Journal of Molecular Liquids 302 (2020) 112586

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Journal of Molecular Liquids

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

In vitro and in silico investigation of anti-biofilm activity of Citrus

macroptera fruit extract mediated silver nanoparticles
Moumita Majumdar a, Shamim Ahmed Khan a, Suresh Chandra Biswas a, Dijendra Nath Roy b,
Anindya Sundar Panja c, Tarun Kumar Misra a,⁎
a
Department of Chemistry, National Institute of Technology Agartala, Agartala, Tripura 799046, India
b
Department of Bioengineering, National Institute of Technology Agartala, Tripura 799046, India
c
Department of Biotechnology, Molecular Informatics Laboratory, OIST, Vidyasagar University, Midnapore, West Bengal 721102, India

a r t i c l e i n f o a b s t r a c t

Article history: Bacteria produce biofilm not only to infect the host and spread into a new substratum but also protect themselves
Received 2 September 2019 against conventional antibiotics. Green silver nanoparticles (AgNPs), having exceptional antimicrobial activity,
Received in revised form 7 January 2020 have developed as an alternative to conventional synthetic medications to treat numerous issues concerning bac-
Accepted 26 January 2020
terial infections mainly caused by biofilm development. In the present study, we have therefore described the
Available online 28 January 2020
green synthesis of Citrus macroptera (CM) fruit extract stabilized silver nanoparticles (CM-AgNPs) and explored
Keywords:
their potential antibiofilm activity. The particles have been conventionally characterized. Moreover, protein-
Citrus macroptera corona and antibiofilm activity are evident from numerous studies including DLS and AFM (e.g. reduction of
Silver nanoparticles the roughness of biofilm N90%). CM-AgNPs shows surface plasmon resonance (SPR) peak at 434 nm. They are
Antibiofilm activity spherical (16 nm), crystalline (FCC) and anionic (zeta potential (ζ) −27.5 mV) in nature. Raman spectral data
Protein corona suggests that the phytochemicals in the juice extract are chemically adsorbed onto the surface of AgNPs. CM-
Molecular docking AgNPs effectively inhibit the growth of biofilm, formed by Bacillus subtilis and Pseudomonas aeruginosa. Molecular
docking studies indicate strong binding interactions of CM-AgNPs with biofilm-forming proteins of B. subtilis and
P. aeruginosa and the formation of the protein corona. Thus, the experimental findings and theoretical supports
make the whole study informative regarding bacterial biofilm and suggest that the CM-AgNPs could be potential
alternative therapeutics against infections involving biofilms.
© 2020 Published by Elsevier B.V.

1. Introduction
Bacterial infections have become a serious threat to the environment
as well as human health due to their increasing adaptability towards an-
tibiotic drugs. Biofilm formation is the prime way by which bacteria not
only spread infections but also protect themselves against the action of
antibiotic drugs. Biofilm formation by a microbial population with in-
creasing cell density protects them from adverse environmental or anti-
biotic effects. Biofilm act as a protective shield to the bacterial
community as it's almost impossible for an antibiotic to invade the mul-
tilayer structure, termed extracellular polymeric substances (EPS) [1]
and eradicate those bacteria [2]. Consequently, biofilm increases the
chances of intra- and inter-bacterial species communication and facili-
tates mutations by the exchange of genetic materials, leading to antibi-
otic resistance [3]. Thus, it is an urgent human need and an intriguing
topic of research to develop alternative antibiotics. As phytochemicals
⁎ Corresponding author.
E-mail address: tkmisra.chem@nita.ac.in (T.K. Misra).
are very effective in treating bacterial and fungal infections at primary
stages, scientists have developed a way to combine them with nanopar-
ticles as nanoparticles have a unique property to target a huge number
of cells within a short period [4].
The green concept of synthesis and stabilization of metal nanoparti-
cles (MNPs) was invoked most likely by Raveendran and his associate
researchers [5]. For the development of MNPs, researchers primarily
focus on noble metals: copper, silver, and gold. Consequently, different
parts of numerous plant extracts of medicinal importance have been
exploited as synthetic media, reducing and stabilizing agents [6–17].
Citrus extracts have extensively been used for these cited purposes
[6–10] due to their available species varieties, nutritional and medicinal
importance [18], and contained phytochemicals, like citric and ascorbic
acids. Of the seven Indian Citrus species that are considered to be en-
dangered, as per IUCN norms, Citrus indica and Citrus macroptera have
a high degree of threat perception [19–21] and need to be conserved
immediately. Citrus macroptera (called satkora, hatkor, or chatukora lo-
cally in Tripura, a north-eastern state of India) is available in the North
East region of India and Bangladesh [19–21]. It has huge proven folk

