Influence of clove oil on certain quorum-sensing-regulated functions and biofilm of Pseudomonas aeruginosa and Aeromonas hydrophila

Quorum sensing (QS) plays an important role in virulence, biofilm formation and survival of many pathogenic bacteria including Pseudomonas aeruginosa. This signalling pathway is considered as novel and promising target for anti-infective agents. In the present investigation, effect of the Sub-MICs of clove oil on QS regulated virulence factors and biofilm formation was evaluated against P. aeruginosa PAO1 and Aeromonas hydrophila WAF-38 strain. Sub-inhibitory concentrations of the clove oil demonstrated statistically significant reduction of las- and rhl-regulated virulence factors such as LasB, total protease, chitinase and pyocyanin production, swimming motility and exopolysaccharide production. The biofilm forming capability of PAO1 and A. hydrophila WAF-38 was also reduced in a concentration-dependent manner at all tested sub-MIC values. Further, the PAO1-preinfected Caenorhabditis elegans displayed an enhanced survival when treated with 1.6% v/v of clove oil. The above findings highlight the promising anti-QS-dependent therapeutic function of clove oil against P. aeruginosa.     

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.     

3,6-Di(pyridin-2-yl)-1,2,4,5-tetrazine (pytz)-capped silver nanoparticles (TzAgNPs) inhibit biofilm formation of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>: a potential approach toward breaking the wall of biofilm through reactive oxygen species (ROS) generation

Microbial biofilms are factions of surface-colonized cells encompassed in a matrix of extracellular polymeric substances. Profound application of antibiotics in order to treat infections due to microbial biofilm has led to the emergence of several drug-resistant microbial strains. In this context, a novel type of 3,6-di(pyridin-2-yl)-1,2,4,5-tetrazine (pytz)-capped silver nanoparticles (TzAgNPs) was synthesized, and efforts were given to test its antimicrobial and antibiofilm activities against Pseudomonas aeruginosa, a widely used biofilm-forming pathogenic organism. The synthesized TzAgNPs showed considerable antimicrobial activity wherein the MIC value of TzAgNPs was found at 40 μg/mL against Pseudomonas aeruginosa. Antibiofilm activity of TzAgNPs was also tested against Pseudomonas aeruginosa by carrying out an array of experiments like microscopic observation, crystal violet assay, and protein count using the sub-MIC doses of TzAgNPs. Since TzAgNPs showed efficient antibiofilm activity, thus, in the present study, efforts were put together to investigate the underlying cause of biofilm attenuation of Pseudomonas aeruginosa by using TzAgNPs. To this end, we discerned that the sub-MIC doses of TzAgNPs increased ROS level considerably in the bacterial cell. The result showed that the ROS level and microbial biofilm formation are inversely proportional. Thus, the attenuation in microbial biofilm could be attributed to the accumulation of ROS level. Furthermore, it was also duly noted that microorganisms upon treatment with TzAgNPs exhibited considerable diminution in virulence factors (protease and pyocyanin) in contrast to the control where the organisms were not treated with TzAgNPs. Thus, the results indicated that TzAgNPs exhibit considerable reduction in the development of biofilms and spreading of virulence factors. Taken together, all the results indicated that TzAgNPs could be deemed to be a promising agent for the prevention of microbial biofilm development that might assist to fight against infections linked to biofilm.     

Mechanistic understanding of Phenyllactic acid mediated inhibition of quorum sensing and biofilm development in Pseudomonas aeruginosa

Pseudomonas aeruginosa depends on its quorum sensing (QS) system for its virulence factors’ production and biofilm formation. Biofilms of P. aeruginosa on the surface of indwelling catheters are often resistant to antibiotic therapy. Alternative approaches that employ QS inhibitors alone or in combination with antibiotics are being developed to tackle P. aeruginosa infections. Here, we have studied the mechanism of action of 3-Phenyllactic acid (PLA), a QS inhibitory compound produced by Lactobacillus species, against P. aeruginosa PAO1. Our study revealed that PLA inhibited the expression of virulence factors such as pyocyanin, protease, and rhamnolipids that are involved in the biofilm formation of P. aeruginosa PAO1. Swarming motility, another important criterion for biofilm formation of P. aeruginosa PAO1, was also inhibited by PLA. Gene expression, mass spectrometric, functional complementation assays, and in silico data indicated that the quorum quenching and biofilm inhibitory activities of PLA are attributed to its ability to interact with P. aeruginosa QS receptors. PLA antagonistically binds to QS receptors RhlR and PqsR with a higher affinity than its cognate ligands N-butyryl-l-homoserine lactone (C₄–HSL) and 2-heptyl-3,4-dihydroxyquinoline (PQS; Pseudomonas quinolone signal). Using an in vivo intraperitoneal catheter-associated medaka fish infection model, we proved that PLA inhibited the initial attachment of P. aeruginosa PAO1 on implanted catheter tubes. Our in vitro and in vivo results revealed the potential of PLA as anti-biofilm compound against P. aeruginosa.

