JAC Advance Access published online on August 13, 2008
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkn325
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Original research |
Antimicrobial efficacy of chlorhexidine digluconate alone and in combination with eucalyptus oil, tea tree oil and thymol against planktonic and biofilm cultures of Staphylococcus epidermidis
School of Life and Health Sciences, Aston University, Aston Triangle, Birmingham B4 7ET, UK
* Corresponding author. Tel: +44-121-204-3951; Fax: +44-121-204-4187; E-mail: karpantj{at}aston.ac.uk
Received 1 May 2008; returned 20 May 2008; revised 26 June 2008; accepted 18 July 2008
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Abstract |
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Objectives: Effective skin antisepsis and disinfection of medical devices are key factors in preventing many healthcare-acquired infections associated with skin microorganisms, particularly Staphylococcus epidermidis. The aim of this study was to investigate the antimicrobial efficacy of chlorhexidine digluconate (CHG), a widely used antiseptic in clinical practice, alone and in combination with tea tree oil (TTO), eucalyptus oil (EO) and thymol against planktonic and biofilm cultures of S. epidermidis.
Methods: Antimicrobial susceptibility assays against S. epidermidis in a suspension and in a biofilm mode of growth were performed with broth microdilution and ATP bioluminescence methods, respectively. Synergy of antimicrobial agents was evaluated with the chequerboard method.
Results: CHG exhibited antimicrobial activity against S. epidermidis in both suspension and biofilm (MIC 2–8 mg/L). Of the essential oils thymol exhibited the greatest antimicrobial efficacy (0.5–4 g/L) against S. epidermidis in suspension and biofilm followed by TTO (2–16 g/L) and EO (4–64 g/L). MICs of CHG and EO were reduced against S. epidermidis biofilm when in combination (MIC of 8 reduced to 0.25–1 mg/L and MIC of 32–64 reduced to 4 g/L for CHG and EO, respectively). Furthermore, the combination of EO with CHG demonstrated synergistic activity against S. epidermidis biofilm with a fractional inhibitory concentration index of <0.5.
Conclusions: The results from this study suggest that there may be a role for essential oils, in particular EO, for improved skin antisepsis when combined with CHG.
Key Words: essential oils , chlorhexidine , synergism , skin antisepsis , antimicrobial activity
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Introduction |
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Incision of human skin is a common practice in the clinical setting, for example, during surgery, when taking blood or inserting intravascular devices. Adequate skin antisepsis is therefore essential in avoiding healthcare-associated infections (HAI) which may occur post-incision, commonly from resident microorganisms located within the skin, particularly Staphylococcus epidermidis.1,2 S. epidermidis is common both on the surface of human skin and also within the deeper layers where it may exist as microcolonies, which, like bacterial biofilms, exhibit increased resistance to antimicrobials including antiseptics.3 Unfortunately, HAI do arise following incision of the skin4,5 and are likely to be associated with increased prevalence of microbial resistance to antibiotics and antiseptics, and inadequate skin antisepsis which encompasses both the contact time between skin and antiseptic prior to incision and permeation of the antiseptic within the skin.6–10 Chlorhexidine is one of the most widely used antimicrobials within clinical practice for skin antisepsis and is currently recommended within the Evidence-Based Practice in Infection Control (EPIC)11 and Healthcare Infection Control Practices Advisory Committee (HICPAC)12 guidelines. However, its antimicrobial efficacy is significantly reduced by factors including pH and organic matter.13 Therefore, additional strategies for skin antisepsis or improvement of existing methods need to be considered.