https://doi.org/10.1016/j.molliq.2020.112586
0167-7322/© 2020 Published by Elsevier B.V.
2 M. Majumdar et al. / Journal of Molecular Liquids 302 (2020) 112586
[19,20,22] and conventional medicinal values [23–25]. These may be the
signature of its ample antioxidant constituents including phenolics, fla-
vonoids, tannins, ascorbic acid, and proteins [26].
The scientific community endeavors to make conjugates of MNPs
with biomolecules or drugs for target-specific delivery for treating nu-
merous diseases [27–30]. The conjugation may enhance the selectivity
and efficiency of the molecules by having surface-specific effects of
NPs. In the course of the synthesis of MNPs, chemicals are required to re-
duce metal ions to their atoms and subsequently to stabilize the aggre-
gated atomic forms i.e. NPs in dimensions within a nanometer range,
1–100 nm. In the context of toxicity, it is advantageous to use bio-
friendly molecules/reagents over commercially available or synthesized
chemicals. No doubt, plant or fruit extracts [6–13,15–17,31] having me-
dicinal and nutritional values are bio-friendly media for reductants and
stabilizers for MNPs. Thus, MNPs have been synthesized using plant or
fruit extracts for better biological prospects. Citrus macroptera having
medicinal values [19,20,22–26], is extremely important and demands
not only for conservation but also exploration of its bioactivity in
conjugation with MNPs. The benefits of conjugated products may be
two-fold; first, making MNPs bio-friendly for employment in biological
systems and, second, to have more active plant extracts for folk or con-
ventional medicinal treatment. We reported the green synthesis and
anti-biofilm/anticancer activity of Citrus macroptera stabilized gold
nanoparticles and found promising results [32]. The application of silver
is well known in various fields of researches such as chemosensor [33]
and as a potent antibacterial agent against a wide range of microbes
[34,35]. MNPs and their conjugation with antibiotics have evolved as
promising biomaterials for eradicating microbial populations by
combating their multidrug-resistant natures [36–38]. Exploitation of
Citrus-fruit peel (except sinensis) or juice (except lemon) extracts medi-
ated AgNPs as anti-bacterial agents or their conjugation with antibiotics
are scarcely reported [10,16].
In this paper, we, therefore, report the synthesis of silver nanoparti-
cles (Ag-NPs) using Citrus macroptera fruit extract and in vitro and in
silico exploration of anti-biofilm activity against Bacillus subtilis and
Pseudomonas aeruginosa. Besides, standard methods of anti-biofilm
study [39], we used the AFM technique to calculate roughness parame-
ters to quantitatively evaluate the structural modification of biofilms
after treating bacteria with CM-AgNPs and report our findings in this
paper. For theoretical provision, we have performed molecular simula-
tions to recognize interactions between AgNP and phytochemicals as
well as those between bacterial proteins (responsible for biofilm forma-
tion) and CM-AgNPs, and report the results as well. Molecular interac-
tion simulation studies on such systems are scarcely reported [40].
Thus, the study would provide a potential anti-biofilm agent and impor-
tant insights into the interactions of bacterial proteins with CM-AgNPs.
2. Experimental
2.1. Materials and methods
Citrus macroptera fruit was purchased from the local market. The
chemicals/materials including silver nitrate (Sigma-Aldrich), tryptone
soya broth (TSB) (HiMedia), safranin (HiMedia), and crystal violet
(HiMedia) were used as received in the present study. Milli-Q water
was used for cleaning glassware and preparing solutions.
pH meter (PHM210, Radiometer, Copenhagen), Shimadzu UV–vis-
1800 spectrophotometer, transmission electron microscopy (TEM)
(JEM-2100, JEOL electron microscope working at 200 kV), dynamic
light scattering (DLS) (Nano track Wave W3222), zeta potential ana-
lyzer (Microtrack Zeta Cheak ZC016), powder X-ray diffraction (X-RD)
(BrukarD8 advanced) and Raman spectroscopy tools were used for
characterization. Structural topographical changes in P. aeruginosa and
B. subtilis bacteria in the presence of CM-AgNPs were imaged using an
atomic force microscope (AFM) (Bruker Multimode 8) and the data
were analyzed using NanoScope Analysis v1.40 software. Raman
spectroscopic analysis of the samples was performed on BRUKER RFS
27: Stand-alone FT-RAMAN Spectrophotometer with laser source Nd:
YAG 1064 nm. The resolution of each spectrograph was 2 cm−1. Each
sample (1 ml) was assessed to get the spectrum. The spectra of the
two samples were collected, using the continuously extended scan
from 50 to 4000 cm−1. Back-scattering geometry was used to selectively
collect the pure signal from samples. The measurements were per-
formed at room temperature [41].
2.2. Extraction of fruit juice
Citrus macroptera (CM) fruit extract was collected following the
method described in our earlier report [32]. The collected extract (con-
sidered as 100% juice) was kept at 4 °C for subsequent experiments.
2.3. Synthesis of Citrus macroptera fruit juice mediated silver nanoparticles
(CM-AgNPs)
An aqueous solution of AgNO3 (10 ml, 1 mM) was stirred vigorously
at 40 °C. Citrus macroptera (CM) fruit extract (2 ml) was then added
dropwise into this warmed AgNO3 solution. The reaction condition
was kept unturned for 2 h until the solution turned yellow to orange
in color, signifying the formation of AgNPs. It was then cooled down at
room temperature and centrifuged at 10,000 rpm for 10 min. The resi-
due was extracted with double-distilled water. The process was re-
peated twice to separate excess uncoated plant extract from the
reaction mixture. The ultimate residue of CM-AgNPs was dispersed in
double-distilled water and kept at 4 °C for future experiments.
2.4. Preparation of bacterial growth culture and determination of MIC
Two microorganisms, namely Bacillus subtilis wild type (MTCC 441)
and Pseudomonas aeruginosa wild-type (MTCC 7814) were selected to
evaluate the antibacterial effect of CM-AgNPs. B. subtilis and
P. aeruginosa were plated in Tryptone Soya Agar (TSA) plates from glyc-
erol stock, previously stored at −80 °C. Liquid cultures of the two bacte-
ria on Tryptone Soya Broth (TSB) at pH 7.4 were prepared separately by
a single colony isolation method from TSA plates. 107CFU/ml cell sus-
pensions of the mid-log phase were made from the 24 h broth culture
for biofilm development [32]. The minimum inhibition concentration
(MIC) was evaluated by broth dilution assay [42,43] in terms of opti-
mum dose to inhibit visible microbial growth of the bacteria under
study.
Fig. 1. UV–vis absorption spectra of (a) fruit extract C. macroptera, (b) aqueous solution of
AgNO3 (1 mM), (c) C. macroptera stabilized silver nanoparticle (CM-AgNPs) (photographs
of Citrus macroptera fruits and diffrent solutions (a – c)).
 M. Majumdar et al. / Journal of Molecular Liquids 302 (2020) 112586 3

Fig. 2. Raman spectra of (a) C. macroptera fruit juice and (b) CM-AgNPs obtained at 1064 nm excitation.

