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N-acetylcysteine Inhibits and Eradicates Candida albicans Biofilms

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N-acetylcysteine (NAC) is used in the treatment of chronic bronchitis that attributed to its mucus dissolving properties. Its ability to reduce biofilm formed by different types of bacteria was proven previously in many studies. Therefore we examined its effect on C. albicans biofilms by testing its effect alone and in combination with ketoconazole using Tissue culture plate assay method (TCP). NAC effects on C. albicans morphology and the texture of biofilms were determined using Scanning electron microscope (SEM). It was found that the inhibitory effect of NAC was concentration dependent. NAC reduced C. albicans adherence by ≥32.8% while ketoconazole reduced adherence by ≥25% in comparison to control. Also, it showed higher disruptive effect (50-95%) than ketoconazole (22-80.7%) on mature biofilms. Using NAC and ketoconazole in combination, a significant inhibitory effect (P<0.01) on both adherence and mature biofilms (54-100%) was seen. NAC reduced the amount of biofilm mass in all tested Candida in concentrations at which their growth was not affected. NAC and ketoconazole combinations showed complete eradication to mature biofilms formed in most of the tested strains. NAC can inhibit C. albicans growth, inhibit dimorphism, which is an important step in biofilm formation, and change the texture of the formed biofilms, what makes NAC an interesting agent to be used as an inhibitor for biofilm formation by C. albicans.
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American Journal of Infectious Diseases and Microbiology, 2014, Vol. 2, No. 5, 122-130
Available online at http://pubs.sciepub.com/ajidm/2/5/5
© Science and Education Publishing
DOI:10.12691/ajidm-2-5-5
N-acetylcysteine Inhibits and Eradicates Candida
albicans Biofilms
Rehab Mahmoud Abd El-Baky*, Dalia Mohamed Mohamed Abo El Ela, Gamal Fadl Mamoud Gad
Department of Microbiology, Faculty of Pharmacy, Minia University, Minia, Egypt
*Corresponding author: dr_rehab010@yahoo.com, rehab.mahmoud@mu.edu.eg
Received October 18, 2014; Revised October 30, 2014; Accepted November 04, 2014
Abstract N-acetylcysteine (NAC) is used in the treatment of chronic bronchitis that attributed to its mucus
dissolving properties. Its ability to reduce biofilm formed by different types of bacteria was proven previously in
many studies. Therefore we examined its effect on C. albicans biofilms by testing its effect alone and in combination
with ketoconazole using Tissue culture plate assay method (TCP). NAC effects on C. albicans morphology and the
texture of biofilms were determined using Scanning electron microscope (SEM). It was found that the inhibitory
effect of NAC was concentration dependent. NAC reduced C. albicans adherence by ≥32.8% while ketoconazole
reduced adherence by ≥25% in comparison to control. Also, it showed higher disruptive effect (50-95%) than
ketoconazole (22-80.7%) on mature biofilms. Using NAC and ketoconazole in combination, a significant inhibitory
effect (P<0.01) on both adherence and mature biofilms (54-100%) was seen. NAC reduced the amount of biofilm
mass in all tested Candida in concentrations at which their growth was not affected. NAC and ketoconazole
combinations showed complete eradication to mature biofilms formed in most of the tested strains. NAC can inhibit
C. albicans growth, inhibit dimorphism, which is an important step in biofilm formation, and change the texture of
the formed biofilms, what makes NAC an interesting agent to be used as an inhibitor for biofilm formation by C.
albicans.
Keywords: antifungal, mature biofilm, mucolytics, adherence, SEM
Cite This Article: Rehab Mahmoud Abd El-Baky, Dalia Mohamed Mohamed Abo El Ela, and Gamal Fadl
Mamoud Gad, “N-acetylcysteine Inhibits and Eradicates Candida albicans Biofilms.” American Journal of
Infectious Diseases and Microbiology, vol. 2, no. 5 (2014): 122-130. doi: 10.12691/ajidm-2-5-5.
1. Introduction
Candida albicans is the most prevalent candida species
isolated from healthy and diseased individuals.
Mycological studies have shown that C. albicans
represents over 80% of isolates from all forms of human
candidosis [1,2]. The transition of candida from a
harmless commensal to a pathogenic organism is complex
as it depends on both host and candidal factors [3].
