Quorum Sensing Activity Research Articles

EXPERIMENTAL INVESTIGATIONS ON CAMELLIA KISSI WALL. FOR ANTIOXIDANT, ANTI-QUORUM SENSING AND ANTI-BIOFILM ACTIVITIES

Plants are known for their widespread biological activities with special reference to the use in folkloric medicines for the treatment of several diseases and metabolic disorders from ancient times. The presence of bioactive phytochemicals especially phenolic compounds, tocopherol, phytol etc. are responsible for the potential bioactivities of plants. In the present study, the radical scavenging potential of ethanolic extract of Camellia kissi wall. was evaluated. In addition, the effect of C. kissi wall. on quorum sensing (QS) associated virulence and biofilm formation in Pseudomonas aeruginosa PAO1 was also investigated. The crude extract of C. kissi wall exhibited a significant antioxidant activity against DPPH and hydroxyl radicals with a scavenging percentage of 73.77 ± 3.58 and 75.3 ± 4.45 % respectively. The plant extract also significantly inhibited the QS regulated pyocyanin production, bacterial motility and recalcitrant biofilm formation in P. aeruginosa PAO1. The anti-biofilm activity was confirmed by confocal laser scanning microscopic (CLSM) analysis. The in vitro anti QS activity of C. kissi wall. was further confirmed by molecular docking studies specifically targeting the QS transcriptional regulatory protein, LasR. The present result will provide ample avenues to exploit medicinal plants in attenuating the QS regulated microbial infections and oxidative stress in the post-antibiotic era.

Directed Evolution of the Stringency of the LuxR Vibrio fischeri Quorum Sensor without OFF-State Selection.

Stringency (low leak) is one of the most important specifications required for genetic circuits and induction systems, but it is challenging to evolve without sacrificing the maximum output level. This problem also comes from the absence of truly tunable negative selection methods. This paper reports that stringently switching variants can sometimes emerge with surprising frequency upon mutations. We randomly mutated the previously generated leaky variants of LuxR, the quorum-sensing transcription activator from Vibrio fischeri, to restore the stringency. We found as much as 10-20% of the entire population exhibited significantly improved signal-to-noise ratios compared with their parents. This indicated that these mutants arose by the loss of folding capability by accumulating destabilizing mutations, not by introducing rare adaptive mutations, thereby becoming AHL-dependent folders. Only four rounds of mutagenesis and ON-state selection resulted in the domination of the entire population by the improved variants with low leak, without direct selection pressure for stringency. With this surprising frequency, conversion into the "ligand-addicted folders" should be one of the prevailing modes of evolving stringency both in the laboratory and in nature, and the workflow described here provides a rapid and versatile method of improving the signal-to-noise ratio of various genetic switches.

Designing cyclic competence-stimulating peptide (CSP) analogs with pan-group quorum-sensing inhibition activity in Streptococcus pneumoniae

Streptococcus pneumoniae is an opportunistic human pathogen that utilizes the competence regulon, a quorum-sensing circuitry, to acquire antibiotic resistance genes and initiate its attack on the human host. Interception of the competence regulon can therefore be utilized to study S. pneumoniae cell-cell communication and behavioral changes, as well as attenuate S. pneumoniae infectivity. Herein we report the design and synthesis of cyclic dominant negative competence-stimulating peptide (dnCSP) analogs capable of intercepting the competence regulon in both S. pneumoniae specificity groups with activities at the low nanomolar range. Structural analysis of lead analogs provided important insights as to the molecular mechanism that drives CSP receptor binding and revealed that the pan-group cyclic CSPs exhibit a chimeric hydrophobic patch conformation that resembles the hydrophobic patches required for both ComD1 and ComD2 binding. Moreover, the lead cyclic dnCSP, CSP1-E1A-cyc(Dap6E10), was found to possess superior pharmacological properties, including improved resistance to enzymatic degradation, while remaining nontoxic. Lastly, CSP1-E1A-cyc(Dap6E10) was capable of attenuating mouse mortality during acute pneumonia caused by both group 1 and group 2 S. pneumoniae strains. This cyclic pan-group dnCSP is therefore a promising drug lead scaffold against S. pneumoniae infections that could be administered individually or utilized in combination therapy to augment the effects of current antimicrobial agents.

