Microwave-Assisted Rapid Green Synthesis of Gold Nanoparticles Using Seed Extract of Trachyspermum ammi: ROS Mediated Biofilm Inhibition and Anticancer Activity.

Green synthesis of metal nanoparticles using plant extracts as capping and reducing agents for the biomedical applications has received considerable attention. Moreover, emergence and spread of multidrug resistance among bacterial pathogens has become a major health concern and lookout for novel alternative effective drugs has gained momentum. In current study, we synthesized gold nanoparticles using the seed extract of Trachyspermum ammi (TA-AuNPs), assessed its efficacy against drug resistant biofilms of Listeria monocytogenes and Serratia marcescens, and evaluated its anticancer potential against HepG2 cancer cell lines. Microwave-assisted green synthesis of gold nanoparticles was carried out and characterization was done using UV-vis spectroscopy, X-ray diffraction (XRD), transmission electron microscopy (TEM), and dynamic light scattering (DLS). Most nanoparticles were observed as spherical and spheroidal with few anisotropies with an average crystalline size of 16.63 nm. Synthesized TA-AuNPs demonstrated significant biofilm inhibitory activity against L. monocytogenes (73%) as well as S. marcescens (81%). Exopolysaccharide (EPS), motility, and CSH, key elements that facilitate the formation and maintenance of biofilm were also inhibited significantly at the tested sub-minimum inhibitory concentrations (sub-MICs). Further, TA-AuNPs effectively obliterated preformed mature biofilms of S. marcescens and L. monocytogenes by 64% and 58%, respectively. Induction of intracellular ROS production in TA-AuNPs treated bacterial cells could be the plausible mechanism for the reduced biofilm formation in test pathogens. Administration of TA-AuNPs resulted in the arrest of cellular proliferation in a concentration-dependent manner. TA-AuNPs decrease the intracellular GSH in HepG2 cancer cell lines, cells become more prone to ROS generation, hence induce apoptosis. Thus, this work proposes a new eco-friendly and rapid approach for fabricating NPs which can be exploited for multifarious biomedical applications.

Identifying the Molecular Targets of an Anti-pathogenic Hydroalcoholic Extract of Punica granatum Peel Against Multidrug-resistant Serratia marcescens.

BACKGROUND: Antibiotic-resistant members of the family Enterobacteriaceae are among the serious threats to human health globally. This study reports the anti-pathogenic activity of Punica granatum peel extract (PGPE) against a multi-drug resistant, beta-lactamase producing member of this family i.e. Serratia marcescens. OBJECTIVE: This study aimed at assessing the anti-pathogenic activity of PGPE against the gramnegative bacterial pathogen S. marcescens and identifying the molecular targets of this extract in the test bacterium. METHODS: Effect of PGPE on S. marcescens growth and quorum sensing (QS)-regulated pigment production was assessed through broth dilution assay. In vivo anti-infective and prophylactic activity of PGPE was assessed employing the nematode worm Caenorhabditis elegans as a model host. Differential gene expression in PGPE-exposed S. marcescens was studied through a whole transcriptome approach. RESULTS: PGPE was able to modulate QS-regulated pigment production in S. marcescens without exerting any heavy growth-inhibitory effect at concentrations as low as ≥2.5 µg/mL. It could attenuate the virulence of the test bacterium towards the worm host by 22-42% (p≤0.01) at even lower concentrations (≥0.5 µg/mL). PGPE also exerted a post-extract effect on S. marcescens. This extract was found to offer prophylactic benefit too, to the host worm, as PGPE-pre-fed worms scored better (34-51%; p≤0.001) survival in face of subsequent bacterial attack. Differential gene expression analysis revealed that PGPE affected the expression of a total of 66 genes in S. marcescens by ≥1.5 fold. CONCLUSION: The anti-virulence effect of PGPE against S. marcescens is multifaceted, affecting stress-response machinery, efflux activity, iron homeostasis, and cellular energetics of this bacterium notably. Among the major molecular targets identified in this study are LPS export transporter permease (LptF), t-RNA pseudouridine synthase (TruB), etc.

