1. Petunidin as a competitive inhibitor of acylated
homoserine lactones in Klebsiella pneumoniae
Venkadesaperumal Gopu,a
Chetan Kumar Meena,b
Ayaluru Muralib
and Prathapkumar Halady Shetty*a
Most of the bacterial species communicate with each other through a mechanism called Quorum Sensing
(QS) to regulate their phenotypic characteristics. Therefore interrupting the bacterial communication is an
attractive strategy for the management of bacteria. The present study aims to identify a novel QS-inhibitor
and to investigate its QS inhibitory activity against K. pneumoniae, an opportunistic food borne pathogen.
Initially, 43 phytochemical compounds were docked with LasR receptor protein and the compound
exhibiting the highest score was further screened for its effect on QS regulated phenotypes. Molecular
docking analysis revealed that out of 43 active components, petunidin exhibited the highest ligand
binding with a dock score of À8.38 kcal molÀ1
. In vitro study showed that petunidin at its sub-MIC level,
reduced exopolysaccharide (EPS) production up to 64.41% and biofilm formation up to 67.66% which
was more evident from scanning electron microscopic (SEM) and confocal laser scanning microscopic
(CLSM) imaging. Synergistic activity of conventional antibiotics with petunidin enhanced the susceptibility
up to 90.69%. In addition, simulation studies predicted that QS inhibitory activity of petunidin occurs
through the conformational changes in the 3D structure of LasR receptor protein and petunidin
complex. Our findings suggest that petunidin can act as an effective competitive inhibitor for signaling
compounds towards LasR receptor pathway and can serve as a novel QS-based antibacterial/anti-biofilm
agent for the management of food borne pathogens.
1. Introduction
Klebsiella pneumoniae is a Gram-negative opportunistic path-
ogen, ubiquitous in nature, mainly associated with nosocomial
and urinary tract infections (UTIs). These bacterial infections
may also lead to complexities like pneumoniae and septicemia. A
number of food-borne disease outbreaks have been reported
attributing to K. pneumoniae and also it is recognized as an
important food-borne pathogen in fresh produce.1
Colonization
and biolm formation are the two main physiological process
used by K. pneumoniae for its pathogenicity. Biolms are
complex aggregates of microorganism encased in an EPS matrix
to grow and survive in organized communities and persist
attached to solid surfaces.2
Biolm formation through QS
mechanism is found to be one of the most important causes for
bacterial pathogenesis.3
In K. pneumoniae, LuxS dependent QS
are found to be involved in the biolm formation. These bio-
lms act as efficient barriers against antimicrobial agents and
host immune system resulting in persistent colonization and/or
infection at the site of biolm formation.4
In addition it protects
bacterial cells from stress, biocides and dehydration. Many
other physical and chemical approaches including low-pressure
oxygen plasma5
and water soluble polymers6
to manage the
bacterial infections were found to be effective however,
considering the emergence of multi-resistant bacterial strains,
use of drugs which can inuence its cell–cell communication
mechanism would be more attractive.
Many bacteria used to communicate other through the
mechanism called Quorum Sensing (QS) to regulate their gene
expression by cell density. These bacteria trigger their signal
transduction cascade through the release of small signaling
molecules, referred as autoinducers (AI) to manage their
phenotypes.7
In Gram-negative bacteria, N-acylated homoserine
lactones (AHLs) acts as autoinducer molecule synthesized by
the members of autoinducer synthases (LuxI homologues).
These signal molecules bind to the receptor protein (LuxR
homologues) to trigger the target gene expressions like biolm
formation, bioluminescence, agellar motility, violacein
production, virulence determination and various other pheno-
typic characteristics.
