Introduction
The microbial communication system called quorum sensing (QS) is used by a wide variety of bacteria allowing them to collectively modify their behavior in response to changes in cell density. This communication is mediated by small molecules accumulated during microbial multiplication and involves the production, secretion, and detection of extracellular signaling molecules, known as autoinducers (AI; Mukherjee and Bassler, 2019).
Gram-positive and Gram-negative bacteria exhibit different communication systems to regulate many physiological traits. In Gram-negative bacteria, signaling is usually mediated by acyl homoserine lactone (AHL) molecules, also known as autoinducer-1 (AI-1; Vanetti et al., 2020). In Gram-positive organisms, communication is mediated by autoinducer peptides which are usually secreted by ABC-type carrier proteins (Monnet et al., 2014; Lima et al., 2020).
In addition to these main QS autoinducers, the furanosyl borate diester, also known as autoinducer-2 (AI-2), is associated with both Gram-positive and Gram-negative bacteria allowing intra and interspecific communications (Chen et al., 2002). The autoinducer-3 (AI-3), a metabolite involved in pathogenesis of enterohemorrhagic Escherichia coli (EHEC), was recently elucidated and characterized (Kim et al., 2020). In fact, AI-3 analogs belong to the pyrazinone family, and they are derived from threonine dehydrogenase (Tdh) products and “abortive” tRNA synthetase reactions, being present in a variety of Gram-negative and Gram-positive pathogens (Kim et al., 2020). A number of other extracellular bacterial metabolites that function as signals in a range of microorganisms have also been discovered recently (Vanetti et al., 2020).
The genes and functions regulated by QS are diverse and can be classified into four groups: (1) cellular behavior, such as biofilm formation and dispersion, motility, and adhesion; (2) cell maintenance and proliferation, such as exoenzyme and siderophore synthesis, sporulation, and acid resistance; (3) horizontal gene transfer, such as conjugation in Agrobacterium tumefaciens; and (4) interactions with the host and other microorganisms, such as production of virulence factors, antibiotics and exo-polysaccharides, and bioluminescence, among others (Jimenez et al., 2010; Monnet et al., 2014; Grandclément et al., 2016; Lima et al., 2020).
The communication mediated by QS can be interrupted in several ways: by inhibiting the autoinducer synthesis, through enzymatic degradation of autoinducers, or by competition for binding to receptor proteins, ultimately inhibiting the target gene expression, mediated by interfering molecules called quorum sensing inhibitors (QSI; Zhang and Li, 2015; Grandclément et al., 2016).
Plants are one of the main sources of natural QSI, including medicinal plants, vegetables, and edible fruits. Some compounds derived from the secondary metabolism of plants significantly increase their ability to adapt to unfavorable environments. These metabolites, such as alkaloids, phenols, flavonoids, quinones, tannins, terpenes, and lecithins, are well-known defense mechanisms against herbivores and microorganisms (Nazir et al., 2020). Phenolic compounds are the second largest family of plant nutraceuticals, after terpenoids (Gutiérrez-Del-Rio et al., 2018). Recent studies have shown that phenolic compounds in extracts of edible foods can act as QSI (Quecán et al., 2019; Rivera et al., 2019; Santos et al., 2020).
Food spoilage causes great concern to the food industry as it leads to significant economic losses. The detection of QS signaling molecules in spoiled foods, as well as microbial interactions in fermentation of foods, has added a new dimension to the understanding of the spoilage process (Bai and Rai, 2011; Nazzaro et al., 2013; Martins et al., 2018; Almeida et al., 2020). Interference in QS communication could be an additional target to delay bacterial food spoilage since some phenotypes regulated by QS are also related to food deterioration, such as production of pectinases, lipases and proteases, and biofilm formation.
Antimicrobial resistance is one of the greatest threats to global health (WHO, 2018), and new alternatives are needed to treat infections and stop the spread of multidrug-resistant bacteria (Singh et al., 2019). The rationale for using QS inhibitors as an anti-virulence strategy is the lower selective pressure when compared to traditional antibiotics (Maura et al., 2016; Saeki et al., 2020).
