Mini Review of Phytochemicals and Plant Taxa with Activity as Microbial Biofilm and Quorum Sensing Inhibitors

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.


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][2][3][4][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].
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.

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.

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.

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][24][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].

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].

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].

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 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].

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.

Active Constituents (Source Plants)
Biological Activities Ref.

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.

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].

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.

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 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).

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.