Green synthesis of silver nanoparticles using Carum copticum: Assessment of its quorum sensing and biofilm inhibitory potential against gram negative bacterial pathogens.

Antimicrobial resistance among pathogenic bacteria has become a global threat to human health. Due to poor progress in development of new antimicrobial drugs, there is a need for the development of novel alternative strategies to combat the problem of multidrug resistance. Moreover, there is focus on ecofriendly approach for the synthesis nanoparticles having efficient medicinal properties including antivirulence properties to tackle the emergence of multi-drug resistance. Targeting quorum sensing controlled virulence factors and biofilms has come out to be a novel anti-infective drug target. The silver nanoparticles (Ag@CC-NPs) were synthesized from aqueous extract of Carum copticum and characterized using UV-vis absorption spectroscopy, fourier-transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), transmission electron microscopy (TEM), and scanning electron microscopy (SEM). Ag@CC-NPs were checked for its ability to inhibit quorum sensing-mediated virulence factors and biofilms against three test pathogens at sub-MIC values. There was ~75% inhibition of violacein production by Ag@CC-NPs against C. violaceum. The P. aeruginosa virulence factors such as pyocyanin production, pyoverdin production, exoprotease activity, elastase activity, swimming motility and rhamnolipid production were inhibited by 76.9, 49.0, 71.1, 53.3, 89.5, and 60.0% at sub-MIC. Moreover, virulence factors of S. marcescens viz. prodigiosin production, exoprotease activity, and swarming motility was reduced by 78.4, 67.8, and 90.7%. Ag@CC-NPs also exhibited broad-spectrum antibiofilm activity with 77.6, 86.3, and 75.1% inhibition of biofilms of P. aeruginosa, S. marcescens, and C. violaceum respectively. The biofilm formation on glass coverslip was reduced remarkably as evident from SEM and CLSM analysis. The findings revealed the in vitro efficacy of Ag@CC-NPs against bacterial pathogens and can be exploited in the development of alternative therapeutic agent in management of bacterial infections for topical application, mainly wound infection, or coating of surfaces to prevent bacterial adherence on medical devices.

The Anti-infective Potential of Hydroalcoholic Extract of Phyllanthus emblica Seeds Against Selected Human-pathogenic Bacteria.

INTRODUCTION: In the context of the global threat of antimicrobial resistance (AMR) among bacterial pathogens against conventional bactericidal antibiotics, investigation on complementary/ alternative approaches to manage bacterial infections is warranted. The present study aimed at investigating the anti-pathogenic potential of Phyllanthus emblica seed extract (PESE) against four different pathogenic bacteria. METHODS: Hydroalcoholic extract of P. emblica seeds was tested for its possible in vitro quorummodulatory potential against Chromobacterium violaceum, Serratia marcescens, Pseudomonas aeruginosa, and Staphylococcus aureus through broth dilution assay. In vivo efficacy of PESE was assayed employing Caenorhabditis elegans as the model host for these four pathogens. RESULTS: PESE was found to exert in vitro quorum-modulatory effect on C. violaceum, S. marcescens, P. aeruginosa, and S. aureus at ≥50 µg/mL. This extract could curb the haemolytic activity of all the four test bacteria by 23-65%, inhibit biofilm formation, and was also able to modulate their antibiotic susceptibility (AS) and catalase activity. Susceptibility of P. aeruginosa and S. aureus to lysis by human serum was enhanced under the influence of this extract by 23% and 49%, respectively. Repeated exposure of both these notorious pathogens to PESE did not induce resistance in them. In vivo assay confirmed the anti-virulence effect of this extract in the C. elegans host, wherein the nematode host challenged with the PESE-treated pathogenic bacteria scored better survival. PESE also displayed notable prebiotic potential by promoting the growth of three probiotic strains. CONCLUSION: To the best of our awareness, this is the first report on the quorum-modulatory potential of P. emblica seed extract, validating its anti-infective potential and prebiotic property.

