Biofouling

The Journal of Bioadhesion and Biofilm Research

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Biofilm inhibition and anti-quorum sensing activity

of phytosynthesized silver nanoparticles against
the nosocomial pathogen Pseudomonas aeruginosa

Saloni Shah, Swapnil Gaikwad, Shuchi Nagar, Shatavari Kulshrestha, Viniti

Vaidya, Neelu Nawani & Sarika Pawar

To cite this article: Saloni Shah, Swapnil Gaikwad, Shuchi Nagar, Shatavari Kulshrestha, Viniti
Vaidya, Neelu Nawani & Sarika Pawar (2019) Biofilm inhibition and anti-quorum sensing activity of
phytosynthesized silver nanoparticles against the nosocomial pathogen Pseudomonas�aeruginosa,
Biofouling, 35:1, 34-49, DOI: 10.1080/08927014.2018.1563686

To link to this article: https://doi.org/10.1080/08927014.2018.1563686

View supplementary material Published online: 07 Feb 2019.

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BIOFOULING
2019, VOL. 35, NO. 1, 34–49
https://doi.org/10.1080/08927014.2018.1563686

Biofilm inhibition and anti-quorum sensing activity of phytosynthesized

silver nanoparticles against the nosocomial pathogen
Pseudomonas aeruginosa
Saloni Shaha, Swapnil Gaikwada, Shuchi Nagarb, Shatavari Kulshresthaa, Viniti Vaidyaa, Neelu Nawania and
Sarika Pawara
a
Microbial Diversity Research Centre, Dr. D. Y. Patil Biotechnology and Bioinformatics Institute, Dr. D. Y. Patil Vidyapeeth, Pune, India;
b
Bioinformatics Research Laboratory, Dr. D. Y. Patil Biotechnology and Bioinformatics Institute, Dr. D. Y. Patil Vidyapeeth, Pune, India

ABSTRACT ARTICLE HISTORY

Quorum sensing (QS), the communication signaling network, regulates biofilm formation and Received 25 May 2018
several virulence factors in Pseudomonas aeruginosa PAO1, a nosocomial opportunistic patho- Accepted 18 December 2018
gen. QS is considered to be a challenging target for compounds antagonistic to virulent factors.
KEYWORDS
Biologically synthesized silver nanoparticles (AgNPs) are reported as anti-QS and anti-biofilm
Piper betle; silver
drugs against bacterial infections. The present study reports on the synthesis and characteriza- nanoparticles; anti‒quorum
tion of Piper betle (Pb) mediated AgNPs (Pb-AgNPs). The anti-QS activity of Pb-AgNPs against sensing; biofilm inhibition;
Chromobacterium violaceum and the potential effect of Pb-AgNPs on QS-regulated phenotypes Pseudomonas aeruginosa
in PAO1 were studied. FTIR analysis exhibited that Pb-AgNPs had been capped by phytochem- PAO1; molecular docking
ical constituents of Pb. Eugenol is one of the active phenolic phytochemicals in Pb leaves, there-
fore molecular docking of eugenol-conjugated AgNPs on QS regulator proteins (LasR, LasI and
MvfR) was performed. Eugenol-conjugated AgNPs showed considerable binding interactions
with QS-associated proteins. These results provide novel insights into the development of phy-
tochemically conjugated nanoparticles as promising anti-infective candidates.

Introduction look for alternative approaches to fight against P. aer-

The development of multiple drug resistant (MDR)
bacterial strains is a result of enormous selective pres-
sure exerted by extreme and random applications of
conventional antibiotics. About 14–15 million patients
die yearly because of diseases caused by such MDR
strains (Dye 2014). Nosocomial opportunistic patho-
gens such as Pseudomonas aeruginosa cause pulmon-
ary infection in immuno-compromised persons and
patients suffer from chronic lung diseases such as cys-
tic fibrosis. Moreover, about 10% of nosocomial bac-
terial infections are caused by this bacterium (Gellatly
and Hancock 2013). P. aeruginosa comprises both
cell-associated determinants and several secreted fac-
tors which contribute to its pathogenicity and toxicity
effect (Balasubramanian et al. 2013). In addition, it
adopts a biofilm mode of lifestyle on both abiotic and
biotic substrata which develops high resistance to
both host immune defense and antibiotics (Donlan
and Costerton 2002). Therefore there is a necessity to
uginosa (O’Loughlin et al. 2013; Pawar et al. 2016).
P. aeruginosa facilitates the establishment of infec-
tion via microbial communication by the quorum
sensing (QS) signaling system (Schuster and Greenberg
2006; Castillo-Ju
arez et al. 2015). This system utilizes
small chemical molecules termed autoinducers (AIs) –
Gram-negative bacteria synthesize AHLs (acyl homo-
serine lactones) while Gram-positive bacteria synthesize
small oligopeptides (Atkinson and Williams 2009).
These AI molecules stimulate expression of several
genes in the targeted bacterial cell when a high popula-
tion density is attained and controls various pheno-
types such as bioluminescence, biofilm formation
(Davies et al. 1998), biosurfactant production (Dusane
et al. 2010) and swarming (Eberl et al. 1996) which
contribute to bacterial pathogenesis.
There are four different interconnected QS signal-
ing networks in P. aeruginosa, viz. LasI/LasR, RhlI/
RhlR, the Pseudomonas quinolone signal (PQS)/MvfR
and IQS (integrated QS). All these pathways are well

