1. INTRODUCTION
Pseudomonas aeruginosa, a gram-negative bacteria and an opportunistic pathogen, is well-known for causing severe infections in immunocompromised individuals, such as those with cystic fibrosis, burns, or ventilator-associated pneumonia [1–3]. Its pathogenicity is driven by an arsenal of virulence factors, that includes quorum sensing (QS) regulated toxin secretion (e.g., elastase and pyocyanin), and biofilm formation [4–7]. Traditional antibiotics, which target bacterial growth or viability, impose strong selective pressures that drive resistance [8]. Due to its rapid development of multidrug resistance (MDR), the World Health Organization classifies P. aeruginosa as a “critical priority” pathogen, emphasizing the urgent need for innovative treatments [8].
To combat MDR, anti-virulence strategies have been developed to disarm pathogens by disrupting their ability to colonize, communicate, or damage host tissues [9]. For instance, QS inhibitors can block the production of virulence factors controlled by the las and rhl QS systems, without affecting bacterial survival [10]. Natural products, especially phytochemicals, are a rich source of anti-virulence agents due to their structural diversity and evolutionary optimization for biological interactions [11]. Ammi visnaga (Apiaceae), a Mediterranean medicinal plant, has been used since antiquity for treating renal colic, asthma, and angina, primarily due to its vasodilatory furanochromones, such as khellin (KH) and visnagin [12]. Recent phytochemical studies have identified furobenzopyrans, structurally related to furanochromones as key bioactive constituents with antimicrobial, anti-inflammatory, and calcium channel-modulating properties [13–15]. Emerging evidence suggests that plant-derived molecules can disrupt bacterial virulence by interfering with QS signaling [16,17]. Building on these findings, we hypothesize that KH, a furobenzopyrans from A. visnaga, attenuate P. aeruginosa pathogenicity by targeting virulence factor secretion and this study addresses a critical gap in understanding the anti-virulence potential of A. visnaga phytochemical.
2. MATERIALS AND METHODS
2.1. Chemicals, reagents, and strain details
The chemicals used were of analytical grade from Sigma–Aldrich Chemicals Private Limited, India. The solvents used of spectroscopic grade from Sigma–Aldrich Chemicals Private Limited, India. Luria–Bertani (LB) media, Hexa G-minus 28, agar, and Mueller Hinton (MH) media, were sourced from Hi-Media Laboratories Private Limited, India. Elastin congo red (ECR) was procured from Sigma–Aldrich Chemicals Private Limited, India. Ciprofloxacin and KH were obtained from Tokyo Chemical Industry, India. The experiments were conducted using the P.aeruginosa PAO1 strain (MTCC 2453) from the microbial type culture collection, Institute of Microbial Technology, India.
2.2. Antibiotic Sensitivity Analysis
The susceptibility to antibiotics was assessed in triplicates using Hexa G-28 sensitivity disc as per the reported procedure [15,18]. A bacterial suspension was prepared in sterile saline to achieve a turbidity equivalent to the 0.5 McFarland standard, which corresponds to approximately 1 × 10^8 colony forming unit (CFU)/mL. Using a sterile swab, excess liquid was removed by pressing alongside the walls of the tubes. The swab was then used to spread the bacterial suspension on the surface of MH agar plates, ensuring a uniform bacterial lawn. The plates were rotated 60° and swabbed two more times for even coverage. After allowing the plates to dry for 5–10 minutes at room temperature, Hexa G-28 sensitivity discs were placed on the agar surface using sterile forceps, ensuring even spacing and firm contact. The plates were subsequently incubated at 37°C for 18 hours. Post-incubation, the plates were inspected for zones of inhibition around the discs, indicating bacterial susceptibility to the antibiotics.
