Quorum Sensing Inhibitors Research Articles

Paeonol Interferes With Quorum-Sensing in Pseudomonas aeruginosa and Modulates Inflammatory Responses InVitro and In Vivo.

Developing quorum-sensing (QS) based anti-infection drugs is one of the most powerful strategies to combat multidrug-resistant bacteria. Paeonol has been proven to attenuate the QS-controlled virulence factors of P. aeruginosa by down-regulating the transcription of QS signal molecules. This research aimed to assess the anti-virulence activity and mechanism of paeonol against P. aeruginosa infection in vitro and in vivo. In this study, paeonol was found to reduce the adhesion and invasion of P.aeruginosa to macrophages and resist the cytotoxicity induced by P.aeruginosa. Paeonol reduced the expression of virulence factors of P.aeruginosa by inhibiting QS, thereby reducing the LDH release and damage of P.aeruginosa-infected macrophages. Paeonol can inhibit bacterial virulence and enhance the ability of macrophages to clear P.aeruginosa. In addition, paeonol exerts anti-inflammatory activity by reducing the expression of inflammatory cytokines and increasing the production of anti-inflammatory cytokines. Paeonol treatment significantly inhibited the activation of TLR4/MyD88/NF-κB signaling pathway and decreased the inflammation response of P. aeruginosa-infected macrophages. Paeonol also significantly reduced the ability of P.aeruginosa to infect mice and reduced the inflammatory response. These data suggest that paeonol can inhibit the virulence of P.aeruginosa and decrease the inflammation response in P.aeruginosa-infected macrophages and mice, which can decrease the damage induced by P.aeruginosa infection and enhance the ability of macrophages to clear bacteria. This study supports the further development of new potential anti-infective drugs based on inhibition of QS and virulence factors.

Evaluation of Novel Quorum Sensing Inhibitors Targeting Auto-Inducer 2 (AI-2) for the Control of Avian Pathogenic Escherichia coli Infections in Chickens.

ABSTRACTAvian pathogenic Escherichia coli (APEC) associated with colibacillosis results in high morbidity and mortality, and severe economic losses to the poultry industry. APEC is a zoonotic pathogen and can infect humans through contaminated poultry products. Vaccination and antibiotic treatment are currently used to control APEC infections; however, the limited effect of vaccines and the emergence of antibiotic-resistant strains have necessitated the development of novel therapeutics. Here, we evaluated seven quorum sensing inhibitors (QSI) identified in our previous study, in APEC-infected chickens. QSIs were administered orally (~92 to 120 μg/bird) and chickens were challenged subcutaneously with APEC. Among them, QSI-5 conferred the best protection (100% reduction in mortality, 82% to 93% reduction in lesions [airsacculitis, perihepatitis, lung congestion, pericarditis] severity, and 5.2 to 6.1 logs reduction in APEC load). QSI-5 was further tested in chickens raised on built-up floor litter using an optimized dose (1 mg/L) in drinking water. QSI-5 reduced the mortality (88.4%), lesion severity (72.2%), and APEC load (2.8 logs) in chickens, which was better than the reduction observed with currently used antibiotic sulfadimethoxine (SDM; mortality 35.9%; lesion severity up to 36.9%; and APEC load up to 2.4 logs). QSI-5 was detected in chicken's blood after 0.5 h with no residues in muscle, liver, and kidney. QSI-5 increased the body weight gain with no effect on the feed conversion ratio and cecal microbiota of the chickens. Metabolomic studies revealed reduced levels of 5′-methylthioadenosine in QSI-5-treated chicken serum. In conclusion, QSI-5 displayed promising effects in chickens and thus, represents a novel anti-APEC therapeutic.IMPORTANCE Avian pathogenic Escherichia coli (APEC), a subgroup of ExPEC, is a zoonotic pathogen with public health importance. Quorum sensing is a mechanism that regulates virulence, biofilm formation, and pathogenesis in bacteria. Here, we identified a novel quorum sensing autoinducer-2 inhibitor, QSI-5, which showed higher anti-APEC efficacy in chickens compared to the currently used antibiotic, sulfadimethoxine at a much lower dose (up to 4,500 times). QSI-5 is readily absorbed with no residues in the tissues. QSI-5 also increased the chicken’s body weight gain and did not impact the cecal microbiota composition. Overall, QSI-5 represents a promising lead compound for developing novel anti-virulence therapies with significant implications for treating APEC infections in chickens as well as other ExPEC associated infections in humans. Further identification of its target(s) and understanding the mechanism of action of QSI-5 in APEC will add to the future novel drug development efforts that can overcome the antimicrobial resistance problem.

