# Spondias tuberosa Arruda (Anacardiaceae) ethanolic leaf extract as a source of natural products inhibiting quorum sensing and pyocyanin production in planktonic Pseudomonas aeruginosa

A.S. Tareau, P.E. Bodet, J.R. Guedes da Silva Almeida, Magalie Barreau, O.

Lesouhaitier, A. Tahrioui, R. Gonçalves de Oliveira Junior, R. Grougnet, P.

Sitarek, L. Quintans-Junior, et al.

► To cite this version:

A.S. Tareau, P.E. Bodet, J.R. Guedes da Silva Almeida, Magalie Barreau, O. Lesouhaitier, et al. Spondias tuberosa Arruda (Anacardiaceae) ethanolic leaf extract as a source of natural products inhibiting quorum sensing and pyocyanin production in planktonic Pseudomonas aeruginosa. Phytochemistry Letters, 2025, 70, pp.104078. 10.1016/j.phytol.2025.104078. hal-05379688

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**Spondias tuberosa** Arruda (Anacardiaceae) ethanolic leaf extract as a source of natural products inhibiting quorum sensing and pyocyanin production in planktonic *Pseudomonas aeruginosa*

A.S. Tareau<sup>a,b</sup>, P.E. Bodet<sup>c</sup>, J.R. Guedes da Silva Almeida<sup>d</sup>, M. Barreau<sup>a,b</sup>, O. Lesouhaitier<sup>a,b</sup>,
A. Tahrioui<sup>a,b</sup>, R. Gonçalves de Oliveira Junior<sup>e</sup>, R. Grougnet<sup>e</sup>, P. Sitarek<sup>f</sup>, L. Quintans-Junior<sup>g</sup>,
K. Baranger<sup>h</sup>, S. Chevalier<sup>a,b</sup>, L. Picot<sup>h,*</sup>

ARTICLE INFO

Keywords:

Anti-virulence

Natural products

Pseudomonas aeruginosa

Pyocyanin inhibition

Quorum sensing

Spondias tuberosa

A B S T R A C T

Pseudomonas aeruginosa is a multidrug-resistant pathogen whose virulence is tightly regulated by quorum sensing (QS). Targeting QS offers a promising anti-virulence strategy by disarming pathogens without promoting resistance. Here, we screened Brazilian medicinal plant extracts for QS inhibition and identified the ethanolic leaf extract of Spondias tuberosa Arruda (STF) as a potent suppressor of pyocyanin production in planktonic P. aeruginosa cultures, without affecting bacterial growth. STF disrupted QS signaling, as demonstrated by the decreased production of QS mediators detected using a biosensor strain. Phytochemical profiling revealed candidate compounds potentially responsible for this activity. These findings highlight S. tuberosa as a promising natural source of QS inhibitors capable of attenuating P. aeruginosa virulence, particularly during the early stages of infection, and open perspectives for future research into its potential interest in anti-virulence therapeutic approaches.

1. Introduction

Pseudomonas aeruginosa is a Gram-negative, opportunistic pathogen responsible for a wide range of infections, particularly in immunocompromised individuals (Hernández-Jiménez et al., 2022; Lyczak et al., 2000; Moradali et al., 2017). Its virulence is tightly regulated by a complex QS system that orchestrates the expression of numerous pathogenic determinants (Lee and Zhang, 2015; Nadal Jimenez et al., 2012). The QS network in P. aeruginosa comprises three interrelated signaling systems, Las, Rhl, and PQS, which govern the coordinated production of

key virulence factors such as pyocyanin, elastase, and rhamnolipids (Lee and Zhang, 2015; Williams and Cámara, 2009). Pyocyanin, a redox-active phenazine pigment, promotes the generation of reactive oxygen species (ROS), resulting in oxidative tissue damage and suppression of host immune responses (Lau et al., 2004). Elastase contributes to tissue invasion by degrading structural components of the extracellular matrix, while rhamnolipids facilitate biofilm development and the lysis of host cells (Bever and Iglewski, 1988). Together, these virulence factors enhance the persistence and resilience of P. aeruginosa, positioning it as a critical member of the ESKAPE group—an acronym

<footnote><sup>a</sup> CBSA UR 4312, Univ Rouen Normandie, Université Caen Normandie, Normandie Univ, Rouen F-76000, France</footnote>

<footnote><sup>b</sup> Plateforme de Sécurité Sanitaire Ebroïcienne (PS2E), Univ Rouen Normandie, Normandie Univ, CBSA UR 4312, Rouen F-76000, France</footnote>

<footnote><sup>c</sup> UMR CNRS 7266 LIENSs, Platform for the High Resolution Analysis of Biomolecules, La Rochelle Université, La Rochelle 17042, France</footnote>

<footnote><sup>d</sup> NEPLAME, Universidade Federal do Vale do São Francisco, Petrolina 56304-205, Brazil</footnote>

<footnote><sup>e</sup> UMR CNRS 8038 CITCoM, Université Paris Cité, Paris 75006, France</footnote>

<footnote><sup>f</sup> Department of Medical Biology, Medical University of Lodz, Lodz, Poland</footnote>

<footnote><sup>g</sup> LANEF, Universidade Federal de Sergipe, Aracaju 49100-000, Brazil</footnote>

<footnote><sup>h</sup> UMR CNRS 7266 LIENSs, La Rochelle Université, La Rochelle 17042, France</footnote>

<footnote>* Correspondence to: UMR CNRS 7266 LIENSs, Institut du Littoral et de l'Environnement, 2 rue Olympe de Gouges, La Rochelle 17000, France.</footnote>

<footnote>E-mail addresses: anne-sophie.tareau@univ-rouen.fr (A.S. Tareau), pierreedouard.bodet@univ-lr.fr (P.E. Bodet), jackson.guedes@univasf.edu.br (J.R. Guedes da Silva Almeida), magalie.barreau@univ-rouen.fr (M. Barreau), olivier.lesouhaitier@univ-rouen.fr (O. Lesouhaitier), ali.tahrioui@univ-rouen.fr (A. Tahrioui), raimundo.goncalves-de-oliveira-junior@parisdescartes.fr (R. Gonçalves de Oliveira Junior), raphael.grougnet@parisdescartes.fr (R. Grougnet), przemyslaw.sitarek@umed.lodz.pl (P. Sitarek), lucindo@academico.ufs.br (L. Quintans-Junior), kevin.baranger@univ-lr.fr (K. Baranger), sylvie.chevalier@univ-rouen.fr (S. Chevalier), laurent.picot@univ-lr.fr (L. Picot).</footnote>
denoting highly virulent, antibiotic-resistant microorganisms responsible for severe hospital-associated infections (Boucher et al., 2009; Pendleton et al., 2013). Considering the growing prevalence of multidrug-resistant *P. aeruginosa*, there is an urgent demand for novel therapeutic approaches. A promising alternative lies in targeting bacterial virulence instead of viability, thereby reducing selective pressure and the emergence of resistance. QS inhibition interrupts the regulation of virulence gene expression, offering a strategic means to attenuate pathogenicity (Defoirdt, 2018; Kalia, 2013; LaSarre and Federle, 2013; Rasko and Sperandio, 2010). In this context, natural products derived from medicinal plants represent a valuable reservoir of potential QS inhibitors, that can include various compounds such as alkaloids, polyphenols, terpenoids, and organosulfur compounds. These bioactive molecules have already shown the ability to disrupt QS networks and decrease the production of virulence determinants such as pyocyanin, elastase, and rhamnolipids (Choo et al., 2006; Jakobsen et al., 2012; Tahrioui et al., 2020; Vadakkan et al., 2024). Brazil, a country renowned for its vast biodiversity, contains an extraordinary array of medicinal plants with demonstrated antimicrobial potential, many of which have long been used in traditional medicine for the prevention and treatment of infections (Almeida et al., 2012, 2006; de Lima-Saraiva et al., 2017; Nascimento et al., 2000). This unique ecological biodiversity presents an ideal opportunity for the discovery of novel QS inhibitors capable of disrupting bacterial communication without triggering resistance mechanisms. Identifying such compounds from Brazilian flora could drive opportunities for innovative anti-infective strategies, particularly against multidrug-resistant pathogens like *P. aeruginosa*, and contribute to global efforts to combat antimicrobial resistance. Several plant species from the Myrtaceae, Fabaceae, and Asteraceae families have already exhibited activity against *P. aeruginosa*, primarily through mechanisms such as QS interference, membrane destabilization, and efflux pump inhibition (D'Aquila et al., 2023; Fabri et al., 2011; Masuku et al., 2023; Musthafa et al., 2017; Rosado-Vallado et al., 2000; Tahrioui et al., 2020). In this study, a screening of 20 extracts obtained from 10 Brazilian medicinal plants was conducted to identify candidates capable of suppressing *P. aeruginosa* virulence. Among the extracts tested, the ethanolic leaf extract of *Spondias tuberosa* Arruda (STF) stood out for its significant inhibition of pyocyanin production in planktonic cells. STF markedly decreased pyocyanin levels without impairing bacterial growth, suggesting a mechanism of action based on QS disruption rather than bactericidal activity. Subsequent bioassays confirmed a decrease in the production of QS signaling molecules, supporting the hypothesis of interference with QS-regulated pathways. Phytochemical analysis of STF revealed several candidate compounds potentially responsible for this anti-virulence effect. These findings position *Spondias tuberosa* leaf ethanolic extract as a promising source of natural QS inhibitors and support further investigations to valorize its bioactive compounds for antimicrobial applications.

## 2. Materials and methods

### 2.1. Plant material collection and extract preparation

All plants studied were collected in areas of the Caatinga biome (semiarid vegetation, Brazil) and the botanical identification was made by comparison with voucher specimens deposited at the Herbário Vale do São Francisco (HVASF). All necessary permissions for the collection and use of plant material were obtained. The study was conducted under SisGen authorization (Register #A654697) in accordance with Brazilian biodiversity regulations (Law No. 13.123/2015). *Spondias tuberosa* was harvested on April 2024 at Sítio Asa Branca in Petrolina, state of Pernambuco, Brazil (Coordinates: S 9°30'86.54", W 40°72'07.94"). The Caatinga biome is a semi-arid and unique North-eastern Brazilian ecosystem characterized by high endemism and a rich diversity of medicinal plants traditionally used by local communities for managing infections and inflammatory conditions. The plant material was washed

with water, dried at a temperature of 45 °C for 72 h, and then, pulverized and exhaustively macerated with solvents at room temperature with solvent renewal every 72 h (three repetitions) as detailed in Table 1. Ethanol and methanol were selected to prioritize the extraction of polar compounds, particularly polyphenols and flavonoids, known for their quorum sensing inhibitory potential. The choice of plant part was based on traditional uses and availability; for instance, stem barks were selected for *Hymenaea martiana* and *Piptadenia viridiflora* due to their recognized medicinal value, while leaves were prioritized for Spondias tuberosa and other species because of their metabolic richness and easier renewability. The extractive solutions were concentrated under low pressure using a rotary evaporator (Quimis, Brazil) to obtain the crude extracts. For some extracts, further fractionation was performed using a secondary solvent as detailed in

**Table 1. Summary of plant extracts and fractions tested in this study.** All plant materials were collected in April 2024 from Sítio Asa Branca (Petrolina, Pernambuco, Brazil), within the Caatinga biome. Extracts were prepared by exhaustive maceration of dried and pulverized plant material using appropriate solvents, followed by concentration under reduced pressure. The initial weights of the dried plant materials ranged from 10 to 500 g, depending on the species and the plant part used. Some extracts were further fractionated as indicated. The table details the extract code, corresponding plant species, botanical family, voucher specimen number, type of extract or fraction, physical appearance and final extract weight.