CONTACT Sarika Pawar sarika.pawar@dpu.edu.in

Supplemental data for this article can be accessed here at https://doi.org/10.1080/08927014.2018.1563686.
ß 2019 Informa UK Limited, trading as Taylor & Francis Group

regulated in a hierarchical manner. The two QS path-
ways, Las and Rhl, produce AHL signaling molecules
such as 3-oxododecanoyl-l-homoserine lactone
(3-oxo-C12-HSL) and butyryl-l-homoserine lactone
(C4-HSL), by the LasI and RhlI synthase enzymes,
respectively (Lee and Zhang 2015; Rampioni et al.
2016). The third QS system responds to the PQS sig-
naling molecule 2-heptyl-3-hydroxy-4(1H)-quinolone
which binds to its transcriptional regulator MvfR
(PqsR) (D
eziel et al. 2005). At their appropriate con-
centrations, these HSL and PQS autoinducers bind to
their respective receptor proteins, named LasR, RhlR,
and MvfR, respectively, and induce the transcription
of several genes that encode for different virulence
products like elastases, rhamnolipids, siderophores
(pyoverdine) and pyocyanin (Strateva and Mitov
2011; Moradali et al. 2017). Moreover, the function of
these QS pathways in P. aeruginosa biofilm formation
was described by Davies et al. (1998). P. aeruginosa
which is defective in QS systems is impaired in viru-
lence and produces a flat biofilm sensitive to antibiot-
ics (Jensen et al. 2007; Van Gennip et al. 2009).
To date, several studies have proved that the bac-
terial virulence factors can be targeted by inhibiting
the QS system using compounds isolated from plants
and/or synthetic drugs (Borges et al. 2016). Some of
these QSIs have also demonstrated promising preclin-
ical benefits in conjunction with conventional antibi-
otics (Rasmussen, Bjarnsholt, et al. 2005; Jakobsen
et al. 2012). However, due to inefficient systemic solu-
bility, stability, delivery, and bioavailability, the appli-
cation of these QSIs to clinical studies has shown
limited uses (Defoirdt et al. 2013). In addition, the
pharmacokinetics of these inhibitors, specifically
plant-based QSIs, indicate that even high doses might
not be adequate to attain their real anti-QS effect
(Murugan et al. 2013; Wu et al. 2015). Thus, there is a
need to search for the other potential compounds/drugs
that would efficiently inhibit the QS signaling network.
In recent years, several researchers have started
using nanotechnology for the development of the
next-generation nano-antimicrobials, including ‘QS
nano-inhibitors’. These have many advantages, such
as better mucus and biofilm penetration, higher solu-
bility, effective delivery, and maintenance of the activ-
ity of QSIs (Nafee et al. 2014).
Biological synthesis of metal nanoparticles using
different plant parts (Gade et al. 2010), microorgan-
isms (Konishi et al. 2007; Gaikwad et al. 2013) and/or
enzymes (Willner et al. 2006) is emerging as a poten-
tial eco-friendly and safe option to chemical or phys-
ical methods. Plant-mediated metal nanoparticles can
BIOFOULING 35
be more useful than those obtained by microbe-medi-
ated synthesis, because plants or plant extracts do not
demand any sophisticated method of maintenance or
preservation like microbial cultures (Singh et al. 2016;
Rafique et al. 2017).
Silver nanoparticles (AgNPs) are attracting much
attention in the fields of biomedical technology because
of their antibacterial, anti-biofilm, anti-cancer and anti-
oxidant properties (Taglietti et al. 2014; Ghosh et al.
2015; Zhang et al. 2016). There are reports on anti QS
and the biofilm inhibitory activity of green synthesized
AgNPs/nanocomposites (Masurkar et al. 2012; Singh
et al. 2015; Kulshrestha et al. 2017; Srinivasan et al.
2017). Piper betle L. (Pb) is a widely cultivated plant in
the Indian subcontinent and Southeast Asia and has
long been in use for several medicinal formulations.
There are a few reports on Pb-functionalized AgNPs
(Pb-AgNPs) for their antibacterial, anti-QS and anti-
cancer activities (Shanmuga Prabha et al. 2014; Preethi
and Padma 2016; Srinivasan et al. 2017), but the anti-
QS and anti-biofilm activity of Pb-AgNPs against P.
aeruginosa PAO1 have not been explored so far. Here
one-step synthesis of AgNPs has been demonstrated
using Pb leaf extract and the anti-QS and anti-biofilm
activity of these nanoparticles against P. aeruginosa
PAO1 is evaluated. Further, the mechanism of action
of AgNPs and eugenol-conjugated AgNPs was exam-
ined by computational molecular docking with P. aeru-
ginosa PAO1 quorum sensing regulator proteins.
Materials and methods
Chemicals and reagents
Elastin Congo red (ECR) and dimethyl sulfoxide
(DMSO) were procured from Sigma Aldrich (Mumbai,
India). Silver nitrate (AgNO3) was obtained from
Qualigens (Mumbai, India). Crystal violet, Tris-HCl,
acridine orange, tryptone, yeast extract, glucose and
sodium chloride (NaCl) and media such as Luria
Bertani (LB) broth and agar and nutrient broth No. 2
were procured from Hi Media Laboratories Pvt. Ltd
(Mumbai, India).