2-tert-Butyl-1,4-benzoquinone, a food additive oxidant, reduces virulence factors of Chromobacterium violaceum

Multi-drug resistant bacteria have been paying increasing attentions. Quorum sensing (QS) and QS-associated biofilms are showing the vital reasons for antibiotics resistant. In this study, 2-tert-butyl-1,4-benzoquinone (TBQ), the oxidant of tert-butyl hydroquinone (TBHQ), can potentially be the QS inhibitor (QSI) of Chromobacterium violaceum ATCC12472. After treated with 50 μg/mL of TBQ, the violacein synthesis, chitinase, hemolysin and swimming motility of C. violaceum decreased by 70.6, 72, 70.5, and 88.45%, respectively. Further investigation demonstrated that combined TBQ (50 μg/mL) and ciprofloxacin (sub-MIC, 0.2 μg/mL) treatment not only inhibited biofilm formation by 73.27% but also improved the susceptibility of the biofilm to ciprofloxacin. The underlying mechanisms involved in the reduction of virulence factors and inhibition of biofilm formation were explored by quantitative real-time PCR, and confocal laser scanning microscopy (CLSM). The results showed that the expression levels of cviI, cviR, vioA, hsmP, chiA, and lolA were reduced by 51, 66, 49, 72, 77, and 72%, respectively. In addition, we determined that TBQ treatment could improve the survival rate of animal model Caenorhabditis elegans to 55.6% following infection with C. violaceum. Thus, food additive oxidant TBQ exhibits new QSI activity against C. violaceum and potential anti-biofilm application in food industry.

Andrographolide Inhibits Biofilm and Virulence in Listeria monocytogenes as a Quorum-Sensing Inhibitor.

Listeria monocytogenes is a major foodborne pathogen that can cause listeriosis in humans and animals. Andrographolide is known as a natural antibiotic and exhibits good antibacterial activity. We aimed to investigate the effect of andrographolide on two quorum-sensing (QS) systems, LuxS/AI-2 and Agr/AIP of L. monocytogenes, as well as QS-controlled phenotypes in this study. Our results showed that neither luxS expression nor AI-2 production was affected by andrographolide. Nevertheless, andrographolide significantly reduced the expression levels of the agr genes and the activity of the agr promoter P2. Results from the crystal violet staining method, confocal laser scanning microscopy (CLSM), and field emission scanning electron microscopy (FE-SEM) demonstrated that andrographolide remarkably inhibited the biofilm-forming ability of L. monocytogenes 10403S. The preformed biofilms were eradicated when exposed to andrographolide, and reduced surviving cells were also observed in treated biofilms. L. monocytogenes treated with andrographolide exhibited decreased ability to secrete LLO and adhere to and invade Caco-2 cells. Therefore, andrographolide is a potential QS inhibitor by targeting the Agr QS system to reduce biofilm formation and virulence of L. monocytogenes.

Fabrication of Isopropanolamine-Decorated Coumarin Derivatives as Novel Quorum Sensing Inhibitors to Suppress Plant Bacterial Disease.

Emerging pesticide-resistant phytopathogenic bacteria have become a stumbling block in the development and use of pesticides. Quorum sensing (QS) blockers, which interfere with bacterial virulence gene expression, are a compelling way to manage plant bacterial disease without resistance. Herein, a series of isopropanolamine-decorated coumarin derivatives were designed and synthesized, and their potency in interfering with QS was investigated. Notably, compound A5 exhibited a better bioactivity with median effective concentration (EC50) values of 6.75 mg L-1 against Xanthomonas oryzae pv. oryzae (Xoo) than bismerthiazol (EC50 = 21.9 mg L-1). Further biochemical studies revealed that compound A5 disturbed biofilm formation and suppressed bacterial virulence factors and so forth. Moreover, compound A5 decreased the expression of QS-related genes. Interestingly, compound A5 had the acceptable control effect (53.2%) toward Xoo in vivo. Overall, this study identifies a novel lead compound for the development of bactericide candidates to control plant bacterial diseases by interfering with QS.

