Citral and its derivatives inhibit quorum sensing and biofilm formation in Chromobacterium violaceum

Article abstract of DOI:10.1007/s00203-020-02127-z

With an upsurge in multidrug resistant bacteria backed by biofilm defence armours, there is a desperate need of new antibiotics with a non-traditional mechanism of action. Targeting bacteria by misguiding them or halting their communication is a new appro

Full text of DOI:10.1007/s00203-020-02127-z

Archives of Microbiology (2021) 203:1451–1459
https://doi.org/10.1007/s00203-020-02127-z
ORIGINAL PAPER
Citral and its derivatives inhibit quorum sensing and bioflm formation
in Chromobacterium violaceum
Nikayla Batohi1 · Shabir Ahmad Lone1 · Musa Marimani2 · Mohmmad Younus Wani3
Abdullah Saad Al‑Bogami3 · Aijaz Ahmad1,4
·
Received: 3 August 2020 / Revised: 25 October 2020 / Accepted: 18 November 2020 / Published online: 3 January 2021
© Springer-Verlag GmbH Germany, part of Springer Nature 2021
Abstract
With an upsurge in multidrug resistant bacteria backed by bioflm defence armours, there is a desperate need of new anti-
biotics with a non-traditional mechanism of action. Targeting bacteria by misguiding them or halting their communication
is a new approach that could ofer a new way to combat the multidrug resistance problem. Quorum sensing is considered to
be the achilles heel of bacteria that has a lot to ofer. Since, both quorum sensing and bioflm formation have been related to
drug resistance and pathogenicity, in this study we synthesised new derivatives of citral with antiquorum sensing and bioflm
disrupting properties. We previously reported antimicrobial and antiquorum sensing activity of citral and herein we report
the synthesis and evaluation of citral and its derivatives (CD1-CD3) for antibacterial, antibioflm and antiquorum sensing
potential against Chromobacterium violaceum using standard methods. Preliminary results revealed that CD1 is the most
active of all the derivatives. Qualitative and quantitative evaluation of antiquorum sensing activity at sub-inhibitory concen-
trations of these compounds also revealed high activity for CD1 followed by CD2, CD3 and citral. These compounds also
inhibit bioflm formation at subinhibitory concentrations without causing any bacterial growth inhibition. These results were
replicated by RT-qPCR with down regulation of the quorum sensing genes when C. violaceum was treated with these test
compounds. Overall, the results are quite encouraging, revealing that bioflm and quorum sensing are interrelated processes
and also indicating the potential of these derivatives to impede bacterial communication and bioflm formation.
Keywords Quorum sensing · Bioflm · C. violaceum · Citral
Communicated by Erko Stackebrandt.
Introduction
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s00203-020-02127-z) contains
supplementary material, which is available to authorized users.
Antibiotics have been deemed as one of the greatest dis-
coveries of last century that have saved more lives than
anything else on the planet. Although, a plethora of mech-
anism of action of these drugs are known, fundamentally
these drugs either kill or inhibit the growth of bacteria,
thus imposing and inflicting evolutionary pressure on the
bacteria and therefore promoting and driving their resist-
ance to antibiotics. Among the various defence strate-
gies of bacteria, the development of biofilms as bacterial
defence armour is considered dangerously challenging
(Koo et al. 2017). The multifactorial nature of biofilm
development and drug tolerance imposes great challenges
for the use of conventional antimicrobials and indicates
the need for development of new antibiotics or treatment
strategies (Bisht and Wakeman 2019). An effective strat-
egy that could impose less evolutionary pressure on the
microbes and disrupt the formation of biofilms is to target
* Mohmmad Younus Wani
mwani@uj.edu.sa
* Aijaz Ahmad
Aijaz.Ahmad@wits.ac.za; Aijaz.Ahmad@nhls.ac.za
1
Clinical Microbiology and Infectious Diseases,
School of Pathology, Health Sciences, University
of the Witwatersrand, Johannesburg 2193, South Africa
2
Anatomical Pathology, School of Pathology, Health Sciences,
University of the Witwatersrand, Johannesburg 2193,
South Africa
3
Department of Chemistry, College of Science, University
of Jeddah, Jeddah 21589, Kingdom of Saudi Arabia
4
Division of Infection Control, Charlotte Maxeke
Johannesburg Academic Hospital, National Health
Laboratory Service, Johannesburg 2193, South Africa
Vol.:(0123456789)
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the bacterial cell-to-cell communication, also known as
quorum sensing (Brackman and Coenye 2015; Roy et al.
