Journal of Infection and Public Health 10 (2017) 586–592

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Journal of Infection and Public Health

journal homepage: http://www.elsevier.com/locate/jiph

In vitro synergistic antibacterial activity of the essential oil from

Zingiber cassumunar Roxb against extensively drug-resistant
Acinetobacter baumannii strains
Wongwarut Boonyanugomol a,∗ , Kairin Kraisriwattana a , Kamolchanok Rukseree a ,
Kraisorn Boonsam b , Panchaporn Narachai b
a
Mahidol University, Amnat Chareon Campus, Amnat Chareon, 37000, Thailand
b
Microbiology Laboratory, Division of Clinical Pathology, Amnat Chareon Hospital, Amnat Chareon, 37000, Thailand

a r t i c l e i n f o a b s t r a c t

Article history: In this study, we determined the antibacterial and synergistic activities of the essential oil from Zingiber
Received 1 October 2016 cassumunar against the extensively drug-resistant (XDR) Acinetobacter baumannii strains. The antibacte-
Received in revised form rial and synergistic properties of the essential oil from Z. cassumunar were examined by agar disc diffusion
22 November 2016
tests. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were
Accepted 7 January 2017
evaluated by broth microdilution using the resazurin assay. The in vitro time–kill antibacterial kinetics
was analyzed using the plate count technique. We found that the essential oil from Z. cassumunar had
Keywords:
antibacterial activity against A. baumannii, with MIC and MBC ranging from 7.00 to 9.24 mg/ml. The
Acinetobacter baumannii
Z. cassumunar
essential oil could completely inhibit A. baumannii at 1 h, and coccoid-shaped bacteria were found after
Essential oil treatment. In addition, the essential oil had a synergistic effect when combined with antibiotics, e.g.,
Antibacterial activity aminoglycosides, fluoroquinolones, tetracyclines, and folate pathway inhibitors. Thus, the essential oil
Synergism from Z. cassumunar has strong antibacterial and synergistic activities against XDR A. baumannii, which
may provide the basis for the development of a new therapy against drug-resistant bacteria.
© 2017 The Authors. Published by Elsevier Limited. This is an open access article under the CC
BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Background
Acinetobacter baumannii is a gram-negative, non-motile bac-
terium, which is often isolated in hospital environments, including
hospitalized patients in intensive care units. It has been proposed
that A. baumannii is an opportunistic pathogen associated with
hospital-acquired infections [1]. Because of diverse and frequent
antibiotic treatments, these bacteria have globally emerged as
multidrug-resistant (MDR) and extensively-drug resistant (XDR)
in hospitals [2]. This simultaneous resistance to many drugs by A.
baumannii may lead to failed treatments, increased medical ther-
apy costs, high rates of mortality, and its spread from hospitals to
the community [3]. Currently, potent therapeutic agents to combat
antibiotic-resistant bacteria, particularly XDR A. baumannii need
to be identified. An attractive strategy for treating severely resis-
tant A. baumannii is the use of medicinal plants. Several medicinal
∗ Corresponding author at: Mahidol University, Amnat Chareon Campus, P.O. Box
37000, Amnat Chareon, Thailand.
E-mail addresses: wongwarut.boo@mahidol.ac.th, wongwarutb@gmail.com
(W. Boonyanugomol).
plants are used as vital sources for drug development because they
contain many bioactive compounds that may be therapeutically
effective. Therefore, the investigation of natural products could
help in identifying novel and potent bioactive agents for the treat-
ment of infectious diseases.
The essential oils produced by secondary metabolism in some
plants comprise complex mixtures of volatile molecules [4]. Vari-
ous essential oils have been reported to exhibit different biological
properties, such as anticancer [5], anti-inflammation [6], antimi-
crobial [7], and antioxidant effects [8]. Essential oils are highly
complex natural mixtures of approximately 20–60 components.
Two or three major components are usually present at high concen-
trations, whereas other components are present in trace amounts
[9]. Because of the chemical diversity of essential oils, phytothera-
peutic approaches should be used to investigate essential oils that
are potentially relevant for the development of novel therapeutic
agents.
Zingiber cassumunar Roxb is classified in the family Zingiber-
aceae. In Thai, it is known as “Plai,” and the rhizomes have been
used for a long time in Thai traditional medicine [10] for the treat-
ment of muscle strains, inflammation, and skin disorders. The safety
of Z. cassumunar has been evaluated in rats, where an extract

