Modulation of quorum sensing and biofilm of Gram-negative bacterial pathogens by Cinnamomum zeylanicum L.

The development of antibiotic resistant microbial pathogens has become a global health threat and a major concern in modern medicine. The problem of antimicrobial resistance (AMR) has majorly arisen due to sub-judicious use of antibiotics in health care and livestock industry. A slow progress has been made in last two decades in discovery of new antibiotics. A new strategy in combatting AMR is to modulate or disarm the microbes for their virulence and pathogenicity. Plants are considered as promising source for new drugs against AMR pathogens. In this study, fraction-based screening of the Cinnamomum zeylanicum extract was performed followed by detailed investigation of antiquorum sensing and antibiofilm activities of the most active fraction that is, C. zeylanicum hexane fraction (CZHF). More than 75% reduction in violacein pigment of C. violaceum 12472 was overserved. CZHF successfully modulated the virulence of Pseudomonas aeruginosa PAO1 by 60.46%-78.35%. A similar effect was recorded against Serratia marcescens MTCC 97. A broad-spectrum inhibition of biofilm development was found in presence of sub-MICs of CZHF. The colonization of bacteria onto the glass coverslips was remarkably reduced apart from the reduction in exopolymeric substances. Alkaloids and terpenoids were found in CZHF. GC/MS analysis revealed the presence of cinnamaldehyde dimethyl acetal, 2-propenal, coumarin, and α-copaene as major phytocompounds. This study provides enough evidence to support potency of C. zeylanicum extract in targeting the virulence of Gram -ve pathogenic bacteria. The plant extract or active compounds can be developed as successful drugs after careful in vivo examination to target microbial infections. RESEARCH HIGHLIGHTS: Hexane fraction of Cinnamomum zeylanicum is active against QS and biofilms. The broad-spectrum antibiofilm activity was further confirmed by microscopic analysis. Dimethyl acetal, 2-propenal, coumarin, α-copaene, and so forth are major phytocompounds.

Coumarin Exhibits Broad-Spectrum Antibiofilm and Antiquorum Sensing Activity against Gram-Negative Bacteria: In Vitro and In Silico Investigation.

Quorum sensing (QS) and biofilm inhibition are recognized as the novel drug targets for the broad-spectrum anti-infective strategy to combat the infections caused by drug-resistant bacterial pathogens. Many compounds from medicinal plants have been found to demonstrate anti-infective activity. However, broad-spectrum anti-QS and antibiofilm efficacy and their mode of action are poorly studied. In this study, the efficacy of coumarin was tested against QS-regulated virulent traits of Gram-negative bacteria. Coumarin inhibited the production of violacein pigment in Chromobacterium violaceum 12472 by 64.21%. Similarly, there was 87.25, 70.05, 76.07, 58.64, 48.94, and 81.20% inhibition of pyocyanin, pyoverdin, and proteolytic activity, lasB elastase activity, swimming motility, and rhamnolipid production, respectively, in Pseudomonas aeruginosa PAO1. All tested virulence factors of Serratia marcescens MTCC 97 were also suppressed by more than 50% at the highest sub-minimum inhibitory concentration. Moreover, the biofilms of bacterial pathogens were also inhibited in a dose-dependent manner. Molecular docking and molecular dynamics (MD) simulation gave insights into the possible mode of action. The binding energy obtained by docking studies ranged from -5.7 to -8.1 kcal mol-1. Coumarin was found to be docked in the active site of acylhomoserine lactone (AHL) synthases and regulatory proteins of QS. MD simulations further supported the in vitro studies where coumarin formed a stable complex with the tested proteins. The secondary structure of all proteins showed a negligible change in the presence of coumarin. Computational studies showed that the possible mechanisms of anti-QS activity were the inhibition of AHL synthesis, antagonization of QS-regulatory proteins, and blocking of the receptor proteins. The findings of this study clearly highlight the potency of coumarin against the virulence factors of Gram-negative bacterial pathogens that may be developed as an effective inhibitor of QS and biofilms.

Broad-spectrum quorum sensing and biofilm inhibition by green tea against gram-negative pathogenic bacteria: Deciphering the role of phytocompounds through molecular modelling.

