Colloids and Surfaces B: Biointerfaces 174 (2019) 28–34

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Colloids and Surfaces B: Biointerfaces

journal homepage: www.elsevier.com/locate/colsurfb

Antibiofilm efficacy of curcumin in combination with 2- T

aminobenzimidazole against single- and mixed-species biofilms of Candida
albicans and Staphylococcus aureus
⁎ ⁎
Yulong Tana, , Matthias Leonharda, Doris Moserb, Su Mac, , Berit Schneider-Sticklera
a
Department of Otorhinolaryngology and Head and Neck Surgery, Medical University of Vienna, Vienna, Austria
b
Department of Cranio-Maxillofacial and Oral Surgery, Medical University of Vienna, Vienna, Austria
c
Biocatalysis and Biosensor Research Group, Division of Food Biotechnology, Department of Food Science and Technology, BOKU-University of Natural Resources and Life
Sciences, Vienna, Austria

A R T I C LE I N FO A B S T R A C T

Keywords: Mixed fungal and bacterial biofilm associated infections of implants have been a huge challenge in health care
2-aminobenzimidazole because of the increased resistance to antimicrobials and the critical biological differences between fungi and
Curcumin bacteria. In this study, we evaluated the 2-aminobenzimidazole (2ABI) and curcumin (CUR) alone to inhibit
Polymicrobial biofilm planktonic cell growth, adhesion as well as single and mixed species biofilms of Candida albicans and
Candida albicans
Staphylococcus aureus on silicone. The combined effects between 2ABI and CUR on mixed species biofilm for-
Staphylococcus aureus
mation and pre-formed biofilm were assessed. Our work showed that 2ABI or CUR alone was effective as a sole
agent, inhibiting planktonic growth, adhesion and the biofilm formation of bacteria and fungi on the silicone
surface. The combination of 2ABI and CUR exhibited the enhanced effect on mixed biofilm compared to mono-
drug therapy. The biofilm architecture was investigated by scanning electron microscopy (SEM) and the dis-
tinction of living/dead organisms within biofilm was examined by confocal laser scanning microscopy (CLSM).
The combination activity was most potent on mixed biofilm. These results suggest the potential applicability of
2ABI and CUR to treatment of biofilm related device infections.

1. Introduction antimicrobials, enhanced surface colonization and the critical biolo-

Microbial adhesion and biofilm formation on the surfaces of medical
devices significantly increase the risk of device-related infections with
possible lethal complications. A biofilm can be defined as community of
microbial cells embedded in a self-produced extracellular polymeric
matrix [1], which starts with the initial physical attraction of microbe
to virtually all biomaterial surfaces, grows in size and reproduce, and
then encourages persistence of infection [2,3]. The biofilms on implant
biomaterials are difficult to control with conventional antimicrobial
therapies and lead to dysfunction and finally replacement of the in-
fested medical devices [4,5]. Although some infections are caused by a
single species pathogen, mixed fungal and bacterial biofilms have been
identified more frequently [6,7]. Among the prevalent pathogens iso-
lated from infections, Staphylococcus aureus and Candida albicans are the
major leading bacterial and fungal species. Furthermore, both of them
are often found to be involved in mixed species biofilm-related infec-
tions [7,8]. In mixed species biofilms, increased resistance to
gical differences between fungi and bacteria can be observed [9]. The
efficacy of directly applied antibacterial and antifungal drugs is often
limited. Moreover, emergence of novel drug resistant strains and side
effects due to toxicity reduce the efficiency of these drugs. Therefore,
new antibioflm agents and strategies against mixed species biofilms of
Candida and bacteria are needed to be developed urgently.
Recently, 2-aminobenzimidazole (2ABI) and its derivatives have
been developed as antibiofilm agents. Several efforts have provided that
2ABI can inhibit and disperse Gram-positive and Gram-negative bac-
terial biofilms specifically without affecting free microbial cell growth
[10,11]. However, there are no studies on antibiofilm activity of 2ABI
on Candida biofilms. As an antibiofilm strategy, a non-microbicidal
molecule that disrupts biofilm growth could be used as an additional
compound. Because even if a molecule can inhibit biofilms effectively
without killing the microbial cells, the planktonic cells still could ad-
here on other site and form biofilm. Therefore, it has been reported for
2ABI derivatives and antibiotics against bacterial biofilms [12]. As

⁎
Corresponding authors.
E-mail addresses: yulong.tan@meduniwien.ac.at (Y. Tan), matthias.leonhard@meduniwien.ac.at (M. Leonhard), doris.moser@meduniwien.ac.at (D. Moser),
su.ma@boku.ac.at (S. Ma), berit.schneider-stickler@meduniwien.ac.at (B. Schneider-Stickler).