### 2.2. Bacterial strains and culture conditions

The *P. aeruginosa* H103, a prototroph from the wild-type strain PA01, was provided by Prof. Robert E W Hancock from the University of British Columbia (Hancock and Carey, 1979). The PA01 mini-CTX pqsA::lux biosensor strain was provided by Prof. Miguel Camara from the University of Nottingham (Fletcher et al., 2007). To obtain it, single-copy luxCDABE transcriptional fusions of the pqsA, pvdS, and pvdE promoters were integrated into the chromosome of the PA01 pqsA mutant using the mini-CTXlux system. (Becher and Schweizer, 2000; Diggle et al., 2007). A 532 bp fragment containing the pqsA promoter region was amplified and cloned into pBluescript KS+ digested with *Hind*III and *Bam*HI. The fragment was excised with *Hind*III and *Bam*HI and cloned into similarly digested mini-CTX1ux resulting in the mini-CTX pqsA::lux biosensor strain (Becher and Schweizer, 2000). *P. aeruginosa* H103 was grown in Luria-Bertani broth containing 171 mM (10 g·L⁻¹) NaCl (LB, BD Difco™, Fisher Scientific, Illkirch-Graffenstaden, France), for 18 h at 37 °C under shaking (180 rpm). All experiments in this study were conducted using planktonic cultures of *P. aeruginosa* H103. This model is widely recognized as representative of the bacterial behavior during acute infection phases, where *P. aeruginosa* predominantly exists in a free-living, planktonic state prior to biofilm establishment. This approach ensures that the observed effects of plant extracts on quorum sensing, pyocyanin production, and 4-hydroxy-2-alkylquinolines (HAQ) signaling are relevant to the early stages of infection, where therapeutic interventions targeting virulence factors could be most effective.

### 2.3. Pyocyanin quantification

*P. aeruginosa* H103 exposed to plant extracts at serial concentrations ranging from 8 to 256 µg·mL⁻¹, following standard practices used in antibiogram assays, or DMSO 1 % (control) was grown in LB in a 96-well microtiter plate (Thermo Fisher Scientific, Nunc™, Villebon-sur-Yvette, France) at 37 °C for 24 h with shaking (180 rpm). After incubation for 24 h, A₅₈₀ nm was measured to determine the bacterial growth. The pyocyanin quantification assay was carried out as previously described (Azuama et al., 2020; Tahrioui et al., 2020) and the 64 µg·mL⁻¹ was determined as a discriminant concentration among plant extracts. For pyocyanin quantification, one volume of chloroform was added to one volume of culture supernatants. After centrifugation, the chloroform
<caption>**Table 1**</caption>

Fractionation was performed on selected extracts to improve compound identification and comparative analysis of inactive versus active extracts. Although STF showed the highest activity, fractionation of less active extracts was conducted to explore chemical diversity and verify whether similar compounds were present.

<table><thead><tr><th>Extract code</th><th>Species name</th><th>Botanical Family</th><th>Nature of the extract</th><th>Voucher specimen number</th><th>Weight (g)</th></tr></thead><tbody><tr><td>BC</td><td>Bauhinia cheilantha</td><td>Fabaceae</td><td>Ethanolic extract of the leaves</td><td>2241</td><td>88.0</td></tr><tr><td>EDSAS</td><td>Annona spinescens</td><td>Annonaceae</td><td>Dichloromethane extract of the seeds</td><td>25507</td><td>55.0</td></tr><tr><td>EHFAS</td><td></td><td></td><td>Hexanic extract of the leaves</td><td></td><td>17.0</td></tr><tr><td>EHSAS</td><td></td><td></td><td>Hexanic extract of the seeds</td><td></td><td>15.0</td></tr><tr><td>EMCAS</td><td></td><td></td><td>Methanolic extract of the stembarks</td><td></td><td>33.0</td></tr><tr><td>EMFAS</td><td></td><td></td><td>Methanolic extract of the leaves</td><td></td><td>35.0</td></tr><tr><td>EMSAS</td><td></td><td></td><td>Methanolic extract of the seeds</td><td></td><td>33.0</td></tr><tr><td>EMTAS</td><td></td><td></td><td>Methanolic extract of the twigs</td><td></td><td>33.0</td></tr><tr><td>ES</td><td>Encholirium spectabile</td><td>Bromeliaceae</td><td>Ethanolic extract of the leaves</td><td>6443</td><td>10.0</td></tr><tr><td>HM</td><td>Hymenaea martiana</td><td>Fabaceae</td><td>Ethanolic extract of the stembarks</td><td>6444</td><td>10.0</td></tr><tr><td>MN</td><td>Morus nigra</td><td>Moraceae</td><td>Ethanolic extract of the leaves</td><td>1764</td><td>322.0</td></tr><tr><td>MT</td><td>Mimosa tenuiflora</td><td>Fabaceae</td><td>Ethanolic extract of the stembarks</td><td>16714</td><td>29.0</td></tr><tr><td>PC</td><td>Passiflora cinninata</td><td>Passifloraceae</td><td>Ethanolic extract of the leaves</td><td>22870</td><td>12.0</td></tr><tr><td>FDPC</td><td></td><td></td><td>Dichloromethane fraction of PC</td><td></td><td>2.9</td></tr><tr><td>FEPPC</td><td></td><td></td><td>Petroleum ether fraction of PC</td><td></td><td>0.5</td></tr><tr><td>FMPC</td><td></td><td></td><td>Methanolic fraction of PC</td><td></td><td>5.7</td></tr><tr><td>PF</td><td>Passiflora foetida</td><td>Passifloraceae</td><td>Ethanolic extract of the leaves</td><td>2413</td><td>28.7</td></tr><tr><td>PV</td><td>Piptadenia viridiflora</td><td>Fabaceae</td><td>Ethanolic extract of the stembarks</td><td>28</td><td>105.0</td></tr><tr><td>STC</td><td>Spondias tuberosa</td><td>Anacardiaceae</td><td>Ethanolic extract of the stembarks</td><td>18319</td><td>483.0</td></tr><tr><td>STF</td><td></td><td></td><td>Ethanolic extract of the leaves</td><td></td><td>252.0</td></tr></tbody></table>

blue layer was collected and ½ volume of 0.5 M HCl was added. After a second centrifugation, the red pink HCl layer was collected and the A$_{520}$ nm was recorded (Spark 20 M multimode Microplate Reader, Tecan Group Ltd., Crailsheim, Germany). The data were normalized to the bacterial cell density (A$_{520}$ nm/A$_{580}$ nm) and expressed as a percentage of the control value. This pyocyanin inhibition assay enabled the identification of the most active extract, which was subsequently selected for further experiments.

## 2.4. Evaluation of Spondias tuberosa Arruda ethanolic leaf extract (STF) on Pseudomonas aeruginosa H103 growth kinetics

To assess the effect of STF on *P. aeruginosa* H103 growth kinetics, bacterial cultures were grown in the presence or absence of STF at 64 µg·mL⁻¹. An absorbance measure at 580 nm was performed every 30 min using the Spark 20 M multimode Microplate Reader controlled by Spark Control™ software Version 2.1 (Tecan Group Ltd., Crailsheim, Germany). The data were plotted, and each point indicates the mean ± standard error of the mean (SEM) of A$_{580}$ nm values. Each set of experiments was performed at least three times.

## 2.5. Extraction and quantification of 4-hydroxy-2-alkylquinolines (HAQs)

Measuring the production of HAQ molecules—key quorum sensing mediators in *P. aeruginosa* such as PQS, HHQ, and HQNO—provides a relevant indicator of quorum sensing activity, thereby enabling the evaluation of the STF extract’s impact on this regulatory network. Planktonic cultures of *P. aeruginosa* H103, either untreated (dimethyl sulfoxide (DMSO) 1 % control) or treated with 64 µg·mL⁻¹ of STF, were incubated for 24 h at 37 °C with shaking (180 rpm) and then subjected to HAQ molecule extraction following the protocol previously described by Tahrioui et al. (Tahrioui et al., 2019). The quantification of HAQs was performed using a combined spectrophotometric and luminometric microplate assay with the biosensor strain PAO1 *pqSA* *CTX-lux*: *pqSA* (Diggle et al., 2011). After overnight culture, the biosensor strain was adjusted with fresh LB medium to an optical density (OD$_{580nm}$) of 1. For the assay, 5 µL of crude HAQ extracts were diluted in 100 µL of LB and mixed with 100 µL of a 1:50 dilution of the biosensor strain. Bioluminescence and OD$_{580nm}$ were recorded every 15 min over 24 h at 37 °C using a Spark 20 M multimode microplate reader, managed by Spark-Control™ software Version 2.1 (Tecan Group Ltd.). Bioluminescence

values, expressed as relative light units (R.L.U), were normalized to the OD$_{580}$ nm of the culture suspensions.

