Review of Biofilm Inhibiting Phytochemicals

Review
Mini Review of Phytochemicals and Plant Taxa with

Activity as Microbial Biofilm and Quorum
Sensing Inhibitors
Chieu Anh Kim Ta and John Thor Arnason *
Received: 11 November 2015; Accepted: 17 December 2015; Published: 26 December 2015
Academic Editor: Peter J. Rutledge
Phytochemistry Laboratory, Department of Biology, University of Ottawa, Ottawa, ON K1N 6N5, Canada;
kta074@uottawa.ca
* Correspondence: John.Arnason@uottawa.ca; Tel.: +1-613-562-5262; Fax: +1-613-562-5486
Abstract: Microbial biofilms readily form on many surfaces in nature including plant surfaces.
In order to coordinate the formation of these biofilms, microorganisms use a cell-to-cell
communication system called quorum sensing (QS). As formation of biofilms on vascular plants
may not be advantageous to the hosts, plants have developed inhibitors to interfere with
these processes. In this mini review, research papers published on plant-derived molecules
that have microbial biofilm or quorum sensing inhibition are reviewed with the objectives of
determining the biosynthetic classes of active compounds, their biological activity in assays,
and their families of occurrence and range. The main findings are the identification of
plant phenolics, including benzoates, phenyl propanoids, stilbenes, flavonoids, gallotannins,
proanthocyanidins and coumarins as important inhibitors with both activities. Some terpenes
including monoterpenes, sesquiterpenes, diterpenes and triterpenes also have anti-QS and
anti-biofilm activities. Relatively few alkaloids were reported. Quinones and organosulfur
compounds, especially from garlic, were also active. A common feature is the polar nature of these
compounds. Phytochemicals with these activities are widespread in Angiosperms in temperate and
tropical regions, but gymnosperms, bryophytes and pteridophytes were not represented.
Keywords: biofilm inhibitors; quorum sensing inhibitors; terpenes; quinones;

organosulfur compounds
1. Introduction
Microbial biofilms are organized aggregations of cells attached to a substratum and surrounded
by a self-produced extrapolymeric substance (EPS) matrix. These biofilms develop on many biotic
and abiotic surfaces such as plant leaves and medical devices. The formation of a biofilm is a complex
process, which requires the coordinated expression of many specific genes. The interconversion
between planktonic and biofilm growth is controlled by a cell-to-cell communication system known
as quorum sensing (QS). QS involves signal molecules called autoinducers that are released by
the microbes themselves. Gram-positive bacteria use autoinducing peptides (AIPs) for signaling
and Gram-negative bacteria have lipid-based molecules known as N-acyl-homoserine lactones
(AHLs) [1–5]. In fungi such as Candida albicans, the quorum sensing molecules are farnesol and
tyrosol [6,7]. Once populations reach a specific density or threshold, expression of certain genes
such as virulence factors and adhesion proteins can occur. In many bacterial species, QS induction
is required in order for biofilm formation to occur; in other species such as Staphylococcus aureus,
repression of QS is necessary [8].
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Molecules 2016, 21, 29 2 of 26
Many human pathogens are also biofilm formers; these are responsible for many persistent
nosocomial infections especially in the immunocompromised population. These pathogens include
Pseudomonas aeruginosa [9], Burkholderia cepacia [10], Listeria monocytogenes [11], Staphylococcus
aureus [8] and Candida albicans [12], to name a few. With the rapid increase in antibiotic resistance
to conventional therapies and the development of multi-resistant superbugs, the need for alternative
antimicrobials is of particular interest. Current strategies to combat the increasing antibiotic resistance
include targeting QS to prevent the formation of new biofilms and inhibiting the growth of existing
biofilms. A recent comprehensive review [13] describes many chemical agents that inhibit bacterial
biofilm formation derived from medicinal chemistry and biodiversity sources.
Within this large field of study, we focused in the present mini-review on the presence of quorum
sensing and biofilm inhibitors against both bacteria and fungi in terrestrial plants, which has been
documented in numerous plant species using various model microorganism bioassays. The presence
of biofilms in plants is of interest for a fundamental understanding of the phenomenon in nature and
also for its practical application. First, the discovery of these substances in plants informs chemical
ecologists of potential new defense mechanisms in plants for future studies as well as which taxa
and habitats are involved. In addition, it provides new insight into the use of these substances and
plants in traditional medicine. The identification of the classes of plant derived secondary metabolites
(phytochemicals) provides information on the evolution of these compounds, substances of interest
for mechanistic studies and lead metabolites for analoging, quantitative structure-activity studies and
drug development.
The objective of this mini review was to describe phytochemicals from terrestrial plant taxa
and that can affect microbial quorum sensing and biofilm formation. Plant literature published
before 2015 is summarized from searches that were performed using PubMed and Web of Science.
The results are organized into a discussion of model organism bioassays used for assessing QS and
biofilm inhibition, the different biosynthetic classes of active phytochemicals with representative
substances and an overview of the taxa with activity.
2. Model Organism Bioassays

Bioluminescence or the production of light in marine bacteria such as Vibrio harveyi and
Vibrio fischeri has been widely used to assess the ability of natural and synthetic compounds to
interfere with quorum sensing in Gram-negative bacteria. In these species, light production is
mediated by the luxCDABEGH operon, which is under QS control [14,15]. Similarly, Chromobacterium
violaceum produces a purple pigment, violacein, which is also cell density-dependent. This species
is popular in screening studies as it can be used to visually assess QS inhibition in a disc
diffusion assay [16]. The lux operon is used in many reporter strains along with the lacZ gene
and green fluorescent protein (GFP). These strains include Agrobacterium tumefaciens NTL4 [16],
Escherichia coli MT102 (pSB403) [17], Pseudomonas aeruginosa PAO1 [18], and Vibrio harveyi [19],
among others. The production of autoinducers can be quantified directly using luminescence,
fluorescence, and absorbance of pigments or directly via chromatographic techniques such as
High Performance Liquid Chromatography-Diode Array Detection (HPLC-DAD). For Gram-positive
bacteria, QS inhibition is usually measured directly by quantifying the concentration of autoinducing
peptides using HPLC coupled with mass spectrometry (MS) [20].
Many biofilm models have been developed using various microbial species and surfaces.
The simplest and most popular assay involves growing a biofilm in a microtiter plate in the presence
and absence of the compound of interest and then measuring the stained biofilm mass at the optimal
wavelength [21]. This method can be used with fungi, Gram-positive and Gram-negative bacteria.
In addition, dual-species and mixed-species biofilms has been studied with many different substrates.
Molecules 2016, 21, 29 3 of 26
3. Phytochemicals as QS and Biofilm Inhibitors

Phytochemicals reported in the literature are arranged according to the biosynthetic groups
described in Dewick [22]. A discussion of their bioactivities follows.
3.1. Phenolics
3.1.1. Phenylpropenoids
By far, phenolics represent the highest number of active compounds reported in terms
of their effects on quorum sensing and biofilm formation when compared to all other classes
(Table 1). Eugenol, a phenylpropene present in many plants, has been shown to inhibit
the production of QS-mediated violacein in Chromobacterium violaceum and virulence factors in
Pseudomonas aeruginosa PAO1 by 32% to 56% at concentrations of 50 to 200 µM, respectively [23].
This compound was also effective against biofilms of Pseudomonas aeruginosa, Listeria monocytogenes
and Klebsiella pneumoniae clinical isolates [23–25]. Cinnamaldehyde, another phenylpropene, was
reported by Brackman et al. [26] to interfere with the AI-2-mediated QS system in Vibrio spp.
(65% inhibition at 100 µM). In terms of biofilm formation, cinnamaldehyde was shown to be
effective against both Gram-positive and Gram-negative bacteria such as Listeria monocytogenes [24],
Staphylococcus epidermidis [27], and Cronobacter sakazakii [28]. In these studies, the active concentration
ranged from 946 µM to 38 mM, which were reported to inhibit the formation of new and preformed
biofilms as well as downregulate the expression of biofilm-associated genes [24,27,28].
3.1.2. Benzoic Acid Derivatives

