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South African Journal of Botany 78 (2012) 37 43

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Antibiofilm activity and post antifungal effect of lemongrass oil on clinical

Candida dubliniensis isolate
S. Taweechaisupapong a,b,, P. Ngaonee a , P. Patsuk a , W. Pitiphat c , W. Khunkitti d
a

Biofilm Research group, Faculty of Dentistry, Khon Kaen University, Khon Kaen 40002, Thailand
Center for Research and Development of Herbal Health Products, Khon Kaen University, Khon Kaen 40002, Thailand
c
Department of Community Dentistry, Faculty of Dentistry, Khon Kaen University, Khon Kaen 40002, Thailand
d
Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand
Received 24 July 2010; received in revised form 7 April 2011; accepted 14 April 2011

Abstract
Candidal infections are often difficult to eradicate due to the resistance of biofilms to antifungal agents. This study aimed at determining the
effects of lemongrass (Cymbopogon citratus DC) oil against Candida dubliniensis in both planktonic and biofilms form. The results from broth
microdilution method revealed that the minimum inhibitory and minimum fungicidal concentration of lemongrass oil on C. dubliniensis were 0.43
and 0.86 mg/ml, respectively. Employing a formazan salt (XTT tetrazolium) reduction assay for biofilm study, the results showed that the average
percentage (mean SD) inhibition of lemongrass oil (0.43 mg/ml) on biofilm formation was 91.57 1.31%, while it exhibited more than 80%
killing activity against C. dubliniensis in biofilm at concentrations of 1.7 mg/ml. In addition, a significant reduction (P = 0.03) of candidal adhesion
to acrylic occurred after a 15 min exposure to 1.7 mg/ml of lemongrass oil. Moreover, limited exposure of yeasts to lemongrass oil at subcidal
concentration can suppress growth for more than 24 h. Altogether, the results obtained indicate that lemongrass oil possessed antifungal and
antibiofilm activities and could modulate candidal colonization. Therefore, the efficacy of lemongrass oil merits further development of this agent
for the therapy of oral candidiasis.
2011 SAAB. Published by Elsevier B.V. All rights reserved.
Keywords: Antifungal activity; Biofilm; Broth microdilution; Candida dubliniensis; Essential oils; Post antifungal effect

1. Introduction
Biofilms of Candida species play a growing role in human
medicine. Indeed, the majority of manifestations of candidiasis
at both mucosal and systemic sites are associated in one way or
another with the formation of biofilms on inert or biological
surfaces (Ell, 1996; Cannon and Chaffin, 1999; Crump and
Collignon, 2000). The prevalence of diseases caused by
Candida spp. has increased dramatically, mainly due to an
increase in the number of at-risk individuals, principally those
with impaired immunity, such as transplant recipients, cancer
patients receiving chemotherapy, and human immunodeficiency
Corresponding author at: Biofilm Research group, Faculty of Dentistry,
Khon Kaen University, Khon Kaen 40002, Thailand. Fax: +66 4320 2862.
E-mail address: suvi_taw@kku.ac.th (S. Taweechaisupapong).

virus-infected patients (Eggimann et al., 2003; Presterl et al.,

2007; Ramirez-Amador et al., 2003; Singh et al., 2002).
Oropharyngeal candidiasis (OPC) is the most common opportunistic infection in the immunocompromised. Although
Candida albicans is a well-known etiological agent of OPC,
Candida dubliniensis has emerged as another pathogen known
for its azole resistance. C. dubliniensis, a new species of
Candida, was recovered from oral cavities of HIV-infected
patients, that is phenotypically and genotypically close to
C. albicans (Sullivan et al., 1995). Therefore, it may be
misidentified (Martinez et al., 2002). Evidence of the inducibility of a stable fluconazole resistance in vitro in C. dubliniensis
strains may indicate that it is an emerging pathogen for
immunocompromised patients receiving long-term fluconazole
prophylaxis (Moran et al., 1998). In addition, C. dubliniensis can
adhere to epithelial cells, which may indicate that this species is