Candida can undergo adherence and colonization to host
surfaces or biomaterials surfaces used in medical devices
as silicone and dental prosthesis as acrylic resin [4]. It can
adhere by many adhesins to different types of human cells
including phagocytic, epithelial and endothelial cells. Also,
adhesion may be attributed to cell surface hydrophobicity.
In addition, the hyphal formation is found to be significant
in the pathogenicity of C. albicans and play an important
role in the adherence process [2]. As Baille and Douglas
[5] reported that yeasthypha transition is necessary for
full maturation of biofilm. Strains that are unable to grow
as yeasts or to form hyphae can still form biofilm but are
easily detachable.
Biofilms can develop on different surfaces on three
stages: (i) attachment and colonization of yeast cells to
surfaces that mediated by both nonspecific factors (cell
surface hydrophobicity and electrostatic forces) and by
specific adhesins on the fungal surface, such as serum
proteins (fibrinogen and fibronectin) and salivary factors.
Importantly, biofilm formation correlates with cell surface
hydrophobicity; (ii) growth and proliferation of yeast cells
for the formation of basal layer, and (iii) growth of
pseudohyphae and the extensive production of extracellular
matrix material. After maturation, detachment of yeast
cells act as a foci for the spread of infection to other body
sites [6,7]. On the other hand, biofilm forming cells show
resistance against antimicrobials and host defense
mechanisms which has a considerable impact on the
treatment of biofilm-related infections [8,9].
Studying biofilms is increasing at a high and fast pace,
particularly for bacterial biofilms, but being somewhat
neglected for fungal biofilms. C. albicans biofilm share
several properties with bacterial biofilms, including their
structural heterogeneity, the presence of exopolymeric material
and their decreased susceptibility to antimicrobials [10].
The current treatment options for fungal biofilm
associated infections are not enough due to the increased
tolerance of biofilms to antifungals. Resistance of C.
albicans biofilms to the majority of antifungals were
reported since the mid of 1990s [11]. Patients with devices
containing fungal biofilms are rarely cured with mono-
antifungal therapy and their devices need to be removed
[12]. Some catheters as percutaneous vascular catheters
American Journal of Infectious Diseases and Microbiology 123
can be removed quickly while the removal of infected
voice prostheses, heart valves, joint prostheses, central
nervous system shunts and other implanted medical
devices is problematic because these implants generally
have a life-supportive function. So, successful treatments
are urgently needed in clinical practice for retaining the
implanted devices [13].
N-Acetyl-L-cysteine (NAC) is used in the treatment of
chronic bronchitis, cancer, and paracetamol intoxication
[14,15]. Also, it is thiol containing antioxidant. So, it may
disrupt disulfide bonds in mucus inhibiting amino acid
(cysteine) utilization [16,17]. In addition, it has antibacterial
properties [18]. Many studies proved the anti-biofilm
activity of NAC against bacterial biofilms [19,20]. So, we
thought to study its effect on Candida biofilms. In this
study we determined the effect of N-acetyl cysteine alone
and in combination with ketoconazole on both adherence
and preformed biofilms. Also we used scanning electron
microscope to determine the morphological changes
occurred for C. albicans cells and the changes in the
intensity of biofilm formed on a surface model in the
presence of the tested agents.
2. Methods
2.1. Microbial Strains
Ten Candida albicans, recently isolated from patients
suffering from vaginal and oral infections were used in
this study. The identification of organisms were based on
the following: colony morphology, germ tube test,
chlamydospores on Tween 80 cornmeal agar (Difco) and
by the pattern of assimilation of a variety of carbon and
nitrogen sources [21]. Biofilm production ability were
tested using tissue culture plate assay (TCP) method [22].
2.2. Drugs
We have followed the guidelines of CLSI [23] for
preparing of stock solution of ketoconazole (2 mg/ml)
(Amriya, Egypt). Two mg of ketoconazole was dissolved
in 1 mL of methanol according to manufacturer's
instructions. Working solution concentrations were ranged
from 0.025-12.8 µg/ml. For N-acetylcysteine (Sedico,
Egypt), stock solution of 40 mg/ml was prepared by
dissolving 4 gm of NAC in 100 ml of water. Working
solution concentrations were ranged from 40-0.3125
mg/ml.
2.3. Determination of Minimum Inhibitory
Concentrations (MIC) of Ketoconazole
Minimum inhibitory concentrations of ketoconazole
were determined by agar dilution method [23].