Benzoic acid derivatives such as vanillin and gallic acid showed mixed effects depending
on the organism and concentration tested. In a study by Ponnusamy et al. [29], 250 µg/mL of
vanillin inhibited QS in Chromobacterium violaceum and biofilm formation in Aeromonas hydrophila.
The anti-biofilm activity of vanillin was also confirmed by Kappachery et al. [30] using different
abiotic surfaces. At a concentration of 0.18 mg/mL, pre-treatment with vanillin reduced the biofilm
formation of Aeromonas hydrophila on membrane filters by 90% [30]. In another study, at 200 µg/mL
vanillin enhanced AHL production in Escherichia coli JDL271/pAL105 and biofilm formation in
Pseudomonas aeruginosa PAO1 and Agrobacterium tumefaciens C58 by at least two-fold [31]. Similarly,
gallic acid at 200 µg/mL had no effect on P. aeruginosa PA14 biofilms [32] but inhibited P. aeruginosa
PAO1 biofilm formation by 30% [31] and enhanced Staphylococcus epidermidis biofilms by three-fold
at a similar concentration [33]. At a much higher concentration of 1 mM, gallic acid was shown to
inhibit Eikenella corrodens biofilm formation by 80% [34]. In terms of QS, Borges et al. [35] saw a 59%
reduction in violacein production at 1 mg/mL using Chromobacterium violaceum.
Other benzoic acid derivatives have also been reported to have anti-QS and anti-biofilm effects.
Ellagic acid at 36 µg/disc showed greater QS inhibition in C. violaceum when compared to the
positive control Delisea pulchra (Greville) Montagne extract [32]. This activity was confirmed by
Huber et al. [17] who observed QS inhibition in E. coli MT102 and Pseudomonas putida at concentrations
of 40 µg/mL and 30 µg/mL, respectively. This compound was not effective against P. aeruginosa
PA14 biofilms at 10 µg/mL [32]. In another study, ellagic acid inhibited biofilm formation in various
Streptococcus dysgalactiae strains by 70% at 4 µg/mL [36]. At higher concentrations of 15 to 40 µg/mL,
ellagic acid was inhibitory against Escherichia coli, Burkholderia cepacia, Staphylococcus aureus,
and Candida albicans biofilms [37].
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Table 1. Phenolics and derivatives affecting microbial quorum sensing (QS) and/or biofilm formation. 44 of
of 26
26
Molecules 2016, 21, 29 4 of 26
Table 1.
Table 1. Phenolics
Phenolics and
and derivatives
derivatives affecting
affecting microbial
microbial quorumquorum sensing
sensing (QS) (QS) and/or
and/or biofilm
biofilm formation.
formation.
Active Constituents (Source Table Plants)1. Phenolics and derivatives affecting microbial quorum sensing Biological(QS)Activities
and/or biofilm formation. Ref.
Active Constituents
Active Constituents (Source
(Source Plants)
Plants) Biological Activities
Biological Activities Ref.
Ref.
Phenylpropenoids
Active Constituents (Source Plants)
Phenylpropenoids Biological Activities Ref.
Phenylpropenoids
Phenylpropenoids
(various species)
eugenol (various • ‚ Inhibited
Inhibited las las QS-mediated
QS-mediated elastase
elastase production
production (32%(32% at at
200 200
μM) µM)
and and
• Inhibited las QS-mediated elastase production (32% at 200 μM) and pqs QS-mediated pyocanin productionpqs pqs QS-mediated
QS-mediated pyocanin
pyocanin production
production
eugenol species) • Inhibited
(56% atlas
50QS-mediatedP. aeruginosa,
in aeruginosa, QS-mediated violacein production pqsin C. violaceum (48% at
eugenol (various species) (56%
(56% at 50
at 50 μM)
μM)
µM)
in P.
in P. elastase productionviolacein
QS-mediated
aeruginosa, QS-mediated
(32% at 200
violacein production
μM) andin
production C.QS-mediated
in C. violaceum
violaceum (48%
(48% 150150
pyocanin
at 150
at μM)
μM)
µM)
production [23]
[23] [23]
• ‚ (56%Decreased
at 50 μM)
Decreased biofilm
in P.
biofilm formation
aeruginosa,
formation in P. aeruginosa
in aeruginosa
QS-mediated
P. PAO1
violacein
PAO1 (43%(43% at
production
at 400 400
μM)µM)
in C. violaceum (48% at 150 μM) [23]
• Decreased biofilm formation in P. aeruginosa PAO1 (43% at 400 μM) [23] [23]
• ‚ Decreased
Inhibited
Inhibited formation
biofilm
formation of new
formation
of new
new and inand
P. inactivated
aeruginosa
inactivated preformed
PAO1
preformed (43% atbiofilms
400 μM)
biofilms in L.
L. L. monocytogenes
in monocytogenes [23]
[24] [24]
• Inhibited formation of and inactivated preformed biofilms in monocytogenes [24]
(2.5
• Inhibited
(2.5 mM mM
mM and and
formation
and 25 25
25 mM, mM,
of respectively)
new and
mM, respectively)
respectively) inactivated preformed biofilms in L. monocytogenes [24] [24]
(2.5 [24]
OCH3
OCH • ‚ (2.5Downregulated
mM and
Downregulated 25 mM, genes
genes critical
respectively)
critical to L. L. monocytogenes
tomonocytogenes biofilm
biofilm formation
formation (2.5 (2.5
mM)mM) [24]
[25] [25]
OCH3
3 • Downregulated genes critical to L. monocytogenes biofilm formation (2.5 mM) [25]
OH Inhibited
• ‚ Downregulated
Inhibited biofilm
biofilm genesformation
critical
formation in to
K. K.monocytogenes
pneumoniae
inL.pneumoniae clinical
biofilm
clinical isolates
formation
isolates (MIC(MIC= = 63.5
(2.5
63.5 mM) µg/mL)
μg/mL) [25]
OH • Inhibited biofilm formation in K. pneumoniae clinical isolates (MIC = 63.5 μg/mL)
OH
• Inhibited biofilm
formation formation
of new in K.inactivated
and pneumoniaepreformed
clinical isolates
• Inhibited formation of new and inactivated preformed biofilms in L. monocytogenes biofilms(MIC in = 63.5
L. μg/mL)
monocytogenes
cinnamaldehyde
cinnamaldehyde • ‚ Inhibited
Inhibited
(0.75 andformation
mM formation 10 mM,
mM, of new
andand
of respectively)
new inactivated
preformed biofilms
biofilms in L. L. monocytogenes
in monocytogenes [24]
cinnamaldehyde
(Cinnamomum sp.—Lauraceae) (0.75 mM and 10 respectively) [24]
(Cinnamomum sp.—Lauraceae) (0.75 mM and 10 mM, respectively)
•• Inhibited
(0.75 mM and 10
Inhibited AI-2-mediated mM,
AI-2-mediated QS respectively)
QS inin Vibrio
Vibrio spp.
spp. (65%
(65% at at 100
100 μM)μM) [24]
[26] [24]
(Cinnamomum sp.—Lauraceae) [26]
• ‚ Inhibited
Inhibited
Downregulated AI-2-mediated
AI-2-mediated
genes QS QS
involved in
in Vibrio
in Vibrio
L. spp.spp.
(65%(65%
monocytogenes atbiofilm
at 100 100
μM) µM)formation (0.75 mM) [26]
[24]
O
O • Downregulated genes involved in L. monocytogenes biofilm formation (0.75 mM) [24] [26]
• ‚ Downregulated
Downregulated genes
genes involved
involved L. monocytogenes
inmonocytogenes
inepidermidis
L. biofilm
biofilm formation
formation (0.75
(0.75 mM)mM) [24]
O Inhibited biofilm formation in S.
• Inhibited biofilm formation in S. epidermidis (MIC = 125 μg/mL)
Inhibited
‚ Inhibited biofilm formation S. epidermidis
ininactivated
(MIC
(MIC
= 125
= 125
μg/mL)
µg/mL)
[27]
[27] [24]
• biofilm
formation formation
of new in S.
and epidermidis (MIC
mature
• Inhibited formation of new and inactivated mature biofilms in C. sakazakii
= biofilms
125 μg/mL) in C. sakazakii [27]
[28]
[28] [27]
• ‚ Inhibited
(750Inhibited
μM and formation
formation
38 mM, of new
andand
ofrespectively)
new inactivated
inactivated
and mature
mature
downregulated biofilms
biofilms in C.
expression C. sakazakii
in sakazakii
biofilm-related genes [28] [28]
(750(750
μMµM andand 38 mM,
38 mM, respectively)
respectively) andanddownregulated
downregulated expression
expression biofilm-related
biofilm-related genes
genes
Benzoic Acid
Acid Derivatives
Derivatives (750 μM and 38 mM, respectively) and downregulated expression biofilm-related genes
Benzoic
Benzoic Acid Derivatives
vanillin
vanillin
(Vanilla vanillin Jacks. ex
planifolia
(Vanilla planifolia
vanillin Jacks. ex
(Vanilla planifolia
Andrews—Orchidaceae)
(Vanilla planifolia Jacks. Jacks. ex
ex Andrews—Orchidaceae)
Andrews—Orchidaceae) •• Inhibited
Inhibited violacein
production in in C.C. violaceum
violaceum CV026
CV026 (69%) (69%) andand biofilm
biofilm formation
formation inin A.
A. hydrophila
hydrophila (50%)
(50%) at
at
O
Andrews—Orchidaceae)
O • ‚ 0.25Inhibited
Inhibited
mg/mL violacein
production in C. C. violaceum
in violaceum CV026CV026 (69%)
(69%) andand biofilm
biofilm formation
formation in A. A. hydrophila
in hydrophila (50%)
(50%) at at [29]
0.25 mg/mL [29]
O 0.25 mg/mL
•• ‚ Inhibited
0.25 mg/mL
Inhibited biofilm
biofilm formation
formation in in A.A. hydrophila
hydrophila on on membrane
membrane filters filters byby 90%
90% with
with pre-treatment
pre-treatment of of 0.18
0.18 mg/mL
mg/mL [29]
[30] [29]
Inhibited biofilm formation in A. hydrophila on membrane filters by 90% with pre-treatment of 0.18 mg/mL [30]
Enhanced
• ‚ Enhanced biofilm
biofilm
formation
formation
formation
inin
in
A.
P.
P.
hydrophila
aeruginosa
aeruginosa
on membrane
PAO1
PAO1 (3-fold
(3-fold
filters
at
at 200
200
by 90% with
μg/mL)
μg/mL) and
and
pre-treatment
A.
A. tumefaciens
tumefaciens
of
C58
C58
0.18 mg/mL [30]
[31]
[31]
[30]
Enhanced
• Enhanced biofilm
biofilm formation
formation P. aeruginosa
in aeruginosa
inproduction
P. PAO1PAO1 (3-fold
(3-fold at at
200200 µg/mL)
μg/mL) and and A. tumefaciens
A. tumefaciens C58C58 [31] [31]
(2-fold
(2-fold at 25
at 25 μg/mL) and AHL in E. coli JDL271/pAL105 at 40 μg/mL
(2-fold at μg/mL)
25 µg/mL) andand AHL AHL production
productionin E.incoli JDL271/pAL105
E. coli JDL271/pAL105 at 40atμg/mL
40 µg/mL
OCH3
OCH (2-fold at 25 μg/mL) and AHL production in E. coli JDL271/pAL105 at 40 μg/mL
3
OH OCH3
OH
OH acid
gallic
gallic acid
gallic acid
gallicspecies)
(various acid
species)
(various
O
(various species) •• ‚ Enhanced
Enhanced
Enhanced biofilm formation
biofilm
biofilm formation
formation in P.
in P. aeruginosa
inaeruginosa PAO1
P. aeruginosa PAO1
PAO1 (2-fold
(2-fold
(2-fold at 25
at 25 25
at μg/mL)
µg/mL)
μg/mL) andand
and A. tumefaciens
A. tumefaciens C58C58
A. tumefaciens
C58 [31]
O • Enhanced biofilm formation in biofilm
P. aeruginosa PAO1in(2-fold at 25 μg/mL) andby
A.30%
tumefaciens C58 [31]
O (2-fold
(2-fold at 100
(2-fold
at 100
at μg/mL);
100 µg/mL);
μg/mL); reduced
reduced
reduced formation
biofilm
biofilm formation
formation P.inaeruginosa
in P. aeruginosa PAO1
P. aeruginosa
PAO1 PAO1by
by 30%
30% at 200
at 200 μg/mL
at 200 µg/mL
μg/mL [31]
HO [31]
[33]
HO • (2-fold
Increasedat 100
Increased μg/mL);
biofilm reduced
formation
biofilm in
formation biofilm
S. formation
epidermidis by
S. epidermidis
inepidermidis in P.
3-fold aeruginosa
at 188 μg/mLPAO1 by 30% at 200 μg/mL [33] [33]
HO OH
OH • ‚ Increased biofilm formation in S. by by 3-fold
3-fold at 188
at 188 µg/mL
μg/mL [33]
[34]
• Increased
Inhibited biofilm
biofilm
Inhibited formation
formation
biofilm in E.
in
formation S. corrodens
in epidermidisbyby
E. corrodens 80%
by 3-fold
at 11atat
80% 1188
mM mMμg/mL [34] [34]
OH • ‚ Inhibited biofilm formation in E. corrodens by 80% at mM [34]
[35]
violacein
Inhibited formation
violacein in E.
production
productionincorrodens
C. by 80%
violaceum
in violaceum ATCC
C. violaceum at 112472
ATCC mM (59%
12472 (59%at 11atmg/mL)
mg/mL)
1 mg/mL) [35] [35]
HO
HO • ‚ Inhibited violacein production in C. ATCC 12472 (59% at [35]
HO • Inhibited violacein production in C. violaceum ATCC 12472 (59% at 1 mg/mL)
OH
OH
OH
Molecules 2016, 21, 29 5 of 26
Molecules 2016, 21, 29 5 of 26
Table 1. Cont.
Table 1. Cont.
Active Constituents (Source Plants) Biological

Biological Activities
Activities Ref. Ref.
Benzoic
Acid Derivatives
acid
ellagic acid
(various species)
O •‚ Inhibited
Inhibited biofilm
biofilm formation
formation (50%(50% at μg/mL)
at 40 40 µg/mL)andand swarming
swarming motility
motility (100%
(100% atμg/mL)
at 20 20 µg/mL) B. cepacia
in B.incepacia
[17]
•‚ Inhibited
Inhibited QS QS
in E. E. coli
incoli MT102
MT102 (pSB403)
(pSB403) by by
40%40% at μg/mL
at 40 40 µg/mL andand P. putida
P. putida (pKR-C12)
(pKR-C12) by 40%
by 40% at μg/mL
at 30 30 µg/mL [17]
OH [17]
O •‚ Inhibited
Inhibited violacein
production in C. C. violaceum:
in violaceum: 18.318.3
mmmm at μg
at 36 36 µg [17]
[32] [32]
HO •‚ Reduced
Reduced biofilm
biofilm formation
formation indysgalactiae
in S. S. dysgalactiae strains
strains by by
70%70%
at 4atμg/mL
4 µg/mL
OH [36] [36]
•‚ Inhibited
Inhibited biofilm
biofilm formation
formation in S. S. aureus
inaureus ATCCATCC 11632
11632 (60%
(60% at 15
at 15 µg/mL),
μg/mL), MRSAMRSA ATCC
ATCC 33591
33591
(70% at μg/mL),
20 µg/mL), E. coli ATCC 10536 (60% atμg/mL),
15 µg/mL), C. albicans [37] [37]
O (70% at 20 E. coli ATCC 10536 (60% at 15 and and
C. albicans ATCC
ATCC 90028
90028 (50%(50%
at 20atμg/mL)
20 µg/mL)
HO
O
Tannins
Tannins
1,2,3,4,6-penta-O-galloyl-ß-D-glucopyranose
(various species)
OH
HO OH
OH
O
O HO OH
Inhibited
• ‚ Inhibited biofilm
biofilm formation
inaureus (IC(IC 3.6= μM)
50 =50 3.6 µM) [38] [38]
O
H O
O
HO O O OH
O H O
H H
O
HO OH
OH
OH
HO OH
OH HO
Molecules 2016, 21, 29 6 of 26
Molecules 2016, 21, 29 6 of 26
Table 1. Cont.
Table 1. Cont.
Active Constituents (Source Plants) Biological

