Journal of Ethnopharmacology 178 (2016) 125–136

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jep

Antimicrobial activity of Eucalyptus camaldulensis essential oils and

their interactions with conventional antimicrobial agents against
multi-drug resistant Acinetobacter baumannii
Petar Knezevic a,n, Verica Aleksic a, Natasa Simin b, Emilija Svircev b, Aleksandra Petrovic a,
Neda Mimica-Dukic b
a
University of Novi Sad, Faculty of Sciences, Department of Biology and Ecology, Trg Dositeja Obradovica 3, Novi Sad, Serbia
b
University of Novi Sad, Faculty of Sciences, Department of Chemistry, Biochemistry and Environmental Protection, Trg Dositeja Obradovica 3, Novi Sad,
Serbia

art ic l e i nf o a b s t r a c t

Article history: Ethnopharmacological relevance: Traditional herbal medicine has become an important issue on the
Received 10 July 2015 global scale during the past decade. Among drugs of natural origin, special place belongs to essential oils,
Received in revised form known as strong antimicrobial agents that can be used to combat antibiotic-resistant bacteria. Eucalyptus
2 December 2015
camaldulensis leaves are traditional herbal remedy used for various purposes, including treatment of
Accepted 4 December 2015
Available online 6 December 2015
infections. The aim of this study was to determine antimicrobial potential of two E. camaldulensis es-
sential oils against multi-drug resistant (MDR) Acinetobacter baumannii wound isolates and to examine
Keywords: possible interactions of essential oils with conventional antimicrobial agents.
Acinetobacter baumannii Materials and methods: Chemical composition of essential oils was determined by gas chromatography–
Multi-drug resistance
mass spectrometry analysis (GC–MS). MIC values of essential oils against A. baumannii strains were es-
Eucalyptus camaldulensis
timated by modified broth microdilution method. The components responsible for antimicrobial activity
Essential oils
Antibiotics were detected by bioautographic analysis. The potential synergy between the essential oils and anti-
Synergistic interactions (synergy) biotics (ciprofloxacin, gentamicin and polymyxin B) was examined by checkerboard method and time kill
curve.
Results: The dominant components of both essential oils were spatulenol, cryptone, p-cimene, 1,8-ci-
neole, terpinen-4-ol and β-pinene. The detected MICs for the E. camaldulensis essential oils were in range
from 0.5 to 2 μl mL
1. The bioautographic assay confirmed antibacterial activity of polar terpene com-
pounds. In combination with conventional antibiotics (ciprofloxacin, gentamicin and polymyxin B), the
examined essential oils showed synergistic antibacterial effect in most of the cases, while in some even
re-sensitized MDR A. baumannii strains. The synergistic interaction was confirmed by time-kill curves for
E. camaldulensis essential oil and polymyxin B combination which reduced bacterial count under de-
tection limit very fast, i.e. after 6 h of incubation.
Conclusions: The detected anti-A. baumannii activity of E. camaldulensis essential oils justifies traditional
use of this plant. The proven E. camaldulensis essential oil synergistic interactions with conventional
antibiotics could lead to the development of new treatment strategies of infections caused by MDR A.
baumannii strains in the term of antibiotic dose reduction.
& 2015 Elsevier Ireland Ltd. All rights reserved.

1. Introduction
Acinetobacter baumannii has emerged as one of the most
threatening pathogens on a global scale, particularly in health care
institutions. The hospital acquired A. baumannii infections prolong
the length of hospital stays and subsequently increase health care
n
Corresponding author.
E-mail address: petar.knezevic@dbe.uns.ac.rs (P. Knezevic).
costs. Its clinical significance was propelled at the beginning of this
century as a result of its remarkable ability to up-regulate existing
genes or acquire resistance determinants, making it one of the
organisms threatening usability of the conventional antibiotics
(Peleg et al., 2008). The main factors most likely contributing to
the persistence of A. baumannii in the hospital environment are
resistance to desiccation and a high level of resistance to disin-
fectants and antimicrobial drugs (Fournier et al., 2006; Smith et al.,
2007). Significant increase in mortality was noted for A. baumannii