Glycosylation increases the anti-QS as well as anti-biofilm and anti-adhesion ability of the cyclo (L-Trp-L-Ser) against Pseudomonas aeruginosa

Pseudomonas aeruginosa infections pose a huge threat to cystic fibrosis patients, as well as those suffering from immunodeficiency. Antimicrobial resistance, especially multi-drug resistance, due to its ability to aggregate the compact biofilm, makes it more inefficient to treat this pathogen with traditional antibiotics. Biofilm and quorum sensing (QS) have become the alternative targets for treating P. aeruginosa infections. Previously, a cyclic dipeptide cyclo(L-Trp-L-Ser) has been identified as a QS inhibitor of P. aeruginosa. On the other hand, some monosaccharides have been proved lectin-targeting behavior and to mediate biofilm formation and adhesion of P. aeruginosa. We constructed novel cyclic dipeptide-carbohydrate conjugates as a low molecular weight dual-functional QS inhibitor, which can not only enhance its anti-QS activity but also enable good anti-biofilm and anti-adhesion ability. The IC50 of galactosylated c(WS) on biofilm formation and glass adhesion was 1/6 and 1/4 of that of the unmodified cyclic dipeptide, respectively. And the ability to eliminate the preformed biofilm was increased 10-fold. Furthermore, the carbohydrate conjugates can increase the germicidal efficiency of clinical antibiotic azithromycin when used synergistically. Our results provide a novel scaffold for developing anti-virulence adjuvants when taken with clinical antibiotics.

Competitive interaction of thymol with cviR inhibits quorum sensing and associated biofilm formation in Chromobacterium violaceum.

Biofilm formation associated with quorum sensing (QS) is a community behaviour displayed by many gram-negative pathogenic bacteria that provide survival advantages in hostile conditions. The inhibitors of QS interrupt bacterial communication and coordinated cell signalling for community aggregation in the biofilm. Thymol, a natural monoterpenoid, was tested against QS in Chromobacterium violaceum. As the first step, the interaction of thymol with cviR protein was investigated using in silico approach followed by validation using detailed in vitro experiments. The QS and biofilm studies were performed using the wild type of strain C. violaceum ATCC 12,472 and a mini-Tn5 mutant CV026. The MIC of thymol was established by the broth micro-dilution method, and IC50 value for violacein inhibition was quantified spectrophotometrically by extracting the violacein from the treated cells. Inhibitory effect of thymol on the biofilm was quantified by the crystal violet staining method, and scanning electron microscopy (SEM) was employed for biofilm visualization. The expression of biofilm associated genes (hmsH, hmsR, pilB, and pilT) was evaluated by qRT-PCR analysis. The in silico molecular interactions of thymol with cviR exhibited a G-score of - 5.847kcal/mol, binding with TYR-80 and SER-155 by Pi-Pi stacking and H-bond, respectively. The MIC of thymol was 160µg/mL, and the IC50 for violacein inhibition was estimated to be 28µg/mL. The thymol treatment significantly reduced the biofilm viability and biomass by > 80% along with disruption of the well-organized biofilm architecture. QS inhibitory activity of thymol resulted in the reduction of exopolysaccharide production, swarming motility, and downregulation of biofilm-associated hmsH, hmsR, pilB, and pilT genes. This data establishes the QS inhibitory role of thymol in the biofilm formation in C. violaceum.

