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ORIGINAL ARTICLE
Effect of Jujube Honey on Candida albicans Growth and Biofilm Formation
Mohammad Javed Ansari,
a
Ahmad Al-Ghamdi,
a
Salma Usmani,
b
Noori S. Al-Waili,
c
Deepak Sharma,
a
Adgaba Nuru,
a
and Yehya Al-Attal
a
a
Chair of Engineer Abdullah Ahmad Bugshan for Bee Research, Department of Plant Protection, College of Food and Agriculture Sciences,
King Saud University, Riyadh, Saudi Arabia
b
Department of Biochemistry, D.K.M. College for Women, Thiruvalluvar University, Vellore, Tamilnadu, India
c
Al-Waili Foundation for Science, Queens, New York
Received for publication December 15, 2012; accepted June 19, 2013 (ARCMED-D-12-00176).
Background and Aims. Candida species, especially Candida albicans, are major fungal
pathogens of humans that are capable of causing superficial mucosal infections and sys-
temic infections in humans. The aim of this study was to evaluate the jujube (Zizyphus
spina-christi) honey for its in vitro inhibitory activity against pre-formed biofilm and
its interference with the biofilm formation of C. albicans.
Methods. The XTT reduction assay, scanning electron microscopy (SEM) and atomic
force microscopy (AFM) were employed to determine the inhibitory effect of Jujube
honey on C. albicans biofilm. Changes in the infrared spectrum after treatment with
honey were also determined by Fourier transform infrared (FTIR) spectroscopy.
Results. Jujube honey affects biofilms by decreasing the size of mature biofilms and by
disruption of their structure. At a concentration of 40% w/v, it interferes with formation
of C. albicans biofilms and disrupts established biofilms. The SEM and AFM results
indicated that this type of honey affected the cellular morphology of C. albicans and
decreased biofilm thickness.
Conclusions. The present findings show that jujube honey has antifungal properties
against C. albicans and has the ability to inhibit the formation of C. albicans biofilms
and disrupt established biofilms. Ó2013 IMSS. Published by Elsevier Inc.
Key Words: Jujube honey, Candida albicans, Biofilm, Scanning electron microscopy, Atomic force
microscopy.
Introduction
Some Candida species are found as endosymbionts in most
healthy individuals. C. albicans is the most common yeast
found on the mucosal membranes of humans including in
the oral cavity, esophagus, gastrointestinal tract, urinary
bladder and genitalia (1). In immunocompromised individ-
uals, C. albicans has emerged as a true opportunistic path-
ogen. This yeast adheres to and colonizes epithelial tissues
and causes superficial and life-threatening infections.
C. albicans has become one of the main causes of mor-
bidity and mortality worldwide among immunocompro-
mised individuals (2). Importantly, Candida has been
shown to be the third most commonly isolated blood path-
ogen from patients in U.S. hospitals (3).
According to the National Institutes of Health (USA),
more than 60% of all microbial infections are associated with
biofilms (4). Biofilms are particularly problematic in the
clinical environment and, like bacteria, various fungal spe-
cies can form biofilms in vivo and in vitro (5). Among fungi,
C. albicans is the most common pathogen associated with
fungal biofilm infections, especially infections related to im-
planted medical devices (6). A common issue associated
with C. albicans biofilms is the increased resistance of these
biofilms to antifungal agents such as azole drugs and their
derivatives and to host immune defenses. The increased
resistance is due to the extracellular matrix secreted by the
Candida cells, which shields the Candida cells from anti-
bodies and prevents drugs from penetrating the biofilm
(7,8). The emergence of resistant C. albicans has a major
Address reprint requests to: Noori S. Al-Waili, MD, PhD, FACP, Waili
Foundation for Science, 134 St, Queens, NY 11418; Phone: 347-666-1144;
E-mail: drnoori6@yahoo.com
0188-4409/$ - see front matter. Copyright Ó2013 IMSS. Published by Elsevier Inc.
http://dx.doi.org/10.1016/j.arcmed.2013.06.003
Archives of Medical Research 44 (2013) 352e360
impact on public health and the economy. Because of the
increasing prevalence of drug-resistant C. albicans, there is
an urgent need to develop alternative treatments for Candida
infections that are safe, effective and inexpensive.