2.4. Determination of Minimum Inhibitory
Concentrations (MIC) of N-acetyl Cysteine
Microorganisms (0.5 ml) of 1.5×106 CFU/ml (0.5
Mcfarland turbidity) were plated in sterile petri dishes
then 20 ml of sterile, molten and cooled (45°C) Muller
Hinton agar media with the addition of methylene blue-
glucose (to enhance zone diameter visualization) was
added to all petri dishes. The plates then were rotated
slowly to ensure uniform distribution of the
microorganisms and then allowed to solidify on a flat
surface. After solidification, four equidistant and circular
wells of 10 mm diameter were carefully punched using a
sterile cork bore.
Two fold serial dilutions were performed on the tested
N-acetyl cysteine. Equal volumes of each dilution were
applied separately to each well in three replicates using a
micropipette. All plates were incubated overnight at 37°C,
then collected and zones of inhibition that developed were
measured. The average of the zones of inhibition was
calculated. The minimum inhibitory concentration (MIC)
was calculated by plotting the natural logarithm of the
concentration of each dilution against the square of zones
of inhibition. A regression line was drawn through the
points. The antilogarithm of the intercept on the logarithm
of concentration axis gave the MIC value [24].
2.5. Effect of the Tested Agents (ketoconazole
and NAC) Each alone and in Combination on
the Adherence and the Preformed Biofilm of
Candida albicans on Plastic Surfaces
The TCP assay is most widely used and was considered
as a standard test for the detection of biofilm formation
[22]. Different materials have been used for the growth of
biofilms in-vitro, including polystyrene, polyvinyl
chloride, silicone elastomer and polymethyl methacrylate
[25]. Although polystyrene is not used in the manufacture
of indwelling medical device, it is widely used for testing
biofilm production as it is considered as an excellent
material for promoting adherence of cells [26]. So, a
standardized method for biofilm formation based on
polystyrene 96-well plates has been established [27].
2.5.1. Preparation of Inoculums
All strains were first streaked onto YEPD agar (1%
yeast extract, 2% Bacto peptone, 2% D-glucose, 1.5%
agar) then, incubated at 25°C for 48 h. A large loop of
actively growing cells (for each strain) was transferred to
sterile trypticase soy broth (TSB) (Difco Laboratories)
containing 0.9% D-glucose. After incubation at 25°C for
24 h, the cells were centrifuged and washed twice with 0.5
ml PBS (phosphate buffer saline), followed by vortexing
and centrifugation at 5000 g for 5 min. The washed cells
were suspended in 1 ml TSB broth and adjusted to a final
OD600 nm value of 1.0 with TSB broth. These cell
suspensions were then used to grow biofilms.
2.5.2. For Testing the Effect of Ketoconazole and N-
Acetylcysteine on the Adherence of EACH STRAIN
100 µl of the suspension (OD600) was inoculated into
individual wells of polystyrene 96-well plates (flat bottom;
Nunc). TSB broth was used as a negative control. The
plates were incubated at 25°C for 90 min (adhesion
period). Supernatants including planktonic cells were
discarded and wells were gently washed with PBS twice
to remove any non-adherent cells. 100 µl of fresh TSB
broth containing one of the following solutions:
ketoconazole (MIC and 2x MIC), N-acetylcysteine (4 and
8 mg/ml) and Ketoconazole/N-acetylcysteine (MIC/4
mg/ml and 2x MIC /8 mg/ml) was added to each well. The
plates were covered to prevent evaporation and incubated
124 American Journal of Infectious Diseases and Microbiology
at 25°C for 24 h. Liquid media containing the non
adherent cells were discarded through two rounds of
washing with 200 µl sterile PBS buffer. Adherent cells to
the plastic surfaces were quantified using Crystal violet
assay [28]. Experiment was performed in triplicate and
repeated three times, the data was then averaged and
standard error was calculated.
2.5.3. The Effect of the Tested Agents on the
Preformed Biofilms
100 µl of the suspension (OD600) was inoculated into
individual wells of polystyrene 96-well plates (flat bottom;
Nunc). The plates were incubated at 25°C for 48 h. After
the incubation period, the supernatants from each well
were aspirated and the wells washed twice with PBS
without disturbing the biofilms at the bottom of the wells,
then one of the following solutions: ketoconazole (MIC
and 2xMIC), N-acetylcysteine (4 and 8 mg/ml) and
Ketoconazole / N-acetylcysteine (MIC/4 mg/ml and
2xMIC /8 mg/ml) was added to each well. Normal saline
without any agents was added to the control wells. The
plates were incubated at 25°C for 24 h. Supernatants were
discarded through two rounds of washing with 200 µl
sterile PBS buffer. Cells adherent to the plastic surfaces
were quantified using Crystal violet assay. Experiment
was performed in triplicate and repeated three times, the
data was then averaged and standard error was calculated.