Tannins
Tannins
punicalagin
punicalagin
(Punica granatum L. (Lythraceae) and
Combretaceae species)
OH
HO O
HO O • ‚ Inhibited
Inhibited violacein
production in C. C. violaceum
inviolaceum andand swimming/swarming
swimming/swarming motility
motility S. typhimurium
in S.intyphimurium SL1344
SL1344
O OH
HO
O
at 15.6
at 15.6 µg/mL
μg/mL [39] [39]
O OH
OH • ‚ Downregulated
Downregulated expression
expression of motility
of motility andand
QSQS related
related genes
genes in S. S. typhimurium
in typhimurium at 15.6
at 15.6 µg/mL
μg/mL
HO O
O OH OH
O
OH O OH
O
O
HO
O OH
O
OH
HO
OH
hamamelitannin
(Hamamelis virginiana L.—Hamamelidaceae)
OH
HO OH • ‚ Inhibited agr agr
Inhibited QS QS regulator
regulator RNAIII
RNAIII andand δ-hemolysin
δ-hemolysin production
production at μg/mL
at 50 50 µg/mL
• ‚ Reduced
Reduced cellcell attachment
attachment of MRSA
of MRSA to polystyrene
to polystyrene plate
plate at 4atμg/mL
4 µg/mL
HO [40] [40]
• ‚ In mice
In mice infection
infection model,
model, treatment
treatment of grafts
of grafts with
with 30 mg/mL
30 mg/mL showed
showed no detectable
no detectable MRSAMRSA
andand MRSE
MRSE loadload
OH
O after
after 7 days
7 days
O O OH
O OH
HO
HO
O
Molecules 2016, 21, 29 7 of 26
Molecules 2016, 21, 29 7 of 26
Molecules 2016, 21, 29 7 of 26
Table
Table1. Cont.
1. Cont.
Table 1. Cont.
Active Constituents (Source Plants) Biological Activities Ref.
(Source Plants)
Tannins
Active Constituents Biological
tannic acid
Tannins
Tannins
(various
tannicspecies)
acid
HO OH
(various species)
HO HO OH
HO O
HO OH • Enhanced biofilm formation in P. aeruginosa PA14 at 100 μg/mL [17]
O HO OH OH •‚ Inhibited
Enhanced QS
Enhanced in P. formation
putida
biofilm
biofilm (pKR-C12)
formationin P. P.by 40% at
aeruginosa
inaeruginosa 30
PA14 μg/mL
PA14 at 100
at 100 and E. coli MT102 (pSB403) by 20% at 60 μg/mL
µg/mL
μg/mL [17]
HO O O [17]
HO
HO O O OH •‚ Inhibited violacein
Inhibited
QS QS
in P. production
P.
inputidaputida in C .violaceum
(pKR-C12)
(pKR-C12) by by
40% 40%at (21.8
at 30
30 mm
µg/mL
μg/mL at 500
and E.μg)
and E. coli
coli MT102
MT102 (pSB403)
(pSB403) by 20%
by 20% at μg/mL
at 60 60 µg/mL [32]
[17] [17]
HO O
HO HO
OO
O O
O
•‚ Inhibited biofilm
violacein
Inhibited formation
production
violacein in P.
production inaeruginosa
Cin.violaceum PA14
C .violaceum (21.8(72%
mmmm
(21.8 atat200
500 μg/mL)
μg)µg)
at 500 [32] [32]
HO
HO OO O
HO O
O O
O
O OH
•‚ Inhibited S. aureus
Inhibited biofilm
biofilm biofilm
formation formation
formation (60% atPA14
P. aeruginosa
inaeruginosa
in P. 2 PA14
μM) and
(72%
(72% atincreased
at 200
200 isaA expression (a transglycosylase)
µg/mL)
μg/mL) [41]
[32] [32]
HO O
HO O O OH OH •‚ Reduced
Inhibited expression
Inhibited S. aureus
S. aureus of biofilm
genes
biofilm responsible
formation
formation (60% foratQS
(60% and
2atμM)
2 µM)virulence
andand in S. isaA
increased
increased aureus
isaA at 20 μg/mL
expression
expression (a transglycosylase)
(a transglycosylase) [42]
[41] [41]
O O O OO
HO
HO
HO OH OH • Reduced
Inhibited
Reduced expression
biofilm
expression of genes
formation
of genes in S.responsible
aureus by
responsible for
QSQS
for>50% atand
and 20 virulence
μg/mL
virulence in S. S. aureus
in aureus at 20
at 20 µg/mL
μg/mL [42] [42]
O O O O
HO
HO
HO
O
OH OH •‚ Inhibited
Inhibited biofilm
biofilm formation
inaureus by by
>50%>50% at μg/mL
at 20 20 µg/mL [42] [42]
HO O O
HO O
O OH OH
HO HO HO OH
O
O HO
O OH
HO HO OH
O
HO
Stilbenes
Stilbenes
resveratrol
Stilbenes
(various species)
resveratrol
OH • Inhibited S. aureus biofilm formation (30%) and enhanced S. epidermidis biofilm formation (1.5-fold) at
(various species) [33]
OH •‚ 100Inhibited S. aureus
μg/mLS. aureus
Inhibited biofilm
biofilm formation
formation (30%)
(30%) andand enhanced
enhanced S. epidermidis
S. epidermidis biofilm
biofilm formation
formation (1.5-fold)
(1.5-fold) at at
HO [42]
[33] [33]
• 100 µg/mL
Inhibited
100 μg/mL biofilm formation in P. aeruginosa PA14 and E. coli O157:H7 at 50 μg/mL
HO [43]
[42] [42]
•‚ Inhibited
Inhibited biofilm
P. acnes
biofilm formation
biofilm
formation formation
in P. P. aeruginosa
inaeruginosa at PA14
by 80% PA140.32%
and E. E.
and colicoli O157:H7
O157:H7 at at
50 50 µg/mL
μg/mL
Inhibited P. acnes biofilm formation [43] [43]
•‚ Inhibited P. acnes biofilm formation by by
80%80% at 0.32%
at 0.32%
OH
OH Stilbenes
pterostilbene
Stilbenes
(Vitis sp.—Vitaceae and Ericaceae species)
pterostilbene •‚ Inhibited
Inhibited formation
formation of new
of new andand mature
mature C. albicans
C. albicans SC5314,
SC5314, Y0109,
Y0109, 0304103,
0304103, andand 01010
01010 biofilms
biofilms at 16atμg/mL
16 µg/mL
OH
(Vitis sp.—Vitaceae and Ericaceae species) •‚ Inhibited
Inhibited hyphal
hyphal
formation formation
formation
of in C.
new and C. albicans
inmature
albicans 4atμg/mL
4 µg/mL
C.atalbicans SC5314, Y0109, 0304103, and 01010 biofilms at 16 μg/mL
OH
H3CO •‚ Treatment
Treatment
Inhibited of μg/mL
of 16
hyphal 16 µg/mL
formation altered
altered expression
expression
in C. albicans of genes
atof4 genes
μg/mL involved
involved in morphological
in morphological transition,
transition, ergosterol
ergosterol [44] [44]
H3CO • biosynthesis,
biosynthesis,
Treatment of 16 filamentation,
filamentation,
μg/mL andand
altered cellcell
expression surface
surface proteins;
ofproteins;
genes alsoalso
involved effective
effective
in in rat
in rat central
central
morphological venous
venous catheter
catheter
transition, ergosterol [44]
infection
infection model
model
biosynthesis, filamentation, and cell surface proteins; also effective in rat central venous catheter
OCH3 infection model
OCH3
Molecules 2016, 21, 29 8 of 26
Molecules 2016, 21, 29 8 of 26
Molecules
Molecules 2016,
2016, 21,
21, 29
29 88 of
of 26
26
Table 1. Cont.
Table 1. Cont.
Table
Table 1.
1. Cont.
Cont.
Active Flavonoids
Active Constituents
Constituents (Source Plants) Biological Activities
Biological
Activities Ref.
Ref. Ref.
quercetin
Flavonoids
Flavonoids
Flavonoids
(various species)
quercetin
quercetin
OH
(various
(various species) • Inhibited AI-induced bioluminescence in V. harveyi BB886, MM32 (75% at 6.25 μg/mL) and biofilm
OH
OH OH [45]
•• ‚ formation
Inhibited in AI-induced
Inhibited
Inhibited E. coli O157:H7,
AI-induced
AI-induced V. harveyi in
bioluminescence
bioluminescence
bioluminescence BB120
in V.
V. V.(60%
in harveyi at
harveyi
harveyi 6.25
BB886, μg/mL)
MM32
BB886,
BB886, MM32MM32 (75% at
at 6.25
(75%
(75% μg/mL)
at 6.25
6.25 μg/mL) and biofilm
andand
µg/mL) biofilm
biofilm
OH [46]
[45]
[45]
HO O OH • Inhibited
formation biofilm
in
in E.
formation
formation E. E.formation
incoli
colicoli
O157:H7, inV.
V.MRSA
O157:H7,
O157:H7, (>80%)
V. harveyi
harveyi
harveyi BB120 and
BB120
BB120 (60%
(60%MSSA
at at (>50%)
at 6.25
(60% 6.25 μg/mL)
6.25 strains at 1 μg/mL
µg/mL)
μg/mL) [45]
[46]
[46]
[46]
HO O
••• ‚ Reduced
Inhibited
Inhibited expression
Inhibited biofilm
biofilm
biofilm of genesin
formation
formation ininvolved
formation in MRSA
MRSA
MRSA in(>80%)
QS and
(>80%)
(>80%) and virulence
andand MSSA
MSSA
MSSA in
(>50%)
(>50%)S.strains
(>50%)aureus at
at 110
strains
strains at μg/mL
1atμg/mL
1 µg/mL
μg/mL
[46]
HO O [46]
•• ‚ Reduced
Reduced expression of genes involved in QS S. aureus [46] [46]
OH Reduced expression
expression of
of genes
genes involved
involved in
in QS
QS andand
and virulence
virulence
virulence in inaureus
in S.
S. aureus at at μg/mL
at 10
10 10 µg/mL
μg/mL
OH O OH
OH
(−)-catechin
OH O
OH O
(Camellia sinensis (L.) Kuntze—
(−)-catechin
(−)-catechin
(´)-catechin
(Camellia Theaceae
(Camellia
sinensis and
(L.) others)
(Camellia sinensis (L.)
sinensis Kuntze—
Kuntze—Theaceae
(L.) Kuntze—
OH
Theaceae and
and others)
and others)
Theaceae others) • Inhibited violacein production in C. violaceum CV026 (50% at 2 mM), pyocyanin (50% at 0.25 mM) and
OH
OH OH
•• ‚ elastase
Inhibitedproduction
violacein
Inhibited
Inhibited (30%
violacein
violacein at 4 mM)
production
production
production in ininviolaceum
in C.
C. C.P.violaceum
aeruginosa
violaceum CV026PAO1
CV026
CV026 (50% at
at 22atmM),
(50%
(50% pyocyanin
2 mM),
mM), pyocyanin
pyocyanin (50% at
at 0.25
(50%
(50% mM)
at 0.25
0.25 mM)mM) and
andand [47]
OH
OH • Reduced biofilm formation
HO O elastase
elastase
elastase production
production
production (30%
(30% at and
(30%
at 4downregulated
44atmM)
mM) mM)in inaeruginosa
in P.
P. QSPAO1
aeruginosa genes
P. aeruginosa PAO1
PAO1 expression in P. aeruginosa PAO1 by at 4 mM [47]
[47] [47]
HO
HO O
O
•• ‚ Reduced
Reduced
Reduced biofilm
biofilm
biofilm formation
formation
formation andand
and downregulated
downregulated
downregulated QSQS
QS genes
genes
genes expression
expression
expression in
in P.
P. P. aeruginosa
in aeruginosa
aeruginosa PAO1
PAO1
PAO1 byby
by at at
at 4 mM
44 mM
mM
OH
OH OH
OH
OHFlavonoids
OH
(−)-epicatechin
Flavonoids
(´)-epicatechin
Flavonoids
(Camellia
(Camellia sinensis
sinensis (L.) Kuntze—
(L.) Kuntze—Theaceae
(−)-epicatechin
(−)-epicatechin • ‚ Increased AHL
Increased AHLproduction
productionin E.incoli JDL271/pAL105
E. coli JDL271/pAL105at 40atto40200 μg/mL
to 200 µg/mL
Theaceae
(Camellia and
and others)
sinensis others)
(L.) Kuntze— [31]
OH ••• ‚ Enhanced
Increased AHL production in E. coli JDL271/pAL105 at 40 to 200 μg/mL andand
Enhanced
Increased biofilm
AHL formation
biofilm formation
production ininE.P.
in aeruginosa
P.
coli PAO1
aeruginosa PAO1
JDL271/pAL105 (2-fold
(2-fold
at 40 at
to 200
at
200 μg/mL)
200 µg/mL)
μg/mL A. tumefaciens C58C58
A. tumefaciens [31]
Theaceae
Theaceae and
and others)
others) [31]
[31]
[31] [31]
(2-fold
•• (2-fold
Enhanced at
at 400 400 µg/mL)
μg/mL)
biofilm formation
OH
OH
OH Enhanced biofilm formation in in P.
P. aeruginosa
aeruginosa PAO1
PAO1 (2-fold
(2-fold atat 200
200 μg/mL)
μg/mL) andand A.
A. tumefaciens
tumefaciens C58
C58 [35]
[31]
• ‚ Inhibited
Inhibited biofilm
biofilm formation
formation in E. E. coli
incoli JM109
JM109 by by
40%40%
at 1atmg/mL
1 mg/mL [31] [35]
OH
OH
(2-fold
(2-fold at
at 400
400 μg/mL)
μg/mL) [35]
HO O [35]
[35] [35]
••• ‚ Inhibited
Inhibited
Inhibited violacein
Inhibited violacein
biofilm production
production
biofilm formation
formation in
in E.incoli
E. C.
coli
C. violaceum
in violaceum
JM109 ATCC
byATCC
JM109 by 40% 12472
at12472
40% at 11 mg/mL
mg/mL
(33%
(33% at 1atmg/mL)
1 mg/mL)
[47]
[35]
[35] [47]
HO
HO O
O ••• ‚ Inhibited
Inhibited
Inhibited elastase
elastase
violacein activity
activity in
production inin
P. P. C.
aeruginosa
aeruginosa PAO1
PAO1
violaceum by
ATCC by
40% 40%at
12472 at
4
Inhibited violacein production in C. violaceum ATCC 12472 (33% at 1 mg/mL)
4 mM
mM
(33% at 1 mg/mL)
OH [47]
[47]
•• Inhibited
Inhibited elastase
elastase activity
activity in
in P.
P. aeruginosa
aeruginosa PAO1
PAO1 by
by 40%
40% atat 44 mM
mM
OH OH
OH
OH
OH
Molecules 2016, 21, 29 9 of 26
Molecules 2016, 21, 29 9 of 26
Molecules 2016, 21, 29 9 of 26
Table 1. Cont.
Table 1. Cont.
Table 1. Cont.
(Source
(Source Plants)
(–)-gallocatechin
Active Constituents Plants) Biological
(Camellia sinensis (L.) Kuntze—Theaceae)
(–)-gallocatechin
(–)-gallocatechin
OH
OH
OH OH
OH
• Inhibited biofilm formation in E. corrodens by >60% at 1 mM (MIC = 0.5 mM) [34]
OH
HO O
OH • ‚ Inhibited biofilm
Inhibited formation
biofilm in E.
formation incorrodens by by
E. corrodens >60% at 1atmM
>60% (MIC
1 mM = 0.5
(MIC mM)
= 0.5 mM) [34] [34]
HO
HO O
O
OH
OH
OH
OH OH
OH
(–)-epigallocatechin
OH
OH
Theaceae
(Camellia and
sinensis
(Camellia sinensis (L.) others)
(L.) Kuntze—
Kuntze—Theaceae
OH
Theaceae and others)
and others)
OH
OH OH
OH
OH
HO O • ‚ Inhibited
Inhibited biofilm
biofilm formation
formation in E. E. corrodens
incorrodens by by >60%
>60% at 1atmM
1 mM (MIC
(MIC = 0.25
= 0.25 mM) mM) [34] [34]
OH
HO
HO O
O
OH
OH
OH
OH OH
OH
OH
OH
Flavonoids
(–)-catechin gallate
Flavonoids
(–)-catechin gallate
OH
OH
OH OH
OH
OH
HO O
HO O
• ‚ Inhibited
Inhibited biofilm
biofilm formation
>60% at 1atmM
1 mM (MIC
(MIC = 0.1
= 0.1 mM)mM) [34] [34]
HO O
O • Inhibited biofilm formation in E. corrodens by >60% at 1 mM (MIC = 0.1 mM) [34]
OH O
O OH
O
OH
OH OH
OH
O
O
OH
OH OH
OH
OH
OH
Molecules 2016, 21, 29 10 of 26
Molecules 2016, 21, 29 10 of 26
Molecules 2016, 21, 29 10 of 26
Table 1. Cont.
Table 1. Cont.
Table 1. Cont.
Constituents (Source
Active (–)-epicatechin gallatePlants) Biological
(Camellia sinensis (L.)gallate
(–)-epicatechin
(–)-epicatechin Kuntze—
gallate
(Camellia Theaceae
(Camellia
sinensis and
sinensis
(L.) others)
(L.) Kuntze—
Kuntze—Theaceae
OH
Theaceae and others)
and others)
OH OH
OH
HO O
HO O
• ‚ Inhibited
Inhibited biofilm
biofilm formation
>60% at 1atmM
1 mM (MIC
(MIC = 0.1
= 0.1 mM)mM) [34] [34]
O
OH O OH
O
OH OH
O
OH
OH OH
Flavonoids OH
(–)-gallocatechin
Flavonoidsgallate
(Camellia(–)-gallocatechin
sinensis (L.) Kuntze—Theaceae)
gallate
OH
OH
OH
HO O OH
OH • ‚ Inhibited
Inhibited biofilm
biofilm formation
>60% at 1atmM
1 mM (MIC
(MIC = 0.1
= 0.1 mM)mM) [34] [34]
HO O
O OH • Inhibited biofilm formation in E. corrodens by >60% at 1 mM (MIC = 0.1 mM) [34]
OH OH
O O
OH OH
O OH
OH
OH
(−)-epigallocatechinOH
(´)-epigallocatechin gallate
gallate
(−)-epigallocatechin gallate
OH
OH OH
HO O
OH •‚ Inhibited
Inhibited QS QS
in E. E. coli
incoli MT102
MT102 (pSB403)
(pSB403) andand P. putida
P. putida (pKR-C12)
(pKR-C12) (>50%
(>50% andand 40%
40% at μg/mL,
at 40 40 µg/mL, respectively)
respectively) [17] [17]
OH B. cepacia
HO O
•‚ Reduced
Reduced
Inhibited QSbiofilm
biofilm formation
in E.formation
coli MT102 (30%)
(30%) andand
(pSB403) andswarming
swarming
P. putida motility
motility in cepacia
in B.
(pKR-C12) (>50% (100%)
(100%)
and at40
at at
40% 40 40μg/mL,
µg/mLrespectively)
μg/mL [17] [17]
O
OH •‚ Inhibited
Inhibited
Reduced biofilm
biofilm
biofilm formation
formation
formation in E.
(30%) E.
andcorrodens
incorrodens by by
swarming >60%>60%
at 1atmM
motility 1inmM
(MIC
B. (MIC =(100%)
= 0.1
cepacia 0.1
mM) mM)
at 40 μg/mL [34]
[17] [34]
OH O OH • Inhibited biofilm formation in E. corrodens by >60% at 1 mM (MIC = 0.1 mM) [34]
O
OH OH
O
OH
OH OH
OH
Molecules 2016, 21, 29 11 of 26
Molecules 2016, 21, 29 11 of 26

