Antimicrobial Activity of Several Cineole-Rich Western Australian Eucalyptus Essential Oils

Essential oils from the Western Australian (WA) Eucalyptus mallee species Eucalyptus loxophleba, Eucalyptus polybractea, and Eucalyptus kochii subsp. plenissima and subsp. borealis were hydrodistilled from the leaves and then analysed by gas chromatography–mass spectrometry in addition to a commercial Eucalyptus globulus oil and 1,8-cineole. The main component of all oils was 1,8-cineole at 97.32% for E. kochii subsp. borealis, 96.55% for E. kochii subsp. plenissima, 82.95% for E. polybractea, 78.78% for E. loxophleba 2, 77.02% for E. globulus, and 66.93% for E. loxophleba 1. The Eucalyptus oils exhibited variable antimicrobial activity determined by broth microdilution, with E. globulus and E. polybractea oils showing the highest activities. The majority of microorganisms were inhibited or killed at concentrations ranging from 0.25% to 8.0% (v/v). Enterococcus faecalis and Candida albicans were the least susceptible organisms, whilst Acinetobacter baumannii was the most sensitive. In conclusion, all oils from WA Eucalyptus species showed microorganism inhibitory activity, although this varied according to both the Eucalyptus species and the microorganism tested. These data demonstrate that WA Eucalyptus oils show activity against a range of medically important pathogens and therefore have potential as antimicrobial agents.


Introduction
Eucalyptus is a genus of plants native to Australia and some islands to the north of Australia. It comprises over 700 species, most of which are endemic to Australia [1,2]. Since Eucalyptus species are able to grow under a variety of climatic and edaphic conditions, they have been extensively introduced in areas outside Australia, including the United States, the Middle East, India, and South America [2]. Eucalyptus oil is obtained from the leaves by steam distillation and contains predominantly volatile terpenes and aromatic compounds, the most abundant typically being the monoterpenoid 1,8-cineole [3,4]. British and European pharmacopoeias specify that Eucalyptus oil must contain at least 70% 1,8-cineole when the oil is used for medicinal purposes [5].
Eucalypt plants have been used in traditional medicine in Australia for thousands of years. The Australian Aborigines use the leaves for medicinal purposes to treat a range of ailments including wounds and fungal infections [6]. The leaf extracts, including the essential oil, are currently widely used in perfumery and cosmetic products and to a lesser extent as a therapeutic agent. The current medicinal use is based on the range of biological effects exhibited by the oils in vitro, including antioxidant [7], anti-inflammatory, analgesic [8], and antimicrobial activities [9][10][11][12][13]. Clinical trials with Eucalyptus oil and the major component 1, 8-cineole (eucalyptol) have been performed to evaluate their efficacy in the treatment of a diverse range of conditions and diseases, including respiratory disorders [14,15], oral hygiene [16], and head lice infestation [17].
Although a number of researchers have previously investigated the antimicrobial activities of Eucalyptus essential oils, relatively little is known about the composition and activity of several Western Australian (WA) Eucalyptus oils. The aim of this work was therefore to determine the chemical composition of the essential oil of WA Eucalyptus species, namely, Eucalyptus loxophleba, Eucalyptus polybractea, Eucalyptus kochii subsp. plenissima, and E. kochii subsp. borealis, also known as "oil mallees", and to examine their antimicrobial activities against a range of common pathogenic bacteria.

Extraction of Essential Oils
A portion of ca. 150 g of leaf material was cut into small pieces and added to 400 mL of de-ionised water in a blender (Waring, HGB2WTS3, New Hartford, CT, USA). The material was macerated for 1 min and then combined with two additional portions (ca. 450 g total) and subjected to hydrodistillation in a Clevenger-type apparatus for approximately 3 h. The oil/water emulsion produced was collected and stored at 4 • C overnight to separate the essential oil from the residual water. The essential oil was then removed and stored in an amber glass bottle at room temperature until further use.