2.6. Scanning Electron Microscopy (SEM)
Polyurethane segments were used as a surface for
testing the effect of the tested agents on biofilm formed by
C. albicans. Polyurethane segments (1 cm length) were
incubated in 5 ml of Trypticase soy broth (BBL, USA)
that contained 5×106 cfu/ml of C. albicans for 90 min. To
test the effect of the tested agents on fungal adherence to
the polyurethane surfaces, One of the following solutions:
Ketoconazole (MIC and 2x MIC), N-acetylcysteine (4 and
8 mg/ml) and ketoconazole/NAC (MIC/2 mg/ml and
2MIC/4 mg/ml) were added to each tube, normal saline
was added to control tubes and incubated at 25°C for 24 h.
To test the effect of the tested agents on the preformed
mature biofilms, polyurethane segments were incubated
with C. albicans cultures at 25°C for 48 h. After
incubation, stents washed twice with normal saline
without disturbing the biofilms, Then placed in new test
tubes containing TSB medium with Ketoconazole (MIC
and 2x MIC), N-acetylcysteine (4 and 8 mg/ml) and
Ketoconazole/NAC (MIC/2 mg/ml and 2MIC/4 mg/ml).
Normal saline was added to control tubes and incubated
for 24 h.
2.6.1. Scanning Electron microscope Examination
Polyurethane segments were fixed in 2.5% (vol/vol)
glutaraldehyde in Dulbecco PBS (PH 7.2) for 1.5 h, rinsed
with PBS, and then dehydrated through an ethanol series.
Samples were dried and gold-palladium coated. SEM
examinations were made on a JSM-840 SEM (JEOL Ltd.,
Tokyo, Japan) (Soboh et al., 1995).
2.7. Statistical Analysis
One-Way ANOVA as employed to evaluate any
significant difference between the values obtained without
the drug (controls) and the values obtained in the presence
of different drug concentrations. Differences were done
using SPSS version19 (SPSS Inc., Chicago, IL) for
windows was used. Descriptive statistics were calculated.
Kruskal-Wallis test was used to compare groups. A significant
P-value was considered when it was less than 0.05.
3. Results
One hundred thirty-eight samples were collected from
patients suffering from oral thrush and vaginities. Twenty
three samples (16.7%) were positive for Candida albicans.
As out of 99 vaginal swabs and 39 oropharyngeal swabs,
14 (14%) and 9 (23%) were positive for Candida albicans,
respectively. Out of 23 Candida albicans isolates, 10
(43.5%) biofilm-producing strains were identified (7 from
vaginal swabs and 3 from oropharyngeal swabs) (Figure 1).
By examining polyurethane segments using SEM, It was
found that Candida biofilm contains both the yeast and the
hyphal forms (Figure 2).
Figure 1. Screening of the extent of biofilm production by Tissue culture
plate method (TCP): high, moderate and non slime producers
differentiated with crystal violet staining in 96 well tissue culture plate
Figure 2. SEM images of a mature (48 h) C. albicans biofilm. Arrows refers to Candida cells and hyphal and pseudohyphal elements in both images (a)
5,000X, Bar represents 5µm (b) 1,000X, Bar represents 10µm
American Journal of Infectious Diseases and Microbiology 125
3.2. Minimum Inhibitory Concentrations of
Ketoconazole and NAC
All the tested C. albicans strains were sensitive to
ketoconazole (9 strains showed MIC of ketoconazole of
0.4 µg/ml while one had MIC of 0.8 µg/ml). Nine strains
showed MIC of NAC of 20 mg/ml but one showed MIC
of 16.5 mg/ml.