http://dx.doi.org/10.1016/j.jiph.2017.01.008
1876-0341/© 2017 The Authors. Published by Elsevier Limited. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-
nd/4.0/).
 W. Boonyanugomol et al. / Journal of Infection and Public Health 10 (2017) 586–592
produced no signs of toxicity or mortality in any of the animals
tested [11]. The active chemical ingredients in Z. cassumunar essen-
tial oil are sabinene, ␥-terpinene, ␣-terpinene, terpinene-4-ol, and
(E)-1–(3,4-dimethoxyphenyl)butadiene (DMPBD) [12]. These phy-
tochemicals have various pharmacological properties, including
anti-inflammation, antifungal, and antibacterial effects [10,13].
However, there have been no previous studies on their antibacterial
and synergistic activities against antibiotic-resistant bacteria.
In this study, we aimed to determine the antibacterial activity
of Z. cassumunar essential oil against XDR A. baumannii strains and
susceptible A. baumannii strains. Combinations of Z. cassumunar
essential oil and standard antibiotics were screened to determine
the presence of any synergistic activity. The results of our study
may support the clinical applications of Z. cassumunar in modern
medicine.
Methods
Plant material and essential oil extraction
Rhizomes of Z. cassumunar Roxb cultured in Northeast Thailand
were collected for use in this study. Fresh Z. cassumunar Roxb rhi-
zomes were washed and chopped before hydro-distillation using a
Clevenger apparatus to obtain the essential oil. The essential oil was
stored at −20 ◦ C in dark bottles until it was used in the experiments.
Preparation of bacterial strains
A. baumannii strains were isolated from clinical blood samples
by conventional methods. Antimicrobial susceptibility was deter-
mined by disc diffusion assay. The extensively drug-resistant (XDR)
A. baumannii was defined as the isolate resistant to cephalosporins,
fluoroquinolones, aminoglycosides, and carbapenems. Nine clin-
ical strains of A. baumannii were used in this study, comprised
of six XDR A. baumannii strains and three non-XDR A. bauman-
nii strains (susceptible strains; sensitive to all tested antibiotics).
Other opportunistic pathogens were also tested in this study, i.e.,
Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans,
and Cryptococcus neoformans. Brain-heart infusion agar (Himedia
Laboratory, Ltd, India) and Sabouraud dextrose agar (Himedia Lab-
oratory, Ltd, India) were used to culture the bacteria (at 37 ◦ C for
18 h) and yeast (at 30 ◦ C for 48 h), respectively.
Standard antibiotics
The standard antibiotics used in this study included eight
groups of standard antibiotics recommended by the Clinical
and Laboratory Standards Institute (CLSI) [14]: (1) penicillin
(amoxicillin), (2) ␤-lactamase inhibitor combinations (piperacillin-
tazobactam and amoxicillin-clavulanic acid), (3) cephems (cef-
tazidime, cefepime, cefotaxime, and ceftriaxone), (4) carbapen-
ems (imipenem, meropenem, and ertapenem), (5) aminogly-
cosides (gentamicin and amikacin), (6) tetracyclines (tetracy-
cline and doxycycline), (7) fluoroquinolones (ciprofloxacin and
levofloxacin), and (8) folate pathway inhibitors (trimethoprim-
sulfamethoxazole) (Oxoid, Ltd., Basingstoke, Hampshire, England).
Antibacterial activity screening using agar disc diffusion assays
The antibacterial activity of Z. cassumunar essential oil was
screened using disc diffusions assays to determine the sensitivity
of A. baumannii, S. aureus, P. aeruginosa, C. albicans, and C. neofor-
mans to Z. cassumunar essential oil. A microbial suspension with
a turbidity of 0.5 McFarland units was spread over a Petri plate
containing Mueller-Hinton agar (MHA) (Himedia Laboratory, Ltd,
India) using a sterile cotton swab. Sterilized discs (6 mm diameter)
587
