Antivirulence Activity of a Dietary Phytochemical: Hibiscus Acid Isolated from Hibiscus sabdariffa L. Reduces the Virulence of Pseudomonas aeruginosa in a Mouse Infection Model

Abstract

Hibiscus sabdariffa L. (Hs) calyxes, rich in organic acids, are included in diets in different countries. In recent years, some phytochemicals have been shown to reduce bacterial virulence at sublethal concentrations by interfering with quorum sensing (QS) systems. Therefore, in this study the antivirulence properties of Hs calyxes and two γ-lactones (hibiscus acid [HA] and its methyl ester) in Pseudomonas aeruginosa were analyzed. Acetone and methanol extracts of Hs showed anti-QS activity by inhibiting violacein production (60% to 80% with 250 μg/mL). In molecular docking analysis, the γ-lactones registered a good binding score, which suggests strong interaction with the active site of LasR protein. To verify their effect in vitro, they were isolated from Hs and evaluated in six QS-regulated phenotypes, as well as in ExoU toxin that is released by the type III secretion system (T3SS). At 500 μg/mL they reduced alkaline protease (29–52%) and elastase (15–37%) activity, biofilm formation (∼75%), and swarming (50%), but there was no effect on pyocyanin production, hemolytic activity, or type III secretion. In a mouse abscess/necrosis model, HA at sublethal concentrations (15 and 31.2 μg/mL) affected infection establishment and prevented damage and systemic spread. In conclusion, HA is the first molecule identified with antivirulence properties in Hs with the potential to prevent infections caused by P. aeruginosa.

Introduction

H ibiscus sabdariffa L. ( Hs) is a subshrub native to Africa but grown and cultivated in several tropical and subtropical regions of the world.¹ Calyxes are consumed predominantly in aqueous infusions for food and for their medicinal properties.² The bactericidal properties of various constituents of plants have been widely reported,³ but it has recently been shown that when evaluated at sublethal concentrations, they can exhibit antivirulence properties.^4,5

Antivirulence is a broad concept that consists in interrupting the production of factors responsible for damage and establishment of pathogenic bacteria, without affecting bacterial viability.⁶ Different targets have been proposed, such as biofilms, swarming, two-component systems, fimbriae, pili, secretion systems, pigments, and proteases, among others.⁷ However, one of the most promising targets is the inhibition of quorum sensing (QS) systems, as it has a central role in the regulation of several virulence factors.⁸ QS is the mechanism that allows bacteria to communicate and exhibit multicellular behaviors.⁹ Bacterial communication is necessary for virulence factors to be expressed in a collective and coordinated manner in the population, maximizing success of infectious processes.⁸ Pseudomonas aeruginosa is one of the main bacterial models where QS and its role in virulence have been studied.¹⁰ Four QS systems have been identified in this bacterium that activate the expression of virulence genes through an intricate regulatory network between them.¹¹ Two systems use acyl-homoserine-lactone type autoinducers (LasI-LasR and RhlI-RhlR), whereas the rest use 2-heptyl-3-hydroxy-4-quinolone (PQS) and cis-2-decenoic acid (CDA).¹² These QS systems regulate some virulence mechanisms involved in motility (swarming), iron clearance (proteases, pyoverdine), immune evasion (elastase, alkaline protease), cytotoxicity (pyocyanin), and biofilm formation. However, there are other essential virulence factors, such as the type III secretion system (T3SS), for which regulation by QS is not yet completely clear.¹³

Furthermore, it is known that diet plays an important role in health.^14,15 In this regard, it has been suggested that antivirulence molecules (especially QS inhibitors) contained in edible plant species could prevent the establishment and development of chronic infections.¹⁶

Fresh or dried Hs calyxes are used in the preparation of hot and cold beverages and is a common ingredient in several foods.¹ Although their bactericidal properties have been reported,^2,17,18 it is only recently that its antivirulence potential has been investigated.^19,20

Organic extracts of the calyx are characterized by containing a high percentage of organic acids, among which hibiscus acid (HA) (γ-lactone) is the most abundant.¹⁵ It should be noted that, despite the high abundance of acids in the calyx of the plant, not much attention has been paid to them, apart from the fact that HA is not commercially available.²¹ Therefore, the objective of this research was to analyze the antivirulence properties of Hs calyxes, as well as HA and its dimethyl ester against P. aeruginosa.

Materials and Methods

Plant material

Two varieties of Hs (“criolla de Guerrero” and “alma blanca”) were analyzed in 2016. Both varieties are cultivated in the state of Guerrero, Mexico, at 18° 20′ 36.6″ N; 99° 30′ 12.6″ W. The calyxes were dried at 50°C (Lab-Line, Ambi Hi-Low Chamber, USA). One hundred grams of powder material was extracted with 900 mL acetone or methanol (JT Baker, USA) for 24 h at room temperature. The process was repeated two more times, supernatant was collected by filtration and the solvent was evaporated.

