Antibacterial Activity of Long Chain Fatty Alcohols Against Staphylococcus aureus. Molecules 2007

The antibacterial activity against Staphylococcus aureus of long-chain fatty alcohols was investigated, with a focus on normal alcohols. The antibacterial activity varied with the length of the aliphatic carbon chain and not with the water/octanol partition coefficient. 1-Nonanol, 1-decanol and 1-undecanol had bactericidal activity and membrane-damaging activity. 1-Dodecanol and 1-tridecanol had the highest antibacterial activity among the long-chain fatty alcohols tested, but had no membrane-damaging activity. Consequently, it appears that not only the antibacterial activity but also the mode of action of long-chain fatty alcohols might be determined by the length of the aliphatic carbon chain.


Introduction
Staphylococcus aureus is a pathogenic microorganism that is responsible for serious problems, in particular in medical facilities, such as nosocomial infection and resistance to antibiotics.Novel compounds with antibacterial activity are needed to solve these problems.We have studied the antibacterial activities of plant-derived compounds, such as essential oils, against S. aureus [1][2][3][4][5] and have reported that two sesquiterpenes with an aliphatic carbon chain, namely, farnesol and nerolidol, have effective activity [3].Methodical investigation of anti-staphylococcus activity of terpene alcohols suggested that mono-and diterpenes with an aliphatic carbon chain have lower activity [3].Our results indicated that there might be a relationship between antibacterial activity and chemical structure.In this study, we selected the length of the carbon chain as the characteristic of the chemical structure and used fatty alcohols with a carbon chain of various lengths.
In an effort to investigate the antibacterial activities of long-chain fatty alcohols against S. aureus in detail, we used a broth dilution with shaking (BDS) method, a time-kill assay and an assay of the leakage of K + ions from cell.The BDS method was an unique method to investigate the antibacterial activity, such as minimum inhibitory concentration (MIC), of hydrophobic compounds.The time kill assay was carried out to observe the antibacterial activity in detail in combination with the BDS method.Leakage of K + ions from cells reflects the cell membrane damage activity.Since farnesol and nerolidol showed effective antibacterial activity and also cell membrane damaging activity, it might be important to measure the leakage of K + ions from cells in response to reagents.

Growth-inhibitory activity
Table 1 summarizes the antimicrobial activities of long-chain fatty alcohols on the growth of S. aureus FDA209P, as determined by two different methods.Long-chain fatty alcohols with aliphatic carbon chains with fewer than seven (data not shown) and eight carbon atoms (Table 1) had no antimicrobial activity.Long-chain fatty alcohols with an aliphatic carbon chain with more than 17 carbon atoms had hardly any antimicrobial activity.The results obtained by the broth dilution method indicated that the most effective number of carbon atoms in long-chain fatty alcohols, in terms of growth-inhibitory activity, ranged from 13 to 15.We obtained similar results when we determined the bactericidal activity by the broth dilution method.The BDS method indicated that long-chain fatty alcohols with an aliphatic carbon chain of 12 or 13 carbons had the greatest growth-inhibitory activity (Figure 1).The highest bactericidal activity was observed with an aliphatic carbon chain of 11 carbon atoms.The growth-inhibitory and bactericidal activities depended on concentration (Figure 2).The viable cell count barely changed over 24 h in the presence of C 8 -OH, while C 9 -OH and C 12 -OH decreased the viable cell count from 10 7 to approximately 10 4 colony forming units (cfu) mL -1 within 4 h.Contact with C 12 -OH decreased the viable cell count to beneath the limit of detection within 24 h.In the presence of C 10 -OH and C 11 -OH, the viable cell count decreased rapidly.The viable cell count fell below the limit of detection within only 2 and 4 h in the presence of C 10 -OH and C 11 -OH, respectively.