     

Food color ‘Azorubine’ interferes with quorum sensing regulated functions and obliterates biofilm formed by food associated bacteria: An in vitro and in silico approach

Quorum sensing (QS) plays a crucial role in different stages of biofilm development, virulence production, and subsequently to the growth of bacteria in food environments. Biofilm mediated spoilage of food is one of the ongoing challenge faced by the food industry worldwide as it incurs substantial economic losses and leads to various health issues. In the present investigation, we studied the interference of quorum sensing, its regulated virulence functions, and biofilm in food-associated bacteria by colorant azorubine. In vitro bioassays demonstrated significant inhibition of QS and its coordinated virulence functions in Chromobacterium violaceum 12472 (violacein) and Pseudomonas aeruginosa PAO1 (elastase, protease, pyocyanin, and alginate). Further, the decrease in the production EPS (49–63%) and swarming motility (61–83%) of the pathogens was also recorded at sub-MICs. Azorubine demonstrated broad-spectrum biofilm inhibitory potency (50–65%) against Chromobacterium violaceum, Pseudomonas aeruginosa, E. coli O157:H7, Serratia marcescens, and Listeria monocytogenes. ROS generation due to the interaction between bacteria and azorubine could be responsible for the biofilm inhibitory action of the food colorant. Findings of the in vitro studies were well supported by molecular docking and simulation analysis of azorubine and QS virulence proteins. Azorubine showed strong binding to PqsA as compared to other virulent proteins (LasR, Vfr, and QscR). Thus, it is concluded that azorubine is a promising candidate to ensure food safety by curbing the menace of bacterial QS and biofilm-based spoilage of food and reduce economic losses.     

Effect of Cinnamon Oil on Quorum Sensing-Controlled Virulence Factors and Biofilm Formation in Pseudomonas aeruginosa

Quorum sensing (QS) is a system of stimuli and responses in bacterial cells governed by their population density, through which they regulate genes that control virulence factors and biofilm formation. Despite considerable research on QS and the discovery of new antibiotics, QS-controlled biofilm formation by microorganisms in clinical settings has remained a problem because of nascent drug resistance, which requires screening of diverse compounds for anti-QS activities. Cinnamon is a dietary phytochemical that is traditionally used to remedy digestive problems and assorted contagions, which suggests that cinnamon might contain chemicals that can hinder the QS process. To test this hypothesis, the anti-QS activity of cinnamon oil against P. aeruginosa was tested, measured by the inhibition of biofilm formation and other QS-associated phenomena, including virulence factors such as pyocyanin, rhamnolipid, protease, alginate production, and swarming activity. To this end, multiple microscopy analyses, including light, scanning electron and confocal microscopy, revealed the ability of cinnamon oil to inhibit P. aeruginosa PAO1 biofilms and their accompanying extracellular polymeric substances. This work is the first to demonstrate that cinnamon oil can influence various QS-based phenomena in P. aeruginosa PAO1, including biofilm formation.     

Eugenol exhibits anti-virulence properties by competitively binding to quorum sensing receptors

The primary objective of this study was to ascertain the anti-biofilm and anti-virulence properties of sub-minimum inhibitory concentration (MIC) levels of eugenol against the standard strain PAO1 and two multi-drug resistant P. aeruginosa clinical isolates utilizing quorum sensing inhibition (QSI). Eugenol at 400 μM significantly reduced biofilm formation on urinary catheters and the virulence factors (VF) including extracellular polysaccharides, rhamnolipid, elastase, protease, pyocyanin, and pyoverdine (p < 0.001). Further, eugenol exhibited a marked effect on the production of QS signals (AIs) (p < 0.001) without affecting their chemical integrity. In silico docking studies demonstrated a stable molecular binding between eugenol and QS receptor(s) in comparison with respective AIs. Investigation on reporter strains confirmed the competitive binding of eugenol to a QS receptor (LasR) as the possible QSI mechanism leading to significant repression of QS associated genes besides the VF genes (p < 0.001). This study provides insights, for the first time, into the mechanism of the anti-virulence properties of eugenol.     

