Anti-virulence Activity Research Articles

Design, Synthesis, and Antimicrobial Evaluation of New Thiopyrimidine-Benzenesulfonamide Compounds.

Bacterial infection poses a serious threat to human life due to the rapidly growing resistance of bacteria to antibacterial drugs, which is a significant public health issue. This study was focused on the design and synthesis of a new series of 25 analogues bearing a 5-cyano-6-oxo-4-substituted phenyl-1,6-dihydropyrimidine scaffold hybridized with different substituted benzenesulfonamides through the thioacetamide linker M1-25. The antimicrobial activity of the new molecules was studied against various Gram-positive, Gram-negative, and fungal strains. All the tested compounds showed promising broad-spectrum antimicrobial efficacy, especially against K. pneumoniae and P. aeruginosa. Furthermore, the most promising compounds, 6M, 19M, 20M, and 25M, were subjected to minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays. In addition, the antivirulence activity of the compounds was also examined using multiple biofilm assays. The new compounds promisingly revealed the suppression of microbial biofilm formation in the examined K. pneumoniae and P. aeruginosa microbial isolates. Additionally, in silico ADMET studies were conducted to determine their oral bioavailability, drug-likeness characteristics, and human toxicity risks. It is suggested that new pyrimidine-benzenesulfonamide derivatives may serve as model compounds for the further optimization and development of new antimicrobial and antisepsis candidates.

Membrane alteration, anti-virulence properties and metabolomic perturbation of a chionodracine-derived antimicrobial peptide, KHS-Cnd, on two bacteria models

Antarctic fishes, living in an extreme environment and normally exposed to pathogens, are a promising source of antimicrobial peptides (AMPs). These are emerging as next-generation drugs due to their activity against multidrug resistant (MDR) bacteria. To infect hosts, beyond intrinsic/acquired resistance, MDR species also use virulence factors such as protease secretion. Hence, AMPs targeting virulence factors could represent a novel strategy to counteract the antimicrobial resistance (AMR).In this paper, we focused on a mutant peptide, named KHS-Cnd, that was obtained from the scaffold of the chionodracine (Cnd), a natural peptide identified in the icefish Chionodraco hamatus. We studied different effects caused by the peptide interaction with the cell membrane of two model bacteria, E. coli and B. cereus. First, we investigated its membranolytic activity revealing that the peptide action is more evident on E. coli, with a 69% uptake of the used dye at 3 μM, whereas for B. cereus we found only a 65% uptake at 6 μM. Successively, we determined the impact of this lysis on total protein concentration in the medium and an increase was estimated for both bacteria (84% after 1h for E. coli and 90% for B. cereus, respectively). Moreover, we evaluated the changes in the proteolytic activity of the supernatant, that is an important aspect of bacterial resistance, showing that there was a significant reduction for both bacteria, although at higher level in the case of E. coli. The membranolytic activity was evidenced also morphologically with TEM analysis and a different alteration was evidenced for the two bacteria.Moreover, NMR metabolomics analysis showed that peptide induces changes in E. coli and B. cereus extracellular metabolites especially at the higher tested concentrations: this metabolic variation could be used as a fingerprinting of the peptide action on bacteria physiology due to its interaction with cell wall.Finally, we determined the KHS-Cnd cytotoxicity on human primary cell lines to verify its selectivity toward bacterial cell membranes and we found low toxicity until a concentration of 5 μM. Considering that the peptide exerts both membranolytic and anti-virulence activity on E. coli at 1.5 μM, we confirmed the interesting potential of this AMP as a new drug to counteract AMR.

Anti-bacteria, anti-biofilm, and anti-virulence activity of the synthetic compound MTEBT-3 against carbapenem-resistant Klebsiella pneumoniae strains ST3984

Anti-bacteria, anti-biofilm, and anti-virulence activity of the synthetic compound MTEBT-3 against carbapenem-resistant Klebsiella pneumoniae strains ST3984

Design, synthesis, and molecular dynamic simulations of some novel benzo[d]thiazoles with anti-virulence activity against Pseudomonas aeruginosa