Isolation and characterization of γ-lactones

The methanol (27.4 g) and acetone (2.58 g) extracts of the variety “criolla de Guerrero” were subjected to a separation process by liquid column chromatography (silica gel, 60 Å; Sigma-Aldrich), using hexane, ethyl acetate, and methanol with increasing polarity (JT Baker) as the mobile phase (Supplementary Table S1).

Infrared spectroscopy

Infrared (IR) spectra were obtained in a PerkinElmer Spectrum 200 Fourier transform spectrophotometer (FT-IR) with universal attenuated total reflectance sampling accessory, at a temperature of 25°C ± 2°C. ¹H and¹³C nuclear magnetic resonance (NMR) spectra were recorded using deuterated chloroform (CDCl₃, to dissolve HA) or dimethyl sulfoxide (DMSO-d₆, to dissolve hibiscus dimethyl ester [HDM]) at room temperature using a Varian NMR, 400 MHz spectrometer. Tetramethylsilane was used as the internal standard.

Molecular docking

The crystal structure of LasR from P. aeruginosa bound to 3-oxo-C₁₂-HSL was obtained from the protein data bank (accession no. 31X3). HA and HDM three-dimensional models were obtained from the PubChem database (National Center for Biotechnology Information) (https://pubchem.ncbi.nlm.nih.gov/). For docking analysis, the 3-oxo-C₁₂-HSL molecule was removed from the LasR-binding site using the software UCFS Chimera Ver. 1,13,1 (developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with support from NIH P41-GM103311).²² Protein and ligand structures were prepared for docking using the software ADT 1.5.2.²³ Docking analysis was carried out using the software Autodock 4.2.5.1.²⁴ After docking, the 100 best conformations for each ligand were selected and then clustered for analysis using the software ADT 1.5.2. Conformations selected were within the most represented cluster and corresponded to those showing the lowest values of binding energy and theoretical Ki values. Model analyses and figure drawing were carried out with PYMOL 2.1.0 (The PyMOL Molecular Graphics System, Version 2.1.0 Schrödinger, LLC).

Anti-QS activity of organic extracts

Anti-QS activity was determined by the production of the violacein pigment in Chromobacterium violaceum by a protocol previously reported (Supplementary Data).²⁵

Virulence factor determination in P. aeruginosa

Pyocyanin inhibition,²⁶ swarming,²⁷ biofilm formation,²⁸ as well as elastase, alkaline protease,²⁶ and hemolytic activities²⁹ were evaluated as previously reported (Supplementary Data).

Type III secretion assay

The type III protein secretion assay was performed as previously reported.³⁰ Proteins secreted to the supernatant were precipitated, concentrated, loaded onto a 15% sodium dodecyl sulfate polyacrylamide gel, transferred to a nitrocellulose membrane, and tested against anti-ExoU polyclonal antibodies. The proteins were detected by immunoblot analysis using the Immobilon Western Chemiluminescent HRP Substrate Kit (Millipore) on X-ray films.

Animals and experimental design

Female CD1 mice (4–8 weeks old) were obtained from the Facultad de Estudios Superiores-Cuautitlán, UNAM. Animals were kept in standard conditions (23°C ± 2°C) with a 12-h light–12-h dark cycle and allowed free access to food and water. The indications of regulation for use and care of animals destined for research at the Hospital Infantil de México-Federico Gómez were always followed (HIM2018-002).

Ethics statements

Studies involving animal subjects Generated Statement: The animal study was reviewed and approved by Hospital Infantil de México-Federico Gómez (HIM2018-002).

Bacterial inoculum and preincubation with γ-lactones

Five milliliters of P. aeruginosa overnight culture were adjusted to an O.D_600nm of 0.06 and incubated at 6000 g at 37°C until reaching an O.D_600nm of 1.0–1.5. Subsequently, 1 mm of the culture was adjusted to an O.D_600nm of 0.08 (≈10⁷ colony-forming unit [CFU]/60 μL), centrifuged at 13,000 g for 2 min, and washed twice with phosphate-buffered saline (PBS). Finally, the bacterial pellet was homogenized in 1 mL of injectable solution (Pisa^®), the γ-lactones were added (final concentrations of 7.5, 15, 31.25, 125, and 500 μg/mL), and incubated for 25 min for its subsequent inoculation into animals.