were impregnated with 0.05, 0.5, 5, or 10 mg/ml of Z. cassumunar
essential oil, and the discs were then placed on the inoculated agar
plates. The Petri plates were incubated at 37 ◦ C for 18 h. The antibac-
terial activity of Z. cassumunar essential oil was determined based
on the diameter of the transparent zone around the disc (zone of
inhibition). The zone of inhibition was interpreted as described pre-
viously by Ponce et al. [15]: not sensitive = total diameter <8.00 mm;
sensitive = total diameter of 8–14 mm; very sensitive = total diam-
eter of 15–19 mm; extremely sensitive = total diameter >20 mm.
Each experiment was performed as independent triplicates and
the results were expressed as the mean ± standard errors of mean
(SEM).
Determination of the minimum inhibitory concentration
(MIC) and minimum bactericidal concentration (MBC)
After screening the antibacterial activity of Z. cassumunar essen-
tial oil, the MIC was determined using a broth microdilution
susceptibility assay, as recommended by the National Committee
for Clinical Laboratory Standards [16]. A. baumannii strains were
cultured in Mueller-Hinton broth at 37 ◦ C for 18 h. The bacterial
suspension was adjusted to a turbidity of 0.5 McFarland units and
diluted to 1:100 before use. Z. cassumunar essential oil dilutions
ranging from 1–100 mg/ml were prepared in Mueller-Hinton broth
(supplemented with a final concentration of 0.5% Tween 80 v/v).
Next, 50 ␮l of diluted essential oil and 50 ␮l of A. baumannii suspen-
sion were added to a 96-well plate and incubated at 37 ◦ C for 18 h.
Afterwards, 10 ␮l of 1 mg/ml resazurin (Sigma, UK) solution was
poured into the 96-well plate to assess the bacterial viability. The
MIC value was defined as the lowest concentration of Z. cassumu-
nar essential oil that prevented the color of resazurin from changing
from blue to pink. The MBC was determined by plating 10 ␮l of the
bacterial suspension from the well where no color change occurred.
After 18 h of incubation, the lowest concentration with no colony
growth was defined as the MBC. Three independent replicates were
performed and the results were expressed as the mean ± SEM.
Time–kill assay
Time–kill assays were performed as described by Aliva et al.
[17]. A. baumannii strains were adjusted to 106 CFU/ml. The MBC
values for Z. cassumunar essential oil were selected for testing in the
time–kill assays. After incubation for 1–6 h, 50 ␮l of treated bacteria
was collected from the test solutions. Ten-fold serial dilutions of
the treated bacteria were spread on MHA to obtain colony counts.
A solution containing 0.5% v/v Tween 80 was used as a control.
Time–kill curves were obtained by plotting the bacterial numbers
against time. These tests were performed in triplicate.
Screening the synergistic effect of the essential oil with antibiotic
discs
An A. baumannii suspension with a turbidity of 0.5 McFar-
land units was spread on plates containing MHA. To evaluate for
synergistic effects, 17 standard antibiotic discs were individually
impregnated with 5 ␮l of Z. cassumunar essential oil (at the MBC
value) and placed onto the inoculated agar plates. After incubat-
ing overnight, the zones of inhibition produced by the essential oil
combined with standard antibiotics were evaluated as described
previously by Toroglu [18]: zone of combination treatment > zone
of essential oil + zone of the corresponding antibiotic, was inter-
preted as synergism; zone of combination treatment = zone of
essential oil + zone of corresponding antibiotic, was interpreted
as additive; zone of combination treatment < zone of essential
oil + zone of corresponding antibiotic, was interpreted as antago-
nism.
588 W. Boonyanugomol et al. / Journal of Infection and Public Health 10 (2017) 586–592