http://dx.doi.org/10.1016/j.jep.2015.12.008
0378-8741/& 2015 Elsevier Ireland Ltd. All rights reserved.
126 P. Knezevic et al. / Journal of Ethnopharmacology 178 (2016) 125–136
bacteremia compared to bacteremia with other Gram negative
organisms (Jerassy et al., 2006; Robenshtok et al., 2006). Further-
more, infections with multi-drug resistant (MDR) A. baumannii
showed a significant increase in mortality compared to those with
MDR Pseudomonas aeruginosa (Gkrania-Klotsasal and Hershow,
2006). A. baumannii is a well-known pathogen in burn units and it
is difficult to eradicate it (Trottier et al., 2007). According to Gaynes
and Edwards, 2005 it caused 2.1% of ICU-acquired skin and soft
tissue infections, while in other assessment of combat victims with
open tibial fractures A. baumannii was the most commonly iso-
lated organism (32.5% of cases) (Johnson et al., 2007).
Great scientific attention has been brought to A. baumannii
because of emerging wide array of its intrinsic and acquired re-
sistance determinants (Peleg et al., 2008). According to the In-
fectious Diseases Society of America, A. baumannii is one of the
“red alert” pathogens that seriously threaten the benefit of cur-
rently used antibacterial agents (Talbot et al., 2006). Due to the
increasing resistance of A. baumannii, even older, previously dis-
carded drugs (e.g. polymyxin B) are again being put in use against
it, in spite of their significant side effects (Boucher et al., 2009).
Thus the novel antimicrobial agents and strategies for combating
MDR A. baumannii infections are urgently required.
Phytochemicals, i.e. essential oils (EOs), could be a possible
solution to combat the MDR bacteria. The antimicrobial efficacy of
many essential oils, such as Eucalyptus camaldulensis Dehnh. oil,
has been known for many years (Takahashi et al., 2004; Akin et al.,
2010; Elaissi et al., 2012). The E. camaldulensis (red river gum) is a
species from genus Eucalyptus, belongs to Myrtaceae family com-
prising about 900 species (Brooker et al., 2002). It is a perennial,
single-stemmed, medium-sized to tall tree (Bren and Gibbs, 1986).
This species is an autochthonous Australian plant, but it has been
naturalized in all other parts of the world with appropriate climate
(Rejmanek and Richardson, 2011).
E. camaldulensis was traditionally used to prepare herbal re-
medies by aborigines in Australia, indicating the plant anti-
microbial properties. Making incisions in the tree trunks they
obtained red kino, i.e. red gum, and applied it directly to abrasions
and cuts. Across Australia dried kino was prepared by mixing fresh
kino with water and subsequently dehydrated. The dried kino was
used in the same way as fresh, but previously was softened in
water (Williams, 2011; Clarke, 2014). The young leaves were used
to prepare smoke bath, with a patient sitting surrounded with
smoke from burning leaves. The smoking medicine was used for
fevers, colds, flu and general sickness (Williams, 2011; Pennacchio
et al., 2010). After being naturalized in Africa, E. camaldulensis was
also used in folk medicine. Its gum was used for sore throat and
diarrhea, while the smoke of burnt leaves was inhaled in case of
respiratory problems in Sudan; decoctions from leaves were pre-
pared with sugar for stomach-ache in Senegal or a decoction of E.
camaldulensis leaves in combination with Citrus limon (L.) Burm. f.
fruits and Psidium guajava L. leaves were used for cough, flu and
fever in Zimbabwe (Doran and Wongkaev, 2008; Maroyi, 2013);
teeth cleaning sticks made from tree have been used to prevent
tooth decay and periodontitis in Nigeria (Bukar et al., 2004); fi-
nally, poultice of leaves containing eucalyptus oil have been used
in traditional medicines to heal wound infections (Adeniyi et al.,
2006).
E. camaldulensis leaf extracts have recently been proven to be
active against multi-drug resistant bacteria, including A. baumannii
(Jazani et al., 2012). Also, its essential oil is reported to be a good
antimicrobial agent against both Gram positive and Gram negative
bacteria (Oyedeji et al., 1999; Adeniyi et al., 2006; Rasooli et al.,
2009; Owlia et al., 2009; Akin et al., 2010; Lima et al., 2013). Its
essential oil is safe for use when administered alone with maximal
adult oral dose 300–600 mg and maximal dermal application level
5–20% (Blumenthal et al., 1998; Tisserand and Young, 2014., Jazani
et al., 2012). This indicates that it could be used as a replacement
for conventional antimicrobials, with minimal side effects. How-
ever, there is still a lack of data on its essential oil activity against
MDR A. baumannii, as well as the oil activity in combination with
conventional antimicrobial agents. Synergistic studies of essential
oils and conventional antibiotics are of a great importance in re-
vealing new strategies for combating the MDR strains. It has been
proven that synergistic effects enable lowering the dosage for one
or both agents, retaining the antimicrobial activity and/or re-
sensitizing bacterial strains (Rosato et al., 2007; Langeveld et al.,
2014; Aleksic et al., 2015). Taking into account traditional E. ca-
maldulensis application in treatment of various diseases caused by
bacteria, including wound infections, the objective of this study
was to determine antimicrobial potential of two E. camaldulensis
essential oils against multi-drug resistant (MDR) A. baumannii
wound isolates and to examine possible interactions of essential
oils with conventional antimicrobial agents to obtain more effi-
cient anti-A. baumannii activity by combining traditional and
conventional agents.
2. Materials and methods
2.1. Essential oil isolation
Plant material of E. camaldulensis was collected from two
coastal areas of Montenegro (Herceg Novi and Bar). Voucher spe-
cimens were prepared and identified by Goran Anackov, PhD, and
deposited at the Herbarium of the Department of Biology and
Ecology, Faculty of Sciences, University of Novi Sad (No 2–1816 and
No 2—1812, BUNS Herbarium).
Air-dried leaves were ground and subjected to hydrodistillation
with n-hexane as a recipient solvent according to the European
Pharmacopeia (2002). The procedure included topping of 100 g of
plant material with 1000 mL of distilled water in a round-bottom
flask and distilling for 3 h. The obtained essential oil solutions
underwent drying with anhydrous sodium sulfate, filtering and
removing n-hexane under reduced pressure. The obtained E. ca-
maldulensis essential oils were labeled as EuHN (originating from
Herceg Novi) and EuB (originating from Bar) and stored at
20 °C
prior to analysis.
2.2. GC–MS analysis
Qualitative and semi-quantitative chemical characterization of
essential oils was performed by GC–MS, using Agilent Technolo-
gies 6890 N gas chromatograph coupled with Agilent Technologies
5975B electron ionization mass-selective detector. A 1 μL aliquot
of essential oil dissolved in hexane (10 μL mL
1) was injected into
a split/splitless inlet at 250 °C, with a split ratio 1:10. Helium
(purity 5.0) was used as a carrier, with a constant flow of 1 mL/
min. Components were separated on a non-polar Agilent Tech-
nologies HP-5 ms column (30 m  0.25 mm, 0.25 μm), using the
temperature program starting at 50 °C, increasing 8 °C/min to
120 °C, then 15 °C/min to 230 °C, and finally 20 °C/min to 270 °C,
and holding at 270 °C for 16.9 min (total run time 35 min). Effluent
was delivered to the mass spectrometer via a transfer line held at
280 °C. Ion source temperature was 230 °C, electron energy 70 eV,
and quadrupole temperature 150 °C. To achieve better correlation
between experimental and library spectra, standard spectra tune