## 2.6. Compounds identification by ultra-performance liquid chromatography coupled with diode array detection and high-resolution mass spectrometry (UPLC-DAD-MS/MS)

An Acquity UPLC H-class system (Waters, Milford, CT, USA) coupled to a photodiode array (Waters 2996) and a high-resolution mass spectrometer (a XEVO G2-S Q-Tof equipped with an electrospray ionization source (Waters, Manchester, UK)) were used. The STF extract was solubilized in DMSO at 0.1 g·L⁻¹ and diluted tenfold with 5 % acetonitrile (ACN). Prior to injection, the solution was centrifuged at 13,000 rpm for 5 min and filtered through a 0.22 µm polyvinylidene fluoride (PVDF) membrane. The injection volumes were 5 µL for MS analysis and 10 µL for MS/MS and UV detection. Chromatographic separation was performed using a BEH C18 column (2.1 × 50 mm, 1.7 µm particle size, Waters, Manchester, UK) with a mobile phase comprising solvent A (H₂O + 0.001 % formic acid) and solvent B (ACN + 0.001 % formic acid) at a column oven temperature of 25°C. The flow was set at 0.3 mL/min and the gradient for solvents A and B was as follows: 0–3 min, 95 % A and 5 % B; 3–7 min 90 % A and 10 % B; 7–10 min 90 % A and 10 % B; 10–12 min, 85 % A and 15 % B; 10–12 min 85 % A and 15 % B; 12–14 min, 85 % A and 15 % B; 14–16 min, 80 % A and 20 % B; 16–19 min, 80 % A and 20 % B; 19–21 min, 70 % A and 30 % B; 21–23 min, 70 % A and 30 % B; 23–26 min, 10 % A and 90 % B; 26–29 min, 10 % A and 90 % B; 29–30 min, 95 % A and 5 % B and 30–35 min, 95 % A and 5 % B. The extraction process was carried out using solvents primarily targeting polar compounds, which are particularly relevant because many polar metabolites such as phenolic acids and flavonoid glycosides are known to interfere with quorum sensing in *Pseudomonas* by disrupting AHL-mediated communication, thereby justifying the chromatographic gradient optimized for their early resolution and the limited consideration given to apolar compounds or contaminants eluting at high acetonitrile concentrations. Mass spectrometry data were acquired in both polarities (capillary voltages of +3 kV and −2.5 kV) using an ESI-QToF setup, with *m/z* ranging from 50 to 1200 and scan time of 0.1 s. MSMS analysis were conducted based on an ion intensity threshold of 1000,000·s⁻¹ with collision energies ranging from 10 to 40 V, and a UV analysis was performed in a detection range from 220 to 800 nm and 10 spectra·s⁻¹. These parameters provided high-resolution separation and detection, enabling detailed
identification and characterization of compounds in the STF extract. After MS/MS acquisition, the resulting ion spectra were processed using the identification software Sirius4© (Dührkop et al., 2019). Molecular formula determination was first performed in Sirius, allowing the elements C, H, O, N, P, and S, with an MS² mass accuracy set to 20 ppm. The 'Predict FPs' module was then applied to infer molecular fingerprints, while 'Search DBs' compared these predicted fingerprints with reference MS/MS data from natural compound databases such as the Human Metabolome Database (HMDB), Chemical Entities of Biological Interest (ChEBI), COleCtion of Open NatUral producTs (COCONUT), and the Global Natural Products Social Molecular Networking (GNPS) repository, among others. Finally, the 'CANOPUS' module was used to predict compound classes. Molecular formulas were retained when Sirius scores exceeded 75 %, and only compound structures showing a minimum similarity of 60 % were considered for annotation.

## 2.7. Statistical analysis

Data analysis was performed using GraphPad Prism version 10.2.2. Statistical significance was assessed using Kruskal-Wallis test followed by Dunn's multiple comparaison and Wilcoxon test, as specified in the figure legends.

# 3. Results

## 3.1. Reduction of pyocyanin production

Pyocyanin is a blue phenazine pigment and a key virulence factor of *P. aeruginosa*, responsible for the generation and accumulation of reactive oxygen species (ROS), leading to oxidative stress in both prokaryotic and eukaryotic cells (*Gonçalves and Vasconcelos, 2021*; Lau et al., 2004). This virulence factor enhances *P. aeruginosa*'s competitive advantage over other microorganisms in diverse environments (*Gonçalves and Vasconcelos, 2021*) and induces apoptosis and senescence in eukaryotic cells (Allen et al., 2005; Managò et al., 2015). Due to its significant role in pathogenicity, pyocyanin is widely recognized as a biomarker of *P. aeruginosa* infections. In this study, we evaluated the effect of several Brazilian plant extracts on pyocyanin production by planktonic cells of *P. aeruginosa*. As shown in Fig. 1, eight extracts significantly reduced pyocyanin levels, with inhibition ranging from

15 % (PC) to 70 % (STF) at 64 µg.mL⁻¹, selected as a discriminating concentration. Notably, STF exhibited the most pronounced effect on pyocyanin production. Based on these findings, STF was selected for further investigation in subsequent assays.

## 3.2. Effect of the STF extract on Bacterial Growth

The impact of STF on *P. aeruginosa* H103 growth was monitored at 37 °C for 24 h, showing that the leaf extract at 64 µg.mL⁻¹ had no impact on planktonic cell growth of H103 strain as compared to the untreated culture (H103 strain grown in presence of 1 % v/v DMSO) (Fig. 2). This result was particularly interesting, as the study aimed to identify extracts that modulate quorum sensing without affecting bacterial viability, thus ensuring that the observed effects on virulence-related phenotypes are not a consequence of impaired growth.

## 3.3. Effect of the STF extract on Quorum Sensing Modulation

We next sought to assess whether the inhibition of pyocyanin production was directly due to the activity of STF on the Pqs QS system that is known to tightly regulate pyocyanin biosynthesis. *P. aeruginosa* H103 cultures were exposed to STF at 64 µg.mL⁻¹, and then HAQs molecules were extracted twice by ethyl acetate. The production of HAQs was determined using a PAO1 *pqsA* CTX-*pqsA::lux* biosensor strain, which does not produce HAQs molecules but shows proportional quantitative response to exogenous HAQs. The H103 cultures treated with STF showed significant reduced HAQs molecules production, demonstrating that STF exhibited its anti-pyocyanin production effect at least through an anti-QS activity (Fig. 3.).

## 3.4. Composition of Spondias tuberosa Arruda ethanolic leaf extract and correlation with its anti-virulence activity

The UHPLC-HRMS analysis of the STF extract revealed a complex mixture of 77 compounds across 30 chemical subclasses. Four major chemical families, flavonoids, carbohydrates, carboxylic acids, and amino acid derivatives, accounted for 50.6 % of the extract's composition (Fig. 5., Tables 2. and 3.). Flavonoids, the most abundant class (14.3 %), included glycosides such as rutin (C27H30O16), nicotifolin (C27H30O15), and cinaroside (C21H20O11), as well as O-methylated

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Inhibitory effect of plant extracts on pyocyanin production in *P. aeruginosa* H103. Plant extracts and fractions were initially tested at concentrations ranging from 8 to 256 µg.mL⁻¹. The figure shows the effect of a selected concentration (64 µg.mL⁻¹), chosen for its discriminating potential, on pyocyanin production by *P. aeruginosa* H103 after 24 h of incubation at 37 °C. Pyocyanin production was quantified spectrophotometrically and expressed as a percentage relative to the untreated control. Statistical significance was determined using Kruskal-Wallis test followed by Dunn's multiple comparison test. NS indicates non-significant differences; **p < 0.01, ***p < 0.001, ****p < 0.0001. Data represent the mean ± SEM of three independent experiments.</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Effect of STF leaf extract on the planktonic growth of *P. aeruginosa* H103. Growth kinetics of *P. aeruginosa* H103 in LB medium at 37 °C in the presence of 1 % DMSO (H103, black curve) or STF leaf extract at 64 µg·mL⁻¹ (grey curve). A~580nm~ was measured every 30 min over 24 h. Data are expressed as the mean ± SEM of the neperian logarithm of absorbance values from four independent experiments. Error bars were very small, indicating a high reproducibility of the assay.</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>**Fig. 3.** STF leaf extract inhibits Pqs quorum sensing system activity in *P. aeruginosa* H103. Bioluminescence kinetics (left) and corresponding maximal values (right) of the *P. aeruginosa* *pqsA::lux* reporter strain exposed to 1 % DMSO (negative control, black curve and bars) or 64 µg·mL⁻¹ of the ethanolic leaf extract of *Spondias tuberosa* (STF). Luminescence was monitored over time, and peak values were extracted for comparative analysis. Data represent the mean ± SEM of four independent experiments. Statistical significance was assessed using the Wilcoxon signed-rank test; *p* < 0.05 was considered significant (*).</figcaption></figure>

derivatives such as sakuranetin (C₁₆H₁₄O₅) and naringenin dimethyl ether (C₁₇H₁₈O₅). Carbohydrates and carbohydrate conjugates (14.3 %) included cerelose (C₆H₁₂O₆) and various complex glycosides. Carboxylic acids (13 %) comprised mono-, di-, and tricarboxylic acid derivatives, notably hibiscus acid methyl ester (C₇H₈O₇) and hydroxycitric acid dimethyl ester (C₈H₁₂O₈). Amino acids, peptides, and analogues represented 9.1 %, including methyl pyroglutamate and ethyl pyroglutamate. Other classes included hydroxycinnamic acids (e.g., ethyl caffeate), fatty acids (e.g., linoleic acid and 10,15-octadecadienoic acid), benzenediols (e.g., loliolide), glycerophosphocholines (e.g., lysolecithins), and pyrididoindoles (e.g., harman-3-carboxylic acid). (Figs. 4, 5 and Tables 2 and 3).

## 4. Discussion

This study provides compelling evidence that the ethanolic leaf extract of *Spondias tuberosa* Arruda (STF) exhibits strong anti-virulence activity against planktonic *Pseudomonas aeruginosa* by specifically targeting quorum sensing (QS)-regulated pathways controlling pyocyanin production. In particular, STF was shown to significantly inhibit the Pqs system, a key regulator of virulence in *P. aeruginosa* through the production of HAQs molecules such as HHQ and PQS, which control the expression of key factors including pyocyanin (Diggle et al., 2011; Lee and Zhang, 2015). The observed reduction in HAQs was confirmed through bioluminescence assays using a *pqsA::lux* biosensor strain and was not accompanied by any adverse effects on bacterial growth, supporting a non-bactericidal and therefore resistance-limiting mode of action. Phytochemical analysis of STF revealed a chemically diverse
<caption>**Table 2**</caption>

UPLC-HRMS data obtained after analysis of the STF extract, listing compounds identified in positive ionization mode. The table includes retention time (RT), mass-to-charge ratio (m/z), molecular formula, adduct type, identification score, proposed molecular structure, similarity index, and chemical subclass. The identified metabolites belong to diverse biochemical classes, including carbohydrates, amino acids, flavonoids, fatty acids, and phospholipids, offering insights into the chemical composition and potential bioactivity of the extract.