https://doi.org/10.1016/j.colsurfb.2018.10.079
Received 16 April 2018; Received in revised form 28 September 2018; Accepted 31 October 2018
Available online 02 November 2018
0927-7765/ © 2018 Published by Elsevier B.V.
Y. Tan et al.
discussed above, direct application of antifungal drugs is not acceptable
in clinic. Alternatively, naturally derived chemotherapeutic agents have
been attractive. Curcumin (CUR), isolated from spice Curcuma longa,
has been widely used in pharmaceutical and medical applications be-
cause of its broad spectrum of biological actions and non-toxicity, in-
cluding antibacterial, antiviral, antifungal and anti-inflammatory ac-
tivity [13,14]. The antibiofilm activities of CUR on bacteria or fungi
also have been reported [15–18]. However, no studies have been per-
formed to clarify the effect of CUR against mixed species biofilms.
Therefore, in this work, we firstly studied the ability of 2ABI or CUR
alone to inhibit planktonic cell growth, adhesion as well as single and
mixed species biofilms of S. aureus and C. albicans on medical grade
silicone. As a follow-up study, we investigated combined effects be-
tween 2ABI and CUR on mixed species biofilm formation and pre-
formed biofilm. Biofilm architecture was investigated by scanning
electron microscopy (SEM) and confocal laser scanning microscopy
(CLSM) was used to examine living/dead organisms within biofilm.
2. Materials and methods
2.1. Strains, media and reagents
S. aureus ATCC 6538 and C. albicans DAY185 were used in this study
for monomicrobial and polymicrobial biofilm formations. Yeast
Peptone Dextrose (YPD, Sigma-Aldrich, Austria) medium or Tryptic Soy
Broth (TSB, Sigma-Aldrich, Austria) medium was used for culturing C.
albicans and S. aureus, respectively. RPMI1640 medium (GlutaMAX™
Supplement, Thermo Fisher Scientific, USA) was chosen for supporting
biofilms growth. All other reagents were purchased from Sigma-Aldrich
(Austria).
29
Colloids and Surfaces B: Biointerfaces 174 (2019) 28–34
Fig. 1. Inhibition of cell-surface initial
interaction by 2ABI or CUR. (A)
Inhibition activity of 2ABI on S. aureus
adhesion; (B) Inhibition activity of
2ABI on C. albicans adhesion; (C)
Inhibition activity of CUR on S. aureus
adhesion; (D) Inhibition activity of
CUR on C. albicans adhesion. Values
obtained are given as the percentage of
adhesion. Results are expressed as
mean ± SD (* p < 0.05).
2.2. Minimum inhibitory concentration (MIC) assay
200 μl of overnight bacteria or fungi cultures diluted to 1 × 106
CFU/ml was added into a polystyrene 96-well microplate (Sarstedt AG
& Co, Germany). 2ABI or CUR with different concentrations (final
concentrations 200, 100, 50, 25 or 0 μg/ml) was then added to each
well. The microplate was incubated at 37 °C for 24 h without shaking.
The MIC was defined as the lowest concentration at which no visible
growth was detected.
2.3. Adhesion assay
Microbial adhesion on silicone plates was performed according to
our previous method with minor modifications [19]. Bacteria and fungi
were diluted to 1 × 106 CFU/ml with medium. 200 μl of the suspension
was added to 96-well microplate which contains sterilized medical
grade silicone discs (1.5-mm-diameter; Websinger, Austria), and in-
cubated for 90 min at 37 °C. The silicone discs were rinsed with PBS.
The adhesion of microbial cells on the silicone was assessed by Cell
Counting Kit-8 (CCK-8, Dojindo Molecular Technologies, Gaithersburg,
MD, USA) according to manufacturer’s protocol, which is based on
bioreduction of 2-(2- methoxy- 4- nitrophenyl)- 3- (4- nitrophenyl)- 5-
(2,4- disulfophenyl)- 2H-tetrazolium (WST-8) that produces a water-
soluble formazan dye. The amount of the formazan dye is directly
proportional to the number of living microorganisms.
2.4. Growth of mixed biofilms on silicone
Single and mixed species biofilm formations on silicone discs were
performed as described previously [20]. Medical grade silicone discs
were sterilized and placed into wells of a 96-well microplate. Bacterial
and fungal suspensions (1 × 106 CFU/ml) were added to the wells of a
96-well microplate. For mixed species assay, equal volumes of these cell
Y. Tan et al.
Fig. 2. Inhibition effect of 2ABI or CUR on single species biofilm formations. (A) Inhibition activity of 2ABI on S. aureus biofilm formation; (B) Inhibition activity of
2ABI on C. albicans biofilm formation; (C) Inhibition activity of CUR on S. aureus biofilm formation; (D) Inhibition activity of CUR on C. albicans biofilm formation.