was used. Data were acquired in scan mode (m/z range 35–400),
with solvent delay of 2.30 min.
Data were processed using Agilent Technologies MSD Chem-
Station software (revision E01.01.335) combined with AMDIS (ver.
2.64) and NIST MS Search (ver. 2.0d). AMDIS was used for de-
convolution, i.e. co-eluting compounds peak area determination
 P. Knezevic et al. / Journal of Ethnopharmacology 178 (2016) 125–136
and pure spectra extraction, and NIST MS Search provided search
algorithm complementary to PBM algorithm of ChemStation. The
compounds were identified by comparison of mass spectra with
data libraries (Wiley Registry of Mass Spectral Data, 7th ed. and
NIST/EPA/NIH Mass Spectral Library 05) and confirmed by com-
parison of linear retention indices with literature data (Adams,
2001). Diesel oil, containing C8–C28 n-alkanes, was used as a
standard for determination of retention indices. Relative amounts
of components, expressed in percentages, were calculated by
normalization procedure according to peak area in total ion
chromatogram.
2.3. A. baumannii strains
Twenty-three A. baumannii strains were used in this study: two
strains from the American Type Culture Collection (ATCC19606
and ATCC BAA747, Rockville, MD, USA), one from the National
Collection of Type Cultures (NCTC 13420, Public Health England,
UK) and previously characterized twenty multi-drug resistant
(MDR) A. baumannii outpatient and clinical wound isolates
(Aleksic et al., 2014). The strains were stored in Luria Bertani broth
(LB) supplemented with glycerol (10% v/v) at
70 °C. The over-
night cultures in LB were used for the experiments. All the ex-
periments were conducted using Muller Hinton agar or broth,
unless stated otherwise.
2.4. Antibacterial assays
2.4.1. Validation of the essential oils antibacterial activity
The antibacterial activity of two E. camaldulensis essential oils,
EuHN and EuB, against A. baumannii was validated in this study.
The minimal inhibitory concentration (MIC) was determined by
modified broth microdilution susceptibility testing method (Clin-
ical and Laboratory Standards Institute, 2007). The essential oils
antibacterial activity was examined using two-fold serial dilutions
ranging from 8 to 0.0625 μL mL
1. In order to enhance oil solu-
bility, DMSO was incorporated into the broth medium to a final
concentration of r0.8% v/v (Sigma, USA). The minimal bacter-
icidal concentration (MBC) was determined by plating of 10 μL
from wells without bacterial growth, in order to define the type of
inhibition (reversible or permanent). MBC was determined as the
lowest essential oil concentration at which reduction of initial
bacterial count was 4 99.9%. Escherichia coli ATCC 25922, S. aureus
ATCC 25923 and appropriate antibiotics (ABs) were used as in-
ternal quality control. Each experiment was performed in triplicate
and in three independent occasions and the results are re-
presented as geometrical means of replications.
2.4.2. Bioautographic agar overlay method
The detection of E. camaldulensis essential oils components
exhibiting antibacterial activity was performed using the modified
bioautographic agar overlay method previously described by Ra-
halison et al., 1991. The TLC aluminum sheets coated with silica gel
60 F254 (Merck, Germany), dimensions 20  20 cm2, were used for
separation of the essential oils compounds. The sterilization of
sheets was done by heating at 120 °C for 3 h before use. Essential
oils (EuHN and EuB) were dissolved in ethanol to obtain con-
centration of 20 μL mL
1, and 4 μL aliquots of the solutions were
applied in triplicate on a TLC plate. The TLC plates were developed
with toluene-ethyl acetate (93:7, v/v) at room temperature
(Wagner and Bladt, 2001) and dried in an oven at 90 °C for 5 min
to remove the solvent. One set of chromatograms was used for
visualization of the spots, by spraying with alcoholic vanillin-sul-
phuric acid reagent followed by heating at 100 °C for 5 min. De-
tection of the compounds was conducted by comparison of Rf
127
values and spot colors with literature data (Wagner and Bladt,
2001). The two remaining sets of chromatograms were used for
direct bioautography assays. One set was cut into pieces in the
form of strips, and another in the form of squares, where one strip
represented one essential oil and one square represented the spot
on TLC plate. The strips and squares of essential oils were placed
onto MH agar plates, topped with melted LB-top semisolid agar
supplemented with 0.1% triphenyl tetrazolium chloride (TTC) so-
lution and inoculated with A. baumannii ATCC 19606 overnight
culture with approx. 107 colony forming units per mL (CFU mL
1).
The plates were incubated at 37 °C for 18 h. The inhibition zones
were colorless due to the lack of bacterial dehydrogenase activity,
which is necessary for transformation of TTC into the red for-
mazan. The TLC plates without essential oils, treated identically as
plates with essential oils, were used as controls.
2.4.3. Interaction of EOs and ABs
Broth microdilution checkerboard method (Verma, 2007) was
used to determine the potential synergistic interactions between
E. camaldulensis essential oils and antibiotics. Three conventional
antibiotics, ciprofloxacin (CIP), gentamicin (GEN) and polymyxin B
(PMB) (Sigma, USA) were used in combinations with essential oils
EuHN and EuB. In this experiment, four A. baumannii strains were
used, one reference strain (ATCC19606) and three MDR wound
isolates (Aba-4914, Aba-5055, Aba-6673), selected according to the
previously determined MIC values for antibiotics, which were
above the break points (Aleksic et al., 2014). Essential oils con-
centrations were in range from 0.03  MICEO to 1  MICEO, while
antibiotic concentrations ranged from 0.03  MICAB to 4  MICAB.
Each combination was tested at least twice in independent
repeats.
The results of the checkerboard assay were used for calculation
of the fractional inhibitory concentration (FIC index) for two an-
timicrobials in combination. The FIC index was calculated by
equation FICI ¼FICEO þ FICAB, where:
MICEOinthecombination
FICEO =
MICEOalone
MICABinthecombination
FICEO =
MICABalone
According to calculated index, the distinction of effects was
performed as follows: FICIr 0.5, a synergistic effect; 0.5 o FICIr1,
an additive effect; 1 oFICI an indifferent or antagonistic effect
(Mulyaningsih et al., 2010). Finally, the obtained FIC indexes were
averaged, expressed as mean þstandard error, and shown graphi-
cally as isobolograms.
2.4.4. Time effect of the EO-AB synergistic interactions
The time-kill assay (Verma, 2007) was used to elucidate the
time effect of EO-AB combinations which was proved to be sy-
nergistic according to the results obtained by checkerboard mi-
crodilution assay for strains ATCC 19606 and Aba-4914. Combi-
nations of 0.5 ml mL
1 EuHN essential oil and PMB subinhibitory
concentrations (1 mg mL
1 for strain Aba-4914 and 0.25 mg mL
1
for ATCC 19606) were investigated by monitoring the changes in
bacterial CFU during the incubation period using four test tubes,
i. e. four different treatments. The first test tube contained only
bacteria (approx. 1  108 CFU mL
1). The second and third test
tube contained bacteria and subinhibitory concentration of AB or
EO, respectively. The forth tube contained bacteria, a subinhibitory
concentration of both examined antimicrobial agents, AB and EO.
The subinhibitory concentrations of the essential oils and anti-
biotics in all test tubes were those that exhibited synergistic effect
in combination. The test tubes were incubated at 37 °C. After 0, 3,
128 P. Knezevic et al. / Journal of Ethnopharmacology 178 (2016) 125–136