3.3. The Effect of Ketoconazole and N-
Acetylcysteine on C. albicans Adherence
Figure 3. Effect of ketoconazole and N-acetylcysteine each alone and in combination on biofilm production and preformed biofilm formed by C.
albicans strains. Most of the tested concentrations had higher inhibitory effect on preformed biofilm than biofilm production (adherence)
Table 1. Effects of ketoconazole and N-acetylcysteine each alone on biofilm production (adherence) by the tested Candida albicans
Candida
albicans
Ketoconazole
N-acetyle cystiene
Conc. (µg/ml)
Mean±S.E
Conc. (mg/ml)
Mean±S.E
% OF reduction
1
CTR
0.128±0.00
1 a
0.048±0.002*
4
0.086±0.002*
32.80%
2 b
0.040±0.002*
8
0.038±0.002*
70.30%
2
CTR
0.150±0.002
1
a
0.040±0.001*
4
0.093±0.001*
38%
2
b
0.035±0.001*
8
0.045±0.001*
70%
3
CTR
0.247±0.000
1 a
0.035±0.458*
4
0.034±0.003*
86.25%
2 b
0.031±0.001*
8
0.030±0.001*
87.80%
4
CTR
0.134±0.003
1
a
0.083±0.001*
4
0.055±0.005*
58.90%
2
b
0.065±0.001*
8
0.025±0.006*
81.30%
5
CTR
0.152±0.002
1
a
0.040±0.001*
4
0.093±0.001*
38.80%
2
b
0.035±0.001*
8
0.045±0.001*
70.30%
6
CTR
0.137±0.002
1
a
0.092±0.002*
4
0.064±0.005*
53.2%
2
b
0.065±0.001*
8
0.037±0.002*
72.90%
7
CTR
0.155±0.000
1
a
0.092±0.003*
4
0.092±0.001*
40.60%
2
b
0.065±0.002*
8
0.055±0.000*
64.50%
8
CTR
0.124±0.000
1
a
0.093±0.003*
4
0.083±0.001*
33.06%
2
b
0.079±0.002*
8
0.070±0.000*
43.50%
9
CTR
0.125±0.000
1
a
0.085±0.002*
4
0.062±0.001*
50.40%
2
b
0.071±0.000*
8
0.053±0.003*
57.60%
10
CTR
0.135±0.000
1 a
0.085±0.002*
4
0.062±0.001*
54.07%
2
b
0.071±0.001*
8
0.053±0.003*
60.70%
CTR: without drug (control).
a: At MIC. b: At 2 MIC. * P<0.05: Significant value, compared to controls.
** P<0.01: Significant value, compared to controls, Ketoconazole group and NAC group.
Mean±S.E.M= Mean values ± Standard error of means of 3 experiment.
126 American Journal of Infectious Diseases and Microbiology
Table 2. Effects of ketoconazole and N-acetylcysteine combinations
on biofilm production (adherence) by the tested Candida albicans
Candida
albicans
Ketoconazole/N-acetyle systiene
Conc.
Mean±S.E
% of reduction
1
CTR
0.128±0.00
1\4a
0.042±0.001**
67.10%
2\8b
0.016±0.001**
87.50%
2
CTR
0.150±0.002
1\4 a
0.018±0.001**
88%
2\8 b
0.008±0.003**
94.60%
3
CTR
0.247±0.000
1\4 a
0.009±0.001**
96.30%
2\8 b
0.000±0.000**
100%
4
CTR
0.134±0.003
1\4 a
0.025±0.001**
81.30%
2\8 b
0.002±0.006**
94.70%
5
CTR
0.152±0.002
1\4 a
0.018±0.001**
88.10%
2\8 b
0.008±0.003**
94.70%
6
CTR
0.137±0.002
1\4 a
0.052±0.003**
62.04%
2\8 b
0.042±0.002**
69.30%
7
CTR
0.155±0.000
1\4 a
0.041±0.001**
73.50%
2\8 b
0.037±0.002**
76.10%
8
CTR
0.124±0.000
1\4 a
0.053±0.001**
57.20%
2\8 b
0.043±0.001**
65.30%
9
CTR
0.125±0.000
1\4 a
0.042±0.001**
65.80%
2\8 b
0.033±0.000**
73.60%
10
CTR
0.135±0.000
1\4 a
0.042±0.001**
68.80%
2\8 b
0.033±0.000**
75.50%
CTR: without drug (control).
a: MIC/2 mg/ml; b: 2X MIC/4 mg/ml; * P<0.05: Significant value,
compared to controls.
** P<0.01: Significant value, compared to controls, Ketoconazole group
and NAC group. Mean±S.E.M= Mean values ± Standard error of means
of 3 experiment.
The inhibitory effects of ketoconazole and N-
acetylcysteine were found to be concentration dependent.
