Archives of Oral Biology 107 (2019) 104481

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Archives of Oral Biology

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

Antimicrobial and biofilm anti-adhesion activities of silver nanoparticles T

and farnesol against endodontic microorganisms for possible application in
root canal treatment
⁎
Gisselle Moraima Chávez-Andradea, Mário Tanomaru-Filhoa, , Maria Inês Basso Bernardib,
Renato de Toledo Leonardoa, Gisele Fariaa, Juliane Maria Guerreiro-Tanomarua
a
Department of Restorative Dentistry, São Paulo State University (UNESP), School of Dentistry, Araraquara, SP, Brazil
b
Institute of Physics, University of Sao Paulo (USP), São Carlos, SP, Brazil

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

Keywords: Objective: This study aimed to evaluate the antimicrobial and biofilm anti-adhesion activities of poly(vinyl al-
Biofilm cohol)-coated silver nanoparticles (AgNPs-PVA) and farnesol against Enterococcus faecalis, Candida albicans or
Endodontics Pseudomonas aeruginosa.
Farnesol Design: Minimum inhibitory concentration (MIC) and minimum microbicidal concentration (MMC) of the so-
Silver nanoparticles
lutions, as well as the effect on the biofilm biomass were evaluated. The biofilm anti-adhesion activity was
evaluated using bovine root dentine treated with the solutions after 3 min of contact and analyzed by scanning
electron microscopy (SEM) and by colony-forming units per milliliter (CFU mL−1) counting. Data were analyzed
using ANOVA and Tukey's, the paired Student’s t-test or Kruskal-Wallis and Dunn’s tests (α = 0.05).
Results: The MIC and MMC values (MIC/MMC) of the AgNPs-PVA and farnesol against E. faecalis were 42.5/
50 μM and 0.85/1.0%, respectively. For C. albicans, the values were 27.5/37.5 μM and 1.75/2.5%; and for P.
aeruginosa, 32.5/32.5 μM and 2.5/2.75%, respectively. Both solutions showed reduced biofilm biomass
(p < 0.05). SEM analysis showed that dentine blocks treated with both solutions had lower biofilm formation
than the control (saline), except for C. albicans. In the CFU mL−1 counting, biofilm cells were viable in the all
groups in comparison with control (p > 0.05).
Conclusions: AgNPs-PVA and farnesol showed antimicrobial and biofilm anti-adhesion activities, as well as
potential for use as coadjuvant in endodontic treatment, and may be an option as auxiliary procedure for root
canal disinfection or to inhibit biofilm formation.

1. Introduction
Irrigating solutions are used to promote root canal cleaning and
disinfection (Holland, Gomes, Cintra, Queiroz, & Estrela, 2017;
Siqueira, 2002; Tong, Zhang, & Wei, 2019). The sodium hypochlorite
(NaOCl) solution is the most used root canal irrigating solution due to
its antimicrobial effect (Estrela et al., 2002; Poggio, Colombo,
Scribante, Sforza, & Bianchi, 2012; Tong et al., 2019). However, its
cytotoxicity can promote complications when in contact with the
periapical tissues (Chavez-Andrade et al., 2017; Gondim, Setzer, Dos
Carmo, & Kim, 2010). Furthermore, NaOCl significantly decreases the
elasticity and flexural strength of human dentin by proteolytic activity
in the collagen matrix, thus interfering with the formation of the hybrid
layer (Zehnder, 2006). New substances and approaches have been
studied as alternatives to NaOCl (Alves, Silva, Rocas, & Siqueira, 2013;
Chavez-Andrade et al., 2017; Estrela et al., 2018; Holland et al., 2017;
Moghadas, Shahmoradi, & Narimani, 2012).
Silver nanoparticles (AgNPs) present bactericidal and antifungal
properties, and show potential for biomedical applications (de Castro
et al., 2016; Franci et al., 2015; Kalishwaralal, BarathManiKanth,
Pandian, Deepak, & Gurunathan, 2010; Monteiro et al., 2012; Shrestha
& Kishen, 2016). The antimicrobial activity is attributed to the release
of Ag+ ions, increasing permeability and causing damage to the bac-
terial cytoplasm and DNA (El-Batal & Ahmed, 2018; Konigs, Flemming,
& Wingender, 2015; Shrestha & Kishen, 2016). AgNPs of 1–10 nm
particle size adhere to the cell membrane surface and interfere in the

⁎
Corresponding author at: Department of Restorative Dentistry, Araraquara Dental School, São Paulo State University-UNESP, Rua Humaitá 1680, CEP 14.801-
903, Araraquara, SP, Brazil.