Relative assessment of anti-quorum sensing in Piper betle leaves extract via pyoverdin assay

Anti-quorum sensing property has been a popular alternative approach over bactericidal/bacteriostatic property in combating bacterial infection while simultaneously tackling the dilemma of antibiotic resistance. We presented pyoverdin assay as an initial screening to qualitatively determine the anti-quorum sensing activity in Piper betle by measuring the loss of absorbance at wavelength 630 nm. Growth of Pseudomonas aeruginosa was proven to be unaffected by the presence of the P. betle leaves extract. The regression value (R2) of the quorum sensing (QS) activity in untreated supernatant of P. aeruginosa was 0.9636 and we presented the QS activity in fold-change, normalized to untreated sample for a fair comparison between batch of assays. We further assessed the QS activity in the extracts of P. betle leaves and found the QS activity of P. aeruginosa grown in the presence of ethyl acetate extract at 200 μg/ml was reduced to 0.6-fold. As the concentrations went lower, higher fold of QS activity was observed, suggesting that P. betle leaves extract is demonstrating anti-QS activity at a higher concentration. Further fractionate of ethyl acetate crude extracts resulted in three fractionates with high anti-QS activity with >50% reduction in QS activity and five fractionates with intermediate anti-QS activity. The use of pyoverdin assay to qualitatively portray the anti-QS activity could shorten the lengthiness of extracting and measuring the signaling molecule yet, produces reliable information to screen for anti-QS activity and guide for further fractionation and purification of bioactive compound.

Disappearance of Quorum Sensing in Burkholderia glumae During Experimental Evolution.

The plant pathogen Burkholderia glumae uses quorum sensing (QS) that allows bacteria to share information and alter gene expression on the basis of cell density. The wild-type strain of B. glumae produces quorum-sensing signals (autoinducers) to detect their community and upregulate QS-dependent genes across the population for performing social and group behaviors. The model organism B. glumae was selected to investigate adaptation, estimate evolutionary parameters, and test diverse evolutionary hypotheses by using experimental evolution. The wild-type B. glumae virulent strain showed genotypic changes during regular subculture due to oxygen limitation. The laboratory-evolved clones failed to produce the signaling molecule of C8-HSL/C6-HSL for activation of the quorum-sensing system. Further, the laboratory-evolved clones failed to produce catalase and oxalate for protecting themselves from the toxic environment at stationary phase and phytotoxins (toxoflavin) for infecting rice grain, respectively. The laboratory-evolved clones were completely sequenced and compared with the wild-type. Sequencing analysis of the evolved clones revealed that mutations in QS-responsible genes (iclR), sensor genes (shk, mcp), and signaling genes (luxR) were responsible for quorum-sensing activity failure. The experimental results and sequencing analysis revealed quorum-sensing process failure in the laboratory-evolved clones. In conclusion, the wild-type B. glumae strain was often exposed to oxidative stress during regular subculture and evolved as an avirulent strain (quorum-sensing mutant) by losing the phenotypic and genotypic characteristics.

Fungal Diversity in Intertidal Mudflats and Abandoned Solar Salterns as a Source for Biological Resources.

Intertidal zones are unique environments that are known to be ecological hot spots. In this study, sediments were collected from mudflats and decommissioned salterns on three islands in the Yellow Sea of South Korea. The diversity analysis targeted both isolates and unculturable fungi via Illumina sequencing, and the natural recovery of the abandoned salterns was assessed. The phylogeny and bioactivities of the fungal isolates were investigated. The community analysis showed that the abandoned saltern in Yongyudo has not recovered to a mudflat, while the other salterns have almost recovered. The results suggested that a period of more than 35 years may be required to return abandoned salterns to mudflats via natural restoration. Gigasporales sp. and Umbelopsis sp. were selected as the indicators of mudflats. Among the 53 isolates, 18 appeared to be candidate novel species, and 28 exhibited bioactivity. Phoma sp., Cladosporium sphaerospermum, Penicillium sp. and Pseudeurotium bakeri, and Aspergillus urmiensis showed antioxidant, tyrosinase inhibition, antifungal, and quorum-sensing inhibition activities, respectively, which has not been reported previously. This study provides reliable fungal diversity information for mudflats and abandoned salterns and shows that they are highly valuable for bioprospecting not only for novel microorganisms but also for novel bioactive compounds.