The Development of an Antimicrobial Contact Lens - From the Laboratory to the Clinic.

Contact lens wear is generally safe and provides excellent vision. However, contact lens wear is often associated with the risk of developing ocular surface infection and inflammation, and in severe cases, the infection can result in loss of vision. Antimicrobial peptide-coated contact lenses have been made to help reduce the incidence of infection and inflammation. This paper reviews the research progress from conception, through the laboratory and preclinical tests to the latest information on clinical testing of an antimicrobial contact lens. We provide insights into the pathways followed and pitfalls that have been encountered. The journey has not always been linear or smooth, but has resulted in some of the first published clinical testing of antimicrobial peptide-coated contact lenses in humans. We hope this may help lead to the development and commercialisation of antimicrobial contact lenses in the future.

Gut bacteria of Cuora amboinensis (turtle) produce broad-spectrum antibacterial molecules.

Antimicrobial resistance is a major threat to human health, hence there is an urgent need to discover antibacterial molecule(s). Previously, we hypothesized that microbial gut flora of animals are a potential source of antibacterial molecules. Among various animals, Cuora amboinensis (turtle) represents an important reptile species living in diverse ecological environments and feed on organic waste and terrestrial organisms and have been used in folk medicine. The purpose of this study was to mine turtle's gut bacteria for potential antibacterial molecule(s). Several bacteria were isolated from the turtle gut and their conditioned media were prepared. Conditioned media showed potent antibacterial activity against several Gram-positive (Bacillus cereus, Streptococcus pyogenes and methicillin-resistant Staphylococcus aureus) and Gram-negative (neuropathogenic Escherichia coli K1, Serratia marcescens, Pseudomonas aeruginosa, Salmonella enterica and Klebsiella pneumoniae) pathogenic bacteria. Conditioned media-mediated bactericidal activity was heat-resistant when treated at 95°C for 10 min. By measuring Lactate dehydrogenase release, the results showed that conditioned media had no effect on human cell viability. Tandem Mass Spectrometric analysis revealed the presence of various secondary metabolites, i.e., a series of known as well as novel N-acyl-homoserine lactones, several homologues of 4-hydroxy-2-alkylquinolines, and rhamnolipids, which are the signature metabolites of Pseudomonas species. These findings are significant and provide the basis for rational development of therapeutic interventions against bacterial infections.

Biofibers and biocomposites from sabai grass: A unique renewable resource.

Natural cellulose fibers were extracted from a fast growing perennial grass Eulaliopsis binata (commonly known as Sabai) and characterized for their structure and properties. The untreated sabai grass has been used as reinforcement for polypropylene composites and properties of the composites have been investigated. Although the composition of the sabai grass is typical to other lignocellulosic sources, there is a high content of flavonoids (630 mg/g) and phenols (510 mg/g) which provides high antibacterial, and antifungal properties to the fibers and composites developed. Fiber bundles extracted from the grass had tensile strength of 493 MPa and tensile modulus of 21 GPa, similar to common natural cellulose fibers. Both tensile and flexural properties of polypropylene composites increased with increasing ratio of sabai grass. Polypropylene composites reinforced with sabai grass show high noise insulation and thermal resistance properties suggesting their suitability for automotive and building applications.

Effect of Capsicum Frutescens Extract, Capsaicin, and Luteolin on Quorum Sensing Regulated Phenotypes.