0254-6299/$ - see front matter 2011 SAAB. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.sajb.2011.04.003

38

S. Taweechaisupapong et al. / South African Journal of Botany 78 (2012) 3743

particularly adapted to colonize the oral cavity (Gilfillan et al.,

1998).
Formation of biofilms by Candida spp. has been demonstrated
on almost any medical device. The most commonly involved
systemic devices include joint prostheses, cardiac valves,
vascular and urinary catheters (Kojic and Darouiche, 2004;
Ramage et al., 2006). In addition, there are many topical devices
at risk, including contact lens and dentures. Different Candida
spp. differ in their capacities to form biofilms (Hawser and
Douglas, 1994; Kuhn et al., 2002). Formation of Candida
biofilms has important clinical repercussions because of their
increased resistance to antifungal therapy and the ability of cells
within biofilms to withstand host immune defenses (Douglas,
2003; Ramage et al., 2006).
Increasing awareness of hazards associated with the use of
antibiotic and chemical agents has accelerated investigations
into plants and their extracts as new sources of antimicrobial
agents. Essential oils are odorous, volatile products of plant
secondary metabolism, found on many leaves and stems. They
have a wide application in folk medicine, food flavoring and
preservation as well as in fragrance industries. The antimicrobial properties of essential oils have been known for many
centuries. In recent years, more than 500 reports stated that
essential oils and their constituents possessed antimicrobial
properties against some bacteria and fungi (Kalemba and
Kunicka, 2003). However, little is known about the spectrum of
action of essential oils against Candida biofilm. Moreover, the
post antifungal effect (PAFE) of lemongrass (Cymbopogon
citratus DC) oil on C. dubliniensis has never been reported. The
aim of current research was to investigate the antifungal activity
of the lemongrass oil against both sessile and planktonic clinical
C. dubliniensis isolate. The PAFE and the effect of lemongrass
oil on the adhesion of C. dubliniensis to denture acrylic were
also evaluated. With the results as reported in this paper, the
lemongrass oil exhibited effective killing activity and possessed
the strong inhibitory effect on Candida biofilm formation. A
significant reduction of candidal adhesion to denture acrylic
occurred after a 15 min exposure to lemongrass oil. In addition,
limited exposure of C. dubliniensis to lemongrass oil at subcidal
concentration can suppress growth for more than 24 h.
Therefore, it is a promising essential oil to combat candidal
colonization and infection.

cine, Mahidol University, Bangkok, Thailand) was maintained

on Sabouraud-dextrose agar (BBL Microbiology Systems,
Cockeysville, MD) and grown in the yeast phase in Sabourauddextrose broth (Pronadisa, Hispanlab, S.A.) for 18 h.
Preparation of the yeast suspension was different in each
assay according to the optimal concentration of the inoculum.
For antifungal and antibiofilm assay, C. dubliniensis was
adjusted to give an optical density (OD) at 600 nm = 0.1 and
plate count to determine the CFU/ml. The suspension of OD at
600 nm of 0.1 possessed 1 106 CFU/ml and was used as
inoculum in antifungal and antibiofilm assay.
To determine the effect of the lemongrass oil and nystatin on
candidal adhesion to denture acrylic, the yeast suspension of
1 108 cells/ml was used according to our previous study
(Taweechaisupapong et al., 2006) which demonstrated that the
optimal concentration of the inoculum for adhesion was
1 108 cells/ml. For this study, the organisms were washed
three times in sterile phosphate buffered saline (PBS) by
centrifugation at 3000 g for 10 min and finally suspended in
PBS to 1 108 cells/ml determined by hemocytometer.
For post antifungal effect assay, a suspension of OD at
600 nm of 1.5 was used according to previous study (Ellepola
and Samaranayake, 1998).