In vivo assay

Based on a previous report.³¹ Mice were shaved with an electric shaver and excess hair was removed with a topical hair remover (Nair^®). Mice were anesthetized with an intraperitoneal injection of sodium pentobarbital (Pisa) 50 mg/kg, then 60 μL of the inoculum (≈10⁷ CFU) incubated with the different treatments was injected into the subcutaneous space on their right flank. Abscess and necrotic areas were measured every 24 h for a period of 96 h. Four days after inoculation, the livers and the soft tissues containing the necrotic area of the mice were excised and homogenized with 3 mL of PBS. Serial dilutions were done in Luria-Bertani plates to count colony-forming units.

Results

Anti-QS activity of Hs calyxes

None of the extracts affected bacterial growth at the concentrations evaluated, but they did reduce violacein production. The acetone extract of the variety “alma blanca” (AB-A) showed the best effect by inhibiting 86% pigment production at 250 μg/mL, similar to that of the positive control treated with the anacardic acid mixture (AA) (95% with 250 μg/mL) (Fig. 1).

FIG. 1.

Quorum sensing inhibitory activity of organic extracts from Hs calyxes. The black bars indicate violacein production and white bars indicate growth calculated by the viable cell counting method. Control, dimethyl sulfoxide, (vehicle).²⁵ Bars represent mean ± standard error. Significant statistical difference between mean values was determined by two-tailed Student's t-test, *P < .05. AA, anacardic acid mixture; AB-A, “alma blanca”-acetone extract; AB-M, “alma blanca”-methanol extract; CG-A, “criolla de Guerrero”-acetone extract; CG-M, “criolla de Guerrero”-methanol extract; Hs, Hibiscus sabdariffa L.

Docking analysis of LasR protein with γ-lactones

Molecular docking analysis suggested that γ-lactones can efficiently interact with the LasR protein-binding site (Fig. 2). To determine the theoretical binding affinity and stability of the complexes coupled between lactones and the LasR protein, energy minimization was performed revealing that the LasR protein had the lowest binding energy when coupled to HA (B.E. −7.39 kCal/mol, Ki 1.66 μM), even lower than that of the natural autoinducer (3-oxo-C12-HSL). While HA deprotonation further facilitates coupling (B.E. −7.26 to −9.06 kCal/mol, Ki 0.228 to 4.51 μM) (Fig. 2 and Table 1). These results indicate that HA can interact with the LasR protein efficiently and suggests a possible mechanism of action related to competition for the LasR active site. Interestingly, the analysis indicated that HA methylation reduces the binding efficiency (B.E. −6.63 kCal/mol, Ki 13.79 μM), however, its binding energy value remains in the same order of magnitude than that recorded for 3-oxo-C12-HSL.

FIG. 2.

Slice of the binding site of the LasR receptor protein showing the interactions with the autoinducer 3-oxo-C12 homoserine lactone (I), HA (II), and HDM (III). HA deprotonated at position: γ (IV), β (V), and γ/β (VI). HA, hibiscus acid; HDM, hibiscus dimethyl ester.

Table 1.

Molecular Docking Parameters of Hibiscus Acid and Hibiscus Dimethyl Ester Isolated from Hibiscus sabdariffa L. Against LasR Receptor Protein of Pseudomonas aeruginosa

Compound	Docking score
Compound	B.E. (kCal/mol)	Ki (μM)
Natural ligand 3-oxo-C12-HSL	−7.16	5.67
HA	−7.39	1.66
HDM	−6.63	13.79
HA deprotonated at position
γ	−7.26	4.51
β	−8.49	0.594
γ and β	−9.06	0.228

B.E., binding energy (more negative scores indicate higher binding affinities); HA, hibiscus acid; HDM, hibiscus dimethyl ester.

Isolation and characterization of γ-lactones

Since HA and HDM are not commercially available, they were isolated from Hs calyxes. Identification was made by comparative analysis of the NMR and IR values previously reported.^17,32

HA, (2S,3R)-tetrahydro-3-hydroxy-5-oxo-2,3-furandicarboxylic acid: white solid; m.p. 188.74–193.03°C. IR νmax cm⁻¹: 3410, 1797, 1742.¹HNMR (acetone-d₆, 400 MHz) δH: 5.34 (1H, s, CH-COOH), 3.25 (1H, d, J = 17.2 Hz, CH_a H_b COHCOOH), 2.80 (1H, d, J = 17.2 Hz, CH_a H_b COHCOOH).¹³C-NMR (acetone-d₆, 400 MHz) δC: 173.2, 172.3, 167.1, 82.9, 78.4, 42.2.