Capsicum peppers have not been investigated as sources of quorum sensing (QS) inhibitors. This study aimed to identify compounds in pimenta-malagueta (Capsicum frutescens) and red pepper (Capsicum annuum) extracts and to evaluate their effect on violacein production in Chromobacterium violaceum ATCC 12472 and C. violaceum CV026, as well as biofilm formation (BF) in Pseudomonas aeruginosa PAO1 and Serratia marcescens MG1. Among the extracts, pimenta-malagueta methanolic extract (PMME) was chosen because it contained capsaicin, dihydrocapsaicin, and luteolin in greater amount than the other extracts. In general, PMME partially inhibited bacterial growth at 2.5 and 5.0 mg/mL, as well as capsaicin at 100 µg/mL and luteolin at 62.5, 125, and 250 µg/mL. At lower concentrations, PMME and luteolin reduced violacein production in C. violaceum ATCC 12472 without affecting growth, a result that was not observed with capsaicin. We show that violacein inhibition by PMME is likely due to luteolin. In silico docking evaluation showed that luteolin binds to the CviR QS regulator. Crystal violet staining and confocal microscopy revealed that BF was increased by PMME and capsaicin, being remarkably superior for P. aeruginosa PAO1 at 30 °C. Capsaicin is not an effective QS inhibitor, while luteolin should be further investigated for its potential effects in QS regulated phenotypes. PRACTICAL APPLICATION: Quorum sensing (QS) is a form of bacterial communication targeted for studies aiming to inhibit bacterial virulence. QS regulates phenotypes that influence microbial activities across many areas, including Food Science. Capsicum frutescens is a type of chili pepper consumed in Brazil, rich in bioactive compounds such as capsaicin (which gives its pungency) and luteolin (a phenolic compound). We show that C. frutescens extract and luteolin inhibit QS in a model bacterium, along with the possible molecular mechanism of inhibition. Capsaicin did not inhibit QS neither biofilm formation. Luteolin should be further investigated for its QS inhibition properties and biotechnological applications.

N-Acyl-Homoserine Lactone Quorum Sensing Switch from Acidogenesis to Solventogenesis during the Fermentation Process in Serratia marcescens MG1.

N-acyl-homoserine lactone quorum sensing (AHL-QS) has been shown to regulate many physiological behaviors in Serratia marcescens MG1. In the current study, the effects of AHL-QS on the biosynthesis of acid and neutral products by S. marcescens MG1 and its isogenic ∆swrI with or without supplementing exogenous N-hexanoyl-L-homoserine lactone (C6-HSL) were systematically investigated. The results showed that swrI disruption resulted in rapid pH drops from 7.0 to 4.8, which could be restored to wild type by supplementing C6-HSL. Furthermore, fermentation product analysis indicated that ∆swrI could lead to obvious accumulation for acidogenesis products such as lactic acid and succinic acid, especially excess acetic acid (2.27 g/l) produced at the early stage of fermentation, whereas solventogenesis products by ∆swrI appeared to noticeably decrease by an approximate 30% for acetoin during 32-48 h and by an approximate 20% for 2,3-butanediol during 24-40 h, when compared to those by wild type. Interestingly, the excess acetic acid produced could be removed in an AHL-QS-independent manner. Subsequently, quantitative real-time PCR was used to determine the mRNA expression levels of genes responsible for acidogenesis and solventogenesis and showed consistent results with those of product synthesis. Finally, by close examination of promoter regions of the analyzed genes, four putative luxI box-like motifs were found upstream of genes encoding acetyl-CoA synthase, lactate dehydrogenase, α-acetolactate decarboxylase, and Lys-like regulator. The information from this study provides a novel insight into the roles played by AHL-QS in switching from acidogenesis to solventogenesis in S. marcescens MG1.

Anti-pathogenic potential of a classical ayurvedic Triphala formulation.