Among all of the strategies that have been exploited to
overcome drug resistance, the use of natural substances
has shown particular promise, and many natural substances
have been found to have antifungal properties (9). Bee
products such as honey and propolis are rich sources of
essential bioactive compounds. Because of its medicinal
qualities, honey has been used for the management of many
diseases throughout the ages and has become a traditional
remedy for treating microbial infections and wounds
(10e14). The Talmud, the Old and New Testaments of
the Bible, and the Holy Qur’an (1400 years ago) mentioned
honey as a cure for diseases. A large chapter (SORA) pre-
sents in the Holey Qur’an named BEE (Al Nahl) and part of
it says (And thy Holy LORD taught the bee to build its cells
in hills, on trees and in men’s habitations, then to eat of all
the produce of the earth and find with skill the spacious
paths of its LORD, there issues from within their bodies
a drink of varying colors, wherein is healing for men, verily
in this is a sign for those who give thought).
The antimicrobial properties of honey depend on its
type, flower source, botanical and geographical origins
and the harvesting, processing and storage conditions used
(12,15,16). Honey is widely used in the Arabian peninsula
for nutritional and therapeutic purposes; however, no
research has been conducted on the antimicrobial activity
of regional honey collected in the Arabian peninsula. The
antimicrobial effects of honey on Staphylococcus aureus,
Pseudomonas aeruginosa and other bacterial biofilms have
been studied (17e20). Honey also reduces the production
of an extracellular polysaccharide matrix while promoting
the disruption of mature biofilms (21,22). The effect of hon-
ey on C. albicans biofilms has not been extensively studied
(23e29). To our knowledge, no research has been conduct-
ed on the effect of honey on C. albicans biofilms. A better
understanding of C. albicans responses to honey may facil-
itate its use as a biofilm inhibitor. The aim of this study was
to use broth dilution assay followed by the determination of
the minimum inhibitory concentration (MIC) of jujube
honey and use of new techniques like scanning electron mi-
croscopy (SEM), atomic force microscopy (AFM) and
Fourier transform infrared (FTIR) spectroscopy to investi-
gate the in vitro effects of jujube honey on planktonic states
of C. albicans and detachment of biofilm-embedded states.
Materials and Methods
Honey
Natural jujube honey was used throughout this study. This
honey was obtained from the beekeepers’ association of
Al-Baha, Saudi Arabia in a 1-kg sterile container. The
honey was obtained directly from the honeycomb by press-
ing and was filtered to remove the wax and other impurities.
This natural honey was passed through 45-mm-pore-size
filters and stored at 4C until use.
Microorganisms and Culture Conditions
The test organism used in this study, C. albicans ATCC
10231, was provided by the College of Medicine, King
Saud University Riyadh, Saudi Arabia. The strain was
cultured in yeast peptone dextrose broth (YEPD) medium
containing 10 g l
1
yeast, 20 g l
1
peptone and 20 g l
1
dextrose. The cultures were incubated for 36 h at 35C with
agitation (120 rev min
1
).
Minimum Inhibitory Concentration (MIC)
MICs of the natural jujube honey against planktonically
grown C. albicans ATCC 10231 were determined using a
macrobroth dilution assay (30). The honeys were serially
diluted (80e5% w/v) in YPD broth. The cultures were
incubated for 36 h at 35C with agitation (120 rev min
1
).
Following incubation, the broth was used to aseptically
inoculate Petri dishes containing Sabouraud dextrose agar
(Oxoid) with 10
3
CFU of Candida. The growth of the col-
onies was assessed after 48 h, and the MIC was the lowest
concentration of honey (w/v) that inhibited the visible
growth of C. albicans ATCC 10231.
