JAC Advance Access originally published online on January 22, 2007
Journal of Antimicrobial Chemotherapy 2007 59(3):425-432; doi:10.1093/jac/dkl519
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Sesquiterpene farnesol inhibits recycling of the C55 lipid carrier of the murein monomer precursor contributing to increased susceptibility to ß-lactams in methicillin-resistant Staphylococcus aureus
Department of Microbiology, Graduate School of Comprehensive Human Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan
* Corresponding author. Tel/Fax: +81-29-853-3928; E-mail: makokuro{at}md.tsukuba.ac.jp
Received 11 August 2006; returned 29 September 2006; revised 22 November 2006; accepted 24 November 2006
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Abstract |
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Background and objectives: The sesquiterpene farnesol, a natural plant metabolite, is known to intensify the effect of antimicrobial agents. However, the mode of action of its antimicrobial synergism has remained poorly understood. In this study, we investigated farnesol's synergistic effects on commonly used antimicrobials, ß-lactams in particular, to explore its potential inhibitory effect on cell wall synthesis.
Methods: We investigated farnesol's effects on: (i) antimicrobial susceptibilities of methicillin-susceptible and -resistant Staphylococcus aureus (MSSA and MRSA) to ampicillin, oxacillin, cefoxitin, bacitracin, teicoplanin, amikacin, ciprofloxacin and clarithromycin by MIC determination using the Etest; (ii) penicillin-binding protein PBP2' (2a) expression by western-blot analysis; (iii) ß-lactamase secretion and activity by in vivo and in vitro farnesol inhibition assays; (iv) staphyloxanthin production by thin-layer chromatography (TLC); and (v) cell wall synthesis by [14C]GlcNAc (where GlcNAc stands for N-acetylglucosamine) and [14C]mevalonate incorporation assays, and TLC-based lipid extract profile analysis.
Results: Farnesol induced variable degrees of increased susceptibility to all antimicrobials except clarithromycin in both MSSA and MRSA. A remarkable increase in susceptibilities to ampicillin, oxacillin and cefoxitin was observed in both MRSA strains, N315 and COL, whereas a moderate increase in susceptibility to bacitracin was observed in all the strains. Although no apparent suppression of PBP2' expression was observed, ß-lactamase secretion and ß-lactamase activity were significantly reduced by farnesol. In addition, farnesol completely suppressed staphyloxanthin production. Farnesol reduced the incorporation of GlcNAc, but significantly increased that of mevalonate. Farnesol induced accumulation of C55-PP, lipid I and lipid II.
Conclusions: Farnesol increased ß-lactam susceptibility of MRSA by inhibition of cell wall biosynthesis through reduction of free C55 lipid carrier with subsequent retardation of murein monomer precursor transport across the cell membrane.
Keywords: skin diseases , plant extracts , antimicrobial synergism , cell wall synthesis inhibitors
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Introduction |
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Increasing multiple resistance of Staphylococcus aureus to antibiotics has made the development of new treatment options for serious infections a matter of urgent concern.1 In addition to hospital-acquired methicillin-resistant S. aureus (MRSA), community-acquired MRSA is becoming an important public health problem.2 Staphylococcus aureus is often isolated from the dry skin areas of patients with atopic dermatitis or impetigo, but is rarely observed on healthy skin.3 In this regard, folk remedies using essential oils or other plant products may be useful in treating or controlling the dissemination of MRSA.
Historically, plant extracts such as essential oils have been used for therapeutic purposes. In recent years, much research has been devoted to investigating such plant extracts: their active components, modes of action and synergistic effects with other antimicrobial compounds.4 Terpenoids are highly complex compounds based on an isoprene structure that are found in essential oils and used in perfumery, cosmetics, food flavourings, food preservatives and for medical purposes.5,6
Although terpenoids have been found to show antimicrobial activity against S. aureus and other bacteria, their mode of action is not fully understood. Among commonly used sesquiterpenes such as farnesol, bisabolol and nerolidol, farnesol is less toxic to humans and likely to be the most potent giving effective K+ leakage from cytoplasm.7 Since membrane damage facilitates penetration of antibiotics such as macrolides, aminoglycosides and quinolones, farnesol is believed to enhance antimicrobial activity.8 In a recent report, it was demonstrated that farnesol inhibits oxidation–reduction reactions and also increases the susceptibility of S. aureus to gentamicin which requires ATP-dependent transport to enter the cells, suggesting that the membrane integrity is disrupted by farnesol.9
In addition to causing increased membrane permeability, farnesol is postulated to possess additional inhibitory actions against S. aureus, because ß-lactams seem to be the most potent antimicrobial agents showing synergism with farnesol.10 ß-Lactams inhibit the transpeptidation of the murein monomer precursor by penicillin-binding proteins (PBPs) outside the membrane. Staphylococcus aureus can become resistant to ß-lactams by horizontal acquisition of the mecA gene encoding PBP2' (PBP2a) showing low-affinity binding to ß-lactam.11–13 Tea tree oil can affect the expression of PBP2' as well as secretion of ß-lactamase,14 also implying that essential oils might possess an uncharacterized specific inhibitory action. Based on the chemical structure of farnesol, it possesses structural similarity to farnesyl pyrophosphate (FPP) which is an indispensable substrate for synthesis of an undecaprenyl (C55) lipid carrier via the mevalonate pathway.15 This C55 lipid carrier plays a role in translocating the murein monomer precursor from the cytoplasm to outside of the membrane for subsequent peptidoglycan synthesis by PBPs.16
In this study, we attempted to investigate farnesol's synergistic effects on commonly used antimicrobials, ß-lactams in particular, and to explore its potential inhibitory effect on cell wall synthesis.