Bacterial culture conditions
P. aeruginosa PAO1 (MTCC 3541, Chandigarh) and
Chromobacterium violaceum 12472 (MCC 2290,
Pune) were routinely grown in sterile Luria Bertani
(LB) (Hi Media Laboratories) medium with 120 rpm
agitation in a laboratory shaker. P. aeruginosa PAO1
was cultured at 37
C and C. violaceum at 30
C. The
cultures were preserved at 80
C in LB medium
36 S. SHAH ET AL.
containing 40% (v v–1) glycerol. All the laboratory
experiments were performed in triplicate.
Preparation of Pb leaf aqueous extract
Pb fresh leaves were purchased from a local market,
cleaned with sterile deionized water (DDW) to remove
dust and other impurities, and ensuring that no particu-
late impurities were left. After washing, the leaves were
allowed to dry in shade and in the dark for 3‒4 days at
room temperature. For extract preparation, 1 g of dried
leaf powder was boiled with DDW (100 ml) at 90
C for
15 min. The solution was filtered through Whatman No.
1 paper and centrifuged at 5,000 g for 20 min in order
to separate any residual materials. The extract solution
was kept at 4
C for further experiments.
Synthesis of Pb-AgNPs
For synthesis of Pb leaf-derived AgNPs (Pb-AgNPs),
AgNO3 from Qualigens was used without further puri-
fication. Pb-AgNPs synthesis was performed by adding
10 ml of dry leaf extract to 0.1 ml of AgNO3 solution
(100 mM). The reaction mixture was then exposed to
direct sunlight until the appearance of a brown color
(Pb-AgNPs). The bio-reduction of the Ag þ ions in
reaction mixture was checked by sampling at regular
intervals using the UV-vis spectrum of the reaction
mixture. The colloidal nanoparticles were centrifuged
(REMI, Mumbai, India) at 5,000 g for 15 min. The pel-
let obtained was washed thoroughly with DDW and
dried to get Pb-AgNPs powder.
Characterization of Pb-AgNPs
AgNPs synthesized using aqueous extracts of Pb leaves
were analyzed using UV-vis spectroscopy (BioTek
Epoch, Winooski, Vermont, USA) in the range 300–
900 nm. TEM (transmission electron microscope) ana-
lysis of the Pb-AgNPs was performed using a Philips
CM 200 TEM, Eindhoven, The Netherlands operating
at 20–200 kV and nanoparticle size was analyzed by
examining a TEM image by Image J software (Image J
1.46r; Java 1.6.0_20, Collins, 2007) by adding  2 mg
of Pb-AgNPs on a carbon-coated copper grid of 300
mesh size. The surface topography of Pb-AgNPs was
checked by scanning electron microscopy (SEM). The
Pb-AgNPs samples were coated with platinum and
examined by SEM (Nova NanoSEM, Hillsboro, State of
Oregon, Washington) at 15 KV. Furthermore, the XRD
patterns of the powdered Pb-AgNPs samples were eval-
uated on an XRD system (Rigaku Corporation, Tokyo,
Japan) operating at 40 kV. For FTIR spectroscopy ana-
lysis the Pb-AgNPs samples were mixed with KBr
(potassium bromide) and spectra were measured in the
wave number range 400–4,000 (cm1) on a JASCO
FTIR 6100 model (Tokyo, Japan).
Evaluation of the minimum inhibitory
concentration (MIC) of Pb-AgNPs
For this assay, the stock solution of Pb-AgNPs was
prepared by dissolving 1 mg of Pb-AgNPs powder in
1 ml of sterile LB broth. The MICs of Pb-AgNPs
against P. aeruginosa PAO1 and C. violaceum 12472
strains were evaluated according to CLSI guidelines
(Clinical and Laboratory Standards Institute), by the
double dilution method using 96-well microtiter plates
(MTPs) (Pattnaik, Ahmed, et al. 2018). Briefly, overnight
grown cultures were inoculated into sterile LB broth with
different concentrations of Pb-AgNPs and incubated for
20 h at 37
C for PAO1 and 30
C for C. violaceum. The
concentration of Pb-AgNPs at which there was no visible
bacterial growth was considered as its MIC.
Anti-QS activity of Pb-AgNPs against
C. violaceum 12472
The effect of sub-lethal Pb-AgNPs concentrations on
the QS mediated purple color violacein production in
the biomarker strain, C. violaceum 12472, was quanti-
tatively analyzed as described earlier (Pattnaik,
Ahmed, et al. 2018). One ml of Pb-AgNPs treated and
untreated bacterial cells was centrifuged (6,000 g for
15 min) to precipitate insoluble pigment. The super-
natant was discarded and 1 ml of dimethyl sulfoxide
was added to the pellet. The solution obtained was
vortexed for 60 s to solubilize violacein and recentri-
fuged at 6,000 g for 15 min. The absorbance of viola-
cein-containing supernatant was measured at 585 nm.
Simultaneously, the effect of the tested concentrations
of Pb-AgNPs on C. violaceum 12472 viability was
determined by the standard plate count method.
Growth assay
A growth assay was performed to check the effect of
Pb-AgNPs on the growth of P. aeruginosa PAO1.
0.1 ml of overnight grown P. aeruginosa PAO1 cells
(0.5 at OD600) was inoculated into 20 ml of sterile LB
medium in the absence and presence of 8 mg ml1 of
Pb-AgNPs. All the samples were incubated at 37
C at
200 rpm. The optical density (OD600 nm) was meas-
ured at regular intervals up to 18 h. The assay was