This study evaluated the ability of some phenolic compounds to inhibit bacterial quorum sensing regulated phenotypes. First, we applied molecular docking with the CviR QS regulator of C. violaceum in order to select potential QSI, and then, we tested these compounds against several quorum sensing-regulated phenotypes.
Discussion
Phenolic compounds are known for their antimicrobial effects as a consequence of structural or functional damages to the bacterial cell membrane (Silva et al., 2018). Besides, phenolic acids can affect the physicochemical properties of the bacterial cell surface, especially hydrophobicity, in addition to altering electron receptors and polar and non-polar components of bacteria (Borges et al., 2013). Furthermore, phytochemicals, such as phenolic compounds, may compete with AHL signaling molecules for binding to QS receptors (Nazzaro et al., 2013; Almeida et al., 2018; Aswathanarayan and Vittal, 2018; Quecán et al., 2018; Santos et al., 2020), suggesting possible interactions of these compounds with LuxR-type proteins, like the CviR QS regulator of Chromobacterium violaceum (Figure 1).
Curcumin, one of the selected phenolic compounds for the in vitro studies, is the main polyphenol in the rhizome of Curcuma longa. Beyond its numerous pharmaceutical properties, such as antitumor, anti-HIV-1, and antibacterial activity, this compound also presents anti-QS activity, inhibiting several phenotypes in Pseudomonas aeruginosa PAO1, Escherichia coli, Serratia marcescens, and Proteus mirabilis (Ding et al., 2017). Resveratrol is also active against various pathogens and presents anti-QS activity, being capable of inhibiting biofilm formation and dispersing established biofilms (Duarte et al., 2015). Gallic acid is a phenolic acid present in numerous foods, and also an antimicrobial agent, affecting bacterial cell membranes and causing irreversible changes in permeability, rupture, and pore formation (Sorrentino et al., 2018). Capsaicin and luteolin are polyphenols abundant in several pepper species, especially those of the Capsicum genus, and have the ability to inhibit microbial growth, in addition to antioxidant, anti-inflammatory, cardioprotective, neuroprotective, and anticancer activity (Basith et al., 2016; Rivera et al., 2019).
The antimicrobial activity of phloridizin in the tested concentrations against the target microorganisms was lower when compared to the other phenolic compounds. Barreca et al. (2014) evaluated the antimicrobial activity of phloridizin and phloretin, its aglycone form, against several Gram-positive and Gram-negative bacteria, and observed that the presence of glucose in the basic chalcone structure caused a reduction in antimicrobial activity. Furthermore, they also observed that phloretin is particularly active against Gram-positive bacteria. The low activity against the tested Gram-negative microorganisms observed in this study corroborate the findings of Barreca et al. (2014). Interestingly, Quecán et al. (2019) also observed that glycosylation of quercetin reduces its antimicrobial and anti-QS effect.
Regarding the determination of the MIC of phenolic compounds, it is crucial to perform QS assays in concentrations that do not interfere with bacterial growth, since this would affect quantification of QS regulated phenotypes due to cell density differences (Defoirdt et al., 2013; Maura et al., 2016). Bali et al. (2019) evaluated the MIC of curcumin (1,500μg/ml, equivalent at 4.1mM) and gallic acid (>1,500μg/ml, equivalent to >8.8mM) in C. violaceum ATCC 12472, corroborating the results of the present work (Table 1). Packiavathy et al. (2014) also evaluated anti-QS activity of curcumin and reported an MIC of 384μg/ml, equivalent to 1.04mM, for C. violaceum 026, a value higher than that tested in the present study (0.1mM). Rivera et al. (2019) studied compounds present in Capsicum frutescens pepper extract and found that MIC of capsaicin was higher than 100μg/ml (equivalent to 0.34mM). The growth of C. violaceum ATCC 12472 and 026 was partially inhibited by the compound at 0.1mM, which is similar to the results found in the present work.
Curcumin inhibited violacein production by C. violaceum ATCC 12472 (Figure 2), as also observed by Bali et al. (2019). However, those authors evaluated higher concentrations (2mM, 1.02mM, and 0.51mM) than those used in the present work (0.012mM, 0.006mM and 0.003mM), indicating that even at lower concentrations, curcumin consistently inhibits this QS regulated phenotype. It should be noted that in concentrations higher than 0.050mM, a partial inhibition of microbial growth was observed in our study (results not shown), which should affect violacein production in a QS-independent manner.