Inhibition of quorum sensing (QS) is an impending approach for targeting bacterial infection. Fourteen benzo[d]thiazole and 2-pyrazolo[1,5-a]pyrimidin-3-yl)benzo[d]thiazoles analogues were designed and synthesized as promising LasR antagonists with QS inhibition activity. Among the investigated compounds, compounds 3c, 3e, and 8d exhibited the highest percentage inhibition in biofilm formation (77 %, 63.9 %, 69.4 %), pyocyanin production (74.6 %, 64.9, 69.4 %), and rhamnolipids production (58.5 %, 51 %, 54.3 %) in P. aeruginosa, respectively. Additionally, compounds 3c, 3e and 8d achieved IC50 values against Las R equal 1.37 ± 0.35, 1.55 ± 0.24, 1.1 ± 0.15 μM respectively. Also, molecular docking of the target compounds into the LasR binding site co-crystalized “odDHL” revealed their binding with the essential residues for protein inhibition. Additionally, molecular dynamics simulation (MDS) experiments over 200 ns of compound 3c showed its ability to interact with the LasR binding site with dissociation of the protein's dimer confirming its action as a LasR antagonist. The obtained findings inspire further investigation for benzo[d]thiazole and 2-pyrazolo[1,5-a]pyrimidin-3-yl)benzo[d]thiazoles aiming to design and synthesize more potential QS inhibitors.

Antivirulence activities of Rutin-loaded chitosan nanoparticles against pathogenic Staphylococcus aureus

BackgroundStaphylococcus aureus is an infectious bacterium that is frequently found in healthcare settings and the community. This study aimed to prepare rutin-loaded chitosan nanoparticles (Rut-CS NPs) and assess their antibacterial activity against pathogenic strains of S. aureus.ResultsThe synthesized Rut-CS NPs exhibited an amorphous morphology with a size ranging from 160 to 240 nm and a zeta potential of 37.3 mV. Rut-CS NPs demonstrated significant antibacterial activity against S. aureus strains. Following exposure to Rut-CS NPs, the production of staphyloxanthin pigment decreased by 43.31–89.63%, leading to increased susceptibility of S. aureus to hydrogen peroxide. Additionally, visual inspection of cell morphology indicated changes in membrane integrity and permeability upon Rut-CS NPs exposure, leading to a substantial increase (107.07–191.08%) in cytoplasmic DNA leakage in the strains. Furthermore, ½ MIC of Rut-CS NPs effectively inhibited the biofilm formation (22.5–37.5%) and hemolytic activity (69–82.59%) in the S. aureus strains.ConclusionsOur study showcases that Rut-CS NPs can serve as a novel treatment agent to combat S. aureus infections by altering cell morphology and inhibiting virulence factors of S. aureus.

Proven anti-virulence therapies in combating methicillin- and vancomycin-resistant Staphylococcus aureus infections.

Despite years of efforts to develop new antibiotics for eradicating multidrug-resistant (MDR) and multi-virulent Methicillin-Resistant Staphylococcus aureus (MRSA) and Vancomycin-Resistant Staphylococcus aureus (VRSA) infections, treatment failures and poor prognoses in most cases have been common. Therefore, there is an urgent need for new therapeutic approaches targeting virulence arrays. Our aim is to discover new anti-virulence therapies targeting MRSA and VRSA virulence arrays. We employed phenotypic, molecular docking, and genetic studies to screen for anti-virulence activities among selected promising compounds: Coumarin, Simvastatin, and Ibuprofen. We found that nearly all detected MRSA and VRSA strains exhibited MDR and multi-virulent profiles. The molecular docking results aligned with the phenotypic and genetic assessments of virulence production. Biofilm and hemolysin productions were inhibited, and all virulence genes were downregulated upon treatment with sub-minimum inhibitory concentration (sub-MIC) of these promising compounds. Ibuprofen was the most active compound, exhibiting the highest inhibition and downregulation of virulence gene products. Moreover, in vivo and histopathological studies confirmed these results. Interestingly, we observed a significant decrease in wound area and improvements in re-epithelialization and tissue organization in the Ibuprofen and antimicrobial treated group compared with the group treated with antimicrobial alone. These findings support the idea that a combination of Ibuprofen and antimicrobial drugs may offer a promising new therapy for MRSA and VRSA infections. We hope that our findings can be implemented in clinical practice to assist physicians in making the most suitable treatment decisions.