<table><thead><tr><th>N°</th><th>m/z</th><th>RT (min)</th><th>Mass formula</th><th>Adduct</th><th>Score</th><th>Proposed structure</th><th>Similarity</th><th>Subclass</th></tr></thead><tbody><tr><td>2</td><td>203.0536</td><td>0.44</td><td>C<sub>6</sub>H<sub>12</sub>O<sub>6</sub></td><td>[M+Na]<sup>+</sup></td><td>100 %</td><td>Cerelose</td><td>94.55 %</td><td>Carbohydrates and carbohydrate conjugates</td></tr><tr><td>3</td><td>134.1185</td><td>0.48</td><td>C<sub>6</sub>H<sub>15</sub>NO<sub>2</sub></td><td>[M+H]<sup>+</sup></td><td>100 %</td><td>2-((2-Methoxyethyl)(methyl)amino)ethanol</td><td>81.48 %</td><td>Amines</td></tr><tr><td>7</td><td>300.1446</td><td>0.83</td><td>C<sub>14</sub>H<sub>21</sub>NO<sub>6</sub></td><td>[M+H]<sup>+</sup></td><td>96.20 %</td><td>(2 R,3S,4 R,5 R,6S)-2-(hydroxymethyl)-6-[[[(4-hydroxyphenyl)methylamino]methyl] oxane-3,4,5-triol</td><td>73.29 %</td><td>Carbohydrates and carbohydrate conjugates</td></tr><tr><td>10</td><td>144.0664</td><td>1.23</td><td>C<sub>6</sub>H<sub>9</sub>NO<sub>3</sub></td><td>[M+H]<sup>+</sup></td><td>100 %</td><td>Méthyl L-pyroglutamate</td><td>98.95 %</td><td>Amino acids, peptides and analogues</td></tr><tr><td>15</td><td>158.0836</td><td>3.5</td><td>C<sub>7</sub>H<sub>11</sub>NO<sub>3</sub></td><td>[M+H]<sup>+</sup></td><td>100 %</td><td>Ethyl pyroglutamate</td><td>98.10 %</td><td>Amino acids, peptides and analogues</td></tr><tr><td>17</td><td>287.0742</td><td>5.04</td><td>C<sub>10</sub>H<sub>16</sub>O<sub>8</sub></td><td>[M+Na]<sup>+</sup></td><td>100 %</td><td>[[2 R,3 R,4S,5 R)-5-acetyloxy-2,3,4-trihydroxy-6-oxohexyl] acetate</td><td>72.67 %</td><td>Carbohydrates and carbohydrate conjugates</td></tr><tr><td>27</td><td>227.0819</td><td>9.05</td><td>C<sub>13</sub>H<sub>10</sub>N<sub>2</sub>O<sub>2</sub></td><td>[M+H]<sup>+</sup></td><td>100 %</td><td>Harman-3-Carboxylic Acid</td><td>90.38 %</td><td>Harmana alkaloids</td></tr><tr><td>29</td><td>197.1174</td><td>12.95</td><td>C<sub>11</sub>H<sub>16</sub>O<sub>3</sub></td><td>[M+H]<sup>+</sup></td><td>100 %</td><td>Loliolide</td><td>67 %</td><td>Benzenediols</td></tr><tr><td>32</td><td>611.1609</td><td>14.85</td><td>C<sub>27</sub>H<sub>30</sub>O<sub>16</sub></td><td>[M+H]<sup>+</sup></td><td>100 %</td><td>Rutine</td><td>99.72 %</td><td>Flavonoid glycosides</td></tr><tr><td>34</td><td>315.1058</td><td>16.54</td><td>C<sub>10</sub>H<sub>14</sub>N<sub>6</sub>O<sub>6</sub></td><td>[M+H]<sup>+</sup></td><td>100 %</td><td>2-Amino-8-(hydroxyamino)-9-pentofuranosyl-3,9-dihydro-6h-purin-6-one</td><td>72.34 %</td><td>Amino acids, peptides and analogues</td></tr><tr><td>35</td><td>595.1656</td><td>16.77</td><td>C<sub>27</sub>H<sub>30</sub>O<sub>15</sub></td><td>[M+H]<sup>+</sup></td><td>97.69 %</td><td>Nicotifolin</td><td>97.41 %</td><td>Flavonoid glycosides</td></tr><tr><td>40</td><td>449.1079</td><td>17.48</td><td>C<sub>21</sub>H<sub>20</sub>O<sub>11</sub></td><td>[M+H]<sup>+</sup></td><td>100 %</td><td>Cinaroside</td><td>96.98 %</td><td>Flavonoid glycosides</td></tr><tr><td>43</td><td>299.1108</td><td>20.39</td><td>C<sub>12</sub>H<sub>20</sub>O<sub>7</sub></td><td>[M+Na]<sup>+</sup></td><td>100 %</td><td>Triéthyl citrate</td><td>100 %</td><td>Fatty acid esters</td></tr><tr><td>48</td><td>241.0979</td><td>23.82</td><td>C<sub>14</sub>H<sub>12</sub>N<sub>2</sub>O<sub>2</sub></td><td>[M+H]<sup>+</sup></td><td>100 %</td><td>9H-pyrido[3,4-b]indole-3-carboxylic acid ethyl ester</td><td>100 %</td><td>Pyridoindoles</td></tr><tr><td>51</td><td>202.087</td><td>25</td><td>C<sub>12</sub>H<sub>11</sub>NO<sub>2</sub></td><td>[M+H]<sup>+</sup></td><td>100 %</td><td>Ethyl quinoline-3-carboxylate</td><td>80.25 %</td><td>Fatty acid esters</td></tr><tr><td>54</td><td>343.2968</td><td>25.65</td><td>C<sub>19</sub>H<sub>38</sub>N<sub>2</sub>O<sub>3</sub></td><td>[M+H]<sup>+</sup></td><td>100 %</td><td>Cocamidopropyl betaine</td><td>98.63 %</td><td>Amino acids, peptides and analogues</td></tr><tr><td>55</td><td>287.0913</td><td>25.84</td><td>C<sub>16</sub>H<sub>14</sub>O<sub>5</sub></td><td>[M+H]<sup>+</sup></td><td>100 %</td><td>Sakuranetin</td><td>97.66 %</td><td>O-methylated flavonoids</td></tr><tr><td>59</td><td>307.2246</td><td>25.8</td><td>C<sub>17</sub>H<sub>32</sub>O<sub>3</sub></td><td>[M+Na]<sup>+</sup></td><td>99.97 %</td><td>7-Oxoheptadecanoic acid/9-keto margaric acid</td><td>68.75 %</td><td>Fatty acids and conjugates</td></tr><tr><td>63</td><td>699.3566</td><td>26.16</td><td>C<sub>33</sub>H<sub>56</sub>O<sub>14</sub></td><td>[M+Na]<sup>+</sup></td><td>98.74 %</td><td>(2S)-3-[[6-O-β-L-glucopyranosyl-β-L-galactopyranosyl)oxy]-2-hydroxypropyl (9Z,12Z,15Z)-octadeca-9,12,15-trienoate</td><td>97.24 %</td><td>Glycosylglycerols</td></tr><tr><td>64</td><td>518.3235</td><td>26.23</td><td>C<sub>26</sub>H<sub>48</sub>NO<sub>7</sub>P</td><td>[M+H]<sup>+</sup></td><td>100 %</td><td>1-alpha-Linolenoyl-glycero-3-phosphocholine (LysoPC(18:3(9Z,12Z,15Z)))</td><td>98.98 %</td><td>Glycerophosphocholines</td></tr><tr><td>65</td><td>476.2785</td><td>26.31</td><td>C<sub>23</sub>H<sub>42</sub>NO<sub>7</sub>P</td><td>[M+H]<sup>+</sup></td><td>100 %</td><td>PE(18:3(6Z,9Z,12Z)/0:0)/PE(18:3(9Z,12Z,15Z)/0:0)/PE(0:0:18:3(6Z,9Z,12Z))/PE(0:0:18:3(9Z,12Z,15Z))</td><td>100 %</td><td>Amino acids, peptides and analogues</td></tr><tr><td>66</td><td>312.1132</td><td>26.53</td><td>C<sub>20</sub>H<sub>13</sub>N<sub>3</sub>O</td><td>[M+H]<sup>+</sup></td><td>100 %</td><td>Pityriacitrin</td><td>80.00 %</td><td>Pyridoindoles</td></tr><tr><td>67</td><td>277.2162</td><td>26.53</td><td>C<sub>18</sub>H<sub>28</sub>O<sub>2</sub></td><td>[M+H]<sup>+</sup></td><td>100 %</td><td>10,15-Octadecadiynoic acid</td><td>71.43 %</td><td>Lineolic acids and derivatives</td></tr><tr><td>68</td><td>478.3376</td><td>26.53</td><td>C<sub>24</sub>H<sub>47</sub>NO<sub>8</sub></td><td>[M+H]<sup>+</sup></td><td>100 %</td><td>1-Glucopyranosyl-2-amino-3,17-dihydroxyoct-4-ène</td><td>87.10 %</td><td>Glycosphingolipids</td></tr><tr><td>69</td><td>301.1075</td><td>26.61</td><td>C<sub>17</sub>H<sub>16</sub>O<sub>5</sub></td><td>[M+H]<sup>+</sup></td><td>99.10 %</td><td>Naringenin 7,4'-Dimethyl Ether</td><td>100 %</td><td>O-methylated flavonoids</td></tr><tr><td>70</td><td>520.3417</td><td>26.74</td><td>C<sub>26</sub>H<sub>50</sub>NO<sub>7</sub>P</td><td>[M+H]<sup>+</sup></td><td>100 %</td><td>2-linoleoyl-sn-glycero-3-phosphocholine (LysoPC (18:2(9Z,12Z)))</td><td>93.07 %</td><td>Glycerophosphocholines</td></tr><tr><td>71</td><td>496.3392</td><td>26.79</td><td>C<sub>31</sub>H<sub>58</sub>O<sub>14</sub></td><td>[M+H]<sup>+</sup></td><td>100 %</td><td>1-16:0-lysoPC</td><td>100 %</td><td>Glycerophosphocholines</td></tr><tr><td>72</td><td>522.3547</td><td>27.05</td><td>C<sub>26</sub>H<sub>52</sub>NO<sub>7</sub>P</td><td>[M+H]<sup>+</sup></td><td>100 %</td><td>Lysolecithin</td><td>100 %</td><td>Glycerophosphocholines</td></tr><tr><td>73</td><td>291.233</td><td>27.3</td><td>C<sub>19</sub>H<sub>30</sub>O<sub>2</sub></td><td>[M+H]<sup>+</sup></td><td>100 %</td><td>cis-Parinaric acid methyl ester</td><td>88.75 %</td><td>Lineolic acids and derivatives</td></tr><tr><td>74</td><td>321.24</td><td>27.35</td><td>C<sub>18</sub>H<sub>34</sub>O<sub>3</sub></td><td>[M+Na]<sup>+</sup></td><td>95.22 %</td><td>12-Hydroxyoctadecanoic acid</td><td>88.81 %</td><td>Fatty acids and conjugates</td></tr></tbody></table>