Acyl Homoserine Lactone Analogs as Potential Inhibitors of Quorum Sensing

Gram negative bacteria are difficult to treat and are becoming increasingly resistant to conventional antibiotics. Pseudomonas aeruginosa is one such bacterium that causes opportunistic infections in hospital settings and has been recognized by the CDC and WHO as a looming threat to public health due to few treatment options and the rise of antibiotic resistance. A promising strategy to combat antibiotic resistance is to focus on virulence, rather than growth. In an antivirulence approach, the goal is to compromise the bacterium’s pathogenicity so that it is easier for the host’s immune system to clear the infection. Many Gram negative bacteria like P. aeruginosa and Chromobacterium violaceum use a LuxIR quorum sensing system to control virulence, where the LuxI synthase makes an acyl homoserine lactone signal that is detected by the LuxR transcriptional regulator, leading to the upregulation of virulence genes. One way to block virulence is to disrupt the production of the quorum sensing signaling molecules. In this study we have designed, synthesized and structurally characterized a series of potential small molecule inhibitors of the CviI synthase from C. violaceum as a model system for the more complex P. aeruginosa system. CviI converts a hexanoyl‐acyl carrier protein (hexanoyl‐ACP) and S‐adenosylmethionine (SAM) to a C6‐homoserine lactone (C6‐HSL). The potential inhibitors were designed to mimic the structural moieties of the acyl‐ACP starting material and the C6‐HSL product. The appropriate chain length should give specificity for the CviI synthase over other LuxI homologs. By varying the head group, the hope is to find an inhibitor that maximizes the affinity for the acyl‐ACP binding pocket, inhibiting the enzyme from making the C6‐HSL signal. An advantage of our methodology is that the synthesis only requires one step, which enables a quick production of a library to elucidate structure‐activity relationships. In future experiments, these compounds will be assayed in C. violaceum to determine if they possess anti‐quorum sensing and, specifically, anti‐CviI activity. The information gained could serve as a guide for future quorum sensing inhibitor development for P. aeruginosa and other Gram negative pathogens.

Bacterial biofilms: role of quorum sensing and quorum quenching

Bacterial biofilms provide an adjustable strategy to manage themselves in the existing conditions. Biofilms of pathogenic bacteria act as a reservoir for various device and non-device related diseases which are tough to cure. Exposure to a high dose of antibiotics is not an appropriate solution to this problem as high antibiotic concentrations lead to the generation of Multi-drug resistant strains as well as affect the human body. So, it is needed to bypass the use of antibiotics to prevent bacterial biofilms. In this context, Quorum Sensing (QS) may be a potential target since biofilm formation is regulated by QS. N-acyl homoserine lactones (N-AHL) act as predominant QS signal molecules in Gram-negative bacteria. Counteraction of the QS-regulated activities using quorum quenching may be an alternative way to combat biofilm formation in bacteria. Quorum sensing inhibitors (QSIs) and QQ enzymes play a significant role in this regard either by interference with the signal generation, perception, or by degradation, and chemical modification, respectively. Many quorum quenching enzymes have been reported from bacteria. Extremophilic bacteria have also been reported to produce potent quorum quenching enzymes which can effectively break down N-AHLs.

In vitro Activity of Antivirulence Drugs Targeting the las or pqs Quorum Sensing Against Cystic Fibrosis Pseudomonas aeruginosa Isolates.

The chronic lung infection caused by Pseudomonas aeruginosa is a major cause of morbidity and mortality in cystic fibrosis (CF) patients. Antivirulence drugs targeting P. aeruginosa quorum sensing (QS) systems are intensively studied as antibiotics substitutes or adjuvants. Previous studies, carried out in non-CF P. aeruginosa reference strains, showed that the old drugs niclosamide and clofoctol could be successfully repurposed as antivirulence drugs targeting the las and pqs QS systems, respectively. However, frequent emergence of QS-defective mutants in the CF lung undermines the use of QS inhibitors in CF therapy. Here, QS signal production and susceptibility to niclosamide and clofoctol have been investigated in 100 P. aeruginosa CF isolates, with the aim of broadening current knowledge on the potential of anti-QS compounds in CF therapy. Results showed that 85, 78, and 69% of the CF isolates from our collection were proficient for the pqs, rhl, and las QS systems, respectively. The ability of both niclosamide and clofoctol to inhibit QS and virulence in vitro was highly variable and strain-dependent. Niclosamide showed an overall low range of activity and its negative effect on las signal production did not correlate with a decreased production of virulence factors. On the other hand, clofoctol displayed a broader QS inhibitory effect in CF isolates, with consequent reduction of the pqs-controlled virulence factor pyocyanin. Overall, this study highlights the importance of testing new antivirulence drugs against large panels of P. aeruginosa CF clinical isolates before proceeding to further pre-clinical studies and corroborates previous evidence that strains naturally resistant to QS inhibitors occur among CF isolates. However, it is also shown that resistance to pqs inhibitors is less frequent than resistance to las inhibitors, thus supporting the development of pqs inhibitors for antivirulence therapy in CF.