Values obtained are given as the percentage of biofilm formation. Results are expressed as mean ± SD (* p < 0.05).
suspensions were mixed. After the adhesion phase (90 min), non-ad-
herent cells were removed by rinsing with PBS. The plates were then
incubated at 37 °C for 48 h without shaking.
2.5. Effect on biofilm formation
Single and mixed species biofilms were grown as described above.
200 μl of the cell suspension containing 2ABI alone, CUR alone or in
combination of 2ABI and CUR together were added to the wells.
Afterwards, biofilms were formed and detected by CCK-8 as described
above.
2.6. Effect on preformed biofilm
Single and mixed biofilms were carried out without agents as de-
scribed above. Mature biofilms were treated with 2ABI alone, CUR
alone or in combination of 2ABI and CUR together for 24 h. The silicone
discs were washed with PBS and determined by CCK-8.
2.7. Influence of metal ions on 2ABI antibiofilm activity
Candida biofilm was grown as described above. 200 μl of the cell
suspension containing metal ions (1 mM), including Ca(II), Cu(II), Fe
(II), Fe(III) and Zn(II), were added to the wells. Afterwards, biofilm was
formed and detected by CCK-8.
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Colloids and Surfaces B: Biointerfaces 174 (2019) 28–34
2.8. Live/Dead staining assay
Mixed biofilms treated with 2ABI alone, CUR alone or in combi-
nation of 2ABI and CUR together were washed with PBS and then
stained with a Live/Dead® BacLight™ Bacterial Viability and Counting
Kit (L34856, Invitrogen) for 30 min at 37 °C in the dark. Cell fluores-
cence was evaluated with confocal laser scanning microscopy (CLSM).
2.9. Scanning electron microscope (SEM)
Mixed biofilms were formed as described above on silicone.
Afterwards, the biofilm was fixed with 3% glutaraldehyde and dehy-
drated in a dilution series of ethanol (70%, 80%, 96% and 100%).
Following chemical dehydration with Hexamethyldisilazane (HMDS)
(Sigma-Aldrich, St. Louis US), the samples were sputter coated with
gold and analyzed with SEM (JSM 6310, Jeol Ltd., Akishima, Tokyo,
Japan).
2.10. Statistical analysis
All the experiments were done in triplicate. Statistical analyses were
performed using GraphPad Prism software program (GraphPad
Software, CA, USA). For each experiment, means ± standard devia-
tions (SD) were calculated. Statistical significance was determined by t-
test analysis with p < 0.05 found to be significantly different.
Y. Tan et al.
Fig. 3. Effects of combined use of 2ABI and CUR on monomicrobial and poly-
microbial biofilm formations. (A) Inhibition activity of combined use of 2ABI
and CUR on S. aureus biofilm formation; (B) Inhibition activity of combined use
of 2ABI and CUR on C. albicans biofilm formation; (C) Inhibition activity of
combined use of 2ABI and CUR on mixed species biofilm formation. Values
obtained are given as the percentage of biofilm formation. Results are expressed
as mean ± SD (* p < 0.05).
3. Results and discussion
3.1. Antibacterial and antifungal activity of curcumin
In order to determine biologically active working concentrations for
the following analyses, the antibacterial and antifungal activities of
CUR against S. aureus and C. albicans were examined by MIC assay.
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Colloids and Surfaces B: Biointerfaces 174 (2019) 28–34
Fig. 4. Effects of combined use of 2ABI and CUR on monomicrobial and poly-
microbial preformed biofilms. (A) Effect of combined use of 2ABI and CUR on S.
aureus biofilm; (B) Effect of combined use of 2ABI and CUR on C. albicans
biofilm; (C) Effect of combined use of 2ABI and CUR on polymicrobial biofilm.
Values obtained are given as the percentage of biofilm. Results are expressed as
mean ± SD (* p < 0.05).
2ABI showed no microbicidal effect, which is consistent with previous
studies [10]. The MIC was shown to be 200 μg/ml of CUR for both S.
aureus and C. albicans.
As reported earlier, the MIC of CUR on S. aureus ranges from 20 to
216 μg/mL [21,22]. while varies from 4.2 to 2000 μg/mL on Candida
[15,23]. Comparable antibacterial and antifungal activity was found in
Y. Tan et al.
Fig. 5. Influence of metal ions on 2ABI antibiofilm activity on Candida biofilm.
Values obtained are given as the percentage of biofilm. Results are expressed as
mean ± SD (* p < 0.05).
our work. Several studies have described the antimicrobial potential of