So far, several natural and chemically synthesized
compounds have been reported to have the QS inhibitory
activity. This includes malvidin,8
aspirin,9
furanones,10
and so
on. However, many of these compounds may be highly reactive
and hence cannot be used in mammalian cells; this led to the
search of novel QS inhibitor for their potential use in various
a
Department of Food Science and Technology, Pondicherry University, Pondicherry,
India. E-mail: pkshalady@yahoo.co.uk; Fax: +91-413-2656743; Tel: +91-413-2656625
b
Centre for Bio-informatics, Pondicherry University, Pondicherry, India
Cite this: RSC Adv., 2016, 6, 2592
Received 6th October 2015
Accepted 23rd December 2015
DOI: 10.1039/c5ra20677d
www.rsc.org/advances
2592 | RSC Adv., 2016, 6, 2592–2601 This journal is © The Royal Society of Chemistry 2016
RSC Advances
PAPER

2. applications. Several active components from plants offer
a repertoire of antimicrobial agents and have attracted consid-
erable interest among the scientic community. Even though
the antimicrobial nature of these compounds has been proved
the underlying mechanism of its functionality is not yet clearly
understood.10,11
Though reports on the antimicrobial property
of phytochemicals were available studies on their QS inhibitory
activity are scarce. Considering above with interest the current
study is aimed to nd out the compounds with potent QS
inhibitory activity. Based on molecular docking with LasR
receptor protein, the top ranking compound was screened for
its QS inhibitory potential in regulating the QS dependent
phenotypes like EPS production and biolm formation in an
opportunistic pathogen K. pneumoniae. Further to understand
the mechanism of QS inhibitory activity, in silico analysis
including molecular docking and simulation studies were
conducted to show the conformational changes in the LasR
receptor protein.
2. Materials and methods
2.1 Bacterial strains and culture conditions
Bacterial cultures used in this study include Chromobacterium
violaceum strain CV026 (CECT 5999), C. violaceum MTCC2656
and K. pneumoniae strain PUFST23 (GenBank: KF817575) dry
sh isolate from the departmental culture collection. All the
cultures were selected based on their QS dependent pheno-
types. CECT 5999 and MTCC 2656 were grown at LB medium
and K. pneumoniae was grown in the nutrient broth. CECT 5999
and MTCC 2656 was routinely cultured aerobically in Luria–
Bertani (LB) broth supplemented with kanamycin (20 mg mlÀ1
)
in shaking incubator at 37
C prior to experiments. N-Octanoyl-
DL-homoserine lactone (OHL) was added to induce the violacein
production in CV026, when required.
2.2 Evaluation of QSI compound using molecular docking
analysis
The phytochemical compound petunidin screened for its
QS inhibitory activity was selected based on the molecular
docking analysis of 43 active components against LasR
receptor protein in our earlier report.8
Briey, compound
structure of LasR receptor protein (PDB ID 2UV0) was ob-
tained from protein data bank, which was docked
with the three-dimensional structures of 43 active compo-
nents obtained from Pubchem database (http://
www.pubchem.ncbi.nlm.nih.gov). PDB 2UV0 structure
contains four chains (E, F, G, and H) whose conrmation
was similar which was analyzed by superimposing with
chimera. Since, the H chain is longest and contained
the preferred binding site for the natural ligand N-octanoyl-
DL-homoserine lactone, H chain was used for docking. All the
water molecules and other chains were removed from the LasR
receptor protein for the analysis to select the potential QS
inhibitory compound. Docking studies were performed with the
Schrodinger 2012. Petunidin was selected for further investi-
gations (Section 2.9).
2.3 Minimal inhibitory concentration (MIC)
The MIC for petunidin was determined against C. violaceum and
K. pneumoniae as per the guidelines of Clinical and Laboratory
Standards Institute, USA. Stock solution was prepared by dis-
solving 10 mg of petunidin in 1 ml of 70% methanol. One
percentage of overnight test pathogen was added to appropriate
growth medium supplemented with the test compound to
attain the nal concentrations ranging from (10 to 200 mg mlÀ1
).
Microtiter plates were then incubated at 37
C for 24 h. MIC was
recorded as the lowest concentration which inhibited the visible
growth. All further experiments in the present study were per-
formed only at sub-MIC level.
2.4 QSI bioassay
Quantitative analysis of violacein inhibition was performed
with violacein-negative mutant C. violaceum CV026, which
requires exogenous AHLs to induce violacein production.
