Antibacterial Activity of the Mixed Systems Containing 1,2-Dodecanediol against Staphylococcus aureus and Staphylococcus epidermidis

checkerboard Abstract: 1,2-Alkanediols are characteristic cosmetic ingredients because these moisturizers exhibit the antibacterial activity against Staphylococcus aureus ( S. aureus ) and Staphylococcus epidermidis ( S. epidermidis ). However, the antimicrobial behavior in mixed systems containing several active ingredients is unclear because previous reports focus on an antibacterial system containing only 1,2-alkanediol. In this study, the minimal inhibitory concentration (MIC) and the fractional inhibitory concentration (FIC) were evaluated for 1,2-dodecanediol/lactic acid, 1,2-dodecanediol/myristic acid, 1,2-dodecanediol/methylparaben, and 1,2-dodecanediol/isopropyl methylphenol mixed systems to show the effect of the addition of other antimicrobial components to 1,2-dodecanediol. The antibacterial property of 1,2-dodecanediol/lactic acid mixed system was almost similar compared to 1,2-dodecanediol monomeric system. On the other hand, the antimicrobial activity of 1,2-dodecanediol against S. epidermidis was inhibited in the 1,2-dodecanediol/ myristic acid mixed system. Because the selective antimicrobial activity of myristic acid against S. aureus was demonstrated in the mixed system. The present findings are useful for designing formulations of cosmetics and body cleansers containing 1,2-dodecanediol.


Introduction
1,2-Alkanediols exert a moisturizing effect and exhibit antibacterial activity against Escherichia coli and Aspergillus niger 1 5 . In a previous study, we evaluated the antimicrobial activity of 1,2-alkanediols against Staphylococcus aureus S. aureus and Staphylococcus epidermidis S. epidermidis and found that the antimicrobial activity of these surfactants was stronger against both Staphylococci, if the alkyl chain length was longer 6 . We selected these bacteria, as S. aureus causes atopic dermatitis and skin roughness, whereas S. epidermidis enhances innate immunity and maintains healthy epidermis 7 11 . Lee et al. reported that 1,2-alkanediols exhibit antibacterial properties as the results of the disruption of the cell membrane 1 . We also considered that 1,2-dodecanediol can penetrate the cell membrane and inhibit the growth of S. aureus and the antimicrobial activity of systems containing multiple ingredients. The synergistic or antagonistic effects of two antimicrobial agents can be determined by calculating the fractional inhibitory concentration FIC index based on the growth inhibition effect of the combination of the two agents 17,18 . The activity of antibiotic combinations, such as imipenem and vancomycin, against methicillin-resistant bacteria has been evaluated using this method 19 22 .
In the present study, the checkerboard method was used to evaluate the antimicrobial activity of a mixture of 1,2-dodecanediol and other antimicrobial substances against S. aureus and S. epidermidis. Lactic acid and myristic acid, which are antimicrobial substances on the surface of the skin, and methylparaben and isopropyl methylphenol, which are typical preservatives and bactericidal ingredients in cosmetics, were selected as additional ingredients 23 26 . The antibacterial activity of these substances was determined using minimal inhibitory concentration MIC , which is the minimum drug concentration required to suppress bacterial growth, and the combined effect of the two-component mixture was evaluated based on the FIC index. The findings of the present study are useful for designing skin care and cleansing products focusing on human skin flora.

Materials
The molecular structures of the materials used in this study are shown in Fig. 1 145 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 MIC against S. aureus and S. epidermidis
The MIC was defined as the minimum concentration at which turbidity did not increase when the antibacterial agents were added to the bacterial dispersion. First, lactic acid was diluted with phosphate buffer, while 1,2-dodecanediol, myristic acid, methylparaben, and isopropyl methylphenol were diluted with ethanol to prepare the following sample solutions: 3.13 400 µg mL 1 1,2-dodecanediol , 300 32000 µg mL 1 lactic acid , 100 2500 µg mL 1 myristic acid , 21.9 3200 µg mL 1 methylparaben , and 9.4 1000 µg mL 1 4-isopropyl-3-methylphenol . We confirmed that the addition of 2 wt ethanol did not affect the antibacterial properties in preliminary tests. Next, 1000 µL a liquid medium containing beef extract 0.01 g , polypeptone 0.02 g , and sodium chloride 0.01 g , 940 µL phosphate buffer pH 6 , and 20 µL bacterial dispersion 3 10 5 2 10 6 CFU mL 1 were added to 40 µL ethanol solution of antimicrobial agents.
The optical density OD 660 of the prepared sample 100 µL was evaluated using a 96-well TrueLine Cell Culture Plate Japan Genetics Co., Ltd., Tokyo, Japan and a grating microplate reader SH-1200 Lab Corona Electric Co., Ltd., Ibaraki, Japan . The evaluated conditions were as follows: wavelength, 660 nm; number of flashes, 10. The S. aureus medium was shaken at 1000 rpm by a microplate shaker PSU-2T Waken B Tech Co., Ltd., Kyoto, Japan during incubation for 24 h at 37 . On the other hand, the dispersion containing S. epidermidis was shaken for 48 h under the same conditions because the growth rate of S. epidermidis is slower compared with that of S. aureus.