Fig. 3. C. macroptera capped silver nanoparticles (A) Distribution of particles recorded through DLS (B) TEM image of CM-AgNPs, (C) AFM 2D representation of CM-AgNPs and (D) X-RD
peak intensity indexing.
4 M. Majumdar et al. / Journal of Molecular Liquids 302 (2020) 112586

Fig. 4. (A) Percentage of inhibition of biofilm of B. subtilis after treatment with CM-AgNPs in dose dependent manner (15 nM to 260 nM) (B) Effect of different concentrations (15 nM to
260 nM) of CM-AgNPs on inhibition (percentage) of biofilm of P. aeruginosa (MTCC7814). Percentage of inhibition was estimated in comparison with control (untreated) group. Data
demonstrated as the mean of five replicates with ± SD. ns = non-significant;* = p b 0.05; ** = p b 0.01; *** = p b 0.001; **** = p b 0.0001: Significance was evaluated in respect of
control. In addition the significance levels between 15 nM and 40 nM, 40 nM and 100 nM, 100 nM and 200 nM were calculated.

2.5. Estimation of biofilm development under the exposure of CM-AgNPs 2.7. Study of CM-AgNPs clustering in bacterial culture using DLS

The bacterial cell suspensions (107CFU/ml) of B. subtilis and To determine interaction and if any aggregation of CM-AgNPs with
P. aeruginosa bacteria were inoculated on a sterile 96-well plate (poly- microbial growth culture had occurred, a time-dependent study was
propylene tissue culture plates with flat-bottom) to develop the biofilm performed using the DLS particle-size-analyzer. DLS data of CM-AgNPs
of these bacteria following well-established methods [32,44]. In our ex- treated B. subtilis and P. aeruginosa cell suspension were recorded after
perimental setup, the wells were incubated at 37 °C for 48 h after the ad- 24 h incubation. Tests were implemented in triplicates for the reproduc-
dition of 200 μl of TSB media for optimum growth with varying tion of data.
concentrations of CM-AgNPs (260 to 15 nM). After incubation, the
wells were washed with sterile distilled water thoroughly and air-
dried for 1 h to get the attached biofilm. The wells of B. subtilis and 2.8. Molecular docking studies
P. aeruginosa biofilms were then incubated with crystal violet solution
(0.05% w/v) for 20 min and safranin 0.5% (w/v) for 10 min, rinsed The AutoDock 4.2 tool was used for molecular docking studies to
with 0.9% saline to remove the excess stain and air-dried again. To eval- find the favorable binding sites of CM-AgNPs with the selected proteins
uate the efficacy of CM-AgNPs against the biofilm growth of the bacte- of B. subtilis (AbbA, sinI, and swrC) and P. aeruginosa (LasR, PelB, and
ria, the absorbances of the biofilms in glacial acetic acid 30% (w/v) BswR). The unit cell (FCC) structure of CM-AgNPs was built in a
were determined at 550 nm for B. subtilis [45,46] and 492 nm for
P. aeruginosa [44] using a microplate reader (Company: Diatek, Model:
LWR96).
In an alternative way, the bacterial growth inhibitions by CM-AgNPs
were also determined by performing quite different experiments. Typi-
cally, 24 h culture of approximately 1 × 107 CFU/ml cells of B. sublitis and
P. aeruginosa, was diluted to 100 fold in TSB and incubated with differ-
ent concentrations (260 nM–15 nM) of CM-AgNPs for 48 h at 37 °C in
shaking condition at 150 rpm. The treated and untreated cultures
were plated in the TSA plate and incubated at 37 °C for 24 h followed
by counting the colonies. The test was performed in triplicates [47].