2018; Lan et al. 2019). Quorum sensing controls a mul-
titude of different physiological functions such as antibi-
otic production (Slater et al. 2003), biofilm differentia-
tion (Coenye 2010), cell division (Chan and Chua 2010),
sporulation (Jabbari et al. 2011), conjugation (White
and Winans 2007), bioluminescence (Bassler and Losick
2006), secretion of virulence factors; including exopro-
teases, siderophores and exotoxins (Falcão et al. 2004;
Rasmussen and Givskov 2006) as well as many other pri-
mary and secondary metabolites (Van Houdt et al. 2006;
Rasmussen and Givskov 2006). Thus, quorum sensing
allows a single prokaryote to behave like a multicellu-
lar organism (Zhu et al. 2011), and therefore targeting
quorum sensing is now considered to be an effective and
long-lasting strategy to tackle the multi-drug resistance
and biofilm formation.
Material and methods
Chemistry
Organic reagents and solvents were purchased from Sigma
Aldrich and Merck (Germany) and used without further
purifcation. Thin Layer Chromatography (TLC) was done
using commercially available precoated aluminium sheets
(silica gel 60 F254, Merck Germany) and visualization done
under UV light. Heraeus Vario EL III analyser was used for
elemental analysis. FTIR spectra were recorded on Bruker
ALPHA FT-IR spectrometer (Eco-ATR). NMR spectra (1H
and 13C) were recorded on Bruker AVANCE 400 spectrom-
eter using DMSO-d6 or CDCl3 as solvent with TMS as inter-
nal standard. Splitting patterns are designated as follows;
s, singlet; d, doublet; dd, doublet of doublets; t, triplet; m,
multiplet. Chemical shift values are given in ppm. MICRO-
MASS QUATTRO II triple quadrupole mass spectrometer
was used to get the ESI–MS spectra.
So far different natural and synthetic compounds have
been successfully studied for the antiquorum sensing
ability, however most of these quorum sensing inhibitory
molecules have been identified as unsuitable for human
use due to their high toxicity (Zhu et al. 2011). There-
fore, developing semi-synthetic analogues or derivatives
of natural products with desired properties is gaining
much interest (Lan et al. 2019). In our previous study,
we reported that citral possess good antimicrobial and
antiquorum sensing activity that could be further ame-
liorated by synthesizing its derivatives with different
functionalities (Ahmad et al. 2014). In continuation of
that work, we herein report the synthesis and evaluation
of three citral derivatives to develop new non-traditional
antibacterial molecules that could target the bacterial
quorum sensing and disrupt the bacterial biofilms.
Synthesis of citral derivatives (CD1‑CD3)
The outline synthesis of citral derivatives (CD1-CD3) is
given in Scheme 1.
3,7‑dimethylocta‑2,6‑dienal oxime (CD1)
To a solution of citral (1 eq) and hydroxylamine hydro-
chloride (1.25 eq) in water (5 ml), a solution of sodium
bicarbonate (1.25 eq) in water (10 ml) was added gradu-
ally with stirring, and the mixture was stirred for further
5 h. An organic layer was formed, which was separated and
the aqueous layer was extracted with ether and added to the
organic layer. The organic layer was dried with NaSO4 and
evaporated.
Scheme 1. Synthesis of citral
derivatives (CD1–CD3). Reac-
tion Conditions: (a) NH2OH.