Evaluation of the combined effect of antibacterial
components based on checkerboard method The effects of lactic acid, myristic acid, methylparaben, and isopropyl methylphenol on the antibacterial properties of 1,2-dodecanediol were evaluated using the checkerboard method, a typical evaluation method for mixed antibacterial systems. In general, a combined effect is determined based on the FIC index, which is calculated from the MIC of a mixture containing two types of drugs 15,27 . Here, the antimicrobial activity of the mixtures containing 1,2-dodecanediol and the additives in the concentrations of 1/64 to 4 times the MIC was evaluated: the concentration ranges of 1,2-dodecanediol and additives were as follows: 0.78 200 µg mL 1 1,2-dodecanediol; S. aureus, S. epidermidis , 62.5 16000 µg mL 1 lactic acid; S. aureus , 50.0 12800 µg mL 1 lactic acid; S. epidermidis , 2.45 628 µg mL 1 myristic acid; S. aureus , 6.25 1600 µg mL 1 myristic acid; S. epidermidis , 25.0 6400 µg mL 1 methylparaben; S. aureus, S. epidermidis , 2.16 552 µg mL 1 isopropyl methylphenol; S. aureus , and 2.70 700 µg mL 1 isopropyl methylphenol; S. epidermidis . To evaluate the combined effect, 20 µL of 1,2-dodecanediol 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 was added to 40 µL of 1,2-dodecanediol 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 concentration 28 .
The prepared sample 100 µL was added to a 96-well TrueLine cell culture plate. The plate diagram used for the evaluation of the 1,2-dodecanediol/lactic acid mixed system against S. aureus is shown in Fig. S1 see supporting information . The OD 660 of these samples was evaluated by grating microplate reader SH-1200 Lab . The conditions were as follows: wavelength, 660 nm; 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 checkerboard 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 antimicrobial combination as the sum of the individual FICs. In the board diagram, the mean turbidity values are shown in five levels, OD 660 0.000 0.150, 0.151 0.300, 0.301 0.450, 0.451 0.600, and 0.601 0.750.

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: 1 synergistic; when ΣFIC ≤ 0.5, the effect of the two drugs combined with much stronger than the sum of the effects of the single drug, 2 additive; when 0.5 ΣFIC ≤ 1.0, two drugs combined are slightly more effective than a single drug, 3 indifferent; when 1.0 ΣFIC ≤ 2.0, the combined effect is neither improved nor inferior to that of the single agent, 4 antagonistic; when 2.0 ΣFIC, the effect of the combination is inferior to the action of the single agent.
Figures S2 a d in supporting information are examples of board diagrams when the combined effects of drugs A and B are synergistic, additive, indifferent, or antagonistic, respectively. The horizontal and vertical axes of the board diagram indicate the concentration of drugs A and B, respectively. Turbidity was increased with bacterial growth under the colored conditions in the board diagram. In these examples, the ΣFIC is a 0.194, b 0.661, c 1.164, and d 3.10, respectively.