A classical ayurvedic polyherbal formulation namely Triphala was assessed for its anti-pathogenic potential against five different pathogenic bacteria. Virulence of four of them towards the model host Caenorhabditis elegans was attenuated (by 18-45%) owing to pre-treatment with Triphala (≤20 µg/ml). Triphala could also exert significant therapeutic effect on worms already infected with Chromobacterium violaceum, Serratia marcescens or Staphylococcus aureus. Prophylactic use of Triphala allowed worms to score 14-41% better survival in face of subsequent pathogen challenge. Repeated exposure to this formulation induced resistance in S. marcescens, but not in P. aeruginosa. It also exerted a post-extract effect (PEE) on three of the test pathogens. Triphala was able to modulate production of quorum sensing (QS)-regulated pigments in three of the multidrug-resistant gram-negative test bacteria. Haemolytic activity of S. aureus was heavily inhibited under the influence of this formulation. P. aeruginosa's lysozyme-susceptibility was found to increase by ~25-43% upon Triphala-pretreatment. These results validate therapeutic potential of one of the most widely used polyherbal ayurvedic formulations called Triphala.

Molecular dynamics simulation analysis of conessine against multi drug resistant Serratia marcescens.

Ornithine decarboxylase (ODC) is an immediate precursor of polyamine biosynthesis in Serratia marcescens and a potential target for inhibition of its growth. We predicted the 3D structural conformation of ODC enzyme and validated it using MDS in our previous study. In this current study, the potential inhibitors of ODC were obtained by virtual screening of potential inhibitors from ZINC database and studied in depth for their different binding pose. Among the ten virtually screened inhibitors, Conessine exhibited the best binding with ODC and its inhibition property was studied further by MDS studies. The natural compound conessine is isolated from plant Holarrhena antidysenterica and it is studied against ODC of Serratia marcenses for its inhibitory potentials. This revealed unforeseen twisted position in root mean square fluctuation (RMSF) and ODC modelled conformation that influenced ligand binding. Both predicted model and ligand bound model were compared and found to be stable with Root Mean Square Deviation (RMSD) of approximately 7 nm and 0.25 nm to that of crystallographic structure over simulation time of 55 ns and 70 ns respectively. This work paves the way for future development of new drugs against nosocomial diseases caused by Serratia marcescens.

Polymicrobial antibiofilm activity of the membranotropic peptide gH625 and its analogue.

This work illustrates a new role for the membranotropic peptide gH625 and its derivative gH625-GCGKKK in impairing formation of polymicrobial biofilms. Mixed biofilms composed of Candida and bacterial species cause frequently infections and failure of medical silicone devices and also show a major drug resistance than single-species biofilms. Inhibition and eradication of biofilms were evaluated by complementary methods: XTT-reduction, and crystal violet staining (CV). Our results indicate that gH625-GCGKKKK, better than the native peptide, strongly inhibited formation of mixed biofilms of clinical isolates of C. tropicalis/S. marcescens and C. tropicalis/S. aureus and reduced the biofilm architecture, interfering with cell adhesion and polymeric matrix, as well as eradicated the long-term polymicrobial biofilms on silicone surface.

Selection of hyperproduction of AmpC and SME-1 in a carbapenem-resistant Serratia marcescens isolate during antibiotic therapy.

Objectives: Antibiotic selective pressure may result in changes to antimicrobial susceptibility throughout the course of infection, especially for organisms that harbour chromosomally encoded AmpC ß-lactamases, notably Enterobacter spp., in which hyperexpression of ampC may be induced following treatment with cephalosporins. In this study, we document a case of bacteraemia caused by a blaSME-1-harbouring Serratia marcescens that subsequently developed resistance to expanded-spectrum cephalosporins, piperacillin/tazobactam and fluoroquinolones, over the course of several months of treatment with piperacillin/tazobactam and ciprofloxacin. Methods: Susceptibility testing and WGS were performed on three S. marcescens isolates from the patient. ß-Lactamase activity in the presence or absence of induction by imipenem was measured by nitrocefin hydrolysis assays. Expression of ampC and blaSME-1 under the same conditions was determined by real-time PCR. Results: WGS demonstrated accumulation of missense and nonsense mutations in ampD associated with stable derepression of AmpC. Gene expression and ß-lactamase activity of both AmpC and SME-1 were inducible in the initial susceptible isolate, but were constitutively high in the resistant isolate, in which total ß-lactamase activity was increased by 128-fold. Conclusions: Although development of such in vitro resistance due to selective pressure imposed by antibiotics is reportedly low in S. marcescens, our findings highlight the need to evaluate isolates on a regular basis during long-term antibiotic therapy.

