Quorum Sensing-regulated Virulence Factors Research Articles

Myrtus communis leaf compounds as novel inhibitors of quorum sensing-regulated virulence factors and biofilm formation: In vitro and in silico investigations

Antibiotic resistance of the Gram-negative bacterium Pseudomonas aeruginosa and its ability to form biofilm through the Quorum Sensing (QS) mechanism are important challenges in the control of infections caused by this pathogen. The extract of Myrtus communis (myrtle) showed strong anti-QS effect on Chromobacterium. violaceum 6267 by inhibiting 80 % of the production of violacein pigment at a sub-MIC concentration of 1/8 (31.25 μg/mL). In addition, the extract exhibited an inhibitory effect on virulence factors of P. aeruginosa PAO1 at half MIC (125 μg/mL), significantly reducing the formation of biofilms (72.02 %), the swarming activity (75 %), and the production of protease (61.83 %) and pyocyanin (97 %). The active fraction also downregulated the expression of selected regulatory genes involved in the biofilm formation and QS in the P. aeruginosa PAO1 strain. These genes included the autoinducer synthase genes (lasI and rhlI), the genes involved in the expression of their corresponding receptors (lasR and rhlR), and the pqsA genes. The analysis of the active fraction by HPLC/UV/MS and NMR allowed the identification of three phenolic compounds, 3,5-di-O-galloylquinic acid, myricetin 3-O-α-l-rhamnopyranoside (myricitrin), and myricetin 3-O-(2″-O-galloyl)-ß-d-galactopyranoside. In silico studies showed that 3,5-di-O-galloylquinic acid, with an affinity score of −9.20 kcal/mol, had the highest affinity to the active site of the CviR protein (3QP8), a QS receptor from C. violaceum. Additionally, myricetin 3-O-α-l-rhamnopyranoside (myricitrin) and myricetin 3-O-(2″-O-galloyl)-ß-d-galactopyranoside interact to a lesser extent with 3QP8. In conclusion, this study contributed significantly to the discovery of new QS inhibitors from M. communis leaves against resistant Gram-negative pathogens.

Attenuation of Las/Rhl quorum sensing regulated virulence and biofilm formation in Pseudomonas aeruginosa PAO1 by Artocarpesin

The emergence of multidrug resistance and increased pathogenicity in microorganisms is conferred by the presence of highly synchronized cell density dependent signalling pathway known as quorum sensing (QS). The QS hierarchy is accountable for the secretion of virulence phenotypes, biofilm formation and drug resistance. Hence, targeting the QS phenomenon could be a promising strategy to counteract the bacterial virulence and drug resistance. In the present study, artocarpesin (ACN), a 6-prenylated flavone was investigated for its capability to quench the synthesis of QS regulated virulence factors. From the results, ACN showed significant inhibition of secreted virulence phenotypes such as pyocyanin (80%), rhamnolipid (79%), protease (69%), elastase (84%), alginate (88%) and biofilm formation (88%) in opportunistic pathogen, Pseudomonas aeruginosa PAO1. Further, microscopic observation of biofilm confirmed a significant reduction in biofilm matrix when P. aeruginosa PAO1 was supplemented with ACN at its sub-MIC concentration. Quantitative gene expression studies showed the promising aspects of ACN in down regulation of several QS regulatory genes associated with production of virulence phenotypes. Upon treatment with sub-MIC of ACN, the bacterial colonization in the gut of Caenorhabditis elegans was potentially reduced and the survival rate was greatly improved. The promising QS inhibition activities were further validated through in silico studies, which put an insight into the mechanism of QS inhibition. Thus, ACN could be considered as possible drug candidate targeting chronic microbial infections.