Ketoconazole at MIC inhibited biofilm synthesis by ≥
25%. At 2xMIC, reduction of biofilm synthesis was
≥36.2%. In this study we used two concentrations that
were lower than MICs of NAC to test its effect on the
adherence and mature biofilms of tested strains without
affecting their growth. N-acetylcysteine showed a
significant inhibitory effect (p<0.05) on adherence and
more than that observed by ketoconazole. As reduction of
biofilm synthesis was ≥32.8% in a concentration of 4
mg/ml, at 8 mg/ml, biofilm synthesis was reduced by
≥43.5% in all tested strains. Ketoconazole/N-
acetylcysteine combinations were found to have the
highest inhibitory effect on adherence (p<0.01) (54-100%)
in comparison to controls (Table 1 and Table 2) (Figure 3)
3.4. Disruption of Preformed Mature Biofilms
Ketoconazole and N-acetylcysteine were found to have
significant inhibitory effects (p<0.05) on preformed mature
biofilm. Results showed that N-acetylcysteine has a higher
disruptive effect on mature biofilms than ketoconazole. As
N-acetylcysteine showed a reduction in the optical density
that ranged from 50 to 95.2% while ketoconazole showed
a reduction ranged from 22 to 80.7% in comparison to
controls. On the other hand, ketoconazole combined to N-
acetylcysteine showed the highest ability to disrupt
preformed biofilms (p<0.01) (54.07-100%, in comparison
to controls) (Table 3 and Table 4) (Figure 3 and Figure 4).
Our results revealed that ketoconazole/N-acetylcysteine
combination showed the highest ability to reduce biofilm
production and to disrupt preformed mature biofilms.
Table 3. Effects of ketoconazole and N-acetylcysteine each alone on preformed biofilm by the tested Candida albicans
Candida
albicans
Ketoconazole
N-acetyle cystiene
Conc.(µg/ml)
Mean±S.E
% of reduction
Conc.(mg/ml)
Mean±S.E
% of reduction
1
CTR
0.128±0.000
1a
0.049±0.003*
61.70%
4
0.033±0.001*
74.20%
2b
0.030±0.000*
76.50%
8
0.030±0.000*
76.50%
2
CTR
0.150±0.000
1 a
0.052±0.001*
65.30%
4
0.030±0.000*
80%
2 b
0.030±0.000*
80.00%
8
0.008±0.000*
95%
3
CTR
0.247±0.000
1 a
0.084±0.000*
65.90%
4
0.038±0.001*
84.60%
2 b
0.048±0.000*
80.50%
8
0.018±0.000*
77.32%
4
CTR
0.134±0.001
1 a
0.085±0.000*
36.50%
4
0.020±0.000*
85.07%
2 b
0.065±0.000*
51.40%
8
0.015±0.000*
88.80%
5
CTR
0.152±0.000
1 a
0.102±0.001*
32.80%
4
0.022±0.000*
85.50%
2 b
0.081±0.065
46.70%
8
0.018±0.000*
88.10%
6
CTR
0.137±0.000
1 a
0.098±0.000
28.40%
4
0.044±0.001*
67.80%
2 b
0.048±0.000*
64.90%
8
0.033±0.03*
76.60%
7
CTR
0.155±0.001
1 a
0.082±0.000*
47.09%
4
0.045±0.000*
70.90%
2 b
0.043±0.000*
72.20%
8
0.029±0.000*
81.20%
8
CTR
0.124±0.000
1 a
0.084±0.011
32%
4
0.062±0.000
50.00%
2 b
0.061±0.000*
50.80%
8
0.034±0.000*
72.50%
9
CTR
0.125±0.000
1 a
0.097±0.000
22%
4
0.048±0.030*
61.60%
2 b
0.031±0.000*
75.20%
8
0.006±0.000*
95.20%
10
CTR
0.135±0.000
1 a
0.027±0.000*
80%
4
0.037±0.004*
72.50%
2 b
0.026±0.000*
80.70%
8
0.025±0.000*
81.40%
CTR: without drug (control).
a: At MIC. b: At 2 MIC. * P<0.05: Significant value, compared to controls.
** P<0.01: Significant value, compared to controls, Ketoconazole group and NAC group. Mean±S.E.M=Mean values±Standard error of means of 3 experiment
American Journal of Infectious Diseases and Microbiology 127
Figure 4. Scanning electron micrographs showing the effect of Ketoconazole, N-acetylcysteine each alone and in combination on a performed C.
albicans biofilm developed in vitro on a polyurethane segment
A. Candida albicans with a high biofilm mass (Cotton like mass) on the surface of a polyurethane stent incubated with Candida albicans suspension for
48 h (control).