Briey, LB broth supplemented with OHL (5 mM) and petunidin
(20–60 mg mlÀ1
) was inoculated with 1% of overnight test
pathogens (adjusted to 0.4 OD at 600 nm) in sterile microtiter
plates (MTP). Microtiter plates were incubated at 37
C over-
night and observed for the diminution in violacein
production.12
2.5 Biolm formation by microplate assay
Microtiter plate assay was performed to quantify the effect of
petunidin on the biolm formation of K. pneumoniae.13
LB broth
with and without petunidin (50–150 mg mlÀ1
) were inoculated
with 1% of bacterial cultures and incubated at 37
C. Aer
incubation, plates were carefully rinsed with double-distilled
water to remove loosely attached cells. Adhered cell on the
walls were stained with 100 ml of crystal violet solution (HiMe-
dia, India) for 10 min. Excess stain was removed by rinsing with
distilled water and washed with 100 ml decolorizer. Intensity was
measured at OD650 nm by using microplate reader (Biotek, USA),
for quantication of biolm biomass.
2.6 In situ visualization of biolm
For scanning electron microscopy, sample preparation was
done as described by Lembke et al. (2006).14
Briey, biolms
formed on the glass slides were xed with 2.5% glutaraldehyde
for 1 h. The xed glass slides were washed using 0.1 M sodium
acetate buffer (pH 7.3). Slides were then dehydrated with
ethanol; air dried, carbon sputtered and analyzed using a scan-
ning electron microscope (Hitachi S-3000H, Japan).
Bacterial biolms were allowed to develop on the glass slides
1 Â 1 cm with and without petunidin for confocal laser scan-
ning microscopy (CLSM). Aer 24 h of incubation, glass slides
were stained with acridine orange (1%) for 1 min. Stained glass
slides were rinsed with distilled water to remove the excess
stain. Glass slides were then dried and visualized under the
advanced confocal microscope at 10Â (LSM 710, Zeiss,
Germany).
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3. 2.7 Reduction in exopolysaccharide (EPS)
Klebsiella pneumoniae was grown at 30
C; late-log phase cells
adhered to the walls of the test tubes were harvested to
obtain the crude EPS. Briey, late log phase cells were
removed by centrifugation at 8000 g for 30 min at 2
C.
Filtered supernatant was added with three volumes of chil-
led ethanol and incubated overnight at 2
C to precipitate
the dislodged EPS. Precipitated EPS was collected by
centrifugation at 8000g for 30 min which was then dissolved
in 1 ml of HPLC water, and stored at À40
C until further
use.15
EPS was quantied by mixing 1 ml of EPS solution with
an equal volume of 5% phenol and 5 ml of concentrated
sulfuric acid to develop red color, glucose (0.25 to 1 mg mlÀ1
)
was used as a standard to determine R2
value in the cali-
bration and for quantication of crude EPS. The intensity of
the color developed was measured using a microplate reader
(Biotek, USA) at 490 nm.
2.8 Synergistic effects of petunidin with antibiotics
Twelve well microtiter plate containing 1 ml LB broth with
petunidin added at different concentrations (50–150 mg mlÀ1
)
was added with 1% test pathogen and antibiotics, wells without
petunidin was maintained as control.16
Antibiotics that were
tested include erythromycin (10 mcg), kanamycin (30 mcg) and
tetracycline (30 mcg). Plates were incubated at 37
C and the cell
density was measured at OD600 using microplate reader (Biotek,
USA).
Synergistic effect resulted from combining quercetin with
antibiotics were assessed by determining fractional inhibi-
tory concentration (FIC) index. FIC was determined by the
formula: FIC index ¼ FIC A + FIC B ¼ [A]/MIC A + [B]/MIC B,
where [A] is the concentration of drug A, MICA is it's MIC
and FICA is the FIC of drug A for the organism, while [B],
MICB, and FICB are dened in the same fashion for drug B.
The FIC index thus obtained was interpreted as follows: 0.5,
synergy; 0.5 to 0.75, partial synergy; 0.76 to 1.0, additive
effect; 1.0 to 4.0, indifference; and 4.0, antagonism.
Finally, the varying rates of synergy between the agents were
determined.
2.9 Molecular docking analysis
Molecular docking studies were performed to study the
interaction mechanism of LasR receptor protein with auto
inducer (N-octanoyl-DL-homoserine lactone) molecule and the
active component petunidin. The study of interacting mech-
anism helps us to enhance the efficacy of active compounds.