Repurposing albendazole as a potent inhibitor of quorum sensing-regulated virulence factors in Pseudomonas aeruginosa: Novel prospects of a classical drug

Pseudomonas aeruginosa has emerged as a critical superbug that poses a serious threat to public health. Owing to its virulence and multidrug resistance profiles, the pathogen demands immediate attention for devising alternate intervention strategies. In an attempt to repurpose drugs against P. aeruginosa, this preclinical study was aimed at investigating the antivirulence prospects of albendazole (AbZ), an FDA-approved anti-helminthic drug, recently predicted to disrupt quorum sensing (QS) in Chromobacterium violaceum. AbZ was scrutinized for its quorum quenching (QQ) prospects, effect on bacterial virulence, different motility phenotypes, and biofilm formation in vitro. Additionally, in silico analysis was employed to predict the molecular interactions between AbZ and QS receptors. At sub-inhibitory levels, AbZ demonstrated anti-QS activity and significantly abrogated AHL biosynthesis in P. aeruginosa. Moreover, AbZ significantly downregulated the transcript levels of QS- (lasI/lasR, rhlI/rhlR, and pqsA/pqsR) and QS-dependent virulence (aprA, lasA, lasB, plcH, and toxA) genes in P. aeruginosa. This coincided with reduced hemolysin, alginate, pyocyanin, rhamnolipids, total protease, and elastase production, thereby lowering phenotypic virulence. Molecular docking with AbZ further revealed strong associations and high binding energies with LasR (−8.8 kcal/mol), RhlR (−6.5 kcal/mol), and PqsR (−6.3 kcal/mol) receptors. AbZ also impeded bacterial motility and abolished EPS production, severely compromising pseudomonal biofilm formation. For the first time, AbZ was shown to interfere with QS circuitry and consequently disarming pseudomonal virulence. Hence, AbZ can be exploited for its antivirulence properties against P. aeruginosa.

Periodically disturbing biofilms reduces expression of quorum sensing-regulated virulence factors in Pseudomonas aeruginosa

Pseudomonas aeruginosa uses quorum sensing to regulate the expression of virulence factors. In static environments, spatial structures, such as biofilms, can increase the expression of these virulence factors. However, in natural settings, biofilms are exposed to physical forces that disrupt spatial structure, which may affect the expression of virulence factors regulated by quorum sensing. We show that periodically disturbing biofilms composed of P.aeruginosa using a physical force reduces the expression of quorum sensing-regulated virulence factors. At an intermediate disturbance frequency, the expression of virulence factors in the las, rhl, and pqs regulons is reduced. Mathematical modeling suggests that perturbation of the pqsR receptor is critical for this reduction. Removing the lasR receptor enhances the reduction in the expression of virulence factors as a result of disturbance. Our results allow identification of environments where virulence is reduced and implicate the lasR receptor as having a buffering role against disturbance.

Evaluation of Biofilm Inhibitory Efficacy of Bacopa monnieri, Acalypha indica and Calotropis gigantea Extracts and their Combination Against Wound Colonizing Bacteria Pseudomonas aeruginosa and Staphylococcus Aureus

Key factors in compromised wound healing primarily include bacterial colonization and infection. Extensive use of systemic antibiotics, preferred choice of treatment for clinically infected wounds, is often accompanied with emergence of bacterial resistance. Quorum sensing (QS), density dependent chemical communication, is emerging as a promising area of research since most infectious microorganisms operate this mechanism to realize their pathogenic potential. Foregoing research indicates the potential of studying medicinal plant extracts/components and their combinations as vital inhibitors of QS regulated virulence factors’ production. Our study aims to understand the potential of three plant extracts and their combinations as vital inhibitors of QS regulated virulence factor’s production by two wound pathogens and to achieve this our objectives are to perform their violacein inhibitory (on C. violaceum) and biofilm inhibitory properties (on Pseudomonas aeruginosa and Staphylococcus aureus). Minimum inhibitory concentration (MIC) of aqueous plant extracts against P aeruginosa and S aureus was performed (two-fold serial microdilution). Violacein inhibition was assayed using agar well diffusion while biofilm inhibition by crystal violet method. Organisms presented 5mg/ml (Bacopa monnieri ), 2.5mg/ml (Acalypha indica), and 2.5mg/ml (Calotropis gigantea) as MIC. Three sets of individual extract concentrations (sub-MIC in μg/ml) and their corresponding combinations were used for this study. Extracts exhibited more significant (P<0.0001) violacein inhibition in combination than individually. Biofilm inhibition by extracts' combination was also significantly (P<0.001) higher than that of individual extracts indicating a possible positive herb-herb interaction of phytoconstituents (synergistic or complementary). The relative decrease in response to individual extracts in higher concentrations (set 'c') by both organisms point to possible recalcitrance behavior generally exhibited by bacteria on exposure to higher antibacterial agents. The improved antibiofilm efficacy exhibited by this novel combination may serve as an alternative approach in managing wound colonization by biofilm-producing bacteria and hence faster-wound healing.

