Effect of Jujube Honey on Candida albicans Growth and Bioﬁlm Formation
Mohammad Javed Ansari,
Noori S. Al-Waili,
and Yehya Al-Attal
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
Department of Biochemistry, D.K.M. College for Women, Thiruvalluvar University, Vellore, Tamilnadu, India
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
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;
0188-4409/$ - see front matter. Copyright Ó2013 IMSS. Published by Elsevier Inc.
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
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
yeast, 20 g l
peptone and 20 g l
dextrose. The cultures were incubated for 36 h at 35C with
agitation (120 rev min
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
Following incubation, the broth was used to aseptically
inoculate Petri dishes containing Sabouraud dextrose agar
(Oxoid) with 10
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
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
. 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
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
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
. A constant
force of 0.58 N m
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
at a 4 cm
resolution in absorbance
mode. Each ﬁnal spectrum was the average of 48 scans.
ANOVA test was used to compare between different means
of bioﬁlm biomass (absorbance). Data analysis was carried
out using GraphPad software.
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
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
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
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
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
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
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
(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
. C-C ring
stretching at 1465 cm
and C-H stretching of primary aro-
matic amines at 1235 cm
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
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.
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
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
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
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.
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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