carried out in triplicate with suitable controls
(Adonizio et al. 2008).
Effect of Pb-AgNPs on QS-controlled virulence
factors in PAO1
Pyocyanin assay
Pyocyanin was extracted by the method of Imperi et al.
(2013) where a P. aeruginosa PAO1 culture was grown
at different concentrations of Pb-AgNPs (0, 2, 4, 6, and
8 mg ml1) and incubated for 20 h at 37
C. After 20 h,
pyocyanin was extracted in three parts of chloroform
(v v–1); the lower layer of chloroform was then re-
extracted with 0.2 M HCl. The absorbance of the
obtained pink colored solution was measured at 520 nm.
Elastase assay
The elastase activity of P. aeruginosa PAO1 was eval-
uated by a previously reported method, with slight
modifications (Vasavi et al. 2016). P. aeruginosa
PAO1 culture supernatant (100 ml) treated with 0, 2,
4, 6, and 8 mg ml1 of Pb-AgNPs was mixed with
900 ml of ECR buffer (100 mM Tris, 1 mM CaCl2, pH
7.5 with 10 mg of ECR) and then the tubes were incu-
bated at 37
C for 4 h. The reaction mixtures were
then centrifuged at 5,000 g for 20 min to remove the
insoluble ECR. The absorbance of the supernatant
obtained was determined by reading at 495 nm. The
percentage change in absorbance with respect to an
untreated sample was then estimated to get the reduc-
tion in elastase activity.
Swarming assay
The swarming motility of P. aeruginosa PAO1 is a
behavioral and synchronized surface translocation of
cells across a semi-solid medium (Datta et al. 2016). A
PAO1 culture grown in LB for 16 h was spotted at the
center of swarming agar plates (0.8% nutrient broth
No.2, 0.5% filter sterilized glucose, 0.5% agar). The
plates were supplemented with 6 and 8 mg ml1 con-
centrations of Pb-AgNPs. After incubation for 18 h at
37
C, swarming motility was monitored at the air–a-
gar interface. Control plates were without Pb-AgNPs.
Swimming assay
An overnight grown culture of P. aeruginosa
PAO1was spot inoculated onto swimming agar plates
(0.1% tryptone, 0.5% NaCl, 0.05% yeast extract, 0.3%
agar) by using a sterile wooden toothpick. Plates were
BIOFOULING 37
supplemented with 6 and 8 mg ml1 concentrations of
Pb-AgNPs as described in the swarming assay. Plates
without Pb-AgNPs were used as a control for the
experiments (Imperi et al. 2013).
Effect of Pb-AgNPs on PAO1 biofilm
Crystal violet assay
The effect of Pb-AgNPs on P. aeruginosa PAO1 biofilm
formation was analyzed by the crystal violet (CV) assay
in 96-MTP as performed by Anjugam et al. (2018) with
a few modifications. Briefly, an overnight grown and
then diluted P. aeruginosa PAO1 culture in the absence
and presence of increasing concentrations (0, 2, 4, 6,
and 8 mg ml1) of Pb-AgNPs was incubated for 18 h at
37
C. After this, planktonic cells were measured at
OD600 while the attached bacterial cells were washed
with sterile phosphate buffer. Then the plate was air
dried and stained with crystal violet (1% w v–1) solution
for the next 20 min at room temperature. Then the wells
were washed with sterile DDW and the surface associ-
ated CV stain was solubilized with ethanol for another
30 min. The OD570 was measured in an Epoch plate
reader (Anjugam et al. 2018). The percentage of PAO1
biofilm inhibition was calculated using the formula: %
of inhibition ¼ [(ControlOD570 nm –TreatedOD570 nm)/
ControlOD570 nm]  100
Biofilm detection using fluorescence microscopy
P. aeruginosa PAO1 biofilm formation was monitored
on sterile glass coverslips placed in 12-well plates sup-
plemented with 0, 6 and 8 mg ml1 of Pb-AgNPs and
incubated under static conditions for 20 h at 37
C
(Rajesh and Rai, 2014). After 20 h, the coverslips were
removed carefully from the wells without disturbing
the biofilm and then rinsed with sterile 1 mM MOPS
buffer (3-N-morpholino-propane-sulfonic acid), pH 7.
The rinsing was repeated three to four times to remove
freely suspended planktonic cells from the coverslips.
This was followed by air drying at room temperature.
The coverslips were then stained using 0.1% acridine
orange for 3 min and were gently rinsed with MOPS
buffer. Both treated and control coverslips were
observed under a fluorescence microscope (Olympus
CX41, Hamburg, Germany) at 100 magnification.
Computational molecular docking studies
The Patchdock online tool was used to independently
study the interaction between natural ligand, furanone
C30, AgNP, eugenol and eugenol þ AgNP with LasI
38 S. SHAH ET AL.
(AHL synthase) and with LasR/MvfR (transcriptional
receptor proteins) (Schneidman-Duhovny et al. 2005).
PatchDock determines the best protein ligand inter-
action based on the shape complementarity of soft
molecular surfaces. The crystallographic structures of
the three QS proteins LasI synthase (PDB ID: 1RO5,
resolution ¼ 2.3 Å), LasR transcriptional protein
(PDB ID: 2UV0 chain E, resolution ¼ 1.8 Å) and
MvfR transcriptional protein (PDB ID: 4JVC, reso-
lution ¼ 2.5 Å) were obtained from the RCSB Protein
Data Bank (PDB) [https://www.rcsb.org/pdb]. The
compound eugenol was obtained from the Pubchem
database (NCBI PubChem CID: 3314) and silver ion
was obtained from the protein databank. Eugenol was
modified to add Ag ion thus forming a complex of
eugenol-Ag in ChemOffice (ChemOffice, 2002). All
the ligands were energy minimized with ChemOffice.
The water molecules and the native ligand were
deleted from the protein files. The ligand files were
prepared as per the requirement of the tool and the
docking parameters were kept as default. The relative
square mean distance (RMSD) clustering was kept at
< 4.0 Å as suggested by the tool. The result files
obtained from the Patchdock server after docking
were analyzed and the figures were generated in a
Discovery Studio Visualizer v17.2.0. The active sites
of these three proteins were retrieved from previous
literature (Parai et al. 2018; Ali et al. 2017).
Statistical analysis
The Student’s t-test was used to calculate the differen-
ces between two mean values. Comparative analysis
of multiple means was performed by one-way analysis
of variance (ANOVA) using the online link http://
www.physics.csbsju.edu/stats/anova.html. p values <
0.05 were considered as statistically significant.
Results and discussion
Synthesis of Pb-AgNPs
Pb-AgNPs were synthesized using the aqueous extract
of Pb leaves with the help of natural sunlight. Pb
belongs to family Piperaceae and is widely used in
India as a post-meal mouth freshener. It shows
diverse pharmacological activity along with stomachic,
antihelminthic, antifungal, tonic, aphrodisiac, and
laxative activities (Pradhan et al. 2013; Pawar et al.
2017). There are some recent studies on the synthesis
of different metal nanoparticles by using the Pb leaf.
These studies suggest that the phytochemicals present
in a crude extract of the Pb leaf assist in the
formation of nanoparticles by acting as reducing and
capping agents (Rajasekharreddy and Rani 2014). The
schematic representation of Pb-AgNP synthesis is
shown in Supplemental Figure S1 (Supplemental
material available online). This procedure did not
require any specific chemical and/or sophisticated
instruments. In short, this method indicates a green,
facile and rapid method for Pb-AgNPs synthesis.
Characterization of green synthesized Pb-AgNPs
UV-vis is common method used to characterize the
synthesized AgNPs in a given sample. Therefore, syn-
thesized Pb-AgNPs were observed using a UV-vis
spectrophotometer. As shown in Supplemental Figure
1b, Pb-AgNPs showed an absorbance peak at 445 nm,
which corresponds to the excitation of surface plas-
mon vibration in AgNPs. The results corroborate pre-
vious studies by Gaikwad et al. (2013).
SEM and TEM techniques were used for morpho-
logical analysis of synthesized Pb-AgNPs. SEM ana-
lysis revealed poly-dispersed Pb-AgNPs with a
roughly spherical shape having in the size range
20–70 nm (Figure 1a). TEM micrographs further con-
firmed that the synthesized Pb-AgNPs were nearly
spherical with a size range of 14–48 nm (Figure 1b
and c). The SAED (Selected area electron diffraction)
pattern of the synthesized Pb-AgNPs showed precise
diffraction spots in the form of rings, which confirms
the polycrystalline nature of silver (Figure 1d).
The crystalline structure of dried Pb-AgNPs was
analyzed using the XRD technique (Figure 2a). The
diffracted intensities were recorded from 20 to 80, 2h
angles. XRD analysis exhibits the number of Bragg
reflections with 2h values of 37.9
, 46.3
, 64.3
and
76.6
sets of lattice planes which correlate to (111),
(200), (220), and (311) facets of a face centered cube
(FCC) crystalline formation of metallic silver, respect-
ively. The results are in accord with the literature
report of the Joint Committee on Powder Diffraction,
standard file no. 04-0783. Thus, the results clearly
showed that the synthesized Pb-AgNPs were crystal-
line in nature (Gade et al. 2010). FTIR spectroscopic
analysis confirmed the different functional groups
present on the surface of the Pb-AgNPs. In FTIR ana-
lysis of the Pb plant extract, peaks were observed at
1,628, 1,404 and 1,073 cm1 (Figure 2b, spectrum i),
and after synthesis of silver nanoparticles these peaks
were switched to 1,638, 1,309 and 1,025 cm1,
respectively (Figure 2b, spectrum ii). Peaks observed
at 1,638, 1,309 and 1,025 cm1 associate with N-H
bending, C-O stretch and C-N stretch, respectively.
 BIOFOULING 39