B: The effect of N-acetylcysteine (4 mg/ml) on C. albicans biofilm. Cotton like mass was highly decreased and cells appeared swollen with disrupted
membranes.
C. The effect of N-acetylcysteine (8 mg/ml) on C. albicans biofilm. Cells appeared scattered with disrupted membranes and no biofilm mass.
D. C. albicans biofilm exposed to Ketoconazole at MIC concentration. Cells with irregular membranes. A decrease in the amount of biofilm mass.
E. Effect of Ketoconazole at 2X MIC. Cells appeared swollen, scattered. No biofilm mass observed.
F. Effect of Ketoconazole-N-acetylcysteine combination (MIC/4 mg/ml). No biofilm mass
G. Effect of Ketoconazole/NAC combination (2 X MIC/8mg/ml) on C. albicans biofilm. No biofilm mass or cells observed.
128 American Journal of Infectious Diseases and Microbiology
SEM showed the disappearance of the hyphael forms
which are essential elements for the integrity and the
formation of highly structured mature/fully developed
biofilms and the presence of the yeast form (Figure 4).
Also, Figure 4 showed the effect of the tested agents on
cells morphology and the texture of biofilm mass. SEM
images showed that the decrease in biofilm mass was
observed at concentrations of 4 and 8 mg/ml (lower than
MICs of NAC) that didn’t affect C. albicans growth.
Table 4. Effects of ketoconazole and N-acetylcysteine combinations
on preformed biofilm by the tested Candida albicans
Candida
albicans
Ketoconazole/N-acetyle systiene
Conc.
Mean±S.E
% of reduction
1
CTR
0.128±0.000
1\4
a
0.013±0.001**
89.80%
2\8
b
0.008±0.000**
93.70%
2
CTR
0.150±0.000
1\4
a
0.012±0.000**
92%
2\8
b
0.000±0.000**
100%
3
CTR
0.247±0.000
1\4
a
0.069±0.05**
72.06%
2\8
b
0.037±0.06**
92.70%
4
CTR
0.134±0.001
1\4 a
0.046±0.000**
65.60%
2\8
b
0.043±0.000**
67.90%
5
CTR
0.152±0.000
1\4
a
0.042±0.001**
72.30%
2\8
b
0.040±0.001
73.60%
6
CTR
0.137±0.000
1\4 a
0.012±0.057
91.20%
2\8
b
0.007±0.000**
94.80%
7
CTR
0.155±0.001
1\4
a
0.042±0.000**
72.90%
2\8
b
0.038±0.000**
75.40%
8
CTR
0.124±0.000
1\4
a
0.031±0.001**
75.00%
2\8 b
0.025±0.000**
79.80%
9
CTR
0.125±0.000
1\4
a
0.018±0.000**
85.60%
2\8
b
0.009±0.004
92.80%
10
CTR
0.135±0.000
1\4
a
0.062±0.006**
54.07%
2\8 b
0.055±0.001**
59.20%
CTR: without drug (control).
a: MIC/2 mg/ml; b: 2X MIC/4 mg/ml; * P<0.05: Significant value,
compared to controls.
** P<0.01: Significant value, compared to controls, Ketoconazole group
and NAC group.. Mean±S.E.M= Mean values ± Standard error of means
of 3 experiment.
4. Discussion
Surface-associated Candida can grow incorporated in
extracellular matrix which consists of carbohydrates,
proteins and some unknown components, known as a
biofilm. Biofilms can be formed readily on biotic (mucous
membranes) and abiotic surfaces (Foley catheters and
intrauterine devices (IUDs)), that render the embedded
Candida isolates resistant to antifungal agents, especially
azoles and promote persistence of fungal infections [30].
The ability of C. albicans to adhere to host mucosal
surfaces is a prerequisite for subsequent biofilm formation
and colonization of the host mucosal surfaces such as
buccal and vaginal mucosa [31].
Many studies have shown that Candida biofilm
development is associated with the generation of an
extracellular matrix and that mature biofilms show a
highly heterogeneous structure and grow variably
depending on the topography of the substrate [25,32].
Scanning electron microscope examination of biofilms
revealed the presence of both adherent yeast form and
invasive hyphal form cells forming the basal and upper
layers, respectively. Both yeast and the hyphal forms were
enclosed in an extracellular polymer matrix consisting of
polysaccharides, proteins and forming a three-dimensional
structures with water channels [33].