2.6. Swarming motility assay

Swarming motility assays of B. subtilis and P. aeruginosa were

achieved in small 35 × 10 mm polystyrene plates. Swarming motility
media was prepared by nutrient agar (8 g/l), supplemented with glu-
cose (5.0 g/l). The treated and untreated bacterial suspensions (2 μl)
were point inoculated at the center of the plates containing freshly pre-
pared swarming motility media. The plates were then air-dried for
10 min at room temperature followed by 48 h incubation at 37 °C. The Fig. 5. Growth curve of two bacteria B subtilis and P. aeruginosa were expressed in terms of
distance from the inoculation point to the extreme point of bacterial colony formation unit/ml (log10 CFU/ml) after treatment with different concentrations (15
growth was measured [39]. to 260 nM) of CM-AgNPs.
 M. Majumdar et al. / Journal of Molecular Liquids 302 (2020) 112586
Fig. 6. UV–vis spectra of CM-AgNPs in presence of bacteria and photographs of the
solutions (A) CM-AgNPs + B. subtilis; (B) CM-AgNPs + P. aeruginosa.
Chemdraw Ultra 8.0 and optimized after energy minimization by the
combined use of Hyperchem 8.0.7 and NanoEnginner-1 software
(Nanorex, Inc. Bloomfield Hills, MI) [40,48]. The crystal structures of
the above-mentioned proteins (AbbA-2lzf, SinI-5TMX, LasR-3ix3,
BswR-4O8B, and PelB-5wft) were obtained from the Protein Data
Bank (http://www.rcsb.org./pdb). The structure of SwrC protein was
obtained by homology modeling using SWISS-MODEL [49]. Before
performing docking analysis, Hetam was deleted from the protein
followed by the selection of any of the chain. Lamarckian genetic algo-
rithms (LGA) were used to calculate the docking results. Phosphoryla-
tion sites were predicted with the help of the NetPhosYeast 1.0 server
(http://www.cbs.dtu.dk/CBS/services/NetPhosYeast/) [50]. The output
of each protein with 10 best docking models, Maestro (Schrodinger)
software was used for visualization of docking results [51].
Fig. 7. DLS image of B. subtilis and P. aeruginosa after treatment with CM-AgNPs; (A) after 24 h incubation of B. subtilis with CM-AgNPs, (B) after 48 h incubation of B. subtilis treated with
CM-AgNPs, (C) after 24 h incubation of P. aeruginosa with CM-AgNPs, (D) after 48 h incubation of P. aeruginosa treated with CM-AgNPs.
5
3. Results and discussion
3.1. Synthesis
The Citrus macroptera Mont. (CM) fruit juice was extracted from a
raw fruit following our reported method [32] (Fig. 1, insert) and used
for synthesizing AgNPs. UV–vis spectra of all the solutions including
fruit juice, an aqueous solution of AgNO3, the mixture of juice and
AgNO3 and as-formed CM-AgNPs are given in Fig. 1. Photographs are
also included in the Fig. 1 (insert). The aspect of the spectrum of
orange-colored solution of the CM-AgNPs is different than the spectra
of other components and exhibits a broad absorption peak with a max-
imum at 434 nm, which can be attributed to the surface plasmon reso-
nance (SPR) band of AgNPs, occurring from the coherent oscillations of
conduction electrons near the NP's surfaces. As is the broad SPR band, it
may be the result of resonance frequencies of dipolar and some sort of
quadrupolar plasmons [52]. The particles' solution stability was verified
by studying UV–vis spectroscopy with time (data not included) and it
reveals that the spectrum features don't change with time, indicating
the formation of highly stable CM-AgNPs. The CM juice contains various
phytochemicals that may perform the dual roles of reducing and stabi-
lizing agent. They may chemically adsorb onto the surface of the parti-
cles and impart high stability to the particles.
3.2. Raman spectroscopy
To get information about the surface adsorbed functionalities, the
Raman spectroscopy was studied. The Raman spectra of the extracted
raw juice (CM) (Fig. 2A) and CM-AgNPs (Fig. 2B) are shown in Fig. 2.
Both the spectra show similar types of closely placed broad peaks.
Paul et al. evaluated constituents of peel and pulp extracts CM-fruits
and found good quantities of polyphenols, flavonoids, tannins, proteins,
ascorbic and citric acids present in it [26]. Moreover, carbohydrates and
6 M. Majumdar et al. / Journal of Molecular Liquids 302 (2020) 112586

in this region may be due to the effect of resonance Raman scattering

[61,62]. There are numerous vibrational modes in the wide region
1106–778 cm−1 for CM and 1184–795 cm−1 for CM-AgNPs due to
stretching vibrations of C\\C & C\\N, rocking vibrations of NH+ 3 &
CH2, bending and out-of-plane vibrations of CO–2, bending vibrations of
CNH and OCH3 and vibrations related to aromatic rings [53–57]. The
very weak peaks in the region of 1694–1340 for CM and
1678–1454 cm−1 for the particles may be assigned to the stretching vi-
brations of C_O & C_C, antisymmetric stretching vibrations of CO–2
groups, bending vibrations of NH+ 3 & CH3, deformation vibrations of
-OH and –CH2, and ring C\\C stretching vibrations [53–57]. The band
at 80 cm−1 might be the lattice mode of crystals [56] of some com-
pounds, formed during preservation of CM and CM-AgNPs. We there-
fore conclude that the CM juice and CM-AgNPs contain the stated
bioactive compounds. Our synthetic strategy makes the concentration
of the juice containing compounds onto the surface of the particles or
their dispersed solution very low. Instead of that, in comparison with
the juice extract Raman spectrum, the intensity of –O-H, -N-H and –C-
N stretching frequencies in the region of 3000–3530 cm−1 and
b1200 cm−1 enhance noticeably in the case of CM-AgNPs. It may be
the cause of the LSPR effect of AgNPs as well as their SERS property
[15,59,60], indicating the formation of Ag\\O and Ag\\N bond forma-
tions. The compounds, polyphenols, ascorbic acid and citric acid being
antioxidants, play crucial roles in reducing Ag+ ions followed by

Fig. 8. AFM 2D and 3D illustration of structural sights of biofilm of the B. subtilis: (A.i–ii)
organization of biofilm structure of B. subtilis; (B.i–ii) structural modification of biofilm
of B. subtilis in presence of fruit extract of C. macroptera; (C.i–ii) structural modification
of biofilm of B. subtilis in presence of 260 nM of CM-AgNPs.

sugars ingredients are also present in the juice. CM and CM-AgNPs ex-
hibit multiple peaks in the region of 3346–2956 cm−1 (3346, 3224,
3161, 3103, 3005, and 2956 cm−1) and 3375–2951 cm−1 (3375, 3328,
3202, 3025, and 2951 cm−1), respectively. These peaks could be corrob-
orated to the overlapping symmetric and antisymmetric stretching vi-
bration frequencies of –O-H, -N-H, and -C-H groups [53–58]. These are
the prime functionalities of polyphenols, carbohydrates and protein
substrates and expected to be present in CM-fruit extracts and on the
surface of CM-AgNPs. The obtained data are comparable with the re-
ported FT-IR spectral data of citrus peel, pear juice and mango leaf ex-
tracts and support the presence of sugars and polyphenols as well as
the compounds containing N- and O- functionalities [15,59,60] in CM
as well as CM-AgNPs. The intense broad band with multiple peaks in
the region 598–224 cm−1 for CM and 600–211 cm−1 for CM-AgNPs is
attributed to the deformation modes of skeletal vibrations of C\\C, C-
C-C, C\\O, and C-C-O groups [53–57]. Rocking vibrational mode of CO–2
and, an out-of-plane and in-plane vibrations of CCH3 groups contribute
Fig. 9. AFM 2D and 3D illustration of structural sights of biofilm of the aeruginosa: (A.i–ii)
in this region as well. It is worthy to mention that the CM and CM-AgNPs organization of biofilm structure of P. aeruginosa; (B.i–ii) structural modification of biofilm
exhibit absorption bands in this region (CM, λabs b 430 nm and CM- of P. aeruginosa in presence of fruit extract of C. macroptera; (C.i–ii) structural modification
AgNPs, λSPR = 434 nm, Fig. 1). Thus, the extremely high intense bands of biofilm of P. aeruginosa in presence of 260 nM of CM-AgNPs.
 M. Majumdar et al. / Journal of Molecular Liquids 302 (2020) 112586 7