Gas Chromatography-Mass Spectrometry (GC-MS) Analysis of Essential Oils
GC-MS was performed with a Shimadzu GCMS-QP2010 (Kyoto, Japan). GC columns used included a Rtx-5 column (5% diphenyl-dimethyl-polysiloxane, 30 m × 0.25 mm × 0.1 µm film thickness, Restek, Bellefonte, PA, USA), and a DB-wax column (polyethylene glycol, 30 m × 0.25 mm × 0.25 µm film thickness, J&W Scientific, Folsom, CA, USA). Helium was used as the carrier gas with a constant flow rate of 1.0 mL/min on both columns. A scan range of m/z 45-400 and a solvent delay of 5 min were used with splitless injections of 1.0 µL for 1 min. The ion source was set to 230 • C, and the transfer line temperature to 250 • C. The oven temperature program was 40 • C, held for 1 min then ramped at 7 • C/min to 250 • C and held for 10 min. Retention indices (RI) were calculated on both columns using the same linear gradient method with comparison to an n-hydrocarbon mixture (Sigma-Aldrich, St Louis, MO, USA, p/n 46827-U). The main peaks in the total ion chromatogram of each oil were then integrated using the MS software, and the relative percentage abundance of peaks was determined.

Microorganisms
Microorganisms included a range of Gram-positive and Gram-negative bacteria and a yeast. Gram-positive bacteria were Enterococcus faecalis ATCC 29212, Enterococcus faecalis (vancomycin-resistant enterococci VRE) ATCC 51299, Staphylococcus aureus ATCC 29213, methicillin-resistant S. aureus (MRSA) NCTC 10442, and Staphylococcus epidermidis NCTC 11047. Gram-negative bacteria were Escherichia coli ATCC 25922, Salmonella enterica subsp. enterica serovar Typhimurium ATCC 13311, Acinetobacter baumannii NCTC 7844, and Pseudomonas aeruginosa ATCC 27853. Candida albicans ATCC 90028 was included as a representative yeast. The strains were cultured on blood agar at 35 • C, for 24 h for the bacteria and for 48 h for C. albicans prior to use in antimicrobial testing.

Evaluation of Antimicrobial Activity
Eucalyptus oils and 1,8-cineole were initially screened for activity against one Gram-positive strain (S. aureus ATCC 29213) and one Gram-negative reference strain (E. coli ATCC 25922) using an agar diffusion assay. The inocula were prepared by inoculating the organisms onto the blood agar and incubating overnight at 35 • C. the colonies were then suspended in 0.85% saline, and the suspension was adjusted to a turbidity of 0.5 McFarland (10 8 colony forming units (CFU)/mL) using a nephelometer. The suspension was then swab-inoculated onto Mueller-Hinton agar, and 8 mm diameter wells were punched into the agar. Volumes of 25 µL and 50 µL of each Eucalyptus oil and 1,8-cineole were then aliquoted into the wells. Trimethoprim (5 µg/disc) was used as a positive control. After incubation at 35 • C for 24 h, zones of inhibition were measured, and the results were reported in millimetres (mm). Each oil was tested at least three times on separate occasions, and mean values were calculated.
The minimum inhibitory concentration (MIC) of each oil was determined using a broth microdilution method based on protocols published by the Clinical and Laboratory Standard Institute (CLSI) [18,19]. The method was modified slightly by incorporating a final concentration of 0.001% Tween 80 to enhance oil solubility. In brief, each Eucalyptus oil was serially diluted two-fold in 100 µL volumes in a 96-well microtitre tray so that after inoculation with 100 µL of inoculum per well, the final concentrations ranged from 8.0% to 0.0016% (v/v). Using the known density of 1,8-cineole (0.9267 g/mL at 20 • C) [20] as a conversion factor, these percentage values corresponded to a range of 74.136 mg/mL-0.145 mg/mL of Eucalyptus oil. A positive growth control well containing growth medium and 0.001% Tween 80 but without Eucalyptus oil was included. The inocula were prepared from overnight cultures as described above and adjusted to 0.5 McFarland for bacteria, which corresponded to approximately 10 8 CFU/mL, or 1.0 McFarland for C. albicans, which corresponded to approximately 10 7 CFU/mL [18,19]. The suspensions were diluted as required to result in final inocula concentrations of approximately 5 × 10 5 CFU/mL. After inoculation and incubation for 24 h for bacteria and for 48 h for C. albicans, the MIC was determined visually as the lowest concentration of the Eucalyptus oil preventing microbial growth.
The minimum bactericidal concentration (MBC) or minimum fungicidal concentration (MFC) was determined by removing 10 µL volumes from each well showing no visible growth and spot-inoculating onto Mueller-Hinton agar. After incubation, colonies were counted and the MBC/MFC was identified as the lowest concentration of oil that killed ≥99.9% of the inoculum. The assay was conducted three times on separate occasions, and modal MIC/MBC/MFC values were selected.