Figure 1. (a) SEM and (b) TEM micrographs of the synthesized Pb-AgNPs. (c) Histogram showing particle size distribution from
TEM micrographs of Pb-AgNPs. (d) SEAD diffraction pattern of Pb-AgNPs.

The peaks revealed binding of an amide linkage with
Pb-AgNPs, which is clearly evident in the IR region of
the electromagnetic spectrum, demonstrating the exist-
ence of biomolecules as a capping agent for Pb-AgNPs
(Ramachandran et al. 2015; Bhople et al. 2016). Pb
leaves contain a large number of phytochemicals such
as amino acids, proteins, hydroxychavicol, eugenol,
polyphenols, and several alkaloids and terpenoids,
which have reduction properties (Guha 2006). From
overall consideration, it is believed that the phytochem-
icals present in Pb extract have adsorbed over the
AgNPs, which stabilizes the synthesized Pb-AgNPs.
Antibacterial concentrations of Pb-AgNPs
The antibacterial properties of silver nanoparticles
have been widely acknowledged because of their less
toxic nature than silver ions (Singh et al. 2017). The
observed MIC of Pb-AgNPs against P. aeruginosa
PAO1 and the biomarker C. violaceum was 12.5 and
25 mg ml1 respectively, which was relatively less than
in previous studies (Singh et al. 2015; Srinivasan et al.
2017). Concentrations below this MIC which did not
inhibit viable growth of the PAO1 and C. violaceum
was used for all further experiments.
Anti QS activity of Pb-AgNPs in C.
violaceum 12472
C. violaceum 12472 has been widely used as a model
biomarker strain for the identification of quorum
sensing inhibitors (QSIs). This bacterium produces
the AHL-based purple colored pigment violacein
40 S. SHAH ET AL.

(Mcclean et al. 1997). Inhibition of this pigment with

no effect on bacterial growth indicates the attenuation
of the AHL signaling molecule. In this assay, violacein
inhibition was used to assess the anti-QS activity of
Pb-AgNPs against C. violaceum and concentration
dependent inhibitory activity of Pb-AgNPs on viola-
cein production was examined. Pb-AgNPs potentially
reduced violacein production by the biomarker strain
compared to the control with 75.25 ± 4.1, 88.69 ± 4.59,
and 97.48 ± 1.31% inhibition at 10, 15, and 20 lg ml1,
respectively (Figure 3). In the present study, violacein
inhibition was comparatively higher than that in a pre-
vious report, where chemically synthesized AgNPs
inhibited violacein production by 60% at a concentra-
tion of 500 lg ml1 (Wagh et al. 2013).

Growth assay
To confirm that the observed anti-QS activity of Pb-
AgNPs was not due to their bactericidal activity, the
effect of Pb-AgNPs on the growth of P. aeruginosa
PAO1 was checked (Ravindran et al. 2018). PAO1
growth assay was performed in the absence and pres-
ence (8 mg ml1) of Pb-AgNPs. There was no differ-
ence in the growth pattern of P. aeruginosa PAO1
treated with Pb-AgNPs compared to the control cells
not treated with Pb-AgNPs (Supplemental Figure 2,
Supplemental material available online). Pb-AgNPs at
the above mentioned concentrations had no effect on
the growth of the test strain, suggesting its possible Figure 2. (a) XRD pattern and (b) FTIR spectrum of the syn-
application as a true anti-QS agent (Li et al. 2018). thesized Pb-AgNPs. Spectra: (i) Control (Pb extract) and (ii)
experiment (Expt; Pb-AgNPs).