In the study of Rivera et al. (2019), capsaicin did not inhibit violacein production when the concentration was similar to the one used in this study. However, those authors did not test the concentration of 70μM, which was the one with best anti-QS activity detected here. When working with resveratrol, Duarte et al. (2015) found positive results for violacein inhibition, corroborating the results of this work and the potential of this compound. They further suggested that the anti-QS activity of resveratrol is due to its ability to mimic QS signals and disrupt bacterial communication (Duarte et al., 2015). In the present study, phloridizin and gallic acid did not show anti-QS activity in C. violaceum ATCC 12472. Borges et al. (2014) and Bali et al. (2019) also reported that gallic acid did not exhibit anti-QS activity in the biosensor strain.
Tests performed with only wild-type C. violaceum strain are not enough to detect the QS inhibitory mechanism, since the inhibition of violacein production may be due to reduced production of the autoinducer (inhibition of AHL synthesis by CviI) or by interference with the AHL-dependent transcriptional activator, the CviR protein (Rivera et al., 2019). Thus, besides C. violaceum ATCC 12472, complementary assays performed with additional strains, such as C. violaceum 026, a mutant unable to produce AHLs but still capable to respond to exogenous AHLs, can be informative (Rivera et al., 2019). If the tested compounds interfere with C6-HSL detection by CviR, a reduced violacein production by C. violaceum 026 can be expected (LaSarre and Federle, 2013). Thus, curcumin and capsaicin likely present anti-QS activity via AHL detection by CviR since these compounds reduced violacein production in C. violaceum 026 (Figure 3). Brackman et al. (2009) and Packiavathy et al. (2014) also observed inhibition of violacein production by curcumin in C. violaceum 026, even though they have used higher concentrations than those used in this study.
Swarming motility is an organized microbial movement on surfaces, dependent on extensive flagellation, cell–cell contact, and QS. This type of motility is associated with virulence and antibiotic resistance of various microorganisms, and is considered a favorable adaptation to the challenges that arise in dynamic environments, contributing to biofilm formation and infection (Rütschlin and Böttcher, 2019; Carette et al., 2020). Interference with this phenotype is an important alternative to reduce or prevent biofilm-based infections. In this study, curcumin effectively inhibited swarming motility (Table 2 and Supplementary Figure S1), which corroborates results reported by Packiavathy et al. (2014), who observed the inhibition of swarming motility of S. marcescens, E. coli, P. aeruginosa, and P. mirabilis by curcumin at 100μg/ml (135μM). One explanation for the observed inhibition is the ability of curcumin in reducing the production of extracellular polysaccharides by some pathogens, negatively affecting their motility (Zheng et al., 2020).
Swimming-type inhibition by curcumin liposomes was also observed for A. hydrophila and Serratia grimesii (Ding et al., 2018) and for S. marcescens, E. coli, P. aeruginosa, and P. mirabilis (Packiavathy et al., 2014). Other studies also observed motility inhibition by phenolic compounds, like proanthocyanidins and tannins that completely inhibited swarming, but did not block swimming (O’May and Tufenkji, 2011), and trans-resveratrol, that had greater activity on swarming than on swimming motility, indicating that these movement phenotypes have different activation mechanisms.
Regarding the inhibition of biofilm formation by A. hydrophila (Figure 4), only resveratrol showed a significant effect. Similar results were found in other studies using different bacteria: L. monocytogenes (Vasquez-Armenta et al., 2018); Campylobacter spp. and Arcobacter butzleri (Duarte et al., 2015); methicillin-resistant S. aureus (MRSA; Qin et al., 2014); and E. coli O157: H7 (Lee et al., 2013), evidencing the broad inhibitory effect of resveratrol.