Table 1. Cont.
Table 1. Cont.
Active Constituents
(Source Plants)
Plants) Biological
Diarylheptanoids
• ‚ Inhibited biofilm
Inhibited biofilmformation
formationin S.
inepidermidis (MIC
S. epidermidis (MIC= 25= μg/mL)
25 µg/mL)
• ‚ Inhibited biofilm formation and AHLs production
Inhibited biofilm formation and AHLs production in P.in aeruginosa
P. aeruginosaat 1atμg/mL
1 µg/mL
• ‚ Altered expression
Altered expression of QS-related
of QS-related genes, reduced
genes, reduced virulence
virulencefactors production
factors production (60%
(60%to 80%)
to 80%) and mortality
and mortality in
in infection
infectionmodels
models(28%(28%toto80%)
80%)with
withtreatment
treatment of of 33 μg/mL
µg/mLininP.P.aeruginosa
aeruginosaPAO1PAO1 [27]
• ‚ Completely
Completelyinhibited
inhibitedbiofilm formation
biofilm formation in H.
in pylori
H. pyloriATCCATCC 43504 andand
43504 other clinical
other isolates
clinical isolatesat 8atμg/mL
8 µg/mL for for [27]
[48]
up up
to 10
to days
10 days [48]
curcumin [48]
• ‚ Inhibited sortase
Inhibited A activity
sortase (IC(IC
A activity 50 =50
10=μM) andand
10 µM) biofilm
biofilmformation
formationin S.inmutans UA159
S. mutans UA159(at 15
(atμM)
15 µM) [48]
(Curcuma longa L.—Zingiberaceae) [49] [49]
O O
• ‚ Completely
Completelyeradicated
eradicated48-h andand
48-h 14-day biofilms
14-day biofilmsandandreduced
reducedbiomass
biomass of 8-week
of 8-weekbiofilm
biofilmin E.
in faecalis
E. faecalis
[50] [50]
ATCCATCC 29212
29212 (625(625 µg/mL)
μg/mL)
[51] [51]
• Inhibited biofilm formation (50%), alginate production (20% to 70%), and motility in V. harveyi,
‚ Inhibited biofilm formation (50%), alginate production (20% to 70%), and motility in V. harveyi,
V. parahaemolyticus, V. vulnificus [52] [52]
HO OH
V. parahaemolyticus, andand
V. vulnificus at 75
at 75 µg/mL
μg/mL
Inhibited violacein production C. violaceum
in violaceum CV026 P. aeruginosa [53] [53]
OCH3 OCH3
• ‚ Inhibited violacein production in C. CV026 andand virulence
virulence factors
factors production
production in aeruginosa
in P. [53]
PAO1 and S. marcescens FJ584421 at 100 µg/mL (56%–63%) [53]
PAO1 and S. marcescens FJ584421 at 100 μg/mL (56%–63%) [53]
‚ Inhibited swimming motility by 50% in E. coli ATCC 10536 (50 µg/mL), P. aeruginosa PAO1 (50 µg/mL), [53]
• Inhibited swimming motility by 50% in E. coli ATCC 10536 (50 μg/mL), P. aeruginosa PAO1 (50 μg/mL), [54]
P. mirabilis ATCC 7002 (75 µg/mL), and S. marcescens FJ584421 (75 µg/mL) [54]
P. mirabilis ATCC 7002 (75 μg/mL), and S. marcescens FJ584421 (75 μg/mL)
‚ Inhibited biofilm formation in E. coli (52%), P. aeruginosa PAO1 (89%), P. mirabilis (52%), and S. marcescens
• Inhibited biofilm formation in E. coli (52%), P. aeruginosa PAO1 (89%), P. mirabilis (52%), and
(76%) at 100 µg/mL
S. marcescens (76%) at 100 μg/mL
‚ Inhibited biofilm formation (50%) and surface adhesion (15%) in C. albicans at 50 µg/mL
• Inhibited biofilm formation (50%) and surface adhesion (15%) in C. albicans at 50 μg/mL
Molecules 2016, 21, 29 12 of 26
3.1.3. Tannins
Tannins including ellagitannins and proanthocyanidins are another type of phenolic that has
documented biofilm and quorum sensing inhibitory activities. At a concentration of ~4 µM,
1,2,3,4,6-penta-O-galloyl-β-D-glucopyranose (a common precursor of gallotannins) inhibited biofilm
formation in Staphylococcus aureus by 50% [38]. Punicalagin [39] and hamamelitannin [40] are
other tannins that can interfere with QS in Gram-negative and Gram-positive bacteria, respectively.
In a study by Li et al. [39], punicalagin (an ellagitannin found in pomegranate and Combretaceae
species) inhibited violacein production in C. violaceum as well as swimming and swarming motility
in Salmonella typhimurium SL1344 at 15.6 µg/mL. Further analyses by these authors showed a
downregulation of QS and motility-related genes in S. typhimurium at the same concentration.
Similarly, hamamelitannin (a gallotannin from American witch-hazel) was shown to reduce cell
attachment of methicillin-resistant Stapylococcus aureus in vitro at 4 µg/mL [40]. At a higher
concentration of 50 µg/mL, δ–hemolysin production and QS regulator RNAIII in S. aureus were
inhibited by hamamelitannin. Furthermore, this decrease in virulence was confirmed in vivo
with a graft infection model using rats. At pre-treatment of 30 mg/mL, implanted grafts
showed no detectable methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant
Staphylococcus epidermidis (MRSE) loads after 7 days [40]. Tannic acid was reported to be active
against both Gram-negative at Gram-positive bacteria. At 100 µg/mL, Huber et al. [17] showed an
enhancement of biofilm formation in Pseudomonas aeruginosa PA14; however, a 72% inhibition was
observed at 200 µg/mL against PA14 biofilms in another study [32]. In other studies, anti-biofilm
activities were shown at lower concentrations from 3.4 to 20 µg/mL against S. aureus [41,42]. In terms
of QS, inhibition was observed in Pseudomonas putida at 30 µg/mL [17] and S. aureus at 20 µg/mL [42].
3.1.4. Stilbenes and Flavonoids