Establishment of Biofilms
The growth of the biofilms was evaluated using the 2,3-bis
(2-methoxy-4-nitro-5-sulfophenyl)-2H-5-tetrazolium-car-
boxanilide (XTT) reduction assay in 96-well flat-bottomed
polystyrene microtiter plates (Jet Biofil, China) using a
method based on that described by Lal et al. (31). To deter-
mine whether the jujube honey could prevent the formation
of Candida biofilms and to determine the lowest concentra-
tion of honey capable of preventing biofilm formation,
different MIC dilutions of honey in YEPD broth (80%
w/v, 40% w/v, 20% w/v, 10% w/v and 5% w/v) were used
to study the kinetics of biofilm inhibition. Each MIC dilu-
tion was tested in at least seven wells in each microtiter
plate. Aliquots of 190 ml of each dilution were dispensed
into the wells of the microtiter plate. C. albicans was
cultured for 48 h in 10 ml of YEPD broth containing 5 x
10
8
CFU ml
1
. Ten microliters of this 48-h culture was
added to each well and incubated for 1.5 h at 37Cinan
orbital shaker at 75 rpm to create a homogeneous distribu-
tion and adherence to surface of the wells. After 1.5 h, non-
adherent cells were removed by gently washing two times
with sterilized phosphate buffered saline (PBS) (pH 7.4)
without disturbing the adherent cells. After the plates were
washed, another aliquot of the same honey dilution in ster-
ile YEPD broth with a final volume of 200 ml was added to
each well, and the plates were incubated for 48 h under the
353Effect of Jujube Honey on Candida albicans
same conditions to allow the colonization and maturation of
the biofilms. As a control, 200 ml of autoclaved YEPD broth
with Candida (positive control) or without Candida (nega-
tive control) was added to each of seven wells of the micro-
titer plate, which was then incubated at 37C for 48 h.
To determine whether jujube honey could disrupt estab-
lished biofilms of C. albicans, biofilms were cultured in 96-
well microtiter plates by adding 10 mlof5x10
8
CFU ml
1
C. albicans in YPD to the microtiter plate. The plate incu-
bated for 1.5 h at 37C in an orbital shaker at 75 rpm to
create a homogeneous distribution and adherence to surface
of the wells. After 1.5 h, nonadherent cells were removed
by gently washing two times with sterilized phosphate buff-
ered saline (PBS) (pH 7.4). One hundred fifty ml of steril-
ized YEPD broth was added to the each well and the
plate was then reincubated for 24e48 h at 37C to allow
proper adhesion and the establishment of biofilms in the
absence of jujube honey. Different concentrations of honey
in YEPD broth (80% w/v, 40% w/v, 20% w/v, 10% w/v and
05% w/v) were added to each well in final volumes of
200 ml. The plate was then incubated at 37C for 48 h.
All experiments were performed in triplicate, and quantifi-
cation was performed using the XTT reduction assay.
Evaluation of Biofilms Using the XTT Reduction Assay
A sodium 30-[1-[(phenylamino)-carbonyl]-3,4-tetrazolium]-
bis(4-methoxy-6-nitro)benzene-sulfonic acid hydrate
(XTT) assay was used to quantify the cells in the biofilms
after treatment with the jujube honey (32). The XTT (Sigma,
St. Louis, MO) solution (1 mg ml
1
in PBS) was prepared,
filtered and sterilized using a 0.22-mm-pore size filter. Prior
to each assay, the XTT solution was thawed and mixed with
menadione solution at a ratio of 5:1 (v/v). The biofilms on
the microtiter plate wells were washed three times with
PBS, and all remaining adherent biofilms were fixed with
2.5% glutaraldehyde (Fluka, UK) for 5 min to prevent
further growth. After the fixative was removed, the wells
were washed twice with PBS. Then, 1 mL of PBS containing
60 ml of the XTT-menadione solution was added to each
well, including the control well without a biofilm. The MTPs
were then incubated for 2 h at 37C in the dark. Following
incubation, 75 ml of XTT-menadione solution from each
well was transferred to a new microtiter plate, and its absor-
bance was determined spectrophotometrically at 490 nm
(Perkin Elmer, Waltham, MA).