Mangoudehi et al. (2020) evaluated curcumin at different concentrations against biofilm formation of A. hydrophila ATCC 7966 and other three strains isolated from fish (silver carps) and found 67% reduction of biofilm formation at 43.4μM. These results differ from those observed in the present study (Figure 4A), where the maximum concentration was 12μM, indicating that biofilm inhibition is concentration-dependent. On the other hand, curcumin inhibited the biofilm formation by S. Montevideo (Figure 5). The efficacy of curcumin against various infectious organisms results from multiple mechanisms, including its ability to disrupt bacterial membranes, inhibit replication, and alter gene expression (Vaughn et al., 2017). Packiavathy et al. (2014) observed a visible reduction in microcolonies, disruption of biofilm architecture, and reduced biofilm biomass of P. aeruginosa, S. marcescens, E. coli, and P. mirabilis treated with curcumin.
Biofilm formation by S. Montevideo was also inhibited by capsaicin (Figure 5). This compound is used as a food additive and flavoring, and has important pharmacological actions, such as bacteriostatic activity against Gram-negative bacteria and anti-inflammatory effects (Zhou et al., 2014). Some studies have shown that capsaicin inhibits adhesion, growth, and biofilm formation of Gram-negative bacteria, such as Pseudomonas putida, Vibrio matriegens, Vibrio parahaemolyticus (Xu et al., 2005), and Porphyromonas gingivalis (Zhou et al., 2014).
Regarding the inhibition of biofilm formation of S. marcescens (Figure 6) by capsaicin at 1,000μM and resveratrol at 25μM or higher, interesting effects were observed. The capability of trans-resveratrol to inhibit biofilm formation by enterohemorrhagic E. coli O157:H7 ATCC43895 (Lee et al., 2013), P. aeruginosa PAO1 and PA14 (Cho et al., 2013), and S. aureus (Morán et al., 2014) has already been described. Thus, resveratrol can be considered a potential anti-biofilm compound for several biofilm-forming microorganisms and its use should be further investigated on food-contacting materials.
Antimicrobial resistance is a serious concern in clinical practice. One strategy that is useful in combating multidrug-resistant bacteria is the coadministration of antibiotics with adjuvants that modify bacterial resistance. Several medicines of great utility have been approved using this concept, such as the use of amoxicillin with beta-lactamase inhibitors. Other studies also show the effective combination of antibiotics with efflux-pump inhibitors (Ayaz et al., 2019). As biofilm formation is associated with reduced sensitivity to antibiotics, and it is known that QS regulates the expression of some genes associated with this phenotype, QS interference can be explored as an alternative to reduce bacterial virulence and increase the efficacy of antibiotic treatment (Mion et al., 2019). This study showed that curcumin at 6μM reduced the MIC of kanamycin by half in S. Montevideo (Figure 7B). These results corroborate those of Packiavathy et al. (2014) who observed increased sensitivity of S. marcencens to different antibiotics when combined with curcumin. Mun et al. (2013) and Kali et al. (2016) also observed a decrease in the MIC of several antibiotics against MRSA and other bacteria, when applied in combination with curcumin. These studies demonstrate that this phenolic compound can increase the sensitivity of several pathogens to antibioticos and is a potential agent to be coadministered with these drugs.
Serratia marcescens may be resistant to several antibiotics, including beta-lactam, aminoglycosides, and fluoroquinolones, hindering the treatment against infections caused by this bacterium (Yang et al., 2012). Like other Enterobacteriaceae, the production of beta-lactamase enzyme which inactivate beta-lactam antibiotics is the most common resistance mechanism in this micro-organism (Yang et al., 2012). The increased sensitivity of S. marcescens to colistin when combined with resveratrol reported by Cannatelli et al. (2018), the potentiation of the effects of aminoglycosides against biofilms of P. aeruginosa PAO1 (Zhou et al., 2018), and the increased sensitivity of multidrug resistant Klebsiella pneumoniae and E. coli (Liu et al., 2020b) and S. aureus (Liu et al., 2020a) deserve to be better explored as effective therapeutic alternatives.
The combination of bioactive compounds with antimicrobials seems to be a promising approach to improve the efficacy of these drugs. However, there are several challenges to be faced before translating these in vitro findings into real-life applications. Some of the challenges we envision are related to interactions and metabolization of these compounds in the human body, which could hinder their antimicrobial and anti-quorum sensing activities in vivo. Additionally, as pointed out by Vipin et al. (2020) and observed in the present work, strain variation in response to bioactive compounds is another challenge in treating infections.