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Effect of Jujube Honey on Candida albicans Growth and Biofilm Formation

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Candida species, especially Candida albicans, are major fungal pathogens of humans that are capable of causing superficial mucosal infections and systemic 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. 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. 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. 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.
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Effect of Jujube Honey on Candida albicans Growth and Biofilm Formation
Mohammad Javed Ansari,
Ahmad Al-Ghamdi,
Salma Usmani,
Noori S. Al-Waili,
Deepak Sharma,
Adgaba Nuru,
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
CFU ml
. 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
CFU ml
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
500e1800 cm
at a 4 cm
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.
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
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
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
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
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
(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.
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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... Indeed, more than 80% of human bacterial infections with increased mortality are due to biofilm development (Dongari-Bagtzoglou, 2008). Several publications have reported that different types of honey prevent biofilm formation by Gram-negative, Gram-positive bacteria, and Candida species (Merckoll et al., 2009;Ansari et al., 2013;Sojka et al., 2016;Halstead et al., 2017;Liu et al., 2018;Lu et al., 2019;Fernandes et al., 2021). Unifloral honeys have antibacterial and antibiofilm effects against food-borne pathogens; P. aeruginosa and S. aureus, as well as respiratory tract bacteria, Haemophilus spp., P. aeruginosa, and S. pneumonia (Farkas et al., 2022;Balázs et al., 2021). ...
... Moreover, another studies showed that Kelulut, Manuka and Sidr honeys can inhibit the biofilms of S. aureus, P. aeruginosa, and E. coli even at low concentrations (Alandejani et al., 2009;Al-kafaween et al., 2021). Further, the anticandidal activity of Portuguese honey (heather honey) and Jujube honey against C. tropicalis and C. albicans biofilm has been mentioned in several studies (Ansari et al., 2013;Fernandes et al., 2021). It is believed that the high sugar concentration, osmotic action, the presence of hydrogen peroxide, and defensin-1 peptide in honey generates hypertonic conditions that lead to the suppression of biofilm formation (Lee et al., 2011;Nassar et al., 2012;Proaño et al., 2021). ...
Ethnopharmacological relevance: Man has used honey to treat diseases since ancient times, perhaps even before the history of medicine itself. Several civilizations have utilized natural honey as a functional and therapeutic food to ward off infections. Recently, researchers worldwide have been focusing on the antibacterial effects of natural honey against antibiotic-resistant bacteria. Aim of the study: This review aims to summarize research on the use of honey properties and constituents with their anti-bacterial, anti-biofilm, and anti-quorum sensing mechanisms of action. Further, honey's bacterial products, including probiotic organisms and antibacterial agents which are produced to curb the growth of other competitor microorganisms is addressed. Materials and methods: In this review, we have provided a comprehensive overview of the antibacterial, anti-biofilm, and anti-quorum sensing activities of honey and their mechanisms of action. Furthermore, the review addressed the effects of antibacterial agents of honey from bacterial origin. Relevant information on the antibacterial activity of honey was obtained from scientific online databases such as Web of Science, Google Scholar, ScienceDirect, and PubMed. Results: Honey's antibacterial, anti-biofilm, and anti-quorum sensing activities are mostly attributed to four key components: hydrogen peroxide, methylglyoxal, bee defensin-1, and phenolic compounds. The performance of bacteria can be altered by honey components, which impact their cell cycle and cell morphology. To the best of our knowledge, this is the first review that specifically summarizes every phenolic compound identified in honey along with their potential antibacterial mechanisms of action. Furthermore, certain strains of beneficial lactic acid bacteria such as Bifidobacterium, Fructobacillus, and Lactobacillaceae, as well as Bacillus species can survive and even grow in honey, making it a potential delivery system for these agents. Conclusion: Honey could be regarded as one of the best complementary and alternative medicines. The data presented in this review will enhance our knowledge of some of honey's therapeutic properties as well as its antibacterial activities.
... However, in the present study, both treated K. pneumoniae strains showed shortened rod and filamentous forms. Moreover, regarding the antifungal effect on C. albicans, the identified changes were regarding the size regularity and morphology of the membrane and similar findings were also observed with Melipona becchei honey (Hau-Yama et al., 2020) and Jujube honey (Ansari et al., 2013). Although several studies have confirmed that honey's antifungal effect is strongly linked to the floral and entomological origin Boorn et al., 2010;Fernandes et al., 2021;Irish et al., 2006;Morroni et al., 2018;Ramón-Sierra et al., 2019;Suntiparapop et al., 2015;Zamora et al., 2015), little is known about the antifungal properties of stingless bee honeys. ...