Quantitation of K + ion leakage
As shown in Figure 4, addition of long-chain fatty alcohols to bacterial suspensions led to the immediate leakage of K + ions from the cells.We investigated initial rates of leakage of K + ions and amounts of K + ions leaked from bacterial cells (Figure 5).In the presence of C 12 -OH and C 11 -OH, both values were larger than in the presence of other long-chain fatty alcohols.The long-chain fatty alcohols used in this study each possess a long aliphatic carbon chain and one hydroxyl group and they differ in terms of the length of the aliphatic carbon chain.The activity against S. aureus was most evident when the carbon chain length ranged from 10 to 13.When there were 10 or 11 aliphatic carbon atoms in the carbon chains, bactericidal activity was both evident and potent (Table 1).The bactericidal activity results were supported by the time-kill assay results (Figure 3).K + ion leakage assays indicated that C 10 -OH and C 11 -OH induced the leakage of K + ions more rapidly than the other tested alcohols.We reported previously that the initial rate of K + ion leakage reflects the damage to cell membranes when a reagent that affects cell membranes is added to a bacterial suspension [19].than those induced by TTO.The total amount of K + ions that leaks from bacterial cells reflects the antibacterial activity that is mediated by cell membrane damage.
Our results indicate that the antibacterial activity of C 10 -OH and C 11 -OH were mediated by damage to cell membranes that allowed leakage of K + ions, with subsequent reactions that induced the further leakage.MIC and MBC measurements (Table 1) confirmed that C 12 -OH and C 13 -OH had the most effective bacteriostatic activity among the long-chain fatty alcohols tested.K + ion leakage induced by C 12 -OH and C 13 -OH was less conspicuous than that observed in the presence of C 10 -OH and C 11 -OH and similar to that observed in the presence of long-chain fatty alcohols that did not have effective antibacterial activity.Thus, the expression of bacteriostatic or bactericidal activity was dependent on the length of the aliphatic carbon chain.We are now studying the nature of the antibacterial effects of C 12 -OH and C 13 -OH.
Some authors have reported that the antibacterial activity of hydrophobic compounds depended on their water/octanol partition coefficients [17,18].The water/octanol partition coefficients of the long-chain fatty alcohols used in this study are listed in Table 1.If activity were dependent on the partition coefficient, the activity would be expected to increase with elongation of the aliphatic carbon chain.Our results do not support such a conclusion.There is a defined range of chain lengths for induction of effective inhibition of the growth of S. aureus.The expression of antibacterial activity was determined by the length of the aliphatic carbon chain rather than by the water/octanol partition coefficient.
MIC and MBC measurements confirmed that C 12 -OH and C 13 -OH had the most effective bacteriostatic activity among the long-chain fatty alcohols tested.Leakage of K + ions in response to these alcohols was smaller than those in response to C 10 -OH and C 11 -OH and similar to that obtained with long-chain fatty alcohols that did not exhibit effective antibacterial activity.Therefore, it appears that the length of the aliphatic carbon chain might determine the nature of the antibacterial activity.
Compounds with alkyl chains are attracting attention since they have some novel attributes [22][23][24][25], for example, the ability to promote antibacterial activity and to resensitize methicillin-susceptible and -resistant S. aureus to antibiotics.Their properties are probably related to their alkyl groups since the effective length of the carbon chain in each alkyl group is in harmony with our results.Although there have been many studies of the antibacterial activities of fatty alcohols, some variations in activity have been missed, perhaps because of the use of high concentrations, on the order of mg mL -1 , in determinations of MICs.A detailed reevaluation is clearly needed of the antibacterial activities of hydrophobic compounds at appropriate concentrations.

General
All long-chain fatty alcohols used in this study were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).Staphylococcus aureus FDA209P was used as the standard strain [1].

Broth dilution method
The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of each alcohol were determined by the micro broth dilution method.The MIC was determined by the NCCLS method [26].The compound to be tested was added to aliquots of Mueller Hinton broth (100 µL, Difco, Detroit, MI, USA).An aliquot of an overnight culture of S. aureus (ca. 1 x 10 5 cfu mL -1 ) was added to each sample.Each culture was incubated in air without shaking at 37°C for 24 h.

Figure 4 .
Figure 4. Changes in the concentration of K + ions in a suspension of S. aureus FDA209P in response to 1-decanol.The arrow indicates the addition of 1-decanol.The results are typical of three assays that gave similar results.See text for full details.

Table 1 .
Antimicrobial effects of 1-alkanols on the growth of S. aureus and n-octanol/water partition coefficients.