E-mail address: tanomaru@uol.com.br (M. Tanomaru-Filho).

https://doi.org/10.1016/j.archoralbio.2019.104481
Received 4 March 2019; Received in revised form 10 July 2019; Accepted 11 July 2019
0003-9969/ © 2019 Elsevier Ltd. All rights reserved.
G.M. Chávez-Andrade, et al.
metabolism of bacteria, such as the permeability and respiration
(Shrestha & Kishen, 2016).
Farnesol is a sesquiterpene alcohol naturally obtained from propolis
and essential oils of seeds, aromatic plants and citric fruits, which
presents antitumor, antimicrobial and antibiofilm activities (Alves,
Silva et al., 2013; Chavez-Andrade et al., 2017). Farnesol has antifungal
effect, damages the fungal cell membrane by activating the caspases
(Cordeiro et al., 2013), and may be used as an adjuvant antibiofilm
agent against C. albicans (Katragkou et al., 2015).
The persistence of microorganisms in the root canal system favors
the development and maintenance of apical periodontitis (Sakamoto,
Siqueira, Rocas, & Benno, 2007; Siqueira, 2002). Therefore, it is im-
portant to research and evaluate substances with antimicrobial effect
and minimal toxic effects on the periapical tissues. Enterococcus, Pseu-
domonas and Candida are isolated from endodontic infections, as well as
in cases of endodontic treatment failure (Pourhajibagher,
Ghorbanzadeh, & Bahador, 2017; Ranta, Haapasalo, & Ranta, 1988;
Siqueira & Rocas, 2004). E. faecalis (Gram-positive anaerobic bac-
terium) has ability to produce biofilm, to invade dentinal tubules, and
to develop a mono-infection (Tennert et al., 2014; Wilson, Cathro,
Rogers, Briggs, & Zilm, 2015). C. albicans (fungi) form biofilm, invade
the dentinal tubules, and resist to antimicrobial agents (Siqueira & Sen,
2004). P. aeruginosa (Gram-negative aerobic bacterium) has also been
isolated from root canals and persistent infection (Pourhajibagher et al.,
2017; Ranta et al., 1988), and has been used to evaluate the effec-
tiveness of antimicrobial agents (Gurunathan, Han, Kwon, & Kim, 2014;
Konigs et al., 2015). Microorganisms organized in biofilms are more
resistant to conventional endodontic procedures and disinfectants
(Fernandes et al., 2016; Konigs et al., 2015).
AgNPs and farnesol have potential to be used as coadjuvant in en-
dodontic treatment (Abbaszadegan et al., 2015; Alves, Neves, Silva,
Rocas, & Siqueira, 2013; Alves, Silva et al., 2013; Chavez-Andrade
et al., 2017). In a previous study, these antimicrobial agents showed
low cytotoxicity, without genotoxic effects on fibroblast cells, and had
ex vivo antimicrobial activity against E. faecalis (Chavez-Andrade et al.,
2017). Thus, the aim of this study was to evaluate the antimicrobial
potential of AgNPs-PVA and farnesol for application in the root canal
treatment. The null hypothesis is that there are no differences in the
antimicrobial and biofilm anti-adhesion activities of solutions against E.
faecalis, C. albicans or P. aeruginosa when compared to NaOCl.
2. Materials and methods
2.1. Synthesis and characterization of AgNPs-PVA
Synthesis and characterization of the poly(vinyl alcohol)-coated
silver nanoparticles (AgNPs-PVA) (50 μM) was performed at the
Institute of Physics, USP (São Carlos, SP, Brazil) (Chavez-Andrade et al.,
2017; Paino & Zucolotto, 2015). AgNPs-PVA were synthesized by re-
duction of silver nitrate salts (AgNO3) with sodium borohydride
(NaBH4). 20 mL of a 0.5 g L−1 poly(vinyl alcohol)-PVA was added in
20 mL of a 1.0 mmolL−1 silver nitrate solution and under magnetic
stirring for 5 min. Following, 1 mL of a 0.1 mol L−1 freshly prepared
sodium borohydride solution was quickly added to the reactional
medium. This synthesis was done in an ice bath at room temperature.