2. Materials and methods

2.4. Determination of antifungal activities of the lemongrass

oil and nystatin

2.3. Identification of lemongrass oils components

The chromatographic analyses were carried out with a Trace
GC ultra gas chromatograph (Model K05200B20000070, Italy)
coupled with a DSQ mass spectrometer (Model Trace DSQ-Mass
spectrophotometry, USA). A Tr-5 capillary column
(30 m 0.25 mm i.d.) coated with 0.25 m film 5%phenyl95%
dimethylpolysiloxane was used for separations. The GC was
operated under temperature programmed condition started with
70 C for 1 min, then programmed at 6 C/min to 280 C for
3 min. One l of 1% solution in hexane was manually injected in a
purged split mode (1:100) using high-purity helium as a carrier
gas at 1 ml/min flow rate. The scan range was 35550 m/z and the
scan rate was 1000 amu/s. The identification of the components
was based on comparison of their mass spectra with those of a
computer library (NIST MS Search library). Further confirmation
was done by referring to retention index (RI) data generated from a
series of alkanes (C10C23).

2.1. Essential oil

Lemongrass oil (Thai China Flavours & Fragrances Industry
Co. Thailand) was dissolved in 95% ethanol to an initial
concentration of 720 mg/ml and further diluted with the
solution contained 5% ethanol and 5% Tween 80 to a
concentration of 55 mg/ml before used.
2.2. Preparation of yeast suspension
C. dubliniensis kindly provided by Dr. P. Chongtrakool
(Microbiology Unit, Ramathibodi Hospital, Faculty of Medi-

Antifungal activities of the lemongrass oil and nystatin

towards C. dubliniensis were determined by the broth dilution
method (NCCLS, 2002). Fifty microliters of the lemongrass oil
(55 mg/ml) and 50 l of nystatin (64 g/ml) were two-fold
serially diluted with Sabouraud's dextrose broth in a microtiter
plate. Fifty microliters of the Candida suspension was added
and mixed with the lemongrass oil and nystatin. C. dubliniensis
cultured in the broth without the tested agents served as a
positive control and the mixture of broth and the tested agents
without microorganism served as a negative control. The plates
were incubated for 24 h, at 37 C. Then C. dubliniensis growth

S. Taweechaisupapong et al. / South African Journal of Botany 78 (2012) 3743

39

was examined and the lowest concentration of the tested agents

which inhibited the visible growth of C. dubliniensis was
recorded as the minimum growth inhibitory concentration
(MIC).
Ten microliters of the mixture of the tested agents and
C. dubliniensis which showed negative-visible growth after the
first 24 h of incubation was dropped onto the surface of
Sabouraud's dextrose agar. The lowest concentration of the
tested agents giving negative growth of the Candida was
recorded as the minimum fungicidal concentration (MFC). All
experiments were repeated on three separate occasions, with
triplicate determinations on each occasion.

(27.6 to 0.22 mg/ml) and incubated for a further 48 h at 37 C. A

series of the essential oil-free wells was also included to serve as
positive control. The effect of the oil against preformed Candida
biofilm was determined by using the XTT-reduction assay
described below.
The effects of nystatin against preformed Candida biofilm
were also determined by the same method as above. Percentage
killing of each agent was calculated using the formula [1 (OD492
sample / OD492 control)] 100%. All experiments were repeated
on three separate occasions, with triplicate determinations on each
occasion.