HDM, dimethyl (2S,3R)-tetrahydro-3-hydroxy-5-oxo-2,3 furandicarboxylate: white solid: m.p. 128–129.54°C. IR νmax cm⁻¹: 3527, 1798, 1741.¹HNMR (CDCl₃, 400 MHz) δH: 5.14 (1H, s, CH-COOMe), 3.95 (3H, s, CH-COOMe), 3.84 (3H, s, COH-COOMe), 3.09 (1H,d, J = 17.5 Hz, H _aH_bC-C = O), 2.88 (1H,d, J = 17.5 Hz, H_a H_b C-C = O).¹³C-NMR (DMSO-d₆): 172.7, 170.8, 166.1, 81.94, 77.93, 53.11, 52.39, 41.55.

Effect of γ-lactones on production of virulence factors

γ-lactones did not exhibit bactericidal activity in any of the evaluated doses (Fig. 3A), but they did affect production of some virulence factors. HA inhibited protease activity by 29.1% at 500 μg/mL (P < .05), but contrary to what was expected, HDM methylations favored a better activity, inhibiting it by 36.2% and 52.4% at 250 and 500 μg/mL, respectively (P < .05) (Fig. 3B). Only HA reduced elastase activity by 37% at 500 μg/mL (P < .05), whereas with HDM the effect was halved at the same concentration and did not show a dose–response effect (Fig. 3C). In contrast, there was no inhibitory effect of γ-lactones on hemolytic activity, pyocyanin production, and the secretion of the effector protein ExoU (Fig. 4).

FIG. 3.

Concentration-dependent effects of γ-lactones on inhibiting Pseudomonas aeruginosa growth and virulence factors. (A) Growth, (B) elastase activity, (C) alkaline protease activity, and (D) biofilm formation. For swarming motility (E), representative photographs are shown (F). Data represent mean of three independent experiments and presented as mean ± SD. *P < .05 (two-tailed Student's t-test).

FIG. 4.

Effect of γ-lactones on the production of P. aeruginosa virulence factors. (A) Pyocyanin production, (B) hemolytic activity, (C) bacterial growth curve with the different treatments, and (D) secretion of the ExoU protein (Western blot). MBX1641 (50 μM) is a T3SS inhibitor used as a positive control. T3SS, type III secretion system.

The best antivirulence activities of the entire study were observed on swarming motility and biofilm formation. γ-lactones significantly reduced biofilm formation by about 75% at 500 μg/mL (P < .05) (Fig. 3D), whereas for swarming, HA showed better activity with respect to its methylated form, reducing it by 50% at 250 μg/mL and 24.9% at 125 μg/mL (P < .05) (Fig. 3E, F). Interestingly, HDM at concentrations of 125 and 250 μg/mL stimulated swarming motility by about 20% (Fig. 3E).

HA displays an antibacterial effect and reduces damage and dispersal of P. aeruginosa

Subcutaneous inoculation of ∼10⁷ CFU induced the formation of 163.4 mm² abscesses and 22.9 mm² necrotic area, on average (Fig. 5A, B), as well as 7% mortality (data not shown). Incubation of the bacterial inoculum with 15 μg/mL HA for 25 min before inoculation, reduced the area of the abscesses to an average of 44.1 mm² (P < .05), whereas with 31.2 μg/mL, it prevented their formation (P < .05) (Fig. 5A). Similarly, with these concentrations of HA, necrotic tissue did not develop (P < .05) (Figs. 5B and 6), and preincubation of the bacteria with HA did not affect inoculum viability (Fig. 5D).

FIG. 5.

Effect of HA on P. aeruginosa infection in an animal model. CD1 mice were infected in the subcutaneous space with ∼10⁷ CFU of PA14, previously incubated for 25 min with HA in injectable solution. (A) Abscess size at 24 h after infection and (B) necrosis at 48 h. In both graphs, each symbol represents the value of a single mouse. Five to seven mice per group from two independent experiments were used. (C) Establishment and spread of bacteria. Injured tissues and liver were harvested 96 h after infection, homogenized, and the number of CFU/g of tissue was calculated. Data were analyzed using the bilateral Mann–Whitney U test, *P < .05. (D) Viability of the inoculum after 25 min preincubation with HA. Error bars represent standard errors of the means and were analyzed using an unpaired t-test *P < .05. CFU, colony-forming unit.

FIG. 6.

Images of animals from a representative experiment subcutaneously infected with P. aeruginosa treated with HA. Maximum abscess formation occurred 24 h postinoculation, whereas necrosis formation occurred at 48 h. In the control group (PA14-V), the bacteria were inoculated through an injectable solution, whereas in the experimental groups (PA14-HA) the inoculum was incubated for 25 min with the different concentrations of HA.