Table 1
Antimicrobial activity defined as the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) for the essential oil from Z. cassumunar against
A. baumannii and other opportunistic pathogens.

Strains Number of strains Zone of inhibition with essential oil from Z. cassumunar (mm) MIC (mg/ml) MBC (mg/ml)

0.05 mg/ml 0.5 mg/ml 5 mg/ml 10 mg/ml

Susceptible A. baumannii 3 NZ NZ 14.33 ± 1.51 22.33 ± 1.86 7.00 ± 1.67 7.33 ± 2.06
XDR A. baumannii 6 NZ NZ 13.14 ± 2.56 20.33 ± 1.98 8.67 ± 1.93 9.24 ± 1.95
S. aureus 1 NZ NZ NZ 11.33 ± 0.57 17.33 ± 1.15 18.67 ± 1.15
P. aeruginosa 1 NZ NZ NZ 14.00 ± 1.73 15.33 ± 1.15 15.33 ± 1.15
C. albicans 1 NZ NZ NZ 13.00 ± 1.73 16.67 ± 1.15 18.67 ± 1.15
C. neoformans 1 NZ NZ NZ 12.66 ± 1.15 14.67 ± 1.15 15.33 ± 1.15

Results represent the mean ± SEM.

NZ = No inhibition zone.

Results susceptible A. baumannii strains. Fig. 3 shows the synergistic effect

Antibacterial activity and MIC of Z. cassumunar essential oil
The essential oil from Z. cassumunar rhizome showed a pale
amber color. The essential oil yield was 6.00 ml/kg based on the
fresh weight. The antibacterial activity of the essential oil against
A. baumannii is summarized in Table 1. Various concentrations of
Z. cassumunar essential oil (0.05, 0.5, 5, and 10 mg/ml) were tested
using agar disc diffusion assays. A zone of inhibition >8 mm in diam-
eter was interpreted as sensitive. All of the susceptible and XDR A.
baumannii strains were sensitive to Z. cassumunar essential oil at 5
and 10 mg/ml. The average zones of inhibition with the essential oil
at 5 mg/ml measured 14.33 ± 1.51 mm and 13.14 ± 2.56 mm in the
susceptible and XDR A. baumannii strains, respectively. Sensitive
results were not obtained with discs containing 0.05 and 0.5 mg/ml
of essential oil. The average MICs for Z. cassumunar essential oil
were 7.00 ± 1.67 and 8.67 ± 1.93 mg/ml with susceptible and XDR A.
baumannii strains, respectively. The average MBCs for Z. cassumunar
essential oil were 7.33 ± 2.06 mg/ml for the susceptible A. bauman-
nii strains and 9.24 ± 1.95 mg/ml for the XDR A. baumannii strains.
The essential oil from Z. cassumunar also produced zones of inhi-
bition with other opportunistic pathogens (S. aureus, P. aeruginosa,
C. albicans, and C. neoformans) at 10 mg/ml, but zones of inhibition
were not obtained at 0.05, 0.5, and 5 mg/ml. Comparisons of the
MICs and MBCs produced by the essential oil using those pathogens
showed that the MICs and MBCs were smaller in the A. baumannii
strains than the other four opportunistic pathogens.
We also observed the bacterial cell morphology of A. baumannii
strains exposed to Z. cassumunar essential oil (Fig. 1). After treat-
ment with MBC values of the Z. cassumunar essential oil for 18 h,
coccoid-shaped bacterial were observed, whereas the control bac-
teria had the usual rod shape.
Time–kill assay
The results of the time–kill assays using Z. cassumunar essential
oil are shown in Fig. 2. After incubating A. baumannii with Z. cas-
sumunar essential oil at the MBC values, the susceptible and XDR
A. baumannii strains were killed completely after 1 h.
Screening for the synergistic effects of Z. cassumunar essen-
tial oil
The results obtained using combinations of Z. cassumunar essen-
tial oil and standard antibiotic discs against A. baumannii strains
are shown in Table 2. All of the susceptible A. baumannii strains
were sensitive to eight groups of standard antibiotics, but all of the
XDR A. baumannii strains showed resistant to all tested antibiotics.
We detected synergistic effects of Z. cassumunar essential oil with
aminoglycosides, fluoroquinolones, tetracyclines, and folate path-
way inhibitors in both susceptible and XDR A. baumannii strains.
The combinations of Z. cassumunar essential oil with penicillin, ␤-
lactamase inhibitor, cephems, and carbapenems had antagonistic
effects against XDR A. baumannii strains, but synergistic effects on
of Z. cassumunar essential oil combined with a standard antibiotic
(gentamicin) against A. baumannii.
Discussion
Z. cassumunar is widely available throughout South and South-
east Asia and has been used for the treatment of various conditions,
such as inflammation, rheumatism, and muscular pain[19]. It has
also been employed as a carminative and mild laxative, antidysen-
teric agent, and wound healing agent in several countries, including
India, Indonesia, Malaysia, and Thailand [20–22]. In Thailand, this
plant is used widely as an embrocation, as a poultice, or a decoc-
tion, because the oil can relieve muscle pain during massage or
when eaten fresh [22]. Therefore, this plant could be an important
natural source for drug development in the future.
Many plant essential oils are used as alternative medicines and
the antimicrobial properties of essential oils have been recognized
for several years [23–25]. The various pharmacological proper-
ties of Z. cassumunar suggest that this plant has the potential to
inhibit pathogenic bacteria. However, its antimicrobial effect on
drug-resistant bacteria, such as XDR A. baumannii strains, has not
been studied previously.
The emergence and spread of antibiotic-resistant bacteria that
cause infections requiring hospitalization is of great concern for
clinicians. Resistance to multiple antibiotic groups makes treat-
ment difficult, particularly for patients in intensive care units
and immunocompromised patients [10]. At present, A. baumannii
infections are emerging as MDR or XDR types [11]. Thus, novel nat-
ural agents need to be developed as drugs for treating MDR and
XDR A. baumannii. Previous studies suggest that gram-negative
bacteria are less susceptible to hydrophobic antibiotics or toxic
substances than gram-positive bacteria because their outer mem-
brane contains hydrophilic lipopolysaccharides, which provide a
barrier against the hydrophobic compounds found in essential oils
[9]. However, other reports suggest that the hydrophobic con-
stituents of essential oils allow them to access the periplasm of
gram-negative bacteria via porin proteins in the outer membrane
[10].
In the present study, we demonstrated the antibacterial activity
of Z. cassumunar essential oil against susceptible and XDR A. bau-
mannii strains, and showed that the essential oil exhibited strong
activity against both susceptible and XDR strains. According to the
dose-response study, the zone of inhibition increased with the con-
centration of the essential oil. The zones of inhibition formed by
this essential oil at 5 and 10 mg/ml were active, whereas the zones
of inhibition produced at 0.05 and 0.5 mg/ml were interpreted as
not active. The MICs and MBCs for this essential oil against suscep-
tible and XDR A. baumannii strains ranged from 7 to 9.24 mg/ml.
We also tested other opportunistic pathogens, including S. aureus,
P. aeruginosa, C. albicans, and C. neoformans, with the essential oil
from Z. cassumunar. We found that the essential oil had lower
Table 2
Effects of combinations of Z. cassumunar essential oil and standard antibiotic discs against A. baumannii strains.

