Application of orange essential oil as an antistaphylococcal agent in a dressing model

Methicillin-susceptible S. aureus strain SH1000[38], methicillin-resistant strains COL[39], 13136 p^-m⁺[40], and N315[41], and vancomycin intermediate-resistant strains 13136 p^-m⁺ V₂₀[42], and Mu50[41] were used in this study. Depending on the experimental condition described in the following sections bacterial strains were grown in either tryptic soya broth (TSB) or tryptic soya agar (TSA) media (Difco Laboratories, Inc. Detroit, MI) and incubated at 37°C for 18 h.

Commercially available terpeneless cold pressed Valencia orange oil was obtained from Firmenich Citrus Center, Safety Harbor, FL, USA. CPV is derived from mechanical extraction of the orange oil which is further concentrated under vacuum[43]. The major components of CPV are Linalool 20.2%, Decanal 18%, Geranial 9.1%, α-Terpineol 5.8%, Valencene 5.2%, Neral 5%, Dodecanal 4.1%, Citronellal 3.9%, and Limonene 0.3%[36]. The most predominant compounds are the alcohol linalool (20.2%) followed by decanal (18%), and geranial (9.1%), the amount of limonene is much lower at 0.3%[36].

Disc diffusion vapor assay was carried out by the method described by Goñi et al.[44] and Edwards-Jones et al.[45]. Overnight cultures of the S. aureus (7 log CFU/mL) were streaked on sterile TSA (Difco Laboratories, Inc.) using a cotton swab dipped into the culture. The swab was used to streak the agar plate to produce a lawn of growth by streaking the plate in 3 different directions. Ten μL of 100% CPV was aseptically pipetted onto sterile 6-mm paper discs (Becton Dickson, Franklin Lakes, NJ) and subsequently the CPV impregnated paper discs were aseptically placed in the centre of the lid of the Petri dish. Control plates were prepared by adding 10 μL of sterile water to the filter discs. The Petri dishes were subsequently sealed using parafilm and incubated at 37°C in an inverted position. The diameters of zones of inhibitions were measured in mm after 24 h of incubation. The assays were carried out on three different occasions in duplicate.

In vitro dressing model was designed by a modification of the method described by Edwards-Jones et al.[45]. Briefly, 10 μL aliquot of CPV (100%) was spotted in four different areas on the sterile 10.2 x 10.2 cm, 12 ply cotton gauze dressing pad (Duka Corp. Happauge, NY) (Figure1a). For control plates 10 μL of sterile deionized water was spotted in the gauze dressing pad. TSA plates were seeded with a suspension of 7 log CFU/mL of MRSA strains COL, Mu50 or VISA strain 13136 p^-m⁺ V₂₀ and covered with single layer of sterile bandage (QMD Medical, Quebéc, Canada) without touching the inoculated agar surface. As shown in Figure1a CPV spotted gauze dressing pad was placed on the bandage by CPV spots facing towards the inoculated agar surface and wrapped with bandage to hold the gauze dressing pad on the Petri plate (Figure1b). The dressing model Petri plates were subsequently incubated at 37°C for 24 h and the treated plates were compared with untreated control to identify the visible inhibition zones. This study was carried out on three different occasions in duplicate.

Homo sapiens skin keratinocyte (HEK001) ATCC CRL-2404™ cells were grown in keratinocyte serum free media (K-SFM) with two additives, bovine pituitary extracts (BPE) and human recombinant epidermal growth factor (EGF) (Invitrogen, Carlsbad, CA). Cells were grown routinely in a 75 cm² flask at 37°C in a 5% CO₂-humidified incubator. Ninety percent confluent cultures were trypsinized, and new cultures were prepared by seeding with 10⁵ cells/ml in 75 cm² flasks. For cytotoxicity and infection assays, BD Falcon™ 24-well tissue culture plates (BD, Franklin Lakes, NJ) were seeded with 10⁵ cells/ml/well and incubated at 37°C in a humidified 5% CO₂ incubator for 18 to 20 h to obtain a semi-confluent monolayer. Prior to assays, the monolayers were washed and incubated with K-SFM medium without antibiotic.

The MIC of CPV for the 6 S. aureus strains used in this study was previously determined as 0.18% for the strains 13136 p^-m⁺ and 13136 p^-m⁺V₂₀ and 0.2% for strains COL, Mu50, and N315[37]. Based on our previous results, 0.1% and 0.2% of CPV was prepared using dimethyl sulfoxide (DMSO) as a dispersing agent and added to the HEK001 cell monolayers and incubated for 24 h. After incubation, the cell monolayer was observed under phase-contrast microscope (Olympus, Japan). One set of HEK001 cell monolayer without any treatment and another set with equal volume of DMSO used in the CPV treatment were used as controls. To compare a typical cytotoxicity one set of HEK001 cell monolayer was treated with a known skin irritant SDS (25 μg/mL)[46].