Potential antivirulence and antibiofilm activities of sub-MIC of oxacillin against MDR S. aureus isolates: an in-vitro and in-vivo study

BackgroundMulti-drug resistant Staphylococcus aureus is one of the most common causes of nosocomial and community-acquired infections, with high morbidity and mortality. Treatment of such infections is particularly problematic; hence, it is complicated by antibiotic resistance, and there is currently no reliable vaccine. Furthermore, it is well known that S. aureus produces an exceptionally large number of virulence factors that worsen infection. Consequently, the urgent need for anti-virulent agents that inhibit biofilm formation and virulence factors has gained momentum. Therefore, we focused our attention on an already-approved antibiotic and explored whether changing the dosage would still result in the intended anti-virulence effect.MethodsIn the present study, we determined the antibiotic resistance patterns and the MICs of oxacillin against 70 MDR S. aureus isolates. We also investigated the effect of sub-MICs of oxacillin (at 1/4 and 1/8 MICs) on biofilm formation using the crystal violet assay, the phenol-sulphuric acid method, and confocal laser scanning microscopy (CLSM). We examined the effect of sub-MICs on virulence factors and bacterial morphology using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and electron microscopy, respectively. Moreover, we studied the effect of sub-MICs of oxacillin (OX) in-vivo using a wound infection model.ResultsOxacillin at 1/2 MIC showed a significant decrease in bacterial viability, while 1/4 and 1/8 MICs had negligible effects on treated bacterial isolates. Treatment of MDR isolates with 1/4 or 1/8 MICs of oxacillin significantly reduced biofilm formation (64% and 40%, respectively). The treated MDR S. aureus with sub-MICs of OX exhibited a dramatic reduction in several virulence factors, including protease, hemolysin, coagulase, and toxic shock syndrome toxin-1 (TSST-1) production. The sub-MICs of OX significantly decreased (P < 0.05) the gene expression of biofilm and virulence-associated genes such as agrA, icaA, coa, and tst. Furthermore, oxacillin at sub-MICs dramatically accelerated wound healing, according to the recorded scoring of histological parameters.ConclusionThe treatment of MDR S. aureus with sub-MICs of oxacillin can help in combating the bacterial resistance and may be considered a promising approach to attenuating the severity of S. aureus infections due to the unique anti-biofilm and anti-virulence activities.

Attenuation of Quorum Sensing Mediated Virulence Factors and Biofilm Formation in Pseudomonas Aeruginosa PAO1 by Substituted Chalcones and Flavonols.

Flavonoids epitomize structural scaffolds in many biologically active synthetic and natural compounds. They showcase a diverse spectrum of biological activities including anticancer, antidiabetic, antituberculosis, antimalarial, and antibiofilm activities. The antibiofilm activity of a series of new chalcones and flavonols against clinically significant Pseudomonas aeruginosa PAO1 strain was studied. Antivirulence activities were screened by analysing the effect of compounds on the production of virulence factors like pyocyanin, LasA protease, cell surface hydrophobicity, and rhamnolipid. The best ligands towards the quorum sensing proteins LasR, RhlR, and PqsR were recognised using a molecular docking study. The gene expression in P. aeruginosa after treatment with test compounds was evaluated on quorum sensing genes including rhlA, lasB, and pqsE. The antibiofilm potential of chalcones and flavonols was confirmed by the efficient reduction in the production of virulence factors and downregulation of gene expression.

Cilostazol is a promising anti-pseudomonal virulence drug by disruption of quorum sensing

Resistance to antibiotics is a critical growing public health problem that desires urgent action to combat. To avoid the stress on bacterial growth that evokes the resistance development, anti-virulence agents can be an attractive strategy as they do not target bacterial growth. Quorum sensing (QS) systems play main roles in controlling the production of diverse virulence factors and biofilm formation in bacteria. Thus, interfering with QS systems could result in mitigation of the bacterial virulence. Cilostazol is an antiplatelet and a vasodilator FDA approved drug. This study aimed to evaluate the anti-virulence activities of cilostazol in the light of its possible interference with QS systems in Pseudomonas aeruginosa. Additionally, the study examines cilostazol’s impact on the bacterium’s ability to induce infection in vivo, using sub-inhibitory concentrations to minimize the risk of resistance development. In this context, the biofilm formation, the production of virulence factors and influence on the in vivo ability to induce infection were assessed in the presence of cilostazol at sub-inhibitory concentration. Furthermore, the outcome of combination with antibiotics was evaluated. Cilostazol interfered with biofilm formation in P. aeruginosa. Moreover, swarming motility, biofilm formation and production of virulence factors were significantly diminished. Histopathological investigation revealed that liver, spleen and kidney tissues damage was abolished in mice injected with cilostazol-treated bacteria. Cilostazol exhibited a synergistic outcome when used in combination with antibiotics. At the molecular level, cilostazol downregulated the QS genes and showed considerable affinity to QS receptors. In conclusion, Cilostazol could be used as adjunct therapy with antibiotics for treating Pseudomonal infections. This research highlights cilostazol’s potential to combat bacterial infections by targeting virulence mechanisms, reducing the risk of antibiotic resistance, and enhancing treatment efficacy against P. aeruginosa. These findings open avenues for repurposing existing drugs, offering new, safer, and more effective infection control strategies.