W. Boonyanugomol et al. / Journal of Infection and Public Health 10 (2017) 586–592

Groups Antibiotics Susceptible A. buamannii strains XDR A. buamannii strains

(A) Mean of (B) Mean of Inhibition zone Inhibition zone Interpretation (A)a Inhibition (B) Mean of Inhibition zone Inhibition zone Interpretation
inhibition zone inhibition zone with synergism for essential oil zone with inhibition zone with synergism for essential oil
with antibiotic with essential (mm) combined with antibiotic with essential (mm) combined with
(mm) oil (mm) standard disc (mm) by CLSI oil (mm) standard disc
(mm) (mm)

Penicillin AML 21 14 >35 36.65 ± 0.49 S 21 13 >34 28.86 ± 1.40 A

␤-lactamase inhibitor TZP 21 14 >35 38.30 ± 1.41 S 21 13 >34 29.14 ± 2.54 A
AMC 21 14 >35 36.15 ± 1.20 S 20 13 >33 27.85 ± 1.46 A
Cephems CAZ 25 14 >39 43.00 ± 1.84 S 18 13 >31 28.52 ± 1.38 A
FEP 18 14 >32 36.50 ± 1.70 S 18 13 >31 28.43 ± 1.48 A
CTX 23 14 >37 40.85 ± 1.63 S 23 13 >36 27.86 ± 1.59 A
CRO 22 14 >36 37.35 ± 1.91 S 21 13 >34 27.33 ± 1.99 A
Carbapenems IPM 24 14 >38 40.00 ± 1.84 S 22 13 >35 28.33 ± 1.97 A
MEM 22 14 >36 40.25 ± 1.77 S 18 13 >31 29.44 ± 1.62 A
ETP 20 14 >34 35.00 ± 2.12 S 18 13 >31 28.24 ± 1.72 A
Aminoglycosides CN 20 14 >34 36.75 ± 1.06 S 15 13 >28 31.76 ± 1.72 S
AK 22 14 >36 40.85 ± 1.63 S 17 13 >30 31.50 ± 1.75 S
Tetracycline DO 20 14 >34 43.50 ± 2.12 S 13 13 >26 29.91 ± 1.90 S
TE 25 14 >39 46.50 ± 2.12 S 15 13 >28 30.07 ± 1.65 S
Fluoroquinolones CIP 21 14 >35 37.00 ± 1.41 S 21 13 >34 35.54 ± 1.27 S
LEV 25 14 >39 43.00 ± 1.41 S 17 13 >30 31.34 ± 2.21 S
Folate pathway inhibitor SXT 18 14 >32 37.00 ± 1.41 S 16 13 >29 32.10 ± 1.83 S

