 .

Conclusion
The antibacterial activity of the two-component mixed system of lauric acid, palmitoleic acid, and lactic acid with myristic acid was evaluated against S. aureus and S. epidermidis using the checkerboard method. The antibacterial activity against S. aureus was enhanced in the myristic acid/lauric acid and myristic acid/palmitoleic acid mixed systems. The myristic acid/lactic acid mixed system did not significant change in the antibacterial properties of each substances. On the other hand, myristic acid/palmitoleic acid mixed system did not show clear antibacterial activity against S. epidermidis. However, myristic acid/lauric acid mixed system and myristic acid/lactic acid mixed system exhibited the antibacterial behavior against S. epidermidis when the concentration of lauric acid and lactic acid above MIC, respectively. These results suggest that the property of myristic acid, which shows the selective antibacterial activity against S. aureus as a single component, may be altered depending on the coexisting components. Antibacterial activity of mixed fatty acids must be related to the physical properties of mixed fatty acids. It has been reported that a decrease in Kraft point, pKa, and water evaporation through molecular membrane occurs in mixed systems containing two types of fatty acids 36 38 . These physical phenomena are caused by the packing change, which increases the intermolecular distance. In the future, systematic analysis between molecular structure, interfacial chemistry, and biological properties will lead to a better understanding of the behavior of molecules on biological surfaces. These findings of this study are useful for cosmetic and body cleansers formulation design containing fatty acids.