extract, comprising 77 metabolites across 30 subclasses. This compositional profile is consistent with prior analyses of *S. tuberosa* leaves, which likewise reported phenolic/flavonoid predominance and specifically identified rutin, quercetin, and ellagic acid as major constituents; those studies also linked these metabolites to antioxidant and antimicrobial effects (da Silva et al., 2012; Thassya Lucas Dos Santos et al., 2019). Moreover, the limited intrinsic antifungal activity but potentiation of fluconazole and inhibition of *Candida* morphogenesis seen with *S. tuberosa* leaf extracts (Thassya Lucas Dos Santos et al., 2019) align with our non-bactericidal, anti-virulence profile against *P. aeruginosa*, reinforcing the relevance of phenolic-rich fractions as modulators of pathogen fitness rather than direct killers. Among these compounds, flavonoids were prominent, including rutin, a compound previously reported to interfere with QS and biofilm formation in *Escherichia coli* (Peng et al., 2018). However, rutin at 64 µg·mL⁻¹ did not significantly affect pyocyanin production by *P. aeruginosa* in our model (data not shown), suggesting that the anti-virulence activity of the STF extract is likely attributable to other constituents or to synergistic interactions among multiple compounds, rather than to rutin alone. In contrast,

related glycosides such as nicotifolin and cinaroside have not yet been studied in this context, making them attractive candidates for further exploration. The presence of O-methylated flavonoids such as sakuranetin and naringenin dimethyl ether, whose role in bacterial communication remains unknown, highlights the presence of structurally diverse compounds with unexplored bioactivity deserving further investigation. Other compound classes likely contributed to the observed anti-QS effect. Carbohydrates and their conjugates (14.3 %), including cerelose and complex glycosides, may mimic or disrupt sugar-based autoinducer signaling pathways. Carboxylic acid derivatives (13 %) such as hibiscus acid methyl ester and hydroxycitric acid dimethyl ester, known to stimulate metabolism in fungi (Sumang et al., 2024), could also interfere with bacterial communication or metabolism. Amino acid derivatives like methyl and ethyl pyroglutamate may disrupt protein synthesis or regulatory proteins involved in QS. Hydroxycinnamic acids, particularly ethyl caffeate, are recognized for antioxidant activity, and similar compounds have also been linked to QS inhibition (Shariati et al., 2024). Fatty acids such as linoleic acid and 10,15-octadecadienoic acid may alter bacterial membrane integrity or
<caption>**Table 3**</caption>

UPLC-HRMS data obtained after analysis of the STF extract, listing compounds identified in negative ionization mode. The table includes retention time (RT), mass-to-charge ratio (m/z), molecular formula, adduct type, identification score, proposed molecular structure, similarity index, and chemical subclass. The identified metabolites belong to diverse biochemical classes, including carbohydrates, amino acids, flavonoids, fatty acids, and phospholipids, offering insights into the chemical composition and potential bioactivity of the extract.

<table><thead><tr><th>N°</th><th>m/z</th><th>RT (min)</th><th>Mass formula</th><th>Adduct</th><th>Score</th><th>Proposed structure</th><th>Similarity</th><th>Subclass</th></tr></thead><tbody><tr><td>1</td><td>317.0551</td><td>0.4</td><td>C<sub>9</sub>H<sub>18</sub>O<sub>10</sub>S</td><td>[M-H]<sup>+</sup></td><td>99.94 %</td><td>6-Sulfo-quinovosyl-Glycerin</td><td>73.71 %</td><td>Carbohydrates and carbohydrate conjugates</td></tr><tr><td>4</td><td>203.0191</td><td>0.52</td><td>C<sub>7</sub>H<sub>8</sub>O<sub>7</sub></td><td>[M-H]<sup>+</sup></td><td>100 %</td><td>Hibiscus acid 1-methylester</td><td>76.73 %</td><td>Tricarboxylic acids and derivatives</td></tr><tr><td>5</td><td>189.0037</td><td>0.65</td><td>C<sub>6</sub>H<sub>6</sub>O<sub>7</sub></td><td>[M-H]<sup>+</sup></td><td>99.99 %</td><td>Hibiscus lactone</td><td>99.44 %</td><td>Tricarboxylic acids and derivatives</td></tr><tr><td>6</td><td>235.0451</td><td>0.71</td><td>C<sub>8</sub>H<sub>12</sub>O<sub>8</sub></td><td>[M-H]<sup>+</sup></td><td>99.99 %</td><td>2-(2-Ethoxy-2-oxoethyl)-2-hydroperoxybutanedioic acid</td><td>70.68 %</td><td>Tricarboxylic acids and derivatives</td></tr><tr><td>8</td><td>315.0727</td><td>0.86</td><td>C<sub>13</sub>H<sub>16</sub>O<sub>9</sub></td><td>[M-H]<sup>+</sup></td><td>89.13 %</td><td>2,5-dihydroxybenzoic acid 5-O-β-D-glucoside</td><td>98.91 %</td><td>Carbohydrates and carbohydrate conjugates</td></tr><tr><td>9</td><td>235.0457</td><td>0.86</td><td>C<sub>8</sub>H<sub>12</sub>O<sub>8</sub></td><td>[M-H]<sup>+</sup></td><td>98.36 %</td><td>(2 R,3S)-2-Hydroxycitric acid 3,5-dimethyl ester</td><td>71.81 %</td><td>Dicarboxylic acids and derivatives</td></tr><tr><td>11</td><td>323.1335</td><td>1.52</td><td>C<sub>13</sub>H<sub>24</sub>O<sub>9</sub></td><td>[M-H]<sup>+</sup></td><td>100 %</td><td>2,2-Dimethyl-4-beta-[(beta-D-glucopyranosyloxy) methyl]-1,3-dioxolane-5-alpha (ou beta)-methanol</td><td>79.43 %</td><td>Fatty acyl glycosides</td></tr><tr><td>12</td><td>249.0605</td><td>1.77</td><td>C<sub>9</sub>H<sub>14</sub>O<sub>8</sub></td><td>[M-H]<sup>+</sup></td><td>100 %</td><td>2-Hydroxy-2-[2-(3-hydroxypropoxy)-2-oxoethyl] butanedioic acid</td><td>64.86 %</td><td>Carboxylic acids derivatives</td></tr><tr><td>13</td><td>249.061</td><td>2.07</td><td>C<sub>9</sub>H<sub>14</sub>O<sub>8</sub></td><td>[M-H]<sup>+</sup></td><td>100 %</td><td>erythro-Pentaric acid, 2-deoxy-3-C- (methoxycarbonyl)-, dimethyl ester</td><td>77.65 %</td><td>Tricarboxylic acids and derivatives</td></tr><tr><td>14</td><td>249.061</td><td>2.14</td><td>C<sub>9</sub>H<sub>14</sub>O<sub>8</sub></td><td>[M-H]<sup>+</sup></td><td>100 %</td><td>2-Hydroxy-2-[2-(2-methoxyethoxy)-2-oxoethyl] butanedioic acid</td><td>72.97 %</td><td>Tricarboxylic acids and derivatives</td></tr><tr><td>16</td><td>327.1081</td><td>4.63</td><td>C<sub>15</sub>H<sub>20</sub>O<sub>8</sub></td><td>[M-H]<sup>+</sup></td><td>98.84 %</td><td>3-(4-hydroxyphenyl)-3-oxopropyl β-D- glucopyranoside</td><td>97.29 %</td><td>Carbonyl compounds</td></tr><tr><td>18</td><td>263.0768</td><td>5.7</td><td>C<sub>10</sub>H<sub>16</sub>O<sub>8</sub></td><td>[M-H]<sup>+</sup></td><td>99.92 %</td><td>2-Hydroxy-2-[2-(4-hydroxybutoxy)-2-oxoethyl] butanedioic acid</td><td>64.14 %</td><td>Carboxylic acids derivatives</td></tr><tr><td>19</td><td>263.0771</td><td>5.8</td><td>C<sub>10</sub>H<sub>16</sub>O<sub>8</sub></td><td>[M-H]<sup>+</sup></td><td>100 %</td><td>2-Hydroxy-2-[2-(4-hydroxybutoxy)-2-oxoethyl] butanedioic acid</td><td>68.91 %</td><td>Carboxylic acids derivatives</td></tr><tr><td>20</td><td>263.0764</td><td>5.86</td><td>C<sub>10</sub>H<sub>16</sub>O<sub>8</sub></td><td>[M-H]<sup>+</sup></td><td>100 %</td><td>2-Hydroxy-2-[2-(4-hydroxybutoxy)-2-oxoethyl] butanedioic acid</td><td>70.97 %</td><td>Carboxylic acids derivatives</td></tr><tr><td>21</td><td>133.0285</td><td>6.1</td><td>C<sub>8</sub>H<sub>6</sub>O<sub>2</sub></td><td>[M-H]<sup>+</sup></td><td>100 %</td><td>4-éthynolbenzenol</td><td>72.57 %</td><td>1-hydroxy-2-unsubstituted benzenoids</td></tr><tr><td>22</td><td>359.0981</td><td>6.61</td><td>C<sub>15</sub>H<sub>20</sub>O<sub>10</sub></td><td>[M-H]<sup>+</sup></td><td>94.31 %</td><td>[3,4,5-Trihydroxy-6-(hydroxymethyl)oxan-2-yl] 3- hydroxy-4,5-dimethoxybenzoate</td><td>99.01 %</td><td>Hydrolysable tannins</td></tr><tr><td>23</td><td>325.0925</td><td>6.69</td><td>C<sub>15</sub>H<sub>18</sub>O<sub>8</sub></td><td>[M-H]<sup>+</sup></td><td>99.93 %</td><td>1-p-Coumarylglucose</td><td>94.83 %</td><td>Hydroxycinnamic acid esters</td></tr><tr><td>24</td><td>355.1029</td><td>8.08</td><td>C<sub>16</sub>H<sub>20</sub>O<sub>9</sub></td><td>[M-H]<sup>+</sup></td><td>99.99 %</td><td>1-O-feruloyl-β-D-glucose</td><td>96.67 %</td><td>Hydroxycinnamic acids and derivatives</td></tr><tr><td>25</td><td>447.1511</td><td>8.54</td><td>C<sub>19</sub>H<sub>28</sub>O<sub>12</sub></td><td>[M-H]<sup>+</sup></td><td>77.51 %</td><td>(2 R,3S,4 R,5S,6 R)-3,4,5-trihydroxy-6- (hydroxymethyl)oxan-2-yl]oxymethoxy] oxane-3,4,5-triol</td><td>76.02 %</td><td>Carbohydrates and carbohydrate conjugates</td></tr><tr><td>26</td><td>447.15</td><td>8.95</td><td>C<sub>19</sub>H<sub>28</sub>O<sub>12</sub></td><td>[M-H]<sup>+</sup></td><td>98.40 %</td><td>2-[hydroxy(phenyl)methoxy]-6-[(3,4,5- trihydroxy-6-(hydroxymethyl)oxan-2-yl)oxy] methyl]oxane-3,4,5-triol</td><td>83.40 %</td><td>Carbohydrates and carbohydrate conjugates</td></tr><tr><td>28</td><td>197.045</td><td>9.64</td><td>C<sub>9</sub>H<sub>10</sub>O<sub>5</sub></td><td>[M-H]<sup>+</sup></td><td>100 %</td><td>Phyllemblin</td><td>100 %</td><td>Benzoic acids and derivatives</td></tr><tr><td>30</td><td>439.182</td><td>13.66</td><td>C<sub>18</sub>H<sub>32</sub>O<sub>12</sub></td><td>[M-H]<sup>+</sup></td><td>90.45 %</td><td>6-O-Hexanoyl-alpha, alpha-D-trehalose</td><td>61.94 %</td><td>Fatty acyls</td></tr><tr><td>31</td><td>433.2068</td><td>13.74</td><td>C<sub>20</sub>H<sub>34</sub>O<sub>10</sub></td><td>[M-H]<sup>+</sup></td><td>99.91 %</td><td>Famille des alkylglycosides</td><td>76.51 %</td><td>Fatty acyls</td></tr><tr><td>32</td><td>609.1445</td><td>14.98</td><td>C<sub>27</sub>H<sub>30</sub>O<sub>16</sub></td><td>[M-H]<sup>+</sup></td><td>92.76 %</td><td>Rutine</td><td>100 %</td><td>Flavonoid glycosides</td></tr><tr><td>33</td><td>413.1808</td><td>16.03</td><td>C<sub>20</sub>H<sub>30</sub>O<sub>9</sub></td><td>[M-H]<sup>+</sup></td><td>99.86 %</td><td>Ptesculentoside</td><td>73.95 %</td><td>Sesquiterpenoids</td></tr><tr><td>35</td><td>593.151</td><td>16.74</td><td>C<sub>27</sub>H<sub>30</sub>O<sub>15</sub></td><td>[M-H]<sup>+</sup></td><td>83.98 %</td><td>Nicotifolin</td><td>95.79 %</td><td>Flavonoid glycosides</td></tr><tr><td>36</td><td>593.151</td><td>16.81</td><td>C<sub>27</sub>H<sub>30</sub>O<sub>15</sub></td><td>[M-H]<sup>+</sup></td><td>89.22 %</td><td>Kaempferol 3-neohesperidoside</td><td>90.44 %</td><td>Flavonoid glycosides</td></tr><tr><td>37</td><td>447.0922</td><td>17.04</td><td>C<sub>22</sub>H<sub>16</sub>N<sub>4</sub>O<sub>7</sub></td><td>[M-H]<sup>+</sup></td><td>77.53 %</td><td>Trifolin</td><td>100 %</td><td>Flavonoid glycosides</td></tr><tr><td>38</td><td>503.1769</td><td>17.11</td><td>C<sub>22</sub>H<sub>32</sub>O<sub>13</sub></td><td>[M-H]<sup>+</sup></td><td>100 %</td><td>Coniferinoside</td><td>76.71 %</td><td>Carbohydrates and carbohydrate conjugates</td></tr><tr><td>39</td><td>417.2121</td><td>17.24</td><td>C<sub>20</sub>H<sub>34</sub>O<sub>9</sub></td><td>[M-H]<sup>+</sup></td><td>100 %</td><td>(5 R,8Z,11Z)-5-(beta-D-Glucopyranosyloxy)-6- hydroxy-8,11-tetradecadienoic acid</td><td>60.93 %</td><td>Carbohydrates and carbohydrate conjugates</td></tr><tr><td>41</td><td>615.0981</td><td>17.67</td><td>C<sub>28</sub>H<sub>24</sub>O<sub>16</sub></td><td>[M-H]<sup>+</sup></td><td>100 %</td><td>Quercétin 3-(2""galloylglucoside)</td><td>100 %</td><td>Flavonoid glycosides</td></tr><tr><td>42</td><td>503.1763</td><td>17.73</td><td>C<sub>22</sub>H<sub>32</sub>O<sub>13</sub></td><td>[M-H]<sup>+</sup></td><td>99.89 %</td><td>Coniferinoside</td><td>76.25 %</td><td>Carbohydrates and carbohydrate conjugates</td></tr><tr><td>44</td><td>207.0656</td><td>21.07</td><td>C<sub>11</sub>H<sub>12</sub>O<sub>4</sub></td><td>[M-H]<sup>+</sup></td><td>100 %</td><td>Ethyl caffeate</td><td>97.95 %</td><td>Hydroxycinnamic acids and derivatives</td></tr><tr><td>45</td><td>355.1182</td><td>22.08</td><td>C<sub>20</sub>H<sub>20</sub>O<sub>6</sub></td><td>[M-H]<sup>+</sup></td><td>99.28 %</td><td>Dimethyl 2,4-bis(4-hydroxyphenyl)cyclobutane-1,3- dicarboxylate</td><td>80.54 %</td><td>Lignans, neolignans ans related compounds</td></tr><tr><td>46</td><td>455.1914</td><td>22.94</td><td>C<sub>22</sub>H<sub>32</sub>O<sub>10</sub></td><td>[M-H]<sup>+</sup></td><td>98.61 %</td><td>Glycoconjugué</td><td>66.25 %</td><td>Carbohydrates and carbohydrate conjugates</td></tr><tr><td>47</td><td>191.0708</td><td>23.62</td><td>C<sub>11</sub>H<sub>12</sub>O<sub>3</sub></td><td>[M-H]<sup>+</sup></td><td>99.55 %</td><td>p-coumaric acid ethyl ester</td><td>100 %</td><td>Hydroxycinnamic acids and derivatives</td></tr><tr><td>49</td><td>242.1755</td><td>24.28</td><td>C<sub>13</sub>H<sub>25</sub>NO<sub>3</sub></td><td>[M-H]<sup>+</sup></td><td>90.18 %</td><td>13-Amino-13-oxotridecanoic acid</td><td>68.94 %</td><td>Amino acids, peptides and analogues</td></tr></tbody></table>

