microorganisms

Article
Antimicrobial Activity of Several Cineole-Rich
Western Australian Eucalyptus Essential Oils
Fahad S. Aldoghaim 1 , Gavin R. Flematti 2 and Katherine A. Hammer 3, *
1 Infection Control Department, Prince Sultan Military Medical City, Riyadh 12233, Saudi Arabia;
[email protected]
2 School of Molecular Sciences, The University of Western Australia, Crawley, WA 6009, Australia;
[email protected]
3 School of Biomedical Science, The University of Western Australia, Crawley, WA, 6009, Australia
* Correspondence: [email protected]; Tel.: +61-08-6457-2137




Received: 8 November 2018; Accepted: 29 November 2018; Published: 3 December 2018


Abstract: 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.

Keywords: minimum inhibitory concentration; monoterpenes; volatile oil; oil mallee;

1,8-cineole; eucalyptol

1. 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 [9–13]. Clinical trials with Eucalyptus oil

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Microorganisms 2018, 6, 122 2 of 11

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.

2. Materials and Methods

2.1. Plant Material

Fresh leaves of E. loxophleba 1, E. polybractea (subspecies not identified) (grown in Armadale,
Western Australia), E. loxophleba 2, E. kochii subsp. plenissima, and E. kochii subsp. borealis (grown
in Kalannie in the Wheatbelt Region of Western Australia) were harvested in March 2015 (about
700 g each) and immediately transported to the School of Molecular Sciences at The University of
Western Australia (UWA), Crawley, Western Australia. Commercial Eucalyptus oil from Eucalyptus
globulus (Thursday Plantation, Australia) and 1,8-cineole (99.0% purity; Fluka Chemika) were used
for comparison.

2.2. 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.

2.3. 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.

2.4. 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
Microorganisms 2018, 6, 122 3 of 11

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.

2.5. 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 (108 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 108 CFU/mL, or 1.0 McFarland for C. albicans,
which corresponded to approximately 107 CFU/mL [18,19]. The suspensions were diluted as required
to result in final inocula concentrations of approximately 5 × 105 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.

2.6. 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.

3. Results
A total of 21 distinguishable compounds were detected across the leaf oil samples by GC–MS
(Table 1; Figure 1). The most abundant compound was 1,8 cineole, ranging from the lowest value
of 66.93% for E. loxophleba 1 to 97.32% for E. kochii subsp. borealis. Other compounds detected in
Microorganisms 2018, 6, 122 4 of 11

proportions greater than 5.0% were limonene (7.52%), p-cymene (5.53%), and γ-terpinene (5.34%) in E.
globulus oil, and 4-methyl-2-pentyl acetate in both E. loxophleba 1 (9.86%) and E. loxophleba 2 (5.53%) oils.
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.
Microorganisms 2018, 6, 122 5 of 11

Table 1. Relative percentage of Eucalyptus oil components present at concentrations greater than 0.1%, determined using GC–MS.

Eucalyptus Eucalyptus Eucalyptus Eucalyptus kochii Subsp. E. kochii subsp.