Fig. 10. (A) Roughness height distribition curve of B. subtilis; (a) untreated control, (b) C. macroptera juice treated biofilm of B. subtilis, (c) CM-AgNPs treated biofilm of B. subtilis.
(B) Roughness height distribition curve of P. aeruginosa; (a) untreated control, (b) C. macroptera juice treated biofilm of P. aeruginosa, (c) CM-AgNPs treated biofilm of P. aeruginosa.

stabilizing Ag-NPs [15,59,60]. The stability of the particles, as revealed
from the UV–vis study, is due to the adsorption of chemical species pres-
ent in the CM fruit juice.
3.3. Size, shape and crystallinity of CM-Ag-NPs
We have ensemble DLS, TEM, AFM and X-RD images in Fig. 3. The 2D
images of AFM (Fig. 3C) reveal that the particles are pseudo-spherical.
The same morphological information is also obtained from the TEM
image (Fig. 3B). The average size of the CM-AgNPs was estimated
from the TEM image, which is found to be 16 nm with a standard devi-
ation (σ) of 2.96. The hydrodynamic diameter (HD) is 30 nm (Fig. 3A)
which is greater than the size obtained from TEM image, indicating
the adsorption of phytochemicals of the juice extract onto the surface
of the particles. The particles have a negative charge as the zeta poten-
tial value is −27 mV. Our synthesized particles, i.e. Citrus macroptera
stabilized AgNPs are much smaller in size (16 ± 2.96 nm) than the re-
ported Citrus (limon, limetta, and sinensis) fruit extract stabilized parti-
cles (25–50 nm) [13,35,63,64] but higher than Citrus limon AgNPs
(2–10 nm) [16]. Moreover, to demonstrate that the synthesized nano-
particles are made of silver and have a certain crystallographic structure,
we have executed X-RD analysis. The analysis was done from the X-RD
2θ (°) vs intensity plot, as shown in Fig. 3D. The graph consists of four
well-defined peaks at 2θ (°): 37.48, 43.74, 64.23, and 77.41, which are
attributed to the planes (111), (200), (220), and (331) present in the
core of a particle. The result is comparable with JCPDS (file no: 04-
0783) and with the reported results [65].
3.4. Antibiofilm activity of CM-AgNPs
The anti-biofilm activity of CM-AgNPs is thus extremely important
and was evaluated against biofilm formation by two groups of bacteria,
gram-positive Bacillus subtilis, and gram-negative Pseudomonas
aeruginosa. The bacteria were treated with CM-AgNPs at different con-
centrations (15 nM to 260 nM) and the percentages of biofilm inhibi-
tions against concentrations are depicted in Fig. 4. The results reveal
that the biofilm growths of the two bacteria are significantly inhibited
by the CM-AgNPs. The degree of inhibition of the biofilm growth in-
creases with the increasing concentration of CM-AgNPs and reaches
highest at the concentration of 260 nM. A similar trend can be observed
from the plots of absorbance vs concentrations (Fig. S1, Supplementary
materials). While treating both the bacteria with an identical dose of
CM-AgNPs, different trends of inhibition against biofilm production
were observed. The percentage of biofilm inhibition shown by
B. subtilis is approximately 70% whereas in the case of P. aeruginosa ap-
proximately 80% biofilm is inhibited at the highest concentration
(260 nM) of CM-AgNPs (see Fig. 4). There are numerous reports
where it had been demonstrated that AgNPs with a size N20 nm are
not suitable for potential antibacterial agents [66–68]. Some reports
suggested that AgNPs with size b20 nm can interact with bacterial
cells and could be used as potent antibacterial agents [69]. Our synthe-
sized particles, CM-AgNPs are more active against biofilm growth of
bacteria at lower concentrations (25–260 nM) than the reported re-
sults; though the particle size is about 16 nm [68]. It is also notable
that our results are superior to the antibacterial activity reported on of
Citrus-fruit peel (sinensis) or juice (lemon) extract-mediated AgNPs

Table 1
Roughness parameters of biofilm by B. subtilis and P aeruginosa.