Aspergillus ochraceopetaliformis SSP13 modulates quorum sensing regulated virulence and biofilm formation in Pseudomonas aeruginosa PAO1

Pseudomonas aeruginosa is an opportunistic nosocomial pathogen causing the majority of acute and persistent infections in human beings. The ability to form biofilm adds a new dimension to its resistance to conventional therapeutic agents. In the present study, down-regulation of quorum sensing regulated virulence and biofilm development resulting from exposure to Aspergillus ochraceopetaliformis SSP13 extract was investigated. The in vitro results inferred impairment in the production of LasA protease, LasB elastase, chitinase, pyocyanin, exopolysaccharides and rhamnolipids. In addition, motility and biofilm formation by P. aeruginosa PAO1 was significantly altered. The in vitro results were further supported by molecular docking studies of the metabolites obtained from GC-MS analysis depicting the quorum sensing attenuation by targeting the receptor proteins LasR and RhlR. The in vitro and in silico studies suggested new avenues for the development of bioactive metabolites from A. ochraceopetaliformis SSP13 extract as potential anti-infective agents.     

Anti-pathogenic Potential of Coral Associated Bacteria Isolated from Gulf of Mannar Against Pseudomonas aeruginosa

Infections of Pseudomonas aeruginosa are of great concern because of its increasing resistance towards conventional antibiotics. Quorum sensing system of P. aeruginosa acts as a global regulator of almost all the virulence factors and majorly its biofilm formation. In the present study, quenching of QS system of P. aeruginosa has been explained with bioactives from bacteria associated with the coral Acropora digitifera. Isolated bioactives inhibited the expression of various virulence traits of P. aeruginosa like biofilm formation, and the production of extracellular enzymes like protease and elastase. This study also emphasises the potential of coral associated bacteria in producing bioactive agents with anti-pathogenic properties.     

Doxycycline interferes with quorum sensing-mediated virulence factors and biofilm formation in Gram-negative bacteria

Inhibition of quorum sensing (QS)-regulated virulence factors including biofilm is a recognized anti-pathogenic drug target. The search for safe and effective anti-QS agents is expected to be useful to combat diseases caused by multidrug-resistant bacteria. In this study, effect of a commonly used antibiotic, doxycycline on QS was evaluated using sensor strains of Chromobacterium violaceum (ATCC 12472 and CVO26) and Pseudomonas aeruginosa PAO1. Sub-MICs of doxycycline reduced QS-controlled violacein production in C. violaceum to a significant degree (70 %) and showed a significant reduction of LasB elastase (67.2 %), pyocyanin (69.1 %), chitinase (69.8 %) and protease (65 %) production and swarming motility (74 %) in P. aeruginosa PAO1 over untreated controls. Similar results were also recorded against a clinical strain of P. aeruginosa (PAF-79). Interestingly, doxycycline at respective sub-MICs (4 and 32 μg ml^?1) significantly reduced the biofilm-forming capability and exopolysaccharide production in both the strains of P. aeruginosa (PAO1 and PAF-79) over untreated controls. The results of this study highlight the multiple actions of doxycycline against QS-linked traits/virulence factors and its potential to attenuate virulence of P. aeruginosa.     

Inhibitory effects of 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF) on acyl-homoserine lactone-mediated virulence factor production and biofilm formation in Pseudomonas aeruginosa PAO1

4-Hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF), a non-halogenated furanone found in a variety of fruits, has been shown to have antimicrobial activity. However, few studies have focused on its inhibitory effect on bacterial quorum sensing (QS) at levels below the non-inhibitory concentration. In this study, 0.1 μM HDMF decreased the production of QS signal molecules and inhibited QS-controlled biofilm formation by Pseudomonas aeruginosa PAO1 without causing growth inhibition. In the presence of 0.1 and 1.0 μM HDMF, biofilm production by PAO1 was reduced by 27.8 and 42.6%, respectively, compared to that by untreated control cells. HDMF (1.0 μM) also significantly affected virulence factor expression (regulated by the las, rhl, and pqs system), resulting in a significant reduction in the production of LasA protease (53.8%), rhamnolipid (40.9%), and pyocyanin (51.4%). This HDMF-dependent inhibition of virulence factor expression was overcome by increasing the levels of two QS signal molecules of P. aeruginosa, N-(3-oxo-dodecanoyl)-L-homoserine lactone and N-butyryl-L-homoserine lactone, suggesting reversible competitive inhibition between HDMF and these molecules. The results of this study indicate that HDMF has great potential as an inhibitor of QS, and that it may be of value as a therapeutic agent and in biofilm control, without increasing selective pressure for resistance development.     