Table 1
Qualitative and quantitative chemical composition of E. camaldulensis essentilal oils.

a
Essential oil compounds LRI EuHNb EuBb

Monoterpene hydrocarbons
α-Thujene 929 0.41 0.41
α-Pinene 937 1.58 1.35
Sabinene 977 0.37 0.30
β-Pinene 982 3.27 5.02
β-Myrcene 992 0.24 0.11
α-Phellandrene 1008 0.55 0.53
p-Cymene 1028 5.35 7.56
β- Phellandrene 1034 3.38 4.36
γ-Terpinene 1062 0.20 0.13
α-Terpinolene 1093 0.23 ndc
Oxygenated monoterpenes
1,8-Cineole 1036 7.62 1.95
Linalool 1101 1.37 2.02
cis-p-Menth-2-en-1-ol 1127 1.11 1.28
trans-p-Menth-2-en-1-ol 1146 0.75 1.04
trans-Pinocarveol 1148 1.22 1.08
Pinocarvone 1172 0.33 0.20
Terpinen-4-ol 1185 3.73 5.92
p-Cymen-8-ol 1192 0.63 0.76
Cryptone 1195 7.59 12.15
α-Terpineol 1197 0.99 nd
Myrtenol 1203 0.79 0.89
Myrtenal 1204 0.45 0.61
Phellandrene epoxide 1209 0.34 0.20
trans-Piperitol 1214 0.44 0.41
trans-Carveol 1226 0.13 nd
Cumin aldehyde 1250 1.98 2.90
Phellandral 1286 2.57 3.17
Cumic alcohol 1297 1.08 1.02
Thymol 1304 1.25 0.94
Myrtenyl-acetate 1334 nd 0.18
α-Terpenyl-acetate 1358 0.38 nd
Geranyl-acetate 1386 0.10 0.11
Sesquiterpene hydrocarbons
β-Elemene 1403 0.28 0.13
α-Gurjunene 1426 0.26 0.12
Aromadendrene 1458 0.19 nd
Allo-Aromadendrene 1481 2.56 3.23
Sesquiterpene hydrocarbon d 1499 0.31 0.16
Bicyclogermacrene 1516 2.83 4.19
δ-Cadinene 1539 0.18 0.11
Oxygenated sesquiterpene
Spathulenol 1605 18.90 21.39
Globulol 1610 2.66 2.43
Sesquiterpene d 1662 2.23 1.54
Isobicyclogermacrenal 1767 2.31 1.83
Chemical classes
Monoterpene hydrocarbons 15.58 19.77
Oxygenated monoterpenes 34.85 36.83
Sesquiterpene hydrocarbons 6.61 7.94
Oxygenated sesquiterpene 26.1 27.2
Others 2.23 1.54
Total area % of identified compoundsb 83.14 91.73

a
LRI – Linear Retention Indices.
b
The amount of the EOs compounds is expressed in % of total peak area.
c
nd - Below the detection limit.

6, 9, 12, 15 and 24 h of incubation bacterial CFU mL
1 was de- 3. Results

termined by scattering appropriate dilutions on Muller Hinton
agar, with detection limit 102 CFU mL
1. The plates were in-
cubated at 37 °C overnight and bacterial colonies were counted.
Time-kill experiments were carried out at least twice, and the
obtained results were averaged and expressed as logarithms with
corresponding standard errors. The interaction of the EO–AB in
combination was considered to be synergistic if the starting bac-
terial count after 24 h of incubation was decreased for Z2 log in
comparison to the more active single agent (EO or AB) (Knezevic
et al., 2013).
3.1. Chemical composition of Eucalyptus camaldulensis essential oils
The results of GC–MS analysis of E. camaldulensis essential are
given in Table 1. A total of 43 volatile compounds were identified
in two E. camaldulensis essential oils originating from Herceg Novi
(EuHN) and Bar (EuB). The identified compounds represent 83.14%
of EuHN and 91.73% of EuB total essential oil components, re-
spectively. Oxygenated monoterpens were the most dominant
chemical class of compounds in the samples, while oxygenated
 P. Knezevic et al. / Journal of Ethnopharmacology 178 (2016) 125–136 129

Table 2 values, i.e. MBC values were in the range of 0.71–4 μL mL
1 for
Antibacterial activity of the E. camaldulensis essential oils against MDR A. bauamnnii EuHN, and 0.71–2 μL mL
1 for EuB.
wound isolates.