The bacteria established in the inoculation area at an average of log 8.7 CFU/g of tissue 4 days postinoculation, whereas with previous incubation of HA at 7.5 μg/mL it was reduced to log 8 CFU/g and with 15 μg/mL to log 4.33 CFU/g of tissue (P < .05). Interestingly, at a concentration of 31.2 μg/mL, P. aeruginosa establishment completely halted (Fig. 5C).

HA prevented systemic dispersion in all the concentrations evaluated since bacteria were not found in the liver of the animals; in contrast, the control group had an average of log 3.8 CFU/g of tissue (Fig. 5C). Likewise, 100% survival of the inoculated animals previously treated with HA was recorded (Fig. 6). Finally, it should be noted that HA at concentrations of 125 and 500 μg/mL also counteracted the damage caused by the bacteria (Fig. 7A, B), as well as their establishment and dispersion (Fig. 7C), while HDM, at the same concentrations, was not significantly different from the control group without treatment (Fig. 7A–C). However, the effect of HA at these concentrations was attributed to a bactericidal effect caused by a decrease in pH (2.7) in the bacteria preincubation process (Fig. 7D and Supplementary Table S2).

FIG. 7.

Effect of γ-lactones on P. aeruginosa infection in an animal model at concentrations of 125 and 500 μg/mL. The same experimental conditions mentioned in Figure 5 were used. (A) Abscess size and (B) necrosis. (C) Establishment and spread of bacteria. (D) Viability of the inoculum after 25 min preincubation with γ-lactones. Five to seven mice per group were used. The data shown are representative of at least two independent experiments. Data were analyzed using the bilateral Mann–Whitney U test, *P < .05. Error bars represent standard errors of the mean and were analyzed using an unpaired t-test *P < .05.

Discussion

Many anti-QS molecules have been reported to act by interfering with acyl-homoserine lactones bonding to homologous LuxR receptors.^5,33 In this study, our docking analysis suggested a strong interaction of HA with the active site of the LasR protein. This prediction was partially corroborated in vitro with elastase activity, biofilm formation, and swarming motility. However, for alkaline protease activity, this trend was not observed and there was no effect on hemolytic activity and pyocyanin production. In this regard, although the antivirulence properties of γ-lactones have been poorly explored, an HDM-like molecule called pantolactone ((3R)-3-hydroxy-4,4-dimethyloxolan-2-one) was reported to inhibit the production of pyocyanin.³⁴ Therefore, this indicates that small structural changes (the position of the methyl groups) favor reduction of some virulence factors in vitro. However, in swarming, acid groups in γ-lactone need to exert a better effect.

Swarming is a phenomenon of collective behavior controlled by QS, which involves the movement of bacteria by flagella on semisolid surfaces using biosurfactants.³⁵ In P. aeruginosa virulence is higher under swarming conditions, where production of QS-regulated phenotypes (proteases, siderophores, and phenazines) increases, and positive regulation of T3SS gene expression and its effectors is induced,³⁶ although this regulation seems to be independent of QS systems.³⁰ Our results suggest that HA acts mainly on some elements of the swarming regulation cascade and has no effect on T3SS activity. In this sense, although it is feasible for HA to inhibit swarming by interference with the LasR active site, it is possible for it to act at other levels of regulation beyond QS,³⁷ because it is known that other virulence regulators are involved in the expression of swarming, such as two-component systems³⁸ and temperature sensor systems,³⁹ among others.^40,41

The difference in activity between HA and its methylated form was also recorded in in vivo assays, where only HA showed antibacterial activity. At the highest evaluated doses, it has a bactericidal effect, which is attributed to a strong decrease in pH. However, at concentrations below 31.25 μg/mL, there is no effect of acidity on the viability of the bacterial inoculum (Supplementary Table S2). Clearly, at these sublethal concentrations, pretreatment of the inoculum with HA blocks the virulence of P. aeruginosa, as it reduces damage and systemic spread in animals. We must mention that, to achieve this effect, HA needs to dissociate partially in the solution, since if the pretreatment is carried out in a buffered solution, the antivirulence activity does not appear (PBS) (Supplementary Table S2 and Supplementary Fig. S1). Docking evaluations in the LasR-binding site with the protonated structure of HA (mainly in position β) support these results (Fig. 2 and Table 1). When the formal charge was assigned to both carboxylates of HA, the form that would be expected at physiological pH, the theoretical Ki diminished seven-fold with respect to the protonated form. Thus, these results suggest that the charge may play an important role in the binding of HA to LasR receptor. In this regard, it has been reported that a slightly acidic environment can enhance the bactericidal effect of some essential oils⁴² and organic acids,⁴³ but in the case of the activity of an antivirulence molecule, as far as we know, this is the first report.