CUR on bacteria or Candida. The potential mechanisms underlying the
antimicrobial effect of CUR could be disruption of cell membrane or
wall, leading to cells lysis [15,17,21,24].
3.2. Adhesion assay
The effects of 2ABI and CUR on initial adhesion of the single species
of bacteria and fungi to medical grade silicone were shown in Fig. 1.
After 90 min incubation, 2ABI and CUR significantly inhibited the ad-
hesion of bacteria and Candida. At the concentration of 200 μg/ml, both
2ABI and CUR inhibited the adhesion of single species completely.
In a normal conjunction, the formation of biofilm includes initial
adhesion, cell-cell interactions and maturation phases [25]. The surface
colonization or adhesion is the initial stage, which provides the bene-
ficial contact between the medical devices surface and planktonic mi-
crobial cells. After adherence of microbe to the surface, the
Fig. 6. CLSM images of biofilm on medical grade silicone surface treated (A) without 2ABI or CUR, (B) with 2ABI alone, (C) with CUR alone or (D) with combination
of 2ABI and CUR. Biofilms were stained with the Live/Dead® BacLight™ Bacterial Viability and Counting Kit.
32
Colloids and Surfaces B: Biointerfaces 174 (2019) 28–34
microorganisms start the formation of biofilms with secretion of poly-
meric matrix. In our work, both 2ABI and CUR can interfere with ad-
hesion and inhibit the further development of biofilms.
3.3. Effect on biofilm formation
At first, we tested the inhibition activities of 2ABI or CUR alone on
single species biofilm formation. As shown in Fig. 2, both 2ABI and CUR
significantly reduced biofilm formations of bacteria and fungi. With the
increase of 2ABI or CUR concentration, a progressive inhibition of
biofilms was observed. C. albicans is more sensitive than S. aureus with
treatment of 2ABI or CUR. At the concentration of 200 μg/ml, 2ABI
inhibited 88.1% and 97.5% biofilm formation of S. aureus and C. albi-
cans, respectively (Fig. 2A and B). With the concentration of CUR in-
creased to 200 μg/ml, almost no biofilm formations of bacteria and
fungi could be observed (Fig. 2C and D).
Then the inhibition activities of combination of 2ABI and CUR on
single and mixed species biofilm formation were evaluated. Based on
the results above, here we used the concentration of 100 μg/ml for both
2ABI and CUR (0.5 × MIC) to assess the combination effect. 2ABI and
CUR alone could reduce mixed species as well as single species biofilm
formation (Fig. 3). When 2ABI and CUR were added together, the
biofilm formations of S. aureus, C. albicans and mixed species were in-
hibited by 92.3% (Fig. 3A), 96.5% (Fig. 3B) and 97.6% (Fig. 3D), re-
spectively.
The results showed that the use of CUR in combination with 2ABI
enhanced the inhibition effect on both single and mixed species biofilm
formations. The possible mechanism of enhancement can be bi-func-
tion: killing microorganism and preventing the adhesion and biofilm
formation simultaneously. On one hand, CUR kills the planktonic cell
directly in the medium. On the other hand, 2ABI prevents the biofilm
formation through inhibiting the interaction between microbe and
material surface, resulting in the microbe in the mixture as planktonic
forms. Thus, CUR can act on the single cell continuously.
3.4. Effect on preformed biofilm
To quantify the effect of 2ABI or CUR alone and the combined ef-
fects between 2ABI and CUR toward established biofilm, we used the
Y. Tan et al.
Fig. 7. CLSM images of biofilm on medical grade silicone surface treated (A) without 2ABI or CUR, (B) with 2ABI alone, (C) with CUR alone or (D) with combination
of 2ABI and CUR.
highest concentration of 2ABI or CUR in this work (200 μg/ml). The
combination of 2ABI and CUR reduced the mature biofilms of S. aureus,
C. albicans and mixed species by 47.7% (Fig. 4A), 90.6% (Fig. 4B) and
73.3% (Fig. 4C), which are more effective than that of 2ABI or CUR
alone.
It is known that microbial cells embedded in biofilm can increase
resistance to antimicrobials compared to planktonic forms. Recently,
the enhanced antibiofilm effect with drug combination has attracted
more attention [26,27]. Studies have reported that the combination of
biofilm dispersal agent and cell-killing agent could enhance the anti-
biofilm activity, based on different antibiofilm mechanisms [28,29]. In
this work, the combination of 2ABI and CUR enhanced the antibiofilm
effect against established biofilm, although both single and mixed