(continued on next page)
<table><caption>**Table 3 (continued)**</caption><thead><tr><th>N°</th><th>m/z</th><th>RT (min)</th><th>Mass formula</th><th>Adduct</th><th>Score</th><th>Proposed structure</th><th>Similarity</th><th>Subclass</th></tr></thead><tbody><tr><td>50</td><td>188.0709</td><td>24.68</td><td>C<sub>11</sub>H<sub>11</sub>NO<sub>2</sub></td><td>[M-H]<sup>-</sup></td><td>100 %</td><td>Lindole-3-éthyl formiate</td><td>97.03 %</td><td>Indolecarboxylic acids and derivatives</td></tr><tr><td>52</td><td>399.1446</td><td>25.21</td><td>C<sub>22</sub>H<sub>24</sub>O<sub>7</sub></td><td>[M-H]<sup>-</sup></td><td>100 %</td><td>(10-acetyloxy-3,8,8-trimethyl-2-oxo-9,10-dihydropyrano[2,3-f]chromen-9-yl) (Z)-2-methylbut-2-enoate</td><td>63.35 %</td><td>1-hydroxy-2-unsubstituted benzenoids</td></tr><tr><td>53</td><td>423.1302</td><td>25.53</td><td>C<sub>20</sub>H<sub>24</sub>O<sub>10</sub></td><td>[M-H]<sup>-</sup></td><td>100 %</td><td>{3,4,5-trihydroxy-6-[(3-phenylprop-2-enoyl)oxy]oxan-2-yl}methyl 3,4-dihydroxy-2-methylidenebutanoate</td><td>70.92 %</td><td>Hydroxycinnamic acids and derivatives</td></tr><tr><td>55</td><td>285.0766</td><td>25.67</td><td>C<sub>16</sub>H<sub>14</sub>O<sub>5</sub></td><td>[M-H]<sup>-</sup></td><td>100 %</td><td>Sakuranetin</td><td>97.27 %</td><td>O-methylated flavonoids</td></tr><tr><td>56</td><td>474.2622</td><td>25.76</td><td>C<sub>23</sub>H<sub>42</sub>NO<sub>7</sub>P</td><td>[M-H]<sup>-</sup></td><td>84.13 %</td><td>1-18:3:lysoPE</td><td>92.22 %</td><td>Amino acids, peptides and analogues</td></tr><tr><td>58</td><td>283.1372</td><td>25.76</td><td>C<sub>15</sub>H<sub>24</sub>O<sub>3</sub>S</td><td>[M-H]<sup>-</sup></td><td>100 %</td><td>Nonylbenzenesulfonic acid</td><td>87.82 %</td><td>Benzenesulfonic acids and derivatives</td></tr><tr><td>57</td><td>593.2746</td><td>25.76</td><td>C<sub>27</sub>H<sub>47</sub>O<sub>12</sub>P</td><td>[M-H]<sup>-</sup></td><td>84.80 %</td><td>1-acylglycerophosphoinositol/PI(18:3(9Z,12Z,15Z)/0:0)</td><td>95.19 %</td><td>Glycerophosphoinositols</td></tr><tr><td>61</td><td>297.1536</td><td>25.99</td><td>C<sub>16</sub>H<sub>26</sub>O<sub>3</sub>S</td><td>[M-H]<sup>-</sup></td><td>99.72 %</td><td>4-decylbenzenesulfonate</td><td>93.46 %</td><td>Benzenesulfonic acids and derivatives</td></tr><tr><td>60</td><td>571.2892</td><td>25.99</td><td>C<sub>25</sub>H<sub>49</sub>O<sub>12</sub>P</td><td>[M-H]<sup>-</sup></td><td>100 %</td><td>1-palmitoylglycerophosphoinositol</td><td>100 %</td><td>Glycerophosphoinositols</td></tr><tr><td>62</td><td>265.1488</td><td>25.99</td><td>C<sub>12</sub>H<sub>26</sub>O<sub>4</sub>S</td><td>[M-H]<sup>-</sup></td><td>99.96 %</td><td>dodecyl sulfate</td><td>100 %</td><td>Sulfuric acid esters</td></tr></tbody></table>