2.3. Effect of KH on P. aeruginosa Growth
The broth microdilution method as per standard guidelines was used to determine the MIC of KH against P.aeruginosa PAO1 [18,19]. The MH broth was used to prepare the working dilutions of KH between 0 µg/ml and 900 µg/ml. These concentrations were selected based on a broad range of preliminary screenings to identify the optimal dosage that could inhibit bacterial growth. The MH broth containing various concentrations of KH and control (no treatment) was inoculated with the overnight-grown culture of P.aeruginosa in triplicates. Then, the mixture was incubated at 37°C overnight and the optical density (OD) was measured at 600 nm (SpectraMax® Plus 384 Absorbance Plate Reader, Molecular Devices, US).
2.4. Effect on Pyocyanin Content
Pyocyanin production was measured following the established methods [20]. The P.aeruginosa culture was incubated at 37°C overnight, with 20 μl of the overnight culture then added to 2 ml of fresh medium (2% peptone, 0.14% magnesium chloride, 1% potassium sulphate, and 1% glycerine, pH 7.4) treated with the KH (9 µg/ml) at 37°C along with shaking at 150 rpm in triplicates. The cells were then centrifuged at 12,000 rpm for 15 minutes at 4°C. The cell-free supernatants were then analyzed for pyocyanin production at 695 nm using a spectrophotometer (SpectraMax® Plus 384 Absorbance Plate Reader, Molecular Devices, US).
2.5. Effect on Elastase Activity
To analyze the elastinolytic activity, a reaction mixture was prepared by adding ECR buffer (900 μL) to the KH(9 µg/mL) treated culture supernatant (100 μL) in triplicates [21]. The ECR buffer comprised 100 mM Tris, 1 mM CaCl2, and 20 mg ECR at a pH of 7.5. Following the incubation period (37°C for 3 hours), centrifugation was carried out at 10,000 rpm for 10 minutes to remove the insoluble ECR. The elastinolytic activity was then estimated by measuring the OD at 495 nm.
2.6. Impact on Secretion of 3-oxo-C12-HSL
The potential anti-QS activity of KH was evaluated by measuring the levels of N-3-oxo-dodecanoyl-L-homoserine lactone (3-oxo-C12-HSL) secreted by P. aeruginosa PAO1 [21]. Briefly, 0.1% overnight cultures of P. aeruginosa PAO1 were inoculated into 50 ml of LB medium with or without KH(9 µg/mL) in triplicates and incubated at 37°C for 48 hours. After incubation, cells were removed by centrifugation at 4°C for 15 minutes. The supernatant underwent three successive extractions using ethyl acetate. The ethyl acetate was evaporated under reduced pressure, and the residues were dissolved in methanol. Quantification of 3-oxo-C12-HSL was performed using high-performance liquid chromatography coupled with an ultraviolet detector [21]. Peaks corresponding to 3-oxo-C12-HSL were identified based on retention times of 3-oxo-C12-HSL standard (Sigma-Aldrich, India). Peak areas were calculated using the extracted chromatograms and the results were quantified.
2.7. Quantitative real-time polymerase chain reaction (qRT-PCR)
qRT-PCR was performed as per the method described by Zhou et al. [22]. The PAO1 strain was cultured in LB medium, with or without the addition of KH(9.0 µg/ml) in triplicates, at 37°C and 180 rpm for 24 hours. After incubation, the cells were washed three times with sterile PBS (pH 7.2) and collected by centrifugation at 4°C for 10 minutes. Total RNA was extracted, genomic DNA was removed and cDNA was synthesized as per the protocol briefed by Zhou et al. [22]. The primers listed in Table 1 were sourced from the previous studies and a SYBR Green Master Mix kit (Vazyme Biotech, Nanjing, China) was used to run qRT-PCR. The fold changes of target genes were calculated using the 2−??Ct method, as previously described [23].
 | Table 1. Primers for quantitative real-time polymerase chain reaction. [Click here to view] |
2.8. Statistical Analysis
Statistical analysis was performed using a one-way analysis of variance (ANOVA) with the STATGRAPHICS® Centurion XVI software (Version 16.2.04, Statgraphics Technologies, Inc., The Plains, VA) to assess statistical significance. Post-hoc comparisons were carried out using Fisher's least significant difference test to identify specific differences between groups after the ANOVA. All experiments were performed in triplicate, and at a 95% confidence interval, a statistically significant was considered if the p-value was less than 0.05.