... Although several studies have confirmed that honey's antifungal effect is strongly linked to the floral and entomological origin Boorn et al., 2010;Fernandes et al., 2021;Irish et al., 2006;Morroni et al., 2018;Ramón-Sierra et al., 2019;Suntiparapop et al., 2015;Zamora et al., 2015), little is known about the antifungal properties of stingless bee honeys. Several studies have found that some phytochemical compounds, especially terpenes and flavonoids, present in natural products including honey, can inhibit morphological transitions in Candida species (Al-Ghanayem, 2022; Ansari et al., 2013;Calixto Júnior et al., 2015;da Silva et al., 2021;Prasath et al., 2020;Priya and Pandian, 2022;Soliman et al., 2017). In our case, the evidence obtained was not sufficient to reinforce this idea. ...
Full-text available
Biofilms are associated with infections that are resistant to conventional therapies, contributing to the antimicrobial resistance crisis. The need for alternative approaches against biofilms is well-known. Although natural products like stingless bee honeys (tribe: Meliponini) constitute an alternative treatment, much is still unknown. Our main goal was to evaluate the antibiofilm activity of stingless bee honey samples against multidrug-resistant (MDR) pathogens through biomass assays, fluorescence (cell count and viability), and scanning electron (structural composition) microscopy. We analyzed thirty-five honey samples at 15% (v/v) produced by ten different stingless bee species (Cephalotrigona sp., Melipona sp., M. cramptoni, M. fuscopilosa, M. grandis, M. indecisa, M. mimetica, M. nigrifacies, Scaptotrigona problanca, and Tetragonisca angustula) from five provinces of Ecuador (Tungurahua, Pastaza, El Oro, Los Ríos, and Loja) against 24h biofilms of Staphylococcus aureus, Klebsiella pneumoniae, Candida albicans, and Candida tropicalis. The present honey set belonged to our previous study, where the samples were collected in 2018–2019 and their physicochemical parameters, chemical composition, mineral elements, and minimal inhibitory concentration (MIC) were screened. However, the polyphenolic profile and their antibiofilm activity on susceptible and multidrug-resistant pathogens were still unknown. According to polyphenolic profile of the honey samples, significant differences were observed according to their geographical origin in terms of the qualitative profiles. The five best honey samples (OR24.1, LR34, LO40, LO48, and LO53) belonging to S. problanca, Melipona sp., and M. indecisa were selected for further analysis due to their high biomass reduction values, identification of the stingless bee specimens, and previously reported physicochemical parameters. This subset of honey samples showed a range of 63–80% biofilm inhibition through biomass assays. Fluorescence microscopy (FM) analysis evidenced statistical log reduction in the cell count of honey-treated samples in all pathogens (P <0.05), except for S. aureus ATCC 25923. Concerning cell viability, C. tropicalis, K. pneumoniae ATCC 33495, and K. pneumoniae KPC significantly decreased (P <0.01) by 21.67, 25.69, and 45.62%, respectively. Finally, scanning electron microscopy (SEM) analysis demonstrated structural biofilm disruption through cell morphological parameters (such as area, size, and form). In relation to their polyphenolic profile, medioresinol was only found in the honey of Loja, while scopoletin, kaempferol, and quercetin were only identified in honey of Los Rios, and dihydrocaffeic and dihydroxyphenylacetic acids were only detected in honey of El Oro. All the five honey samples showed dihydrocoumaroylhexose, luteolin, and kaempferol rutinoside. To the authors’ best knowledge, this is the first study to analyze stingless bees honey-treated biofilms of susceptible and/or MDR strains of S. aureus, K. pneumoniae, and Candida species.
... The FTIR spectral peaks in the range between 1700 cm −1 and 1400 cm −1 representing Amide I and Amide II in C. albicans biofilm has been reported by [18]. On the other hand, the FTIR spectral peaks in the range between 1100 cm −1 and 800 cm −1 representing carbohydrates in C. albicans biofilm have been reported by [19]. In addition, several lines of work have shown that the biochemical composition of microbial biofilm represents an important aspect for the study of the potential antibiofilm mode of action [12,20]. ...