<figure><img src="image_7.png" /><figcaption>**Fig. 4.** UV and MS chromatograms of the STF extract. (A) UV chromatogram recorded at 260 nm; (B) MS chromatogram in negative ionization mode (MS⁻); (C) MS chromatogram in positive ionization mode (MS⁺). Chromatograms show the distribution and intensity of compounds detected, illustrating the complexity of the STF extract composition.</figcaption></figure>

interfere with DSF-type signaling systems (Kim et al., 2021), suggesting possible cross-talk mechanisms. Additional compounds, including the benzenediol loliolide, lysolecithins, and the β-carboline alkaloid harman-3-carboxylic acid, offer further mechanistic hypotheses. Loliolide has demonstrated broad antimicrobial activity (Morais et al., 2024), while lysolecithins, being amphipathic, could indirectly affect QS through membrane destabilization. Harman-3-carboxylic acid, although not directly linked to QS, may possess unexplored bioactivity relevant to bacterial signaling. Together, these structurally varied molecules likely exert their effects through complementary and possibly synergistic

mechanisms. While flavonoids and hydroxycinnamic acids appear central to QS modulation, other families—including carboxylic acids, fatty acids, and amino acid derivatives—may enhance the overall antimicrobial potential of the extract. Importantly, most of these compounds have not been characterized for their impact on bacterial QS, underscoring the originality and potential of STF as a reservoir of novel QS inhibitors. These findings are particularly relevant in the context of early-stage infections, where QS-regulated virulence factors are essential for bacterial colonization and immune evasion. Moreover, although the potential synergy between STF and conventional antibiotics was not
<figure><img src="image_8.png" /><figcaption>**Fig. 5.** Chemical subclass distribution of metabolites identified in the STF leaf extract by UHPLC-HRMS. Pie chart representing the relative proportions of the 77 identified compounds in STF leaf extract, highlighting predominant subclasses: carbohydrates and carbohydrate conjugates (14 %), flavonoid glycosides (10 %), carboxylic acid derivatives (5 %), glycerophosphocholines (5 %), and hydroxycinnamic acid derivatives (6 %).</figcaption></figure>

explored in this study, the extract's non-bactericidal profile makes it a promising candidate for combination therapies, which have been shown to restore antibiotic susceptibility in resistant *P. aeruginosa* strains (Wang et al., 2024). Finally, STF is derived from *Spondias tuberosa*, a plant endemic to the Caatinga biome. This highlights the pharmacological richness of underexplored ecosystems and the value of bio-prospecting efforts grounded in traditional knowledge. Such initiatives not only support innovative anti-infective strategies but also contribute to the sustainable valorization of local biodiversity in relation to their use for traditional medicine as previously reported (de Oliveira-Júnior et al., 2018).

## 5. Conclusion

This study demonstrates that *Spondias tuberosa* Arruda ethanolic leaf extract effectively inhibits pyocyanin production and disrupts QS in planktonic *P. aeruginosa* without affecting bacterial growth. These findings underscore the potential of STF as a source of anti-virulence compounds and open perspectives for further research into its bioactive molecules, synergistic applications with antibiotics, and possible therapeutic development.

## CRediT authorship contribution statement

S. Chevalier: Investigation, Funding acquisitions. K. Baranger: Investigation, Funding acquisitions. L. Quintans-Junior: Investigation. J.R. Guedes da Silva Almeida: Investigation. R. Gonçalves de Oliveira Junior: Investigation. P. Sitarek: Investigation. R. Grougnet: Investigation. A. Tahrioui: Investigation. O. Lesouhaitier: Investigation. M. Barreau: Investigation. P.E. Bodet: Investigation. A.S. Tareau: Investigation. Picot L.: Writing – original draft, Investigation, Data

curation, Conceptualization.

## Declaration of Competing Interest

The authors declare they have no conflict of interests.

## Acknowledgments

The authors thank the CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico), the French National Research Agency (ANR) HeparInfSkin (ANR-21-CE44-0004), La Région Nouvelle-Aquitaine, La Région Normandie, Evreux Portes de Normandie and European FEDER funds for financial support. AST was supported by a grant from the French ministry (MENRT). This study was conducted as part of an international collaboration within the framework of the Franco-Brazilian Network on Natural Products (FB2NP). The funders had no role in study design, data collection and interpretation, or the decision to submit this work for publication. We gratefully acknowledge Prof. Paul Williams (Centre for Biomolecular Sciences, University of Nottingham) for providing PAO1 pqsA CTX-pqsA::lux biosensor strain.

## Data availability

Data will be made available on request.

## References

Allen, L., Dockrell, D.H., Pattery, T., Lee, D.G., Cornelis, P., Hellewell, P.G., Whyte, M.K. B., 2005. Pyocyanin production by Pseudomonas aeruginosa induces neutrophil apoptosis and impairs neutrophil-mediated host defenses in vivo. J. Immunol. 174, 3643-3649. https://doi.org/10.4049/jimmunol.174.6.3643.
Almeida, J.R.G. da S., Santana, C.R.R., Oliveira-Jãoior, R.G. de, Araújo, C. de S., Souza, G.R., Lima-Saraiva, S.R.G. de, Guimarães, A.L., Oliveira, A.P. de, Filho, J.A. de S., Pacheco, A.G.M., 2012. Phytochemical screening, antioxidant and antibacterial activity of enolpirium spectabile (Bromeliaceae). Int. J. Sci. 1, 96–114.

Almeida, J.R.G. da S., Silva-Filho, R.N. da, Nunes, X.P., Dias, C. da S., Pereira, F. de O., Lima, E. de O., 2006. Antimicrobial activity of the essential oil of Bowdichia illigioioides Kunt. Rev. Bras. Farm. 16, 638–641. https://doi.org/10.1590/S0102-695X200600050009.

Azuama, O.C., Ortiz, S., Quirós-Guerrero, L., Bouffartigues, E., Tortuel, D., Maillot, O., Feuillelo, M., Cornelis, P., Lesouhaitier, O., Grougnet, R., Boutefnouchet, M., Wolfender, J.-L., Chevalier, S., Tahrioui, A., 2020. Tackling pseudomonas aeruginosa virulence by mulinane-like diterpenoids from Azorella atacamensis. Biomolecules 10, 1626. https://doi.org/10.3390/biom10121626.

Becher, A., Schweizer, H.P., 2000. Integration-proficient pseudomonas aeruginosa vectors for isolation of single-copy chromosomal lacZ and lux gene fusions. *BioTechniques* 29, 948–952. https://doi.org/10.2144/00295bm04.

Bever, R.A., Iglewski, B.H., 1988. Molecular characterization and nucleotide sequence of the Pseudomonas aeruginosa elastase structural gene. J. Bacteriol. 170, 4309–4314. https://doi.org/10.1128/jb.170.9.4309-4314.1988.

Boucher, H.W., Talbot, G.H., Bradley, J.S., Edwards, J.E., Gilbert, D., Rice, L.B., Scheld, M., Spellberg, B., Bartlett, J., 2009. Bad Bugs, no drugs: No eskapel! an update from the infectious diseases society of America. Clin. Infect. Dis. 48, 1–12. https://doi.org/10.1086/595011.

Choo, J.H., Rukayadi, Y., Hwang, J.-K., 2006. Inhibition of bacterial quorum sensing by vanilla extract. Lett. Appl. Microbiol. 42, 637–641. https://doi.org/10.1111/j.1472-765X.2006.01928.x.

D'Aquila, P., Sena, G., Crudo, M., Passarino, C., Bellizzi, D., 2023. Effect of Essential Oils of Apiaceae, Lamiaceae, Lauraceae, Myrtaceae, and Rutaceae Family Plants on Growth, Biofilm Formation, and Quorum Sensing in Chromatolium violaceum, Pseudomonas aeruginosa, and Enterococcus faecalis. Microorganisms 11. https://doi.org/10.3390/microorganisms11051150.

Defordit, T., 2018. Quorum-sensing systems as targets for antivirulence therapy. Trends Microbiol. 26, 313–328. https://doi.org/10.1016/j.tim.2017.10.005.

Diggle, S.P., Fletcher, M.P., Câmera, M., Williams, P., 2011. Detection of 2-Alkyl-4-Quinolones Using Biosensors. In: Rumbaugh, K.P. (Ed.), Quorum Sensing: Methods and Protocols. Humana Press, Totowa, NJ, pp. 21–30. https://doi.org/10.1007/978-1-60761-971-02.

Diggle, S.P., Matthijs, S., Wright, V.J., Fletcher, M.P., Chhabra, S.R., Lamont, I.L., Kong, X., Hider, R.C., Cornelis, P., Câmera, M., Williams, P., 2007. The Pseudomonas aeruginosa 4-quinolone signal molecules HQQ and PQS play multifunctional roles in quorum sensing and iron entrapment. Chem. Biol. 14, 87–96. https://doi.org/10.1016/j.chembiol.2006.11.014.

Fabri, R.L., Nogueira, M.S., Dutra, L.B., Bouzada, M.L.M., Scio, E., 2011. Antioxidant and antimicrobial potential of asteraceae species. Rev. Bras. de Plantas Med. 13, 183–189. https://doi.org/10.1590/S1516-05722011000200009.

Fletcher, M.P., Diggle, S.P., Cruz, S.A., Chhabra, S.R., Câmera, M., Williams, P., 2007. A dual biosensor for 2-alkyl-4-quinolone quorum-sensing signal molecules. Environ. Microbiol. 9, 2683–2693. https://doi.org/10.1111/j.1462-2920.2007.01380.x.

Gonçalves, T., Vasconcelos, U., 2021. Colour me: the history and the biotechnological potential of pyocyanin. Molecules 26, 927. https://doi.org/10.3390/molecules26040927.

Hancock, R.E., Carey, A.M., 1979. Outer membrane of Pseudomonas aeruginosa: heat-2-mercaptoethanol-modifiable proteins. J. Bacteriol. https://doi.org/10.1128/jb.140.3.902-910.1979.

Hernández-Jiménez, P., López-Medrano, F., Fernández-Ruiz, M., Silva, J.T., Corbella, L., San-Juan, R., Lizaosain, M., Díaz-Regañón, J., Viedma, E., Aguado, J.M., 2022. Risk Factors and Outcomes for Multidrug Resistant Pseudomonas aeruginosa Infection in Immunocompromised Patients. Antibiotics 11. https://doi.org/10.3390/antibiotics1111459.