Methyl gallate attenuates virulence and decreases antibiotic resistance in extensively drug-resistant Pseudomonasaeruginosa

Pseudomonas aeruginosa infections have become a serious threat to public health due to the increasing emergence of extensively antibiotic-resistant strains and high mortality rates. Therefore, the search for new therapeutic alternatives has become crucial. In this study, the antivirulence and antibacterial activity of methyl gallate was evaluated against six clinical isolates of extensively antibiotic-resistant P. aeruginosa. Methyl gallate exhibited minimal inhibitory concentrations of 256–384 μg/mL; moreover, the use of subinhibitory concentrations of the compound inhibited biofilm formation, swimming, swarming, proteolytic activity, and pyocyanin production. Methyl gallate plus antipseudomonal antibiotics showed a synergistic effect by reduced the MICs of ceftazidime, gentamicin and meropenem. Furthermore, the potential therapeutic effect of methyl gallate was demonstrated in an infection model. This study evidenced the antivirulence and antimicrobial activity of methyl gallate as a therapeutic alternative against P. aeruginosa.

Antibacterial and anti-virulence potential of plant phenolic compounds against Vibrio parahaemolyticus.

Background: Vibrio parahaemolyticus is a pathogenic bacterium that affects shrimp aquaculture; its infection can lead to severe production losses of up to 90%. On the other hand, plant phenolic compounds have emerged as a promising alternative to combat bacterial infections. The antibacterial and anti-virulence activity of the plant phenolic compounds quercetin, morin, vanillic acid, and protocatechuic acid against two strains of V. parahaemolyticus (Vp124 and Vp320) was evaluated. Methods: The broth microdilution test was carried out to determine phenolic compounds' antibacterial activity. Moreover, the biofilm-forming ability of V. parahaemolyticus strains in the presence of phenolic compounds was determined by total biomass staining assay using the cationic dye crystal violet. The semisolid agar displacement technique was used to observe the effect of phenolic compounds on the swimming-like motility of V. parahaemolyticus. Results: Results showed that phenolic compounds inhibited both strains effectively, with minimum inhibitory concentrations (MICs) ranging from 0.8 to 35.03 mM. Furthermore, at 0.125 - 0.5 × MIC of phenolic compounds, V. parahaemolyticus biofilms biomass was reduced by 63.22 - 92.68%. Also, quercetin and morin inhibited the motility of both strains by 15.86 - 23.64% (Vp124) and 24.28 - 40.71% (Vp320). Conclusions: The results suggest that quercetin, morin, vanillic, and protocatechuic acids may be potential agents for controlling V. parahaemolyticus.

Arctigenin from Burdock Root Exhibits Potent Antibacterial and Anti-Virulence Properties against Pseudomonas aeruginosa.

Arctium lappa (Burdock) root is used in various culinary applications especially in Asian Cuisine. Arctigenin (ARC) is a polyphenolic compound abundant in the roots of the burdock plant from which it derives its name. The emergence of bacterial resistance is a growing global worry, specifically due to the declining availability of new antibiotics. Screening for the antibacterial candidates among the safe natural products is a promising approach. The present study was aimed to assess the antibacterial activity of ARC against Pseudomonas aeruginosa exploring its effect on the bacterial cell membrane. Furthermore, the anti-virulence activities and anti-quorum sensing (QS) activities of ARC were in vitro, in vivo and in silico assessed against P. aeruginosa. The current results showed the ARC antibacterial activity was owed to its disruption effect of the cell membrane. ARC at sub-MIC significantly decreased the formation of biofilm, motility, production of extracellular enzymes and in vivo protected mice against P. aeruginosa. These anti-virulence activities of ARC are owed to its interference with bacterial QS and its expression. Furthermore, ARC showed mild effect on mammalian erythrocytes, low probability to induce resistance and synergistically combined with antibiotics. In summary, the promising anti-virulence properties of ARC indicate its potential as an effective supplement to conventional antibiotics for treating severe P. aeruginosa infections.