Name Categories Roughness Parameter of biofilm (nm)

Ra % of reduction Rq % of reduction Rt % of reduction Rmax % of reduction

Bacillus subtilis Untreated control 148.6 0.00 179.4 0.00 620 0.00 363.8 0.00
C. macroptera crude juice 42.32 71.5 57.38 68 178.1 71.2 93.71 74.2
CM-AgNPs(260 nM) 9.11 93.9 11.88 93.3 44.3 93.8 21.95 94
Pseudomonas aeruginosa Untreated control 210.3 0.00 262.3 0.00 971.6 439 0.00
C. macroptera crude juice 38.04 81.91 49.23 81.23 157.5 83.7 84.65 80.71
CM-AgNPs 14.93 92.8 18.78 92.8 80.07 91.8 37.74 91.4
(260 nM)
8 M. Majumdar et al. / Journal of Molecular Liquids 302 (2020) 112586

[11,16]. The antibacterial activity of AgNPs does not only depend on the
size but also other many factors, especially the chemical nature of cap-
ping agents. The CM-fruit extract itself is an antibacterial agent
[24,25]. Thus, the higher activity of CM-AgNPs may be due to the com-
bined effect of both the juice extract components and AgNPs. The parti-
cles may have the ability to damage the cell membrane and get into the
cell, causing CM-AgNPs to show great activity. Nevertheless, the parti-
cles are somehow more active against gram-negative than that of
gram-positive bacteria. This may be due to the chemical nature of
their cell wall and cell membrane compositions. Gram-positive bacteria
possess multiple layers of peptidoglycan approximately 20 nm thick,
whereas gram-negative bacteria possess a single layer of peptidoglycan
of 5 to 7 nm [71]. When silver nanoparticles interact with the gram-
negative bacteria, there is less cellular obstruction to prevent the parti-
cle from reaching the primary cell membrane and disrupting it to a
Fig. 11. Effect of CM-AgNPs on swarming motility of B. subtilis and P. aeruginosa. (A) The
higher degree than that of gram-positive ones. CM-AgNPs more signifi- motility movement of untreated B. subtilis, (B) the motility movement of C. macroptera
cantly inhibit biofilm at lower concentrations than that of the gold juice treated B. subtilis, (C) motility movement of CM-AgNPs treated B. subtilis; (D) the
nanoparticle, as we have reported in our previous study [32]. motility movement of untreated P. aeruginosa, (B) the motility movement of
C. macroptera juice treated P. aeruginosa, (C) motility movement of CM-AgNPs treated
P. aeruginosa.
3.5. Effect of CM-AgNPs on bacterial growth

Biofilm formation takes place within a specific bacterial population the increased size is somewhat less, 213 nm after 24 h and 266 nm
when a certain threshold of bacterial cell number has been achieved. (Fig. 7C and D) after 48 h. Thus, the study provides a piece of evidences
To evaluate the effect of CM-AgNPs on the viability of bacterial cells, that the proteins get adsorbed onto the surface of particles to form the
the treated culture was grown on plates for 24 h followed by CFU protein corona, leading to aggregation of the particles.
counting in both B. subtilis and P. aeruginosa. The CFU/ml values (see
Fig. 5) drop sharply in respect to the untreated control, with increasing
concentration of CM-AgNPs (15 nM–260 nM). Thus, the effect of parti-
cles to inhibit bacterial growth is observed at 15 nM concentration
(see Fig. 5). Maximum inhibition in CFU count was observed at
200 nM concentration of CM-AgNPs in both the bacteria, compared to
the untreated control group after 48 h incubation. The cell density of un-
treated control was approximate 1 × 108 cells whereas after treatment
the CFU/ml count drops dramatically to 1 × 106 at after 200 nM particle
treatment. However, the cell viability of B. subtilis is somewhat higher
than P. aeruginosa. The MIC values of CM-AgNPs against both the bacte-
ria are 192 ng/ml and 211 ng/ml. Catechin polymer-stabilized AgNPs
showed MIC value against P. aeruginosa at 1.25 μg/ml [70] whereas
MIC of CM-AgNPs for the same bacteria is 211 ng/ml. CM-AgNPs are
thus extremely effective for inhibition of bacterial biofilm growth.

3.6. Effect of CM-AgNPs on protein corona formation

The interplay of MNPs with cell proteins has grown to be an integral

part of research in the field of applied biochemistry. Immediately after
exposure to a biological cell, proteins from the surrounding cell niche
start to adsorb onto the surface of NPs and forms a canopy of protein
which is designated as the protein corona. Such particle-protein interac-
tions often depend on the surface charge of either proteins or NPs
[59,72]. To understand the protein corona formation, a quite different
experiment was performed. The particle solution (25 nM) was incu-
bated with bacterial suspensions for 24 h followed by recording of the
UV–vis spectra and the measurement of HD sizes (DLS tool). The UV–
vis spectra along with photographs of the solutions are ensemble in
Fig. 6. The SPR band maximum at 434 nm bleaches almost completely
and becomes flattened at a long wavelength region. The color of the par-
ticles' solution changes to brown-yellow. The color change of the parti-
cles in the presence of bacteria indicated that the proteins of bacteria
may interact with the particles strongly, leading to aggregation of the
particles. The HD size of CM-AgNPs is 30 nm, Fig. 3. The aggregation
can also be observed in the increased HD size value of CM-AgNPs in
the presence of bacteria. The HD size of the particles in both the bacterial
suspension was measured using DLS after 24 and 48 h time intervals.
The HD size distributions are given in Fig. 7. The size of the particles in Fig. 12. Docking of CM-AgNPs with AbbA protein of B. subtilis. (A) Three dimentional
B. subtilis increases 30 nm to 270 nm after 24 h and further increases structure of binding site of protein with CM-AgNPs, (B) 2D representation showing
to 391 nm (Fig. 7A and B) after 48 h. However, in the case of P. aeruginosa chemical inteactions.
 M. Majumdar et al. / Journal of Molecular Liquids 302 (2020) 112586 9

Fig. 13. Docking of CM-AgNPs with LasR protein of P. aeruginosa. (A) Three dimentional structure of binding site of protein with CM-AgNPs, (B) 2D representation showing chemical
inteactions.