Compound RI (DB-wax) RI (Rtx-5) E. loxophleba 2
globulus polybractea loxophleba 1 plenissima plenissima borealis
4-Methyl-2-pentyl acetate (1) 1109 (1110 a ) – – – 9.86 5.53 – –
β–Pinene (2) * 1110 (1116 b ) 974 (981 b ) – 1.07 – – – –
Sabinene (3) 1123 (1123 b ) – – 1.98 – – – –
β-Myrcene (4) * 1164 (1160 c ) 992 (992 b ) – 0.59 0.57 – – –
α-Phellandrene (5) * 1166 (1166 b ) 1003 (1007 b ) 0.43 – 2.75 – – –
Limonene (6) * 1201 (1201 b ) – 7.52 3.67 3.52 1.58 0.53 –
1,8-Cineole (7) * 1214 (1213 b ) 1032 (1030 b ) 77.02 82.95 66.93 78.78 96.55 97.32
γ-Terpinene (8) * 1248 (1238 b ) 1058 (1074 b ) 5.34 – 0.37 – – –
p-Cymene (9) * 1272 (1261 b ) 1026 (1027 b ) 5.53 1.50 1.11 1.77 1.39 1.34
α-Gurjunene (10) 1541 (1536 d ) 1409 (1412 e ) – – 1.48 – – –
3-Pinanone (11) 1558 (1576 a ) 1173 (1163 a ) – – – 0.89 – –
Pinocarvone (12) * 1578 (1565 a ) 1162 (1160 a ) – – – 0.79 – –
Terpinen-4-ol (13) * 1609 (1618 d ) 1177 (1176 e ) 0.56 1.39 0.52 0.39 0.61 0.12
Aromadendrene (14) * 1619 (1625 d ) 1440 (1446 e ) – – 4.37 0.3 – –
allo-Aromadendrene (15) * 1659 (1667 d ) 1462 (1466 e ) – – 0.94 – – –
trans-Pinocarveol (16) 1664 (1675 d ) 1137 (1127 f ) – – – 4.66 – –
α-Terpineol (17) * 1702 (1709 a ) 1190 (1189 a ) 1.49 3.67 1.45 1.22 0.33 0.11
Ledene (18) * 1708 (1706 d ) 1498 (1504 e ) – – 1.87 – – __
Verbenone (19) 1722 (1728 d ) 1210 (1228 a ) – – – – – 0.36
epi-Globulol (20) 2025 (2039 d ) 1561 (1566 e ) – – 0.44 – – –
Globulol (21) 2091 (2103 d ) 1585 (1595 e ) – – 1.90 0.3 – –
Total identified compounds 97.89 96.82 98.08 96.21 99.41 99.25
Notes: – not detected. * confirmed with commercial standard. Compound numbers correlate with Figure 1. Retention indices (RI) values in parenthesis indicate literature values from a P.J.
Linstrom and W.G. Mallard, Eds., NIST Chemistry WebBook, NIST Standard Reference Database Number 69, National Institute of Standards and Technology, Gaithersburg MD, 20899,
http://webbook.nist.gov, (retrieved 28 June 2016). b Flavornet at http://www.flavornet.org (retrieved 28 June 2016). c LRI and Odour database at http://www.odour.org.uk, (retrieved 28
June 2016). d Reference [21]. e Reference [22]. f Reference [1].
Microorganisms 2018, 6, 122 6 of 11

Overlaid GC–MS
Figure 1. Overlaid
Figure GC–MStotal
totalion
ionchromatograms
chromatograms(TIC)
(TIC)of ofthe Eucalyptusoils
theEucalyptus oilsused
usedininthis
thisstudy.
study.
Each number
Each number correlates
correlates with specific compounds identified in Table
Table 1.
1.

When the oils were screened for activity using the semi-quantitative agar diffusion assay, all
Table 2. Zones of bacterial growth inhibition (mean and standard deviation in mm) resulting from agar
Eucalyptus oils and 1,8-cineole produced zones of inhibition against the two test bacteria (Table 2).
diffusion of two different volumes of Eucalyptus oils.
The largest zone of inhibition was observed for 50 μL of E. polybractea oil against S. aureus ATCC
29213. For the remaining oils, zone
Eucalyptus Oils
sizes were relatively
Staphylococcus aureus ATCCmodest
29213andEscherichia
ranged fromcoli 11.0
ATCC to25922
16.7 mm.
On the basis of this data, more comprehensive
25 µL antibacterial
50 µL studies were
25 µL conducted, using an
50 µL
expanded range of test
E. globulus organisms. 13.0 ± 1.0 15.3 ± 0.6 11.3 ± 0.6 15.0 ± 0.0
E. loxophleba 1 15.3 ± 0.6 16.7 ± 0.6 14.7 ± 0.6 16.7 ± 0.6
Table 2.E.Zones of bacterial
loxophleba 2 growth13.0
inhibition
± 0.6 (mean and15.0standard
± 0.0 deviation
12.3in±mm)
0.6 resulting
15.3 ±from
0.6
E. polybractea
agar diffusion 28.0 ±
of two different volumes of 0.0 29.5 ± 0.7
Eucalyptus oils. 14.0 ± 1.0 16.7 ± 0.6
E. kochii subsp. plenissima 13.0 ± 0.0 15.7 ± 0.6 13.0 ± 0.0 13.0 ± 0.0
Eucalyptus Oilsborealis Staphylococcus
E. kochii subsp. 12.7 ± 0.6 aureus ATCC 29213 Escherichia
14.7 ± 0.6 11.3 ± 0.6 coli ATCC
13.0 ± 25922
0.0
1,8 Cineole 25 µL± 0.0
11.0 12.7 ± 0.6
50 µL 13.3
25 µL± 0.6 50±µL
14.3 0.6
E. globulus 5 µg
Trimethoprim 13.0 ± 1.0 27.7 ± 0.6
15.3 ± 0.6 26.3 ± 0.6 15.0 ± 0.0
11.3 ± 0.6
E. loxophleba 1 15.3 ± 0.6 16.7 ± 0.6 14.7 ± 0.6 16.7 ± 0.6
E. loxophleba 2 13.0 ± 0.6 15.0 ± 0.0 12.3 ± 0.6 15.3 ± 0.6
E. polybractea 28.0 ± 0.0 29.5 ± 0.7 14.0 ± 1.0 16.7 ± 0.6
E. kochii subsp. plenissima 13.0 ± 0.0 15.7 ± 0.6 13.0 ± 0.0 13.0 ± 0.0
E. kochii subsp. borealis 12.7 ± 0.6 14.7 ± 0.6 11.3 ± 0.6 13.0 ± 0.0
1,8 Cineole 11.0 ± 0.0 12.7 ± 0.6 13.3 ± 0.6 14.3 ± 0.6
Trimethoprim 5 μg 27.7 ± 0.6 26.3 ± 0.6