Study on Antibacterial and Quorum-Sensing Inhibition Activities of Cinnamomum camphora Leaf Essential Oil.

Many essential oils (EOs) regulate the quorum-sensing (QS) system of pathogens and inhibit the virulence expression. Interference with QS can potentially reduce bacterial multidrug resistance and aid the biological control of bacterial disease. In the present work, the antibacterial and anti-QS activities of Cinnamomum camphora leaf EO were investigated. A total of 23 chemical components with relative levels ≥0.11%, including a large number of terpene compounds, were identified in C. camphora leaf EO by gas chromatography–mass spectrometry (GC-MS). The principal component was linalool, followed by eucalyptol, with relative levels of 51.57% and 22.07%, respectively. The minimum inhibitory concentration (MIC) and antibacterial activity of C. camphora EO were examined, and P. aeruginosa and E. coli ATCC25922 showed the highest and lowest sensitivity to C. camphora EO, respectively. Tests of QS inhibitory activity revealed that C. camphora EO significantly decreased the production of violacein and biofilm biomass in C. violaceum, with the maximum inhibition rates of 63% and 77.64%, respectively, and inhibited the biofilm formation and swarming movement, independent of affecting the growth of C. violaceum. Addition of C. camphora EO also resulted in downregulation of the expression of the acyl-homoserine lactones (AHL) synthesis gene (cviI) and transcription regulator (cviR), and inhibited the expression of QS-regulated virulence genes, including vioA, vioB, vioC, vioD, vioE, lasA, lasB, pilE3, and hmsHNFR. Collectively, the prominent antibacterial activity and anti-QS activities clearly support that C. camphora EO acts as a potential antibacterial agent and QS inhibitor in the prevention of bacterial contamination.

Development of Streptococcus pneumoniae Pan-Group Quorum-Sensing Modulators.

The rapid increase in multidrug-resistant pathogens is a major health concern that could bring mankind back to the pre-antibiotic era. Streptococcus pneumoniae is a highly recombinogenic opportunistic pathogen that causes a variety of deadly diseases and rapidly develops resistance to current antibiotic treatments. S. pneumoniae pathogenicity is dependent on a cell-density communication mechanism, or quorum sensing (QS), termed the competence regulon. In this work, we set out to design signal-based QS modulators capable of affecting the two specificity groups found in S. pneumoniae. Through systematic analysis and rational design, we were able to construct peptide-based pan-group QS activators and inhibitors with activities in the nanomolar range. These novel analogues are privileged scaffolds for the development of anti-virulence therapeutics against S. pneumoniae infections.

In silico Analysis Reveals Distribution of Quorum Sensing Genes and Consistent Presence of LuxR Solos in the Pandoraea Species

The most common quorum sensing (QS) system in Gram-negative bacteria consists of signaling molecules called N-acyl-homoserine lactones (AHLs), which are synthesized by an enzyme AHL synthase (LuxI) and detected by a transcriptional regulator (LuxR) that are usually located in close proximity. However, many recent studies have also evidenced the presence of LuxR solos that are LuxR-related proteins in Proteobacteria that are devoid of a cognate LuxI AHL synthase. Pandoraea species are opportunistic pathogens frequently isolated from sputum specimens of cystic fibrosis (CF) patients. We have previously shown that P. pnomenusa strains possess QS activity. In this study, we examined the presence of QS activity in all type strains of Pandoraea species and acquired their complete genome sequences for holistic bioinformatics analyses of QS-related genes. Only four out of nine type strains (P. pnomenusa, P. sputorum, P. oxalativorans, and P. vervacti) showed QS activity, and C8-HSL was the only AHL detected. A total of 10 canonical luxIs with adjacent luxRs were predicted by bioinformatics from the complete genomes of aforementioned species and publicly available Pandoraea genomes. No orphan luxI was identified in any of the genomes. However, genes for two LuxR solos (LuxR2 and LuxR3 solos) were identified in all Pandoraea genomes (except two draft genomes with one LuxR solo gene), and P. thiooxydans was the only species that harbored no QS-related activity and genes. Except the canonical LuxR genes, LuxIs and LuxR solos of Pandoraea species were distantly related to the other well-characterized QS genes based on phylogenetic clustering. LuxR2 and LuxR3 solos might represent two novel evolutionary branches of LuxR system as they were found exclusively only in the genus. As a few luxR solos were located in close proximity with prophage sequence regions in the genomes, we thus postulated that these luxR solos could be transmitted into genus Pandoraea by transduction process mediated by bacteriophage. The bioinformatics approach developed in this study forms the basis for further characterization of closely related species. Overall, our findings improve the current understanding of QS in Pandoraea species, which is a potential pharmacological target in battling Pandoraea infections in CF patients.