3. RESULTS
3.1. Antibiotic Sensitivity
The antibiotic sensitivity profile of P. aeruginosa was studied, revealing susceptibility to a range of antibiotics (Fig. 1). Specifically, P. aeruginosa demonstrated good sensitivity to the tested antibiotics such as gentamicin, meropenem, cefepime, cefotaxime, levofloxacin, piperacillin/tazobactam, amikacin, and cefoperazone. This broad spectrum of sensitivity indicates that the strains used for the studies were not antibiotic-resistant strains.
 | Figure 1. Effect of antibiotics (gentamicin, meropenem, cefepime, cefotaxime, levofloxacin, piperacillin/tazobactam, amikacin, and cefoperazone) against Pseudomonas aeruginosa showing top(left image) and back (right image) side of the plates. [Click here to view] |
3.2. Effect of KH on Growth
The antibacterial activity of KH was inferred based on the growth visually, measuring OD at 600 nm, and colony count (CFU/ml) at various concentrations, and the results are summarized in Table 2. In the control group (0 µg/ml), there was significant bacterial growth, with an OD of 0.73 and a colony count of 351*10^6 CFU/ml, indicating normal bacterial proliferation without any antimicrobial intervention. The positive control, ciprofloxacin at 0.1 µg/ml, effectively inhibited bacterial growth, as evidenced by an OD of 0.09 and a colony count of less than 10 CFU/ml, demonstrating the antibiotic’s strong antibacterial activity. When tested with KH at 0.9 µg/ml, bacterial growth was still observed, with an OD of 0.75 and a colony count of 368 × 10^6 CFU/ml, which was similar to the control group, suggesting that this concentration of KH did not inhibit bacterial growth. At KH concentrations of 9.0 µg/ml and 90.0 µg/ml, bacterial growth remained evident, with OD values of 0.74 and 0.71, respectively, and colony counts of 361 × 10^6 and 348 × 10^6 CFU/ml. These results indicate that KH, at these concentrations, did not substantially reduce bacterial growth. However, at a higher concentration of KH (900.0 µg/ml), growth was significantly inhibited, with an OD of 0.29 and a reduced colony count of 136 × 10^3 CFU/ml. This suggests that at high concentrations, KH exhibits antibacterial activity, although it was not as potent as ciprofloxacin in fully preventing bacterial growth. Overall, the results demonstrate that while KH has some antibacterial potential at higher concentrations, its effectiveness at lower concentrations is limited, and it is less potent than the positive control.
 | Table 2. Effect of khellin on P. aeruginosa growth. Each experimental data represents the average of triplicates. [Click here to view] |
3.3. Effect On Pyocyanin Content
The ability of KH to inhibit pyocyanin production was studied and the results are shown in Figure 2 and Figure 3. Pseudomonas aeruginosa produces pyocyanin which is in green color. As shown in Figure 2, the KH-treated group showed less green color compared to the strain control.
 | Figure 2. Culture flask showing the grown cultures of Pseudomonas aeruginosa. A) Positive control treated with ciprofloxacin; B) Strain control with no treatment; C) P. aeruginosa treated with Khellin. [Click here to view] |
 | Figure 3. The effect of Khellin on pyocyanin content in P. aeruginosa was evaluated, with ciprofloxacin (0.1 μg/ml) serving as the positive control (A) and the control group receiving no treatment (B). Each experimental data is expressed as mean (n = 3) and the error bar denotes standard deviation (n = 3). Different lowercase alphabets in superscripts are used to indicate significant differences between groups (p < 0.05). [Click here to view] |
The overnight-grown cultures were subjected to pyocyanin content estimation, and the results are shown in Figure 3. We could see a marked reduction in the concentrations from 2.74 to 1.43 µg/ml in the KH-treated group(p < 0.05).