An Immunochemical Approach to Detect the Quorum Sensing-Regulated Virulence Factor 2-Heptyl-4-Quinoline N-Oxide (HQNO) Produced by Pseudomonas aeruginosa Clinical Isolates.

ABSTRACTUnderstanding quorum sensing (QS) and its role in the development of pathogenesis may provide new avenues for diagnosing, surveillance, and treatment of infectious diseases. For this purpose, the availability of reliable and efficient analytical diagnostic tools suitable to specifically detect and quantify these essential QS small molecules and QS regulated virulence factors is crucial. Here, we reported the development and evaluation of antibodies and an enzyme-linked immunosorbent assay (ELISA) for HQNO (2-heptyl-4-quinoline N-oxide), a QS product of the PqsR system, which has been found to act as a major virulence factor that interferes with the growth of other microorganisms. Despite the nonimmunogenic character of HQNO, the antibodies produced showed high avidity and the microplate-based ELISA developed could detect HQNO in the low nM range. Hence, a limit of detection (LOD) of 0.60 ± 0.13 nM had been reached in Müeller Hinton (MH) broth, which was below previously reported levels using sophisticated equipment based on liquid chromatography coupled to mass spectrometry. The HQNO profile of release of different Pseudomonas aeruginosa clinical isolates analyzed using this ELISA showed significant differences depending on whether the clinical isolates belonged to patients with acute or chronic infections. These data point to the possibility of using HQNO as a specific biomarker to diagnose P. aeruginosa infections and for patient surveillance. Considering the role of HQNO in inhibiting the growth of coinfecting bacteria, the present ELISA will allow the investigation of these complex bacterial interactions underlying infections.IMPORTANCE Bacteria use quorum sensing (QS) as a communication mechanism that releases small signaling molecules which allow synchronizing a series of activities involved in the pathogenesis, such as the biosynthesis of virulence factors or the regulation of growth of other bacterial species. HQNO is a metabolite of the Pseudomonas aeruginosa-specific QS signaling molecule PQS (Pseudomonas quinolone signal). In this work, the development of highly specific antibodies and an immunochemical diagnostic technology (ELISA) for the detection and quantification of HQNO was reported. The ELISA allowed profiling of the release of HQNO by clinical bacterial isolates, showing its potential value for diagnosing and surveillance of P. aeruginosa infections. Moreover, the antibodies and the ELISA reported here may contribute to the knowledge of other underlying conditions related to the pathology, such as the role of the interactions with other bacteria of a particular microbiota environment.

Nano-Formulation Endows Quorum Quenching Enzyme-Antibiotic Hybrids with Improved Antibacterial and Antibiofilm Activities against Pseudomonas aeruginosa.