... In the present study, econazole nitrate also altered the FTIR spectral peaks in the carbohydrate region (FIGURE 4). This corroborates Ansari et al. [19] studying the effects of usnic acid on C. albicans biofilm. They demonstrated that usnic acid was able to significantly inhibit C. albicans biofilm and reduce various sugars present in the exopolysaccharide layer which was confirmed by the FTIR spectral peaks in the range between 1128 cm −1 and 800 cm −1 . ...
Conference Paper
Full-text available
Candida albicans is a fungus that exists as a commensal constituent of the human microbiome and an opportunistic pathogen. Biofilm formation by this fungal pathogen frequently occurs in the mucosa or endothelium associated with candidiasis. Econazole nitrate is an antifungal cream commonly used to treat C. albicans infection, however, little is known about its mode of action against C. albicans biofilm. Therefore, the present study was performed to investigate the mode of action of econazole nitrate against C. albicans biofilm by determining the change in biochemical composition of the biofilm. Biofilm biomass and viability in the presence of econazole nitrate were assessed using crystal violet and resazurin assay, respectively. Biochemical composition of the biofilm was determined by Fourier transform infrared (FTIR) spectroscopy. The results demonstrated that econazole nitrate substantially inhibited the biomass (52% - 68%) and viability (10% - 16%) of C. albicans biofilm. Typical FTIR spectrum of C. albicans biofilm showed a total of 12 peaks in the range between 1800 cm-1 - 600 cm-1. Treatment with econazole nitrate was found to alter the FTIR spectral peaks at 625 cm-1 (O=C–N bending), 720 cm-1 (C=C bending), 762 cm-1 (C-H bending), 817 cm-1 (α-mannans), 920 cm-1 (C=C bending), 1017 cm-1 (β-glucans), 1220 cm-1 (PO2-asymmetric stretching), 1375 cm-1 (N-H bending vibration), 1465 cm-1 (C-C ring stretching), 1534 cm-1 (CONH bending), 1625 cm-1 (C=O stretching), and 1735 cm-1 (C=O stretching). Our findings suggest that econazole nitrate may inhibit C. albicans biofilm by modifying its biochemical composition.
... To validate the inhibitory efect of L. javanica (ethyl acetate) on EPS production, in situ visualization was performed using the AFM. Te AFM allows for the quantifcation of EPS, revealing their roughness and height at the nanometre scale [71,76]. In this study, the AFM micrographs of EPS treated with L. javanica (ethyl acetate) at MIC value exhibited distinguishable changes in surface roughness and height observed when compared to the untreated EPS. ...
Full-text available
The emergence of multidrug-resistant (MDR) Klebsiella pneumoniae remains a global health threat due to its alarming rates of becoming resistant to antibiotics. Therefore, identifying plant-based treatment options to target this pathogen’s virulence factors is a priority. This study examined the antivirulence activities of twelve plant extracts obtained from three South African medicinal plants (Lippia javanica, Carpobrotus dimidiatus, and Helichrysum populifolium) against carbapenem-resistant (CBR) and extended-spectrum beta-lactamase (ESBL) positive K. pneumoniae strains. The plant extracts (ethyl acetate, dichloromethane, methanol, and water) were validated for their inhibitory activities against bacterial growth and virulence factors such as biofilm formation, exopolysaccharide (EPS) production, curli expression, and hypermucoviscosity. The potent extract on K. pneumoniae biofilm was observed with a scanning electron microscope (SEM), while exopolysaccharide topography and surface parameters were observed using atomic force microscopy (AFM). Chemical profiling of the potent extract in vitro was analysed using liquid chromatography-mass spectrometry (LC-MS). Results revealed a noteworthy minimum inhibitory concentration (MIC) value for the C. dimidiatus dichloromethane extract at 0.78 mg/mL on CBR- K. pneumoniae. L. javanica (ethyl acetate) showed the highest cell attachment inhibition (67.25%) for CBR- K. pneumoniae. SEM correlated the in-vitro findings, evidenced by a significant alteration of the biofilm architecture. The highest EPS reduction of 34.18% was also noted for L. javanica (ethyl acetate) and correlated by noticeable changes observed using AFM. L. javanica (ethyl acetate) further reduced hypermucoviscosity to the least length mucoid string (1 mm-2 mm) at 1.00 mg/mL on both strains. C. dimidiatus (aqueous) showed biofilm inhibition of 45.91% for the ESBL-positive K. pneumoniae and inhibited curli expression at 0.50 mg/mL in both K. pneumoniae strains as observed for H. populifolium (aqueous) extract. Chemical profiling of L. javanica (ethyl acetate), C. dimidiatus (aqueous), and H. populifolium (aqueous) identified diterpene (10.29%), hydroxy-dimethoxyflavone (10.24%), and 4,5-dicaffeoylquinic acid (13.41%), respectively, as dominant compounds. Overall, the ethyl acetate extract of L. javanica revealed potent antivirulence properties against the studied MDR K. pneumoniae strains. Hence, it is a promising medicinal plant that can be investigated further to develop alternative therapy for managing K. pneumoniae-associated infections.