Stilbenes such as resveratrol and pterostilbene have been reported to interfere with the formation
of both fungal and bacterial biofilms. In a study by Cho et al. [42], 50 µg/mL of resveratrol
inhibited P. aeruginosa PA14 and E. coli O157:H7 biofilms. In another study, Coenye et al. [43]
showed that resveratrol at 0.32% also inhibited biofilm formation in Propionibacterium acnes. At a
higher concentration of 100 µg/mL, this compound was inhibitory to S. aureus biofilms but actually
enhanced biofilm formation in S. epidermidis [33]. For pterostilbene, Li et al. [44] showed that
treatment of 16 µg/mL inhibited new and mature biofilms in various Candida albicans strains. In the
same study, at a concentration of 4 µg/mL pterostilbene prevented hyphal formation in the same
fungal strains. Transcriptomic analyses showed that this compound altered the expression of genes
involved in morphological transition, ergosterol biosynthesis, filamentation and cell surface proteins.
Furthermore, in a rat central venous catheter infection model, treatment of pterostilbene showed
anti-biofilm effects in a dose-dependent manner [44].
Flavonoids are another type of phenolic that showed inhibitory activities in quorum sensing and
biofilm formation. In a study by Vikram et al. [45], quercetin was shown to inhibit bioluminescence
in Vibryo harveyi strains by 75% at a concentration of 6.25 µg/mL. Lee et al. [46] reported anti-biofilm
activities of this compound against E. coli O157:H7 and V. harveyi BB120 at the same concentration
as well as inhibition of S. aureus biofilms at 1 µg/mL. Microarray analyses by the same authors
showed that quercetin reduced the expression of genes involved in QS and virulence of S. aureus.
Other flavonoids such as catechins from green tea (Camellia sinensis L.) also showed similar effects.
In a study by Matsunaga et al. [34], (´)-gallocatechin, (´)-epigallocatechin, (´)-catechin gallate,
(´)-epicatechin gallate, (´)-gallocatechin gallate, and (´)-epigallocatechin gallate all inhibited
biofilm formation in Eikenella corrodens at 1 mM. (´)-catechin, a related compound, also inhibited
violacein and virulence factors production in Chromobacterium violaceum and Pseudomonas aeruginosa
PAO1, respectively [47]. However, (´)-epicatechin (another related compound) showed different
activity depending on the tested organism. At 200 µg/mL, (´)-epicatechin enhanced biofilm
formation in P. aeruginosa PAO1 and Agrobacterium tumefaciens C58 [31]. At higher concentrations
Molecules 2016, 21, 29 13 of 26
of 1 mg/mL, this compound inhibited Escherichia coli JM109 biofilms by 40% [35]. In terms of QS,
(´)-epicatechin inhibited violacein production in C. violaceum at 1 mg/L [35] but increased AHL
production in E. coli DL271/pAL105 at concentrations of 40 to 200 µg/mL [31]. (´)-epigallocatechin
gallate also showed QS inhibitory activities against E. coli MPT102 and Pseudomonas putida as well as
swarming motility in Burkholderia cepacia at 40 µg/mL [17].
3.1.5. Diarylheptanoids
Another noteworthy phenolic in terms of QS and biofilm activities is curcumin
(a diarylheptanoid found in turmeric). This compound has been well studied and showed various
pleiotropic biological effects. Karaman et al. [55] showed that this compound enhanced biofilm
formation in Staphylococcus aureus at 16 µg/mL. The opposite effect was seen by Rudrappa
and Bais [48] and Pattiyathanee et al. [49] where curcumin inhibited AHL production and
biofilm growth in P. aeruginosa PAO1 at 1 µg/mL and completely inhibited biofilm formation
in Helicobacter pylori clinical isolates at 8 µg/mL, respectively. At higher concentrations from 25
to 625 µg/mL, this compound displayed anti-biofilm activities against Staphylococcus epidermidis,
Vibrio sp., Escherichia coli, Pseudomonas aeruginosa PAO1, Proteus mirabilis, Serratia marcescens,
Klebsiella pneumoniae, Enterococcus faecalis, Streptococcus mutans and Candida albicans [25,27,50–54].
In terms of QS, curcumin inhibited violacein production in C. violaceum as well as virulence factors
production in Vibrio sp., P. aeruginosa PAO1, S. marcescens at concentrations ranging from 3 µg/mL to
100 µg/mL [48,52,53].
3.2. Terpenoids
Different types of terpenes such as monoterpenes, limonoids, and triterpenes have also
been reported to have anti-biofilm and/or anti-QS activities (Table 2). Thymol and carvacrol
(monoterpenes) were shown to be effective against new and existing biofilms of Gram-positive
and Gram-negative bacteria [24,56,57]. In a study by Upadhyay et al. [24], thymol inhibited
the formation of new Listeria monocytogenes biofilms and inactivated preformed ones at 0.5 mM
and 5 mM, respectively. At the lower concentration, genes critical to L. monocytogenes biofilm
development were downregulated [24]. Using the same model organism, the authors showed that
carvacrol also effective at concentrations of 0.65 mM and 10 mM against new and existing biofilms,
respectively. This compound also downregulated biofilm-associated genes in L. monocytogenes at
0.65 mM [24]. In another study, Soumya et al. [56] reported inhibitory activities of these monoterpenes
against different strains of Pseudomonas aeruginosa. At a concentration of 0.1%, thymol reduced
the biofilm mass of P. aeruginosa strains ATCC 27853, CIP A22 and IL5 by 86%, 54% and 70%,
respectively. Similarly, 0.04% of carvacrol inhibited biofilms of the same strains by more than 90% [56].
The inhibitory activity of thymol was also confirmed by Qiu et al. [57] where treatment with 64 µg/mL
resulted in more than five-fold reduction of enterotoxin genes expression in Staphylococcus aureus.
Sesquiterpenoids such as salvipisone and acanthospermolides were shown to reduce biofilm
growth in P. aeruginosa, S. aureus, and Staphylococcus epidermidis [58–60]. At a concentration of
37.5 µg/mL, salvipisone (from the roots of Salvia sclarea L.) inhibited preformed S. epidermidis biofilms
by at least 85% and preformed S. aureus biofilms by 85% [58,59]. In another study, a number of
acanthospermolides isolated from Acanthospermum hispidum DC. showed inhibitory activities against
P. aeruginosa biofilms by at least 70% at 2.5 µg/mL [60].
Molecules 2016, 21, 29 14 of 26
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Molecules 2016,
2016, 21,
21, 29
29 14
14 of
of 26
26
Table 2. Terpenoids
Table
affecting microbial quorum sensing (QS) and/or
and/or biofilmformation.
formation.
Table 2.
2. Terpenoids
Terpenoids affecting
affecting microbial
microbial quorum
quorum sensing
sensing (QS)
(QS) and/or biofilm
biofilm formation.
Active
Constituents (Source Plants) Biological
Activities Ref.
Ref.
Monoterpenes
Monoterpenes
Monoterpenes
thymol
thymol
(Thymus
(Thymus vulgaris L.—Lamiaceae)
vulgaris L.—Lamiaceae) ••‚ Inhibited formation of new and inactivated preformed biofilms in L. monocytogenes
OH Inhibited formation
Inhibited of of
formation newnewandandinactivated preformed
inactivated preformed biofilms
biofilms in in
L. L.
monocytogenes
monocytogenes
OH (0.5 mM and 5 mM, respectively) [24]
(0.5
(0.5 mM mMandand 5 mM,
5 mM, respectively)
respectively) [24]
[24]
••‚ Downregulated
Downregulated
Downregulated genes
genes
genes critical to
critical
critical L.
to to monocytogenes
L. L.
monocytogenes biofilm
biofilm formation
formation
formation (0.5 mM)
(0.5
(0.5 mM)
mM) [24]
[24]
[24]
••‚ Inhibited formation
Inhibited
Inhibited of
formation
formation biofilm
of of biofilm
biofilm in P.
in in aeruginosa
P. P.
aeruginosa ATCC
aeruginosa ATCC27853,
ATCC 27853,CIP
27853, CIPA22,
CIP A22,and
A22, andIL5
and IL5at
IL5 atat0.1%
0.1%by
0.1% byby86%,
86%,54%,
86%, 54%,
54%, [56]
[56]
[56]
OH
and
and 70%,
and
70%, respectively
70%, respectively
respectively [57]
[57]
OH
••‚ Downregulated
Downregulated
Downregulated expression
expression
expression of enterotoxin
of of enterotoxin
enterotoxin genes in
genes
genes S.
in in aureus
S. S. aureus
aureus at 64
at at
6464μg/mL
µg/mL
μg/mL (>5-fold)
(>5-fold)
(>5-fold)
H
H33C
C CH
CH33
carvacrol
carvacrol
(Thymus
(Thymus vulgaris L.
vulgaris L.
and other Lamiaceae species)
and other Lamiaceae species)
OH
••‚ Inhibited
Inhibited formation
Inhibited
formation of
formation new
of of
newnewand
andandinactivated preformed
inactivated
preformed biofilms in
biofilms
biofilms L.
in in monocytogenes
L. L. monocytogenes
monocytogenes
OH [24]
[24]
(0.65
(0.65 mM
(0.65
mM mMand
and 10
and mM,
1010 mM,
mM, respectively)
respectively)
respectively) [24]
OH
OH [24]
[24]
••‚ Downregulated genes critical to L. monocytogenes biofilm formation (0.65 mM) [24]
Downregulated
Downregulated genes
genes critical
critical L. L.
to to monocytogenes
biofilm formation
formation (0.65
(0.65 mM)
mM) [56]
••‚ Inhibited formation of biofilm in P. aeruginosa
P. ATCC
aeruginosa 27853, CIP A22, and IL5
IL5at [56]
Inhibited formation of biofilm in ATCC 27853, CIP A22,
Inhibited formation of biofilm in P. aeruginosa ATCC 27853, CIP A22, and IL5 and atat0.04%
0.04%by
0.04% byby>90%
>90%
>90%
OH
OH
H
H333C
C CH
CH333
Sesquiterpenes
Sesquiterpenes
salvipisone
salvipisone
(Salvia sclarea
(Salvia sclarea L.—Lamiaceae)
L.—Lamiaceae)
OH
OH CH
CH333
O
O
CH
CH333 ••‚ Reduced
Reduced
Reduced preformed
preformed
preformed biofilm
biofilm
biofilm of S. S.
of of
S. epidermidis
epidermidis
epidermidis RP12
RP12
RP12 (>90%),
(>90%),
(>90%), 6756/99
6756/99
6756/99 (85%)
(85%)
(85%) andand
and S. S. aureus
S. aureus
aureus 1474/01
1474/01
1474/01 (85%)
(85%)
(85%)
at 37.5 µg/mL [58,59]
[58,59]
at
at 37.5
37.5 μg/mL
μg/mL
O
O
H
H333C
C
H
H333C
C
CH
CH222
Molecules 2016, 21, 29 15 of 26
Molecules
Molecules 2016,
2016, 21,
21, 29
29 15
15 of
of 26
26
Table 2. Cont.
Table
Table 2.
2. Cont.
Cont.
Active
Active Constituents
Constituents (Source Plants)
(Source Plants) Biological
Activities
Ref.
acanthospermolide
acanthospermolide
acanthospermolide
(Acanthospermum
(Acanthospermum hispidum
(Acanthospermum hispidum
DC.—Asteraceae)
DC.—Asteraceae)
CHO
CHO
CH
CH33
OR
OR
••‚ Inhibited
Inhibited biofilm
Inhibited
biofilm formation
biofilm in
formation
formation P.
in in aeruginosa
P. P.
aeruginosa (70%
aeruginosa at
(70%
(70% 2.5
atat μg/mL)
2.52.5 µg/mL)
μg/mL) [60]
[60]
[60]
CH
CH22
O
O O
O
O
O Ac
Ac
Triterpenoids
Triterpenoids
isolimonic
isolimonic acid
acid
(Citrus
(Citrus ×× aurantium
(Citrus ˆ aurantium L.—Rutaceae)
aurantium L.—Rutaceae)
L.—Rutaceae)
O
O
CH
CH33 ••‚ Inhibited
Inhibited
Inhibited AI-induced
AI-induced
AI-induced bioluminescence
bioluminescence
bioluminescence in V. V.
in in
V. harveyi
harveyi
harveyi BB170
BB170
BB170 (60%
(60%
(60% at at
at 6.25
6.25
6.25 µg/mL)
μg/mL)
μg/mL) and
and
and biofilm
biofilm
biofilm formation
formation
formation in in
in
OH
OH [61]
[61]
O
O V. V. harveyi
harveyi BB120
BB120 (40%
(40% at at
100100 µg/mL)
μg/mL)
V. harveyi BB120 (40% at 100 μg/mL)
HOOC
HOOC
O
O
O
O O
O
H O
O
H33C
C
CH
CH33
ichangin
ichangin
(Citrus
(Citrus aurantium L.—Rutaceae)
(Citrus × aurantium L.—Rutaceae)
×
ˆ aurantium L.—Rutaceae)
O
O
O
O O
O
H O CH
HO CH33
••‚ Inhibited
OH
OH
Inhibited
Inhibited AI-induced
AI-induced
AI-induced bioluminescence
bioluminescence
bioluminescence in V. V.
in in
V. harveyi
harveyi
harveyi BB170
BB170
BB170 (90%)
(90%)
(90%) and
andand biofilm
biofilm
biofilm formation
formation
formation in in
in V. V. harveyi
V. harveyi
harveyi BB120
BB120
BB120 (40%)
(40%) [61]
H
H33C
C [61]
O
O at (40%)
at 100 at 100 µg/mL
100 μg/mL
μg/mL
H
H33C
C
HO
HO H
H
O
O
O
O
Molecules 2016, 21, 29 16 of 26
Molecules 2016,
Molecules 2016, 21,
21, 29
29 16 of
16 of 26
26
Table 2. Cont.
Molecules 2016, 21, 29 16 of 26
Table 2.
Table 2. Cont.
Cont.
Active Constituents
Constituents (Source
(Source Plants)
Plants) Table 2. Cont. Biological Activities Ref.
Active Triterpenoids Biological Activities Ref.
Triterpenoids
Triterpenoids
betulinic acid Biological Activities Ref.
betulinic acid (various
(various
Triterpenoids
(various
betulinic acid species)
species)species)
OH
OH
betulinic acid (various species)
H
H H
H CH
OH33
CH
CH2
CH 2
H3C
H C H H CH3
CH
3 H2 CH3 CH3 3 ‚ Enhanced biofilm formation in P. aeruginosa PA14 at 100 µg/mL
CH
•• Enhanced
H CH CH Enhanced biofilm
biofilm formation
formation in
in P.
P. aeruginosa
aeruginosa PA14
PA14 at
at 100
100 μg/mL
μg/mL [42]
[42]
H3 C
3 3
CH3 [42]
H CH3 CH3 • Enhanced biofilm formation in P. aeruginosa PA14 at 100 μg/mL [42]
H
H CH3
CH 3
H CH3
HO
HO O
O
ursolic acid
HO acid
ursolic (various
O (various
ursolic species)
acid species)
H3C
H C H
ursolic(various
acid (various H
3species)
species)
H3C
H C H3C H
3
H3C •• Inhibited biofilm
Inhibited biofilm formation
formation in
in P.
P. aeruginosa
aeruginosa PAO1
PAO1 (>87%),
(>87%), E.
E. coli
coli JM109
JM109 (50%),
(50%), and
and V.
V. harveyi
harveyi BB120
BB120 (57%)
(57%)
‚ at Inhibited
10 μg/mL biofilm formation in P. aeruginosa PAO1 (>87%), E. coli JM109 (50%), and V. harveyi BB120 (57%) [21]
• Inhibited
at 10 μg/mL biofilm formation in P. aeruginosa PAO1 (>87%), E. coli JM109 (50%), and V. harveyi BB120 (57%) [21]
OH at 10 µg/mL
CH3 CH3 CHCH3
OH ••‚ Induced
at 10 μg/mL
Induced expression of
expression of chemotaxic
chemotaxic and
and motility
motility genes
genes and
and repressed
repressed sulphur
sulphur metabolism
metabolism inin [21]
[21]
[21]
CH3 CH3 3 Induced expression of chemotaxic and motility genes and repressed sulphur metabolism in [21]
H
H OH • E.
E. coli K-12
Induced
coli K-12 at 10
at 10 μg/mL
μg/mL
expression of chemotaxic and motility genes and repressed sulphur metabolism in [62]
[21]
[62]
CH3 CH3 CH3 O E. coli K-12 at 10 µg/mL [62]
O
H CH3
CH ••‚ Inhibited
E. Inhibited
coli K-12
Inhibited biofilm
at formation in
10 μg/mL
biofilm
biofilmformation in P.
formation in
P. aeruginosa
aeruginosa PAO1
PAO1 by
P. aeruginosa
by 35% at
PAO1 by35%
at 10 μg/mL
35% at1010μg/mL
µg/mL [62]
3 O
HO CH3 • Inhibited biofilm formation in P. aeruginosa PAO1 by 35% at 10 μg/mL
HO
H3C
HO H C CH3
CH
3 3
H3C Triterpenoids
CH
Triterpenoids
3
gymnemic acids
Triterpenoids
gymnemic acids
(Gymnema
(Gymnema sylvestre
gymnemic (Retz.)
acids
sylvestre (Retz.)
R.Br. ex Sm.—Apocynaceae
(Gymnema
R.Br. sylvestre (Retz.)and
ex Sm.—Apocynaceae and
Asclepiadaceae
R.Br.Asclepiadaceae species) and
ex Sm.—Apocynaceae
species)
H C CH
••‚
H3C
3
Asclepiadaceae species) CH33OR
OR11
Mixture
Mixture of of
Mixture
of 44 acids
acids at at
4 acids
at 40 40
40 μg/mL
µg/mL
μg/mL inhibited
inhibited yeast-to-hypha
inhibited yeast-to-hypha
yeast-to-hypha transition in C.
transition
transition in C. albicans
inalbicans SC5314
C. albicans SC5314
SC5314 andand
and induced conversion
induced
induced conversion
H3C conversion oftohyphae back (100%
to yeastafter
form
CH3
OR1 • of hyphae
Mixture
of hyphae back
ofback to
4 acids yeast form
at 40 μg/mL
yeast form (100% after
inhibited 11(100%
h) after 11transition
yeast-to-hypha
11 h) h) in C. albicans SC5314 and induced conversion
••‚
CH Aspergillus fumigates
CH
CH33
CH
CH33
H
H
CH33
of Inhibited
Inhibited conidial
conidial
hyphaeconidial
Inhibited back germination
germination
to germination
yeast and
form (100%
andand hyphal
hyphal
after
hyphal h)growth
growth
11growth in in
in Aspergillus
Aspergillus fumigates
fumigates byby
by 74%
74%74%atat
at 4040
40 µg/mL
μg/mL
μg/mL [63]
[63]
[63]
OH
••‚
H
OH
HO H
H
H
CH3H CH3H
CH3CH
OR
CH
OHOR
OH 2
23 Treatment
Treatment
Inhibited
Treatment ofof4040
conidial
of 40 µg/mL
μg/mL mixture
mixture
germination
μg/mL mixture and improved
improved survival
survival
hyphalsurvival
improved growth in C. C.
in
C. albicans-fed
in Aspergillus
albicans-fed C. C.
fumigates
albicans-fed C. elegans
elegans
by 74%
elegans infection
infection model
model
at 40 model
infection μg/mL (100%
(100%
(100% rescue)
rescue)
rescue) by
by [63]
H HO H
H OH
OH 3
by inhibiting
inhibiting formation
formation of of invasive
invasive hyphaehyphae
O H H
HO
H OHO
O H
OR
OH 2 • Treatmentformation
inhibiting of 40 μg/mL mixture improved
of invasive hyphae survival in C. albicans-fed C. elegans infection model (100% rescue) by
H O H H3C H H CH3
OH H 3C OH
HO H
H O
OH inhibiting formation of invasive hyphae
O H HO H
GA-III,
GA-III, R1 =
R1 = (S)-2-methylbutyroyl,
(S)-2-methylbutyroyl,
H3C OH R2 =
R2 =HH
GA-IV, R1= HO H
R1= tigloyl,
tigloyl, R2
R2 =
=H H
GA-IV,
GA-III, R1
GA-XIII, = (S)-2-methylbutyroyl,
R1= H, R2
R2 = R2 = H
= (S)-2-methylbutyroyl
(S)-2-methylbutyroyl
GA-XIII, R1= H,
GA-IV, R1=
GA-XIV, R1=tigloyl,
R2 R2
H, R2 =H
= tigloyl
tigloyl
GA-XIV, R1= H, =
GA-XIII, R1= H, R2 = (S)-2-methylbutyroyl
GA-XIV, R1= H, R2 = tigloyl
Molecules 2016, 21, 29 17 of 26
Limonoids (derived from triterpenes) isolated from Citrus ˆ aurantium L. (bitter orange) showed
anti-QS activities at low concentrations [61]. These include isolimonic acid, ichangin and several other
compounds; a 17% to 83% inhibition of bioluminescence in Vibryo harveyi was observed at 6.25 µg/mL
and dose-dependent activity was seen for all compounds tested [61]. Other triterpenoids are reported
to have differing activities. Betulinic acid, a lupane triterpene, showed no activity at 5 µg/mL
in a bioassay with P. aeruginosa PA14 [32]; at 100 µg/mL biofilm formation is greatly enhanced
in the same strain [42]. Ursane triterpenoids, on the other hand, have been documented to have
good inhibitory activities. Ursolic acid has been shown to inhibit P. aeruginosa PAO1, E. coli JM109,
and V. harveyi BB120 biofilms by 35% to 87% at 10 µg/mL [21,62]. This compound was not active
against P. aeruginosa PA14 biofilm at a concentration of 5 µg/mL [32]. Furthermore, gymnemic acids
(ursane triterpene glycosides) isolated from Gymnema sylvestre (Retz.) R.Br. ex Sm. (Apocynaceae)
have been reported by Vediyappan et al. [63] to inhibit yeast-to-hypha transition in Candida albicans
SC5314. At 40 µg/mL, the four-compound mixture inhibited hyphal transition and induced the
conversion of hyphae to yeast form; 100% conversion was observed after 11 h. These compounds also
inhibited conidial germination and hyphal growth in Aspergillus fumigates at the same concentration.
Furthermore, treatment of the gymnemic acids mixture, at 40 µg/mL, significantly improved the
survival rate of Candida albicans-infected Caenorhabditis elegans and resulted in 100% rescue [63].
3.3. Sulfur-Containing Phytochemicals