HCl, NaHCO3, H2O, rt, 5 h. b
C2H5OH, Semicarbazide, 10%
NaOH, refux, 12 h. c C2H5OH,
Thiosemicarbazide, 10% NaOH,
refux, 12 h
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Oil; Yield 80%; Anal. Calc. For C10H17NO: C
71.81, H 10.25, N 8.37%; found: C 71.79, H 10.26, N
8.39%; IR maxcm−1: 3250 (NO–H), 2864 (C–H), 1632
(C = N), 936 (N–O stretch); 1H NMR (DMSO-d6)
(ppm): 8.95 (s, 1H, N–OH), 7.81 (s, 1H, CH = N–OH),
6.01 (d, 1H, = CH–CH=N, J = 11.7 Hz), 5.13–5.09 (t,
1H,=CH–CH2), 2.36–2.32 (t, 2H, CH2-CH2), 2.14–2.11 (t,
2H, CH2–CH2), 1.81 (s, 3H, CH3), 1.65 (s, 3H, CH3), 1.61
(s, 3H, CH3); 13CNMR (DMSO-d6) (ppm): 146.2 (C=N),
144.9, 132.2, 123.4, 121.7, 39.2, 26.2, 24.6, 21.2, 19.8;
ESI–MS m/z: [M+ +1] 168.15.
(s, 3H, CH3);13CNMR (DMSO-d6) d(ppm): 179.9, 144.1,
141.6, 132.2, 123.4, 121.9, 39.4, 26.5, 24.9, 19.8, 17.9;
ESI–MS m/z: [M+ +1] 226.15.
Bacterial strain and growth conditions
An existing stock culture of Chromobacterium violaceum
ATCC12472 stored at -80 °C supplemented with 20% glyc-
erol in the Department of Clinical Microbiology and Infec-
tious Diseases, University of the Witwatersrand, Johannes-
burg, was used in this study. For experimental purpose, the
bacterial culture was revived by growing in Luria–Bertani
(LB) agar plates at 30 °C for 24 h. All culture media and
other chemicals used in this study are of high analytical
grade.
3,7‑dimethylocta‑2,6‑dien‑1‑ylidene)
hydrazine‑1‑carboxamide (CD2)
Citral (1 eq) was mixed with semicarbazide (1 eq) in ethanol
with few drops of 10% NaOH under refux for 5 h. Comple-
tion of the reaction was monitored by TLC. After comple-
tion of the reaction, the organic layer was extracted, washed,
dried with NaSO4 and evaporated under vacuo.
Antimicrobial susceptibility testing
To determine the antibacterial activity of citral and its newly
synthesised derivatives, minimum inhibitory concentration
(MIC) for C. violaceum ATCC12472 was determined by
broth microdilution susceptibility testing as per the recom-
mended guidelines of Clinical and Laboratory Standards
Institute (CLSI) reference document M07-A10 (Clinical and
Laboratory Standards Institute 2015). The stock solutions of
250 µg/ml all the test compounds were prepared using 1%
dimethyl sulfoxide (DMSO) and the range of concentrations
tested was 62.5–0.031 µg/ml. In every set of experiment, cell
free (sterility) and compound free (growth) controls were
included. The MICs were defned as the lowest concentra-
tion of test compounds that resulted in the inhibition of vis-
ible growth. Results were calculated as a mean of experi-
ments performed in triplicate.
Yield 75%; Anal. Calc. For C11H19N3O: C 63.13, H 9.15,
N 20.08%; found: C 63.25, H 9.20, N 20.25%; IR νmaxcm−1:
3550, (NH), 3360, 3180 (NH2), 3010 (C–H) 1530 (C=N),
1650 (C=C), 1165 (C–N), 1085 (C=O); 1H NMR (DMSO-
d6) d(ppm): 9.26 (1H, s, −NH), 6.95 (1H, d, –CH=N,
8.0 Hz), 6.90 (s, 2H, NH2), 5.97 (d, 1H, − CH, 4.0 Hz),
5.13–5.09 (t, 1H,=CH–CH2), 2.36–2.32 (t, 2H, CH2–CH2),
2.15–2.11 (t, 2H, CH2–CH2), 1.81 (s, 3H, CH3), 1.64 (s,
3H, CH3), 1.61 (s, 3H, CH3); 13CNMR (DMSO-d6) d(ppm):
156.9, 144.0, 141.8, 132.2, 123.4, 122.0, 39.4, 26.2, 24.9,
19.8, 17.9; ESI–MS m/z: [M+ +1] 210.20.
3,7‑dimethylocta‑2,6‑dien‑1‑ylidene)
hydrazinecarbothioamide (CD3)
Antiquorum sensing activity
Qualitative evaluation of antiquorum activity in C.