Substantial inhibition of QS-mediated virulence

factors by Pb-AgNPs
QS is a chemical signaling system utilized by P. aerugi-
nosa to regulate several virulence products that support
successful establishment as a source of infection
(Rutherford and Bassler 2012; Lee and Zhang 2015).
Hence, the inhibition of this signaling system has
emerged as a promising approach to target infections
caused by P. aeruginosa (Bjarnsholt et al. 2010;
Rampioni et al. 2014). To date, several natural and syn-
thetic compounds have been documented as potential
inhibitors of QS in P. aeruginosa, including 4-nitro-pyri-
dine-N-oxide (NPO) (Rasmussen, Skindersoe, et al.
2005), meta-bromo-thiolactone (mBTL) (O’Loughlin
et al. 2013), organosulfur (Cady et al. 2012), cinnamon
oil (Kalia et al. 2015), zingerone (Kumar et al. 2015)
and others. These compounds do not kill the bacterial
cell and are likely to develop less resistance towards
conventional antibiotics (Bhardwaj et al. 2013). Keeping
this in view, the effect of sub-lethal Pb-AgNPs concen-
trations on QS controlled virulence products of P. aeru-
ginosa was studied.
P. aeruginosa PAO1 produces a blue color pigment,
pyocyanin, a redox active hydroxyphenazine, which is
one of the toxic virulence factors secreted by this bac-
terium. It produces reactive oxygen species (ROS) in
mammalian cells and increases oxidative stress. It
interferes with several host cellular processes such as
energy metabolism, innate immune responses and gene
expression (Lau et al. 2004; Rada and Leto 2013). The
effect of sub-lethal concentrations of Pb-AgNPs on
pyocyanin production by PAO1 is shown in Figure 4a.
A significant decline in pyocyanin production was
detected, which corresponded with Pb-AgNP concen-
tration; the maximum reduction (82.43 ± 0.64%) was
seen at 8 mg ml1, which was significantly higher than
in an earlier report (Ali et al. 2015).

Figure 3. Violacein inhibition in C. violaceum by different concentrations of Pb-AgNPs.
Figure 4. Effect of Pb-AgNPs on QS-regulated virulence factor
production in P. aeruginosa PAO1. (a) Effect of Pb-AgNPs on
pyocyanin production; (b) effect of Pb-AgNPs on elastase activ-
ity. (Error bars indicate the SD for three samples. p < 0.05
was compared with the control).
BIOFOULING 41
Another important QS-mediated virulence factor is
elastase. The influence of Pb-AgNPs on the activity of
elastase was determined. Elastase is a zinc metallopro-
tease that degrades several host tissue components
including elastin, collagen and fibrin, and also interferes
with the host defense system by preventing the secretion
of immune components such as immunoglobulins and
cytokines (Pearson et al. 1997; Bjarnsholt et al. 2010).
Figure 4b shows the reduction in elastase activity of
PAO1 with increased sub-lethal concentrations of Pb-
AgNPs with reductions of 18.45 ± 7.12, 36.64 ± 5.53,
56.43 ± 2.48 and 77.40 ± 1.02% at 2, 4, 6 and 8 lg ml1,
respectively. A significant reduction in elastase activity
due to Pb-AgNPs provides good evidence of their
potential to make P. aeruginosa susceptible to host
immune responses (Prateeksha et al. 2017).
Inhibition of biofilm formation
Motility inhibition assay
P. aeruginosa forms antibiotic resistant biofilms which
are a source of persistent nosocomial infections.
Biofilm formation in this bacterium requires QS-
controlled factors such as exopolysaccharide, rhamnoli-
pids and swarming and swimming motility. Therefore,
targeting these factors could be a promising approach
for the inhibition of biofilm in PAO1 (Kalia 2013;
Rasamiravaka et al. 2015).
Flagella-mediated motility of P. aeruginosa PAO1
regulates the initial attachment of this bacterium to
an extensive range of surfaces and also facilitates
infection through biofilm formation (Kohler et al.
42 S. SHAH ET AL.

2000; Shrout et al. 2006). Therefore, the migration ability the formation of a well-defined biofilm (Figure 7a).
of PAO1 over the swimming and swarming agar surface However, Pb-AgNP treated biofilms (6 mg ml1 and
in the presence and absence of Pb-AgNPs was observed 8 mg ml1) displayed a substantially less well-defined
by the motility test. Interestingly, sub-lethal concentra- biofilm architecture (Figure 7b and c), which is in
tions (6 and 8 mg ml1) of Pb-AgNPs significantly inhib- agreement with a previous report (Srinivasan et al.
ited both motilities of PAO1 over the agar surface 2017). It is evident that Pb-AgNPs displayed a sub-
compared to the untreated controls (Figure 5a and b), bactericidal concentration dependent anti-biofilm
suggesting its function in biofilm interruption which is in effect against P. aeruginosa. The results corroborate
agreement with a previous report (Prateeksha et al. 2017). earlier studies on the inhibition of biofilm formation
in P. aeruginosa by anti-QS nanomaterials (Singh
et al. 2015; Loo et al. 2016; Pattnaik, Barik, et al.
Biofilm formation assay (Crystal violet method)
2018). Flagella-mediated motility is a key factor in
Pb-AgNPs significantly reduced biofilm formation biofilm formation, initiating the planktonic bacterial
without inhibiting planktonic cell growth compared cell-to-surface attachment. Therefore, inhibition of
to the control in a concentration dependent manner.
The biofilm inhibition was 14.33 ± 4.6, 36.10 ± 5.4,
55.09 ± 2.62 and 78.20 ± 3.1% at concentrations of 2,
4, 6 and 8 lg ml1, respectively. Figure 6 shows that
Pb-AgNPs strongly reduced bacterial cell attachment
to a plastic substratum (Zhang et al. 2014). The pre-
sent biofilm inhibition was significantly higher than
AucoreAgshell nanoparticles synthesized by the medi-
cinal plant Dioscorea bulbifera, which inhibited biofilm
formation by 18.93% in P. aeruginosa at 100 mg ml1
(Ghosh et al. 2015).

