title: Piper betle and its bioactive metabolite phytol mitigates quorum sensing mediated virulence factors and biofilm of nosocomial pathogen Serratia marcescens in vitro.
abstract: BACKGROUND
  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.
  
  
  OBJECTIVE
  To evaluate the anti-quorum sensing (anti-QS) and antibiofilm efficacy of P. betle and its bioactive metabolite phytol against Serratia marcescens.
  
  
  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-1concentrations. 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-1concentration, 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Biotechnology, Science Campus, Alagappa University, Karaikudi 630004, Tamil Nadu, India.
authors.forename: Ramanathan
authors.initials: R
authors.lastname: Srinivasan
authors.affiliation: Department of Biotechnology, Science Campus, Alagappa University, Karaikudi 630004, Tamil Nadu, India.
authors.forename: Kannan Rama
authors.initials: KR
authors.lastname: Devi
authors.affiliation: Department of Biotechnology, Science Campus, Alagappa University, Karaikudi 630004, Tamil Nadu, India.
authors.forename: Arunachalam
authors.initials: A
authors.lastname: Kannappan
authors.affiliation: Department of Biotechnology, Science Campus, Alagappa University, Karaikudi 630004, Tamil Nadu, India.
authors.forename: Shunmugiah Karutha
authors.initials: SK
authors.lastname: Pandian
authors.affiliation: Department of Biotechnology, Science Campus, Alagappa University, Karaikudi 630004, Tamil Nadu, India. Electronic address: aveeraravi@rediffmail.com.
authors.forename: Arumugam Veera
authors.initials: AV
authors.lastname: Ravi
mesh_terms: D018441:Biofilms / Q000187:drug effects* / Q000254:growth & development; D018533:Biomass; D003428:Cross Infection / Q000382:microbiology / Q000652:urine; D004305:Dose-Response Relationship, Drug; D006801:Humans; D018613:Microscopy, Confocal; D008855:Microscopy, Electron, Scanning; D010836:Phytol / Q000302:isolation & purification / Q000494:pharmacology*; D031721:Piper betle / Q000737:chemistry* / Q000378:metabolism; D018515:Plant Leaves / Q000737:chemistry / Q000378:metabolism; D011353:Prodigiosin / Q000037:antagonists & inhibitors; D053038:Quorum Sensing / Q000187:drug effects*; D012706:Serratia marcescens / Q000187:drug effects* / Q000254:growth & development / Q000378:metabolism / Q000472:pathogenicity; D014774:Virulence
chemical_list: D010836:Phytol; D011353:Prodigiosin
keywords: Biofilm; Nosocomial infections; Phytol; Piper betle; Quorum sensing; Serratia marcescens