Results
3.1 MIC of 1,2-dodecanediol and additives against S. aureus and S. epidermidis Liquid medium containing S. aureus or S. epidermidis was added to 1,2-dodecanediol, lactic acid, myristic acid, methylparaben, and isopropyl methylphenol solutions and incubated at 37 for 24 or 48 h, respectively. The temporal change of the OD 660 was as follows: a 1,2-dodecanediol. The antimicrobial activity of 1,2-dodecanediol against S. aureus or S. epidermidis was evaluated in a previous study 6 . Figures 2 a and b show the temporal changes in the OD 660 of the liquid medium containing 1,2-dodecanediol and S. aureus or S. epidermidis. The white circles indicate the OD 660 immediately after the addition of the bacteria to the liquid medium, and black ones are that at 24 or 48 h, respectively. In these profiles, the growth of the bacteria is inhibited if the turbidity does not increase. Here, the OD 660 increased when the 1,2-dodecanediol concentration was less than 50.0 µg mL 1 for both S. aureus and S. epidermidis; the MIC of 1,2-dodecanediol was 50.0 µg mL 1 where bacterial growth was not observed against S. aureus and S. epidermidis.
b Lactic acid. Figures 3 a and b show the temporal changes in the OD 660 of the liquid medium containing lactic acid and S. aureus or S. epidermidis. Here, turbidity increased at less than 4000 µg mL 1 for S. aureus and less than 3200 µg mL 1 for S. epidermidis: the MICs against S. aureus and S. epidermidis were 4000 µg mL 1 and 3200 µg mL 1 , respectively. c Myristic acid. Figures 4 a and b show the temporal changes in the OD 660 of the liquid medium containing myristic acid and S. aureus or S. epidermidis. The turbidity increased with the increase of the concentration at 0 h, because insoluble myristic acid was dispersed in the medium. In the case of S. aureus, turbidity increased at myristic acid concentrations less than 180 µg mL 1 , whereas for S. epidermidis, turbidity increased at all fatty acid concentrations. Furthermore, the MICs against S. aureus were 140, 150, and 180 µg mL 1 in the three times antimicrobial tests. Therefore, the MIC of myristic acid against S. aureus was set to 157 µg mL 1 in this study, which is the average of the obtained MICs. Significant antimicrobial activity was not observed against S. epidermidis; the MIC was above 2500 µg mL 1 .   Figures 5 a and b show the temporal changes in the OD 660 of the liquid medium containing methylparaben and S. aureus or S. epidermidis. The OD 660 increased when the concentration was less than 1600 µg mL 1 for both S. aureus and S. epidermidis: the MICs were 1600 µg mL 1 against both S. aureus and S. epidermidis.
e Isopropyl methylphenol. Figures 6 a and b show the temporal changes in the OD 660 of the liquid medium containing isopropyl methylphenol and S. aureus or S. epidermidis. Here, turbidity increased at less than 150 µg mL 1 for S. aureus and less than 200 µg mL 1 for S. epidermidis. The MIC against S. aureus varied from 125 to 150 µg mL 1 in the three times antimicrobial tests. Similarly, the MIC against S. epidermidis varied from 150 to 200 µg mL 1 . Therefore, the MIC of isopropyl methylphenol against S. aureus and S. epidermidis were set to 138 and 175 µg mL 1 , respectively, which were the averages of the obtained MICs.