Docking studies were performed in three different interac-
tions: (1) with signaling molecule; (2) with active component
and (3) with signaling molecule and active components
together. The third interaction was performed to screen the
competitive inhibitory nature of active components against
signaling molecule. Docking was performed by using
prepared protein (as explained in 2.2), signaling molecule,
and active components with the help of Schrodinger suite
2012.
2.10 Molecular dynamics simulation
The molecular docking study is not able to nd out the
conformational changes of the complete structure of receptor
protein because the changes made at the time of docking are
restrained to a dened region. To nd out the effect of this
interaction globally, molecular dynamics study needs to be
done and this study helps to verify the changes appear aer
interaction. If this interaction is making any unwanted change
in the topology of protein then this interaction will break in
simulation studies. Therefore, these molecular docking studies
were followed by the docking analysis, molecular dynamics
simulation studies were performed to study the conformational
changes in the receptor due to binding of signaling molecule
and petunidin. Protein-signaling molecule and protein-active
compound complex were simulated by administering gromos
force eld17
in the Gromacs 4.5.3 18
simulation package. Both
the complexes were solvated by using SPCE water molecules as
solvent into the cubic box of volume 436.24 nm3
. The pressure
of the system at 1 atm was maintained by Parrinello–Rahman
while V-rescale was used to regulate the temperature at 310 K.
Equilibration of the entire system was carried out for 50 ps each
for position restrains of both NVT (constant number of parti-
cles, volume, and temperature). Equilibration of the entire
ensemble conrmed the uniformity of temperature; pressure;
density and total energy of the system. Finally, these well-
equilibrated systems were simulated for 20 ns with the time
step of 2 ps.
2.11 Statistical analysis
All the experimental data represents the mean of triplicate
values. Differences between control and test were analyzed by
one-way ANOVA.
3. Results
3.1 Evaluation of QS inhibitory compound using molecular
docking analysis
The molecular docking analysis of phytochemical compounds
with LasR receptor protein exhibited that active components
like malvidin, petunidin, and cyanidin displayed docking score
more than À7 kcal molÀ1
against LasR receptor protein
(Table 1). Among the selected active components, malvidin has
been already reported for its anti-quorum activity in our earlier
report.8
Hence in the present study petunidin that exhibited
the docking score of À8.38 kcal molÀ1
was screened for its
quorum quenching activity against the reporter strain and K.
pneumoniae.
3.2 MIC of petunidin
Minimum inhibitory concentration of petunidin was deter-
mined against bio-sensor strain C. violaceum and K. pneumo-
niae. Petunidin exhibited bacterial growth inhibition, and the
MIC was determined as the lowest concentration which showed
complete inhibition of visible growth. The MIC of petunidin was
80 mg mlÀ1
for C. violaceum and 200 mg mlÀ1
for K. pneumoniae.
Hence, in the present study, sub-MIC concentrations at 50–150
2594 | RSC Adv., 2016, 6, 2592–2601 This journal is © The Royal Society of Chemistry 2016
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4. mg mlÀ1
and 20–60 mg mlÀ1
were used for experimental analysis
with K. pneumoniae and C. violaceum respectively.
3.3 Quantitative inhibition of violacein production
In ask incubation assay, petunidin at all tested concentration
(20–60 mg mlÀ1
) showed a signicant drop in violacein
production without inhibition of bacterial growth. At the
concentration of 20 mg mlÀ1
, 25.85% inhibition was observed
when compared with the control (P 0.05). Concentration-
dependent increase in the inhibitory activity was observed
with increasing concentration of petunidin and maximum
of 82.43% inhibition was observed at the concentration of
60 mg mlÀ1
(Fig. 1).
3.4 Inhibition of biolm formation and EPS production
Fig. 2A represents the quantitative assay for screening the anti-
biolm activity of petunidin against K. pneumoniae. It was
observed that there was a concentration-dependent reduction
in the biomass of tested pathogens. Petunidin at 150 mg mlÀ1
efficiently extricated the biolm biomass by 67.66% in K.
pneumoniae, when compared with control (P 0.05). As EPS
production is correlated with biolm forming potential of the
bacterium, the ability of petunidin to reduce EPS production
was also tested. Analysis of precipitated EPS revealed that
a maximum of 64.41% of inhibition was observed at the
concentration of 150 mg mlÀ1
. Gradual decreases in the EPS
production was also observed with the increase in the concen-
tration of petunidin (Fig. 2B).