The bactericidal mechanism of some weak organic acids is related to its high lipid permeability in its uncharged state,⁴⁴ which favors its entry into the bacteria where it dissociates and causes a decrease in the pH of the cytoplasm.⁴⁵ The accumulation of anions inside the bacteria generates osmotic stress and the inhibition of some metabolic processes.⁴⁶

In the case of antivirulence activity, it is also necessary for HA to diffuse into the cytoplasm so it can interact with the LasR receptor protein or other possible virulence regulatory elements not analyzed in this investigation. Therefore, other possible antivirulence mechanisms involved in the HA effect at sublethal concentrations in vivo remain to be explored. In this context, some organic acids, such as lactic, acetic, and propionic acids, have recently been reported to inhibit various QS-regulated virulence factors, mainly in Salmonella and Escherichia coli.^47,48

Furthermore, it has been suggested that any natural food source is unlikely to have biologically relevant amounts of quorum quenching (or antivirulence) molecules to eliminate established infections.¹⁶ However, if consumed constantly, the natural food source may help prevent development of chronic infections.^14,49 Hence, we are in the process of understanding the antimicrobial properties of Hs at sublethal concentrations. For example, the antivirulence activity of the aqueous extract of Hs calyxes has recently been reported on Staphylococcus aureus ¹⁹ and enteropathogenic E. coli.²⁰ Finally, unlike other reported antivirulence phytochemicals, it is likely that HA can exert this property when ingested in the diet, since it is soluble in water, it is in high concentrations¹⁵ and its intake is considered safe.⁵⁰

In conclusion, HA is the first antivirulence molecule identified in Hs calyxes, whose biofilm and swarming inhibitory effects, as well as antibacterial properties observed in vivo, indicate that it has potential for preventing bacterial infectious diseases.

Footnotes

Acknowledgments

I.C.-J. was supported by Cátedras-CONACyT program. The authors acknowledge Héctor Quezada-Pablo, PhD and facilities granted by the Hospital Infantil de México-Federico Gómez (HIM2018-002). They also acknowledge Norma Espinosa Sánchez, PhD for technical assistance.

Author Disclosure Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationship that could be construed as a potential conflict of interest.

Funding Information

This work was supported by grants from Scientific Development Projects for Solving National Problems/CONACyT Mexico no. 2015-01-402. H.C.-L. thanks CONACYT for her doctoral scholarship (449277). R.G.-C. is funded by CONACYT grant CB 2017–2018 number A1-S-8530.

Supplementary Material

Supplementary Data

Supplementary Figure S1

Supplementary Table S1

Supplementary Table S2

References

Da-Costa-Rocha

, Bonnlaender

, Sievers

, Pischel

, Heinrich

: Hibiscus sabdariffa L.—A phytochemical and pharmacological review. Food Chem, 2014; 165:424–443.

Borrás-Linares

, Fernández-Arroyo

, Arráez-Roman

, et al.: Characterization of phenolic compounds, anthocyanidin, antioxidant and antimicrobial activity of 25 varieties of Mexican Roselle (Hibiscus sabdariffa). Ind Crops Prod, 2015; 69:385–394.

Khameneh

, Iranshahy

, Soheili

, Bazzaz

BSF

: Review on plant antimicrobials: A mechanistic viewpoint. Antimicrob Resist Infect Control, 2019; 8:118.

Muñoz-Cazares

, García-Contreras R, Pérez-López

, Castillo-Juárez

: Phenolic compounds with anti-virulence properties. In: Phenolic Compounds-Biological Activity (Soto-Hernández M, ed.). In Tech, Croatia, 2017, pp. 139–167.

Deryabin

, Galadzhieva

, Kosyan

, Duskaev

: Plant-derived inhibitors of AHL-mediated quorum sensing in bacteria: Modes of action. Int J Mol Sci, 2019; 20:5588.

Totsika

: Benefits and challenges of antivirulence antimicrobials at the dawn of the post-antibiotic era. Curr Med Chem, 2016; 6:30–37.

Defoirdt

: Quorum-sensing systems as targets for antivirulence therapy. Trends Microbiol, 2018; 26:313–328.

Castillo-Juárez

, Maeda

, Mandujano-Tinoco

, et al.: Role of quorum sensing in bacterial infections. World J Clin Cases, 2015; 3:575–598.

Cortes-López

, Juárez-Rodríguez

, García-Contreras

, Soto-Hernández

, Castillo-Juárez

: “Old acquaintances in a new role: Regulation of bacterial communication systems by fatty acids.” In: Trends in Quorum Sensing and Quorum Quenching: New Perspectives and Applications (Ravishankar RV, ed.). CRC Press/Taylor & Francis, New York, 2020, pp. 47–57.

10.