The emergence of antibiotic resistant bacteria coupled with the shortage of efficient antibacterials is one of the most serious unresolved problems for modern medicine. In this study, the nano-hybridization of the clinically relevant antibiotic, gentamicin, with the bacterial pro-pathological cell-to-cell communication-quenching enzyme, acylase, is innovatively employed to increase its antimicrobial efficiency against Pseudomonas aeruginosa planktonic cells and biofilms. The sonochemically generated hybrid gentamicin/acylase nano-spheres (GeN_AC NSs) showed a 16-fold improved bactericidal activity when compared with the antibiotic in bulk form, due to the enhanced physical interaction and disruption of the P. aeruginosa cell membrane. The nano-hybrids attenuated 97 ± 1.8% of the quorum sensing-regulated virulence factors’ production and inhibited the bacterium biofilm formation in an eight-fold lower concentration than the stand-alone gentamicin NSs. The P. aeruginosa sensitivity to GeN_AC NSs was also confirmed in a real time assay monitoring the bacterial cells elimination, using a quartz crystal microbalance with dissipation. In protein-enriched conditions mimicking the in vivo application, these hybrid nano-antibacterials maintained their antibacterial and antibiofilm effectiveness at concentrations innocuous to human cells. Therefore, the novel GeN_AC NSs with complementary modes of action show potential for the treatment of P. aeruginosa biofilm infections at a reduced antibiotic dosage.

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.

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.

Azithromycin Exhibits Activity Against Pseudomonas aeruginosa in Chronic Rat Lung Infection Model.

Pseudomonas aeruginosa forms biofilms in the lungs of chronically infected cystic fibrosis patients, which are tolerant to both the treatment of antibiotics and the host immune system. Normally, antibiotics are less effective against bacteria growing in biofilms; azithromycin has shown a potent efficacy in cystic fibrosis patients chronically infected with P. aeruginosa and improved their lung function. The present study was conducted to evaluate the effect of azithromycin on P. aeruginosa biofilm. We show that azithromycin exhibited a potent activity against P. aeruginosa biofilm, and microscopic observation revealed that azithromycin substantially inhibited the formation of solid surface biofilms. Interestingly, we observed that azithromycin restricted P. aeruginosa biofilm formation by inhibiting the expression of pel genes, which has been previously shown to play an essential role in bacterial attachment to solid-surface biofilm. In a rat model of chronic P. aeruginosa lung infection, we show that azithromycin treatment resulted in the suppression of quorum sensing-regulated virulence factors, significantly improving the clearance of P. aeruginosa biofilms compared to that in the placebo control. We conclude that azithromycin attenuates P. aeruginosa biofilm formation, impairs its ability to produce extracellular biofilm matrix, and increases its sensitivity to the immune system, which may explain the clinical efficacy of azithromycin in cystic fibrosis patients.

Inhibition of biofilm and quorum sensing-regulated virulence factors in Pseudomonas aeruginosa by Cuphea carthagenensis (Jacq.) J. F. Macbr. Leaf extract: An in vitro study

Ethnopharmcological relevanceMicrobial biofilm formation, a quorum sensing (QS) regulated process, is one of the major causes of nosocomial and chronic infections, foodborne diseases, and associated deaths. Various approaches have been used to eradicate the menace of biofilm. Ethnomedicinal plants as potent antibiofilm agents are gaining a lot of interest in an era where the drug resistance is increasing and the availability of potent antibiotics is no longer promised. In this context, the methanol extract of Cuphea carthagenensis (CCMD), an ethno-medicinal and culinary herb, was evaluated as an antibiofilm and anti-QS agent against Pseudomonas aeruginosa.Aim of the study: The aim of the study is to evaluate the antibiofilm and anti-QS activity of an ethnomedicinal plant against a strong biofilm forming microorganism, P. aeruginosa. MethodsAntibiofilm activity of CCMD was demonstrated at different concentrations by Tissue Culture Plate, Test Tube method and other microscopic techniques. The effect of CCMD on QS and QS-related virulence factors viz. Pyocyanin, exopolymeric substance matrix (EPS), total protease, elastase, pyoverdin and swimming motility in P. aeruginosa were also evaluated. Antioxidant activity (DPPH & FRAP), total phenolic and flavonoid content were also checked. In order to determine the composition of the extract HPLC analysis was also performed. ResultsIn vitro study demonstrated a significant inhibition of biofilm formation (81.88 ± 2.57%) as well as production of QS-dependent virulence factors in P. aeruginosa. The extract also inhibited violacein production (83.31 ± 2.77%) in Chromobacterium violaceum which correlates with the reduction in QS-mediated virulence factors. The extract showed 64.79% ± 0.83% DPPH scavenging activity and reduction of ferricyanide complex (Fe3+) to the ferrous form (Fe2+) in DPPH and FRAP assay, respectively. Furthermore, the extract showed thermal stability and does not have any growth inhibitory effect on P. aeruginosa. The HPLC analysis demonstrated the presence of ellagic acid, ascorbic acid and hippuric acid in the extract. ConclusionThis work is the first to demonstrate that C. carthagenensis can attenuate biofilm formation and QS-mediated virulence factors of P. aeruginosa. Further investigation is required to use this ethnomedicinal plant (CCMD) as an important source of antibiofilm agents.