The solution immediately turned yellow confirming the formation of
silver nanoparticles. The solutions were then stored in dark bottles and
were found to be stable for more than 2 months.
The average size of AgNPs-PVA (4–11 nm) was determined by
Dynamic Light Scattering (DLS, Malvern instruments, UK) (Paino &
Zucolotto, 2015), present and under magnetic stirring (Fig. 1).
2.2. Farnesol solution preparation
Farnesol solution was obtained from the commercial product
3,7,11-Trimethyl-2,6,10-dodecatrien-1-ol, Ref. F203 (Sigma-Aldrich, St
2
Archives of Oral Biology 107 (2019) 104481
Fig. 1. Dynamic light scattering (DLS): size distribution analysis by intensity of
the AgNPs-PVA.
Louis, MO, USA) with concentration of 95%. Farnesol dilutions was
prepared in sterile distilled water immediately before use (Chavez-
Andrade et al., 2017).
2.3. Microorganisms and inoculum
Standardized E. faecalis strains (ATCC 29,212), C. albicans (ATCC
10,231) and P. aeruginosa (ATCC 27,853) were used. E. faecalis strain
was cultured overnight in Tryptic Soy broth (TSb), seeded onto Tryptic
Soy agar (TSa) and incubated at 37 °C for 24 h. The inoculum of C.
albicans and P. aeruginosa were obtained by seeding the strains in plates
with Sabouraud Dextrose agar (SDa) and Brain Heart Infusion agar
(BHIa), respectively. Microbial suspensions were prepared and adjusted
to optical density equivalent to 1 × 107 colony-forming units per mil-
liliter (CFU mL−1) using a spectrophotometer (Model 600 Plus, Femto,
SP, Brazil).
2.4. Determination of minimum inhibitory concentration (MIC) and
minimum microbicidal concentration (MMC)
The antibacterial activity of the AgNPs-PVA (50 μM), 4% farnesol
and 1% NaOCl solutions against the planktonic cells was evaluated by
determining the MIC and MMC. The broth microdilution method was
used in accordance with the M11-A8 standard of the CLSI - Clinical and
Laboratory Standards Institute (CLSI, 2012) and staining with resazurin
(Chhillar & Gahlaut, 2013). From the initial concentrations, micro-
dilutions were made in 96-well microtiter plates (Sarstedt AG & Co.,
Nümbrecht, Germany). Positive control group (C+, culture medium
with inoculum) and two negative control groups (C−, saline solution
and culture medium) were included. Aliquots of the solutions diluted in
culture medium were placed in each well; after, 10 μL of the standar-
dized inoculum was added and the plates were incubated at 37 °C for
24 h under appropriate conditions for each strain. After incubation,
20 μL of 0.01% resazurin solution (Sigma-Aldrich, St. Louis, MO, USA)
was added to each well (duplicate) and the plates were reincubated for
color change for 1 h. The MIC was defined as the lowest concentration
of the solutions that prevented change in color (microbial growth in-
hibition), indicated by the blue/purple resazurin coloring. MMC was
determined after MIC determination, by using aliquots from the wells
without coloring agent corresponding to MIC and two microdilutions,
before and after, which were seeded in Petri dishes with the culture
medium corresponding to each strain. After incubation at 37 °C for 24 h,
the MMC was defined as the lowest concentration that showed micro-
bial growth inhibition. The tests were performed in triplicate and re-
peated in 3 different time intervals.
G.M. Chávez-Andrade, et al.
2.5. Crystal violet assay
The effectiveness of the AgNPs-PVA (50 μM), 2% farnesol and 1%
NaOCl solutions on the biofilm biomass of E. faecalis, C. albicans or P.
aeruginosa was evaluated and quantified by the crystal violet assay
(Alves, Silva et al., 2013; Fernandes et al., 2016). The biofilms were
formed in 96-well microtiter plates (NEST Biotechnology, Wuxi, China)
in the culture medium appropriate for each microorganism, for 48 h at
37 °C in an orbital agitation incubator. After incubation, the content of
each well was aspirated and the wells were rinsed two times with
200 μL of sterile phosphate-buffered saline (PBS, pH 7). Next, 200 μL per
well of the solutions were added for 3 and 5 min. Positive controls
(sterile saline solution and culture medium with standard inoculum)
and negative control (sterile culture medium) were used. After re-
moving the solutions, the wells were rinsed two times with 200 μL of
PBS, and the plates were dried in an oven at 50 °C for 30 min. After-
wards, the wells with biofilm were stained with 200 μL of 0.1% violet
crystal solution (Synth, Diadema, SP, Brazil) at room temperature for
20 min. Excess stain was rinsed off by copious washing with distilled
water. Plates were overturned and air-dried, and the dye attached to the
adherent cells was solubilized with 150 μL of 33% acetic acid for 5 min.