Citral (1 eq) was mixed with thiosemicarbazide (1 eq) in
ethanol with few drops of 10% NaOH under refux for 5 h.
Completion of the reaction was monitored by TLC. After
completion of the reaction, the organic layer was extracted,
washed, dried with NaSO4 and evaporated under vacuo.
Yield 75%; Anal. Calc. For C11H19N3S: C 58.63, H 8.50,
N 18.65%; found: C 58.85, H 8.20, N 18.85%; IR νmaxcm−1:
3545, (NH), 3386, 3180 (NH2), 3021 (C–H) 1534 (C=N),
1650 (C=C), 1168 (C–N), 1080 (C=S); 1H NMR (DMSO-
d6) d(ppm): 9.32 (1H, s, − NH), 8.84 (s, 2H, NH2), 7.29
(1H, d, –CH=N, 8.0 Hz), 5.98 (d, 1H, − CH, 8.0 Hz), 5.13
(t, 1H,=CH–CH2), 2.36–2.33 (t, 2H, CH2–CH2), 2.15–2.11
(t, 2H, CH2–CH2), 1.82 (s, 3H, CH3), 1.64 (s, 3H, CH3), 1.61
violaceum
Citral and its three newly synthesised derivatives
(CD1–CD3) were screened for their antiquorum activity by
qualitatively measuring the violacein pigment production by
biosensor strain C. violaceum ATCC12472, following the
method described previously (Ahmad et al. 2015a). Briefy,
1.5×108 CFU ml−1 C. violaceum ATCC12472 cell suspen-
sion was mixed with molten LB agar at 45 ºC and gently
poured into petri dishes and allowed to solidify. Sterile flter
discs (Mast Diagnostics, Merseyside, UK) infused with 20 µl
(MIC) of each test compound were placed on solid agar and
incubated at 30 °C for 18 h. Sterile disc impregnated with
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20 µl of 1% DMSO was used as a negative control. Fol-
lowing incubation, diameter of the clear zones represent-
ing growth inhibition and the opaque zones against a purple
background representing violacein inhibition were measured
in millimetres. For data analysis, the average diameters of
the clear and turbid (i.e., opaque) zones were recorded. All
the tests were repeated thrice and the results were reported
as mean SD.
Bioflm inhibition assay
Bioflm inhibition assay was done using 96-well fat-bottom
microtiter plate assay (Ravichandran et al. 2018). C. viola-
ceum ATCC12472 cells were grown in LB broth for 24 h
and 48 h at 30 ºC with 0.25×MIC, 0.5×MIC and 1×MIC
values of the test compounds. Following incubation, micro-
titre plates were washed with PBS (pH 7.4) to remove the
free-foating planktonic cells and bioflms were stained using
200 μl of 0.1% crystal violet solution (Sigma—Aldrich St.
Louis, MO, U.S.A.). After 15 min, crystal violet solution
was removed and 200 μl of 95% ethanol was added. The
bioflm was then quantifed by measuring the absorbance at
OD470 nm using iMark microplate reader (Bio-Rad labora-
tories, CA, USA).
Quantitative evaluation of antiquorum activity in C.