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Bacterial strains
Methicillin-susceptible S. aureus (MSSA) ATCC 25923 (oxacillin MIC 0.25 mg/L), homogeneously-resistant MRSA COL strain (oxacillin MIC >256 mg/L) and pre-MRSA N315 strain (oxacillin MIC 12 mg/L) were used for the experiments described below.
Antimicrobial susceptibility tests
Farnesol was purchased from WAKO (Wako Pure Chemical Industries, Osaka, Japan). Farnesol MICs were determined by the agar or broth dilution methods according to CLSI (formerly NCCLS) guidelines17 except for using Mueller–Hinton medium supplemented with 1% Tween 80 (MHT medium), which was used as a farnesol emulsifier. Concentrations of farnesol used in MIC determinations ranged from 4000 to 15.6 mg/L.
To determine synergism between antimicrobial agents and farnesol, MICs were determined using the Etest (AB Biodisk, Solna, Sweden) on MHT agar containing 1 g/L farnesol.
PBP2' (PBP2a) detection by western-blot analysis
PBP2' expression was determined by western-blot analysis using anti-PBP2' mouse monoclonal IgG, which was kindly provided by Denka Seiken (Tokyo, Japan). Staphylococcus aureus ATCC 25923 and COL strains were cultivated on MHT agar with or without 1 g/L farnesol at 37°C for 16 h. Colonies were scraped with a cotton swab and suspended in TE buffer to give a cell optical density (OD600) of 1.5. The cell suspension was treated with 10 µg/mL lysostaphin (WAKO, Osaka, Japan) at 37°C for 30 min, followed by brief sonication to obtain complete cell lysis. Protein concentration was determined using a Bio-Rad Protein Assay Kit (Bio-Rad, CA, USA). Total protein (4 µg) was separated by 7.5% SDS-PAGE, followed by staining with a SeePico CBB stain kit (benebiosis, Seoul Korea) or semi-dry blotting onto a PVDF membrane (Bio-Rad). The blotted membrane was immersed in a blocking solution (5% skimmed milk in PBS) at 4°C overnight and soaked in 1000-fold diluted anti-PBP2' antibody in the blocking solution at room temperature for 1 h, followed by washing three times with PBST (PBS containing 0.05% Tween 20). Subsequently, the membrane was soaked in 3000-fold diluted anti-mouse IgG alkaline phosphatase conjugate (Promega, Madison, WI, USA) in blocking solution at room temperature for 1 h, followed by washing three times each with PBST and PBS. Positive signals were detected by incubating the membrane with Western blue stabilized substrate for alkaline phosphatase (Promega, Madison, WI, USA) for 1 min. Relative expression of PBP2' was determined using NIH Image v. 1.62 software.
Determination of ß-lactamase secretion and activity
To observe the effect of farnesol on ß-lactamase secretion, ß-lactamase-positive S. aureus N315 strain was grown in MHT broth containing farnesol at 37°C for 18 h with shaking. Culture supernatant (4 mL) was dialysed twice against 2 L of PBS at 4°C for 12 h. The dialysed supernatant was mixed with nitrocefin (Merck, Darmstadt, Germany) to give a final concentration of 10 µg/mL, followed by incubation at 37°C for 90 min.18 Colorimetric change from yellow to red was measured at 486 nm. One unit of ß-lactamase specific activity was defined as the amount of enzyme that hydrolysed 1 µmol of nitrocefin per min at 37°C. Each assay was performed in triplicate.