A novel LuxR-type solo of Sinorhizobium meliloti, NurR, is regulated by the chromosome replication coordinator, DnaA and activates quorum sensing.

The genome of Sinorhizobium meliloti, a model for studying plant-bacteria symbiosis, contains eight genes coding for LuxR-like proteins. Two of these, SinR and ExpR, are essential for quorum sensing (QS). Roles and regulation surrounding the others are mostly unknown. Here, we reveal the DNA recognition sequence and regulon of the LuxR-like protein SMc00877. Unlike ExpR, which uses the long-chain acyl homoserine lactones (AHLs) as inducers, SMc00877 functioned independently of AHLs and was even functional in Escherichia coli. A target of SMc00877 is SinR, the major regulator of AHL production in S. meliloti. Disruption of SMc00877 decreased AHL production. A weaker production of AHLs resulted in smaller microcolonies, starting from single cells, and delayed AHL-dependent regulation. SMc00877 was expressed only in growing cells in the presence of replete nutrients. Therefore, we renamed it NurR (nutrient sensitive LuxR-like regulator). We traced this nutrient-sensitive expression to transcription control by the DNA replication initiation factor, DnaA, which is essential for growth. These results indicate that NurR has a role in modulating the threshold of QS activation according to growth. We propose growth behavior as an additional prerequisite to population density for the activation of QS in S. meliloti.

Biocontrol of Soft Rot: Confocal Microscopy Highlights Virulent Pectobacterial Communication and Its Jamming by Rhodococcal Quorum-Quenching.

Confocal laser-scanning microscopy was chosen to observe the colonization and damage caused by the soft rot Pectobacterium atrosepticum and the protection mediated by the biocontrol agent Rhodococcus erythropolis. We developed dual-color reporter strains suited for monitoring quorum-sensing and quorum-quenching activities leading to maceration or biocontrol, respectively. A constitutively expressed cyan or red fluorescent protein served as a cell tag for plant colonization, while an inducible expression reporter system based on the green fluorescent protein gene enabled the simultaneous recording of signaling molecule production, detection, or degradation. The dual-colored pathogen and biocontrol strains were used to coinoculate potato tubers. At cellular quorum, images revealed a strong pectobacterial quorum-sensing activity, especially at the plant cell walls, as well as a concomitant rhodococcal quorum-quenching response, at both the single-cell and microcolony levels. The generated biosensors appear to be promising and complementary tools useful for molecular and cellular studies of bacterial communication and interference.

Characterization and Transcriptome Studies of Autoinducer Synthase Gene from Multidrug Resistant Acinetobacter baumannii Strain 863.

Quorum sensing (QS) is a cell-to-cell communication system that uses autoinducers as signaling molecules to enable inter-species and intra-species interactions in response to external stimuli according to the population density. QS allows bacteria such as Acinetobacter baumannii to react rapidly in response to environmental changes and hence, increase the chances of survival. A. baumannii is one of the causative agents in hospital-acquired infections and the number of cases has increased remarkably in the past decade. In this study, A. baumannii strain 863, a multidrug-resistant pathogen, was found to exhibit QS activity by producing N-acyl homoserine lactone. We identified the autoinducer synthase gene, which we named abaI, by performing whole genome sequencing analysis of A. baumannii strain 863. Using high resolution tandem triple quadrupole mass spectrometry, we reported that abaI of A. baumannii strain 863 produced 3-hydroxy-dodecanoyl-homoserine lactone. A gene deletion mutant was constructed, which confirmed the functionality of abaI. A growth defect was observed in the QS-deficient mutant strain. Transcriptome profiling was performed to determine the possible genes regulated by QS. Four groups of genes that showed differential expression were discovered, namely those involved in carbon source metabolism, energy production, stress response and the translation process.