Statistical Analysis
Geometric means of MICs and MBC/MFCs were determined for each Eucalyptus oil and for each microorganism. Geometric means were also determined to examine the difference in sensitivity between Gram-positive and Gram-negative bacteria. To enable the analyses, values >8.0% were converted to the next highest doubling dilution value of 16.0%. A one-way analysis of variance (ANOVA) was used to compare the MIC results between the two S. aureus strains and the two E. faecalis strains. A p-value of <0.05 was considered significant.
When the oils were screened for activity using the semi-quantitative agar diffusion assay, all Eucalyptus oils and 1,8-cineole produced zones of inhibition against the two test bacteria ( Table 2). The largest zone of inhibition was observed for 50 µL of E. polybractea oil against S. aureus ATCC 29213. For the remaining oils, zone sizes were relatively modest and ranged from 11.0 to 16.7 mm. On the basis of this data, more comprehensive antibacterial studies were conducted, using an expanded range of test organisms.
MIC and MBC/MFC results are shown in Table 3. The Eucalyptus oils showed variable antimicrobial activity against the different test organisms. The MIC geometric means for test organisms ranged from 1.2% for A. baumannii to 14.5% for E. faecalis and the MBC⁄MFC geometric means ranged from 1.6% for A. baumannii to >8.0% for E. faecalis ATCC 29212 and S. epidermidis. The Gram-negative organism A. baumannii was the most sensitive to the Eucalyptus oils, followed by S. enterica Typhimurium and E. coli. The Gram-positive E. faecalis ATCC 29212 was the least susceptible. All the examined Eucalyptus oils, with the exception of E. kochii subsp. plenissima, showed high activity against E. faecalis VRE, with MIC values ranging from 2.0% to 8.0% v/v. Comparison of the two strains of S. aureus showed that the MRSA strain was significantly more susceptible to Eucalyptus oils than the antibiotic-sensitive S. aureus strain (p < 0.05). Similarly, MICs obtained for E. faecalis VRE were also significantly different from those obtained for the susceptible E. faecalis strain (p = 0.00002).
The MIC values also showed that the different Eucalytpus oils tested varied in antimicrobial activity. Comparison of the geometric mean of the MICs for each oil using the results for all test organisms showed that E. polybractea and E. globulus oils displayed the highest activity with geometric means of 4.3% for both oils, followed by E. loxophleba 2 and E. kochii subsp. borealis (4.6%), E. kochii subsp. plenissima (5.6%), 1,8 cineole (7.0%), and E. loxophleba 1 (7.5%) ( Table 3). E. polybractea oil was the only oil that inhibited the growth of all organisms at ≤8.0% (v/v). E. globulus oil inhibited 8/10 organisms at ≤8.0% (v/v), the exceptions being E. faecalis and C. albicans. In contrast, 1,8 cineole inhibited only 4/10 test organisms. The MICs of E. loxophleba 1 and E. loxophleba 2 oils varied substantially from each other. There was a one-to two-fold decrease in MICs with E. loxophleba 2 oil compared to E. loxophleba 1 oil for both S. aureus strains, S. epidermidis, C. albicans, S. enterica Typhimurium, and A. baumannii, and a one-to two-fold increase for E. faecalis VRE and P. aeruginosa. E. kochii subsp. plenissima oil showed strong antibacterial activity against S. aureus, S. enterica Typhimurium, E. coli, and A. baumannii at ≤8.0% (v/v), yet, had no activity against the two strains of E. faecalis, S. epidermidis, and P. aeruginosa. For E. kochii subsp. borealis oil, the most sensitive strain was A. baumannii with an MIC of 1.0% (v/v), followed by S. aureus and E. coli with MICs of 2.0% (v/v); the least susceptible strains were E. faecalis and P. aeruginosa (>8.0%).
Overall, for each organism and oil combination, the MIC values were often identical to, or differed by only one concentration from the MBC or MFC values for that oil (Table 3). This indicates that most oils had activity that was bactericidal or fungicidal in nature. The exceptions were the Gram-positive bacteria S. aureus MRSA, E. faecalis VRE, and S. epidermidis, for which the values differed by more than two concentrations. When the oils were screened for activity using the semi-quantitative agar diffusion assay, all Eucalyptus oils and 1,8-cineole produced zones of inhibition against the two test bacteria ( Table 2). The largest zone of inhibition was observed for 50 μL of E. polybractea oil against S. aureus ATCC 29213. For the remaining oils, zone sizes were relatively modest and ranged from 11.0 to 16.7 mm. On the basis of this data, more comprehensive antibacterial studies were conducted, using an expanded range of test organisms.   Table 1.