Sulfur-containing compounds (Table 3) such as allicin, ajoene, and thiocyanates have been
shown to interfere with biofilm formation and quorum sensing of both Gram-positive and
Gram-negative bacteria [32,64–68]. Allicin (from garlic) inhibited Staphylococcus epidermidis and
Pseudomonas aeruginosa PAO1 biofilm adhesion as well as the expression of QS-regulated virulence
factors in P. aeruginosa PAO1 [64,65]. At a concentration of 1.1 mg/mL, a 74% reduction in biofilm
mass was observed in P. aeruginosa PA14 [32]. In another study using GFP-transformed P. aeruginosa
PAO1, treatment of 128 µg/mL of allicin resulted in a 50% decrease in biofilm thickness and a 70%
decrease of EPS production [64]. For S. epidermidis strains, 4 mg/mL of allicin inhibited biofilm
formation by more than 90% [65]. Similarly, ajoene (also from garlic) inhibited QS in reporter strains
P. aeruginosa lasB-gfp, P. aeruginosa rhlA-gfp and E. coli luxI-gfp with 50% effective concentrations
(EC50 ) ranging from 15 to 100 µM [66]. In the same study, ajoene also downregulated virulence factors
expression (elastase, rhamnolipid, enterotoxins) by five-fold at 80 µg/mL [66]. Furthermore, using
a mouse pulmonary infection model, subcutaneous treatment of ajoene at 25 mg/kg, significantly
improved bacteria clearance after three days [66]. Thiocyanates such as sulforaphane and allyl
isothiocyanate (from Brassicaceae species) have been reported to inhibit QS in Escherichia coli
and Chromobacterium violaceum as well as reduce biofilm formation in Pseudomonas aeruginosa and
Listeria monocytogenes [35,67,68]. In a study by Ganin et al. [67], complete inhibition of QS in E. coli
DH5 (pJN105L) (pSC11) was observed at 100 µM of sulforaphane. This compound also inhibited
biofilm formation and pyocyanin production (QS-mediated) in P. aeruginosa PAO1 by 60% and 70% at
concentrations of 37 µM and 100 µM, respectively. Similarly, allyl isothiocyanate was shown to reduce
violacein production (QS-controlled) in C. violaceum by 70% at 5 µg/mL [66]. The anti-biofilm activity
of this compound was demonstrated in L. monocytogenes, P. aeruginosa and E. coli where treatment
with 1 mg/mL resulted in 61 to 100% inhibition [68].
Molecules 2016, 21, 29 18 of 26
Molecules 2016, 21, 29 18 of 26