Mechanisms of Inhibition of Quorum Sensing as an Alternative for the Control of E. coli and Salmonella.

Quorum sensing (QS) is a process of cell–cell communication for bacteria such as E. coli and Salmonella that cause foodborne diseases, with the production, release, and detection of autoinducer (AI) molecules that participate in the regulation of virulence genes. All of these proteins are useful in coordinating collective behavior, the expression of virulence factors, and the pathogenicity of Gram-negative bacteria. In this work, we review the natural or synthetic inhibitor molecules of QS that inactivate the autoinducer and block QS regulatory proteins in E. coli and Salmonella. Furthermore, we describe mechanisms of QS inhibitors (QSIs) that act as competitive inhibitors, being a useful tool for preventing virulence gene expression through the downregulation of AI-2 production pathways and the disruption of signal uptake. In addition, we showed that QSIs have negative regulatory activity of genes related to bacterial biofilm formation on clinical artifacts, which confirms the therapeutic potential of QSIs in the control of infectious pathogens. Finally, we discuss resistance to QSIs, the design of next-generation QSIs, and how these molecules can be leveraged to provide a new antivirulence therapy to combat diseases caused by E. coli or Salmonella.

Natural bioactives versus synthetic antibiotics for the attenuation of quorum sensing-regulated virulence factors of Pseudomonas aeruginosa.

Aim: To study the influence of plant volatiles, bioactives and synthetic antibiotics on the attenuation of the quorum sensing (QS)-regulated virulence factors of Pseudomonas aeruginosa. Materials & methods: QS inhibition; the QS-regulated virulence factors pyocyanin, hemolysin, elastase, protease, alginate and pyochelin; and motility phenotypes were performed at sub-MIC to check the attenuation effect of 24 agents on the virulence of P. aeruginosa. Results: Eighteen out of 24 assayed compounds exhibited anti-QS activity and reduced the production of all virulence factors. Cinnamaldehyde, zingerone and lavender oil exhibited a significant reduction in motility phenotypes. Conclusion: Natural phytomolecules as a whole or their bioactives could be used to develop antivirulence drugs after in vivo evaluation.

Investigation of interspecies crosstalk between probiotic Bacillus subtilis BR4 and Pseudomonas aeruginosa using metabolomics analysis

Pseudomonas aeruginosa (PA) is an opportunistic pathogen that causes high mortality in cystic fibrosis patients. Treatment failures often occur due to the emergence of antibiotic resistance. Inhibition of virulence factors production without suppressing the growth of the pathogens is a potential alternative strategy to control the antibiotic resistance. In order to accomplish, three different interaction studies were performed using Bacillus subtilis BR4, PA and their extracellular contents. Firstly, co-cultivation was performed with different cell density of BR4 or PA. In co-culture setup (F), high cell density of BR4 significantly inhibits the biofilm formation of PA in a growth-independent manner (p < 0.01). To substantiate the biofilm inhibition, LC-MS/MS was performed and metabolic profile of monocultures and cocultures were compared. Multivariate analysis corroborated that metabolic profile of coculture setup (F) is drastically different from other coculture and monoculture setups. To check the effect of extracellular content of PA on BR4, supernatant of PA was extracted with ethyl acetate and different concentration of that extract (PA-EXT) was supplemented with BR4 culture. Exogenous supplementation PA-EXT (40 μg/mL) led to increased biofilm inhibitory activity (p < 0.01) in BR4. Further, to check the effect of extracellular content of BR4, PA was grown in the supernatant of BR4. PA survives in the spent media of BR4 without biofilm formation. Though 50% spent media of BR4 was replaced with fresh media, PA could not produce biofilm. In support of this, LC-MS/MS analysis has revealed that abundance of quorum sensing (QS) signals was reduced in the spent media grown PA than control. Furthermore, BR4 protects zebrafish larvae (Danio rerio) against PA infection and increases their survival rate (p < 0.05). We found that PA-induced oxidative stress and apoptosis were also significantly reduced in the BR4-pretreated larval group than control group. These results clearly indicate that BR4 exerts growth-independent QS inhibition in PA, suggesting that it could be used as a probiotic for future therapeutic interventions.