Antioxidant, anti-quorum sensing, biofilm inhibitory activities and chemical composition of Patchouli essential oil: in vitro and in silico approach

The interest in naturally occurring essential oils from medicinal plants has increased extremely over the last decade markedly because they possess antimicrobial and antioxidant protective properties against different chronic diseases. Extensive survival of drug-resistant infectious bacteria depends on quorum sensing (QS) signaling network which raises the need for alternative antibacterial compounds. The aim of this study was to examine the phytochemical compounds of patchouli essential oil (PEO) and to assess its antioxidant activity. Antioxidant studies estimated by 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging method showed that the PEO has effective antioxidant activity (IC50 19.53 µg/mL). QS inhibitory activity of PEO was examined by employing the biosensor strain, Chromobacterium violaceum CV12472. At sub-lethal concentrations, PEO potentially reduced the QS regulated violacein synthesis in CV12472 without inhibiting its cell proliferation. Moreover, it also effectively reduced the production of some QS regulated virulence factors and biofilm development in P. aeruginosa PAO1 without hindering its growth. Phytochemical analysis of PEO was done by GC/MS technique. Molecular docking of PEO major compounds with QS (LasR and FabI) and biofilm regulator proteins (MvfR and Sialidase) of PAO1 was evaluated. These phytocompounds showed potential hydrogen binding interactions with these proteins. The overall results, in vitro and in silico, suggest that PEO could be applied as biocontrol agent against antibiotic resistance pathogens. Communicated by Ramaswamy H. Sarma

Inhibition of quorum sensing-associated virulence factors and biofilm formation in Pseudomonas aeruginosa PAO1 by Mycoleptodiscus indicus PUTY1.

Pseudomonas aeruginosa is the second most emerging multidrug-resistant, opportunistic pathogen after Acinetobacter baumannii that poses a threat in nursing homes, hospitals, and patients who need devices such as ventilators and blood catheters. Its ability to form quorum sensing-regulated virulence factors and biofilm makes it more resistant to top most therapeutic agents such as carbapenems and next-generation antibiotics. In the current study, we studied the quorum quenching potential of secondary metabolites of Mycoleptodiscus indicus PUTY1 strain. In vitro observation showed a mitigation in virulence factors such as rhamnolipids, protease, elastase pyocyanin, exopolysaccharides, and hydrogen cyanide gas. Furthermore, a significant reduction in the motility such as swimming, swarming, twitching, and inhibition in biofilm formation by Pseudomonas aeruginosa PAO1 was observed. Results of in vitro studies were further confirmed by in silico studies through docking and molecular dynamic simulation of GC-MS-detected compounds of Mycoleptodiscus indicus employing LasR and RhlR proteins. Both in vitro and in silico observations indicate a new alternative approach for combating virulence of Pseudomonas aeruginosa by targeting its protein receptors LasR and RhlR. Graphical abstract.