... The presence of Pseudomonas sp. and its biofilm was detected by SEM following the previously described method with some modifications [19]. Briefly, the previously described biofilm-formation protocol was used to form biofilm on a 96-microtiter plate. ...
Full-text available
Citation: Islam, S.; Mahmud, M.L.; Almalki, W.H.; Biswas, S.; Islam, M.A.; Mortuza, M.G.; Hossain, M.A.; Ekram, M.A.-E.; Uddin, M.S.; Zaman, S.; et al. Cell-Free Supernatants (CFSs) from the Culture of Bacillus subtilis Inhibit Pseudomonas sp. Biofilm Formation. Microorganisms
... Fungal infections are becoming more widespread in both community and hospital settings, with several causative agents, including yeasts such as Candida spp., which is an opportunistic polymorphic fungus that is thought to be the most important yeast pathogen (Arisawa et al. 2007). Honey has been shown to have antifungal properties against the yeast C. albicans as well as most species of Aspergillus baumannii, Penicillium chrysogenum, Cryptococcus neoformans, and Candida krusei (Ansari et al. 2013). Honey's antifungal properties are hypothesized to be aided by the same reasons as mentioned in the discussion of its antibacterial properties. ...
Honey's broad-spectrum antimicrobial properties, including its antibacterial, antifungal, and antiviral properties, have been showcased in numerous in vitro and limited clinical studies. In this chapter, a brief overview of the research carried out on the antimicrobial properties of honey is presented. Honey has been shown to have antibacterial action (in vitro) in clinical case studies in a number of studies. To fully understand the mechanisms of action of honey against COVID-19, more preclinical and clinical research are needed. In the future, more detailed molecular investigation studies of honey's impact on viral multiplication toward the immune system as well as a thorough and comprehensive examination of the pharmacokinetics of honey-derived phenolic chemicals are essential to unravel its biological functions in a much more detailed manner. Therefore, identifying the bioactive compounds in honey and their clinical assessment and pharmacological standardization is deemed essential and significant in paving the way for any future standardization initiatives.
... cloacae) [134]. Among the monofloral types, manuka honey is one of the most studied, and researchers have demonstrated its ability to inhibit the biofilm formation of Clostridium difficile [135], S. aureus [136], and C. albicans [137]. In a recent study by Lu et al., manuka honey inhibited the formation of P. aeruginosa biofilms, halted the growth of the planktonic cells, and eradicated established (pre-formed) biofilms at a concentration of 8-32% [95]. ...
Full-text available
Microbial pathogens and their virulence factors like biofilms are one of the major factors which influence the disease process and its outcomes. Biofilms are a complex microbial network that is produced by bacteria on any devices and/or biotic surfaces to escape harsh environmental conditions and antimicrobial effects. Due to the natural protective nature of biofilms and the associated multidrug resistance issues, researchers evaluated several natural anti-biofilm agents, including bacteriophages and their derivatives, honey, plant extracts, and surfactants for better destruction of biofilm and planktonic cells. This review discusses some of these natural agents that are being put into practice to prevent biofilm formation. In addition, we highlight bacterial biofilm formation and the mechanism of resistance to antibiotics.