The optical density (DO 590 nm) was measured in a spectrophotometer
UVM 340 (ASYS, Nova Analítica Importação e Exportação Ltda., SP,
Brazil), quantifying the biofilm biomass remaining. Final absorbance
values were obtained, and afterwards the reduction in biofilm biomass
was calculated in percentage (%) in comparison with the positive
control (Gurunathan et al., 2014). This experiment was performed in
triplicate and repeated in 3 different times.
2.6. Biofilm anti-adhesion activity
Bovine root dentine blocks (N = 72) measuring
5 mm × 5 mm × 0.7 mm (width × length × thickness) were prepared
using an Isomet 1000 (Buelher Ltda., Lake Bluff, Il, USA) cutting ma-
chine and used as the substrate for biofilm growth. The dentine blocks
were immersed in 17% EDTA (Odahcan-Herpo; Prod. Dent. Ltda., Rio
de Janeiro, RJ, Brazil) for 3 min, rinsed with sterile saline and sterilized
at 121 °C for 20 min. For each microbial strain (E. faecalis, C. albicans or
P. aeruginosa), dentine blocks (n = 24) were divided into two experi-
mental and one control sub-groups (n = 8). The dentine blocks were
treated with AgNPs-PVA (50 μM), 2% farnesol or saline solutions for
3 min, and were inoculated and incubated at 37 °C for 48 h (E. faecalis
and C. albicans) and 24 h (P. aeruginosa), in an environment appropriate
for each strain.
2.6.1. Scanning electron microscopy analysis
Two dentine blocks from each sub-group were rinsed twice in PBS
and fixed in glutaraldehyde, dehydrated in increasing concentrations of
alcohol and dried in Hexamethyldisilazane-HMDS (Electron Microscopy
Sciences, Industry Road, Hatfield, USA). After drying completely, the
specimens were analyzed by scanning electron microscopy (JEOL,
JSM–6610LV Scanning Electron Microscope, USA) at 15 kV. Images
were acquired in 6 areas of each specimen (n = 12 per group) at 1000×
and 2000× magnifications (for E. faecalis and P. aeruginosa), and at
500× and 1000× magnifications (for C. albicans). The biofilm
Table 1
The minimum inhibitory concentrations (MIC) and minimum microbicidal concentrations (MMC) of the tested solutions against E. faecalis, C. albicans or P. aeru-
ginosa.
Solutions MIC
E. faecalis C. albicans
AgNPs-PVA (μM) 42.5 27.5
Farnesol (%) 0.85 1.75
NaOCl (%) 0.25 0.5
AgNPs-PVA, poly(vinyl alcohol)-coated silver nanoparticles; NaOCl, sodium hypochlorite.
Archives of Oral Biology 107 (2019) 104481
formation was analyzed by scores, as follows: Score 0: 0% of microbial
cells adhered; Score 1: 25% of substrate with microbial cells adhered;
Score 2: 50% of substrate with microbial cells adhered; Score 3: 75% of
substrate with microbial cells adhered; and Score 4: substrate com-
pletely covered with microbial cells.
2.6.2. Analysis by CFU mL−1 counting
After the contact periods, other dentine blocks (n = 6 per group)
were individually placed in microtubes containing 1 mL of sterile saline
and glass pearls (3.5 ± 0.2 mm). The microtubes were agitated in a
vortex mixer (model Q220, Quimis Aparelhos Cientificos Ltda.,
Diadema, SP, Brazil) for 1 min to disrupt biofilm and serial decimal
dilutions were performed. Three 20 μL of aliquots of each suspension
was inoculated onto Petri dishes containing TSa (E. faecalis), SDa (C.
albicans) and BHIa (P. aeruginosa). After incubation at 37 °C for 48 h in
conditions appropriate for each strain, the number of CFU mL−1 was
calculated.
2.7. Statistical analysis
Statistical analysis was performed with the significance level set at
p < 0.05. The crystal violet assay results were analyzed by using
ANOVA and Tukey’s tests. To compare the same solution in the dif-
ferent periods (3 or 5 min), the paired Student’s t-test was used. For the
biofilm anti-adhesion test, data were analyzed by using Kruskal-Wallis
and Dunn’s tests (SEM analysis) and for analysis by CFU mL−1
counting, the data were subjected to logarithmic transformation and
analyzed by using ANOVA and Tukey’s tests.