Scanning Electron Microscopy
For SEM, a microtiter plate with established Candida bio-
films was carefully cut into small pieces using a sterile
knife and washed with 4% (v/v) formaldehyde and 1%
(v/v) PBS at room temperature. These samples were then
treated with 1% osmium tetroxide for 1 h and washed in
distilled water. The samples were dehydrated in a series
of ethanol (30% for 10 min, 50% for 10 min, 70% for
10 min, 95% for 10 min, and absolute alcohol for
20 min). All specimens were air dried to the critical point
using a Polaron critical point drier and then sputter coated
with gold. After sputter coating, the surfaces of the biofilms
were visualized by SEM (Leo 435, Cambridge, UK).
Atomic Force Microscopy
Images of biofilms on MTPs were taken with Nanoscope III
Multi Mode AFM (NTEGRA; NT-MDT, Moscow, Russia).
Biofilms were established in MTPs. After washing the bio-
films with PBS, different concentrations of honey in YEPD
broth (80% w/v, 40% w/v, 20% w/v, 10% w/v and 5% w/v)
were added to each well in final volumes of 200 ml. One
well without any honey was used as a control. After 48 h
of incubation, the liquid medium was withdrawn and the
wells were washed twice with PBS. The biofilms were fixed
with 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH
7.0, at 4C for 4 h. After washing with distilled water,
the biofilms were dried in air. All images were collected
in tapping mode using sharpened silicon NSG10S nitride
cantilevers with a spring constant of |10 N m
1
. A constant
force of 0.58 N m
1
was used. The cantilevers had an
amplitude range of 5e15 nm, a tip radius of 10 nm and a
cone angle of 22. Height and deflection images were
simultaneously acquired at a scan rate of 250 kHz.
Fourier Transform Infrared Spectroscopy
Treated and untreated Candida biofilms were analyzed us-
ing an IR spectrometer (Thermo Electron Corp., Waltham,
MA) using the KBr pellet technique. Biofilm materials
were powdered and added to KBr to form a pellet that con-
tained 1% test material. Purified dextran was used as a stan-
dard, and the spectrum was taken in the frequency range of
500e1800 cm
1
at a 4 cm
1
resolution in absorbance
mode. Each final spectrum was the average of 48 scans.
Statistical Analysis
ANOVA test was used to compare between different means
of biofilm biomass (absorbance). Data analysis was carried
out using GraphPad software.
Results
Determination of the Minimum Inhibitory Concentration
Jujube honey inhibited C. albicans ATCC 10231 growth
in a concentration-dependent manner. The MIC of jujube
honey against biofilm-forming C. albicans ATCC 10231
was 40% (w/v), and the minimal fungicidal concentration
(MFC) was 50% (w/v). The MFC is defined as the lowest
concentration of honey resulting in the death of 99.9% of
the inoculum. In general, the MFC value is greater than
the MIC value. The growth curves of yeast exposed to
354 Ansari et al./ Archives of Medical Research 44 (2013) 352e360
40% (w/v) jujube honey showed a reduced growth rate
and a reduction in the total number of cells (Figure 1)over
a 24-h period relative to cell growth without honey. The
growth assays conducted with 50% (w/v) jujube honey
revealed no C. albicans growth.
Prevention of Biofilm Formation
In this experiment, to determine whether jujube honey
could prevent the formation of Candida biofilms and to
determine the lowest concentration of honey capable of
preventing biofilm formation, different concentrations of
honey in YEPD broth (80% w/v, 40% w/v, 20% w/v,
10% w/v and 5% w/v) were used to study the kinetics of
biofilm inhibition. The inhibition of biofilm formation
was dependent on the concentration of the honey. It was
evident that concentrations of honey below 10% w/v did
not inhibit the biofilm and even encouraged biofilm devel-
opment (Figure 2). However, concentrations more than
10% w/v inhibited significantly the biofilm formation.