A. baumannii isolates E. camaldulensis essential oils 3.2.2. Bioactive constituents of the E. camaldulensis essential oil
TLC chromatograms of investigated essential oils are given in
EuHN EuB Fig. 1A. After spraying with vanilin-sulphuric reagent, six spots
a b
were visible. In comparison with the results of GC–MS analysis and
MIC MBC MIC MBC
literature data (Wagner, 2001), it can be conluded that spot 2
ATTC 19606 c
1 2 1.41 2 (Rf¼0.42) belongs to spathulenol, spot 3 (Rf¼0,53) to 1,8 cineole,
ATCC BAA747 1 1 1 1 spot 4 to thymol (Rf ¼0.58), and spot 5 (Rf¼0.68) probably to
NCTC 13420 1 2 1 1.41 cryptone. Polar terpenoid compounds are distributed below eu-
Aba-2572 0.71 1 1 2
calyptol (Rf ¼0.25–0.35, spot 1), while terpene hydrocarbons are
Aba-2793 1.41 2 1 2
Aba-4156 1.41 4 1 2 located at solvent front (Rf¼ 0.93, spot 6). The results of bioauto-
Aba-4727 1.41 4 1 2 graphic analysis of essential oils strips are presented in Fig. 1B and
Aba-4779 1.41 4 1 2 those of separated spots in Fig. 1C. The antibacterial activity was
Aba-4803 1.41 2 1 1 noted in the lower half of the chromatograms, from the start to the
Aba-4804 1 2 1 1
Aba-4890 1.41 4 1 1
Rf value of 0.43 for EuHN and 0.46 for EuB (Fig. 1B). This was
Aba-4914d 1 2 1 2 confirmed by bioautography of separated spots, where the in-
Aba-5055e 1 1.41 1 2 hibition zone was observed for the polar terpene compounds and
Aba-5074 1 2 1 2 spathulenol (Fig. 1C, spots 1 and 2).
Aba-5081 1 2 0.5 2
Aba-5372 2 1.41 1 1
Aba-6673f 1.41 2 1.41 2 3.2.3. EO-AB synergistic activity
Aba-7860 1.41 2 1 2 The results of combinations of E. camaldulensis essential oils
Aba-8255 2 2 1.41 1.41 and conventional antibiotics are shown in Figs. 1 and 2. When two
Aba-8781 0.71 0.71 0.5 0.71 euclayptus essential oils were combined with ciprofloxacin, sy-
Aba-8833 0.5 0.71 0.5 0.71
Aba-34963 1 1 0.71 0.71
nergism was detected against two out of three tested MDR A.
Aba-40100 1.41 2 1 1.41 baumannii strains (Aba-4914 and Aba-5055), with FIC index value
E. coli ATCC 25922g 1 1 1 2 lower than 0.5 (Fig. 2). This combination against strain Aba-6673
S. aureus ATCC 25923h 1 1 1 1 expressed additive effect (FIC index ¼0.53). However, in all com-
a binations against three A. baumannii strains the MIC value for CIP
MIC, minimum inhibitory concentration.
b
MBC, minimum bactericidal concentration, both values given as μl ml
1 from was reduced to 1/4 MIC. Synergistic effect detected in combination
the geometrical means of triplicate experiments. of EuB essential oil and CIP against Aba-4914 and Aba-5055 was
c
Determined MIC of antibiotics used as controls: GEN 16.0 mg mL
1; CIP 0.25 m the same as in combinations with EuHN essential oil.
g mL
1; PMB 0.5 mg mL
1. Combinations of the essential oils and PMB also expressed sy-
d
Determined MIC of antibiotics used as controls: GEN 4256.0 mg mL
1; CIP
64 mg mL
1; PMB 4 mg mL
1.
nergistic effect against two wound isolates (Aba-4914 and Aba-
e
Determined MIC of antibiotics used as controls: GEN 16 mg mL
1; CIP 64 mg 5055) and reference strain ATCC 19606 (Fig. 3). The effect of the
mL
1; PMB 4 mg mL
1. same combination against strain Aba-6673 was not tested because
f
Determined MIC of antibiotics used as controls: GEN 64 mg mL
1; CIP 32 m this strain was sensitive to PMB when administered alone. The
g mL
1; PMB 0.5 mg mL
1.
combination of EO-PMB against MDR strain Aba-4914 reduced
g
Determined MIC/MBC of antibiotics used as controls: GEN 0.5/1.0 mg mL
1;
CIP o 0.06/ o 0.06 mg mL
1; PMB 0.5/0.5 mg mL
1. MIC value for PMB to 1/2 MIC. The same combination was more
h
Determined MIC/MBC of antibiotics used as controls: GEN 0.5/2.0 mg mL
1; efficient against Aba-5055, lowering the MIC value for PMB to 1/4
CIP 0.25/0.5 mg mL
1; PMB not applicable. in combination with EuHN, while in combination with EuB this
value was reduced to 1/8 MIC for PMB. In all examined combina-
sesquiterpene spatulenol and monoterpene hydrocarbons were tions the MIC values for PMB were reduced to the break point or
also present in considerable amounts (Table 1). The dominant under it.
components of both essential oils were spathulenol, cryptone, p- The results of E. camaldulensis essential oils combination with
cimene, 1,8-cineole, terpinen-4-ol and β-pinene. Williams (2010) gentamicin varied (Fig. 4). Against MDR strain Aba-4914, combi-
proposed two chemotypes of E. camaldulensis essential oil: (1) ci- nations with both eucalyptus oils acted synergistically, lowering
neole-rich (80–90% 1,8-cineole, plus pinene) and (2) cineole-poor the GEN concentrations to 64 μg mL
1 MIC in combination with
(containing significantly less cineol) chemotype. Other con- EuHN and to 32 μg mL
1 MIC in GEN-EuB combination. However,
stituents (β-phellandrene, p-cymene and cryptone) can be present taking into consideration that the MIC value of this strain for GEN
in variable amounts (Brophy and Southwell, 2002). According to was very high ( 4256 μg mL
1), the reduction was not sufficient
the results obtained, E. camaldulensis from Montenegro can be for reaching the therapeutic dosage. The same combination
classified in the chemotype with low 1,8-cineole and high against strain Aba-6673 was interpreted as additive effect with FIC
p-cymene and cryptone content. index close to 0.5, with the MICs reduced to 1/8 MIC in combi-