violaceum
Following the qualitative assay, quantifcation of violacein
pigment inhibition was done in untreated and treated C. vio-
laceum cells, using the microtiter plate method as described
previously (Ahmad et al. 2015a). Briefy, C. violaceum
ATCC12472 cells were sub-cultured on LB agar plates at
30 ºC for 24 h and a single colony from these plates was
inoculated into 5 ml LB broth and incubated at 30 °C for
24 h with shaking. To confrm the capability of the test
compounds to inhibit violacein production, 0.5 × MQSIC
and MQSIC values of all the test compounds were added
to 100 μl of LB broth in a microtiter plate. From the above
prepared C. violaceum suspension, 100 µl were added to
all the wells. In all the experiments, 1% DMSO was added
as negative control. A culture control was included in each
plate to confrm viability along with a media control to con-
frm sterility. Plates were incubated at 30 ºC for 24 h with
shaking and the quantity of violacein was determined by
measuring absorbance at OD585nm. The percentage of viola-
cein suppression was determined using a formula:
Gene expression
Efect of the test compounds on the expression levels of the
genes for quorum sensing (cviL, vioA, vioB, vioD and vioE
gene) was evaluated by RT-qPCR. Briefy, C. violaceum
cells at a concentration of 5×106 cells/ml were exposed to
MIC of test compounds followed by incubation at 30 °C for
3 h. Untreated cells were used as negative control. Total
RNA was extracted using High Pure RNA Isolation Kit
(Roche, Basel, Switzerland), following manufacturer’s
instructions. Concentration of total RNA was measured
using Nanodrop 2000 spectrophotometer (Thermo Scien-
tifc, MA, USA). Thereafter, cDNA was synthesised using
RevertAid™ First Strand cDNA Synthesis Kit (Fermentas,
MA, USA), according to manufacturer’s instructions. Fol-
lowing cDNA synthesis, RT-qPCR was performed using
2 × FastStart Essential DNA Green Master (Roche, Basel,
Switzerland), 10 pmol of each primer (Anatech, Randburg,
South Africa) in RocheLight® Cycler Nano instrument
Real-time PCR system (Roche, Basel, Switzerland). The
primer sequences used to amplify the above-mentioned
genes and housekeeping genes are shown in Table 1. The
following cycling conditions were employed for all amplif-
cations: hot start at 95 °C for 10 min, followed by 45 cycles
of denaturation at 95 °C for 20 s, annealing at 52 °C for
20 s and extension at 72 °C for 20 s. Crossing-point analysis
Percentage of violacein inhibition
control OD585nm − test OD585nm
=
× 100
control OD585nm
The amount of violacein production in the control sample
was employed to calculate the minimum quorum sensing
inhibitory concentration (MQSIC) of test compounds and
was determined as the minimum concentration at which vio-
lacein production was inhibited by≥50%.
Table 1 List of primers employed for RT-qPCR experiment
Gene
Forward primer
Reverse primer
cviL
vioA
vioB
vioD
vioE
16S
5′-GGATCCCCGTAGGCAAAGAACTAA-3’
5′-GGATCCGCCCAAAGCCAGACTA-3’
5′-GGCCTATTACGAGCTGGTCT-3’
5′-GCCGCAACAAGTACATCTGG-3’
5′-TGGAAGCAGAAGGTGGCC-3’
5′-GAATTCTTGTGTCTGAACGCCA-3’
5′-GAATTCTGAACGGCACGATTGA-3’
5′-TGGGCCAGGTATTTGAGGAA-3’
5′-AACACCTTGGCGACGTATTC-3’
5′-GCGGCGTCCAGGTACAAC-3’
5′-GCGCAACCCTTGTCCTTAGTT-3’
5′-TGTCACCGGCAGTCTCCTTAG-3’
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Table 2 Minimum inhibitory concentrations, minimum quorum sens-
ing inhibitory concentrations and zone of turbidity in mm of citral
and its synthetic derivatives against C. violaceum
derivatives (CD2 and CD3) was confirmed from their
spectra. Semicarbazones and thiosemicarbazones may
exhibit keto-enol or thione–thiol tautomerism, since they
have a keto (C=O) and thione (C=S) group adjacent to
a proton. It has been stated that the thione group (C=S)
is relatively unstable in the monomeric form and tends
to turn into a stable C–S single bond by tautomerism, if
there is at least one hydrogen atom adjacent to the C=S
bond. However, CD3 showed intense, strong bands in the
region 1085 and 1088 cm–1 due to ν(C=S) stretch and
no band near 2570 cm–1 due to ν(C–SH), suggesting that
these compounds remain in the thione form. Semicarba-
zone CD2 showed similar spectrum which confirms the
absence of any tautomerism. The spectra of these com-
pounds also exhibit a strong band at 1530 and 1550 cm–1
region due to the ν(C=N) stretch of the azomethine link-
age. The 1HNMR, 13CNMR and ESI MS spectra were also
in good agreement with the structures of the compounds.