The emerging prevalence of multidrug-resistance in Gram-negative pathogens, due to conventional antimicrobial therapeutics, has led the researchers to emphasize on development of alternative novel strategies to suppress the bacterial virulence and pathogenicity through inhibition of quorum sensing (QS) and biofilms. QS is a bacterial communication system to produce density-dependent response via chemical signalling that controls pathogenesis and biofilms formation. Leaves of green tea are used worldwide as beverage which is also known for its broad-spectrum therapeutic efficacy. In this work, we have identified and characterized the most bioactive faction of green tea extract and evaluated the anti-QS and antibiofilm activity of green tea ethyl acetate fraction (GTEF) i.e. most active fraction, on three different Gram-negative bacterial pathogens. GTEF inhibited the violacein production by >75% in C. violaceum 12472. Many virulence factors of P. aeruginosa PAO1 viz. pyocyanin, pyoverdin, exoprotease, elastase, rhamnolipid production, and swimming motility were remarkably reduced in presence of sub-MICs of GTEF. Moreover, prodigiosin, protease activity, cell surface hydrophobicity, and swimming of S. marcescens MTCC 97 were also decreased significantly by the supplementation of GTEF in culture media. GTEF exhibited broad-spectrum antibiofilm action with >80% reduction in biofilm formation of test pathogens. In silico studies gave a mechanistic insight of action of GTEF. Molecular modelling revealed that phytoconstituents detected by GC/MS exhibited affinity (in order of 104 M-1) towards AHL synthases (LasI and EsaI). The molecular binding between phytocompounds and receptor proteins (LasR, RhlR, and PqsR) of QS circuit was also energetically favourable (ΔG°≥ 5.0 kcal mol-1) and supported by hydrogen bonds and hydrophobic interactions. These compounds were found to be docked in ligand binding domain of CviR and occupied same cavity as that of its antagonist. Squalene and thunbergol interacted with LasA at tartaric acid binding pocket and the complex was strengthened with binding energy -5.9 kcal mol-1. Moreover, interaction of thunbergol with biofilm-associated proteins viz. PilT and PilY1, might be disabling the pilus assembly and consequently inhibiting biofilm formation. In vivo validation of results suggested the protective role GTEF against QS-mediated pathogenicity and it might become a novel non-antibiotic QS inhibitor to control bacterial infection.

Genotoxicity inhibition by Syzygium cumini (L.) seed fraction and rutin: understanding the underlying mechanism of DNA protection.

Considering the ethnopharmacological importance of Syzygium cumini's seed and the lack of information on the antimutagenic and DNA-protecting mechanisms, a fraction-based study was conducted. Four different (hexane, chloroform, ethyl acetate, and aqueous) fractions were obtained from the sequential extraction of the methanolic extract of the seed. The most active antioxidant fraction (ethyl acetate) contained significant amount of phenolics and flavonoids. LC-qTOF-MS analysis of the ethyl acetate fraction revealed the presence of rutin, myricetin, naringin, cuscohygrin, and epoxycarryophyllone as constituent phytocompounds. The ethyl acetate fraction (100 µg ml-1) and a selected compound (rutin, 40 µg ml-1) showed remarkable decrease in the revertants frequency range from 74-77% and 66-84%, respectively, against both the mutagens (sodium azide (NaN3) and methyl methane sulfonate (MMS)) in the Salmonella typhimurium tester strains. All the statistical analyses were at a significance level of 0.05 between the different treatment groups. Moreover, the underlying mechanism of antimutagenicity using different treatment regime for rutin was explored. MMS-mediated DNA fragmentation and oxidation in lymphocytes were also shown to be decreased significantly when treated with the ethyl acetate fraction and rutin. Oxidative damage to pBR322 plasmid DNA was also reduced when incubated with different concentration of the ethyl acetate fraction and rutin. Biophysical (UV, fluorescence, ITC, etc.) and computational methods were employed to obtain a closer look at the DNA-rutin interaction. The data obtained clearly revealed that the ethyl acetate fraction exhibited promising antimutagenic and DNA-protective activity and its flavonoid constituents, including rutin, contribute significantly to the observed activity.

Antioxidant properties and anti-mutagenic potential of Piper Cubeba fruit extract and molecular docking of certain bioactive compounds.

Spices and herbs are recognized as sources of natural antioxidants and thus play an important role in the chemoprevention of diseases and aging. Piper cubeba is one among them and known for its medicinal properties for decades. Various biological activities are associated with its extract and phytocompounds. However, the anti-mutagenic activity of antioxidant rich extract is less explored. In this study, we performed the fraction-based antioxidant activity of P. cubeba using four different assays and evaluated the anti-mutagenic activity of most potent antioxidant fraction using Salmonella typhimurium tester strains against four mutagens (methyl methanesulfonate [MMS], sodium azide [SA], benzo(a)pyrene, and 2-aminoflourene) respectively. Among all tested fractions at 25-200 µg/ml, ethanolic extract revealed highest antioxidant activity and significant anti-mutagenicity against both direct and indirect acting mutagens at least one tester strain. Phytochemical analysis by gas chromatography-mass spectrometry (GC/MS) revealed the presence of various phytocompounds including copaene, isocaryophyllene, α-cubebene, etc. Molecular docking studies on DNA binding interactions of GC/MS detected phytocompounds highlight the possible mode of binding. In summary, these in vitro studies have provided the scientific basis for validation of using this plant in the traditional system of medicine and highlighted the need for exploring the role of various compounds for therapeutic efficacy. On the other hand, synergistic interaction among phytocompounds is to be explored to optimize or standardize the extracts for the exploitation in modern phytomedicine.