nation with EuHN and 1/16 with EuB. The EuHN-GEN combination
3.2. Antibacterial activity (MIC value for essential oil and 1/16 MIC for GEN), although not
synergistic, lowered the MIC value to the break point for GEN.
3.2.1. E. camaldulensis essential oils activity
E. camaldulensis essential oils antibacterial activity is shown in 3.2.4. Activity of synergistic antibiotic-essential oil combinations
Table 2. Both essential oils exibited bacteriostatic and bactericidal over time
activity against all examined MDR A. baumannii wound isolates. Time effect of detected synergistic activity for eucalyptus es-
The MIC values for EuHN essential oil ranged from 0.5 μL mL
1 to sential oils and PMB is shown by time-kill curves in Fig. 5. The
2 μL mL
1 and for EuB from 0.5 μL mL
1 to 1.41 μL mL
1, with effect of EuHN essential oil and PMB against reference strain ATCC
median concentrations of 1.41 μL mL
1 and 1 μL mL
1, respec- 19606 was similar or lower than the effect of PMB administred as
tively. The MBC values were the same or one fold greater than MIC single agent, but only till 9 h of incubacion when PMB effect
130 P. Knezevic et al. / Journal of Ethnopharmacology 178 (2016) 125–136
EuHN
EuB
A B
Fig. 1. TLC chromatogram of investigated EOs (1 – polar terpene compounds, 2 – spathulenol, 3–1,8 cineole, 4 – thymol, 5 – probably cryptone, 6 – terpene hydrocarbons)
(A), bioautograms of EOs against A. baumannii ATCC 19606 (essential oil strips (B) and separated spots (C)).
continued to decrease and bacterial CFU started to grow. On the
contrary, the effect of AB-EO combination after 12 h of incubation
reduced bacterial CFU below detection limit (Fig. 5). The effect of
the same combination against MDR A. baumannii strain Aba-4914
was more profound. Antibacterial effect of the combination of
natural agents and PMB alone was the same for the first 3 h of
incubation, and bacterial CFU increased in the treatment with only
PMB after 6 h. However, combination of EuHN and PMB reduced
bacterial CFU below detection limit after 6 h. The combination of
the antibiotic with eucalyptus essential oil decreased bacterial CFU
for more than 5 log compared to more active single agent, i.e. PMB
(Fig. 5).
4. Discussion
Two E. camaldulensis essential oils, EuHN and EuB, originating
from different locations along Montenegro coastal area (Herceg
Novi and Bar) were investigated in this study. GC–MS analysis of
essential oils revealed that they belong to cineole-poor type of
eucalyptus essential oils according to William’s classification
(2011). Apart from 1,8-cineole, the substantial amounts of
p-cymene, cryptone, terpinen-4-ol, spathulenol and β-pinene
were detected. The results correspond to those obtained for eu-
calyptus essential oil from South Florida, where the major iden-
tified constituents included p-cymene (35.0%), cryptone (13.7%),
terpinen-4-ol (5.7%), spathulenol (4.3%), and cuminaldehyde
(3.7%), with a very low amount of 1,8-cineole (2.7%) (Pappas and
Sheppard-Hanger, 2000). It is important to emphasize that cryp-
tone has been reported to be present in low-cineole varieties of E.
camaldulensis essential oils from Australia (Bignell et al., 1996),
C
Benin (Dellacassa et al., 1990) and South Florida (Tsiri et al., 2003),
which is in agreement with the results acquired in this study.
Contrary to this, eucalyptus essential oils originating from Greece,
Pakistan, Mozambique, Nigeria and Taiwan belong to cineole-rich
oils (Tsiri et al., 2003; Ashraf et al., 2010; Pegula et al., 2000;
Oyedeji et al., 1999; Shieh, 1996). Although the chemical compo-
sition of both E. camaldulensis essential oils was similar, the EuB
essential oil lacked five compounds (α-terpinolene, α-terpineol,
trans-carveol, α-terpenyl-acetate and aromadendrene). Since EuB
expressed higher antibacterial activity compared to EuHN, it is
obvious that these five minor components do not play a significant
role in antimicrobial activity of the examined oils. The observed
difference in essential oil composition is probably a consequence
of different biotope and plant genetic factors, which affect the
activation/inhibition of different metabolic pathways of the aro-
matic plants. Although the EuB essential oil demonstrated a
slightly higher anti-MDR strains activity, the antibacterial effect of
both essential oils was promising. The lowest MIC and MBC values
for both essential oils were 0.5 μl ml
1 and 0.71 μl ml
1, respec-
tively. The detected MICs for A. baumannii are similar or even
lower than those previously determined by microdilution method
and reported for EOs of other Eucalyptus species: E. globulus Labill.
1–2 mg mL
1, E. radiata Siber ex DC. 1 mg mL
1 and E. citadora
Hook. 2 mg mL
1 (Mulyaningsih et al., 2010; 2011). Similarly, anti-
A. baumannii effect of E. camaldulensis EOs is comparable to bark
oil of Cinnamomum zeylanicum Ness (0.5–2.5 ml mL
1) (Sienkie-
wicz et al., 2014), Citrus hystrix DC. lime oil (1.1–4.4 mg mL
1)
(Srisukh et al., 2012) or Rosa damascena Mill. petals oil
(2 mg mL
1) (Shohaye et al., 2014). The used E. camaldulensis EOs
are more active against A. baumannii than Citrus hystrix DC. leaf oil
(1–17 mg mL
1) (Srisukh et al., 2012), Pelargonium graveolens Ait.
 P. Knezevic et al. / Journal of Ethnopharmacology 178 (2016) 125–136 131