Screening and anti-virulent study of N-acyl homoserine lactones DNA aptamers against Pseudomonas aeruginosa quorum sensing

In Pseudomonas aeruginosa, a quorum sensing (QS) system regulates the expression of many virulence factors. N-acyl homoserine lactone (HSL) is the signal molecule of QS system. In order to find a novel HSL binder to interfere with QS signaling and to attenuate P. aeruginosa virulence, an amino lactam surrogate (ALS) of HSL was used as a target to screen HSL aptamers with the technique of systematic evolution of ligands by exponential enrichment (SELEX). Eight HSL aptamers with high affinities for 3O-C12-HSL (20 nM ≤ K _d < 35 nM) or C4-HSL (25 nM < K _d < 50 nM) were finally obtained. In vitro QS-inhibiting study of P. aeruginosa showed that HSL aptamers could inhibit virulence in a dose-dependent manner. ALSap-8 which bound C4-HSL primarily acted on the rhl system and inhibited the secretion of pyocyanin. ALSap-5 which bound 3O-C12-HSL not only showed strong inhibitory activity on biofilm formation as well as secretions of LasA protease and LasB elastase, but also reduced pyocyanin secretion. Since the las system is capable of activating the rhl system mildly, we speculated that ALSap-5 can simultaneously interfere with the las and rhl systems. High-affinity aptamers against HSL in this study are novel QS and virulence-inhibitors, and may have potential as drug candidates for the treatment of P. aeruginosa infection.     

Anti-quorum sensing and antibiofilm potential of Alternaria alternata, a foliar endophyte of Carica papaya, evidenced by QS assays and in-silico analysis

In the present study, secondary metabolites from an endophytic fungus, Alternaria alternata, colonizing Carica papaya, demonstrated antiquorum sensing properties against Pseudomonas aeruginosa. This study reports the antagonistic effects of fungal crude extract of A. alternata against the various quorum sensing (QS) associated virulent factors such as percentage decrease in production of pyocyanin, alginate, chitinase and rhamnolipid; significant decrease in proteases activity such as LasA protease activity, staphylolytic activity, Las B elastase; and a marked decrease in biofilm formation and associated factors such as exopolysaccharide (EPS) production and cell surface hydrophobicity (CSH). Further, motility pattern i.e., swimming and swarming was also found to be inhibited. This down regulation of QS and associated factors are further supported by in-silico analysis of interaction between QS receptor LasR and bioactive molecules viz., sulfurous acid, 2-propyl tridecyl ester and 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester present in fungal crude extract, found based on GCMS analysis, sketches the modulating ability of QS expression. This is the first report on an endophytic fungus of C. papaya having a role in QS inhibition against P. aeruginosa and lays a platform to explore further the endophytes for potent therapeutic agents in QS.     

Identification of a Pseudomonas sp. that Inhibits RHL System of Quorum Sensing

The production of many Pseudomonas aeruginosa virulence factors and secondary metabolites is regulated in concert with cell density by quorum sensing (QS). Therefore, strategies designed to inhibit QS are promising for the control of diseases. Here, we succeeded in isolating soil bacteria (56 out of 7,000 isolates) capable of inhibiting violacein production by Chromobacterium violaceum CV026. We focused on an isolate identified as a Pseudomonas sp. based on its 16S rRNA nucleotide sequence. A partially purified inhibitor factor(s) derived from culture supernatants consisted of at least three major components by HPLC analysis. A more highly purified preparation (16 μg/ml) specifically inhibited rhl-controlled pyocyanin and rhamnolipid production by wild type P. aeruginosa PAO1 (PAO1) and a QS double mutant PAO-MW1, without affecting growth. A significant inhibitory effect on elastase, protease and biofilm was also observed. These results provide compelling evidence that the inhibitor(s) interferes with the QS system. The identities of the inhibitors remain to be established.     

Furvina inhibits the 3-oxo-C12-HSL-based quorum sensing system of Pseudomonas aeruginosa and QS-dependent phenotypes

Disruption of cell–cell communication or quorum sensing (QS) is considered a stimulating approach for reducing bacterial pathogenicity and resistance. Although several QS inhibitors (QSIs) have been discovered so far their clinical use remains distant. This problem can be circumvented by searching for QSI among drugs already approved for the treatment of different diseases. In this context, antibiotics have earned special attention. Whereas at high concentrations antibiotics exert a killing effect, at lower concentrations they may act as signaling molecules and as such can modulate gene expression. In this study, the antibiotic furvina was shown to be able to cause inhibition of the 3-oxo-C12-HSL-dependent QS system of Pseudomonas aeruginosa. Furvina interacts with the LasI/LasR system. The data were validated by modeling studies. Furvina can also reduce biofilm formation and decrease the production of QS-controlled virulence factors.