Effect of Honey on Established Biofilms
Similarly, when 24 h established biofilms were treated with
different concentrations of jujube honey (80e5% w/v), the
C. albicans biomass was significantly reduced after 24 h of
contact with honey concentrations greater than 10% w/v,
but biofilm growth was enhanced at 5% w/v. A higher con-
centration of jujube honey was required to disrupt estab-
lished biofilms than to prevent biofilm formation (Figure 3).
Effect of Contact Time on Established Biofilms Exposed to
an Inhibitory Concentration of Honey
To monitor the effectiveness of jujube honey over time, bio-
films that had been established for 24 h were incubated with
and without 40% w/v of jujube honey for varying time
intervals, after which the biofilm biomass was determined.
The biomass of the Candida biofilms was determined after
exposure to 40% w/v of honey for 1, 2, 3, 4, 5, 6, 12 and
24 h. The results show that after 24 h of exposure to jujube
honey, the biofilm biomass detected was significantly
reduced compared with the biomass of the untreated estab-
lished biofilm (Figure 1).
Scanning Electron Microscopy Analysis of C. albicans
Biofilms
To evaluate the prevention and inhibition of C. albicans
biofilm growth, SEM was performed. SEM images of a
control C. albicans biofilms and of a biofilm treated with
40% w/v of jujube honey are shown in Figure 4. Untreated
sessile cells of biofilm showed a smooth cell wall
(Figure 4A, inset) and covered by exopolysaccharide
Figure 1. Growth analyses of established C. albicans biofilms treated with
40% (w/v) jujube honey. F 5612, p!0.0001. (A color figure can be
found in the online version of this article.)
Figure 2. The effect of jujube honey on the formation of C. albicans
biofilms. F 5301, p!0.0001. (A color figure can be found in the online
version of this article.)
Figure 3. The effect of jujube honey on established C. albicans biofilms.
F568.8, p!0.0001. (A color figure can be found in the online version of
this article.)
355Effect of Jujube Honey on Candida albicans
materials. Visualization of the ultrastructure revealed that
reductions in the number of adherent cells and in biofilm
development take place when the biofilm is treated with
40% w/v of honey. When a 24-h established biofilm was
treated with 40% w/v of honey, growth of the established
biofilm was inhibited, and some small pores developed in
the cell walls. These pores may be due to bursting of cell
membrane of C. albicans cells by shrinkage and osmotic ef-
fect of honey, which led to cell death and to a reduction in
the numbers of established cell (Figure 4B). No exopoly-
saccharide material is observed and shrinkage of cell
membrane due to plasmolysis has been observed
(Figure 4B). Biofilm formed in the presence of 40% (w/
v) jujube honey, no exopolysaccharide material and cell ag-
gregation are observed. Shrinkage of the cell membrane in-
dicates cell lysis (Figure 4C and 4D).
Atomic Force Microscopy (AFM) Analysis of C. albicans
Biofilms
The inhibition of C. albicans biofilms was also analyzed
using AFM. AFM images of untreated C. albicans biofilms
Figure 4. Scanning electron microscopy micrographs of the 48 h C. albicans biofilms on microtiter plates. (A) Biofilm formed in the absence of honey,
showing a dense network of cells and hyphae. White arrow indicated exopolysaccharides material (A, inset). White arrow indicates the smooth cell wall
of a normal cell. (B) Inhibition of established biofilm treated with 40% w/v of jujube honey (after 24 h) is illustrated. There is no exopolysaccharide material
observed and white arrow indicates the formation of small pores within the cell walls (B, inset). i, white arrow indicates the rough cell wall; ii, vesicle
formation due to lytic material; iii, shrinkage in cell membrane due to plasmolysis of cell. (C) Prevention of biofilm formation on microtiter plates after
48 h is illustrated. (C, inset). White arrow shows rough cell wall and shrinkage in cell membrane due to plasmolysis of cell.