3.4. Effect on Elastase Activity
The effect of KH on elastase activity was studied and the results are presented in Figure 4. A statistically significant reduction of elastase activity was observed for the KH-treated group (OD of 0.56) compared to the control OD of 1.24 (p < 0.05). These results indicate a general trend of decreasing elastase activity with increasing concentration, after an initial slight increase at the lowest concentration.
 | Figure 4. Khellin’s effect on the elastase activity of Pseudomonas aeruginosa. Ciprofloxacin (0.1 µg/ml) served as the positive control and the control group received no treatment. Each experimental data are expressed as mean (n = 3) and the error bar denotes standard deviation (n = 3). Different lowercase alphabets in superscripts are used to indicate significant differences between groups (p < 0.05). [Click here to view] |
3.5. Impact on Secretion of 3-oxo-C12-HSL
The levels of 3-oxo-C12-HSL produced by P.aeruginosa PAO1 were quantified to assess the potential anti-QS activity of KH. Chromatography analysis revealed the presence of 3-oxo-C12-HSL, in the culture supernatants, and treatment with KH at 9 µg/mL for 24 hours led to a significant reduction of 3-oxo-C12-HSL (Fig. 5). Relative quantification analysis showed that KH reduced 3-oxo-C12-HSL levels by approximately 69% compared to the control (Fig. 6). These results indicate that KH has anti-QS activity, likely by inhibiting the production of signaling molecules.
 | Figure 5. Chromatogram showing the impact of khellin on 3-oxo-C12 HSL signaling molecule in P. aeruginosa. Ciprofloxacin (0.1 µg/mL) served as the positive control and the control group received no treatment. [Click here to view] |
 | Figure 6. Effect of khellin on the secretion of 3-oxo-C12 HSL signaling molecule in Pseudomonas aeruginosa. Ciprofloxacin (0.1 µg/ml) served as the positive control and the control group received no treatment. Each experimental data are expressed as mean (n = 3) and the error bar denotes standard deviation (n = 3). Different lowercase alphabets in superscripts are used to indicate significant differences between groups (p < 0.05). [Click here to view] |
3.6. Effect on QS-Related Gene Expression
A qRT-PCR assay was conducted to assess the effect of khellin on the expression of the QS-related gene lasR in P.aeruginosa PAO1. The results revealed a significant reduction in lasR expression, with a 45% down-regulation observed after exposure to 9 µg/ml of khellin (Fig. 7).
 | Figure 7. Effect of khellin on the lasR gene expression of QS-related circuits in P. aeruginosa. Khellin’s effect on the elastase activity of Pseudomonas aeruginosa. Ciprofloxacin (0.1 µg/ml) served as the positive control and the control group received no treatment. Each experimental data are expressed as mean (n = 3) and the error bar denotes standard deviation (n = 3). Different lowercase alphabets in superscripts are used to indicate significant differences between groups (p < 0.05). [Click here to view] |
4. DISCUSSION
The antibiotic susceptibility profile of P. aeruginosa outlined in Figure 1 demonstrated that P. aeruginosa used for the experiments were sensitive to gentamicin, meropenem, cefepime, cefotaxime, levofloxacin, piperacillin/tazobactam, amikacin, and cefoperazone. These antibiotics are known for their effectiveness against various bacterial infections, and their efficacy against P. aeruginosa aligns with the published reports [24]. In the current dose-response study with KH, the growth of P. aeruginosa cultures was reduced at 900 µg/ml (Table 1). In the low concentration (<900 µg/ml), no visible impact on P. aeruginosa growth was noticed (Table 1), indicating the sub-MIC for KH. Rafiee et al. [25] investigated the anti-QS activity of a ciprofloxacin–copper complex against P. aeruginosa PAO1 [25]. They found that 1/4 and 1/16 of MIC (sub-MIC) of the complex significantly reduced virulence factors while having minimal impact on bacterial growth. Similarly, Similarly Naga et al. studied the effects of methyl gallate (MG) from Mangifera indica on QS in P. aeruginosa [26]. They found that MG at sub-MIC concentrations (1/4 and 1/2 MIC) significantly inhibited QS-regulated virulence factors without affecting bacterial viability. In the current study, at 1/10, 1/100 of MIC, KH had minimal or no impact on the growth of P. aeruginosa supporting the published reports [27–30]. These findings underscore the potential of using sub-MIC concentrations of QS inhibitors to reduce P. aeruginosa’s virulence.