3. Results
3.1. Determination of MIC and MMC
The MIC and MMC values of the new substances against the mi-
croorganisms tested are showed in Table 1. The MIC and MMC values
(MIC/MMC) of AgNPs-PVA and farnesol against E. faecalis were 42.5 /
50 μM and 0.85 / 1.0%, respectively. For C. albicans, the values were
27.5 / 37.5 μM and 1.75 / 2.5%; and for P. aeruginosa, 32.5 / 32.5 μM
and 2.5 / 2.75%, respectively. The new substances demonstrated a
microbicidal effect against the microorganisms tested.
3.2. Crystal violet assay
The effectiveness of the solutions on the biofilm biomass of E. fae-
calis, C. albicans or P. aeruginosa is represented in Fig. 2. All the solu-
tions evaluated showed reduction of biomass in comparison with the
positive control (culture medium with inoculum) and NaOCl was more
effective against all microbial strains (P < 0.05), in the different per-
iods (3 or 5 min). C. albicans was the species most resistant to both
solutions (AgNPs-PVA and farnesol), in the two periods evaluated.
Comparing the new solutions, AgNPs-PVA was more effective against E.
faecalis, in both periods; however, AgNPs-PVA and farnesol were si-
milarly effective against C. albicans. Farnesol was more effective against
P. aeruginosa in 3 min. The longest contact period promoted an increase
of reduction of biofilm for farnesol and NaOCl against C. albicans, and
MMC
P. aeruginosa E. faecalis C. albicans P. aeruginosa
32.5 50 37.5 32.5
2.0 1.0 2.5 2.75
0.55 0.3 0.55 0.55
3
G.M. Chávez-Andrade, et al.
for AgNPs and farnesol against P. aeruginosa (P < 0.05).
3.3. Biofilm anti-adhesion activity
Fig. 3 shows the micrographs (SEM) representative of the dentine
blocks treated with AgNPs-PVA, farnesol and saline. AgNPs-PVA and
farnesol inhibited the formation of E. faecalis and P. aeruginosa biofilms;
however, they did not inhibit C. albicans biofilm formation, but farnesol
changed the fungal cell structure, thereby inhibiting the formation of
hyphae. The results of the analysis in scores are showed in Table 2.
AgNPs-PVA and farnesol showed a lower quantity of biofilm adhered to
the substrate when compared with the control (saline), however,
without statistical difference between the two solutions (p > 0.05).
Against C. albicans, all the groups showed the same quantity of mi-
crobial cells adhered to the substrate (Score 4). Farnesol showed the
lowest score against P. aeruginosa (p < 0.05).
Fig. 4 shows the results of the CFU mL−1 log10 counting, in which
no statistically significant differences was observed between AgNPs-
PVA and farnesol solutions (p > 0.05), being similar to those of the
Fig. 3. Micrographs (SEM) representative of bovine dentine blocks treated with AgNPs-PVA, farnesol and saline.
4
Archives of Oral Biology 107 (2019) 104481
Fig. 2. Violet crystal assay: microbial biofilms
of E. faecalis (A), C. albicans (B) or P. aeruginosa
(C) were stained with 0.1% crystal violet so-
lution after 3 or 5 min of contact with the so-
lutions and quantification was performed by
determining the absorbance at 590 nm.
Different capital letters in same line indicate
statistically significant differences among the
groups in the same period. Different lower case
letters in the column indicate statistically sig-
nificant differences among the periods in the
same group (p < 0.05).
control group (saline), for the three strains evaluated.
4. Discussion
Silver nanoparticles (AgNPs) are considered one of the most pro-
mising nanomaterials, with an efficient antimicrobial effect, in addition
to anti-inflammatory, antifungal, and antiviral effects (de Castro et al.,
2016; El-Batal & Ahmed, 2018). The chemical reaction is one of the
most used method to synthesize AgNPs, and is based on reducing agent
and stabilizer to control particle growth and prevent agglomeration
(Monteiro et al., 2012). This aspect is important to provide anti-
microbial efficacy to the prepared solution, allowing the contact of
nanoparticles with the cellular structure of the microorganism. AgNPs
solution used in this study was synthesized by reduction of AgNO3 in
NaBH4 (reducing agent) and poly(vinyl alcohol)-PVA was used as sta-
bilizer (Paino & Zucolotto, 2015). Poly(vinyl alcohol)-PVA was used in
synthesis of the AgNPs-PVA solution in the present study, due itis
biocompatibility, high solubility in water and chemical resistance,
which allow its use as a stabilizing agent and for coating the AgNPs
G.M. Chávez-Andrade, et al.