The antimicrobial efficacy of essential oils has been known for several years, and many studies have demonstrated activity against bacteria, fungi and viruses.14 More recently, in light of increased antimicrobial resistance within the clinical setting, the potential of essential oils for the prevention and treatment of infection has been researched in several studies.15–19 Indeed, tea tree oil (TTO) has recently been shown to be more effective than chlorhexidine digluconate (CHG) at clearing superficial skin sites and lesions colonized with methicillin-resistant Staphylococcus aureus.17 In addition, preliminary research within our laboratory has demonstrated that several essential oils [eucalyptus oil (EO), TTO, thymol, lavender, juniper and citronella] have antimicrobial efficacy against a wide range of microorganisms associated with HAI.20 Many of the essential oils are thought to disrupt cell membranes by the lipophilic compounds contained within the essential oil, such as terpenes. However, essential oils are mixtures of many heterogeneous compounds, and the main components responsible for their antimicrobial activity and the mode of their activity are not well understood.14 Furthermore, essential oils have been shown to act as effective penetration enhancers, increasing permeation and improving retention of drugs within the skin.21,22
The aim of this study was to investigate the antimicrobial efficacy of aqueous CHG alone and in combination with TTO, EO and thymol against planktonic and biofilm cultures of S. epidermidis.
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Materials
Congo Red agar for demonstrating slime production in the test strain S. epidermidis was prepared by mixing 0.4 g of Congo Red (Hopkins and Williams Ltd, Essex, UK), 25 g of sucrose (Fisher Scientific, Leicestershire, UK) and 5 g of agar No. 1 (Oxoid, Basingstoke, UK) with 490 mL of brain heart infusion (Oxoid) and sterilized according to the manufacturer's recommendations. Mueller–Hinton agar (MHA) and Mueller–Hinton broth (MHB) (Oxoid) were also prepared and sterilized in line with the manufacturer's recommendations. Phosphate-buffered saline (PBS), aqueous CHG (20% in water), TTO (40.2% terpinen-4-ol and 3.5% cineole), EO (82.9% cineole), thymol (>99.5%) and dimethyl sulphoxide (DMSO) were purchased from Sigma-Aldrich (Dorset, UK) and glucose was purchased from Fisher Scientific. White-walled, clear bottom, tissue culture-treated 96-well microtitre plates were from Corning Incorporated (NY, USA) and clear, round bottom 96-well plates were from Barloworld Scientific (Staffordshire, UK).
S. epidermidis RP62A23 and a clinical isolate of S. epidermidis, TK1 (University Hospital Birmingham NHS Trust, Birmingham, UK), were stored on MicroBank beads (Pro-Lab Diagnostics, Cheshire, UK) at –70°C until required.
Preparation of antimicrobial agents
Aqueous CHG was diluted with MHB to obtain a stock solution of 512 mg/L. Thymol, TTO and EO were diluted with MHB to obtain stock suspensions of 512 g/L. Five percent (v/v) DMSO was added to the essential oil stock suspensions to enhance the solubility of the oils in suspension.
Preparation of S. epidermidis inoculum for suspension assay
Suspensions of S. epidermidis for the suspension assays were prepared by inoculating 10 identical colonies of overnight cultures of S. epidermidis from MHA into sterile PBS. The bacterial concentration was adjusted to 1 x 108 cfu/mL by diluting the culture with sterile PBS and measuring the optical density at 570 nm. The suspensions were further diluted with MHB to obtain inocula containing 1 x 106 cfu/mL.
Determination of MIC and MBC of CHG, TTO, EO and thymol against S. epidermidis in suspension
MICs of aqueous CHG and TTO, EO and thymol were determined using a broth microdilution assay in line with CLSI (formerly NCCLS) guidelines.24 The antimicrobial activity of 5% (v/v) DMSO was also studied on a separate microtitre plate alongside the assay. Each well containing 100 µL of antimicrobial agent was inoculated with 100 µL of S. epidermidis suspension containing 1 x 106 cfu/mL. Following 24 h incubation in air at 37°C, the wells were inspected for microbial growth and the MIC was defined as the lowest concentration which did not show visual growth. Controls containing antimicrobial agent in broth and broth with inocula were also included. MBCs were determined by removing the total volume (200 µL) from each of the clear wells into duplicate plates and mixing with 20 mL of cooled molten MHA, which was then allowed to set. Plates were incubated in air at 37°C for 24 h. MBC was defined as the first plate yielding no growth. The assay was performed in triplicate.