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).

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Microorganisms 2018, 6, 122 7 of 11

Table 3. Susceptibility of microorganisms to Eucalyptus oils (MIC % v/v) determined by the broth microdilution assay.

S. aureus Enterococcus E. faecalis Staphylococcus Candida Salmonella E. coli Pseudomonas Acinetobacter Geometric
S. aureus
Essential Oil Parameter a MRSA faecalis VRE ATCC epidermidis albicans Typhimurium ATCC aeruginosa baumannii Mean of
ATCC 29213
NCTC 10442 ATCC 29212 51299 NCTC 11047 ATCC 90028 ATCC 13311 25922 ATCC 27853 NCTC 7844 the MIC
E. globulus MIC 4 2 >8 4 4 >8 0.5 8 8 2 4.3
MBC/MFC 4 >8 >8 >8 >8 >8 0.5 8 8 2
E. loxophleba 1 MIC >8 8 >8 2 >8 >8 8 8 4 2 7.5
MBC/MFC >8 >8 >8 8 >8 >8 8 8 4 4
E. loxophleba 2 MIC 4 4 >8 4 8 8 2 8 >8 0.25 4.6
MBC/MFC >8 4 >8 4 >8 >8 4 8 >8 0.25
E. polybractea MIC 8 4 8 2 2 8 8 4 4 2 4.3
MBC/MFC >8 8 8 >8 >8 >8 8 4 8 2
E. kochii subsp. MIC 2 4 >8 >8 >8 8 2 2 >8 2 5.6
plenissima MBC/MFC 8 4 >8 >8 >8 >8 4 2 >8 4
E. kochii subsp. MIC 2 4 >8 4 8 8 4 2 >8 1 4.6
borealis MBC/MFC 4 8 >8 4 >8 >8 4 2 >8 2
1,8 Cineole MIC >8 >8 >8 8 >8 >8 2 1 >8 1 7.0
MBC/MFC >8 >8 >8 >8 >8 >8 4 4 >8 1
Geometric mean MIC 5.4 4.9 14.5 4.4 8.0 10.8 2.7 3.6 9.7 1.2
MBC/MFC 9.7 8.8 14.5 9.7 >8.0 >8.0 3.6 4.4 9.8 1.6
a MIC, minimum inhibitory concentration; MBC, minimum bactericidal concentration; MFC, minimum fungicidal concentration. Values are expressed as % (v/v).
Microorganisms 2018, 6, 122 8 of 11

4. 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 [23–25]. 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
Microorganisms 2018, 6, 122 9 of 11

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.

5. 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.

Author Contributions: Conceptualization, K.A.H. and G.R.F.; methodology, K.A.H. and G.R.F.; formal analysis,
F.S.A., K.A.H., and G.R.F.; investigation, F.S.A.; data curation, F.S.A.; writing—original draft preparation, F.S.A.;
writing—review and editing, K.A.H. and G.R.F.; supervision, K.A.H. and G.R.F.; project administration, K.A.H.
Funding: The Australian Research Council is gratefully acknowledged for funding for GRF (FT110100304).
Acknowledgments: The authors thank Simon Dawkins, Director of the Oil Mallee Association of Australia,
for providing the Eucalyptus leaf samples used in this study.
Conflicts of Interest: The authors declare no conflict of interest.
Microorganisms 2018, 6, 122 10 of 11

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