Synergism (S) > A + B; Additive (Ad) = A + B; Antagonism (A) < A + B

Antibiotics: AML = amoxicillin, TZP = piperacillin-tazobactam, AMC = amoxicillin-clavulanic acid, CAZ = ceftazidime, FEP = cefepime, CTX = cefotaxime, CRO = ceftriaxone, IPM = imipenem, MEM = meropenem, ETP = ertapenem,
CN = gentamicin, AK = amikacin, DO = doxycycline, TE = tetracycline, CIP = ciprofloxacin, LEV = levofloxacin, SXT = trimethoprim-sulfamethoxazole
Values represent the mean ± SEM.
a
(A) All of the XDR A. baumannii strains were resistant to 17 antibiotics (no inhibition zone), the inhibition zone from CLSI guideline were required.

589
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Fig. 1. Normal rod-shaped A. baumannii in the control group (A) and coccoid-shaped A. baumannii after exposure to Z. cassumunar essential oil (B).

Fig. 2. Time-kill curves obtained after treating A. baumannii strains with Z. cassumunar essential oil for 3 h.

antimicrobial activities against these four opportunistic pathogens
compared with A. baumannii strains. Previous study has reported
the antimicrobial properties of the essential oil from Z. cassumu-
nar, where the oil from the Z. cassumunar rhizome was shown
to exhibit high antimicrobial activity against dermatophytes and
yeasts [26]. Z. cassumunar has also been shown to exhibits antibac-
terial effects against pathogenic bacteria such as Bacillus cereus, P.
aeruginosa, E. coli, and S. aureus, but with very low antibacterial
activity [27]. Our results indicate that the essential oil from Z. cas-
sumunar had weak effects on several opportunistic pathogens, but
strong effects on A. baumannii. We suggest that the antimicrobial
activities of Z. cassumunar are diverse and its varying antimicrobial
effect may depend on the chemical composition, cultivation area,
and pathogen type. Thus, the essential oil from Z. cassumunar could
be applicable as an antibacterial treatment against A. baumannii
strains.
We detected changes in the bacterial morphology after Z. cas-
sumunar essential oil treatment; while the control A. baumannii
bacteria exhibited the normal rod shape, treatment with the essen-
tial oil from Z. cassumunar produced coccoid-shaped bacteria. These
results agree with those of a previous study, which showed that
rod-shaped bacterial cells were more sensitive to essential oils
than coccoid cells [28]. We postulate that the modified morphology
of A. baumannii after treatment with the essential oil is an adap-
tive response to stress and toxic substances where the biological
membrane is a possible target of the essential oil. The chemical
composition of the essential oil from hydro-distilled Z. cassumunar
has been characterized previously in Thailand, revealing that the
 W. Boonyanugomol et al. / Journal of Infection and Public Health 10 (2017) 586–592
Fig. 3. Zones of inhibition against A. baumannii obtained with Z. cassumunar essential oil (A), gentamicin (aminoglycosides group) (B), and gentamicin combined with Z.
cassumunar essential oil (C).
main components are sabinene (36–53%), terpinene-4-ol (21–29%),
␥-terpinene (6–7%), and DMPBD (1–16%), all of which are terpenes
[12]. A previous in vitro study also indicated that terpenes had no
effective antimicrobial activity when used as single compounds
[28]. Thus, we suggest that the antimicrobial activity of the essen-
tial oil from Z. cassumunar against A. baumannii strains may be
attributable to the co-activation of the various components of the
essential oil.
In addition to the direct antimicrobial activity of the essential
oil from Z. cassumunar, we investigated the effects of combination
therapy of the essential oil with standard antibiotics. Combination
therapy of antibiotics with natural agents may be better for treat-
ing various problems due to the resistance mechanisms of bacteria
and the side effects of some antibiotics in patients. The effects of
combined treatment of the Z. cassumunar essential oil with dif-
ferent antibiotics have not been studied previously. However, the
oil from another plant in the same genus, Z. officinale, exhibits a