Discussion
The major compound present in all oils was 1,8-cineole, which is in keeping with previous studies indicating that 1,8-cineole is often the major component of Eucalyptus oils [1,5]. The percentages of 1,8-cineole in E. polybractea, E. loxophleba, and E. kochii subsp. plenissima oils were largely in agreement with those previously reported [23][24][25]. E. kochii subsp. borealis was previously known as both Eucalyptus oleosa var. borealis and Eucalyptus horistes [26], and the levels of 1,8-cineole reported for the oils from these species were 75.5% [27] and 90.17% [23], respectively. The two E. loxophleba oils varied in composition, possibly due to differences in the local climatic and environmental conditions under which the plants grew (given that they were from different regions), as well as the genetic characteristics and the age of the trees [28]. It is also possible that they were different subspecies, as E. loxophleba has three subspecies, including loxophleba, lissophloia, and gratiae. Bignell et al. (1997) found 1,8-cineole levels of 25.2% in E. loxophleba subsp. loxophleba and 63.0% in E. loxophleba subsp. lissophloia, indicating large differences in composition [24]. Regardless, relatively little information is published on the chemical composition of WA Eucalyptus oils, and, as such, a comparison of the current data with previous findings is limited.
With regard to antimicrobial activity, numerous publications have described the activity of Eucalyptus oils against S. aureus and E. coli using the agar diffusion method [11,12]. In the current study, low to modest activity (with the exception of E. polybractea) was observed by agar diffusion, which concurs with these previous studies. However, although E. polybractea oil was the only oil that exhibited a considerable zone of inhibition against S. aureus, Gilles et al. (2010) reported considerable zones of inhibition against S. aureus, ranging from 25.4 to >90 mm, for oils from four other Eucalyptus species [13]. These differences in results may be attributable to variation in the Eucalyptus oils tested, as well as to variations in the experimental conditions. Finally, there was little correlation between the zone of inhibition and the MIC results, particularly for E. polybractea oil which showed the largest zone of inhibition against S. aureus but not the lowest MICs. This could be due to the presence of components in the E. polybractea oil that are relatively more water-soluble and able to diffuse further through the agar. The lack of correlation between the results from the two assays suggests that the two methods are not necessarily comparable. The agar diffusion method has long been regarded as problematic for the antimicrobial testing of natural products [29]. Because of issues with inconsistent diffusion of antimicrobial components through the agar, potential evaporation of volatile components, and lack of standardization between laboratories [30], data generated by agar diffusion must be regarded as largely qualitative, and the assay as useful for screening purposes only.
When more quantitative testing was conducted using the broth microdilution method, all eucalyptus oils showed antimicrobial activity. This is consistent with previous studies, verifying that both eucalyptus oil and 1,8-cineole have activity against a wide range of Gram-positive and Gram-negative bacteria and yeasts [11,12]. The current study found considerable variation in activity between the different eucalyptus oils, which could be a reflection of differences in their chemical composition. Comparison of the activity of Eucalyptus oils to 1,8-cineole alone confirmed the importance of the other moderate and minor components in Eucalyptus oils in relation to the MIC and MBC/MFC values obtained. Comparison of geometric mean MICs for all oils showed that E. globulus and E. polybractea oils had the lowest values, with a geometric mean of 4.3% (w/v), and E. loxophleba 1 and 1,8-cineole had the highest values, with geometric means of 7.5% and 7% (w/v), respectively. These results align with those reported by Cimanga et al. (2002). They showed that oils from Eucalyptus deglupta, Eucalyptus saligna, Eucalyptus urophylla, and Eucalyptus propinque, which all contain relatively high percentages of 1,8 cineole (>30%), exhibited similar or lower antimicrobial activity than oils from Eucalyptus alba, Eucalyptus robusta, Eucalyptus citriodora, and Eucalyptus tereticornis, which contain low percentages of 1,8-cineole (<10%) [31].