ANTI QUORUM SENSING AND ANTI BIOFILM POTENTIAL OF ANOGEISSUS ACUMINATA AND MALLOTUS ROXBURGHIANUS MUELL. AGAINST PSEUDOMONAS AERUGINOSA PAO1

Pseudomonas aeruginosa causes severe persistent infections in immunocompromised patients by showing tolerance conventional antibiotic therapy by virtue of specialized hierarchy of quorum sensing (QS) network. The purpose of the study was to evaluate the efficacy of two Mizoram medicinal plants i.e., Anogeissus acuminata and Mallotus roxburghianus Muell. in attenuating QS regulated P. aeruginosa virulence. The crude leaf extracts were tested for their ability to combat QS associated virulence phenotypes such as pyocyanin production, LasB elastase and Staphylolytic activity and production of biofilm determinants such as alginate and exopolysaccharide in P. aeruginosa PAO1. The gas chromatography mass spectrometry (GC-MS) analysis revealed the presence of phytocompounds, the synergistic activity of which is responsible for the anti QS activity. The in silico studies provide the binding efficacy of obtained phytochemical with QS receptor protein, LasR. The in vitro studies followed by in silico analysis demonstrated the efficacy of phytochemical of A. acuminata and M. roxburghianus Muell. in competitively inhibiting the binding of natural ligand with LasR and thereby altering production of virulence phenotypes. The efficacy in down regulating bacterial virulence shown by A. acuminata and M. roxburghianus Muell. provides promising alternatives to develop next generation anti-pathogenic agents.

Quorum sensing is required for full virulence of Vibrio campbellii towards tiger grouper (Epinephelus fuscoguttatus) larvae.

The link between quorum sensing in Vibrio campbellii and its virulence towards tiger grouper (Epinephelus fuscoguttatus) was investigated using V. campbellii wild type and quorum-sensing mutants with inactive quorum sensing or constitutively maximal quorum-sensing activity, and signal molecule synthase mutants. The results showed that wild-type V. campbellii is pathogenic to grouper larvae, causing more than 50% mortality after 4days of challenge. Furthermore, the mortality of larvae challenged with the mutant with maximally active quorum sensing was significantly higher than that of larvae challenged with the wild type, whereas a higher survival was observed in the larvae challenged to the mutant with a completely inactive quorum-sensing system. Grouper larvae challenged with either the signal molecule synthase triple mutant, the harveyi autoinducer-1 (HAI-1) synthase mutant and the autoinducer-2 (AI-2) synthase mutant showed higher survival than larvae challenged with the wild type. In contrast, larvae challenged with the cholerae autoinducer-1 (CAI-1) synthase mutant showed high mortality. This indicates that HAI-1 and AI-2, but not CAI-1, are required for full virulence of V.campbellii towards grouper larvae. Our data suggest that quorum-sensing inhibition could be an effective strategy to control V. campbellii infections in tiger grouper.

Comparative Study of Inhibitory Potential of Dietary Phytochemicals Against Quorum Sensing Activity of and Biofilm Formation by Chromobacterium violaceum 12472, and Swimming and Swarming Behaviour of Pseudomonas aeruginosa PAO1.

SUMMARYQuorum sensing (QS) and biofilm formation are important mechanisms related to antibiotic resistance of many pathogens. Alternative treatments are needed to prevent recurrent or chronic infections caused by multi-resistant pathogens. Therefore, the aim of this study is to investigate and compare the inhibitory potential of the dietary phytochemicals: curcumin, quercetin, apigenin, pyrogallol, gallic acid and luteolin against QS of and biofilm formation by Chromobacterium violaceum ATCC 12472 and the swimming and swarming abilities of Pseudomonas aeruginosa PAO1. Anti-QS potential of the phytochemicals was evaluated qualitatively and quantitatively using C. violaceum via the disk diffusion assay based on violacein pigment inhibition at the subminimal inhibitory concentrations ranging from 46.87 to 750 µg/mL. The results of anti-QS and antibiofilm activities on C. violaceum demonstrated that all the phytochemicals except pyrogallol and gallic acid inhibited violacein production (from (11.0±0.1) to (88.2±0.1) %) in a concentration-dependent manner. In addition, the biofilm formation was also significantly inhibited (p<0.05) in the presence of all the phytochemicals ((1.38±0.08)–(84.2±0.2) %). In the present study, the results revealed that quercetin, curcumin, apigenin and luteolin could be promising QS and biofilm inhibitory agents against the C. violaceum 12472 biosensor system. Our findings also suggest that all the phytochemicals, especially curcumin, quercetin and pyrogallol, might be anti-pathogenic agents against P. aeruginosa PAO1 infections due to the ability to control QS. However, more comprehensive studies at the molecular level, explaining their anti-QS mechanisms, need to be conducted to confirm these results and identify the genes involved.