Evaluation of the combination effect
The antimicrobial activity was evaluated for a mixed system containing 1,2-dodecanediol and four additives, lactic acid, myristic acid, methylparaben, and isopropyl methylphenol against S. aureus and S. epidermidis. Culture medium containing S. aureus or S. epidermidis was added to a mixture of 1,2-dodecanediol and to each substance, respectively. The OD 660 was measured after incubation at 37 for 24 or 48 h. a 1,2-dodecanediol/lactic acid mixed system. Board diagrams of the antibacterial activity of 1.2-dodecanediol/ lactic acid mixed system against S. aureus and S. epidermidis are shown in Figs. 7 a and b , respectively. In the case of 1,2-dodecanediol/lactic acid mixture, the turbidity did not increase when concentrations of 1,2-dodecanediol and lactic acid were above 50.0 and 4000 µg mL 1 , respectively. These concentrations were consistent with the MICs of 1,2-dodecanediol and lactic acid individually. The ΣFIC for this mixed system was 1.29 0.09, which suggests that the antibacterial behavior of 1,2-dodecanediol and lactic acid against S. aureus was indifferent .
The antimicrobial behavior of the 1,2-dodecanediol/lactic acid mixed system against S. epidermidis was similar to  that against S. aureus. Antimicrobial activity was confirmed as the turbidity did not increase when the concentration of 1,2-dodecanediol and lactic acid was above the respective MICs of 50.0 and 3200 µg mL 1 . In the case of this mixed system, antimicrobial behavior was indifferent against S. epidermidis because ΣFIC was 1.23 0.11. b 1,2-dodecanediol/myristic acid mixed system. Figures 8 a and b show the board diagram of the antibacterial activity of 1,2-dodecanediol/myristic acid mixed system against S. aureus and S. epidermidis, respectively. The 1,2-dodecanediol/myristic acid mixture exhibited an antibacterial against S. aureus as the turbidity did not increase when concentrations of 1,2-dodecaendiol and myristic acid were above 50.0 and 78.5 µg mL 1 , respectively. These results suggest that the antimicrobial behavior of this mixed system is additive as ΣFIC of mixed system was 0.89 0.24.
On the other hand, the antimicrobial behavior of this binary mixture against S. epidermidis was complicated. In this mixed system, the turbidity of the liquid medium in-creased even when it included 50.0 µg mL 1 of 1,2-dodecanediol. This suggests that myristic acid inhibited the antibacterial activity of 1,2-dodecaendiol. The extent of this inhibition intensified when the amount of myristic acid was increased. When the concentration of myristic acid was 6.25 µg mL 1 , the antibacterial phenomenon was confirmed under the condition of more than 100 µg mL 1 of 1,2-dodecanediol. However, the turbidity increased even with the addition of 100 µg mL 1 of 1,2-dodecanediol when the concentration of myristic acid was 200 µg mL 1 . On the other hand, in the system containing 0.78 µg mL 1 of 1,2-dodecanediol and 1600 µg mL 1 of myristic acid, turbidity increased from 0.164 to 0.277 in 48 h. This board diagram suggests that this increase is caused by the growth of bacteria. Therefore, the mixture containing myristic acid did not show antimicrobial activity against S. epidermidis. c 1,2-dodecanediol/methylparaben mixed system. Figures 9 a and b show the board diagram of the antibacterial activity of 1,2-dodecanediol/methylparaben mixed system against S. aureus and S. epidermidis, respectively. The 1,2-dodecanediol/methylparaben mixture exhibited an antibacterial activity against S. aureus as the turbidity did not increase when the concentrations of 1,2-dodecaendiol and methylparaben were above 100 and 1600 µg mL 1 , respectively. ΣFIC was 1.38 0.16, indicating that the antimicrobial properties of methylparaben and 1,2-dodecanediol are indifferent against S. aureus.
On the other hand, turbidity did not increase against S. epidermidis when the concentrations of 1,2-dodecanediol and methylparaben were 100 and 3200 µg mL 1 , respec-tively. The MICs of these components are 50.0 and 1600 µg mL 1 , respectively. It was confirmed that the coexistence of the two components required the addition of a large amount of antimicrobial components. The antimicrobial behavior of 1,2-dodecanediol and methylparaben was antagonistic because ΣFIC for this binary system was 2.14 0.20. d 1,2-dodecanediol/isopropyl methylphenol mixed system. Figures 10 a and b show the board diagram of the antibacterial activity in 1,2-dodecanediol/isopropyl methylphenol mixed system against S. aureus and S. epidermidis, respectively. In this mixture, the turbidity did not increase against S. aureus when concentrations of  The antimicrobial behavior of the 1,2-dodecanediol/isopropyl methylphenol mixed against S. epidermidis was similar to that against S. aureus. Antimicrobial activity was confirmed as the turbidity did not increase when the concentrations of 1,2-dodecanediol and isopropyl methylphenol were above 50.0 and 350 µg mL 1 , respectively. The behavior of this mixed system was indifferent against S. epidermidis as ΣFIC was 1.29 0.30.