Pyocyanin is a key virulence factor that contributes to the P. aeruginosa pathogenicity. Recent studies have explored the potential of quorum-sensing inhibitors to reduce pyocyanin production. For instance, Morkunas et al. tested an inhibitor that competitively inhibits the native autoinducer, leading to a reduction of pyocyanin production in wild-type P. aeruginosa cultures [31]. Fekete-Kertész et al. demonstrated that α-cyclodextrin (ACD) and β-cyclodextrin significantly reduced pyocyanin production, with ACD showing a higher inhibitory effect, highlighting the potential of CDs as antivirulence agents [32]. In the current study, the concentration of pyocyanin pigment was reduced in the KH-treated group. Based on all this information, the findings observed in the current study on the reduction of pyocyanin production by KH (Figs. 2 and 3) could be through suppression of virulence gene expression.
In P. aeruginosa pathogenesis, another major virulence factor that is involved is elastase, especially in lung-related severe infections [33,34]. Elastase breaks down elastin, a key extracellular matrix component, contributing to tissue destruction and inflammation. A recent study by Zhou et al. demonstrated that a phyto-compound (resveratrol), when exposed to P. aeruginosa cultures, significantly reduced elastase production [35]. Ren et al. studied the effects of quercetin on the reduction of elastase activity by 57% when P. aeruginosa was exposed at sub-MIC concentrations [36]. In the current study, KH showed elastase inhibition of 54.83% in comparison to control (p < 0.05, Fig. 4) which suggests that KH would have mediated its action similar to that of quercetin and resveratrol.
Pyocyanin and elastase play crucial roles in colonizing host tissues, and biofilm formation and are regulated by the las QS system mediated through lasR gene [22]. To confirm the mechanism of action of KH’s reduced virulence factors, gene expression studies were performed and the KH-treated group showed suppression of lasR gene expression compared to the control (Fig. 7). These results closely aligned with KH impacting the expression of QS-related genes, confirming that KH treatment inhibits the transcriptional levels of virulence factors.
In P. aeruginosa QS systems, the las QS system modulates the synthesis of the QS signaling molecule (3-oxo-C12 HSL). Through these signaling molecules, P. aeruginosa regulates the secretion of virulence factors [10]. In this study, the KH-treated P. aeruginosa group secreted lower levels of 3-oxo-C12 HSL compared to the control (Fig. 6), likely due to the suppression of lasR gene expression observed in the study. In a normal QS circuit, lasR, upon binding to 3-oxo-C12 HSL, activates genes responsible for virulence factor production and enhances lasI expression, which encodes the enzyme responsible for synthesizing more 3-oxo-C12 HSL [5,10]. KH likely interferes with lasR expression, leading to reduced lasR availability and disrupting this positive feedback loop, ultimately lowering 3-oxo-C12 HSL synthesis and secretion. By targeting lasR, KH effectively disrupts bacterial communication, reducing virulence and potentially enhancing the susceptibility of P. aeruginosa to host defenses or antibiotic treatments, making it a promising anti-virulence agent.
While KH effectively reduces virulence, its bioavailability, in-vivo efficacy, stability, and potential cytotoxicity require further investigation. Findings suggest that KH could be combined with antibiotics to treat resistant strains causing persistent infections, warranting a more detailed study.
5. CONCLUSION
This study demonstrated that KH, at sub-MIC concentrations, effectively reduced virulence factors such as pyocyanin content and elastase activity, which are mediated by the las QS system. Mechanistic studies revealed that KH exerted its anti-virulence activity by suppressing lasR gene expression, as confirmed by the decreased production of 3-oxo-C12 HSL signaling molecules. Hence, by targeting QS pathways, it may be possible to develop novel therapeutic strategies that weaken the pathogen's defenses and improve treatment outcomes.