Vanillic acid from Actinidia deliciosa impedes virulence in Serratia marcescens by affecting S-layer, flagellin and fatty acid biosynthesis proteins.

Serratia marcescens is one of the important nosocomial pathogens which rely on quorum sensing (QS) to regulate the production of biofilm and several virulence factors. Hence, blocking of QS has become a promising approach to quench the virulence of S. marcescens. For the first time, QS inhibitory (QSI) and antibiofilm potential of Actinidia deliciosa have been explored against S. marcescens clinical isolate (CI). A. deliciosa pulp extract significantly inhibited the virulence and biofilm production without any deleterious effect on the growth. Vanillic acid was identified as an active lead responsible for the QSI activity. Addition of vanillic acid to the growth medium significantly affected the QS regulated production of biofilm and virulence factors in a concentration dependent mode in S. marcescens CI, ATCC 14756 and MG1. Furthermore vanillic acid increased the survival of Caenorhabditis elegans upon S. marcescens infection. Proteomic analysis and mass spectrometric identification of differentially expressed proteins revealed the ability of vanillic acid to modulate the expression of proteins involved in S-layers, histidine, flagellin and fatty acid production. QSI potential of the vanillic acid observed in the current study paves the way for exploring it as a potential therapeutic candidate to treat S. marcescens infections.

Green synthesised zinc oxide nanostructures through Periploca aphylla extract shows tremendous antibacterial potential against multidrug resistant pathogens.

To grapple with multidrug resistant bacterial infections, implementations of antibacterial nanomedicines have gained prime attention of the researchers across the globe. Nowadays, zinc oxide (ZnO) at nano-scale has emerged as a promising antibacterial therapeutic agent. Keeping this in view, ZnO nanostructures (ZnO-NS) have been synthesised through reduction by P. aphylla aqueous extract without the utilisation of any acid or base. Structural examinations via scanning electron microscopy (SEM) and X-ray diffraction have revealed pure phase morphology with highly homogenised average particle size of 18 nm. SEM findings were further supplemented by transmission electron microscopy examinations. The characteristic Zn-O peak has been observed around 363 nm using ultra-violet-visible spectroscopy. Fourier-transform infrared spectroscopy examination has also confirmed the formation of ZnO-NS through detection of Zn-O bond vibration frequencies. To check the superior antibacterial activity of ZnO-NS, the authors' team has performed disc diffusion assay and colony forming unit testing against multidrug resistant E. coli, S. marcescens and E. cloacae. Furthermore, protein kinase inhibition assay and cytotoxicity examinations have revealed that green fabricated ZnO-NS are non-hazardous, economical, environmental friendly and possess tremendous potential to treat lethal infections caused by multidrug resistant pathogens.

Properties of halogenated and sulfonated porphyrins relevant for the selection of photosensitizers in anticancer and antimicrobial therapies.

The impact of substituents on the photochemical and biological properties of tetraphenylporphyrin-based photosensitizers for photodynamic therapy of cancer (PDT) as well as photodynamic inactivation of microorganisms (PDI) was examined. Spectroscopic and physicochemical properties were related with therapeutic efficacy in PDT of cancer and PDI of microbial cells in vitro. Less polar halogenated, sulfonamide porphyrins were most readily taken up by cells compared to hydrophilic and anionic porphyrins. The uptake and PDT of a hydrophilic porphyrin was significantly enhanced with incorporation in polymeric micelles (Pluronic L121). Photodynamic inactivation studies were performed against Gram-positive (S. aureus, E. faecalis), Gram-negative bacteria (E. coli, P. aeruginosa, S. marcescens) and fungal yeast (C. albicans). We observed a 6 logs reduction of S. aureus after irradiation (10 J/cm2) in the presence of 20 µM of hydrophilic porphyrin, but this was not improved with incorporation in Pluronic L121. A 2-3 logs reduction was obtained for E. coli using similar doses, and a decrease of 3-4 logs was achieved for C. albicans. Rational substitution of tetraphenylporphyrins improves their photodynamic properties and informs on strategies to obtain photosensitizers for efficient PDT and PDI. However, the design of the photosensitizers must be accompanied by the development of tailored drug formulations.