N-Acyl Homoserine Lactone-Mediated Quorum Sensing System Inhibition of Phenolic Strawberry Extract

Phenolic compounds which have many bioactivities such as antioxidant, antimicrobial and also inhibition of quorum sensing-regulated violacein pigment production properties are one of the plant secondary metabolites. The objective of this study was to determine the antioxidant, antimicrobial activity and the inhibition of quorum sensing (QSI) activity by the phenolic extract obtained from strawberry fruits (Fragaria×ananassa cv. ‘Rubygem‘). The phenolic content of extract was determined as 3089 μg GAE g fw-1. The antioxidant activity determined through the ABTS+ and FRAP method was 28.4 and 26.2 mmol TE L-1 respectively. The phenolic extract was able to inhibit all the evaluated bacteria by the disc diffusion assay in the range of 3.12–18.36 mm and could inhibit the quorum sensing phenomena in bacteria. The strawberry phenolic extract exhibited high antioxidant capacity, antimicrobial and anti-quorum sensing properties. To our knowledge, this is the first report on the quorum sensing inhibition activity of strawberry fruit extract. The discovery of non-toxic novel compounds within antibacterial and QSI activity could lead to the development of antimicrobial therapeutic agents that can be alternative and complementary treatments against antibiotic resistant bacterial pathogens

Relationship Between the Quorum Network (Sensing/Quenching) and Clinical Features of Pneumonia and Bacteraemia Caused by A. baumannii.

Acinetobacter baumannii (Ab) is one of the most important pathogens associated with nosocomial infections, especially pneumonia. Interest in the Quorum network, i.e., Quorum Sensing (QS)/Quorum Quenching (QQ), in this pathogen has grown in recent years. The Quorum network plays an important role in regulating diverse virulence factors such as surface motility and bacterial competition through the type VI secretion system (T6SS), which is associated with bacterial invasiveness. In the present study, we investigated 30 clinical strains of A. baumannii isolated in the “II Spanish Study of A. baumannii GEIH-REIPI 2000-2010” (Genbank Umbrella Bioproject PRJNA422585), a multicentre study describing the relationship between the Quorum network in A. baumannii and the development of pneumonia and associated bacteraemia. Expression of the aidA gene (encoding the AidA protein, QQ enzyme) was lower (P < 0.001) in strains of A. baumannii isolated from patients with bacteraemic pneumonia than in strains isolated from patients with non-bacteraemic pneumonia. Moreover, aidA expression in the first type of strain was not regulated in the presence of environmental stress factors such as the 3-oxo-C12-HSL molecule (substrate of AidA protein, QQ activation) or H2O2 (inhibitor of AidA protein, QS activation). However, in the A. baumannii strains isolated from patients with non-bacteraemic pneumonia, aidA gene expression was regulated by stressors such as 3-oxo-C12-HSL and H2O2. In an in vivo Galleria mellonella model of A. baumannii infection, the A. baumannii ATCC 17978 strain was associated with higher mortality (100% at 24 h) than the mutant, abaI-deficient, strain (carrying a synthetase enzyme of Acyl homoserine lactone molecules) (70% at 24 h). These data suggest that the QS (abaR and abaI genes)/QQ (aidA gene) network affects the development of secondary bacteraemia in pneumonia patients and also the virulence of A. baumannii.