6. ACKNOWLEDGMENTS
The authors would like to acknowledge M/S. Kemin Industries South Asia Private Limited, India for providing support for carrying out this work.
7. CONFLICTS OF INTEREST
The authors report no financial or any other conflicts of interest in this work.
8. FUNDING
The authors declare that no funding was received for this work.
9. ETHICAL APPROVALS
This study does not involve experiments on animals or human subjects.
10. DATA AVAILABILITY
All the data are available with the author and will be provided upon request.
11. AUTHOR CONTRIBUTION STATEMENT
All authors made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; took part in drafting the article or revising it critically for important intellectual content; agreed to submit to the current journal; gave final approval of the version to be published; and agree to be accountable for all aspects of the work. All the authors are eligible to be an author as per the International Committee of Medical Journal Editors (ICMJE) requirements/guidelines
12. PUBLISHER’S NOTE
All claims expressed in this article are solely those of the authors and do not necessarily represent those of the publisher, the editors and the reviewers. This journal remains neutral with regard to jurisdictional claims in published institutional affiliation.
13. USE OF ARTIFICIAL INTELLIGENCE (AI)-ASSISTED TECHNOLOGY
The authors declares that they have not used artificial intelligence (AI)-tools for writing and editing of the manuscript, and no images were manipulated using AI.
REFERENCES
1. Gellatly SL, Hancock REW. Pseudomonas aeruginosa: new insights into pathogenesis and host defenses. Pathog Dis 2013;67:159–73; doi: https://doi.org/10.1111/2049-632X.12033
2. Avinash MG, Aishwarya S, Zameer F, Gopal S. Pseudomonas aeruginosa biofilm and their molecular escape strategies. J App Biol Biotech 2023;11:28–37; doi: https://doi.org/10.7324/JABB.2023.36700
3. Mukhopadhyay S, Narayan R, Gadag S, Shenoy PA, Garg S, Ashwini T, Nayak UY. Development of levofloxacin glycosylated mesoporous silica nanoparticles for urinary tract infections. J App Pharm Sci 2024;14:174–9; doi: https://doi.org/10.7324/JAPS.2024.181547
4. Hancock REW, Speert DP. Antibiotic resistance in Pseudomonas aeruginosa: mechanisms and impact on treatment. Drug Resist Updates 2000;3:247–55; doi: https://doi.org/10.1054/drup.2000.0152
5. Hibbert TM, Whiteley M, Renshaw SA, Neill DR, Fothergill JL. Emerging strategies to target virulence in Pseudomonas aeruginosa respiratory infections. Crit Rev Microbiol 2024;50:1037–52; doi: https://doi.org/10.1080/1040841X.2023.2285995.