2.5. Inhibitory effects of the lemongrass oil on C. dubliniensis

biofilm formation

2.7. XTT-reduction assay

To determine the effects of the oil in inhibiting of biofilm

formation, C. dubliniensis biofilm formation in wells of
microtiter plates was performed as described previously
(Taweechaisupapong et al., 2010). One hundred microliters
of the oil (55 mg/ml) was two-fold serially diluted with
Sabouraud's dextrose broth in a microtiter plate. An equal
volume of C. dubliniensis suspension was added and mixed
with the oil. C. dubliniensis suspension in the broth without
the oil served as a positive control and the broth without
microorganism served as a negative control. The plates were
incubated for 48 h at 37 C. After biofilm formation, the
medium was aspirated, and nonadherent cells were removed
by thoroughly washing the biofilms three times in sterile
PBS. The effect of the oil in inhibiting of biofilm formation
was determined by using the 2,3-bis(2-methoxy-4-nitro-5sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazolium hydroxide (XTT)-reduction assay described below. The effect of
nystatin on Candida biofilm formation was also determined by
the same method as above. Percentage of inhibition of the tested
agents was calculated using the formula [1 (OD492 sample /
OD492 control)] 100%. All experiments were repeated on three
separate occasions, with triplicate determinations on each
occasion.
2.6. Antifungal activities of the lemongrass oil against
preformed Candida biofilm
Antifungal susceptibility testing of sessile cells was performed
as described previously (Taweechaisupapong et al., 2010).
C. dubliniensis was grown in Sabouraud-dextrose broth for 18 h.
Biofilms were formed on commercially available presterilized,
polystyrene, flat-bottom 96-well microtiter plates (Corning Inc.,
Corning, N.Y.) by pipetting standardized cell suspensions (100 l
of the ~1 106 CFU/ml) into selected wells of the microtiter plate
and incubating them for 48 h at 37 C. The wells contained the
broth without microorganism served as a negative control. After
biofilm formation, the medium was aspirated, and nonadherent
cells were removed by thoroughly washing the biofilms three
times in sterile PBS. Residual PBS was removed by blotting with
paper towels before the addition of the lemongrass oil. The oil was
then added to the biofilms in serially double-diluted concentrations

The XTT solution was prepared by dissolved 0.5 mg/ml of a

water soluble tetrazolium salt, XTT (Sigma, St. Louis, USA)
and 40 g/ml of coenzyme Q0 (Sigma) in PBS and diluted 1:6
with PBS before used. A 100-l aliquot of the XTT solution
was added to each prewashed biofilm and to control wells (for
the measurement of background XTT-reduction levels). The
plates were incubated in the dark for 2 h at 37 C. A
colorimetric change in the XTT-reduction assay, a direct
correlation of the metabolic activity of the biofilm, was
measured in a microplate reader (Bio-tek instruments, Inc.,
Vermont, USA) at 492 nm.

2.8. Effect of the lemongrass oil on candidal adhesion to

denture acrylic
Heat-cured denture acrylic sheets were fabricated according to conventional prosthodontic technique. Subsequently,
the acrylic sheet formed was cut into 5 5 mm square strips,
the average thickness of the sheet being 2 mm. These strips
were immersed for up to 4 weeks in water to leach the
excess monomer then washed in running water for 3 h.
Finally, they were sealed and sterilized in petri dishes before
used.
For experiment examining the effect of the lemongrass oil
and nystatin on candidal adhesion to denture acrylic, the acrylic
strips were placed vertically in Eppendorf tubes and 0.4 ml of
the yeast suspension (1 108 cells/ml) was added to each tube,
mixed gently and incubated at 37 C for 4 h with gentle
agitation. After incubation, the acrylic strips were removed
from the tubes and washed three times with 20 ml sterile normal
saline solution (NSS) to remove non-adherent cells. Then the
acrylic strips were placed in new Eppendorf tubes, each with
0.4 ml of the tested agents at concentration that exhibited more
than 80% killing activity against C. dubliniensis in biofilm. The
acrylic strips which were placed in tubes with 0.4 ml of sterile
PBS served as a control. The tubes were incubated at 37 C for
15, 30, 60, 120 min and 24 h. After incubation, the acrylic
strips were removed from the tubes and washed with 20 ml
sterile NSS. The effect of the tested agents on candidal
adhesion was determined by using the XTT-reduction assay
described above.