species biofilm exhibited more resistance to 2ABI and CUR alone even
at the highest concentration of 200 μg/ml. This finding can be attrib-
uted to the presumptive mechanism of two different actions. On one
hand, 2ABI restores the sensitivity of microbial cells to CUR through
disruption of biofilm, which results in more microbial cell being killed
by CUR. On the other hand, CUR can interfere with the planktonic
microbial cells, which were released from biofilm by 2ABI, and further
inhibit biofilm reformation. Therefore, the enhanced antibiofilm effect
was observed with the combination of 2ABI and CUR. Moreover, 2ABI
dosen’t only show antibiofilm activity on bacterial biofilm, but also
exhibits the biofilm dispersal effects on the Candida biofilm and mixed
species biofilms, which expands the application fields of 2ABI.
3.5. Influence of metal ions on 2ABI antibiofilm activity
In this set of experiments, a wide range of metals were selected in
order to examine the influence of metal ions on 2ABI antibiofilm ac-
tivity, including Ca(II), Cu(II), Fe(II), Fe(III) and Zn(II). As shown in
Fig. 5, only Cu(II) exhibited some inhibition response (without 2ABI),
which has been reported in other studies [30,31]. Moreover, the results
revealed that all of the tested metals didn’t change the inhibitory ac-
tivity of 2ABI on Candida biofilm.
Although it has identified 2ABI and derivatives as potent antibiofilm
agents, the antibiofilm mechanisms are different between Gram-posi-
tive and Gram-negative bacteria. Melander and co-workers have shown
that 2ABI derivatives effected on Gram-positive bacterial biofilms
through a zinc-dependent mechanism [10,12]. But the antibiofilm ac-
tivity on Gram-negative bacteria works through a quorum sensing (QS)-
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Colloids and Surfaces B: Biointerfaces 174 (2019) 28–34
dependent mechanism [32]. Here, we screened a wide range of metals
and found no mitigating effects on the antibiofilm activity of 2ABI in
Candida. It suggested that the mechanism of 2ABI antibiofilm activity
on Candida biofilm is not owing to metal sequestration. Therefore, it
might be due to a QS-dependent mechanism, which requires further
investigation.
3.6. Live/Dead assay
CLSM images revealed the cell viability of mixed biofilm treated
with 2ABI alone, CUR alone and combination of both. In the control
group (Fig. 6A) we can observe the multiple layers of green color,
which indicates the mature biofilm with a large number of live cells.
2ABI treatment (Fig. 6B) resulted in less biofilm thickness, but most of
the cells are alive (green). However, the use of CUR (Fig. 6C) didn’t
cause less biomass, but also more red color (dead cells). The treatment
with combination of 2ABI and CUR (Fig. 6D) led to almost complete red
color and full destruction of the biofilm architecture, which means the
synergy of 2ABI and CUR in killing disrupting the biofilm structure and
killing the cells.
3.7. SEM
The mixed biofilm structures on silicone disc with treatment of 2ABI
alone, CUR alone and combination of both were observed using SEM
(Fig. 7). The mixed biofilm grown in control group formed typical
biofilm structures (Fig. 7A). Images showed extensive interactions be-
tween bacteria and fungi. S. aureus clearly adhered to yeast and hyphal
forms of C. albicans. However, treated with 2ABI or CUR alone, biofilm
showed reduction of microbial colonization (Fig. 7B and C). The SEM
image of biofilm treated with combination of 2ABI and CUR displayed
much less cells adhesion and almost no biofilm structure (Fig. 7D).
4. Conclusion
Our work showed the inhibition and dispersion activity of 2ABI and
CUR on the single and mixed biofilm of bacteria and fungi on the sili-
cone surface. The combination of 2ABI and CUR exhibited the enhanced
effect on mixed biofilm. SEM and CLSM images confirmed these results.
Although further evaluation of 2ABI and CUR in vivo is still needed, our
studies indicate the potential applicability of 2ABI and CUR to
Y. Tan et al.
treatment of biofilm related device infections.
Conflict of interest
The authors claim no conflict of interest.
Submission declaration
The work described above has not been published previously or
under consideration for publication elsewhere.
Author contributions
The manuscript was written through contributions of all authors. All
authors have given approval to the final version of the manuscript.
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