3.7. Structural alteration effect of CM-AgNPs on biofilm
AFM is a sophisticated tool from which a surface topographical
image of a sample in nanometer resolution can be documented. There-
fore, the effect of CM-AgNPs on structural features of the biofilms of
B. subtilis and P. aeruginosa were studied. Upon incubation with particles
for 48 h, the structural changes of the biofilms in different dimensions
were recorded by the AFM tool. Two and three-dimensional topological
images of B. subtilis and P. aeruginosa are presented in Figs. 8 and 9. The
topographic images clearly show the rapture of the biofilm surfaces in

Table 2
Molecular docking results of CM-AgNPs with proteins of B. subtilis and P. aeruginosa.

Name of Protein (PDB Binding interactions (force involved) Biding energy

microorganism Code) (kcal/mol)

B. subtilis AbbA (2LZF) Glu15 (H bond), Glu17 (Metal acceptor bond) −1.79
SinI (5TMX) His14 (Conventional hydrogen bond), Glu16 (Metal acceptor) −1.37
SwrC Asn689 (Conventional hydrogen bond) −2.03
P. aeruginosa LasR (3IX3) Asp73 (Conventional hydrogen bond), Ala127 (Conventional hydrogen bond), Ser129 (Metal acceptor) −1.63
PelB (5WFT) Gln430 (Conventional hydrogen bond) −2.51
BswR (4O8B) Leu76 (Metal acceptor), Ser80 (Metal acceptor) −1.21
10 M. Majumdar et al. / Journal of Molecular Liquids 302 (2020) 112586
the presence of particles and change of roughness. The graphical repre-
sentation of roughness distribution is shown in Fig. 10. The roughness
parameters Ra, Rq, Rt and Rmax of treated samples are compared with
the control group to evaluate the structural modification of B. subtilis
and P. aeruginosa biofilm under the exposure of CM-AgNPs. The rough-
ness of a biofilm, which is found maximum height in untreated samples,
drops sharply after treatment with CM-AgNPs in a dose-dependent
manner (Figs. 8–10). In contrast to the control, the roughness of
B. subtilis or P. aeruginosa biofilm is reduced almost completely, to an ex-
tent of ~94% (Figs. 8 and 9A–C). The values of Ra, Rq, Rt, and Rmax are
listed in Table 1. This finding further supports the biofilm growth inhibi-
tion properties of CM-AgNPs.
3.8. Study of swarming motility
We further investigated the effect of CM-AgNPs on bacterial move-
ment at the time of biofilm formation into a new substratum or environ-
ment. Bacteria spread through various translocation mechanisms.
Swarming motility is one of the principle coordinated translocation
technique which bacteria use to grow over solid or semi-solid medium
[73]. Based on that, we further evaluated the swarming motility of the
two bacteria. The cells of B. subtilis and P. aeruginosa, after treatment
with CM-AgNPs exhibit considerably lower swarming motility with re-
spect to the control group. One representative image of 3 replicates of
each treatment was demonstrated in Fig. 11A to F. One interesting result
from this assay is that C. macroptera fruit extract is not able to inhibit
biofilm over 48 h incubation but, interestingly it significantly quenches
the swarming motility (Fig. 11B and E) of both the bacteria.
3.9. Molecular simulation studies of CM-AgNPs
Experimental findings get asserted from theoretical study and vice-
versa. It is our first endeavor to explore theoretical study in accounts
of several interactions responsible for particle stabilization and inhibi-
tory action of the particles towards biofilm growth.
3.9.1. Interactions of C. macroptera with AgNPs
The extract of C. macroptera comprises of certain bioactive com-
pounds such as β carotene, thiamine, riboflavin, ascorbic acid and citric
acid [74,75]. The fruit also contains a good amount of terpenoids like
edulinine, isoplatydesmine, geraniol, lipeol, etc. The antibacterial activ-
ity of AgNPs [34] and C. macroptera (ethyl acetate extract) [25] have al-
ready been reported. Experimentally, we have demonstrated that
C. macroptera extract stabilized AgNPs are superior antibiofilm agents
to the individual ones [25,34]. To get molecular insights we have per-
formed in silico docking studies. As a model of CM-AgNPs for docking
study an FCC crystal unit cell of Ag cluster was permissible to interact
with bioactive molecules (ascorbic acid, riboflavin, edulinine, thiamine),
1:1 manner, present in the extract of C. macroptera (see Fig. S2, Supple-
mentary materials). From the figures, it is revealed the bioactive mole-
cules interact with the Ag cultures, indicating that they adsorb onto
the surface of AgNPs. This fact is also evident from the Raman spectra
(Fig. 2). The model was then set to interact with proteins responsible
for biofilm production to predict possible binding sites of CM-AgNPs
with the proteins of bacteria (Figs. 12 and 13). There are different
types of non-covalent interactions (hydrogen bond, hydrophobic inter-
actions, van der Walls interactions, metal acceptor bond, etc.) exerted
between active phosphorylation sites of proteins of bacterial biofilm
and either AgNPs or phytochemical adsorbed on them. These interac-
tions lead to conformational changes of proteins (Table 2) which in
turn deactivate their biological functions [76].
3.9.2. Interactions of B. subtilis proteins with CM-AgNPs
Upon treatment of B. subtilis with CM-AgNPs, three proteins, AbbA,
SinI and SwrC, directly and indirectly facilitate the process of biofilm for-
mation and are taken into consideration for the docking study. The
proteins are directly and indirectly facilitate the process of biofilm for-
mation. AbrB is the key transcription factor that binds with the DNA of
the bacteria to negatively regulate biofilm formation [77], but AbbA re-
presses the activity of AbrB to accelerate the growth of biofilm. How-
ever, in the case of treated bacteria, particles inhibit the action of
AbbA. As a result, AbrB can show its actual activity to inhibit biofilm for-
mation. This is clearly understood from the docking study (Fig. 12).
AbbA consists of two peptide chains, chain A and B. The first 30 residues
of the protein chain have a binding affinity with AbrB [78]. It is found