356 Ansari et al./ Archives of Medical Research 44 (2013) 352e360
on microtiter plates revealed that the Candida cells were
embedded within a sticky layer of exopolysaccharides
distributed around the cell surface, whereas this layer was
absent in treated Candida biofilms. The 3D images of
C. albicans biofilms revealed that this layer surrounded
the cells residing in the biofilm (Figure 5). The 3D images
provide significantly better image resolution than SEM,
providing both the height and roughness of the biofilm on
the microtiter plate. The roughness analysis of Candida
biofilms treated with 40% w/v of honey compared with un-
treated biofilms was also conducted. The root mean square
(rms) values of the untreated and treated biofilms were
216.29 nm and 431.28 nm, respectively. A significant vari-
ation in the height of the biofilms was observed. The
heights of the untreated and treated biofilms were 200 nm
and 90 nm, respectively (Figure 5A and 5B). A significant
reduction in the height observed in the biofilm formed in
the presence of 40% w/v of jujube honey (Figure 5C).
The thickness of the honey-treated biofilm was reduced to
approximately half of that of the control. The three-
dimensional structure of the Candida biofilms also ex-
hibited significant differences in the Z axis value, with
values of 200 nm/div, 90 nm/div and 14 nm/div for the un-
treated and treated established biofilms and biofilm formed
in the presence of 40% w/v of jujube honey, respectively
(Figure 5).
Fourier Transform Infrared Spectroscopy
To visualize the main spectral differences between untreated
and treated C. albicans biofilm, averages of spectra from all
three experiments were calculated and offset-corrected
(Figure 6). Distinctive absorption maxima in the mid-
infrared region of 800e1200 cm
1
were found to be useful
to study the differences in the absorbance between untreated
and treated C. albicans biofilms. Results from the
Figure 5. Atomic force microscopy micrographs showing the variation in the roughness and height of C. albicans biofilms on microtiter plates: (A) untreated
biofilm after 48 h (height 200 nm). (B) 40% w/v jujube honey-treated established biofilm (48 h) (height 90 nm). (C) Formation of biofilm after treatment with
40% w/v of jujube honey (48 h) (height 14 nm). (A color figure can be found in the online version of this article.)
357Effect of Jujube Honey on Candida albicans
comparison of the FTIR spectra of untreated and treated C.
albicans biofilm showed that there were some differences
in the wave number, shape, and the number of absorption
peaks within the same range of wave number. The FTIR
spectral profile of control (without honey) obtained in
800e1200 cm
1
region mainly reflected the absorption of
sugars present in the exopolysaccharide matrix secreted by
C. albicans cells. Absorbance peaks for sugars in the mid-
infrared region were present at 836, 935, 1017, 1088, 1155
and 1171 cm
1
(Figure 6A). These peaks indicate the pres-
ence of b-glucans and mannans moieties with other sugars
like arabinose, mannose etc. The FTIR spectra also exhibited
specific absorbance bands corresponding to the C 5O
stretching of carboxylate groups at 1636 cm
1
. C-C ring
stretching at 1465 cm
1
and C-H stretching of primary aro-
matic amines at 1235 cm
1
were also observed (Figure 6A).
Comparison of the untreated biofilm spectrum with the
treated biofilm spectrum showed remarkable differences.
Exopolysaccharide sugar specific peaks were not clearly
discernible in treated biofilm to that of untreated biofilm,
apart from the peaks at 1515 and 1465 cm
1
(Figure 6).
The major differences of spectra in this region might result
from the differences in exopolysaccharide sugar composi-
tion. This reflected no production of extracellular polysac-
charides in C. albicans biofilm in the presence of honey.
Discussion
Honey is widely used in a variety of household recipes.