Table 2
Median (Med), maximum/minimum and first/third quartile values (1Q–3Q) of the scores attributed, after the use of different protocols for treatment of dentine
blocks.
Groups AgNPs-PVA Farnesol
Strains Med min-max 1Q–3Q Med
E. faecalis 2.0a 1–3 1.25–2 1.0a
C. albicans 4.0A 4–4 4–4 4.0A
P. aeruginosa 3.5a 3–4 3–4 2.0b
Different letters in same line indicate statistical difference among the groups in each microbial strain (p < 0.05). Med, median; min-max, minimum-maximum; Q,
quartile.
(Chavez-Andrade et al., 2017; Paino & Zucolotto, 2015).
In the present study, the efficacy of NPsAg-PVA and farnesol solu-
tions on planktonic cells and monospecies biofilms of pathogenic en-
dodontic microorganisms (E. faecalis, C. albicans and P. aeruginosa) were
evaluated. MIC and MMC data showed that E. faecalis was less sus-
ceptible to AgNPs-PVA than the other strains, which may be explained
by the different cell structures of the species evaluated. AgNPs had less
effect on the growth of Gram-positive bacteria. The Gram-negative
bacteria present an external layer with negative charges that is at-
tracted by the weak positive charge of the AgNPs. On the other hand,
the cell wall of the Gram-positive bacteria is mainly composed of a thick
layer that reduces the binding of the AgNPs to the bacterial cell wall
(Franci et al., 2015). This may also explain the results of the antibiofilm
assay (crystal violet), in which the AgNPs-PVA promoted greater re-
duction in the biofilm biomass of P. aeruginosa than that of E. faecalis.
Some authors have suggested specific mechanisms of action against
these bacterial species, and thus, against E. faecalis, AgNPs caused
change in the cell wall and cytoplasm (Franci et al., 2015; Wu, Fan,
Kishen, Gutmann, & Fan, 2014). Nevertheless, AgNPs produced irre-
versible damage to the cells of P. aeruginosa, with changes in the
membrane permeability (Franci et al., 2015).
The antifungal activity of AgNPs against C. glabrata and C. albicans
was evaluated and the results showed that these species were sensitive,
however, they were not effective in the reduction of the biofilm biomass
of C. albicans (Monteiro et al., 2012), in agreement with our results. C.
albicans is a polymorphic organism that forms a complex biofilm that is
difficult to eradicate. Moreover, when the biofilm is exposed to stressful
conditions, physiological changes may occur to protect the cells. AgNPs
could interact with the microorganisms in the biofilm, inducing ag-
gregation of the particles in suspension (Kalishwaralal et al., 2010), and
Fig. 4. Biofilm anti-adhesion activity: bovine dentine blocks treated with the solutions after 3 min of contact. Mean and standard deviation of UFC mL−1 log10 values
for the solutions in each microbial strain. Different letters on the same column indicate statistically significant differences (p < 0.05).
5
Archives of Oral Biology 107 (2019) 104481
Control (saline)
min-max 1Q–3Q Med min-max 1Q–3Q
0–2 1–1.75 3.0b 3–4 3–4
4–4 4–4 4.0A 4–4 4–4
2–3 2–2.75 4.0a 4–4 4–4
thus diminishing contact with the fungal cells.
AgNPs inhibited the growth of P. aeruginosa in planktonic cells and
in biofilm formed for 24 h (Konigs et al., 2015). Similarly, studies have
shown that AgNPs presented excellent antibacterial properties against
P. aeruginosa, by inhibiting biofilm formation (Kalishwaralal et al.,
2010). Nanosilver-based endodontic irrigation solution was evaluated
in comparison to NaOCl against E. faecalis and S. aureus. No bacterial
growth was observed, so they concluded that the new nanobased irri-
gant was as effective as NaOCl (Moghadas et al., 2012). However, other
study (Wu et al., 2014) evaluated the antibacterial efficacy of AgNPs
against E. faecalis biofilms. The integrity of the biofilm structure was
not destroyed after 2 min in contact with the AgNPs solution, in
agreement with our results.