Inhibition of biofilm formation, quorum sensing activity and molecular docking study of isolated 3, 5, 7-Trihydroxyflavone from Alstonia scholaris leaf against P.aeruginosa

The current study is to evaluate the inhibition of biofilm formation and quorum sensing activity of isolated 3, 5, 7-Trihydroxyflavone (TF) from A.scholaris leaf extract against Pseudomonas aeruginosa. The effects of isolated TF on quorum sensing-regulated virulence factors production such as swimming motility, pyocyanin production, proteolytic, EPS, metabolic assay and inhibition of biofilm formation against P.aeruginosa was evaluated by standard protocols. In addition, the interaction between the isolated TF and active sites of QS- gene (LasI/rhlI, LasR/rhlR, and AHLase) in P.aeruginosa was evaluated by molecular docking studies using AutoDock Tools version 1.5.6. Based on the structural elucidation of the isolated compound was identified as 3, 5, 7-Trihydroxyflavone. Consequently, the isolated TF shows a significant reduction of biofilm formation through the inhibition of QS-dependent phenotypes such as pyocyanin production, proteolytic, swimming motility, EPS activities against P.aeruginosa in a dose-dependent manner. Molecular docking analysis of isolated TF can interfere the signaling [N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL) and N-butanoyl-L-homoserine lactone (C4-HSL)] molecules in P.aeruginosa by QS genes (LasI, LasR, rhlI, and AHLase) regulation. The isolated TF compound from A.scholaris reveals a greater potential to inhibit biofilm and QS dependent virulence factor production in P.aeruginosa. Docking interaction studies of TF-LasR complex express higher binding affinity than the other QS gene in P.aeruginosa.

Anti quorum sensing and anti biofilm efficacy of cinnamaldehyde encapsulated chitosan nanoparticles against Pseudomonas aeruginosa PAO1

The encapsulation of cinnamaldehyde into biodegradable polymeric systems represents a possible strategy to overcome the bioavailability issues and enhance the sustained release of the drug at the target sites. In the present study, the efficacy of cinnamaldehyde encapsulated chitosan nanoparticles (CANPs) in attenuating the quorum sensing (QS) regulated virulence of P. aeruginosa PAO1 was investigated. CANPs were synthesized by ionic gelation method, characterized by dynamic light scattering (DLS), and transmission electron microscopic (TEM) analysis. The DLS and TEM analysis confirmed the synthesis of CANPs with a mean diameter of 208.12 nm. The encapsulation efficiency was observed to be 65.04 ± 3.14% and in vitro release study confirmed the slow and sustained release of cinnamaldehyde. CANPs showed significant anti quorum sensing activity by down regulating the QS regulated virulence factors and associated biofilm formation as evidenced from microscopic observation. CANPs also significantly altered the swimming and swarming motility of P. aeruginosa PAO1. The present results suggested the greater prospective of the application of CANPs as potential anti quorum sensing agents as compared to native cinnamaldehyde and suggested new avenues for development of novel anti-infective agents in the post antibiotic era.

PA5470 Counteracts Antimicrobial Effect of Azithromycin by Releasing Stalled Ribosome in Pseudomonas aeruginosa.

Pseudomonas aeruginosa causes various acute and chronic infections in humans. Treatment with azithromycin (AZM) has been shown to benefit patients with chronic P. aeruginosa infections. By binding to the exit tunnel of the 50S ribosome, AZM causes ribosome stalling and depletion of the intracellular tRNA pool. It has been shown that AZM is able to kill stationary-phase P. aeruginosa cells and repress quorum sensing-regulated virulence factors as well as swarming motility. In P. aeruginosa, the PA5470 gene encodes a putative peptide chain release factor whose expression is highly induced by macrolide antibiotics. However, its function remains unknown. Here, we found that overexpression of PA5470 increased bacterial tolerance against AZM and alleviated the repression of swarming motility. Ribosome pulldown assays revealed that PA5470 contributes to the release of ribosome stalled by AZM. We further demonstrate that overexpression of PA5470 counteracts AZM-mediated repression of the translation of the quorum sensing regulator RhlR. Overall, our results revealed a novel role of PA5470 in the bacterial response to AZM.