3.5 In situ visualization of biolm
In situ visualization of biolm developed with and without
petunidin was analyzed using scanning electron microscopy
and confocal laser microscopy. SEM analysis revealed that;
there was deterioration in the biolm architecture and
cells were loosely attached to the surface of the slides
treated with petunidin (Fig. 3A and B). Confocal laser
microscopic images showed a thick biolm biomass on the
control slide, whereas treated slides exhibited the dislodged
biolm (Fig. 3C and D). Three-dimensional images of
CLSM clearly showed a reduction in the thickness of the
biolm in the treated slides when compared with untreated
one (Fig. 3E and F).
Table 1 Docking scores of N-octanoyl-DL-homoserine lactone and petunidin with LasR receptor protein
S. no. Pubchem ID Compound Docking score Glide E-model score
1 3474204 N-Octanoyl-DL-homoserine lactone À4.28 À42.2
2 73386 Petunidin À8.38 À72.6
Fig. 1 Inhibition of violacein production in C. violaceum by petunidin
at different concentration (0–60 mg mlÀ1
). Line graph represents the
percentage inhibition. Vertical bars represent the mean values of
triplicates with a standard deviation.
Fig. 2 Quantitative analysis of anti-biofilm activity (A) and EPS
reducing activity (B) by petunidin against K. pneumoniae at the
different concentration (0–150 mg mlÀ1
). Data represented were mean
of triplicate values with a standard deviation.
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5. 3.6 Synergistic effects of petunidin with antibiotics
This experiment was performed to examine the synergistic
activity of petunidin with selected antibiotics against K. pneu-
moniae. On testing the pathogen growth inhibition with anti-
biotics, K. pneumoniae showed a maximum of 60.02% inhibition
against kanamycin. Enhanced susceptibility was observed
towards all tested antibiotics when treated with petunidin at the
different concentration (50–150 mg mlÀ1
). It was also revealed
that increasing concentration of petunidin with antibiotics
enhanced the sensitivity of K. pneumoniae towards relevant
antibiotics in a dose-dependent manner (Table 2). Upon treat-
ment with 150 mg mlÀ1
of petunidin, K. pneumoniae showed
88.76% increase in sensitivity towards kanamycin when
compared with control (P 0.05). Fractional inhibitory
concentration (FIC) index was calculated for each dose of
treatment and different combination of antibiotics. Results
Fig. 3 Scanning Electron Microscopic (SEM) and Confocal Laser Scanning Microscopic (CLSM) images of bacterial biofilm grown in the presence
and/or absence of petunidin (150 mg mlÀ1
). Images A, C, and E – untreated slide; images B, D, and F – treated slide.
2596 | RSC Adv., 2016, 6, 2592–2601 This journal is © The Royal Society of Chemistry 2016
RSC Advances Paper

6. showed that out of nine combinations screened only three
combinations showed to have no difference on the sensitivity of
test bacteria towards any of the tested antibiotics (FICI: 1–4). All
other combinations were found to act synergistic and partial
synergistic with the screened antibiotics (FICI: 0.5–1).
3.7 Docking analysis
Three-dimensional structure of LasR was retrieved from PDB
database. Bottomley et al. (2007)19
reported the crystal structure
of this receptor protein at 1.80 ˚A. The NCBI CD database search
of this protein reveals that it contains auto inducer domain
from residues 20 to 160, which is crucial for the transcription
process.20
PDBSum database was used for its secondary struc-
tural component analysis,21
which is shown in Fig. 4A and B.
Molecular docking studies were performed to nd out the hot-
spot residues of the protein. These residues are interacting with
signaling molecule and active compounds to change its
conformation for activation. Dock score of signaling molecule
with LasR receptor protein is À4.28 kcal molÀ1
. This docked
complex of OHL–LasR was submitted to PDBSum databases for
analysis. The LigPlot module of this server helps to visualize the
H-bond interaction between residues. Here the numbers of H-
bonds are 3 along with 14 hydrophobic interactions. The
information of interacting atoms along with the docking score
is in Table 3.