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Introduction Recurrent vulvovaginal candidiasis (RVVC) affects up to 9% of women worldwide. This amount is expected to increase due to lifestyle changes, increased fungal resistance and biofilm formation. Treatment options are limited and in 57% of the cases, relapses occur within 12 months after starting fluconazole therapy (golden standard). The pathogenesis of RVVC is multifactorial and includes fungal biology, the vaginal microenvironment and the immune system. Fluconazole is antimicrobial and effective in inducing short-term remission but a long-term cure is hard to achieve. Medical grade honey (MGH) has antimicrobial, protective, antioxidative and immunomodulatory activity and may therefore be a good alternative treatment. This study aims to investigate the clinical cure rate and long-term efficacy of MGH compared with fluconazole in patients with RVVC. Methods and analysis This study is a multicentre, randomised controlled trial (Maastricht University Medical Centre+ and Zuyderland Medical Centre). A total of 252 eligible women will be randomly assigned to the fluconazole group (control) or the MGH group (L-Mesitran, treatment). The primary objective is to investigate the mycological cure rate after 1 month assessed through a vaginal culture. Secondary objectives are the clinical cure rate regarding symptoms, the prophylactic activity after 6 months of maintenance therapy and the number of relapses within 12 months. Moreover, information about side effects, discomfort and quality of life will be collected with the use of questionnaires. Ethics and dissemination Ethical approval from the Medical Ethics Review Committee of the academic hospital Maastricht/University Maastricht has been obtained (NL 73974.068.21, V.7 on 8 February 2022). Additional approval was obtained from the Ethics Committee of the Zuyderland Medical Centre Heerlen (Z2021141 on 4 March 2022). The first patient was randomised on 22 August 2022. Results will be made available to researchers and healthcare professionals via conferences, meetings and peer-reviewed international publications. Trial registration number NCT05367089 .
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In a healthy physiological state, the mucous membrane of the oral cavity creates a suitable environment for the colonization of Candida spp. yeasts. The aim of the study was to analyze the nanomechanical properties of C. albicans cells derived from the oral cavity of healthy people in a biofilm produced in laboratory conditions. Candida spp. were sampled from the oral cavity of healthy individuals. The process of biofilm formation was analyzed using classic microscopic observation enriched with SEM (scanning electron microscope) and the nanomechanical properties of the cells were assessed with the use of the atomic force microscopy technique (AFM). From all isolated strains in the samples collected of the oral cavity healthy people was detected 79% C. albicans. Other isolated species belonged to the group "non-albicans". The observations of C. albicans carried out in 24-h cultures revealed a tendency of the cells to form a biofilm structure.
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The available literature regarding the epidemiological or clinical features of vulvovaginal candidiasis (VVC) in most women will experience episodes of vulvovaginal candidiasis in their lifetime, 50% of whom will experience at least a second episode, and 5-10% of all women will experience recurrent vulvovaginal candidiasis (≥ 4 episode / 1 year). Vulvovaginal candidiasis is known as one of the most common yeast infections in women of reproductive age and is considered an important public health problem. In recent years, due to resistance to common antifungal drugs, the use of traditional antifungal medicines and herbal remedies has increased. Therefore this system aimed to investigate the effect of honey vaginal gel/ointment and to compare it with clotrimazole vaginal cream on symptoms of vulvovaginal candidiasis in patients. Vaginal clotrimazole is the drug of choice for the treatment of VVC. However, the increasing drug resistance to these microorganisms has led to greater interest in naturally occurring antifungal drugs. This systematic literature was conducted to compare vaginal honey ointment/gel and clotrimazole vaginal cream for the treatment of VVC.
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Honey is a broad spectrum antimicrobial agent that has been reintroduced into clinical practice to treat wounds. Wounds support polymicrobial communities of bacteria that either colonise or infect wounds. Strains with resistance to antibiotics are difficult to eradicate and pose a risk of transfer to other patients. Manuka honey has been shown to inhibit many of the bacteria commonly associated with wounds, such as staphylococci, pseudomonads, coliforms and anaerobes, but its efficacy against streptococci isolated from wounds has not been reported. Using macro-and micro-dilution in broth and an agar incorporation technique, the susceptibility to manuka honey of 15 cultures of catalase negative, Gram positive cocci that had been isolated from wounds was tested. All cultures were inhibited by 10% (v/v) manuka honey and statistically significant differences between the three test methods were not found. Manuka honey offers clinical potential in eradicating streptococci from wounds.