Sustained Release of a Synthetic Autoinducing Peptide Mimetic Blocks Bacterial Communication and Virulence In Vivo.

AbstractThere is significant interest in approaches to the treatment of bacterial infections that block virulence without creating selective pressures that lead to resistance. Here, we report the development of an “anti‐virulence” strategy that exploits the activity of potent synthetic inhibitors of quorum sensing (QS) in Staphylococcus aureus. We identify peptide‐based inhibitors of QS that are resistant to sequestration or degradation by components of murine tissue and demonstrate that encapsulation of a lead inhibitor in degradable polymer microparticles provides materials that substantially inhibit QS in vitro. Using a murine abscess model, we show that this inhibitor attenuates methicillin‐resistant S. aureus (MRSA) skin infections in vivo, and that sustained release of the inhibitor from microparticles significantly improved outcomes compared to mice that received a single‐dose bolus. Our results present an effective and modular approach to controlling bacterial virulence in vivo and could advance the development of new strategies for skin infection control.

Computational screening of natural compounds as putative quorum sensing inhibitors targeting drug resistance bacteria: Molecular docking and molecular dynamics simulations

Quorum sensing (QS) is a bacterial communication strategy controlling cells density, biofilm formation, virulence, sporulation, and survival. Since QS is considered a virulence factor in drug-resistant pathogenic bacteria, inhibition of QS can contribute to control the spread of these bacteria. We propose in this study to test in silico, 19 natural compounds for their potential to inhibit QS transcriptional regulators of Pseudomonas aeruginosa (LasR and PqsE) and Chromobacterium violaceum (CviR and CviR′). Molecular docking was performed to explore the binding energies between selected compounds, and QS signaling proteins. Additionally, molecular dynamics (MD) simulations of the complexes protein-ligand were tested to evaluate the stability of the complexs throughout the simulation process. The simulation interaction diagram (SID) was achieved to compute the radius of gyration (rGyr), solvent accessible surface area (SASA), intramolecular HBs, molecular surface area (MolSA), and polar surface area (PSA). Additionally, the physicochemical properties, pharmacokinetics, drug-likeness, and toxicity analysis of the best-selected compounds were determined. Among these compounds, catechin and nakinadine B were identified as potent QS antagonists that showed the best XP GScore and stable interaction during molecular dynamic simulation. Catechin interacts with LasR and CviR′ displaying XP GScore −10.969 kcal/mol and −9.936 kcal/mol respectively. Additionally, nakinadine B interacts with PqsE and CviR giving XP GScore −7.442 kcal/mol and −10.34 kcal/mol respectively. RMSD plot analysis showed that both catechin and nakinadine B were stable during 50 ns simulation time with the tested target proteins. The predictive result of toxicity demonstrated that catechin and nakinadine B doesn't induce cytotoxicity, immunotoxicity, carcinogenicity, mutagenicity, hepatotoxicity and were at medium risk for hERG inhibition. Also they were found to be inactive for androgen receptor and aromatase. These results imply that catechin and nakinadine B may be suggested as QS modulators, which may reduce the virulence factors of drug-resistant bacteria.

Inhibition of Quorum-Sensing Regulator from Pseudomonas aeruginosa Using a Flavone Derivative.