Honey is an excellent natural food product rich in minerals,
antioxidants and simple sugars. Honey can prevent deterio-
rative oxidation reactions in foods such as the browning of
fruits and vegetables and lipid oxidation in meat. Honey in-
hibits growth of foodborne pathogens and microorganisms
that cause food spoilage (33,34).
Several studies conducted on the antimicrobial proper-
ties of honey have confirmed that honey is effective at treat-
ing some oral infections such as ulcers, mucositis, and
periodontal diseases (12,35e37). Several reports demon-
strating the effectiveness of honey in the treatment of
various bacterial biofilms have been published
(17e19,38,39). However, little information is available on
the effect of honey on C. albicans biofilms. The primary
aim of this study was to determine whether honey can
prevent the establishment of C. albicans biofilms and/or
disrupt established C. albicans biofilms.
Among several known human pathogens, Candida sp.
are known to be a part of the endosymbiotic community
in humans. However, in immunocompromised patients,
C. albicans can cause severe nosocomial infections (40).
In most of these infections, C. albicans forms a biofilm
and becomes resistant to azole drugs, which are commonly
used as antifungal agents to treat Candida infections (41).
Currently, some Candida strains show resistance to these
drugs, which have a limited ability to penetrate the matrix
of C. albicans biofilms. The increased resistance of
Candida against azole drugs and the few drugs available
for Candida treatment has led to search for new therapeutic
alternatives (42). One of these alternatives is honey, which
has a wide range of antifungal properties.
We selected jujube honey for this study because it is
commonly used as a folk medicine to treat several infec-
tions and diseases in the Arabian peninsula. Some honeys
from different plant sources and geographical origins were
found to be effective against planktonic C. albicans cul-
tures; the most effective was jujube honey, with a 40 %
MIC (w/v) and a 50% MFC (w/v). Jujube honey was thus
selected for further testing against C. albicans biofilms.
The MIC of jujube honey effectively prevented the forma-
tion of C. albicans biofilms and inhibited established
C. albicans biofilms. We further tested different MIC dilu-
tions of jujube honey in YEPD broth (80% w/v, 40% w/v,
20% w/v, 10% w/v and 05% w/v). It was found that 20%
w/v and 40% w/v of jujube honey significantly prevented
biofilm formation, and 80% w/v completely prevented bio-
film formation. In contrast, 5% w/v of honey slightly
increased biofilm formation. This result indicates that the
active antimicrobial ingredients in jujube honey were
diluted to a degree that rendered them ineffective. A
similar effect has been reported previously (20,43).When
evaluating the time- and concentration-dependent effects
of honey at different concentrations on 24-h established
biofilms, we found that 5% w/v of honey had no inhibitory
effect on biofilms and concentrations of 10% w/v and
higher significantly reduced the established biofilm after
12 h of treatment at room temperature. These results are
supported by the study of Cooper et al. (19) in which man-
uka honey at concentrations below 10% (w/v) promoted
the growth of established biofilms of Staphylococcus
aureus.
Figure 6. FTIR spectra of C. albicans biofilms. (a) Untreated biofilm
spectra after 48 h. (b) MIC-treated established biofilm spectra after 48 h
(c) Spectra of biofilm formed with 40% w/v of jujube honey after 48 h.
358 Ansari et al./ Archives of Medical Research 44 (2013) 352e360
The mechanism of the antifungal effect of honey is not
fully understood; however, several potential pathways have
been proposed. One proposed mechanism is that H
2
O
2
,a
potent antimicrobial agent, is produced in honey by glucose
oxidase enzyme (12,44). Flavonoids, a group of plant pig-
ments that are found in honey, are also considered a poten-
tial source of the antimicrobial properties of honey (45).