Dictyostelium discoideum -assisted pharmacognosy of plant resources for discovering antivirulence molecules targeting Klebsiella pneumoniae

The rise of antibiotic-resistant bacterial strains represents an important challenge for global health, underscoring the critical need for innovative strategies to confront this threat. Natural products and their derivatives have emerged as a promising reservoir for drug discovery. The social amoeba Dictyostelium discoideum is a potent model organism in this effort. Employing this invertebrate model, we introduce a novel perspective to investigate natural plant extracts in search of molecules with potential antivirulence activity. Our work established an easy-scalable developmental assay targeting a virulent strain of Klebsiella pneumoniae, with Helenium aromaticum as the representative plant. The main objective was to identify tentative compounds from the Helenium aromaticum extract that attenuate the virulence of K. pneumoniae virulence without inducing cytotoxic effects on amoeba cells. Notably, the methanolic root extract of H. aromaticum fulfilled these prerequisites compared to the dichloromethane extract. Using UHPLC Q/Orbitrap/ESI/MS/MS, 63 compounds were tentatively identified in both extracts, 47 in the methanolic and 29 in the dichloromethane, with 13 compounds in common. This research underscores the potential of employing D. discoideum-assisted pharmacognosy to discover new antivirulence agents against multidrug-resistant pathogens.

Potential antivirulence activity of sub-MIC of aspirin either alone or combined with certain antibiotics against Pseudomonas aeruginosa isolates

Potential antivirulence activity of sub-MIC of aspirin either alone or combined with certain antibiotics against Pseudomonas aeruginosa isolates

Potential antivirulence activity of sub-inhibitory concentrations of ciprofloxacin against Proteus mirabilis isolates: an in-vitro and in-vivo study.

Proteus mirabilis is a significant nosocomial pathogen that is frequently associated with a wide range ofinfections, necessitating heightened attention to mitigate potential health risks. Hence, this study was performed to investigate the impact of sub-minimum inhibitory concentrations (MICs) of ciprofloxacin (CIP) on Proteus mirabilis clinical isolates. The sub-MICs of CIP were selected using the growth curve approach. The untreated and treated isolates with sub-MICs of CIP were assessed for their biofilm development, motilities on agar, and other virulence factors. The cell morphology of untreated and treated isolates with sub-MIC of CIP was explored using electron microscope. Moreover, the expression levels of the virulence genes in isolates were measured using quantitative real-time PCR. Data revealed that sub-MICs of CIP significantly (p < 0.05), in a concentration-dependent manner, inhibited biofilm formation and other virulence factors in the selected isolates. Electron microscope analysis showed cell enlargement and various abnormalities in the cell wall and membrane integrity. Sub-MICs of CIP exhibited inhibition of virulence and alterations in morphological integrity against P. mirabilis isolates.

Antivirulence and antipathogenic activity of Mayan herbal remedies against Pseudomonas aeruginosa

Ethnopharmacological relevanceThe Yucatan Peninsula has a privileged wealth of vascular plants with which various Mayan herbal formulations have been developed. However, studies on their antipathogenic and antivirulence properties are scarce. Aim of the studyIdentify antivirulence properties in Mayan herbal remedies and determine their antipathogenic capacity in burn wounds infected with Pseudomonas aeruginosa. Materials and methodsAn ethnobotanical study was conducted in Mayan communities in central and southern Quintana Roo, Mexico. Furthermore, the antipathogenic capacity of three Mayan herbal remedies was analyzed using an animal model of thermal damage and P. aeruginosa infection. Antivirulence properties were determined by inhibiting phenotypes regulated by quorum sensing (pyocyanin, biofilm, and swarming) and by the secretion of the ExoU toxin. The chemical composition of the most active herbal remedy was analyzed using molecular network analysis. ResultsIt was found that topical administration of the remedy called “herbal soap” (HS) for eleven days maintained 100% survival of the animals, reduced establishment of the bacteria in the burn and prevented its systemic dispersion. Although no curative effect was recorded on tissue damaged by HS treatment, its herbal composition strongly reduced swarming and ExoU secretion. Through analysis of Molecular Networks, it was possible to carry out a global study of its chemical components, and identify the family of oxindole monoterpenoid alkaloids and carboline and tetrahydropyrididole alkaloids. In addition, flavonols, flavan-3-ols, and quinic acid derivatives were detected. ConclusionsThe antipathogenic and antivirulence capacity of ancient Mayan remedies makes them a potential resource for developing new antibacterial therapies to treat burns infected by P. aeruginosa.