Anti-Quorum Sensing Activity of Forsythia suspense on Chromobacterium violaceum and Pseudomonas aeruginosa.

Background:Quorum sensing (QS) plays an important role in the production of virulence factors and pathogenicity in Pseudomonas aeruginosa, and the interruption of QS will be a hopeful pathway to combat bacterial infection.Objective:In this study, we selected Forsythia suspense (Thunb.) Vahl from traditional Chinese herbal medicines for its anti-QS activity.Materials and Methods:Anti-QS of F. suspense extracts (FSE) was monitored using the Chromobacterium violaceum 12472 bioassay. Standard methods were used to investigate the effects of FSE on QS-controlled virulence factors production, swimming motility, and biofilm establishment in P. aeruginosa PAO1.Results:FSE could obviously inhibit the violacein production in C. violaceum 12472 and also could inhibit quorum sensing–regulated virulence factors production and biofilm formation in P. aeruginosa in a concentration-dependent manner. The elastase activity and pyocyanin production were inhibited at a maximum of 40.97 and 47.58% when P. aeruginosa was grown in the presence of 0.25 g/mL FSE, which can also inhibit swimming motility of P. aeruginosa. The biofilm formation ability was decreased about 72.45% when in PAO1 cultured with the 0.25 g/mL FSE. The results suggested that FSE may be used as an alternative drug to control and handle harmful infections caused by bacterial pathogens based on QS inhibition.SUMMARY Forsythia suspense water extract could obviously inhibit the purple pigment production in C. violaceum 12472Forsythia suspense water extract could inhibit QS-regulated virulence factors production and biofilm formation in P. aeruginosa. Abbreviations used: QS: Quorum sensing, Pseudomonas aeruginosa P. aeruginosa, Forsythia suspense F. suspense, FSE: F. suspense extracts, Chromobacterium violaceum 12472 C. violaceum 12472, AIs: autoinducers, AHLs: N-acyl-homoserinelactones, LB: Luria-Bertani, MICs: Minimum inhibitory concentrations, CFU: Colony-Forming Units, ATCC: American Type Culture Collection, PBS: phosphate buffered saline

Understanding the Pathogenicity of Burkholderia contaminans, an Emerging Pathogen in Cystic Fibrosis.

Several bacterial species from the Burkholderia cepacia complex (Bcc) are feared opportunistic pathogens that lead to debilitating lung infections with a high risk of developing fatal septicemia in cystic fibrosis (CF) patients. However, the pathogenic potential of other Bcc species is yet unknown. To elucidate clinical relevance of Burkholderia contaminans, a species frequently isolated from CF respiratory samples in Ibero-American countries, we aimed to identify its key virulence factors possibly linked with an unfavorable clinical outcome. We performed a genome-wide comparative analysis of two isolates of B. contaminans ST872 from sputum and blood culture of a female CF patient in Argentina. RNA-seq data showed significant changes in expression for quorum sensing-regulated virulence factors and motility and chemotaxis. Furthermore, we detected expression changes in a recently described low-oxygen-activated (lxa) locus which encodes stress-related proteins, and for two clusters responsible for the biosynthesis of antifungal and hemolytic compounds pyrrolnitrin and occidiofungin. Based on phenotypic assays that confirmed changes in motility and in proteolytic, hemolytic and antifungal activities, we were able to distinguish two phenotypes of B. contaminans that coexisted in the host and entered her bloodstream. Whole genome sequencing revealed that the sputum and bloodstream isolates (each representing a distinct phenotype) differed by over 1,400 mutations as a result of a mismatch repair-deficient hypermutable state of the sputum isolate. The inferred lack of purifying selection against nonsynonymous mutations and the high rate of pseudogenization in the derived isolate indicated limited evolutionary pressure during evolution in the nutrient-rich, stable CF sputum environment. The present study is the first to examine the genomic and transcriptomic differences between longitudinal isolates of B. contaminans. Detected activity of a number of putative virulence factors implies a genuine pathogenic nature of this novel Bcc species.