Castillo-Juárez

, López-Jácome

, Soberón-Chávez

, et al.: Exploiting quorum sensing inhibition for the control of Pseudomonas aeruginosa and Acinetobacter baumannii biofilms. Curr Top Med Chem, 2017; 17:1915–1927.

11.

Lee

, Zhang

: The hierarchy quorum sensing network in Pseudomonas aeruginosa . Protein Cell, 2015; 6:26–41.

12.

Liu

, Li

, Cheng

, et al.: Structural and functional studies on Pseudomonas aeruginosa DspI: Implications for its role in DSF biosynthesis. Sci Rep, 2018; 8:1–11.

13.

Pena

, Blasco

, Ambroa A, et al.: Relationship between quorum sensing and secretion systems. Front Microbiol, 2019; 10:1–14.

14.

Vattem

, Mihalik

, Crixell

, McLean

: Dietary phytochemicals as quorum sensing inhibitors. Fitoterapia, 2007; 78:302–310.

15.

Beltran-Debon

, Alonso-Villaverde

, Aragones

, et al.: The aqueous extract of Hibiscus sabdariffa calices modulates the production of monocyte chemoattractant protein-1 in humans. Phytomedicine, 2010; 17:186–191.

16.

Givskov

: Beyond nutrition: Health-promoting foods by quorum-sensing inhibition. Future Microbiol, 2012; 7:1025–1028.

17.

Portillo-Torres

, Bernardino-Nicanor

, Gómez-Aldapa

, et al.: Hibiscus acid and chromatographic fractions from Hibiscus sabdariffa calyces: Antimicrobial activity against multidrug-resistant pathogenic bacteria. Antibiotics, 2019; 8:218.

18.

Tolulope

: Cytotoxicity and antibacterial activity of methanolic extract of Hibiscus sabdariffa . J Med Plant Res, 2007; 1:009–013.

19.

Salman

, Aldeen

WRT

: Antibacterial, anti-virulence factors of Hibiscus sabdariffa extracts in Staphylococcus aureus isolated from patients with urinary tract infection. Res J Pharm Technol, 2018; 11:735–740.

20.

Mohamed-Salem

, Rodríguez Fernández

, Nieto-Pelegrín

, Conde-Valentín

, Rumbero

, Martinez-Quiles

: Aqueous extract of Hibiscus sabdariffa inhibits pedestal induction by enteropathogenic E. coli and promotes bacterial filamentation in vitro. PLoS One, 2019; 14:e0213580.

21.

Zheoat

, Gray

, Igoli

, Kennedy

, Ferro

: Crystal structures of hibiscus acid and hibiscus acid dimethyl ester isolated from Hibiscus sabdariffa (Malvaceae). Acta Cryst E, 2017; 73:1368–1371.

22.

Pettersen

, Goddard

, Huang

, et al.: UCSF Chimera—A visualization system for exploratory research and analysis. J Comput Chem, 2004; 25:1605–1612.

23.

Morris

, Huey

, Lindstrom

, et al.: AutoDock 4 and AutoDockTools 4: Automated docking with selective receptor flexibility. J Comput Chem, 2009; 30:2785–2791.

24.

Huey

, Morris

, Olson

, Goodsell

: A semiempirical free energy force field with charge-based desolvation. J Comp Chem, 2007; 28:1145–1152.

25.

Castillo-Juárez

, García-Contreras

, Velázquez-Guadarrama

, Soto-Hernández

, Martínez-Vázquez

: Amphypterygium adstringens anacardic acid mixture inhibits quorum sensing-controlled virulence factors of Chromobacterium violaceum and Pseudomonas aeruginosa . Arch Med Res, 2013; 44:488–494.

26.

Muñoz-Cazares

, Aguilar-Rodríguez

, García-Contreras

, et al.: Phytochemical screening and anti-virulence properties of Ceiba pentandra and Ceiba aesculifolia (Malvaceae) bark extracts and fractions. Bot Sci, 2018; 96:415–425.

27.

Kollaran

, Joge

, Kotian

, et al.: Context-specific requirement of forty-four two-component loci in Pseudomonas aeruginosa swarming. iScience, 2019; 13:305–317.

28.

Lee

, Cho

, Lee

: 3-Indolylacetonitrile decreases Escherichia coli O157: H7 biofilm formation and Pseudomonas aeruginosa virulence. Environ Microbiol, 2011; 13:62–73.

29.

Hofemeister

, Conrad

, Adler

, et al.: Genetic analysis of the biosynthesis of non-ribosomal peptide-and polyketide-like antibiotics, iron uptake and biofilm formation by Bacillus subtilis A1/3. Mol Genet Genomics, 2004; 272:363–378.

30.