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S. Taweechaisupapong et al. / South African Journal of Botany 78 (2012) 3743

Table 1
Chemical compositions of the lemongrass oil.
Chemical composition

Retention time (min)

Area %

RI

Identification method

6-methyl-5-hepten-2-one
Beta-myrcene
Beta-ocimene
Linalool
Ethenyl-cyclohexanone
Beta-citral (Neral)
Geraniol
Alpha-citral (Geranial)
Geranyl acetate

6.77
6.94
8.73
11.47
15.13
17.79
18.32
19.16
24.31

1.405
9.470
0.450
0.725
1.466
32.518
4.362
43.377
2.154

NI
NI
1034
1000
1180
1238
1250
1268
1378

MS
MS
RI, MS
RI, MS
RI, MS
RI, MS
RI, MS
RI, MS
RI, MS

RI: retention index data generated from a series of n-alkanes (C10C23).

MS: mass spectrum.
NI: not identified.

2.9. Post antifungal effect assay

3. Results

The PAFE assay was performed as described previously

(Ellepola and Samaranayake, 1998) with minor modifications. A
cell suspension was prepared in sterile PBS at 600 nm to an OD of
1.5. From this cell suspension, 0.5 ml was added to tubes
containing 2 ml of RPMI broth (control) and 2 ml of RPMI/tested
agents solution (test) in which the tested agents' concentrations
varied from 1 to 2 times the MIC. The tubes were then incubated
at 37 C for a period of 1 h in a rotary incubator. Following this
limited exposure, the tested agents were removed by two cycles of
centrifugation for 10 min at 3000 g. Afterwards the supernatant
was completely decanted and the pellets were resuspended in
2.5 ml of sterile PBS. Then aliquots of 100 l from each cell
suspension were added to a microtiter well containing 150 l of
RPMI broth. Then the microtiter plate was placed in a
computerized spectrophotometric incubator (Varioskan Flash,
Thermo Fisher Scientific, USA) and incubated at 37 C for 24 h.
Growth of yeast cells was automatically monitored by the
computerized instrument in terms of the change in the turbidity
(absorbance at 595 nm), at 30-min intervals, for a period of 24 h.
The duration of PAFE was calculated by using the formula
PAFE = T C (Craig and Gudmundsson, 1996; Lowdin et al.,
1993; Scalarone et al., 1991) where T was the time required for
the relative OD of the tested agents-exposed cell suspension to
reach the 0.05 absorbance level after removal of the tested
agents and C was the time required for the relative OD of the
tested agents free control cell suspension to reach the same
absorbance level. Thus T C expressed the time in which the
antifungal agent was capable of causing growth suppression of
the organism following limited exposure to the tested agents
(i.e. post antifungal effect).

The components of lemongrass oil were identified by GCMS

and the results were shown in Table 1. The major constituents
found in lemongrass oil were - and -citral (75.89%), myrcene
(9.47%) and geraniol (4.36%). The MIC of lemongrass oil and
nystatin on C. dubliniensis by the broth microdilution method
were 0.43 mg/ml and 2 g/ml, respectively, while the MFC were
0.86 mg/ml and 4 g/ml, respectively. Employing a formazan salt

2.10. Statistical analysis

The effect of the tested agents on candidal adherence was
analyzed using Kruskal Wallis with MannWhitney U test to
evaluate the differences in adhesion between the test and control
groups. Bonferroni method was used to adjust for multiple
comparisons. P-values b 0.05 were considered as statistically
significant.

Fig. 1. Effects of different concentrations of lemongrass oil (A) and nystatin

(B) on biofilm formation () and against preformed biofilm () of C.
dubliniensis. The presented percentage of inhibition was calculated from the
formula [1 (OD492 sample / OD492 control)] 100%. Results are from three
experiments performed in triplicate (n = 9).