C. albicans was less susceptible to the action of farnesol than E.
faecalis, with higher MIC/MCC values, a lower percentage of reduction
in biomass in comparison with the two bacteria strains, and without
inhibiting biofilm formation. The cell wall thickness of this fungal
species may make it difficult for farnesol to act. Some studies have
shown the antifungal efficacy of farnesol (Cordeiro et al., 2013;
Fernandes et al., 2016) that could be explained due to the different
dilution protocols and inoculum concentrations. The inoculum was
standardized at 1 × 107 CFU mL−1 in all the tests performed, according
to a previous study (Van den Driessche, Rigole, Brackman, & Coenye,
2014).
Farnesol and associations were effective for the reduction in biofilm
biomass (Alves et al., 2013). All substances affected the bacterial via-
bility in biofilms and farnesol presented efficacy, however, it was not
better than NaOCl, in accordance with our results. Farnesol is an ad-
juvant antibiofilm agent against C. albicans (Cordeiro et al., 2013;
Katragkou et al., 2015) and effective against single and mixed species
G.M. Chávez-Andrade, et al.
biofilms formed by C. albicans and S. mutans. Farnesol reduced the
biofilm biomass of C. albicans, as well as the number of CFUs, in
comparison with the negative control (Fernandes et al., 2016). Fur-
thermore, SEM micrographs showed that farnesol changed the mor-
phology of the adhered fungal cells, in agreement with the present
study.
Farnesol may affect both the viability and antibiotic resistance in a
number of bacterial and fungal species (Cugini et al., 2007). Against P.
aeruginosa, farnesol had various effects, since it induced oxidative
stress, stimulated the production of fenazine and diminished the pyo-
cyanin levels (its main virulence factor) (Cugini, Morales, & Hogan,
2010).
Crystal violet assay is considered simple and effective for quanti-
fying the reduction in total biofilm biomass, which includes the cells
and extracellular matrix. However, in addition to staining the extra-
cellular matrix, it also stains the viable and dead cells (Monteiro et al.,
2015). Consequently, on quantifying the biofilm biomass after treat-
ment with the antimicrobial agents, we could observe that there was no
reduction in the biomass; but perhaps the microbial cells were not vi-
able and had their metabolic activity irreversibly compromised. For this
reason, the use of CFU counting and analysis by SEM in this study al-
lowed to observe the quantity and cell morphology that adhered to the
bovine root dentine substrate. Farnesol was unable to inhibit the for-
mation of biofilm for C. albicans, however, it changed the fungal cell
structure without the formation of hyphae, as demonstrated by SEM.
The characteristic of biofilms, such as the concentration of oxygen
(O2), pH, composition and substrate, may have an influence on ag-
gregation of AgNPs and consequently, on the diffusion and anti-
microbial effect against the biofilm cells (Stewart & Franklin, 2008).
This affirmation may justify our results in the different tests, in which
the AgNPs-PVA showed a reduction of biomass, highlighting their effect
on P. aeruginosa; however, in the CFU mL−1 counting, AgNPs-PVA was
similar to the groups treated with saline solution. Thus, the null hy-
pothesis was rejected. AgNPs-PVA and farnesol showed antimicrobial
and biofilm anti-adhesion activities, but were less effective than NaOCl.
5. Conclusions
In this study, AgNPs-PVA and farnesol showed antimicrobial and
biofilm anti-adhesion activities against E. faecalis, C. albicans and P.
aeruginosa, and may be an option as auxiliary products for disinfection
of root canal system, or to inhibit biofilm formation/adhesion. AgNPs
and farnesol may be an option in cases where NaOCl has limitations of
use and/or side effects, including cases of hypersensitivity. In addition,
these solutions present potential for use in different root canal irrigation
protocols, as final irrigation after root canal biomechanical preparation.
Moreover, these substances can be used in association with other ma-
terials (intracanal medications, root canal sealers and/or repair ce-
ments), to improve the antimicrobial effect of these materials.
Ethical approval
This study has no need for prior approval by an ethics committee.
Declaration of Competing Interest
The authors declare that they have no conflict of interest.
Acknowledgment
This study was supported in part by the Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) -
Finance Code 001, and by FAPESP [São Paulo State Research
Foundation, grant number 2012/11362-8, 2017/14305-9].
6
Archives of Oral Biology 107 (2019) 104481
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