Chequerboard assay to assess the antimicrobial activity of CHG in combination with TTO, EO and thymol against S. epidermidis in suspension
The antimicrobial activity of aqueous CHG in combination with TTO, EO and thymol was assessed in a suspension assay by the chequerboard method.25 In brief, serial double dilutions of the antimicrobial compounds were prepared (256 to 1 g/L for EO and TTO, 64 to 0.25 g/L for thymol and 64 to 0.5 mg/L for CHG). Fifty microlitres of each CHG solution was added to the rows of a 96-well microtitre plate in diminishing concentrations and 50 µL of the essential oil was added to the columns in diminishing concentrations. The wells were then inoculated with 100 µL of S. epidermidis suspension containing 1 x 105 cfu (the final concentrations of EO and TTO ranged from 64 to 0.25 g/L, thymol 16 to 0.06 g/L and CHG 16 to 0.125 mg/L). Columns 10–12 served as controls containing MHB and inoculum alone, and antimicrobial compounds separately with the inoculum. The microtitre plates were incubated in air at 37°C for 24 h and the MIC, of both antimicrobial compounds in combination were determined as described above. To assess the synergistic or antagonistic activity of antimicrobial combinations, the FIC and FIC index (FICI) were determined using the following formulae:
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0.5, synergistic; FICI between >0.5 and 4.0, indifferent; FICI > 4.0, antagonistic. The assay was performed in duplicate microtitre plates. Preparation of S. epidermidis biofilm
The ability of S. epidermidis strains to produce slime was confirmed by culturing the bacteria on Congo Red agar.26 The optimal conditions and incubation period for the production of the bacterial biofilms were established in preliminary experiments over a 72 h period (data not shown). Bacterial biofilms were prepared by aliquotting 200 µL of the bacterial suspension containing 1 x 105 cfu/mL into the wells of white walled, clear bottom, tissue culture-treated 96-well microtitre plates. Four wells in the last column of each plate were left blank to serve as bioluminescence negative controls. Suspensions of S. epidermidis were prepared in MHB supplemented with 2% (w/v) glucose. Microtitre plates containing S. epidermidis suspensions were incubated in air at 37°C for 48 h (optimal conditions for the production of a confluent biofilm with 
5.5 x 106 cfu per well as established in preliminary experiments).
Determination of MICs and MBCs of CHG, TTO, EO and thymol against S. epidermidis in biofilm
Microtitre plates containing S. epidermidis biofilms were washed once with sterile PBS to remove any unbound bacteria. Antimicrobial agents were diluted with MHB to obtain CHG concentrations ranging from 128 to 0.25 mg/L, thymol 128 to 0.25 g/L and EO and TTO 256 to 0.5 g/L. Two hundred and fifty microlitres of each antimicrobial agent was added to each microtitre plate well. Columns 11 and 12 served as controls containing the biofilm and saline alone and MHB alone without bacterial biofilm. Antimicrobial activity of 5% (v/v) DMSO against the bacterial biofilm was also tested on a separate plate. Following incubation in air at 37°C for 24 h, the wells were washed once with sterile PBS and the microbial viability was determined using an ATP bioluminescence assay (ViaLight MDA Bioassay kit, Cambrex, Berkshire, UK). In brief, 100 µL of Bactolyse with 100 µL of saline was added to each well and the plates were sonicated at 50 Hz for 30 min to release and lyse the cells of the bacterial biofilm. Fifty microlitres of ATP-monitoring reagent was added to each well and luminescence measured (Lucy 1, type 16 850 fluorescence measurer, Rosys Anthos Labtech Instruments). MIC was defined as the minimum concentration of antimicrobial agent that inhibited further growth of the initial biofilm (control well containing biofilm treated with saline), and MBC was defined as the concentration that produced below, or equal, to the background level of luminescence (empty well). The assay was performed in duplicate microtitre plates.