Citral and its
derivatives
MIC (μg ml−1
)
MQSIC (μg
ml−1
ZoT (MIC)
)
C
31.25
0.122
1.95
15.62
0.061
0.49
13
22
19
17
CD1
CD2
CD3
3.91
1.95
was employed to calculate the relative mRNA concentra-
tions and specifcity of amplicons was confrmed by melting
curve analysis. Fold diference was calculated using formula
2−(ΔΔCt), where ΔCt was the mean Ct value of the target
gene minus the mean of housekeeping genes, and ΔΔCt was
the ΔCt of the tested cells minus ΔCt of the control cells.
Statistical analysis
Antimicrobial susceptibility testing
GraphPad Prism software, version 8.1.0. was used for
statistical analysis. Two-way ANOVA followed by Dun-
nett’s multiple comparisons test were applied to data. All
the experiments were performed in triplicates (n = 3),
and data were presented as mean standard deviation
(SD). Value of P < 0.0001 was considered as statistically
significant.
Antimicrobial susceptibility testing of citral and its deriva-
tives confrmed that these test compounds inhibit the growth
of C. violaceum within the MIC range of 0.122–31.25 µg/ml
(Table 2). All the test compounds showed high antimicrobial
as well as antiquorum sensing activities with varying MIC
values. Based on the MIC values, order of potency of the test
compounds was CD1>CD2>CD3>C.
Antiquorum sensing
Results
Qualitative antiquorum sensing activity
Chemistry
Qualitative disc difusion assay for antiquorum sensing
activity of citral and its derivatives revealed their antiquo-
rum properties at MIC (Table 2). The clear and turbid zones
formed around sterile discs observed after performing the
qualitative biosensor assay demonstrated inhibition of bac-
terial growth and antiquorum activity of test compounds,
respectively. CD1 demonstrated the highest antiquorum
sensing activity by efecting the formation of opaque zones
with an average diameter of 22 mm, while parent compound
C exerted the lowest antiquorum sensing efcacy resulting
in the formation of turbid zones with an average diameter of
about 13 mm (Table 2).
The outline synthesis of citral derivatives (CD1–CD3)
is given in Scheme 1. Characteristic FTIR bands provide
significant indications for the formation of the oxime
(CD1). The absence of a band at/or around 2665 cm−1 due
to the proton of the aldehyde functional group in citral
and the appearance of characteristic bands at 3260 cm−1
and 1620 cm−1 due to ν(NO–H) and ν(C=N), respectively,
confirmed the formation of the oxime CD1. A singlet at
7.30 ppm due to imine (C–H=N) proton showed conden-
sation between the carbonyl group of citral and amino
group of hydroxylamine hydrochloride. The signal at
8.97 ppm assigned to (N–OH) proton further confirmed
the formation of CD1. The structure of CD1 was further
supported by 13C NMR spectra. The absence of any peak
at 192 ppm which is characteristic of carbonyl carbon
of the aldehyde and the presence of a signal at 149 ppm
due to (C=N) confirmed the formation of CD1. Similarly
the structure of semicarbazone and thiosemicarbazone
Quantitative antiquorum sensing activity
To further confrm and quantify the antiquorum activity of
the test compounds, production of a quorum sensing related
pigment, violacein, by C. violaceum was measured spec-
trophotometrically in treated and untreated cells. From the
results, it is evident that all the test compounds signifcantly
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Bioflm inhibition activity
A signifcant inhibition in bioflm formation was observed
in C. violaceum cells after treated with diferent concen-
trations of test compounds for 24 or 48 h (Fig. 2). After
24 h of incubation, test entities showed inhibition in bioflm
formation in the range of 28.84%–58.08%, 18.8%–35.38%
and 9.7%–23.06% at concentrations of 1×MIC, 0.5×MIC
and 0.25 × MIC, respectively. However, after 48 h of
incubation, the higher rate of inhibition in biofilm for-
mation was observed and the values were in the range of
38.68%–68.27%, 27.81%–49.41% and 20.64%–36.21% at
1 × MIC, 0.5 × MIC and 0.25 × MIC of test compounds,
respectively. At sub-MIC values (0.5×MIC and 0.25×MIC)
of test compounds, no reduction in bacterial growth was
observed indicating that bacterial killing was not responsible
for the lowered bioflm formation, whereas at MIC values
signifcant reduction in bacterial growth was observed. The
rate of inhibition in bioflm formation by these test com-
pounds was depended on concentration and treatment time.