synergistic effect against Helicobacter pylori when combined with
clarithromycin, and has the potential to control H. pylori-associated
gastroduodenal diseases [29]. In this study, we screened for syn-
ergistic effects against A. baumannii by combining the essential
oil from Z. cassumunar with eight groups of standard antibiotics
recommended by CLSI [14]. The synergistic effects of the essen-
tial oil combined with antibiotic discs were examined as described
in a previous study [18]. We found that the essential oil had syn-
ergistic effects against susceptible strains when combined with
penicillin, ␤-lactamase inhibitors, cephems, and carbapenems, but
had antagonistic effects against XDR strains. We also showed that
the essential oil from Z. cassumunar had synergistic effects with
aminoglycosides, tetracycline, fluoroquinolones, and folate path-
way inhibitors against both susceptible and XDR A. baumannii
strains. Our results are consistent with those of a previous study
using Curcuma longa, a similar plant in the family Zingiberaceae,
which had synergistic effects against S. aureus when combined with
aminoglycosides and fluoroquinolones [30]. Thus, essential oils
from members of the family Zingiberaceae, including Z. cassumu-
nar, may have synergistic effects when combined with antibiotics.
According to our results, we conclude that the essential oil from
591
Z. cassumunar could be combined with aminoglycosides, tetracy-
cline, fluoroquinolones, and folate pathway inhibitors to control
drug-resistant A. baumannii strains. The optimum concentration
and the mechanisms of the essential oil against A. baumannii when
combined with antibiotics should be elucidated further.
Conclusions
Our study is the first to show that the essential oil from Z.
cassumunar has antibacterial activities against A. baumannii, partic-
ularly XDR strains. When combined with standard antibiotics, the
essential oil from Z. cassumunar had a synergistic effect against A.
baumannii. Thus, we conclude that the essential oil from Z. cassumu-
nar alone, or in conjunction with some antibiotics, could potentially
be a useful antibacterial agent against XDR A. baumannii. These find-
ings could lead to the development of a new treatment method
for infectious diseases caused by drug-resistant pathogens. Further
research will be required to develop pharmacological agents for
treating drug-resistant bacteria, and the molecular mechanisms of
these antibacterial and synergistic activities also require further
study.
Funding
No funding sources.
Competing interests
None declared.
Ethical approval
Not required.
Acknowledgments
This study was supported by Mahidol University (Talent Man-
agement, TM:CP281). The authors thank the Division of Pathology
592 W. Boonyanugomol et al. / Journal of Infection and Public Health 10 (2017) 586–592
and Microbiology, Amnat Chareon Hospital for providing the clin-
ical bacterial strains of A. baumannii. We also thank the Central
Laboratory of Amnat Chareon Campus, Mahidol University for
access to the instruments used in this study. The authors would like
to thank Enago (www.enago.com) for the English language review.
References
[1] Zhang HZ, Zhang JS, Qiao L. The Acinetobacter baumannii group: a systemic
review. World J Emerg Med 2013;4:169–74.
[2] Maragakis LL, Perl TM. Acinetobacter baumannii: epidemiology, antimicrobial
resistance, and treatment options. Clin Infect Dis 2008;46:1254–63.
[3] Peleg AY, Seifert H, Paterson DL. Acinetobacter baumannii: emergence of a
successful pathogen. Clin Microbiol Rev 2008;21:538–82.
[4] Bakkali F, Averbeck S, Averbeck D. Biological effects of essential oils–a review.
Food Chem Toxicol 2008;46:446–75.
[5] Bayala B, Bassole IH, Scifo R. Anticancer activity of essential oils and their
chemical components—a review. Am J Cancer Res 2014;4:591–607.
[6] Silva J, Abebe W, Sousa SM. Analgesic and anti-inflammatory effects of essential
oils of Eucalyptus. J Ethnopharmacol 2003;89:277–83.
[7] Pattnaik S, Subramanyam VR, Kole C. Antibacterial and antifungal activity of