To observe the effect of farnesol on ß-lactamase activity, the same assay procedure was employed, except that N315 strain was grown without farnesol in MHT broth and farnesol (62.5, 250 or 1000 mg/L) was added during the 90 min incubation at 37°C.
Detection of staphyloxanthin production by thin-layer chromatography (TLC)
Staphylococcus aureus N315 was grown on MHT agar supplemented with sub-MIC concentrations of farnesol (1 g/L), oxacillin (2 mg/L), bacitracin (16 mg/L), teicoplanin (0.5 mg/L), amikacin (4 mg/L) or ciprofloxacin (0.03125 mg/L) at 35°C for 20 h. MHT agar without any supplements was also used to grow strain N315 as a control. The colonies were collected and suspended in distilled water to give a cell density OD600 of 1.0. The cell pellet harvested after centrifugation was resuspended in 1 mL of PBS containing 10 µg/mL lysostaphin (WAKO) and incubated at 37°C for 30 min. The cell lysate was mixed well with 1 mL of ethyl acetate and centrifuged at 10 000 g for 10 min. An aliquot (500 µL) of the ethyl acetate phase was recovered and evaporated in the dark. The dried sample was dissolved again in 10 µL of ethyl acetate and 5 µL of the extract was separated on a 10 x 10 cm HPTLC Silica gel 60 plate (Merck, Darmstadt, Germany) with chloroform/methanol/water (65 : 25 : 4, v/v/v). The pigment spots were visually observed without conventional staining methods.
Alternatively, for visualization of the whole lipid extracts, the plate was soaked in 10% sulphuric acid and baked at 150°C until the reaction was visible.
[14C]GlcNAc and [14C]mevalonate incorporation assays
Staphylococcus aureus N315 was cultivated using either Mueller–Hinton or MHT medium at 37°C to mid-log phase (OD600 0.6). Chloramphenicol (final concentration 50 mg/L) was added to the culture to terminate the de novo protein synthesis. Five hundred microlitres of the culture was mixed with 500 µL of MHT broth containing a 2-fold concentration of farnesol to give a final concentration of 0.5, 1 or 2 g/L, followed by addition of 1 µL of [14C]GlcNAc (where GlcNAc is N-acetylglucosamine) (7.40 MBq/mL) or [14C]mevalonate (1.85 MBq/mL) (Amersham Biosciences, Piscataway, NJ, USA) for each labelling reaction, or MHT broth only for the control reaction. The cell suspension was incubated at 37°C for 60 min. The labelled cells were harvested and washed twice with distilled water. In the case of [14C]GlcNAc, additional washing with 0.1% SDS was carried out. The washed cell pellets were mixed with 1 mL of MicroScinti 40 liquid (PerkinElmer, Wellesley, MA, USA) and the radioactivity was measured.
Analysis of the C55 lipid carrier synthesis by HPTLC
As described above, S. aureus N315 cells were labelled with [14C]mevalonate, but during the labelling reaction, the cells were exposed to 1% Tween 80 as control, 2 g/L farnesol, 32 mg/L bacitracin, or 10 mg/L vancomycin. The labelled cells were harvested and washed twice with distilled water. The cell pellets were suspended with 500 µL of chloroform/methanol (1 : 1, v/v), and the labelled lipids were extracted. An aliquot of 100 µL of the lipid extract was dried and resuspended in 10 µL of chloroform and then separated on a 10 x 10 cm HPTLC Silica gel 60 plate (Merck, Darmstadt, Germany) with chloroform/methanol/water/ammonia (88 : 48 : 10 : 1, v/v/v/v).19 The radioactive spots were detected after exposure to an imaging plate for 3 days and scanning with a BAS5000 scanner (Fuji film, Minamiashigara, Japan).
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MICs of farnesol for S. aureus strains
All tested S. aureus strains, ATCC 25923, COL and N315, showed MICs of farnesol of 2000 mg/L and 125 mg/L by MHT agar and broth dilution methods, respectively.
Synergistic action of farnesol with antimicrobials
Table 1 shows the synergistic action of farnesol with the antimicrobials tested. The MIC values obtained by the agar dilution method using either MHT or MH medium were the same (data not shown), indicating that Tween 80 did not influence the MIC values of the tested antimicrobials.
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Farnesol supplementation induced variable degrees of increased susceptibility to all antimicrobials except clarithromycin in all the S. aureus strains tested. A remarkable increase in susceptibilities to ampicillin, oxacillin and cefoxitin was observed in both MRSA strains, N315 and COL, whereas a moderate increase in susceptibility to bacitracin was observed in all the strains.