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To evaluate the additive action of ginger starch on the antifungal activity of honey against Candida albicans (C. albicans). C. albicans was used to determine the minimum inhibitory concentration (MIC) of four varieties of Algerian honey. Lower concentrations of honey than the MIC were incubated with a set of concentrations of starch and then added to media to determine the minimum additive inhibitory concentration (MAIC). The MIC for the four varieties of honey without starch against C. albicans ranged between 38% and 42% (v/v). When starch was incubated with honey and then added to media, a MIC drop was noticed with each variety. MAIC of the four varieties ranged between 32% honey (v/v) with 4% starch and 36% honey (v/v) with 2% starch. The use of ginger starch allows honey benefit and will constitute an alternative way against the resistance to antifungal agents.
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Background: Propolis and honey are natural bee products with wide range of biological and medicinal properties. The study investigated antimicrobial activity of ethyl alcohol extraction of propolis collected from Saudi Arabia (EEPS) and from Egypt (EEPE), and their synergistic effect when used with honey. Single and polymicrobial cultures of antibiotic resistant human pathogens were tested. Material and methods; Staphylococcus aureus (S. aureus),), Escherichia coli (E. coli) and Candida albicans (C.albicans) were cultured in 10-100% (v/v) honey diluted in broth, or 0.08-1.0% (weight/volume) EEPS and EEPE diluted in broth. Four types of polymicrobial cultures were prepared by culturing the isolates with each other in broth (control) and broth containing various concentrations of honey or propolis. Microbial growth was assessed on solid plate media after 24 h incubation. Results; EEPS and EEPE inhibited antibiotic resistant E.coli, and S.aureus, and C.albicans in single and polymicrobial cultures. S.aureus became more susceptible when it was cultured with E.coli or C.albicans or when all cultured together. C.albicans became more susceptible when it was cultured with S.aureus or with E.coli and S. aureus together. The presence of ethyl alcohol or honey potentiated antimicrobial effect of propolis toward entire microbes tested in single or polymicrobial cultures. EEPS had lower MIC toward E.coli and C.albicans than EEPE. When propolis was mixed with honey, EEPS showed lower MIC than EEPE. In addition, honey showed lower MIC toward entire microbes when mixed with EEPS than when it was mixed with EEPE. Conclusion; 1) propolis prevents the growth of the microorganisms in single and mixed microbial cultures, and has synergistic effect when used with honey or ethyl alcohol, 2) the antimicrobial property of propolis varies with geographical origin, and 3) this study will pave the way to isolate active ingredients from honey and propolis to be further tested individually or in combination against human resistant infections.
Candida albicans is the most prevalent dimorphic pathogen in humans. We investigated the potential protective effect of honey flavonoid extract (HFE) on Candida dimorphism induced by RPMI 1640 medium. Yeast to hyphal transition was dose dependently prevented when Candida cultures were treated with HFE for 6 h (germ-tube formation) and 18 h (hypha elongation). Since during hyphal growth remarkable amounts of reactive oxygen species (ROS) are generated, we investigated whether HFE affects the generation of ROS, the glutathione level and the activity of glutathione-dependent enzymes. Treatment of Candida cells with 48 μg/ml (MIC50) of HFE for 6 or 18 h inhibited the dimorphic conversion by supporting of the intracellular glutathione level. HFE exerts a dual protective effect inhibiting both ROS generation during germ-tube formation and γ-glutamyltranspeptidase activity, responsible for GSH degradation, during hypha elongation. These results show that HFE confers a significant protection against yeast to hyphal transition of C. albicans.
A new strain, exhibiting an intriguing pink-colored cell phenotype, was obtained after an encoding α-glucosidase gene from an archaebacteria Thermococcus hydrothermalis was cloned by functional complementation of a mal11 Saccharomyces cerevisiae mutant TCY70. The possible implications of the α-glucosidase on the cell wall were evaluated by infrared spectroscopy and data indicate a 30% decrease in mannoproteins and an increase in β-glucans. The loss of mannoproteins was confirmed by experiments on cells deprived of peptidomannans. Modifications in the major components of the cell wall did not jeopardize cell viability. Such rapid optical spectroscopic method can be used to screen a wide range of yeast mutants.