Test compound CD1 showed the highest percentage of bio-
flm formation inhibition followed by CD2, CD3 and parent
compound citrus, respectively.
Fig. 1 Quantitative inhibition of violacein production by citral and
its synthesised derivatives. C. violaceum cells were incubated with
0.5×MQSIC and MQSIC values of test compounds for 24 h. Control
bars (CN) indicate untreated cells, accepted as 0% inhibition. Data
are presented from three independent experiments using means S.D.
****p≤0.0001
inhibited the production of violacein pigment at varying con-
centrations (Table 2). CD1 being the most active compound
while inhibiting the growth and quorum sensing activity of
C. violaceum efected the highest violacein suppression at
MQSIC (94.17%) and 0.5×MQSIC (78.1%) (Fig. 1). Other
compounds also displayed high antiquorum sensing ef-
cacy with violacein suppression ranging from 53 to 69%
at 0.5 × MQSIC and 76% to 83% at MQSIC (Fig. 1). At
0.5×MQSIC and MQSIC, where signifcant reduction in the
production of violacein pigment was measured (P≤0.0001),
the numbers of cfu/ml present remained constant. For all
experiments, the solvent controls showed similar cell growth
and violacein production as observed in control cells, denot-
ing that 1% DMSO neither inhibit cell growth nor inhibit
violacein production.
Efect on quorum sensing associated genes
In a parallel investigation, an experiment was conducted to
assess if administration of the test compounds would alter
the expression levels of genes involved in quorum sensing.
For this study, total RNA was extracted at 48 h post-adminis-
tration and the relative expression of mRNAs corresponding
to cviL, vioA, vioB, vioD and vioE genes was evaluated by
RT-qPCR. Relative to untreated cells, compounds-treated
bacterial cells signifcantly decreased expression of genes
implicated in quorum sensing in C. violaceum, with CD1
being the most active compound followed by CD2, CD3 and
Fig. 2 Efect of citral and its synthesised derivatives on C. vio-
laceum bioflm formation. C. violaceum cells were incubated with
0.25×MIC, 0.5×MIC and 1×MIC values of test compounds for 24
and 48 h. Control bars (CN) indicate untreated cells, accepted as 0%
inhibition. Data are presented from three independent experiments
using means S.D. ****p≤0.0001
Fig. 3 Efect of citral and its synthesised derivatives at their respec-
tive MIC values on the expression of genes involved in quorum sens-
ing. Cells without drug treatment (CN) were used as negative control.
Data are presented from three independent experiments using means
of fold changes S.D. ****p≤0.0001
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citral, respectively (Fig. 3). All the test compounds at their
MIC values downregulated the expression of all target genes,
with a fold range of 3.0 to 4.3-fold, 2.3 to 2.8-fold, 1.8 to
2.2-fold and 1.5 to 1.7-folds by CD1, CD2, CD3 and citral,
respectively, compared to control (untreated cells) that were
set to 1.0 (Fig. 3). From these results, it can be concluded
that citral and its derivatives downregulated the expression
of above-mentioned genes which in turn inhibited N-acyl-
L-homoserine lactone (AHL) synthase and thereby quorum
sensing in C. violaceum.
researchers where semi-synthetic modifcations of natural
compounds have been reported to increase their antimicro-
bial activity (Ahmad et al. 2015b; Shi et al, 2016).
Bioflm formation in bacteria is directly linked to the
drug resistance and is currently a serious medical problem.
Our results have shown that these derivatives signifcantly
inhibit the bioflm formation at subinhibitory concentra-
tions without killing the bacteria. This also signifes that
the bacterial killing was not responsible for the reduction
of bioflm formation. Our results are congruent with the
previous fndings, where natural compounds and synthetic
derivatives are reported to inhibit bioflm formation at
lower concentrations (Burt et al. 2014). Similarly these
compounds also inhibit quorum sensing property of C.