Glycosylated Flavonoids Have Fewer Antibacterial Activity than Corresponding Aglycone: Is It True for Antivirulence Activity?

Glycosylated Flavonoids Have Fewer Antibacterial Activity than Corresponding Aglycone: Is It True for Antivirulence Activity?

Progress of Antimicrobial Mechanisms of Stilbenoids.

Antimicrobial drugs have made outstanding contributions to the treatment of pathogenic infections. However, the emergence of drug resistance continues to be a major threat to human health in recent years, and therefore, the search for novel antimicrobial drugs is particularly urgent. With a deeper understanding of microbial habits and drug resistance mechanisms, various creative strategies for the development of novel antibiotics have been proposed. Stilbenoids, characterized by a C6-C2-C6 carbon skeleton, have recently been widely recognized for their flexible antimicrobial roles. Here, we comprehensively summarize the mode of action of stilbenoids from the viewpoint of their direct antimicrobial properties, antibiofilm and antivirulence activities and their role in reversing drug resistance. This review will provide an important reference for the future development and research into the mechanisms of stilbenoids as antimicrobial agents.

Exploring the antifungal, antibiofilm and antienzymatic potential of Rottlerin in an in vitro and in vivo approach

Candida species have been responsible for a high number of invasive infections worldwide. In this sense, Rottlerin has demonstrated a wide range of pharmacological activities. Therefore, this study aimed to evaluate the antifungal, antibiofilm and antivirulence activity of Rottlerin in vitro against Candida spp. and its toxicity and antifungal activity in vivo. Rottlerin showed antifungal activity against all yeasts evaluated, presenting Minimum Inhibitory and Fungicidal Concentration (MIC and MFC) values of 7.81 to > 1000 µg/mL. Futhermore, it was able to significantly inhibit biofilm production, presenting Biofilm Inhibitory Concentration (MICB50) values that ranged from 15.62 to 250 µg/mL and inhibition of the cell viability of the biofilm by 50% (IC50) from 2.24 to 12.76 µg/mL. There was a considerable reduction in all hydrolytic enzymes evaluated, with emphasis on hemolysin where Rottlerin showed a reduction of up to 20%. In the scanning electron microscopy (SEM) analysis, Rottlerin was able to completely inhibit filamentation by C. albicans. Regarding in vivo tests, Rottlerin did not demonstrate toxicity at the therapeutic concentrations demonstrated here and was able to increase the survival of C. elegans larvae infected. The results herein presented are innovative and pioneering in terms of Rottlerin’s multipotentiality against these fungal infections.

Miconazole and phenothiazine hinder the quorum sensing regulated virulence in Pseudomonas aeruginosa

Antibiotic resistance is a major health problem worldwide. Pseudomonas aeruginosa is a Gram-negative pathogen with an arsenal of virulence factors and elevated antimicrobial resistance. It is a leading cause of nosocomial infections with high morbidity and mortality. The significant time and effort required to develop new antibiotics can be circumvented using alternative therapeutic strategies, including anti-virulence targets. This study aimed to investigate the anti-virulence activity of the FDA-approved drugs miconazole and phenothiazine against P. aeruginosa. The phenotypic effect of sub-inhibitory concentrations of miconazole and phenothiazine on biofilm, pyocyanin, protease, rhamnolipid and hemolysin activities in PAO1 strain was examined. qRT-PCR was used to assess the effect of drugs on quorum-sensing genes that regulate virulence. Further, the anti-virulence potential of miconazole and phenothiazine was evaluated in silico and in vivo. Miconazole showed significant inhibition of Pseudomonas virulence by reducing biofilm-formation approximately 45–48%, hemolytic-activity by 59%, pyocyanin-production by 47–49%, rhamnolipid-activity by approximately 42–47% and protease activity by 36–40%. While, phenothiazine showed lower anti-virulence activity, it inhibited biofilm (31–35%), pyocyanin (37–39%), protease (32–40%), rhamnolipid (35–40%) and hemolytic activity (47–56%). Similarly, there was significantly reduced expression of RhlR, PqsR, LasI and LasR following treatment with miconazole, but less so with phenothiazine. In-silico analysis revealed that miconazole had higher binding affinity than phenothiazine to LasR, RhlR, and PqsR QS-proteins. Furthermore, there was 100% survival in mice injected with PAO1 treated with miconazole. In conclusion, miconazole and phenothiazine are promising anti-virulence agents for P. aeruginosa.