The grid parameters used for signaling molecule was reused
for the second iteration with petunidin. Aer completion of
docking, complex with the maximum dock score À8.38 kcal
molÀ1
was used for further in vitro analysis. This complex was
submitted to PDBSum database for further analysis. Three H-
bonds was formed between protein and active compound
along with 16 hydrophobic interactions. The information of
interacting atom of protein and active compounds is given in
Table 3.
The third iteration of docking was performed to check the
competitive nature of petunidin against signaling molecule. All
the ligand including signaling molecules docked to the same
site that was used in earlier docking. In the presence of
signaling molecule, the docking score of docked complexes was
changed. In this attempt, the docking score of petunidin was
À10.10 kcal molÀ1
. This complex was submitted to PDBSum
database for analysis. From the LigPlot analysis, it was revealed
that, there were 3 H-bonds formed between LasR and petunidin
along with 15 hydrophobic interactions, which provided
additional strength to this complex. The information on all
interacting atoms of protein and petunidin along with H-bond
direction and distances is given in Table 3. The pose of petu-
nidin in LasR receptor protein is shown in Fig. 4C. Black dashed
line represents the H-bonds.
3.8 Molecular dynamics simulation
The molecular docking studies of protein and ligand are not
competent to reveal the possible resultant conformational
changes due to interaction. To study those conformational
changes, molecular dynamics simulation was performed aer
molecular docking studies. This study predicts the intermediate
transition states, which take place between activation and
deactivation of LasR receptor protein in complex form. The
simulations were performed with two complexes, LasR–OHL
and LasR–petunidin for 20 ns with the time step of 2 ps.
The RMSD prole was generated for the complex of LasR
with both signaling molecule (AHL) and petunidin (test
compound). The thermal dynamism was observed in the term
of deviation of three-dimensional structure of both complexes
aer simulation from their native states. This deviation was
calculated in terms of Root Mean Square Deviation (RMSD). The
uctuations of RMSD value in case of signaling molecule
complex remained throughout the simulation and did not
stabilize even aer a simulation period of 20 ns. However, the
active molecule complex got stability aer 10 ns, which was
maintained over the remaining period of simulation time. This
conrms that the complex with the signaling molecule is ther-
mally more dynamic than the complex with an active compound
as seen from the RMSD values. The activation of LasR protein
takes place due to the opening of Loop2 (residues 40–51) and
Loop3 (residues 66–80). While in the case of the active molecule,
it closes activation site by closing this loops. This opening and
closing of the pocket were validated by calculating the distance
between the centers of mass of Loop2 and Loop3 for both
complexes. The distance between the two centers of mass also
shows a similar behavior. The two loops Loop2 and Loop3
seems more closer in active compound complex as compared to
the signaling compound complex (Fig. 5B). The difference of
around 1 ˚A in the center of mass of loop atoms is sufficient to
cause many conformational changes in the three-dimensional
structure of the protein which can affect its functioning. This
result supports RMSD prole that the binding of signaling
molecule cause dynamism in the three-dimensional structure of
Table 2 Synergistic effect of petunidin with antibiotics against K. pneumoniaea
Bacterial strain Antibiotics
Growth
inhibition (%)
Increase in the
sensitivity (%)
FICI
Increase in the
sensitivity (%)
FICI
Increase in the
sensitivity (%)
FICI50 mg 100 mg 150 mg
K. pneumoniae Erythromycin 25.51 Æ 1.80 33.75 Æ 3.72 0.50 49.94 Æ 0.49 0.68 65.71 Æ 1.66 0.85
Tetracycline 42.72 Æ 0.49 61.93 Æ 1.87 0.75 70.58 Æ 1.63 0.93 86.92 Æ 0.69 1.10
Kanamycin 60.02 Æ 0.51 68.25 Æ 0.57 1.00 72.31 Æ 0.90 1.18 88.76 Æ 0.71 1.35
a
FICI ¼ fractional inhibitory concentration index: 0.5 synergy; 0.5–0.75 partial synergy; 0.76–1.0 additive effect; 1–4 indifference; 4 antagonism.
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Paper RSC Advances

7. this protein by keeping its active site opened while, petunidin
closes this active site and prevent its access for further
interactions.