Molecules 2016, 21, 29 Table 3. Other biosynthetic classes of phytochemicals affecting microbial quorum sensing (QS) and/or biofilm formation. 18 of 26
Table 3. Other biosynthetic classes of phytochemicals affecting microbial quorum sensing (QS) and/or biofilm formation.
Table 3. Other biosynthetic classes of phytochemicals affecting microbial quorum sensing (QS) and/or biofilm formation.
Sulfur-Containing Compounds Plants) Biological Activities Ref.
Sulfur-Containing Compounds
allicin (Allium sativum L.—Amaryllidaceae) ‚• Inhibited P. aeruginosa PA14
Inhibited P. PA14 biofilm
biofilm (74%
(74% atat 11 µL).
μL)
allicin (Allium sativum L.—Amaryllidaceae) ‚• Inhibited P. aeruginosa PA14 biofilm (74% at 1 μL) PAO1, EPS production (70%), biofilm thickness (50%), [32]
O • Reduced adhesion
Reduced adhesion of GFP-transformed P. aeruginosa PAO1, EPS production (70%), biofilm thickness (50%), [32] [32]
O • Reduced adhesion of GFP-transformed P. µg/mL
aeruginosa PAO1, EPS production (70%), biofilm thickness (50%), [64]
and expression
and expression of virulence
of virulence factors at
factors at 128
128 μg/mL [64] [64]
[64]
S [65] [65]
S S ‚• and expression
Inhibited
Inhibited biofilm
biofilm
offormation
virulence factors
by >90%
formation by >90%atin128
S. μg/mL
in S. epidermidis strains
epidermidis strains at
at 44 mg/mL
mg/mL [65]
[65]
S • Inhibited biofilm formation by >90% in S. epidermidis strains at 4 mg/mL
ajoene (Allium sativum L.—Amaryllidaceae)
ajoene (AlliumOsativum L.—Amaryllidaceae)
O (Z)-ajoene
S (Z)-ajoene • Inhibited QS in P. aeruginosa lasB-gfp (IC50 = 15 μM), rhlA-gfp (IC50 = 50 μM), E. coli luxI-gfp (IC50 = 100 μM)
S ‚• Inhibited
Inhibited QS
QS in
in P. 50 =
P. aeruginosa lasB-gfp (IC50
50 =15
15μM),
µM),rhlA-gfp
rhlA-gfp (IC 50==50
(IC50
50 50μM),
µM),E.E.coli
coliluxI-gfp
luxI-gfp(IC
(IC50 ==
5050 100 μM)
100 µM)
reporter strains [66]
reporter strains
reporter
reporter strains
strains [66] [66]
[66]
S • Downregulated QS-regulated virulence factors (5-fold at 80 μg/mL) and improved bacteria clearance in [66]
S S ‚•• Downregulated QS-regulated
Downregulated
Downregulated QS-regulated virulence
virulence factors (5-fold at
factors (5-fold at 80
80 µg/mL)
μg/mL) and
μg/mL) and improved
and improved bacteria
improved bacteria clearance
bacteria clearance in
clearance in
in [66] [66]
[66]
S mouse infection
mouse infection model
infection model (subcutaneous
model (subcutaneous treatment
(subcutaneous treatment
treatment ofof 25
of 25 mg/kg
25 mg/kg
mg/kgbodybody weight)
bodyweight)
weight)
O (E)-ajoene mouse
O
O (E)-ajoene
S S
S S S
S S
sulforaphane (Brassicaceae species)
sulforaphane
sulforaphane (Brassicaceae
(Brassicaceae species)
species) • Inhibited lasR-mediated QS in E. coli DH5 (pJN105L) (pSC11) completely at 100 μM
O ‚• Inhibited
Inhibited lasR-mediated
lasR-mediated QS
QS in
in E. coli DH5 (pJN105L) (pSC11) completely at 100 μM
µM
O S • Inhibited biofilm formation (60% at 37 μM) and pyocyanin production in P. aeruginosa PAO1 [67]
S C S ‚• Inhibited
Inhibited biofilm
biofilm formation (60% at 37 μM)
(70% at 100 μM)
µM) and pyocyanin production in P.
P. aeruginosa
aeruginosa PAO1
PAO1 [67] [67]
H3C S N C (70% at
(70% at 100
100 μM)
µM)
H3C N
Compounds
allyl Sulfur-Containing
isothiocyanate (Brassicaceae species)
(Brassicaceae species)
allyl isothiocyanate (Brassicaceae species) • Inhibited violacein production in C. violaceum ATCC 12472 (70% at 5 μg/mL) [35]
H2C
H C ‚• Inhibited violacein
production in C. violaceum
in C. violaceum ATCC
ATCC 12472
12472 (70%
(70% at
at 55 µg/mL)
2C
Inhibited μg/mL) [35] [35]
N C ‚•• Inhibited biofilm formation in L. monocytogenes (61%), P. aeruginosa (90%), E.
E. coli (100%)
(100%) at 11 mg/mL
mg/mL [68]
N S
S
Inhibited biofilm
formation in L. monocytogenes
in L. monocytogenes (61%), P. aeruginosa
(61%), P. (90%), E. coli
aeruginosa (90%), coli (100%) at
at 1 mg/mL [68] [68]
Coumarins
Coumarins
aesculetin (various species)
aesculetin (various species)
HO
HO
O species)
O
O
O ‚•• Inhibited QS
Inhibited QS
Inhibited
in
QS in C. violaceum
C. violaceum
in C. CV026,
violaceum CV026,
CV026, P.P. aeruginosa, E. coli
P. aeruginosa,
aeruginosa, E.
coli JB523 (30%
E. coli JB523
(30% to 60%
JB523 (30% to
60% at 500
to 60% at
500 μM)
at 500 µM)
μM)
[18]
[18] [18]
HO O O
‚•• Inhibited
Inhibited biofilm
biofilm formation
formation in
in S.
S. aureus
aureus strains
strains (53%
(53% to
to 77% at
77% at 128
128 µg/mL)
Inhibited biofilm formation in S. aureus strains (53% to 77% at 128 μg/mL)
μg/mL) [36]
[36] [36]
‚•• Reduced expression
Reduced expression
Reduced expression ofof biofilm-related
of biofilm-related genes
biofilm-related genes (motility, adhesion,
genes (motility,
(motility, adhesion, virulence) in
in E. coli
virulence) in E.
adhesion, virulence)
coli O157:H7 at
E. coli O157:H7
at 50 µg/mL
O157:H7 at 50
μg/mL
50 μg/mL
[69]
[69] [69]
HO
HO
HO
umbelliferone (Apiaceae species)
umbelliferone (Apiaceae species)
HO O O ‚••• Inhibited
Inhibited
biofilm
Inhibited biofilm
biofilm
formation
biofilm formation
formation in
formation
in E.
in E.
in E.
coli
E. coli
coli
O157:H7
coli O157:H7 (>80%
O157:H7 (>80%
O157:H7
inhibition
(>80% inhibition
(>80%
at
inhibition at
inhibition
50
at 50
at
μg/mL)
50 μg/mL)
50 µg/mL)
μg/mL)
[69]
[69] [69]
HO O O Inhibited [69]
‚•• Reduced expression
Reduced expression
Reduced
of
expression of biofilm-related
of biofilm-related genes
biofilm-related genes (motility
genes (motility and
(motility and adhesion)
and adhesion)
in E. coli
in E.
adhesion) in
coli O157:H7 at
E. coli O157:H7
at 50 µg/mL
O157:H7 at 50
μg/mL
50 μg/mL
[69]
[69] [69]
‚•• Inhibited formation
Inhibited formation
Inhibited
of
formation of S. aureus
S. aureus
of S. CECT
aureus CECT
CECT 976976 biofilm
976 biofilm by
biofilm by 50%
by 50% at
50% at
800
at 800
μg/mL
800 µg/mL
μg/mL
[70]
[70] [70]
Molecules 2016, 21, 29 19 of 26
Molecules
Molecules 2016,
2016, 21,
21, 29
29 19
19 of
of 26
26
Molecules 2016, 21, 29 Table 3. Cont. 19 of 26
Table
Table 3.
3. Cont.
Cont.
Active Table 3. Cont. Biological Activities
Active Constituents
Constituents (Source Plants)
(Source Plants)
Plants) Biological Activities
Ref.
Ref.
Quninones
Active Constituents
Quninones(Source Plants) Biological Activities Ref.
chrysophanol (various
(various species)
Quninones
chrysophanol species)
chrysophanolO
O (various species)
O CH
CH333 ‚• Inhibited biofilm formation P. aeruginosa PAO1
formation in P. (44%) and S. maltophilia (38%) at 200 µM
CH3 • Inhibited
Inhibited biofilm
biofilm formation in
in P. aeruginosa
aeruginosa PAO1
PAO1 (44%)
(44%) and
and S.
S. maltophilia
maltophilia (38%)
(38%) at
at 200
200 μM
μM [71]
[71][71]
• Inhibited biofilm formation in P. aeruginosa PAO1 (44%) and S. maltophilia (38%) at 200 μM [71]
OH
OH O
O OH
OH
emodin
OH
emodin (various
O OHspecies)
(various species)
O
O
emodin (various species)
HO O CH
HO CH333
‚•• Inhibited biofilm
Inhibited
biofilm formation in
formation in P. aeruginosa
in P.
P. aeruginosa PAO1
aeruginosa PAO1(75%)
PAO1 (75%) andS.
(75%)and
and S.maltophilia
S. maltophilia(43%)
maltophilia (43%)at
(43%) at20
at 20µM
20 μM
μM [71]
[71][71]
HO CH3
OH
OH O
O OH
OH
shikonin (Boraginaceae
shikoninOH O species)
OH species)
(Boraginaceae
OH
OH
shikonin O
O OH
OH
(Boraginaceae species)
OH O OH CH3
CH33
‚ •• Inhibited biofilm
formation in
in P.
P. aeruginosa PAO1
P. aeruginosa
aeruginosa PAO1(44%)
PAO1 (44%)and
(44%) andS.
and S. maltophilia(54%)
S.maltophilia
maltophilia (54%)at
(54%) at 200µM
at200
200 μM
μM [71][71]
[71]
CH3
CH3
CH33
CH3
OH
OH O
O
purpurin
OH(Rubia
purpurin O tinctorum
(Rubia L.—Rubiaceae)
tinctorum L.—Rubiaceae)
O
O OH
OH
purpurin (Rubia tinctorum L.—Rubiaceae) ‚•• Inhibited
Inhibited yeast-to-hypha
yeast-to-hypha transition
transition in
in C.
C. albicans
albicans SC5314
SC5314 atat 33 μg/mL
µg/mL
μg/mL
O OH OH ‚• formation
OH Inhibited formation of
of new
Inhibited yeast-to-hypha and
and preformed
transition
new C.
C. albicans
in C. albicans
preformed SC5314
albicans biofilms
at 3 μg/mL
biofilms [72]
OH • (50% at
at 55 formation
Inhibited μg/mL
µg/mLandand
and 30%
30%
of30% at
newatat10 μg/mL,
10μg/mL,
and respectively)
respectively)
µg/mL,respectively)
preformed C. albicans biofilms [72][72]
(50% μg/mL 10
[72]
‚ •• Downregulated
(50% expression
at 5 μg/mL expression
Downregulated and 30% atof of hypha-specific
10hypha-specific genes
μg/mL, respectively) in C.
genes in C. albicans
albicans at
at 55 to10 μg/mL
to10 µg/mL
μg/mL
OH
OH O
O OH
OH • Downregulated expression of hypha-specific genes in C. albicans at 5 to10 μg/mL
Alkaloids
OHO OH
Alkaloids
berberine
berberine (Berberidaceae
(Berberidaceae
berberine species
Alkaloids
(Berberidaceae and
species
species others)
andand
others)
berberine (Berberidaceae O
O and others)
others)species
O O
O
O • Inhibited biofilm formation in K. pneumoniae clinical
‚• Inhibited
Inhibited biofilm
biofilm formation
formation in
in K.
K. pneumoniae clinical isolates
pneumoniae clinical isolates (MIC
isolates (MIC =
(MIC
== 63.5
63.5 μg/mL)
63.5 μg/mL)
µg/mL)
[25]
[25]
• S.
Inhibited biofilm formation in K. epidermidis
pneumoniaeATCC
clinical35984 (50%
isolates at
(MIC 30 μg/mL)
= 63.5 and SE243
μg/mL) (50% at 45 μg/mL) [73]
[25] [25]
‚ Inhibited biofilm formation in S. epidermidis ATCC 35984 (50% at 30 µg/mL) and
S. epidermidis ATCC 35984 (50% at 30 μg/mL) and SE243
SE243 (50%
(50% at
at 45
45 μg/mL)
µg/mL) [73]
[73]
N+++
N
• Inhibited biofilm formation in S. epidermidis ATCC 35984 (50% at 30 μg/mL) and SE243 (50% at 45 μg/mL) [73]
H
H333CO
CO N+
H3CO OCH3
OCH33
OCH3
Molecules 2016, 21, 29 20 of 26
Molecules 2016, 21, 29 20 of 26