1 1
Aba-4914 Aba-4914

0,5 0,5
FIC E. camaldulensis D. Herceg Novi essential oil

FIC E. camaldulensis D. Bar essential oil

0 0
0 0,5 1 0 0,5 1
1 1
Aba-5055 Aba-5055

0,5 0,5

0 0
0 0,5 1 0 0,5 1

1 1
Aba-6673 Aba-6673

0,5 0,5

0 0
0 0,5 1 0 0,5 1

FIC Ciprofloxacin
Fig. 2. Antibacterial effect of E. camaldulensis essential oils and ciprofloxacin combination against MDR A. baumannii isolates.

herb oil (7.5–9.5 ml mL
1) and Lavandula angustifolia Mill. flower- activity against MDR A. baumannii strains, except the recent report
ing herb oil (10.5–13.0 ml mL
1) (Sienkiewicz et al., 2014). Anti- on E. camaldulensis extracts (Jazani et al., 2012). As in the present
bacterial activity of E. camaldulensis essential oils was previously study the examination focused on essential oils and not extracts,
reported against S. aureus, L. monocytogenes, E. durans, Salmonella the results cannot be compared. The results of the present study
Typhi, E. coli, B. subtilis, P. aeruginosa, Streptococcus mutans and confirm anti-A. baumannii activity of E. camaldulensis EOs and
Streptococcus pyogenes (Akin et al., 2010; Lima et al., 2013; Owlia justify traditional application of this plant for diseases caused by
et al., 2009; Rasooli et al., 2009). However, there are no data on its bacteria.
132 P. Knezevic et al. / Journal of Ethnopharmacology 178 (2016) 125–136

1 1
Aba-4914 Aba-4914

0,5 0,5

0 0
FIC E. camaldulensis D. Herceg Novi essentila oil

0 0,5 1 0 0,5 1

FIC E. camaldulensis D. Bar essential oil

1 1
Aba-5055 Aba-5055

0,5 0,5

0 0
0 0,5 1 0 0,5 1

1 1
ATCC 19606 ATCC 19606

0,5 0,5

0 0
0 0,5 1 0 0,5 1

FIC Polymyxin B
Fig. 3. Antibacterial effect of E. camaldulensis essential oils and polymyxin B combination against MDR A. baumannii isolates.

On TLC plates only dominant compounds of EOs (polar terpene camaladulensis essential oils (polar monoterpenoid alcohols and
alcohols, spathulenol, 1,8-cineole, thymol and terpene hydro- sesquiterpenoid alcohol spathulenol) are responsible for the an-
carbons) were visible. The results of bioautographic analysis tibacterial activity. As the plates were dried at 90 °C for 5 min., this
(the inhibition zones on bioautogram strips and squares – Figs. 1B could lead to evaporation of some highly volatile monoterpene
and C) show that polar terpenoid compounds present in E. hydrocarbons, and their activity may have been overlooked. Well-
 P. Knezevic et al. / Journal of Ethnopharmacology 178 (2016) 125–136 133

1 1
Aba-4914 Aba-4914
FIC E. camaldulensis D. Herceg Novi essential oil

0,5 0,5

FIC E. camaldulensis D. Bar essential oil

0 0
0 0,5 1 0 0,5 1
1 1
Aba-6673 Aba-6673

0,5 0,5

0 0
0 0,5 1 0 0,5 1

FIC Gentamicin
Fig. 4. Antibacterial effect of E. camaldulensis essential oils and gentamicin combination against MDR A. baumannii isolates.