Effect of farnesol on PBP2' (PBP2a) expression
Figure 1 shows the expression of PBP2' (PBP2a) as detected by western-blot analysis using anti-PBP2' monoclonal antibody. A single PBP2'-specific band was detected in the PBP2'-positive COL strain and the expression was slightly lower with farnesol supplementation. No positive band was observed in the PBP2'-negative S. aureus ATCC 25923 strain.
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Protein A non-specifically bound to the Fc region of mouse IgG was identified from its size in both ATCC 25923 and COL strains by western-blot, but its expression was completely suppressed by farnesol.
Effect of farnesol on ß-lactamase secretion and activity
Figure 2(a and b) show the inhibitory effect of farnesol on the secretion and enzyme activity of ß-lactamase, respectively. The reduction in ß-lactamase specific activity was seen in the culture supernatant of the N315 strain that was grown in the presence of farnesol (Figure 2a) and also in the culture supernatant treated with farnesol in vitro (Figure 2b).
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Effect of farnesol on staphyloxanthin production
Treatment with farnesol completely suppressed the production of staphyloxanthin, which was visible as a yellow pigment, whereas treatment with antimicrobials did not affect production (Figure 3a). There was no significant difference in the profile of whole lipid extracts between the samples (Figure 3b).
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Modes of action of farnesol on the antimicrobial susceptibility of MRSA
Modes of action of farnesol on S. aureus susceptibility to the antimicrobials, with regard to peptidoglycan synthesis and C55 lipid carrier synthesis through the mevalonate pathway, were investigated, respectively, by GlcNAc and mevalonate incorporation assays and by HPTLC using the known inhibitors of cell wall synthesis, bacitracin and vancomycin, as references.
Incorporation assays showed that farnesol reduced the incorporation of GlcNAc, the most potent sugar source for peptidoglycan synthesis, whereas it significantly increased the incorporation of mevalonate, a source of the C55 lipid carrier (Figure 4a). TLC analysis of lipid extracts from the [14C]mevalonate-labelled cells showed that supplementation with either farnesol or bacitracin, but not vancomycin, induced accumulation of C55-PP, lipid I and lipid II, but stronger induction was observed with farnesol (Figure 4b).
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Discussion |
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We investigated farnesol's antimicrobial synergism and mode of action for its possible application as a therapeutic agent in combination with commonly used antimicrobial agents. The MICs of farnesol for S. aureus (2000 mg/L and 125 mg/L) obtained by the MHT agar dilution method and the MHT broth dilution method correspond to those of previous reports.8–10 The 16-fold lower MIC by the MHT broth dilution method suggests that the growth conditions apparently affect the inhibitory action of farnesol on S. aureus.
Farnesol had a remarkable effect on ß-lactam resistance even in the homogeneously-resistant MRSA COL strain (Table 1). But, as farnesol did not significantly influence the expression of PBP2' which is a factor responsible for ß-lactam resistance in S. aureus, PBP2' does not seem to be associated with the increased susceptibility to ß-lactam. Intriguingly, farnesol increased the susceptibility to ampicillin by 256-fold even in the ß-lactamase-positive strain N315, which was higher than the 24-fold increase in the ß-lactamase-negative strain COL (Table 1). Such a notable effect suggests that farnesol may inhibit the ß-lactamase instead of PBP2'. Indeed, farnesol decreased ß-lactamase secretion and its enzymatic activity (Figure 2). So far, Yam et al.14 have reported that tea tree oil can affect the secretion of ß-lactamase, but our result is the first report with regard to the inhibition of ß-lactamase activity. The mode of inhibition, however, remains to be elucidated.
So far, one of farnesol's modes of action has been demonstrated to be an increase in membrane permeability and disruption of membrane integrity resulting in a non-specific increase in susceptibility to antimicrobial agents.7,9 These findings demonstrate that a fragile membrane facilitates the influx of antimicrobial agents or certain exogenous chemical compounds. In our experiments, farnesol did not affect the susceptibility to the macrolide clarithromycin, either in the highly macrolide-resistant N315 strain carrying five copies of the erm(A) gene on Tn554,20 or in the macrolide-susceptible COL strain (Table 1). Similar to the observation with macrolides, farnesol did not significantly reduce the susceptibility to amikacin or ciprofloxacin (Table 1). Therefore, we postulated that in addition to increasing membrane permeability, farnesol might possess an additional potential inhibitory action that intensifies antimicrobial activity, particularly that of ß-lactams and bacitracin.