Highly efficient antibacterial diblock copolypeptides based on lysine and phenylalanine.

A series of amphiphilic diblock copolypeptides (K30 -b-F15 , K30 -b-F30 , and K30 -b-F45 ) were synthesized via N-carboxy-α-amino-anhydride ring-opening polymerization. The copolypeptides had excellent antibacterial efficacy to both Gram positive (S. aureus) and Gram negative (E. coli) bacteria. The minimum inhibitory concentrations (MICs) against E. coli and S. aureus are 8 µg mL-1 and 2 µg mL-1 , respectively, lower than most natural and artificial antimicrobial peptides (AMPs). The morphological changes of the bacteria treated with diblock copolypeptides were investigated by transmission electron microscopy; the results proved that the diblock copolypeptides had a similar antibacterial pore-forming mechanism to natural cationic peptides. This was confirmed by laser scanning confocal microscope images. CCK-8 results and the MICs showed that the diblock copolypeptides have high selectivity to bacteria, which suggested that the diblock copolypeptides could be excellent candidates to replace traditional antibiotics in future.

Multi-effect of the water-soluble Moringa oleifera lectin against Serratia marcescens and Bacillus sp.: antibacterial, antibiofilm and anti-adhesive properties.

AIMS: To evaluate the antibiofilm potential of water-soluble Moringa oleifera seed lectin (WSMoL) on Serratia marcescens and Bacillus sp. METHODS AND RESULTS: WSMoL inhibited biofilm formation by S. marcescens at concentrations lower than 2·6 µg ml-1 and impaired bacterial growth at higher concentrations, avoiding biofilm formation. For Bacillus sp., the lectin inhibited bacterial growth at all concentrations. The antibiofilm action of WSMoL is associated with damage to bacterial cells. WSMoL did not disrupt preformed S. marcescens biofilms but was able to damage cells inside them. On the other hand, the lectin reduced the number of cells in Bacillus sp. biofilm treated with it. WSMoL was able to control biofilm formation when immobilized on glass surface (116 µg cm-2 ), damaging S. marcescens cells and avoiding adherence of Bacillus sp. cells on glass. The Bacillus sp. isolate is member of Bacillus subtilis species complex and closely related to species of the conspecific 'amyloliquefaciens' group. CONCLUSION: WSMoL prevented biofilm development by S. marcescens and Bacillus sp. and the antibiofilm effect is also observed when the lectin is immobilized on glass. SIGNIFICANCE AND IMPACT OF THE STUDY: Taking together, our results provide support to the potential use of WSMoL for controlling biofilm formation by bacteria.