4. Discussion
Intervening with the bacterial communication is a prospective
method for managing bacterial pathogenicity. Various natural
compounds have been investigated for its quorum sensing
inhibitory activity. Type-2 QS regulatory molecules and AI-2
transport genes were found to be involved in biolm forma-
tion by K. pneumoniae, and the role of LuxS dependent signal
molecule in the earlier stages of biolm formation was also
elucidated. In the present study, 43 phytochemicals were
selected and docked with LasR receptor protein to identify the
potential anti-quorum compound. Out of 43 compounds tested,
8 compounds showed dock score more than À7 and petunidin
exhibited docking score of À8.38. It has been reported earlier
that competitive molecules showing the nearest or higher
affinity than natural ligand may compete to inhibit the QS
dependent physiological functions.22
Compound which exhibi-
ted the highest score was selected for further experimental
analysis.
Quorum quenching potential of petunidin was screened
initially against C. violaceum MTCC 2656 and CV026. In plate
incubation assay, petunidin exhibited a concentration-
dependent reduction in violacein production, indicated by the
zone of pigmentation loss. In ask incubation assay with
CV026, petunidin reduced the violacein production up to
82.43% at the concentration of 60 mg mlÀ1
. Above results are
comparable with those of Truchado et al. (2012),23
who reported
97.98% of violacein inhibition by resveratrol at the
Fig. 4 Three-dimensional structure of LasR receptor protein [reproduced with permission from microbial pathogenesis (License Number:
3760590787814)]. (A) Three-dimensional structure of petunidin (B), and docked conformation of signaling molecule into the active site of LasR
receptor protein (C). H-bonds are displayed in dashed line. Residues, which are forming hydrophobic interaction, are also labeled.
2598 | RSC Adv., 2016, 6, 2592–2601 This journal is © The Royal Society of Chemistry 2016
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8. concentration of 50 mg mlÀ1
in CV026. Brackman et al. (2008),24
reported that about 56.5% of reduction in violacein production
was observed at the concentration of 200 mM of eugenol.
QS mechanism in K. pneumoniae is known to play a decisive
role in inuencing the biolm formation.25
Treatment with
petunidin effectively reduced the biolm formation without
inhibiting planktonic growth and loosened the attachment of
bacterial cells, which might be due to reduced AHL production
in K. pneumoniae and binding of petunidin on the LasR receptor
protein. Olofsson et al. (2003),26
reported that quorum signals
also regulate the production of EPS, which acts as a protective
barrier for cells27
and enhances biolm formation. Hence,
interference with quorum signals may result in reduced EPS
production. In our study petunidin considerably reduced the
EPS production in K. pneumoniae that were more apparent from
SEM and CLSM images. Our results are in accordance with
Watnick and Kolter (1999),28
who reported the signicance of
EPS production in biolm formation and maturation.
Biolm formation is characterized in part by the production of
highly extensive EPS network. EPS production confers facilitation
of initial attachment of bacteria; enhanced resistance to antimi-
crobial agents and environmental stress; formation of microcolony
structure.29
Thus, inhibition of EPS production may facilitate the
direct exposure of food borne pathogens to the antibiotics, may in
turn facilitate the eradication of biolm. Here, reduced EPS
production was observed in test pathogen, when treated with
petunidin. Abraham et al. (2011),30
reported that Capparis spinosa
reduced EPS production up to 67% in P. mirabilis. As shown in
Fig. 2, petunidin inhibited the biolm formation of K. pneumoniae
in a concentration-dependent manner.
Enhanced susceptibility of microbes towards antibiotics
relies on quorum sensing mechanism which was acknowledged
by Bjarnsholt et al. (2005),31
in an earlier study. K. pneumoniae
cells are less sensitive to antibiotics, such as ooxacin, tetracy-
cline, and chloramphenicol. K. pneumoniae showed enhanced
sensitivity towards all the above-tested antibiotics with added
petunidin. This evidenced that the anti-biolm compound
proved to be non-antibacterial may overcome the resistance by
acting synergistically with conventional antibiotics, as reported
by Rogers et al. (2010).16
Brackman et al. (2008),24
reported that
the cinnamaldehyde enhanced the sensitivity of V. vulnicus
against doxycycline.
In silico analysis also proves the mode of action by which the
test compounds exhibit the quorum quenching potential.