It was not surprising that the two E. loxophleba oils had substantially different antimicrobial activity, given that these two oils varied in composition and that E. loxophleba comprises a complex of several sub-species, rather than a single species [32]. Whilst it is not known which sub-species of E. loxophleba the oils in the current study were from, the variability in activity may be attributed to differences in the composition such as the presence of trans-pinocarveol (4.66%) in E. loxophleba 2 oil, which has been shown to have broad-spectrum antimicrobial activity [32]. These observations also suggest that a high 1,8-cineole content is not necessarily fully responsible for the activity of Eucalyptus oil and that moderate and minor compounds also play a vital role in the overall activity. Components other than 1,8-cineole may contribute to the activity as a result of their individual actions, by acting in combination with each other, or by acting in combination with 1,8-cineole. Such interactions between 1,8-cineole and other essential oil components have been demonstrated in previous studies [32,33].
It was unexpected that, according to the MIC/MBC values, the Gram-negative bacteria were, broadly speaking, more susceptible to Eucalyptus oils than the Gram-positive bacteria. This susceptibility of Gram-negative bacteria may be due to the presence of certain oil components, such as p-cymene, terpinolene, 1,8-cineole, and cis-geraniol, which can cause the discharge of Gram-negative outer membrane lipopolysaccharide and increase the permeability of the cytoplasmic membrane [34]. Of the Gram-negative bacteria, A. baumannii was the most sensitive to the Eucalyptus oils. This result is particularly relevant, given that Acinetobacter spp. are emerging globally as problematic antibiotic-resistant pathogens [35]. Our results are in agreement with several previous studies that also found Acinetobacter spp. to be relatively more sensitive than other Gram-negative species [22,36]. Very few researchers have investigated the potential mechanisms underlying this increased sensitivity; however, it could be due to differences in the outer membrane composition or efflux pumps of Acinetobacter species and Enterobacteriaceae [37,38]. Finally, many previous reports on the antimicrobial activity of Eucalyptus oil are difficult to compare with the current study because of differences in the methods used to assess the antimicrobial activity and differences in the chemical composition of the oils.
A number of clinical studies indicate that Eucalyptus oil and 1,8-cineole have significant potential as therapeutic agents, due to several different properties. For example, the benefits of 1,8-cineole for airway disease [39] and Eucalyptus oil as an insect repellent [40] have recently been reviewed. The toxicity of Eucalyptus oil has been extensively studied both in vitro and in animal studies, with the latter indicating that 1,8-cineole has low toxicity [40]. Furthermore, studies with human volunteers indicate that, when applied correctly, the oil has relatively low allergenicity and toxicity [40]. Whilst many studies have investigated the antimicrobial activity of Eucalyptus oil in vitro, fewer clinical studies have been performed. As such, there is no clear overall picture of the clinical usefulness of Eucalyptus oil as an antimicrobial agent. Further studies are required to determine how the antimicrobial properties of Eucalyptus oil can be best therapeutically utilised.

Conclusions
This study showed that Eucalyptus oils from some selected WA species had moderate antimicrobial activity, which varied according to the Eucalypt species and the test microorganism. The data suggest that WA Eucalyptus oils are potentially a good source of antimicrobial agents, particularly against Gram-negative bacteria. As such, further studies with additional test organisms and additional oil samples are warranted.

Conflicts of Interest:
The authors declare no conflict of interest.