Discussion
As shown in Table 1, four additives showed additive, indifferent, or antagonistic effects to 1,2-dodecanediol. These characteristic behaviors reflected the antibacterial mechanism of each substance. In the case of 1,2-dodecanediol/lactic acid mixed system, no synergistic effect was observed in the antimicrobial activity against both Staphylococci. In particular, the turbidity did not increase when the concentration of lactic acid was above the MIC. This was caused by a decrease in the intracellular pH of the bacteria due to the addition of lactic acid. Some previous studies showed that a reduction of pH inside the bacteria cell is a mainly mechanism of the antibacterial effect of acidic materials 29 32 . At a low pH condition, undissociated acids can permeate the cell membrane and flow into the intracellular regions. In general, hydrogen ion H is dissociated from acidic molecules because the pH of the cytoplasm is higher compared with the environment external to the bacteria. This causes the inhibition of growth of the bacteria because the pH range is beyond the optimal range for biological reactions. The pH of 1,2-dodecanediol/lactic acid mixed system was 3.2 4.4 when the concentration of lactic acid was above 4000 µg mL 1 , which is the MIC against S. aureus. The pH of these mixed systems was out of the buffer region of the phosphate buffer pH 6 , because they contained high concentration of lactic acid. Since the pKa value of lactic acid is 3.83, a system containing more than 4000 µg mL 1 lactic acid would be expected to have many lactic acid molecules in an undissociated form 33 . Table 1 Fractional inhibitory concentration of 1,2-dodecanediol in combination with lactic acid, myristic acid, methylparaben or isopropyl methylphenol against S. aureus or S. epidermidis. 1,2-dodecanediol/myristic acid mixed system showed a slight stronger selective antibacterial activity, because the antimicrobial effects of both surfactants are due to their effects on cell membranes. 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 34 36 . On the other hand, an increase in turbidity was observed for S. epidermidis regardless of the mixture of 1,2-dodecanediol and myristic acid. This reflects the selective antibacterial properties of myristic acid: Myristic acid shows antibacterial activity against S. aureus, but not against S. epidermidis 37 . That is, the fact that antimicrobial activity against S. epidermidis was not observed under many conditions indicated that the selective antimicrobial activity of myristic acid was maintained even in a two-component mixture. Conversely, the antimicrobial activity of 1,2-dodecanediol against S. epidermidis was inhibited. As shown in Fig. 8 a , if 50.0 µg mL 1 of 1,2-dodecanediol, which is the MIC, was added, the antibacterial activity against S. epidermidis was not observed. The exact mechanism of this antimicrobial behavior is unclear.
In the case of 1,2-dodecanediol/methylparaben mixed system, the antimicrobial behavior was indifferent against S. aureus and antagonistic against S. epidermidis. 1,2-dodecanediol/isopropyl methylphenol mixed system showed that synergistic effect was not found against both Staphylococci. It is difficult to understand the exact antibacterial mechanism of these mixed systems. Phenolic compounds such as methylparaben and isopropyl methylphenol are often thought to exhibit antimicrobial properties by damaging cell membranes and causing changes in the membrane function and structure 38 40 . However, these mechanisms are not clear. In this study, the antagonism of 1,2-dodecanediol and methylparaben against S. epidermidis may not only be due to the antimicrobial mechanism, but also to a complicated action, for example, the regulation of bacterial gene transcription in response to change in the external environment 41 .
When the concentration of amphiphilic molecules is more than the critical micelle concentration CMC , the molecules form the self-assembling structure. In most of the cases in this study, antimicrobial activity was observed when the concentration of 1,2-dodecanediol was above MIC. 1,2-dodecanediol may form aggregates at MICs; the MIC against Staphylococci and CMC of 1,2-dodecanediol are 50.0 µg mL 1 and 36.8 µg mL 1 , respectively 42 . These results suggest that 1,2-dodecanediol, which is highly amphiphilic, may penetrate cell membranes and exhibit antimicrobial activity. Previously, some researchers reported that micelle formation affects the antibacterial property of antibacterial agents. For example, the antimicrobial activity of the phenol type preservative is weakened in micellar solu-tion of nonionic surfactants, because the molecules are solubilized in the micelles or form a hydrophobic complex 43 45 . On the other hand, Tobe et al. reported that bactericidal activities of benzalkonium chloride is stronger when the concentration of methyl ester ethoxylates type nonionic surfactant is greater than the CMC 46 .

Conclusion
The antibacterial activity of the mixed system of lactic acid, myristic acid, methylparaben, and isopropyl methylphenol with 1,2-dodecanediol was evaluated against S. aureus and S. epidermidis using the checkerboard method. The antibacterial activities did not significantly change in the 1,2-dodecanediol/lactic acid from that each component monomeric system. In the 1,2-dodecanediol/ myristic acid mixed system, the antibacterial activity against S. aureus was slightly improved, and no clear was observed antimicrobial activity against S. epidermidis. The antibacterial activity of 1,2-dodecanediol against S. epidermidis was inhibited by myristic acid. On the other hand, the antimicrobial activities of mixed systems, including 1,2-dodecanediol and phenolic compounds, were complicated. The 1,2-dodecanediol/methylparaben mixed system showed that no variation in the antibacterial activity of each component against S. aureus, but the two components tended to be antagonistic against S. epidermidis. In the case of 1,2-dodecanediol/isopropyl methylphenol mixture, although FIC showed that the antimicrobial activity was indifferent to both Staphylococci, the turbidity increased with the addition of MIC of isopropyl methylphenol under some conditions. In particular, methylparaben and isopropyl methylphenol are added to cosmetic products and body cleansers as preservative and sterilization agents. These results of this study predict that these antimicrobial properties may be altered when 1,2-dodecanediol is added to the product. These findings are useful for cosmetic and body cleansers formulation design.