Quorum sensing (QS) is a cell-to-cell communication process that controls bacterial collective behaviors. The QS network regulates and coordinates bacterial virulence factor expression, antibiotic resistance and biofilm formation. Therefore, inhibition of the QS system is an effective strategy to suppress the bacterial virulence. Herein, we identify a phosphate ester derivative of chrysin as a potent QS inhibitor of the human pathogen Pseudomonas aeruginosa (P. aeruginosa) using a designed luciferase reporter assay. In vitro biochemical analysis shows that the chrysin derivative binds to the bacterial QS regulator LasR and abrogates its DNA-binding capability. In particular, the derivative exhibits higher anti-virulence activity compared to the parent molecule. All the results reveal the potential application of flavone derivative as an anti-virulence compound to combat the infectious diseases caused by P. aeruginosa.

Antibacterial Properties of Ag-Cu Alloy Nanoparticles Against Multidrug-Resistant Pseudomonas aeruginosa Through Inhibition of Quorum Sensing Pathway and Virulence-Related Genes.

Objective: To evaluate the application of Ag-Cu NPs as quorum sensing (QS) inhibitors and attenuate virulence expression to overcome the global crisis of multidrug-resistant (MDR) P. aeruginosa. Methods: Ag-Cu NPs were synthesized by co-reduction of silver-nitrate and copper-nitrate (Ag:Cu = 1:1 0.75 μM). In this cross-sectional study, a total of eighty clinical strains of P. aeruginosa were collected from patients with burns. The antibacterial and resistance pattern of the clinical isolated was determined using the microdilution and Kirby Bauer disk methods. The effect of sub-MIC of Ag-Cu NPs on the expression of lasI, exoS and toxA in five clinical isolates of imipenem-resistant P. aeruginosa was performed using qRT-PCR. Results: The characterization methods confirm the formation of the Ag-Cu alloy NPs with agglomerated spherical morphology and particle sizes of about 30-40 nm. We observed that the MIC and MBC of Ag-Cu alloy NPs against MDR P. aeruginosa was found to be 2.5 and 5 μg ml-1, respectively. The effects of a sub-inhibitory concentration of Ag-Cu NPs on MDR P. aeruginosa QS and virulence-related genes showed that the expression level of QS regulatory and virulence genes significantly decreased in both MDR P. aeruginosa and reference strain under Ag-Cu sub-MIC treatment. Conclusion: Ag-Cu NPs could be potentially used as a promising QS inhibitor and anti-virulence compound against P. aeruginosa.

Inhibition of Quorum Sensing Regulated Virulence Factors and Biofilm Formation by Eucalyptus globulus against Multidrug-Resistant Pseudomonas aeruginosa.

ObjectivesThe quorum-sensing–inhibitory and anti-biofilm activities of the methanol extract of E. globulus leaves were determined against clinically isolated multidrug-resistant Pseudomonas aeruginosa.MethodsThe preliminary anti-quorum–sensing (AQS) activity of eucalyptus was investigated against a biosensor strain Chromobacterium violaceum ATCC 12472 (CV12472) by using the agar well diffusion method. The effect of sub-minimum inhibitory concentrations (sub-MICs) of the methanol extract of eucalyptus on different quorum-sensing–regulated virulence factors, such as swarming motility, pyocyanin pigment, exopolysaccharide (EPS), and biofilm formation, against clinical isolates (CIs 2, 3, and 4) and reference PA01 of Pseudomonas aeruginosa were determined using the swarm diameter (mm)-measurement method, chloroform extraction method, phenol (5%)-sulphuric acid (concentrated) method, and the microtiter plate assay respectively, and the inhibition (%) in formation were calculated.ResultsThe preliminary AQS activity (violacein pigment inhibition) of eucalyptus was confirmed against Chromobacterium violaceum ATCC 12472 (CV12472). The eucalyptus extract also showed concentration-dependent inhibition (%) of swarming motility, pyocyanin pigment, EPS, and biofilm formation in different CIs and PA01 of P. aeruginosa.ConclusionOur results revealed the effectiveness of the E. globulus extract for the regulation of quorum-sensing–dependent virulence factors and biofilm formation at a reduced dose (sub-MICs) and suggest that E. globulus may be a therapeutic agent for curing and controlling bacterial infection and thereby reducing the possibility of resistance development in pathogenic strains.