violaceum at sub-inhibitory concentrations where growth
is not inhibited and thereby representing diferent mecha-
nism of these compounds for antiquorum sensing other
than growth inhibition or bacterial cell death. Inhibiting
bioflm and quorum sensing at sub-inhibitory concentra-
tions indicated that the mechanism of these compounds
to inhibit bioflm formation can also be linked to quorum
sensing inhibition. In addition, administration of these
compounds to bacterial cells had a marked efect on the
expression levels of genes implicated in quorum sensing
activity in C. violaceum. Specifcally, expression level of
cviL, vioA, vioB, vioD and target genes vioE (Stauf and
Bassler 2011; Liu et al. 2013; Devescovi et al. 2017) was
signifcantly down regulated in treated cells as compared
to untreated bacterial cells. This was in agreement with a
previous study that reported a marked suppression in the
expression of these target genes following administration
of antiquorum sensing compounds in C. violaceum (Liu
et al. 2013).
Discussion and conclusion
Quorum sensing, a communication system used by
microbes based on the secretion and detection of external
signal molecules, is highly regulated process and controls
numerous functions in bacteria including pathogenicity
and bioflm formation. Bioflm-associated bacteria are
less likely to be cleared by our immune cells and they are
more tolerant to antimicrobial therapies than non-bioflm-
associated bacteria (Roy et al. 2018; Lebeaux et al. 2014).
It has therefore become a challenge to eradicate bioflms
using conventional antibiotics and to completely suppress
the infection, despite high dosage and long-term treatment.
Therefore, immediate measures are required for the devel-
opment of antibioflm drugs to fully cure bioflm-related
infections. Since bioflm formation and quorum sensing
are central and often interconnected features of bacterial
lifestyle and it is now known that quorum sensing ena-
bles bacteria to turn on and of the secretion of EPS so as
to increase their competitive ability against other strains
and species within bioflms. Targeting quorum sensing is
therefore an acknowledged strategy to stop bacteria from
communicating and hence developing bioflms as their
defence armour against any antibiotics (Rasko et al. 2008;
Burt et al. 2014).
In contrast to conventional antibacterial regimens which
generally aim to treat disease symptoms, these antiquorum
sensing compounds are advantageous in that they abolish
cell-to-cell communication, thus, preventing establishment
of infection (Williams 2007; Sarkar and Chakraborty 2008;
Swem et al. 2009; Dandekar et al. 2012; Rahman et al.
2017). Additionally, since these compounds are synthesised
from natural plant material, they may potentially serve as a
cheaper source of therapy, particularly for patients in under-
developed and developing countries.
In our previous study, we reported the antimicrobial
and antiquorum sensing properties of citral (Ahmad et al.
2015a). In continuation to that study, we further modi-
fed citral to change its functionality in the quest to get
compounds with improved antimicrobial and antiquorum
sensing properties. In this study, citral showed potent anti-
microbial activity against the growth of C. violaceum and
these results are in congruence with the previous fndings
where citral has been reported to possess high antibacterial
activity (Shi et al. 2016). Modifcation of this compound
further resulted in a signifcant increase of its antibiotic
property by decreasing the MIC value up to 256-fold
against C. violaceum when compared to the parent com-
pound citral. These fndings corroborate the work of other
The major limitations of the current study include the
lack of strain diversity, in which only a single C. violaceum
bacterial pathogen was employed for all experiments as
well as defcit of not performing cell culture and animal
studies. Additionally, future studies will also evaluate the
antiquorum activity of citral and its derivative compounds
against diferent C. violaceum strains. This will include
assessment of antibacterial activity in drug susceptible and
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Archives of Microbiology (2021) 203:1451–1459
drug resistant C. violaceum pathogens. The advantage of
inhibiting cell-to-cell communication in microorganisms
following administration of citral and its derivatives may
also be interrogated in other bacterial pathogens of clinical
signifcance such as Streptococcus pyogenes, Staphylococ-
cus aureus and Vancomycin-Resistant Enterococci. Testing
of antiquorum activity against diverse bacterial strains will
generate reliable and reproducible data. Ultimately, this may
have useful clinical applications with regard to suppressing
the expression of virulence genes and associated proteins,
inhibiting host infection and mitigating bacterial prolifera-
tion particularly in drug-resistant strains.
Compliance with ethical standards
Conflict of interests All authors declare no confict of interest.
Consent for Publication Not applicable.
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