Microscopic observation of PAO1 biofilm

Further, to interpret the anti-biofilm efficacy of Pb-
AgNPs on the architecture of PAO1 biofilms, fluores-
cence microscope analysis was performed (Figure 7).
Closely packed cells of P. aeruginosa forming a thick
green mat were observed in control images, indicating
Figure 6. Inhibition of P. aeruginosa PAO1 biofilm formation
in the presence of different concentrations of Pb-AgNPs.
(Values are presented as means ± SD; number of samples ¼ 3;
p < 0.05 was compared with the untreated control).

Figure 5. Effect of sub-lethal concentrations of Pb-AgNPs on (a) swarming and (b) swimming motility of P. aeruginosa PAO1. (i)
Untreated; (ii) 6 mg ml1 Pb-AgNPs; (iii) 8 mg ml1 Pb-AgNPs.
 BIOFOULING 43

Figure 7. Fluorescence microscopy (at 100) of P. aeruginosa PAO1 biofilms in the absence (a) and presence of Pb-AgNPs at the
concentration of 6 mg ml1 (b) and 8 mg ml1 (c).

Figure 8. Molecular docking analysis of LasR with (a) the natural ligand 3-oxo-C12-HSL, (b) furanone C30, (c) AgNP, (d) eugenol
and (e) Eu þ AgNP. Interactions between amino acid residues and the ligands are depicted as hydrogen bondings and metallic
interactions (in blue), hydrophobic interactions (in pink) and electrostatic interactions (in green).

this motility could be one of the reasons for decreased
biofilm formation. Similarly, Fuente-N
~ez et al.
un
(2012) reported that small peptides successfully
inhibit P. aeruginosa biofilm formation by interfering
with swimming and swarming motilities.
Molecular docking
Eugenol is an active phenolic compound of Pb leaves,
which possess notable chemotherapeutic potential,
and it is non-toxic. Preethi and Padma (2016)
reported the synthesis of silver nanoparticles with Pb
leaf extract and eugenol. In addition, the anti-QS and
anti-biofilm activity of phytochemicals from Pb leaves
and PbAgNPs against several pathogenic bacteria
have been reported earlier (Tan et al. 2013; Datta
et al. 2016; Srinivasan et al. 2017). However, there are
no in silico reports on the interaction of eugenol-
conjugated AgNPs (Eu þ AgNPs) with bacterial QS
regulator proteins. Thus, computational docking of
44 S. SHAH ET AL.

the native ligand, eugenol, AgNP and
eugenol þ AgNP with AHL synthase (LasI) and with
transcriptional regulator proteins (LasR and MvfR)
was performed. The docking geometric score, desolva-
tion energy and the ligand-interacting amino acids of
the QS regulator proteins are shown in Supplemental
Table 1.
LasR
The Las system is the master quorum sensing regulator
which induces expression of both Rhl and Pqs path-
ways in P. aeruginosa (Rathinam et al. 2017). The nat-
ural ligand of LasR is 3-oxo-C12-HSL, which shows a
hydrogen bond interaction with the amino acids
Tyr56, Asp73, Tyr93 and hydrophobic interactions
with Val76, Cys79, Leu125, and Ala127 (Figure 8a)
with a score of 6,056. These interactions are similar to
those reported in previous studies performed by
Bottomley et al. (2007). Docking of known QSI such
as furanone C30 (de Nys et al. 2006) with LasR was
also performed and showed similar interactions as
those with the natural ligand, 3-oxo-C12-HSL (Figure
8b). Docking studies of AgNP (Figure 8c) show elec-
trostatic interaction with the amino acid Asp73 in a
LasR active site which is same as in previously reported
data (Vyshnava et al. 2016). Eugenol displayed hydro-
gen bond interactions with the hydroxyl group of
Tyr56 and Tyr93, hydrophobic interactions with
Trp60, Tyr64, Trp88 and Ala127 and electrostatic
interaction with Asp73 in the LasR active site, with the
docking score 3,290 (Figure 8d). This result corrobo-
rates the observations of Rathinam et al. (2017), where
eugenol showed more stable binding with LasR com-
pared to the natural ligand. The docking result of
Eu þ AgNPs with LasR gave superior binding interac-
tions compared to eugenol alone. The Eu þ AgNP
complex interacted with LasR active site residues simi-
lar to 3-oxo-C12-HSL and eugenol. Strong hydrogen
bonding was observed between Eu þ AgNPs and the
amino acids of the active site – Tyr56 and Ser129.
Thr75 was seen to have a metallic interaction with the
silver atom of the ligand complex. Hydrophobic
interactions with Trp88, Ala105 and Leu110, and
electrostatic interaction with Asp73 were observed
(Figure 8e). A docking study between reserpine and

Figure 9. Molecular docking analysis of LasI with (a) the natural ligand, (b) furanone C30, (c) AgNP, (d) eugenol and (e)
Eu þ AgNP. Interactions between the amino acid residues and the ligands are depicted as hydrogen bonding and acceptor interac-
tions (in blue), hydrophobic interactions (in pink) and electrostatic interactions (in green).
 BIOFOULING 45

Figure 10. Molecular docking analysis of MvfR with (a) ligand (4R)-2-methylpentane-2,4-diol (MDR), (b) furanone C30, (c) AgNP,
(d) eugenol and (e) Eu þ AgNP. Interactions between the amino acid residues and the ligands are depicted as hydrogen bonding
and acceptor interactions (in blue), hydrophobic interactions (in pink) and electrostatic interactions (in green).