S. Taweechaisupapong et al. / South African Journal of Botany 78 (2012) 3743

reduction assay for biofilm study, the results revealed that the
inhibitory effect of lemongrass oil and nystatin on biofilm
appeared to be dose-related (Fig. 1). The lemongrass oil and
nystatin at concentrations between 0.1127.6 mg/ml and
4512 g/ml exhibited 4099% and 2093% inhibition on
biofilm formation, respectively, while the same concentrations of
both agents showed less active against preformed biofilm of
C. dubliniensis. The lemongrass oil and nystatin exhibited more
than 80% inhibitory effect on biofilm formation at concentration
of 0.43 mg/ml and 8 g/ml, respectively, while the MIC of
lemongrass oil and nystatin against preformed Candida biofilm at
80% (SMIC80) were 1.7 mg/ml and 16 g/ml, respectively.
Therefore the SMIC80 of both agents were selected to test their
effects on the adhesion of C. dubliniensis to the acrylic strips at
various time intervals. Compared with the control, a significant
reduction of yeast adhesion to the acrylic strips was observed after
exposure to lemongrass oil for 15 min (P = 0.03), while a
significant reduction of yeast adhesion to the acrylic strips was
evident after exposure to nystatin for 30 min (Fig. 2).
The in vitro PAFE of the lemongrass oil and nystatin on
C. dubliniensis after exposure for 1 h at 1, 1.5 and 2 times MIC in
RPMI broth is shown in Fig. 3 and Table 2. There was continuous
growth suppression following removal of lemongrass oil, which
failed to show a growth rate comparable to that of the unexposed
control during a 24 h period. In contrast, the PAFE of nystatin
which ranged from 1 to 7.5 h was shorter compared to lemongrass
oil.
4. Discussion
In this study, the conventional 96-well microtiter plates
coupled to a colorimetric method were used to assess the effects
of lemongrass oil against biofilm cells. Measurements of
cellular mitochondrial dehydrogenase activity using tetrazolium
salts have been employed with Candida cells for studies of
biofilm formation on catheter materials and plastic in several
studies (Hawser, 1996; Hawser and Douglas, 1994). This
method is rapid, inexpensive, easy to use, accurate, and
reproducible methodology for antifungal susceptibility testing
of Candida biofilms (Ramage et al., 2001).

41

Fig. 3. Growth curves of C. dubliniensis after limited exposure to lemongrass oil

and nystatin for 1 h. Results are from three experiments performed in triplicate
(n = 9).

The present study demonstrated that lemongrass oil

exhibited the strong inhibitory effects against C. dubliniensis
in both planktonic and biofilms form. However, preformed
Candida biofilms were found to be more resistant to lemongrass
oil than their planktonic cells. The observation in this study is
consistent with previous reports that biofilm-associated Candida
cells are resistant to antifungal agents relative to their planktonic
counterparts (Bachmann et al., 2002; Chandra et al., 2001;
Shuford et al., 2007). Although adherent populations were not
completely eradicated by treatment with lemongrass oil, a N80%
reduction in the metabolic activity of adherent cells was detected
at concentrations of 1.7 mg/ml. Moreover, it is interesting to
observe that lemongrass oil at the MIC values (0.43 mg/ml) can
inhibit biofilm formation of C. dubliniensis, while the concentration of nystatin that can inhibit biofilm formation was 4 MIC
(8 g/ml). These results indicated that exposure of Candida cells
to subcidal concentration of lemongrass oil can reduce the
adherence ability of the cells compared with the unexposed
controls, whereas the subcidal concentration of nystatin did not
exhibit inhibitory effect on biofilm formation. Since adherence
represents a major step in biofilm formation, therefore,
lemongrass oil might be used to prevent Candida biofilmassociated infection.
Antifungal activities of lemongrass oil found in this study are
consistent with several reports of lemongrass oil against various
fungi (Abe et al., 2003; Cassella and Cassella, 2002; Hammer
et al., 1999; Helal et al., 2006; Paranagama et al., 2003;
Wannissorn et al., 1996). These activities can be attributed to
the presence of various constituents such as citral, limonene,
citronellal, -myrcene, linalool and geraniol (Rauber Cda et al.,

Table 2
Postantifungal effect of lemongrass oil and nystatin on C. dubliniensis.