A simple method for direct isolation of N-acyl-L-homoserine lactone mediated biofilm-forming rhizobacteria from roots

Plant-associated bacteria produce quorum sensing (QS) signals for community (biofilm) formation and functioning in the rhizosphere. The QS-positive biofilm-forming rhizobacteria that excel benefits to the plants are now gaining increased importance for agricultural use due to their high competitiveness. However, there is no method available to distinguish these bacteria from the roots of a plant to ease the isolation. Currently, all the plant-associated bacteria have to be isolated, purified and subsequently screened for the QS activity using biosensor strains. This study describes a direct isolation method for N-acyl-homoserine lactone (AHL) type quorum sensing signal producing bacteria from the plant root. In this method, the root sample collected from the field was overlaid directly with the bacterial growth medium seeded with the biosensor reporter, Chromobacterium violaceum (CV026). The AHL produced by QS positive rhizobacteria residing on the surface of the root will be recognized by violacein production of CV026. The bacterial isolates recovered from rice root using this method were further confirmed for the QS activity and biofilm formation. All the QS-positive strains produced N-butyryl DL-homoserine lactone (a C4-AHL type) signal in the culture medium and had biofilm formation during in vitro culturing. The 16S rRNA gene sequences of these QS-positive biofilm-forming rhizobacteria revealed that these strains are phylogenetically close to Pseudomonas siluiensis, Aeromonas hydrophila and A. caviae. Therefore, this could be a simple, rapid and straightforward procedure for isolation and characterization of quorum-sensing rhizobacteria from plant roots.

SCREENING FOR ANTI-QUORUM SENSING AND ANTI-BIOFILM ACTIVITY IN Viscum album L. EXTRACTS AND ITS BIOCHEMICAL COMPOSITION

Many opportunistic pathogenic bacteria use the Quorum Sensing (QS) system to coordinate their virulence expressions. Thus, QS can likely be a new approach to control bacterial infections. The aim of this study was to evaluate the antimicrobial, anti-biofilm, and anti-quorum sensing activities of ethanol, chloroform, and dichloromethane: methanol extracts of leaf, stem, and fruits of the European mistletoe Viscum album L. on 2 Gram-positive and 7 Gram-negative pathogenic bacteria. The extracts at concentrations ranging from 50 to 250mg/ml were tested depending on the extracts of the plant parts and the test bacteria. The extract with 50mg/ml concentration, in which no antimicrobial activity was observed, was used for anti-quorum sensing and antibiofilm studies. The dichloromethane: methanol extracts were found to show the highest biological activities. QS activities of the plant extracts were also determined using the recently established Chromobacterium violaceum CV026 reporter strain and the signaling molecule N-(ß-ketocaproyl)-L-homoserine lactone (3-oxo-C6-HSL) agar well diffusion assay. Biofilm was quantified using the microtiter plate test and the crystal violet assay. Anti-microbial, anti-biofilm, and anti-quorum sensing activity of leaf and stem extracts showed higher efficiency than fruit extracts. It was concluded that the extracts of V. album had the potential to treat microbial infections by biofilm inhibition or inhibition of QS.

Characterization of quorum sensing genes and N-acyl homoserine lactones in Citrobacter amalonaticus strain YG6

In the phylum of Proteobacteria, quorum sensing (QS) system is widely driven by synthesis and response of N-acyl homoserine lactone (AHL) signalling molecules. AHL is synthesized by LuxI homologue and sensed by LuxR homologue. Once the AHL concentration achieves a threshold level, it triggers the regulation of target genes. In this study, QS activity of Citrobacter amalonaticus strain YG6 which was isolated from clams was investigated. In order to characterise luxI/R homologues, the genome of C. amalonaticus strain YG6 (4.95 Mbp in size) was sequenced using Illumina MiSeq sequencer. Through in silico analysis, a pair of canonical luxI/R homologues and an orphan luxR homologue were identified and designated as camI, camR, and camR2, respectively. A putative lux box was identified at the upstream of camI. The camI gene was cloned and overexpressed in E. coli BL21 (DE3)pLysS. High-resolution triple quadrupole liquid chromatography mass spectrometry (LC-MS/MS) analysis verified that the CamI is a functional AHL synthase which produced multiple AHL species, namely N‑butyryl‑l‑homoserine lactone (C4-HSL), N‑hexanoyl‑l‑homoserine lactone (C6-HSL), N‑octanoyl‑l‑homoserine lactone (C8-HSL), N‑tetradecanoyl‑l‑homoserine lactone (C14-HSL) and N‑hexadecanoyl‑l‑homoserine lactone (C16-HSL) in C. amalonaticus strain YG6 and camI gene in recombinant E. coli BL21(DE3)pLysS. To our best knowledge, this is the first functional study report of camI as well as the first report describing the production of C14-HSL by C. amalonaticus.