Table 3. Cont.
Table 3. Cont.
chelerythrine (Papaveraceae species)
O
O ‚• Inhibited biofilm
formation in S. aureus
in S. aureus 6538P
6538P (50%
(50% at
at 15
15 μM) and S.
µM) and S. epidermidis
epidermidis RP62A
RP62A (50%
(50% at
at 99 µM)
μM) [74] [74]
N+
N
H3CO CH3
OCH3
sanguinarine (Papaveraceae species)
O
O ‚• Inhibited biofilm
formation in S. aureus
in S. aureus 6538P
6538P (50%
(50% at
at 25
25 μM) and S.
µM) and S. epidermidis
epidermidis RP62A
RP62A (50%
(50% at
at 55 µM)
μM) [74] [74]
N+
N
O CH3
O
Alkaloids
reserpine (Rauwolfia sp.—Apocynaceae)
OH33C OCH33
H33CO O O
H OCH33 ‚• Inhibited biofilm
biofilm formation
formation in K. pneumoniae
in K. pneumoniae clinical
clinical isolates
isolates (MIC
(MIC == 15.6
15.6 μg/mL)
µg/mL)
H Inhibited [25] [25]
N H O
OCH33
N H
H33CO
Molecules 2016, 21, 29 21 of 26
3.4. Coumarins
Coumarins have been documented to possess QS and biofilm inhibitory activities (Table 3).
Aesculetin was reported to inhibit QS in C. violaceum, P. aeruginosa, and E. coli JB523 by 30% to
78% at 500 µM [18]. In other studies, Dürig et al. [36] and Lee et al. [69] showed that aesculetin
was effective against S. aureus biofilms (>50% inhibition at 128 µg/mL) and reduced the expression
of biofilm-related genes (at 50 µg/mL) in E. coli O157:H7, respectively. Furthermore, aesculetin
decreased Shiga-like toxin production in E. coli O157:H7 and reduced virulence in a C. elegans
infection model [69]. Lee et al. [69] also reported that umbelliferone (another coumarin) inhibited
biofilm formation (90%) and expression of motility and adhesion genes expression in E. coli O157:H7
at a concentration of 50 µg/mL. The anti-biofilm activity of umbelliferone was also confirmed in
S. aureus CECT976 by Monte et al. [70] where treatment with 800 µg/mL caused a 50% inhibition in
biofilm formation.
3.5. Quinones
Quinones have also been reported to have anti-biofilm activities against bacterial and fungal
species (Table 3). In a study by Ding et al. [71], quinones such as chrysophanol, emodin, and shikonin
all showed inhibitory activities against biofilms of Pseudomonas aeruginosa PAO1 and Stenotrophomonas
maltophilia. Emodin was more 10 times more active than both chrysophanol and shikonin as treatment
with 20 µM caused a 75% reduction in biofilm growth for P. aeruginosa PAO1 and a 43% reduction in
S. maltophilia. Chrysophanol and shikonin both required a higher concentration of 200 µM to elicit
the same level of activity in both bacterial species. Purpurin, another quinone, was shown to repress
yeast-to-hypha transition in Candida albicans SC5314 at 3 µg/mL [72]. At higher concentrations of 5 to
10 µg/mL, this compound was effective against new and existing C. albicans biofilms (30% to 50%
inhibition). Further analyses showed that purpurin downregulated the expression of hypha-specific
genes [72].
3.6. Alkaloids
In regard to alkaloids, relatively few compounds have been reported to have inhibitory activities
against bacterial biofilm (Table 3). In particular, Wang et al. [73] and Magesh et al. [25] showed
that berberine inhibited the biofilm formation in Staphylococcus eipidermidis and Klebsiella pneumoniae,
respectively. At a concentration of 63.5 µg/mL, berberine decreased biofilm growth in different
K. pneumoniae clinical isolates [25]. Treatment of 30 to 45 µg/mL of berberine resulted in 50%
inhibition of S. epidermidis biofilms [73]. Similarly, chelerythrine and sanguinarine were also effective
against Gram-positive biofilms of S. aureus and S. epidermidis at micromolar concentrations [74];
the 50% inhibitory concentrations were reported to range from 15 to 25 µM for S. aureus and 5 to 9 µM
for S. epidermidis [74] Resperine, an alkaloid from Rauwolfia sp. (Apocynaceae), also showed inhibitory
activity against K. pneumoniae biofilms with a minimum inhibitory concentration of 15.6 µg/mL [25].
Overall the literature shows that the biosynthetic classes of compounds inhibiting biofilms and
QS are diverse. The published literature to date reports activity mostly with phenolics, terpenoids,
organosulfur compounds, and quinones but relatively few alkaloids or other types of secondary
metabolites reported.
4. Taxa and Habitats

A wide variety of plant families are represented in the literature from which active compounds
were isolated. At least 21 families are described in Tables 1–3. They are angiosperms and many
more could be added if the re-occurrence of compounds in different families is considered. However,
this represents a relatively small number of the total 642 plant families described in The Plant List [75].
Both monocots and eudicots are represented but gymnosperms, bryophytes and pteridophytes are
either absent or under-represented in the literature. Many plant families are characteristic of tropical
Molecules 2016, 21, 29 22 of 26
or subtropical areas such as Zingiberaceae, Rubiaceae, Lauraceae and Theaceae or temperate areas
such as Asteraceae, Lamiaceae, Ericaceae, Berberidaceae and Apiaceae. Few records describe plant
families of arid areas or dry habitats. For example, there are no records of common families Cactaceae,
Poaceae, or Crassulaceae. This may be a sampling bias and there are few systematic studies to make
any firm conclusions at this time but this could be a rich area of study. Our own observations
are that tropical plants are a rich source of biofilm and QS inhibitors [76]. In rainforests, the high
humidity and ever-wet conditions are ideal for bacterial biofilm growth. When these occur on leaves,
the exopolysaccharide layer is a perfect habitat for germination of bryophyte spores, which leads to
fouling of leaves and a decline in plant health as fouled leaves cannot performance photosynthesis
(the bryophyte mat prevents sunlight from reaching the mesophyll layer).
5. Conclusions
The results presented in this mini review show that plant species contain many reported
phytochemicals that interfere with microbial quorum sensing and biofilm formation. The biosynthetic
classes of compounds involved is diverse but has not been systematically examined. The identified
inhibitors may be a rich mine of lead compounds to produce drugs to address the growing problem
of antibiotic resistance or to develop adjuvant therapies to impede pathogen success. The chemical
ecology of these compounds as natural defenses is still largely untouched and their role in traditional
medicines is an interesting topic for future investigation.
Acknowledgments: This research was funded by the Natural Science and Engineering Research Council
of Canada.
Author Contributions: Chieu Anh Kim Ta performed the literature searches and draft manuscript.
John Thor Arnason assisted with the development of the concept, analysis of data and writing of the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
AHL N-acyl-homoserine lactone
AIPs autoinducing peptides for signaling
EPS extrapolymeric substance
GFP green fluorescent protein
HPLC-DAD High performance liquid chromatography, diode array detection
QS quorum sensing
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Review

Mini Review of Phytochemicals and Plant Taxa with

Keywords: biofilm inhibitors; quorum sensing inhibitors; terpenes; quinones;

Molecules 2016, 21, 29; doi:10.3390/molecules21010029 www.mdpi.com/journal/molecules

2. Model Organism Bioassays

3. Phytochemicals as QS and Biofilm Inhibitors

3.1.2. Benzoic Acid Derivatives

Molecules 2016, 21, 29 5 of 26

Active Constituents (Source Plants) Biological

Molecules 2016, 21, 29 6 of 26

Active Constituents (Source Plants) Biological

Molecules 2016, 21, 29 11 of 26

3.1.4. Stilbenes and Flavonoids

3.3. Sulfur-Containing Phytochemicals

Molecules 2016, 21, 29 18 of 26

Molecules 2016, 21, 29 20 of 26

4. Taxa and Habitats

28. Amalaradjou, M.A.R.; Venkitanarayanan, K. Effect of trans-cinnamaldehyde on Inhibition and Inactivation

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