Phenotypic heterogeneity of Pseudomonas aeruginosa isolates in the protected nature park ‘Palić’ (Serbia)

Pseudomonas aeruginosa is a globally distributed environmental bacterium, which is also a significant opportunistic pathogen of humans, animals and plants. It is considered that wide distribution of this bacterium is connected with its most significant constitutive property to form biofilms, and that this multicellular mode of growth, predominant in nature, serves as a protective mechanism against unfavourable environmental conditions. The work presented here examines the phenotypic diversity of Pseudomonas aeruginosa environmental isolates with respect to biofilm production capacity under different environmental conditions (temperature, pH, NaCl), production of virulence factors, and motility. The purpose of this work is to present the production of two quorum sensing-regulated virulence factors (rhamnolipids and pyocyanin), explore different motility tests (swimming, swarming and twitching) and discover potential relationship between assessed phenotypic features. Obtained results delineate environmental conditions coinciding with biofilm production and suggest a high correlation between rhamnolipid production levels and biofilm formation. Rhamnolipids affect motility competence, yet only the flagellum-mediated swimming motility has significant impact on the biofilm formation potential. Although it is challenging to demarcate a definitive, clear correlation between parameters tested, rhamnolipid content appears to serve as a link between the tested phenotypic factors.

Proteomic analysis reveals modulation of iron homeostasis and oxidative stress response in Pseudomonas aeruginosa PAO1 by curcumin inhibiting quorum sensing regulated virulence factors and biofilm production

The aim of the present study was to evaluate the effect of known quorum sensing inhibitors (QSIs) against reference strain Pseudomonas aeruginosa PAO1 and 32 clinical isolates. Among the evaluated QSIs, curcumin effectively inhibited the production of quorum sensing (QS) regulated virulence factors and biofilm production in the reference strain as well as all the clinical isolates. Hence, we sought to unearth the underlying molecular mechanism responsible for QS inhibition by curcumin. Proteomic, mass spectrometric and gene ontology analysis revealed that the differentially regulated proteins are indeed involved in iron acquisition, iron storage, detoxification of reactive oxygen species and synthesis of metabolic intermediates for virulence factor production. In vitro assays also confirmed the alterations in catalase, superoxide dismutase, pyocyanin and pyoverdine production and sensitivity of PAO1 to H2O2 upon curcumin exposure. All these results suggest that curcumin attenuates the QS and biofilm formation by affecting iron homeostasis and oxidative stress response of PAO1. Furthermore, successive exposure of PAO1 to curcumin for 20 successive passages does not affect the efficacy of curcumin to inhibit virulence factor and biofilm production. Hence, it is hypothesized that curcumin inhibits the virulence factor production and biofilm formation without obviously inciting any selection pressure from PAO1. Biological significanceMost of the well-known QSIs, which are effective against standard strains of P. aeruginosa, are found ineffective against QS regulated virulence determinants of clinical isolates of P. aeruginosa, suggesting the existence of resistance towards QS inhibitors. The non-toxic nature, wide range of pharmacological benefits and the ability to inhibit the QS regulated virulence factors of all the tested clinical isolates of P. aeruginosa in the current study makes curcumin as a potent antagonistic agent against P. aeruginosa. The study on proteomics of P. aeruginosa against curcumin is expected to hold a greater significance in the development of antipseudomonal regimen. The results revealed the ability of curcumin to attenuate the virulence by targeting antioxidant enzymes, iron transport and biosynthesis of metabolic intermediates involved in virulence factors production.