6. Krell T, Matilla MA. Pseudomonas aeruginosa. Trends Microbiol 2024;32:216–8; doi: https://doi.org/10.1016/j.tim.2023.11.005
7. Sultan M, Arya R, Kim KK. Roles of two-component systems in Pseudomonas aeruginosa virulence. Int J Mol Sci 2021;22:12152; doi: https://doi.org/10.3390/ijms222212152
8. Tacconelli E, Carrara E, Savoldi A, Harbarth S, Mendelson M, Monnet DL, et al. Discovery, research, and development of new antibiotics: the WHO priority list of antibiotic-resistant bacteria and tuberculosis. Lancet Infect Dis 2018;18:318–27; doi: https://doi.org/10.1016/S1473-3099(17)30753-3
9. Clatworthy AE, Pierson E, Hung DT. Targeting virulence: a new paradigm for antimicrobial therapy. Nat Chem Biol 2007;3:541–8; doi: https://doi.org/10.1038/nchembio.2007.24
10. Rutherford ST, Bassler BL. Bacterial quorum sensing: its role in virulence and possibilities for its control. Cold Spring Harb Perspect Med 2012;2:a012427; doi: https://doi.org/10.1101/cshperspect.a012427
11. Silva LN, Zimmer KR, Macedo AJ, Trentin DS. Plant natural products targeting bacterial virulence factors. Chem Rev. 2016;116:9162–236; doi: https://doi.org/10.1021/acs.chemrev.6b00184
12. Khalil N, Bishr M, Desouky S, Salama O. Ammi visnaga L., a potential medicinal plant: a review. Molecules 2020;25:301; doi: https://doi.org/10.3390/molecules25020301
13. El-Hawary SS, Moawad AS, Bahr HS, Abdelmohsen UR, Mohammed R. Natural product diversity from the endophytic fungi of the genus Aspergillus. RSC Adv 2020;10: 22058–79; doi: https://doi.org/10.1039/D0RA04290K
14. El-sayed H, Fahmy Y. Correlation between biofilm formation and multidrug resistance in clinical isolates of Pseudomonas aeruginosa. Microb Infect Dis 2021;2:541; doi: https://doi.org/10.21608/mid.2021.81284.1164
15. Rajendran RM, Parthiban BD. Identification, characterization, and antibacterial studies of furobenzopyrans from Ammi visnaga. J App Pharm Sci 2023;13:084–94; doi: https://doi.org/10.7324/JAPS.2023.138123
16. Vadakkan K, Sathishkumar K, Mapranathukaran VO, Ngangbam AK, Nongmaithem BD, Hemapriya J, et al. Critical review on plant-derived quorum sensing signaling inhibitors in pseudomonas aeruginosa. Bioorg Chem 2024;151:107649; doi: https://doi.org/10.1016/j.bioorg.2024.107649
17. Lu L, Wang J, Wang C, Zhu J, Wang H, Liao L, et al. Plant-derived virulence arresting drugs as novel antimicrobial agents: discovery, perspective, and challenges in clinical use. Phytother Res 2024;38:727–54; doi: https://doi.org/10.1002/ptr.8072
18. Wiegand I, Hilpert K, Hancock REW. Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. Nat Protoc 2008;3:163–75; doi: https://doi.org/10.1038/nprot.2007.521
19. Saqr AA, Aldawsari MF, Khafagy ES, Shaldam MA, Hegazy WAH, Abbas HA. A novel use of allopurinol as a quorum-sensing inhibitor in Pseudomonas aeruginosa. Antibiotics 2021;10:1385; doi: https://doi.org/10.3390/antibiotics10111385
20. Yang R, Guan Y, Zhou J, Sun B, Wang Z, Chen H, et al. Phytochemicals from Camellia nitidissima Chi flowers reduce the pyocyanin production and motility of Pseudomonas aeruginosa PAO1. Front Microbiol 2018;8:2640; doi: https://doi.org/10.3389/fmicb.2017.02640
21. Zhou J, Bi S, Chen H, Chen T, Yang R, Li M, et al. Anti-biofilm and antivirulence activities of metabolites from Plectosphaerella cucumerina against Pseudomonas aeruginosa. Front Microbiol 2017 May 3;8:769; doi: https://doi.org/10.3389/fmicb.2017.00769
22. Zhou JW, Luo HZ, Jiang H, Jian TK, Chen ZQ, Jia AQ. Hordenine: a novel quorum sensing inhibitor and antibiofilm agent against Pseudomonas aeruginosa. J Agric Food Chem 2018;66:1620–8; doi: https://doi.org/10.1021/acs.jafc.7b05035