Jakobsen, T.H., Bragason, S.K., Phipps, R.K., Christensen, L.D., van Gennip, M., Alhede, M., Skindersoe, M., Larsen, T.O., Heiby, N., Bjarnsholt, T., Givskov, M., 2012. Food as a Source for Quorum Sensing Inhibitors: Iberin from Horseradish Revealed as a Quorum Sensing Inhibitor of Pseudomonas aeruginosa. Appl. Environ. Microbiol. 78, 2410–2421. https://doi.org/10.1016/j.aem.05992-11.

Kalia, V.C., 2013. Quorum sensing inhibitors: An overview. Biotechnol. Adv. 31, 224–245. https://doi.org/10.1016/j.biotechadv.2012.10.004.

Kim, H.-S., Cha, E., Ham, S.-Y., Park, J.-H., Nam, S., Kwon, H., Byun, Y., Park, H.-D., 2021. Linoleic acid inhibits Pseudomonas aeruginosa biofilm formation by activating diffusible signal factor-mediated quorum sensing. Biotechnol. Bioeng. 118, 82–93. https://doi.org/10.1002/bit.27552.

LaSarre, B., Federle, M.J., 2013. Exploiting quorum sensing to confuse bacterial pathogens. Microbiol. Mol. Biol. Rev. 77, 73–111. https://doi.org/10.1128/mmbc.00046-12.

Lau, G.W., Hassett, D.J., Ran, H., Kong, F., 2004. The role of pyocyanin in Pseudomonas aeruginosa infection. Trends Mol. Med. 10, 599–606. https://doi.org/10.1016/j.molmed.2004.10.002.

Lee, J., Zhang, L., 2015. The hierarchy quorum sensing network in Pseudomonas aeruginosa. Protein Cell 6, 26–41. https://doi.org/10.1007/s13238-014-0100-x.

de Lima-Saraiva, S.R.G., Oliveira, F.G. da S., Junior, R.G. de O., Araújo, C. de S., de Oliveira, A.P., Pacheco, A.G.M., Rolim, L.A., de Amorim, E.L.C., César, F.C.S., Almeida, J.R.G. da S., 2017. Chemical Analysis and Evaluation of Antioxidant, Antimicrobial, and Photoprotective Activities of Schinopsis brasiliensis Engl. (Anacardiaceae). ScientificWorldJournal 2017, 1713921. https://doi.org/10.1155/2017/1713921.

Lyczak, J.B., Cannon, C.L., Pier, G.B., 2000. Establishment of Pseudomonas aeruginosa infection: lessons from a versatile opportunist1. Microbes 2, 1051–1060. https://doi.org/10.1016/S1286-4579(00)01259-4.

Managò, A., Becker, K.A., Carpinteiro, A., Wilker, B., Soddemann, M., Seitz, A.P., Edwards, M.J., Grassmé, H., Szabó, I., Gubins, E., 2015. Pseudomonas aeruginosa pyocyanin induces neutrophil death via mitochondrial reactive oxygen species and mitochondrial acid sphingomyelinase. Antioxid. Redox Signal 22, 1097–1110. https://doi.org/10.1089/ars.2014.5979.

Masuku, M., Mozirandi, W., Mukanganyama, S., 2023. Evaluation of the antibacterial and antibiofilm effects of ethyl acetate root extracts from Vernonia adonisii (Asteraceae) against pseudomonas aeruginosa. Sci. World J. 2023. https://doi.org/10.1155/2023/5782656.

Moradali, M.F., Ghods, S., Rehm, B.H.A., 2017. Pseudomonas aeruginosa Lifestyle: a paradigm for adaptation, survival, and persistence. Front. Cell. Infect. Microbiol 7. https://doi.org/10.3389/fcimb.2017.00039.

Morais, A.M.M.B., Kumla, D., Martins, V.F.R., Alves, A., Gales, L., Silva, A.M.S., Costa, P. M., Mistry, S., Kijjoa, A., Morais, R.M.S.C., 2024. Monoterpe Hydroxy Lactones Isolated from Thalassiosira sp. Microalga and Their Antibacterial and Antioxidant Activities. Molecules 29. https://doi.org/10.3390/molecules29215175.

Musthafa, K.S., Sianglum, W., Saising, J., Lethongkam, S., Voravuthikunchai, S.P., 2017. Evaluation of phytochemicals from medicinal plants of Myrtaceae family on virulence factor production by Pseudomonas aeruginosa. APMIS 125, 482–490. https://doi.org/10.1111/apm.12672.

Nadal Jimenez, P., Koch, G., Thompson, J.A., Xavier, K.B., Cool, R.H., Quax, W.J., 2012. The Multiple Signaling Systems Regulating Virulence in Pseudomonas aeruginosa. Microbiol. Mol. Biol. Rev. 76, 46–65. https://doi.org/10.1128/mmbr.05007-11.

Nascimento, G.G.F., Locatelli, J., Freitas, P.C., Silva, G.L., 2000. Antioxidant activity of plant extracts and phytochemicals on antibiotic-resistant bacteria. Braz. J. Microbiol 31, 247–256. https://doi.org/10.1590/S1517-83822000000400003.

de Oliveira-Júnior, R.G., Alves Ferraz, C.A., Pontes, M.C., Cavalcante, N.B., da Cruz Araújo, E.C., de Oliveira, A.P., Picot, L., Rolim, L.A., da Silva Almeida, J.R.G., 2018. Antibacterial activity of terpenoids isolated from *Cnidoscolus quercifolius* Pohl (Euphorbiaceae), a Brazilian medicinal plant from Caatinga biome. Eur. J. Integr. Med. 24, 30–34. https://doi.org/10.1016/j.eujim.2018.10.012.

Pendleton, J.N., Gorman, Sean P., Gilmore, B.F., 2013. Clinical relevance of the ESKAPE pathogens. Expert Rev. AntiInfect. Ther. 11, 297–308. https://doi.org/10.1586/er.1312.

Peng, L.-Y., Yuan, M., Cui, Z.-Q., Wu, Z.-M., Yu, Z.-J., Song, K., Tang, B., Fu, B.-D., 2018. Rutin inhibits quorum sensing, biofilm formation and virulence genes in avian pathogenic *Escherichia coli*. Microb. Pathog. 119, 54–59. https://doi.org/10.1016/j.micpath.2018.04.007.

Rasko, D.A., Sperandio, V., 2010. Anti-virulence strategies to combat bacteria-mediated disease. Nat. Rev. Drug Discov. 9, 117–128. https://doi.org/10.1038/ndr3013.

Rosado-Vallado, M., Brito-Loeza, W., Mena-Rejón, G.J., Quintero-Marmol, E., Flores-Guido, J.S., 2000. Antimicrobial activity of Fabaceae species used in Yucatan traditional medicine. Fitoterapia 71, 570–573. https://doi.org/10.1016/S0367-326X(00)00200-8.

Shariati, A., Noei, M., Askarinia, M., Khoshbayan, A., Farahani, A., Chegini, Z., 2024. Inhibitory effect of natural compounds on quorum sensing system in Pseudomonas aeruginosa: a helpful promise for managing biofilm community. Front Pharm. 15, 1350391. https://doi.org/10.3389/fphar.2024.1350391.

da Silva, A.R.A., de Morais, S.M., Marques, M.M.M., de Oliveira, D.F., Barros, C.C., de Almeida, R.R., Vieira, I.G.P., Guedes, M.I.F., 2012. Chemical composition, antioxidant and antibacterial activities of two Spondias species from Northeastern Brazil. Pharm. Biol. 50, 740–746. https://doi.org/10.3109/13880209.2011.627347.

Sumang, F.A., Ward, A., Errington, J., Dashti, Y., 2024. Hibiscus acid and hydroxycitric acid dimethyl esters from Hibiscus flowers production of dithiolpyrrolone antibiotics by Streptomyces Strain MBN2-2. Nat. Prod. Bioprospect 14, 40. https://doi.org/10.1007/s13659-024-00460-0.

Tahrioui, A., Duchesne, R., Bouffartigues, E., Rodrigues, S., Maillot, O., Tortuel, D., Hardouin, J., Taupin, L., Groleau, M.-C., Dufour, A., Déziel, E., Brenner-Weiss, G., Feuillelo, M., Orange, N., Lesouhaitier, O., Cornelis, P., Chevalier, S., 2019. Extracellular DNA release, quorum sensing, and PrrF1/F2 small RNAs are key players in Pseudomonas aeruginosa tobramycin-enhanced biofilm formation. npj Biofilms Micro 5. https://doi.org/10.1038/s41522-019-0088-3.

Tahrioui, A., Ortiz, S., Azuama, O.C., Bouffartigues, E., Benalia, N., Tortuel, D., Maillot, O., Chemat, S., Kritsanida, M., Feuillelo, M., Orange, N., Michel, S., Lesouhaitier, O., Cornelis, P., Grougnet, R., Boutefnouchet, S., Chevalier, S., 2020. Membrane-Interactive Compounds From Pistacia lentiscus Thwart Pseudomonas aeruginosa Virulence. Front. Microbiol 11, 1068. https://doi.org/10.3389/fmicb.2020.01068.

Thassya Lucas Dos Santos, A., Pereira Carneiro, J.N., Pereira da Cruz, R., Lima Sales, D., Cosmo Andrade, J., de Oliveira Almeida, W., Martins da Costa, J.G., Riceli Vasconcelos Ribeiro, P., Sousa de Brito, E., Alves Batista, F.L., Alves Magalhães, F.E., Iriti, M., Morais-Braga, M.F.B., Coutinho, H.D.M., 2019. UPLC-MS-ESI-QTOF Analysis and Antifungal Activity of the Spondias tuberosa Arruda Leaf and Root Hydroalcoholic Extracts. Antibiot. (Basel) 8, 240. https://doi.org/10.3390/antibiotics8040240.

Vadakkan, K., Sathishkumar, K., Mapranathukaran, V.O., Ngangbam, A.K., Nongmaithem, B.D., Hemapriya, J., Nair, J.B., 2024. Critical review on plant-derived
quorum sensing signaling inhibitors in *Pseudomonas aeruginosa*. Bioorg. Chem. 151, 107649. https://doi.org/10.1016/j.bioorg.2024.107649.

Wang, J., Lu, X., Wang, C., Yue, Y., Wei, B., Zhang, H., Wang, H., Chen, J., 2024. Research progress on the combination of quorum-sensing inhibitors and antibiotics against bacterial resistance. Molecules 29, 1674. https://doi.org/10.3390/molecules29071674.

Williams, P., Cámara, M., 2009. Quorum sensing and environmental adaptation in *Pseudomonas aeruginosa*: a tale of regulatory networks and multifunctional signal molecules. Curr. Opin. Microbiol. Cell Regul. 12, 182–191. https://doi.org/10.1016/j.mib.2009.01.005.