Adherence and invasion assays were performed using a modified procedure derived from Harvey et al.[47]. Briefly, one loopful of overnight grown bacterial cells were collected from TSA plates and suspended in K-SFM media with additives. Aliquots (100 μl) of the bacterial suspension, containing approximately 7 log CFU/mL [multiplicity of infection (MOI) 1:100], were inoculated in duplicate in a 24-well tissue culture plate containing semi-confluent HEK001 cell monolayers. The CFUs of bacteria were determined simultaneously by serial dilution plate count on TSA plates. Infected monolayers were incubated for 2 h at 37°C under a 5% CO₂ humidified atmosphere to allow the bacterial cells to adhere and infect the HEK001 cells. Infected HEK001 monolayers were washed five times with K-SFM medium and re-incubated for 3 h in fresh media containing 0.1 or 0.2% CPV. For controls, infected HEK001 monolayers were re-incubated with fresh K-SFM medium alone or K-SFM medium with equal volume of DMSO used in CPV treatment. After 1, 2, and 3 h of incubation both controls and CPV treated HEK001 cells were lysed with 0.1% Triton X (Sigma, St. Louis, MO) in PBS for 15 min to detach the bacterial cells and subsequently the lysate containing bacterial cells were diluted and viable intra- and extra-cellular bacterial numbers were determined by counting the CFU on TSA plate. Results are expressed as the average number of bacterial CFU from three assays.

In an effort to explore the potential use of orange EO against antibiotic resistant S. aureus, in our earlier study inhibitory effects and mode of action of CPV against methicillin-susceptible strain SH1000, MRSA strains COL, 13136 p^-m⁺, and N315, and VISA strains 13136 p^-m⁺ V₂₀ , and Mu50 were studied[37]. Compared to other tested EOs CPV effectively inhibited all the tested S. aureus strains[37]. Therefore, in the present study the effect of CPV vapour on S. aureus was examined on both agar plates and an in vitro dressing model to evaluate the potential of CPV for topical therapy. Generally to study the antimicrobial effect of vapour, EO impregnated paper disc is placed on the lid of Petri dish and subsequently the growth inhibition zone is measured and used to indicate the antimicrobial effect of EO[19, 21].

In our disc diffusion CPV vapour study, growth of all the six S. aureus strains were inhibited at different levels (Table1). The maximum diameter of inhibition zone 78.8 ± 1.8 mm was observed in VISA strain 13136 p^-m⁺V₂₀ followed by the MRSA strains 13136 p^-m⁺, COL, N315, and Mu50. The lower diameter of inhibition zone 17.8 ± 1.4 mm was observed in an antibiotic susceptible strain SH1000. Similar to our observation, strain specific variation in the growth inhibition in S. aureus was previously observed by Edwards-Jones et al.[45]. In their study, the most effective combination of geranium and commercial grapefruit extract Citricidal™ vapour against MRSA did not affect the growth of antibiotic susceptible S. aureus strain NCTC 6571[45]. It has been previously noted that the degree of inhibition of bacterial growth by EOs considerably varies due to the complexity of EOs and characteristics of bacterial strains[48]. Related to results observed in our study Gaunt et al.,[49] demonstrated the antistaphylococcal effect of evaporated volatile compounds from orange oil. In their study, agar plates inoculated with S. aureus strain 8532 were exposed in a large air-tight chamber to candle flames combined with the volatile bactericidal compounds β-pinene and orange oil and results showed that compared to plain candle addition of volatile oils significantly reduced the number of S. aureus colonies. Goñi et al.[44] also reported the susceptibility of various strains of Gram-negative and -positive microorganisms including S. aureus to vapour phase cinnamon, clove and a mixture of cinnamon and clove EOs. In their study they found that vapour phase of cinnamon, clove or mixture of cinnamon and clove EOs showed better inhibition of S. aureus growth than the direct contact disc diffusion method[44].

Our disc diffusion vapor assay proved that the vapor phase of the volatile components in the CPV serves as possible source of antimicrobial agents. Therefore, we considered assessment of the anti-staphylococcal effect of CPV vapor in an in vitro dressing model to evaluate the potential topical application of CPV. In this experiment the inhibitory effect of CPV was qualitatively evaluated by the presence or absence of inhibition zone by comparing the test and control dressing models (Figure1c-h). As we observed in the disc diffusion vapor assay a similar inhibitory effect was observed in the dressing model as well. Growth of MRSA strains COL and Mu50 and VISA strain 13136 p^-m⁺ V₂₀ was inhibited by the vapor released from the CPV spotted gauze dressing pad and exhibited a clear inhibition zones on agar plates (Figure1f-h). Our present study has demonstrated the potential of the CPV as an anti-MRSA agent in the vapor phase in both the disc diffusion assay and the in vitro dressing model. Effectiveness of the vapor phase EOs against microbial growth was shown to be better than the direct contact of EOs with the inoculated culture has already been reported in other studies[44, 50]. In an in vitro dressing model study Edwards-Jones et al.[45] demonstrated the inhibitory effect of combination of geranium and commercial grapefruit extract Citricidal™ vapor against MRSA. Results of their study concluded that EOs can be applied as a natural anti-MRSA agent on the outer layer of the dressing without disturbing the normal wound healing process.