ten essential oils in vitro. Microbios 1996;86:237–46.
[8] Liju VB, Jeena K, Kuttan R. An evaluation of antioxidant, anti-inflammatory,
and antinociceptive activities of essential oil from Curcuma longa. L. Indian J
Pharmacol 2011;43:526–31.
[9] Pandey PS Abhay Kumar, Singh Pooja, Tripathi Nijendra Nath. Chemistry and
bioactivities of essential oils of some Ocimum species: an overview. Asian Pac
J Trop Biomed 2014;4:682–94.
[10] Pithayanukul P, Tubprasert J, Wuthi-Udomlert M. In vitro antimicrobial activ-
ity of Zingiber cassumunar (Plai) oil and a 5% Plai oil gel. Phytother Res
2007;21:164–9.
[11] Koontongkaew SPO, Sireeratawong S, Dechatiwongse Na Ayudhya T, Khonsung
P, Jaijoy K, Soawakontha R, et al. Safety Evaluation of Zingiber cassumunar Roxb.
Rhizome Extract: Acute and Chronic Toxicity Studies in Rats. Int Sch Res Notices
2014;2014:1–14.
[12] Sukatta URP, Punjee P, Chidchenchey S, Keeratinijakal V. Chemical Compo-
sition and Physical Properties of Oil from Plai (Zingiber cassumunar Roxb.)
Obtained by Hydro Distillation and Hexane Extraction. Kasetsart J. (Nat. Sci.)
2009;43:212–7.
[13] Chaiwongsa R, Ongchai S, Boonsing P. Active compound of Zingiber cassumu-
nar Roxb: down-regulates the expression of genes involved in joint erosion
in a human synovial fibroblast cell line. Afr J Tradit Complement Altern Med
2012;10:40–8.
[14] Performance standards for antimicrobial susceptibility testing; twenty-fourth
informational supplement. Pennsylvania: Clinical and Laboratory Standards
Institute; 2014, 230 p.
[15] Ponce AG, Fritz R, del Valle C, Roura SI. Antimicrobial activity of essential oils on
the native microflora of organic Swiss chard. Food Sci Technol 2003;36:679–84.
[16] Standards NCfCL. Methods for dilution antimicrobial susceptibility tests for
bacteria that grow aerobically—fifth edition: approved standard M7-A5.
Wayne, PA, USA: NCCLS; 2000.
[17] Avila JG, de Liverant JG, Martinez A. Mode of action of Buddleja cordata verbas-
coside against Staphylococcus aureus. J Ethnopharmacol 1999;66:75–8.
[18] Toroglu S. In vitro antimicrobial activity and antagonistic effect of essential oils
from plant species. J Environ Biol 2007;28:551–9.
[19] Cb Singh MN, Swapana N, Chanu Sb. Ethnobotany, Phytochemistry and Pharma-
cology of Zingiber cassumunar Roxb. (Zingiberaceae). J Pharmacogn Phytochem
2015;4:1–6.
[20] Tushar, Basak S, Sarma GC. Ethnomedical uses of Zingiberaceous plants of
Northeast India. J Ethnopharmacol 2010;132:286–96.
[21] Taroeno, Brophy JJ, Zwaving JH. Analysis of the essential oil of Zingiber cassumu-
nar Roxb. From Indonesia. Flavour Fragr J 1991;6:161–4.
[22] Youngchiyud PW P, Suthamsmi T, Kanchanapee P, Dechatiwongse T. Clinical
assessment of the efficacy and tolerance of Zingiber cassumunar Roxb. Siriraj
Hosp Gaz 1985;37:435–40.
[23] Bin JI, Yassin MSM, Chin CB, Chen LL, Sim NL. Antifungal activity of the essential
oils of nine Zingiberaceous species. Pharm Biol 2003;41:392–7.
[24] Prakatthagomol W, Sirithunyalug Jakkapan, Okonogi O. Comparison of antibac-
terial activity against food-borne bacteria of Alpinia galanga, Curcuma longa, and
Zingiber cassumunar. CMU J Nat Sci 2012;11:117–86.
[25] Sriphanaa UPS, Kongsaereeb P, Yenjai C. Antimalarial activity and cytotoxicity
of zerumbone derivatives. Sci Asia 2013;39:95–9.
[26] Nakamura S, Iwami J, Matsuda H. Structures of new phenylbutanoids and nitric
oxide production inhibitors from the rhizomes of Zingiber cassumunar. Chem
Pharm Bull (Tokyo) 2009;57:1267–72.
[27] Habsah M, Amran M, Mackeen MM. Screening of Zingiberaceae extracts for
antimicrobial and antioxidant activities. J Ethnopharmacol 2000;72:403–10.
[28] Nazzaro F, Fratianni F, De Martino L. Effect of essential oils on pathogenic
bacteria. Pharmaceuticals (Basel) 2013;6:1451–74.
[29] Nostro A, Cellini L, Di Bartolomeo S. Effects of combining extracts (from propolis
or Zingiber officinale) with clarithromycin on Helicobacter pylori. Phytother Res
2006;20:187–90.
[30] Teow SY, Ali SA. Synergistic antibacterial activity of Curcumin with antibiotics
against Staphylococcus aureus. Pak J Pharm Sci 2015;28:2109–14.