Effect of Autoinducer-2 Quorum Sensing Inhibitor on Interspecies Quorum Sensing.

Bacterial drug resistance caused by overuse and misuse of antibiotics is common, especially in clinical multispecies infections. It is of great significance to discover novel agents to treat clinical bacterial infections. Studies have demonstrated that autoinducer-2 (AI-2), a signal molecule in quorum sensing (QS), plays an important role in communication among multiple bacterial species and bacterial drug-resistance. Previously, 14 AI-2 inhibited compounds were selected through virtual screening by using the AI-2 receptor protein LuxP as a target. Here, we used Vibrio harveyi BB170 as a reporter strain for the preliminary screening of 14 inhibitors and compound Str7410 had higher AI-2 QS inhibition activity (IC50 = 0.3724 ± 0.1091 μM). Then, co-culture of Pseudomonas aeruginosa PAO1 with Staphylococcus aureus ATCC 25923 was used to evaluate the inhibitory effects of Str7410 on multispecies infection in vitro and in vivo. In vitro, Str7410 significantly inhibited the formation of mixed bacterial biofilms. Meanwhile, the combination of Str7410 with meropenem trihydrate (MEPM) significantly improved the susceptibility of mixed-species-biofilm cells to the antibiotic. In vivo, Str7410 significantly increased the survival rate of wild-type Caenorhabditis elegans N2 co-infected by P. aeruginosa PAO1 and S. aureus ATCC 25923. Real-time quantitative PCR analysis showed that Str7410 reduced virulence factor (pyocyanin and elastase) production and swarming motility of P. aeruginosa PAO1 by downregulating the expression of QS-related genes in strain PAO1 in co-culture with S. aureus ATCC 25923. Compound Str7410 is a candidate agent for treating drug-resistant multispecies infections. The work described here provides a strategy for discovering novel antibacterial drugs.

Naturally Occurring Quorum Sensing Inhibitors for Pseudomonas aeruginosa by Molecular Modeling

The quorum-sensing (QS) system allows organisms to communicate and form virulence factors such as biofilm. Pseudomonas aeruginosa is one of the Gram-negative bacteria leaving many casualties every year. In the past years, the bacteria's QS system has gained attention as a valuable target to design and develop new efficient antibiotics. Nature has always been an inspiration source for designing many new medicines, and using natural compounds is always the first approach to discover and design new drugs. Here, we in-silico studied 24,000 natural compounds, including first and secondary metabolites, on five known QS-related receptors of Pseudomonas aeruginosa. After several levels of screening, compounds NP-1 (3,6′-di-O-sinapoylsucrose), NP-2 (ZINC000257412737), NP-3 (3-O-β-D-rutinosin) showed the best inhibition activity on AHL synthase LasI with the docking scores of -8.988, -8.690, and -8.925 kcal/mol, respectively. Also, NP-101 (ZINC000077264779), NP-102 (ZINC000225518732), and NP-103 (ZINC000096269362) were selected for LasR-LBD with the scores of -13.355, -13.038, and -12.917 kcal/mol, respectively. In the case of LasA, docking scores of -13.357, -10.796, and -10.564 kcal/mol were obtained for NP-201 (Parishin), NP-202 (7-O-galloyl-D-sedoheptulose), and NP-203 (1,2-di-O-galloyl-6-O-p-coumaroyl-β-D-glucose), respectively. Furthermore, when the LasR-TP4 was the target, the best results were observed from NP-301 (ZINC000514288841), NP-302 (ZINC000077264754), and again NP-103 (ZINC000096269362), in which they showed docking scores of -13.175, -13.020, and -12.998 kcal/mol, respectively. Finally, the highest docking scores belonged to NP-401 (ZINC000150351649), NP-402 (ZINC000150351636), NP-403 (ZINC000150349056), which interacted with the ATPase Type IV pilus with the docking scores of -16.274, -15.773, and -15.405 kcal/mol, respectively.