Chequerboard assay to assess the antimicrobial activity of CHG in combination with TTO, EO and thymol against S. epidermidis in biofilm
Microtitre plates containing S. epidermidis biofilms were washed once with sterile PBS to remove any unbound bacteria. Antimicrobial agents were diluted with MHB as described previously and 125 µL of each of the antimicrobial dilutions aliquotted into each well in decreasing concentrations. Columns 10 and 11 contained biofilm and antimicrobial compounds alone at various concentrations. Column 12 contained controls (biofilm and saline, and saline alone). The plates were incubated in air at 37°C for 24 h after which the wells were emptied, and the FIC and FICI values were determined by ATP bioluminescence as described previously. The assay was performed in duplicate microtitre plates.
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Determination of MICs and MBCs of CHG, TTO, EO and thymol against S. epidermidis in suspension and in biofilm
CHG, TTO, EO and thymol demonstrated antimicrobial activity against S. epidermidis RP62A and S. epidermidis TK1 in both suspensions and biofilms (Table 1). MIC and MBC of CHG were 4-fold higher against S. epidermidis growing in biofilm compared with cells in suspension (MIC 2 to 8 mg/L and MBC 4 to 16 mg/L). Of the three essential oils tested, thymol demonstrated the most efficacy with MIC ranging from 0.5 to 4 g/L and MBC ranging from 2 to 16 g/L. All antimicrobials tested reduced the luminescence from the bacterial biofilms to below negligible levels with concentrations of 16 mg/L (CHG), 2–8 g/L (thymol), 32–64 g/L (TTO) and 256 g/L (EO). Five percent (v/v) DMSO, which was used as a co-solvent in oil suspension, did not show antimicrobial activity against S. epidermidis in biofilm or suspension.
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Chequerboard assay to assess the antimicrobial activity of CHG in combination with TTO, EO and thymol against S. epidermidis in suspension and in biofilm
Combination of CHG with TTO, EO and thymol demonstrated indifferent activity against S. epidermidis RP62A and TK1 when grown in suspension (Table 2). In biofilm, CHG in combination with EO demonstrated synergistic activity against both strains of S. epidermidis, with an FICI of 0.156 and 0.188 for S. epidermidis RP62A and TK1, respectively (Table 3).
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Discussion |
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The aim of this study was to assess the antimicrobial efficacy of aqueous CHG and three essential oils (TTO, EO and thymol) against the common skin microorganism S. epidermidis and to determine the antimicrobial activity of CHG in combination with the oils.
The results demonstrate that CHG, EO, TTO and thymol exhibit antimicrobial activity against S. epidermidis when growing both in suspension and as a biofilm. However, the concentration of essential oils required to achieve the same level of growth inhibition as CHG is several orders of magnitude higher (g/L for essential oils compared with mg/L for CHG). Thymol showed increased activity against S. epidermidis growing in biofilm compared with planktonic cells. This is unusual, as biofilms are considered to be more resistant to antimicrobial agents compared with planktonic cells.3 Partitioning of oil, especially pure compound thymol, in oil suspension as well as in extracellular matrix of bacterial biofilm, may alter thymol activity. In a previous study by Nostro et al.,27 only small differences between biofilm and planktonic cultures’ susceptibility to thymol was demonstrated. Furthermore, in the study by Al-Shuneigat et al.,15 staphylococci in a biofilm mode of growth demonstrated increased susceptibility to an essential oil-based formulation compared with planktonic cells, which concurs with our findings in relation to thymol. Thymol is a phenolic compound that has both hydrophilic and hydrophobic properties, which may enhance diffusion of this compound in a biofilm and allow its access to bacterial cells where it alters the permeability of plasma membranes.27
Combining CHG with TTO, EO and thymol did not improve its antimicrobial activity against S. epidermidis TK1 and RP62A strains during their planktonic phase of growth; however, reductions in CHG and EO concentrations required to inhibit the growth of both S. epidermidis strains in biofilm were observed. Of the three essential oils used in this investigation, EO demonstrated the best potential for combination with CHG. Synergistic activity between EO and CHG was demonstrated against biofilms of both strains of S. epidermidis (FICI 0.19 and 0.16 for TK1 and RP62A, respectively). To our knowledge, this is the first report of synergism between EO and CHG.