Methylglyoxal, a compound present in manuka honey,
may be responsible for the antimicrobial activity of this
honey (46). The high sugar content has also been thought
to be involved (20), challenging this theory. To propose a
mechanism that explains how honey might affect C. albi-
cans biofilms at the cellular level, we performed SEM,
AFM and FTIR analyses of treated and untreated C. albi-
cans biofilms. The results indicate that jujube honey has
not only prevented C. albicans biofilm formation and dis-
rupted established biofilms but also caused changes to the
cell wall and exopolysaccharides.
In our study, SEM observations demonstrated the inter-
ference of jujube honey with cell membrane integrity,
which was obvious with shrinkage of the cell surface in bio-
film cells. A similar mode of action was also observed
against planktonic cells of C. albicans. Other authors have
also shown that some phytocompounds affect cell mem-
brane integrity of yeast cells (47).
These results also indicate that jujube honey interferes
with the metabolism of the C. albicans biofilm. Honey
may interfere in any step of biofilm formation and thereby
inhibit C. albicans biofilm formation.
In the past decade, AFM has been used to study micro-
bial biofilms without the need for time-consuming sample
preparation steps (48). AFM-based methodology can poten-
tially reveal the effects of subtle changes in cell surface
composition and of interactions with biomaterials. AFM
also provides surface information regarding the exopoly-
saccharides that cover the Candida cells in biofilms.
AFM studies have indicated that the C. albicans biofilm
thickness decreases by more than half after treatment with
honey. At the same time, the roughness of the C. albicans
biofilm also increases significantly. This increase in rough-
ness may be due to the removal of the exopolysaccharides
layer that covers the C. albicans biofilm. This layer main-
tains the smooth texture of the biofilm and inhibits the
penetration of antifungal drugs into the biofilm. These re-
sults are also supported by the data of Lal et al. (31), which
show that C. albicans biofilms secrete a thick layer of
exopolysaccharides in which cells remain embedded and
protected from their outer surroundings.
FTIR spectroscopy allows analysis of molecular compo-
sition through the interaction between the infrared radiation
and the sample (49). FTIR spectroscopy has been proven
very simple to use and very sensitive to small changes in
the composition of cells (50). Here FTIR spectroscopy
analysis was performed for comparative biochemical
composition of exopolysaccharides matrix of treated and
nontreated C. albicans biofilm. The FTIR spectra in the
region of 800e1200 cm
1
primarily reflected the different
sugars present in the C. albicans biofilms. The spectral
differences between the untreated and treated C. albicans
biofilms in this region indicated that honey affected the
formation and secretion of exopolysaccharide matrix by
altering the sugars (major constituents of C. albicans bio-
film exopolysaccharides) composition and deposition.
Thus, there is direct evidence that honey affects the exopo-
lysaccharide composition of C. albicans biofilms.
Mature C. albicans biofilms are very difficult to eradicate
and are recalcitrant to antifungals. The extracellular glucan
present in extracellular matrix is required for C. albicans
biofilm resistance and it acts by sequestering antifungals,
rendering cells resistant to their action (51). Many antimicro-
bials have been isolated from naturally occurring substances
over the years. Our findings indicate that jujube honey
inhibits the initial phase of biofilm formation and has fungi-
static, fungicidal and antibiofilm potential. This potential is
superior to that of most of the commonly used antifungals.
Because biofilms are multifactorial phenomena, multiple
mechanisms that target different steps in biofilm develop-
ment are probably involved in the effects of honey on bio-
films. This intriguing observation may have important
clinical implications that could lead to a new approach for
the management of C. albicans biofilm-related infections.
In conclusion, the findings indicate that jujube honey can
inhibit C. albicans biofilms. The significant antifungal activ-
ity of jujube honey suggests that this could serve as a source
of compounds which have a therapeutic potential for the
treatment of Candida-related infections. Further evaluation
in vivo is required to determine whether these findings can
be exploited in treating biofilm-associated candidiasis.
Acknowledgments
The authors are thankful to National Plan for Science and Tech-
nology (NPST) program by King Saud University Riyadh, Project
No. 11-AGR1748-02 for financial support.
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