Dermal exposure to either synthetic or natural chemical substances can lead to a wide variety of skin reactions. Therefore, it is important to evaluate the new topical therapy agents that can potentially affect the skin cells[51]. Skin is the first-line defense against invading pathogens[52], and is composed of three layers, whereas the outermost epidermis is a squamous epithelium that mainly consists of keratinocytes[53]. Therefore, in this study we used keratinocyte cells as an in vitro model to assess the effect of CPV. Since there are general concerns about the toxicity and adverse effects of the plant derived natural products on the host first we evaluated the toxic effect of CPV on human keratinocyte cell monolayer. In our previous study we have shown that the MIC of CPV for the S. aureus strains used in the present study as 0.18% for the strains 13136 p^-m⁺ and 13136 p^-m⁺V₂₀ and 0.2% for strains COL, Mu50, and N315[37]. Therefore, to examine whether CPV has toxic effect on keratinocyte cells, 0.1% (½ x MIC) and 0.2% (1x MIC ) of CPV was added to the HEK001 keratinocyte cell monolayer and subsequently at regular intervals the cell monolayers were examined under phase-contrast microscope for any morphological changes caused by the toxic effect of CPV. Figure2a is a representative picture of normal keratinocyte cells and DMSO treated cells did not show any toxic effect and looks similar to normal keratinocyte cells (Figure2b). Figure2c represents the cytotoxic effect caused by a known skin irritant SDS on cell monolayer. Compared to controls (Figure2a-c), CPV (0.1% and 0.2%) treated keratinocyte monolayer cells did not exhibit any toxic response even after 24 h of incubation (Figure2d,e). Result of this observation indicated that the concentration (0.1 and 0.2%) of CPV that has previously shown to be bactericidal for MRSA and VISA cells did not produce any adverse effect on human epidermal keratinocyte cells[37]. Host tolerance is one of the issues that must be considered when evaluating natural antibacterial agents[54]. Many currently available antifungal and antibacterial agent possess undesirable toxicity[16, 54]. However, interestingly in our in vitro study the CPV treatment on keratinocyte cells did not exhibit any toxic effect mediated cell death in keratinocyte cells. It indicates the potential suitability of CPV for the topical antimicrobial treatment for dermal S. aureus infections.

Enumeration of total number S. aureus cells infected in HEK001 keratinocyte monolayer indicated that addition of CPV (0.1% or 0.2%) into the infected cells reduced the adhesion of MRSA strain COL and VISA strain 13136p^-m⁺V₂₀ to the keratinocyte cells. Infected HEK001 monolayers treated with fresh K-SFM medium alone or K-SFM medium mixed with equal volume of DMSO used in CPV treatment were used as controls in this experiment. We did not observe any difference between these two controls (data not shown). Compared to the control, CPV treated keratinocyte exhibited gradual decreases in total numbers of attached S. aureus cells within 3 h of treatment. After 3 h of incubation with 0.2% CPV number of viable COL and 13136p^-m⁺V₂₀ cells became undetectable (Table2). There are also several clinical studies[55, 56] and case reports[57, 58] reporting the successful use of EOs in treating MRSA nasal carriage or MRSA infections. Dryden et al.[55] and Caelli et al.[59] reported that a topical tea tree oil treatment was as effective as standard therapy for reducing MRSA nasal colonization. Sherry et al. demonstrated the successful use of the commercial phytochemical mixture Polytoxinol™ containing the extracts from Lemongrass, Eucalyptus, Melaleuca, Clove, Thyme as well as B.H.T. (Butylated Hydroxy Toluene), Triclosan (0.3%) and 95 undematured ethano (69.7%) to treat an intractable MRSA infection of the tibia in an adult patient[58]. Previous case studies have also demonstrated the topical use of Eucalyptus oil extracted from the Eucalyptus globulus leaf together with bioethanol for MRSA wound infection. This case study has reported that without administering any antibiotic application of 0.5 g of the oil per day to the wound for three weeks improved wound healing with clearing the MRSA to an undetectable level[60]. Recently, Palaniappan and Holley showed that natural antimicrobials carvacrol, thymol, and cinnamaldehyde were able to substantially decrease the MIC of antibiotics in a diverse group of bacteria containing genetic elements responsible for drug resistance[61]. They have demonstrated the synergistic effect of carvacrol, thymol, and cinnamaldehyde in the reduced MIC's of ampicillin, penicillin and bacitracin against penicillin-resistant S. aureus[61]. Thus, we speculate it is possible to use the CPV either single topical agent or synergistically with other antibiotics to control the S. aureus skin infections.