Soto-Aceves

, Cocotl-Yañez

, Merino

, et al.: Inactivation of the quorum-sensing transcriptional regulators LasR or RhlR does not suppress the expression of virulence factors and the virulence of Pseudomonas aeruginosa PAO1. Microbiology, 2019; 165:425–432.

31.

Berube

, Murphy

, Torhan

, et al.: Impact of type III secretion effectors and of phenoxyacetamide inhibitors of type III secretion on abscess formation in a mouse model of Pseudomonas aeruginosa infection. Antimicrob Agents Chemother, 2017; 61:e01202–17.

32.

Ibnusaud

, Thomas

, Rani

, Sasi

, Beena

, Hisham

: Chiral γ-butyrolactones related to optically active 2-hydroxycitric acids. Tetrahedron, 2002; 58:4887–4892.

33.

Qin

, Thota

, Singh

, Balamurugan

, Goycoolea

: Synthetic homoserine lactone analogues as antagonists of bacterial quorum sensing. Bioorg Chem, 2020; 98:103698.

34.

Abd-Alla

, Bashandy

: Production of quorum sensing inhibitors in growing onion bulbs infected with Pseudomonas aeruginosa E (HQ324110). ISRN Microbiol, 2012; 2012:161890.

35.

Köhler

, Curty

, Barja

, Van Delden

, Pechère

: Swarming of Pseudomonas aeruginosa is dependent on cell-to-cell signaling and requires flagella and pili. J Bacteriol, 2000; 182:5990–5996.

36.

Overhage

, Bains

, Brazas

, Hancock

: Swarming of Pseudomonas aeruginosa is a complex adaptation leading to increased production of virulence factors and antibiotic resistance. J Bacteriol, 2008; 190:2671–2679.

37.

Francis

, Stevenson

, Porter

: Two-component systems required for virulence in Pseudomonas aeruginosa . FEMS Microbiol Lett, 2017; 364:fnx104.

38.

Sperandio

, Torres

, Kaper

: Quorum sensing Escherichia coli regulators B and C (QseBC): A novel two-component regulatory system involved in the regulation of flagella and motility by quorum sensing in E. coli . Mol Microbiol, 2002; 43:809–821.

39.

LeeYY, Belas

: Loss of FliL alters Proteus mirabilis surface sensing and temperature-dependent swarming. J Bacteriol, 2015; 197:159–173.

40.

Soutourina

, Bertin

: Regulation cascade of flagellar expression in Gram-negative bacteria. FEMS Microbiol Rev, 2003; 27:505–523.

41.

Alford

, Baghela

, Yeung

, Pletzer

, Hancock

: NtrBC regulates invasiveness and virulence of Pseudomonas aeruginosa during high-density infection. Front Microbiol, 2020; 11:773.

42.

Juven

, Kanner

, Schved

, Weisslowicz

: Factors that interact with the antibacterial action of thyme essential oil and its active constituents. J Appl Bacteriol, 1994; 76:626–631.

43.

Bushell

, Tonner

, Jabbari

, Schmid

, Lund

: Synergistic impacts of organic acids and pH on growth of Pseudomonas aeruginosa: A comparison of parametric and Bayesian non-parametric methods to model growth. Front Microbiol, 2019; 9:3196.

44.

Slonczewski

, Fujisawa

, Dopson

, Krulwich

: Cytoplasmic pH measurement and homeostasis in bacteria and archaea. Adv Microb Physiol, 2009; 55:1–317.

45.

Saparov

, Antonenko

, Pohl

: A new model of weak acid permeation through membranes revisited: Does Overton still rule?. Biophys J, 2006; 90:L86–L88.

46.

Hirshfield

, Terzulli

, O'Byrne

: Weak organic acids: A panoply of effects on bacteria. Sci Progress, 2003; 86:245–270.

47.

Burt

, Adolfse

, Ahad

, et al.: Cinnamaldehyde, carvacrol and organic acids affect gene expression of selected oxidative stress and inflammation markers in IPEC-J2 cells exposed to Salmonella typhimurium . Phytother Res, 2016; 30:1988–2000.

48.

Amrutha

, Sundar

, Shetty

: Effect of organic acids on biofilm formation and quorum signaling of pathogens from fresh fruits and vegetables. Microb Pathogenesis, 2017; 111:156–162.

49.

Pérez-López

, García-Contreras

, Soto-Hernández

, et al.: Anti-quorum sensing activity of seed oils from oleaginous plants and protective effect during challenge with Chromobacterium violaceum . J Med Food, 2018; 21:356–363.

50.

Izquierdo-Vega

, Arteaga-Badillo

, Sánchez-Gutiérrez

, et al.: Organic acids from Roselle (Hibiscus sabdariffa L.)—A brief review of its pharmacological effects. Biomedicines, 2020; 8:100.

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