known antibacterial compounds-1,8-cineole and thymol have not analysis because of the low concentration applied.
expressed visible antibacterial activity according to the bioauto- Hendry et al. (2009) pointed out superior antimicrobial efficacy
graphy. However, these compounds were very active against dif- of crude eucalyptus essential oil (2–8 g L
1), compared with
ferent Gram negative and Gram positive bacteria in oprevious dominant component 1,8-cineole (8–64 g L
1/L), suggesting that
studies, with MIC values for 1,8-cineole ranging from minor components also contribute to the final antibacterial ac-
0.93–7.5 mg mL
1 and for thymol from 0.7–1.4 mg mL
1 (Rosato tivity. This is particularly important for cineole-poor eucalyptus
et al., 2007; Sonboli et al., 2006). Also, eugenol expressed anti- essential oils, which are composed of various compounds present
bacterial activity against Sarcina lutea, Bacillus subtilis, Xanthomo- in low concentration, including the oils examined in the present
nas campestries (7.81 μg mL
1) and E. coli (15.6 μg mL
1), while study, in which neither component was present in an amount
spathulenol MICs were even higher against S. lutea, B. subtilis, X. greater than 22%. The synergistic effect between components can
campestries (15.62 μg mL
1) and E. coli ( 420 μg mL
1) (Srisukh also be proposed, because each of the essential oil constituents
et al., 2012). The discrepancy between the previously determined might have its own mechanism of action. The examination of es-
antimicrobial activity of some hydrophobic compounds from the sential oils in preventing bacterial resistance is believed to be very
EOs and the results of TLC bioautography assay in which only polar promising, because many conventional antimicrobials are pure
terpenes were active, might be a consequence of various diffusion compounds having only a single target site of action, while es-
of hydrophylic and hydrophobic compounds in a hydrophilic sential oils, as multi-component mixtures, can act on different
semisolid growth medium used for bioautography. In addition, the levels (Yap et al., 2014). For example, the mechanism of action
amount of the EOs applied on TLC plate was only 0.08 μL, which is determined for p-cymene and γ-terpinene is membrane disrup-
quite below the detected MIC values against A. baumannii ATCC tion (Ultee et al., 2007; Oyedemi et al., 2009), for thymol it is also
19606. This implies that concentrations of some potentially active membrane disruption, but with potential intracellular targets such
compounds, i.e. 1,8-cineole and thymol on TLC plates were prob- as citrate metabolic pathway disruption (Di Pasqua et al., 2007;
ably not sufficient for the activity expression, as they are not Trombetta et al., 2005; Di Pasqua et al., 2010). Spathulenol is
present in high concentrations in the examined EOs. The same can considered active against Gram-positive and Gram-negative bac-
be concluded for other minor components of the investigated EOs- teria (Bougatsosa et al., 2004), and here also, according to the
they have not expressed antibacterial potential in bioautographic bioautographic analysis, this compound expressed anti-A.
134 P. Knezevic et al. / Journal of Ethnopharmacology 178 (2016) 125–136
14
A. baumannii ATCC 19606 count (log CFU mL )
-1
0.25 µg mL-1 polymyxin B + 0.5 µl mL-1 EuHN
12
10
8 authors suggested that association of antibiotics with essential oils
6
4 CIP acts on DNA gyrase by inhibiting the process of DNA replica-
2 EOs-PMB combination is probably due to the similar mode of ac-
0 5 10 15
Time (h)
14
A. bumannii Aba-4914 count (log CFU mL-1)
1 µg mL-1 polymyxin B + 0.5 µl mL-1 EuHN
12
10
8 been sensitive to PMB and thus a lower PMB concentration was
6
4 duction rate for this strain were already reported for PMB com-
2 clinical isolates, especially from wounds, as the determined active
0 5 10 15
Time (h)
Fig. 5. Time effect of E. camaldulensis essential oil and polymyxin B synergistic
intercation against A. baumannii refernece strain ATCC 19606 (left) and MDR strain
Aba-4914 (right); (■) no tretamnent, (●) PMB, (♦) EuHN, (▲) PMB and EuHN.
baumannii activity, but its mechanism of action still remains
unknown.
The anti-MDR strains activity of eucalyptus oils obtained in this
study seems to be very promising for future development of novel
anti- A. baumannii agents of natural origin or novel therapeutic
strategies. The activity of combinations of EOs and conventional
antibiotics against A. bauamnnii were examined previously and
synergism was detected in the following combinations: Melaleuca
alternifolia (Maiden & Betche) Cheel with gentamicin (Rosato et al.,
2010), Cinnamomum zeylanicum L. with amikacin, Citrus limon with
amikacin (Guerra et al., 2012), Coriandrum sativum L. with chlor-
amphenicol, ciprofloxacin, gentamicin and tetracycline (Duarte
et al., 2012) and Myrtus communis L. with ciprofloxacin and poly-
myxin B (Aleksic et al., 2014). Here we examined a possible
strategy for combating MDR A. bauamnnii strains, i.e. effect of the
E. camaldulensis essential oils combination with ciprofloxacin,
gentamicin or polymyxin B against MDR wound isolates. When CIP
was examined in combination with E. camalulensis essential oils,
the synergistic activity was recorded in two strains, while against
Aba-6673 strain this effect was additive with FIC index above, but
close to 0.5. The same results for Aba-6673 strain were obtained in
combination with GEN, but the MIC reduction was higher. It is
important to notice that Aba-6673 was sensitive to PMB, which
represents the current solution for its control. Accordingly, in sy-
nergistic combinations the MIC reduction for CIP and GEN was
significant, but not sufficient because the MIC values for MDR A.
baumannii strains were above therapeutic doses. On the contrary,
the combination of eucalyptus essential oils with PMB was effi-
cient against all examined A. baumannii strains and the renewed
sensitivity of the MDR strains to PMB is very promising. Some
targeting resistant bacteria may have different mechanism of ac-
tion and it may lead to new choices to overcome the problem of
bacterial resistance (Yap et al., 2014). This is the case with com-
bination of E. camaldulesis essential oils and ciprofloxacin, since
tion, while EOs primarily damage bacterial envelopes. Never-
theless, the detected in vitro synergistic activity of the eucalyptus
20 25
tion, as both of the agents act by affecting the bacterial cell
membrane structures.
Since this is the first report so far regarding the antibacterial
efficiency of E. camaladulensis essential oils alone and in combi-
nation with conventional antibiotics against MDR A. baumannii
isolates, we investigated the time effect of eucalyptus EO and PMB
synergistic interaction against A. baumannii reference strain ATCC
19606 and MDR wound isolate Aba-4914. The synergistic combi-
nations were more efficient in CFU reduction of MDR clinical iso-
late Aba-4914, probably because ATCC 19606 strain has already
applied (Fig. 3). The CFU reduction under detectable level using
EuHN-PMB combination was obtained for ATCC 19606 after 15
hours of incubation and in the case of MDR A. baumannii strain
after only 6 h of incubation. Similar results with such a high re-
bination with Myrtus communis essential oils (Aleksic et al., 2014).
These results are very promising in eradication MDR A. baumannii
20 25
concentrations or even higher are possible to be obtained in
wounds during a topical treatment. Sripriya et al. (2007) proposed
the collagen bilayer dressing with CIP, as an effective system for
infected wound healing. However, in the view of continual in-
crease of bacterial resistance to conventional antibiotics, it is ne-
cessary to consider the usage of similar bilayer dressings con-
taining combination of antibiotic and essential oils, such as E. ca-
maldulensis or its components with confirmed antibacterial
activity.
5. Conclusion
In conclusion, this study shows for the first time the anti-
bacterial activity of the essential oil of E. camaldulensis against
multi-drug resistant A. baumannii wound isolates, justifying the
plant traditional application for wound healing. The polar terpene
compounds and spathulenol as constituents of the E. camaldulensis
oil are at least partially responsible for the detected antibacterial
activity. Also, obtained in vitro synergistic interactions of anti-
biotics ciprofloxacin, gentamicin and polymyxin B with E. ca-
maldulsensis essential oils have been detected in this study for the
first time against MDR A. baumannii isolates. The results, therefore,
represent a basis for further ex vivo, in vivo and clinical studies that
could lead to the development of a new treatment based on
combination of traditional and conventional antimicrobial agents
in combating MDR A. baumannii strains.
 P. Knezevic et al. / Journal of Ethnopharmacology 178 (2016) 125–136
Acknowledgments
Research support was provided by the Ministry of Education,
Science and Technological Development of the Republic of Serbia,
grant OI 172058. The authors are grateful to Dr. Ljiljana Knezevic
(Faculty of Sciences, University of Novi Sad) for language revision.
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