Biofilms associated drug resistance is a result of many
factors: (i) The exopolymer matrix of biofilms decrease or
inhibit the penetration of immune system components and
antimicrobials [34]. (ii) In Candida, the matrix consists of
carbohydrates, proteins, hexosamine, phosphorus and
uronic acid [35]. These components have the ability to
bind antifungals, preventing their access to the antifungal
targets in the cell and results in resistance [36]. Also,
extracellular polymeric material may act as an adsorbent
to the antimicrobials. (iii) The growth rate differentials. (iv)
Production of antimicrobial-degrading enzymes [37].
Many studies attributed the anti-biofilm activity of
NAC to a number of factors which are: (i) its ability to
bind surfaces, increasing their wettability that results in
decreasing of microorganisms adhesion. (ii) NAC can
detach the adherent microbial cells to steel surfaces. (iii)
its ability to reduce the amount of exopolysaccharide (EPS)
by direct effect in which a possible reaction of its
sulfydryl group occurs with the disulfide bonds of the
enzymes involved in EPS production or excretion, which
renders these molecules less active. Also, its competitive
inhibition of cysteine utilization and indirect effect by
affecting cell metabolism and EPS production due to its
antioxidant activity [19,20]. So that it is expected that an
antibiofilm/antimicrobial agent combination would be
synergistic which lead us to use it in our study on biofilm
formation by Candida albicans.
Harriott et al. [38] were the first who reported that C.
albicans can form biofilms on vaginal mucosa. As they
found that vaginal C. albicans have biofilm architecture
typical of in-vitro grown C. albicans biofilms, consisting
of yeast and hyphae forming a complex network
surrounded by extracellular matrix. Our results showed
that by examining Candida albicans biofilm developed in
an in-vitro model, it was found that biofilm formed
showing the yeast form and the hyphal form.
The use of ketoconazole at MIC and 2 MIC reduced
biofilm formation or adherence by 25-87.4% (P<0.05) and
disrupted the mature biofilm causing a reduction in optical
density of 22-80.7% but Baillie and Douglas [39] found
that 20 times the MIC of commonly used antifungals such
as amphotericin B, fluconazole, or flucytosine is required
to cause a significant reduction in cell numbers. Chandra
et al. [25] reported that C. albicans required low MICs of
polyenes and fluconazole during the early biofilm
development phase. However, during biofilm maturation,
they became highly resistant to these drugs. The potency
of ketoconazole as anti-biofilm was also reported by
Chebotar and Parshikov [40], who showed that ketoconazole
was the most potent anti-biofilm as it resulted in 100%
American Journal of Infectious Diseases and Microbiology 129
cell death after 48h exposure of Candida biofilms in
comparison to the effect of nystatin and fluconazole.
N-acetylcysteine was previously reported to have a high
antibiofilm activity, a high disruptive effect on mature
biofilms and increase the therapeutic activity of some
antimicrobials as ciprofloxacin and tigecycline against
biofilm producing bacteria [41]. In a study by Olofsson et
al. [19], it was found that the initial adhesion of bacteria to
stainless steel surfaces is dependent on the wettability of
the substratum. Aslam and Darouiche [42] reported that
NAC is fungistatic and has a significant effect on biofilm
formation by Candida albicans which agree with our
results. Also our results agree with those obtained by
Venkatesh et al. [43] who showed that NAC showed
synergistic action in combination with amphotericin B and
fluconazole against C. albicans biofilms.
This study showed that by using NAC and ketoconazole
(each alone or in combination), Candida albicans became
unable to form the hyphal form which lead to the
formation of thin biofilm (easily removed). Similar results
were obtained by Ramage et al. [44] and Lewis et al. [45]
who reported that strains of Candida albicans that unable
to filament form poor biofilms lacking in three-
dimensional structure and composed mainly of sparse
monolayers of elongated cells.
5. Conclusion
Our results showed that NAC has a great anti-biofilm
and can disrupt the preformed mature biofilms and
antifungal properties. In addition, both NAC and
ketoconazole can inhibit the dimorphism of Candida
albicans which plays an important role in the maturation
of biofilm. So, it will be valuable to use NAC in the
treatment of candidal infections as oral candidiasis or to
prevent biofilm formation on different medical devices
surfaces as voice prosthesis or ureteral stents.
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