LasR showed similar amino acid interactions (Parai
et al. 2018). The desolvation energy of the Eu þ AgNP
complex was notably higher (114.11 kcal mol1) than
that of known QSI, furanone C30 (–5.44 kcal mol1),
indicating efficient and stable bindings of the
Eu þ AgNP complex with LasR. Therefore, this finding
suggests that the inactivation of transcriptional regula-
tor LasR can be attributed to the anti-biofilm and anti-
QS activity of phytofabricated AgNPs.
LasI
In P. aeruginosa, LasI is responsible for the synthesis
of the diffusible 3-oxo-C12-HSL molecule which inter-
acts with intracellular LasR transcriptional receptors.
Computational docking was performed on LasI with
its native substrate S-adenosyl-L-mathionine (SAM)
(Maisuria et al. 2016). SAM showed hydrogen bond
interaction with Thr75, Asn135, and hydrophobic
interactions with Tyr52, Asp 73 and Trp100 (Figure
9a). Docking of AgNP with LasI (Figure 9c) showed
an electrostatic interaction with Asp73, which was the
same as reported by Ali et al. (2017). The docking of
LasI with eugenol displayed a docking score of 2,528
and two hydrogen bond interactions with Gln185 and
Glu195 and one hydrophobic interaction with Leu133
(Figure 9d). Eu þ AgNPs showed good interaction
with LasI having a desolvation energy of 71.31 kcal
mol1. It exhibited hydrogen bond interactions with
Asp73 and Tyr52 with a dock score of 2,608 which
was higher than that of eugenol alone. The complex
also forms hydrophobic interactions with Trp100 and
His98 which are present in the active site of LasI
(Figure 9e). The molecular docking of the
Eu þ AgNPs complex with LasI showed promising
binding efficacy to the active site of LasI with a desol-
vation energy being relatively higher than that of fur-
anone C30 (2.39 kcal mol1). This finding suggests
that Eu þ AgNPs can be associated with the inhibition
of AHL production.
MvfR
In addition to LasI/LasR, P. aeruginosa possesses
another type of QS system, named PQS (Pseudomonas
quinolone signal), which significantly contributes to
46 S. SHAH ET AL.
biofilm development (Dubern and Diggle 2008). The
pqsABCDE operon controls the synthesis of the PQS
molecule through MvfR (PqsR), a transcriptional regu-
lator, which in turn controls the production of pyocya-
nin, rhamnolipid and biofilm development. Thus,
inhibition of PQS is considered to be one of the prom-
ising targets to attenuate pathogenic bacteria, especially
in biofilm-associated infections (Rampioni et al. 2016;
Parai et al. 2018). The docking study of the (4R)-2-
methylpentane-2,4-diol (MDR) ligand with MvfR
shows hydrogen bond interaction with Tyr258 with a
bond distance of 2.85 Å and a score of 1,550 (Figure
10a). Figure 10c shows acceptor interactions with
Asn206, Leu207 and Arg209 on docking of MvfR with
AgNP. Docking showed that eugenol interacted with
the MvfR active site with a desolvation energy of
106.46 kcal mol1 (Figure 10d) though hydrogen
bonds (Glu259, Tyr258) and hydrophobic interaction
(Leu207). The docking of the Eu þ AgNPs complex
with MvfR shows that the complex has better inter-
action than the ligand or eugenol alone. The
Eu þ AgNPs complex formed stable interaction with
active site residues of Mvfr showing higher desolvation
energy and score (61.28 kcal mol1 and 2,534) than
the known QSI furanone C30 (41.57 kcal mol1 and
2,246) (Figure 10e). This result was in accordance with
a previous study by Parai et al. (2018) which showed
stable integrations of reserpine within the binding
pocket of MvfR. In summary, computational analysis
demonstrated that the Eu þ AgNPs complex forms
favorable interactions with MvfR which might attenu-
ate the PQS signaling system. Therefore, those phyto-
conjugated AgNPs might be considered a promising
candidate to combat P. aeruginosa biofilm formation.
Conclusions
In this study, the development of an eco-friendly,
rapid and cost-effective method for the synthesis of
silver nanoparticles using the capping and reducing
potential of Pb leaves has been demonstrated. These
phytofabricated silver nanoparticles significantly atte-
nuated QS-regulated virulence factors such as the vio-
lacein of C. violaceum, and pyocyanin and elastase of
P. aeruginosa. In addition, these nanoparticles exhib-
ited considerable anti-biofilm activity against P. aeru-
ginosa, signifying their role in decreasing bacterial
resistance towards traditional antibiotics. Although
there have been previous studies on the in silico ana-
lysis of eugenol and silver nanoparticles using P. aeru-
ginosa QS regulator proteins (Rathinam et al. 2017;
Vyshnava et al. 2016), to the best of the authors’
knowledge, this is the first report which shows a sta-
ble interaction of eugenol-conjugated silver nanopar-
ticle against P. aeruginosa with LasI and LasR/MvfR
quorum sensing regulators.
Acknowledgements
The authors are thankful to the Dr D. Y. Patil
Biotechnology and Bioinformatics Institute, Dr D. Y. Patil
Vidyapeeth, Tathawade, Pune, for providing the necessary
facilities and infrastructure to carry out this work.
Disclosure statement
The authors declare no conflicts of interest in this work.
Compliance with ethics requirements
This article does not contain any studies with human or
animal subjects.
Funding
This research work received funding from Dr D. Y. Patil
Vidyapeeth, Pune (DPU/421/2018).
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