Piper betle and its bioactive metabolite phytol mitigates quorum sensing mediated virulence factors and biofilm of nosocomial pathogen Serratia marcescens in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Piper betle, a tropical creeper plant belongs to the family Piperaceae. The leaves of this plant have been well known for their therapeutic, religious and ceremonial value in South and Southeast Asia. It has also been reported to possess several biological activities including antimicrobial, antioxidant, antinociceptive, antidiabetic, insecticidal and gastroprotective activities and used as a common ingredient in indigenous medicines. In Indian system of ayurvedic medicine, P. betle has been well recognized for its antiseptic properties and is commonly applied on wounds and lesions for its healing effects. AIM OF THE STUDY: To evaluate the anti-quorum sensing (anti-QS) and antibiofilm efficacy of P. betle and its bioactive metabolite phytol against Serratia marcescens. MATERIALS AND METHODS: The P. betle ethyl acetate extract (PBE) was evaluated for its anti-QS efficacy against S. marcescens by assessing the prodigiosin and lipase production at 400 and 500µgml-1 concentrations. In addition, the biofilm biomass quantification assay was performed to evaluate the antibiofilm activity of PBE against S. marcescens. Besides, the influence of PBE on bacterial biofilm formation was assessed through microscopic techniques. The biofilm related phenomenons like exopolysaccharides (EPS) production, hydrophobicity and swarming motility were also examined to support the antibiofilm activity of PBE. Transcriptional analysis of QS regulated genes in S. marcescens was also done. Characterization of PBE was done by separation through column chromatography and identification of active metabolites by gas chromatography -mass spectrometry. The major compounds of active fractions such as hexadecanoic acid, eugenol and phytol were assessed for their anti-QS activity against S. marcescens. Further, the in vitro bioassays such as protease, biofilm and HI quantification were also carried out to confirm the anti-QS and antibiofilm potential of phytol in PBE. RESULTS: PBE inhibits QS mediated prodigiosin pigment production in S. marcescens, which confirmed its anti-QS potential against S. marcescens. At 500µgml-1 concentration, PBE significantly inhibited the production of protease, lipase, biofilm and EPS to the level of 71%, 68%, 65% and 43% in S. marcescens, respectively. Further, their antibiofilm efficacy was confirmed through microscopic techniques. In addition, PBE effectively inhibited the hydrophobicity and swarming motility. Additionally, the results of qPCR analysis validated the downregulation of QS genes. Chromatographic techniques the presence of hexadecanoic acid, eugenol and phytol in PBE and the potential bioactive compound with anti-QS activity was identified as phytol. In vitro assays with phytol evidenced the potent inhibition of QS-controlled prodigiosin, protease, biofilm and hydrophobicity in S. marcescens, without exerting any deleterious effect on its growth. CONCLUSION: This study demonstrates the promising anti-QS and antibiofilm activities of PBE and its active metabolite phytol, and confirms the ethnopharmacological applications of these leaves against S. marcescens infections.

Antibacterial Properties and Effects of Fruit Chilling and Extract Storage on Antioxidant Activity, Total Phenolic and Anthocyanin Content of Four Date Palm (Phoenix dactylifera) Cultivars.

Phoenix dactylifera or date palm fruits are reported to contain natural compounds that exhibit antioxidant and antibacterial properties. This research aimed to study the effect of fruit chilling at 4 °C for 8 weeks, extract storage at -20 °C for 5 weeks, and extraction solvents (methanol or acetone) on total phenolic content (TPC), antioxidant activity and antibacterial properties of Saudi Arabian P. dactylifera cv Mabroom, Safawi and Ajwa, as well as Iranian P. dactylifera cv Mariami. The storage stability of total anthocyanin content (TAC) was also evaluated, before and after storing the extracts at -20 °C and 4 °C respectively, for 5 weeks. Mariami had the highest TAC (3.18 ± 1.40 mg cyd 3-glu/100 g DW) while Mabroom had the lowest TAC (0.54 ± 0.15 mg cyd 3-glu/100 g DW). The TAC of all extracts increased after storage. The chilling of date palm fruits for 8 weeks prior to solvent extraction elevated the TPC of all date fruit extracts, except for methanolic extracts of Mabroom and Mariami. All IC50 values of all cultivars decreased after the fruit chilling treatment. Methanol was a better solvent compared to acetone for the extraction of phenolic compounds in dates. The TPC of all cultivars extracts decreased after 5 weeks of extract storage. IC50 values of all cultivars extracts increased after extract storage except for the methanolic extracts of Safawi and Ajwa. Different cultivars exhibited different antibacterial properties. Only the methanolic extract of Ajwa exhibited antibacterial activity against all four bacteria tested: Staphylococcus aureus, Bacillus cereus, Serratia marcescens and Escherichia coli. These results could be useful to the nutraceutical and pharmaceutical industries in the development of natural compound-based products.