23. Sarabhai S, Harjai K, Sharma P, Capalash N. Ellagic acid derivatives from Terminalia chebula Retz. increase the susceptibility of Pseudomonas aeruginosa to stress by inhibiting polyphosphate kinase. J Appl Microbiol 2015;118: 817–25; doi: https://doi.org/10.1111/jam.12733
24. Mohamed WF, Askora AA, Mahdy MMH, EL-Hussieny EA, Abu-Shady HM. Isolation and characterization of bacteriophages active against Pseudomonas aeruginosa strains isolated from diabetic foot infections. Arch Razi Inst 2022;77:2187–200; doi: https://doi.org/10.22092/ARI.2022.359032.2357
25. Rafiee F, Haghi F, Bikas R, Heidari A, Gholami M, Kozakiewicz A, et al. Synthesis, characterization and assessment of anti-quorum sensing activity of copper(II)-ciprofloxacin complex against Pseudomonas aeruginosa PAO1. AMB Express 2020;10:82; doi: https://doi.org/10.1186/s13568-020-01017-3
26. Naga NG, Zaki AA, El-Badan DE, Rateb HS, Ghanem KM, Shaaban MI. Inhibition of Pseudomonas aeruginosa quorum sensing by methyl gallate from Mangifera indica. Sci Rep 2023;13(1):17942; doi: https://doi.org/10.1038/s41598-023-44063-0
27. Khan F, Lee JW, Javaid A, Park SK, Kim YM. Inhibition of biofilm and virulence properties of Pseudomonas aeruginosa by sub-inhibitory concentrations of aminoglycosides. Microb Pathogenesis 2020;146:104249; doi: https://doi.org/10.1016/j.micpath.2020.104249
28. Chadha J, Harjai K, Chhibber S. Repurposing phytochemicals as anti-virulent agents to attenuate quorum sensing-regulated virulence factors and biofilm formation in Pseudomonas aeruginosa. Microb Biotechnol 2022;15:1695–718; doi: https://doi.org/10.1111/1751-7915.13981
29. Trancassini M, Brenciaglia MI, Ghezzi MC, Cipriani P, Filadoro F. Modification of Pseudomonas aeruginosa virulence factors by sub-inhibitory concentrations of antibiotics. J Chemother 1992;4:78–81; doi: https://doi.org/10.1080/1120009X.1992.11739144
30. Fonseca AP, Extremina C, Fonseca AF, Sousa JC. Effect of subinhibitory concentration of piperacillin/tazobactam on Pseudomonas aeruginosa. J Med Microbiol 2004;53:903–10; doi: https://doi.org/10.1099/jmm.0.45637-0
31. Morkunas B, Galloway WRJD, Wright M, Ibbeson BM, Hodgkinson JT, O’Connell KMG, et al. Inhibition of the production of the Pseudomonas aeruginosa virulence factor pyocyanin in wild-type cells by quorum sensing autoinducer-mimics. Org Biomol Chem 2012;10:8452–64; doi: https://doi.org/10.1039/C2OB26501J
32. Fekete-Kertész I, Berkl Z, Buda K, Fenyvesi É, Szente L, Molnár M. Quorum quenching effect of cyclodextrins on the pyocyanin and pyoverdine production of Pseudomonas aeruginosa. Appl Microbiol Biotechnol 2024;108:271; doi: https://doi.org/10.1007/s00253-024-13104-7
33. Wretlind B, Pavlovskis OR. Pseudomonas aeruginosa elastase and its role in pseudomonas infections. Rev Infect Dis 1983;5:S998–1004; doi: https://doi.org/10.1093/clinids/5.supplement_5.s998
34. Everett MJ, Davies DT. Pseudomonas aeruginosa elastase (LasB) as a therapeutic target. Drug Discovery Today 2021;26:2108–23; doi: https://doi.org/10.1016/j.drudis.2021.02.026
35. Zhou JW, Li PL, Ji PC, Yin KY, Tan XJ, Chen H, et al. Carbon quantum dots derived from resveratrol enhances anti-virulence activity against Pseudomonas aeruginosa. Surf Interfaces 2024;44:103662; doi: https://doi.org/10.1016/j.surfin.2023.103662
36. Ren Y, Zhu R, You X, Li D, Guo M, Fei B, et al. Quercetin: a promising virulence inhibitor of Pseudomonas aeruginosa LasB in vitro. Appl Microbiol Biotechnol 2024;108:57; doi: https://doi.org/10.1007/s00253-023-12890-w