that the riboflavin moiety in the functionalized CM-AgNPs involves
electrostatic interaction using binding energy −1.79 kcal/mol
(Table 2) with amino acids present in chain A of the AbbA protein
such as conventional hydrogen bond (2.69 Å) between NH group of ri-
boflavin and Glu15 and polar interaction with Gly1, Ser2, His3, and
Met4. In addition, the surface Ag-atoms of AgNPs interact with the neg-
atively charged amino acid Glu17 (Ag-Glu17 distances: 2.31 Å and
2.38 Å) (Fig. 12A, B) with strong negative binding energy. This binding
further supports our experimental data, showing that CM-AgNPs signif-
icantly inhibit the biofilm formation by forming an AgNP-Protein com-
plex. A small inhibitor molecule can change the elasticity and
conformation of a protein by binding with its active sites [79]. The inhi-
bition effect of CM-AgNPs may be attributed to the fact that binding CM-
AgNPs in the active site of proteins may cause structural and functional
instability of the conformation of respective proteins which make them
unfavorable to bind with their native ligand for downstream transcrip-
tion and translation processes. This may significantly hamper the subse-
quent biochemical pathways during the formation of biofilm.
SinI represses the binding of SinR to the promoter region of DNA as
an antagonist. SinR is the key molecule that negatively regulates biofilm
matrix formation by inhibition of exopolysaccharide formation of the
bacteria. SinI represses SinR, thereby indirectly facilitating the matrix
formation over a solid or semi-solid substratum which is the primary
step of biofilm growth [80,81]. SwrC is a transmembrane protein of B
subtilis which gives self-resistance to surfactin produced by B. subtilis
and moderates the transition phase towards the swarming motility
[82,83]. The amino group of the thiamine moiety of the functionalized
CM-AgNPs shows binding affinity (−1.37 kcal/mol, Table 2) with
chain A of SinI (RS1 and Fig. S3, Supplementary material) and chain A
of SwrC (binding energy −2.03 kcal/mol (Table 2) (RS2 and Figs. S4,
Supplementary material). Detailed interactions are documented in the
Supplementary materials (RS1 and Fig. S3, Supplementary material).
Strong binding between active sites of proteins and CM-AgNPs confirms
its activity to quench the matrix formation and swarming motility of
bacteria, established in the earlier sections.
3.9.3. Interactions of P. aeruginosa proteins with CM-AgNPs
P. aeruginosa forms biofilm under the regulation of the quorum sens-
ing (QS) system. One of the principal regulatory proteins of the QS sys-
tem is LasR which governs the synthesis of virulence factors of the
bacteria at the time of colonization and biofilm formation. LasR has
been taken into consideration for a docking study to predict the proba-
ble binding sites with CM-AgNPs. As designed, thiamine component of
the functionalized CM-AgNPs interacts with LasR chain B at the nega-
tively charged Asp73 and nonpolar Ala127 through stable hydrogen-
bonds (2.34, 2.72 Å) (Fig. 13A and B) and surface Ag atoms interact
with the Ser129 residue with two metal coordinate bonds (1.66,
2.31 Å) with strong negative binding energy −1.63 kcal/mol (Table 2).
PelB is a structural protein of P. aeruginosa which regulate the secre-
tion of polysaccharide, which is a major constituent of extra polymeric
substances, needed for biofilm matrix formation [84]. Like B. subtilis,
P. aeruginosa also has a monomeric swarming motility protein BswR,
which it uses for translocation to initiate biofilm formation into a new
environment after biofilm maturation. It's a transcription factor that co-
ordinates biofilm development and swarming motility [85]. Functional-
ized CM-AgNPs interacts with PelB through binding energy −2.51 kcal/
mol (Table 2) (RS3 and Fig. S4, Supplementary material) and BswR
 M. Majumdar et al. / Journal of Molecular Liquids 302 (2020) 112586
utilizing binding energy −1.21 kcal/mol (Table 2). Detailed interaction
documented in Supplementary materials (RS4 and Fig. S6, Supplemen-
tary material).
These important molecular interactions validate our experimental
outcomes which have demonstrated the antibiofilm effect of CM-
AgNPs against B. subtilis and P. aeruginosa in both biochemical and struc-
tural aspects.
4. Conclusion
Citrus macroptera fruit extract mediated AgNPs were synthesized
and characterized properly. Extract containing phytochemicals that
adsorbed onto the surface of particles, are evidenced by Raman spectro-
scopic study. CM-AgNPs at a very low concentration significantly inhibit
biofilm formation by B. subtilis and P. aeruginosa. The values of CFU/ml
and quenching of swarming motility reveal that the particles could ef-
fectively inhibit biofilm development. Protein corona formation is evi-
denced by the UV–vis spectra and HD size values. Particles can modify
the biofilm matrix and as evidenced by the AFM study. Molecular
docking studies have shown that CM-AgNPs interact with different
amino acids of biofilm proteins of bacteria of interest through strong
H-bonds and metal coordination bonds which further validate the ex-
perimental findings. Finally, we may conclude that citrus fruit extract
mediated MNPs could be treated as potential antibacterial agents. In
this context, CM-AgNPs set a paradigm whose potentiality is verified ex-
perimentally and theoretically.
Declaration of competing interest
The authors do hereby declare that they have no conflict of interest.
Acknowledgement
Authors are grateful to NIT Agartala, especially CRF for AFM and X–
RD instruments for having infrastructure facilities. MM expresses sin-
cere thanks to NIT Agartala again for institutional fellowship. Authors
are thankful to Dr. Roberto Mendez, post-doctoral associate, Depart-
ment of Surgery, University of Miami for proofreading. TKM is greatly
thankful to DST Nanomission, Govt. of India, grant no. SR/NM/NS-
1176/2012(G&C).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.molliq.2020.112586.
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