Fig. 2. Adhesion of C. dubliniensis to denture acrylic following differential

periods of exposure to lemongrass oil and nystatin. Results are from three
experiments performed in triplicate (n = 9).

Concentration
of tested
agent

Postantifungal effect (h)

Lemongrass oil

Nystatin

1 MIC
1.5 MIC
2 MIC

N24
N24
N24

1
2
7.5

MIC: minimum growth inhibitory concentration.

42

S. Taweechaisupapong et al. / South African Journal of Botany 78 (2012) 3743

2005; Schaneberg and Khan, 2002; Tognolini et al., 2006). It

was observed that citral demonstrated inhibitory effects on both
mycelial and yeast-form growth of C. albicans (Abe et al.,
2003). Geraniol was found to inhibit growth of C. albicans and
possessed antibiofilm activity (Bard et al., 1988; Dalleau et al.,
2008). Moreover, several studies have demonstrated that
terpenes (i.e. citral, geraniol, linalool, menthol, and thymol)
which are the major components of essential oils, alter cell
permeability by penetrating between the fatty acyl chains
making up the membrane lipid bilayers, disrupting lipid packing
and changing membrane fluidity (Bard et al., 1988; Braga and
Dal Sasso, 2005). Braga and Dal Sasso (2005) suggested that
these phenomena led to major surface alterations and
morphological modifications, also reducing the adherence
capacity of C. albicans. Since C. dubliniensis is phenotypically
and genotypically close to C. albicans (Sullivan et al., 1995),
the inhibitory effects on biofilm formation after exposure to
lemongrass oil found in this study could be due to those effects
of terpenes in the oil.
For PAFE assay in this study, a two washing step was used to
reduce antimicrobial concentration of the tested agents because it
has been found by previous investigators that removal of 90% of
the supernatant with two washings reduces antimicrobial
concentration 100-fold, while complete decanting of the supernatant with two washings (as carried out in the current study)
reduces the concentration 10,000-fold (Craig and Gudmundsson,
1996). Hence this method virtually eliminates any carry-over
effect of the tested agents. Previous workers have evaluated the
PAFE of antifungal agents either by using viable counts (Turnidge
et al., 1994), or turbidometric measurement of fungal growth
(Scalarone et al., 1991). In this study, a turbidometric measurement was used for this purpose albeit the evaluation was greatly
facilitated due to an automated technique. The Varioskan Flash
machine (Varioskan Flash, Thermo Fisher Scientific, USA)
enabled easy, automatic and reliable quantification of yeast
growth in terms of increased turbidity as reported for bacteria and
fungi in previous studies (Lowdin et al., 1993; Scalarone et al.,
1991). The sensitivity of the machine was good as replicate
experiments yielded only minimal growth differentials in the
tested organisms, and did not yield high standard errors of means
(s.e.m.) usually witnessed in conventional techniques relying on
CFU determinations (MacKenzie and Gould, 1993).
In the mouth the diluent effect of saliva and the cleansing
action of the oral musculature often tend to reduce the
availability of the drug below that of the effective therapeutic
concentration. Thus the organisms undergo only a limited
exposure to the antifungal agents during treatment and the
concentration of the drug tends to vary in different niches of the
oral cavity. The current observations suggest that limited
exposure to lemongrass oil elicits a significantly high PAFE in
C. dubliniensis even at the MIC value. Therefore, the efficacy of
lemongrass oil merits further development of this agent for the
therapy of oral candidiasis.
In conclusion, the results in this study demonstrated potent in
vitro activity in inhibiting biofilm formation and against
preformed biofilms of C. dubliniensis by lemongrass oil.
These effects could modulate candidal colonization thereby

suppressing the invasive potential of the pathogen. Therefore,

the efficacy of lemongrass oil merits further investigation of this
agent for the therapy of Candida biofilm-associated infection.
Acknowledgments
This work was supported by a grant from the Faculty of
Dentistry, Khon Kaen University, Thailand.
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