Table 3 Details of LasR receptor protein docked with N-octanoyl-DL-homoserine lactone (1), petunidin (2), and petunidin along with N-
octanoyl-DL-homoserine lactone
S. no. Molecules
Hydrogen bonding interactions
Dock score
(kcal molÀ1
)
Glide-Emodel
score Hydrophobic interactions
H-bond
donor
H-bond
acceptor
Length
(˚A)
1 LasR–OHL complex Lig::O2 Trp 60:NH1 3.01 À4.28 À42.2 Leu 110, Phe 101, Tyr 93, Ala 105, Trp 88,
Tyr 56, Ser 129, Tyr 64, Leu 36, Ala 127,
Tyr 47, Ala 50, Val 57, Ile52
Thr 75:OG1 Lig::N1 3.30
Asp 73:OD1 Lig::N1 3.00
2 LasR–petunidin complex Lig::O7 Tyr:47 O 2.95 À8.38 À72.6 Tyr:64, Ile:52, Leu:39, Ala:50, Gly:38,
Ala:127, Val:76, Leu:36, Thr:115, Thr:75,
Ser:129, Leu:110, Trp:88, Tyr:56, Asp:73,
Phe:101
Lig::O5 Tyr:47 O 3.01
Lig::O6 Trp:60 NE1 3.00
3 LasR–petunidin–OHL complex Tyr:47 O LIG::O7 2.87 À10.10 À64.0 Tyr:64, Ala:50, Leu:39, Ile:52, Ala:127,
Val:76, Leu:36, Gly:38, Asp:73, Tyr:56,
Ser:129, Thr:115, Thr:75, Trp:88, Leu:110
Tyr:47 O LIG::O5 3.02
Trp:60 NE1 LIG::O6 3.03
Fig. 5 RMSD profile of LasR–OHL and LasR–petunidin complex (A),
and distance graph between LasR–OHL and LasR–petunidin
complexes (B). Black color indicates the protein and signaling mole-
cules while red color is showing protein-active molecule.
This journal is © The Royal Society of Chemistry 2016 RSC Adv., 2016, 6, 2592–2601 | 2599
Paper RSC Advances

9. Molecular docking analysis with LasR receptor protein showed
that, petunidin binds rigidly to the receptor with high docking
score when compared with signaling molecule in both docking
conditions (signaling molecule docked with/without petuni-
din). The strong interaction between the compounds may be
due to the binding of specic groups, which mediates confor-
mational changes in the receptor protein. RMSD prole showed
that throughout the simulation, LasR–petunidin complex is
more stable than the LasR-signaling molecule complex because
of the thermal stability of the protein three-dimensional struc-
ture. While signaling molecule causes thermal dynamism in
protein structure which is not letting protein to get stable
conformation. Even distance calculation between the centers of
mass of the atoms shows that binding of active compounds to
LasR prevent further binding of other compounds by closing its
activation site. In a recent study Mowafy et al. (2014),32
evi-
denced that docking analysis may suggest the quorum
quenching efficiency of test compounds. It was proved through
molecular docking studies that aspirin can act as an anti-
quorum agent against P. aeruginosa.
5. Conclusion
In summary, the present study evidenced that petunidin effi-
ciently inhibited biolm formation and other QS regulated
phenotypes like violacein inhibition (82.43%), EPS production
(64.41%), and biolm formation (67.66%) in K. pneumoniae.
In silico studies evidenced that petunidin binds more rigidly
with the receptor protein than the signaling molecule. This
proves that petunidin may act as a potential competitive
inhibitor of signaling molecules towards LasR protein activity.
Both in vitro and in silico studies were done to prove the
QS-inhibitory activity of petunidin. As all the experiments con-
ducted were at the sub-MIC level, it is not expected to impose
pressure on test pathogens to develop resistance, which offers
a new hope for combating with multi-antibiotic resistant
bacteria. In future animal studies may be conducted to
demonstrate the activity of petunidin over pathogenic infec-
tions, which may render impressive results.
Conflict of interest
Authors declare that they have no conict of interest.
Acknowledgements
Authors acknowledge Pondicherry University for providing
necessary facilities. Venkadesaperumal G, thankfully acknowl-
edges ICMR for providing nancial assistance in the form of
“ICMR-Senior Research Fellowship” (3/1/2/14/2013-Nut).
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