During antimicrobial susceptibility testing, we observed a reduction in yellow pigmentation in farnesol-treated S. aureus. This bacterium produces a unique yellow pigment called staphyloxanthin, which is classified as a triterpenoid carotenoide possessing a C30 chain instead of the C40 chain found in most other organisms.21 Since the structure of farnesol is a part of farnesyl pyrophosphate (FPP), farnesol might inhibit the terpenoid biosynthesis associated with FPP. Furthermore FPP is a substrate required for staphyloxanthin production, and the pyrophosphate side chain of FPP is indispensable in condensing the C30 carotenoid by the dehydrosqualene synthase, CrtM.22 Thus, farnesol may bind the active domain of CrtM, resulting in the complete suppression of staphyloxanthin biosynthesis at the initial step. Likewise, FPP is an indispensable substrate in the synthesis of C55-PP as a lipid carrier involved in the translocation of the murein monomer precursor.23
The reduction in staphyloxanthin production by farnesol led us to speculate that farnesol might inhibit the biosynthesis of the C55 lipid carrier. Indeed, farnesol caused reduced incorporation of GlcNAc whereas it increased incorporation of mevalonate (Figure 4a). This indicates that farnesol reduces nascent peptidoglycan synthesis, whereas it increases the C55 lipid carriers or the intermediates through the mevalonate pathway. To determine what metabolites from mevalonate are affected in C55 lipid carrier synthesis through the mevalonate pathway, we analysed lipid extracts from the [14C]mevalonate-labelled cells by TLC using the known inhibitors bacitracin and vancomycin as references, and found that the profile of metabolites derived from [14C]mevalonate with farnesol treatment was similar to that with bacitracin treatment (Figure 4b). Bacitracin inhibits peptidoglycan synthesis by inhibiting the dephosphorylation of C55-PP by undecaprenyl phosphatase UppP (BacA), a step essential to the recycling of the lipid carrier.24–26 Thus, its binding leads to accumulation of C55-PP. Vancomycin binds to the D-alanyl-D-alanine residue of lipid II containing pentaglycine outside the cell membrane, leading to the termination of nascent peptidoglycan synthesis.27 Thus, vancomycin treatment results in accumulation of lipid II containing pentaglycine, while it consumes the freely available C55-PP. The band profiles obtained by TLC analysis suggest that farnesol may not inhibit the primary step of C55 biosynthesis but accumulate C55-PP, lipid I and lipid II. It is postulated that bacitracin accumulates only C55-PP by inhibition of its dephosphorylation, but does not influence lipids I and II. Thus, farnesol should affect recycling of C55 lipid carrier from inside to outside, and also both ways, resulting in accumulation of lipids I and II inside of the cytoplasmic membrane. The mechanism of recycling of the C55 lipid carrier has not been well characterized so far. The possible driving force for the recycling may be the proton-motive force, because a recent report demonstrated that farnesol inhibits reactions of oxidation–reduction.9 If so, the accumulation of lipids I and II indicates the inhibition of the recycling both ways due to loss of proton-motive force. Such retardation of the recycling could cause a shortage of murein monomer precursors outside the membrane for sufficient peptidoglycan synthesis, resulting in the remarkable synergism observed with ß-lactam and bacitracin.
We observed significant suppression of protein A by farnesol treatment (Figure 1). In addition to its intensifying effect on antimicrobial agents, farnesol has been found to reduce fibrin-fibre formation by inhibiting plasma coagulation, which is one of the most characteristic virulence properties of S. aureus.10,28,29 Furthermore, farnesol treatment also contributes to the reduced staphylococcal biofilm formation that would also lead to the intensifying effect of antimicrobial agents.9 Such findings imply that down-regulation of multiple colonization factors contributes to preventing bacterial colonization. Further characterization of multiple inhibitory actions by farnesol should promote the development of an alternative therapy in the control of infections associated with multiple antimicrobial-resistant S. aureus.
Intriguingly, isoprenoids have tumour-suppressive potency and initiate apoptotic cell death by the inhibition of phospholipase D signal transduction,30,31 denoting its applicability in cancer chemotherapy and chemoprevention.32,33 In this regard, farnesol is one such promising plant metabolite, acting as it does as an inhibitor of broad targets of lipid metabolism. Further characterization of its extensive inhibitory action will advance the possibility of its clinical applications in treating more bacterial infections.
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None to declare.
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Acknowledgements |
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We thank Ms F. Miyamasu and Dr William Ba-Thein for critical reading and help with the text. No funding was received for this study.
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