Previous research that has investigated the synergistic activity of an essential oil and an antimicrobial agent has suggested that the synergism may be due to their action on both different28 or similar targets on the bacterial cells (i.e. cell membranes).29 EO and its main component 1,8-cineole are thought to act on the plasma membranes, the same target as CHG. However, TTO (and its main antimicrobial component terpinen-4-ol) and thymol also have lipophilic properties and target cellular membranes, without showing synergy in combination with CHG. Therefore, the interaction of EO and CHG requires further studies to establish the mode of action of the potential synergism. It is possible that not only one component is involved in the synergistic interaction between EO and CHG, but a mixture of several components. Moreover, it has been suggested that cationic CHG diffusion in the biofilm is hindered by the negatively charged extracellular matrix, changing the physicochemical properties of the extracellular matrix and its tertiary structure.30 Chlorhexidine is likely to remain in the aqueous phase in the oil suspension (Log P of CHG 0.03731); both EO, which consists of several heterogeneous compounds, and CHG have hydrophilic and hydrophobic properties, and it may be possible that they alter ionic interactions in extracellular matrix of the biofilm, as well as acting on the same target on the bacterial cell. However, further studies are needed to establish the mode of action of EO and CHG in combination.
The use of essential oils for the prevention and treatment of infection has been gaining popularity within the research field over the past decade.19,32–34 Furthermore, the antimicrobial activity of TTO,16,17 thymol27 and EO35 has been reported against several important pathogens. However, there has been little research to assess the efficacy of essential oils in combination with CHG against S. epidermidis, which is the major microorganism associated with skin-related HAI. Chlorhexidine is widely used as a skin antiseptic within the clinical setting and is the recommended antimicrobial within the EPIC and HICPAC guidelines.11,12 However, infection rates associated with surgical incision of the skin remain high.4 Thus, the current strategies adopted for skin antisepsis need to be considered with a view for improvement.
The antimicrobial activity of CHG alone in vivo is reported as being bacteriostatic36 and may be one factor which contributes to the survival of S. epidermidis within the skin following antisepsis and its association with subsequent infection. Furthermore, subinhibitory concentrations of chlorhexidine may increase a biofilm mode of growth of staphylococci,37 which may reduce the efficacy of skin antisepsis if inappropriate levels of antiseptic are used. The synergistic action of CHG in combination with EO may therefore be one way forward for enhancing skin antisepsis and potentially for disinfecting hard surfaces. The environment in the healthcare setting contributes to the spread of pathogens and transfer of microorganisms between patients and healthcare workers.38 Microorganisms may reside on surfaces in aggregates embedded in a biofilm, rendering them less susceptible to cleaning and disinfection. Furthermore, many medical devices such as central venous catheter hubs and needleless connectors also become colonized with microorganisms capable of producing a biofilm.39 At present, chlorhexidine-based compounds or isopropyl alcohol are commonly used for disinfecting these medical devices prior to use. The synergistic activity between CHG and EO in combination may therefore be of benefit in the clinical setting, for example, in improved skin antisepsis and the elimination of S. epidermidis existing as microcolonies which are likely to exhibit increased resistance to CHG alone, and also potentially in hard surface disinfection. However, while much of the research data advocate the potential use of essential oils in the clinical setting for preventing and treating infection, there is little information regarding safety in relation to their use, which needs to be taken into consideration. Therefore, further studies are warranted.
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This research was funded by EPSRC case reward CNA/05/09 with industrial contribution from Insight Health Ltd, UK.
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None to declare.
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Acknowledgements |
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We thank Dr Anna Casey at the University Hospital Birmingham NHS Foundation Trust (UK) for supplying the clinical strain for this study.
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