ABSTRACT
PURPOSE: Streptococcus pyogenes, a notorious human pathogen thatis responsible for various invasive and non-invasive diseases, possesses multiple virulence armaments, including biofilm formation. The current study demonstrates the anti-biofilm and anti-virulence potential of fukugiside, a biflavonoid isolated from Garciniatravancorica, against S. pyogenes. METHODOLOGY: The anti-biofilm activity of fukugiside was assessed and established using microdilution and microscopic analysis. Biochemical assays were performed to assess the effects of fukugiside on important virulence factors, which were further validated using quantitative real-time PCR and in vivo analysis in Caenorhabditis elegans. RESULTS: Fukugiside exhibited concentration-dependent biofilm inhibition (79 to 96â%) against multiple M serotypes of S. pyogenes (M1, M56, M65, M74, M100 and st38) with a minimum biofilm inhibitory concentration of 80 µg ml-1. Electron microscopy and biochemical assay revealed a significant reduction in extracellular polymeric substance production. The results for the microbial adhesion to hydrocarbon assay, extracellular protease quantification and differential regulation of the dltA, speB, srv and ropB genes suggested that fukugiside probably inhibits biofilm formation by lowering cell surface hydrophobicity and destabilizing the biofilm matrix. The enhanced susceptibility to phagocytosis evidenced in the blood survival assay goes in unison with the downregulation of mga. The downregulation of important virulence factor-encoding genes such as hasA, slo and col370 suggested impaired virulence. In vivo analysis in C. elegans evinced the non-toxic nature of fukugiside and its anti-virulence potential against S. pyogenes. CONCLUSION: Fukugiside exhibits potent anti-biofilm and anti-virulence activity against different M serotypes of S. pyogenes. It is also non-toxic, which augurs well for its clinical application.
Subject(s)
Anti-Bacterial Agents/pharmacology , Bacterial Proteins/genetics , Biflavonoids/pharmacology , Biofilms/drug effects , Garcinia/chemistry , Plant Extracts/pharmacology , Streptococcal Infections/microbiology , Streptococcus pyogenes/drug effects , Virulence Factors/genetics , Animals , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/isolation & purification , Bacterial Proteins/metabolism , Biflavonoids/chemistry , Biflavonoids/isolation & purification , Caenorhabditis elegans , Humans , Microbial Sensitivity Tests , Plant Extracts/chemistry , Plant Extracts/isolation & purification , Streptococcus pyogenes/genetics , Streptococcus pyogenes/physiology , Virulence Factors/metabolismABSTRACT
Fruits of Myristica fragrans Houtt. are the source of two valuable spices: nutmeg and mace, traditionally used for its flavoring and medicinal properties and found as an ingredient in many marketed polyherbal formulations and food products. In this study, a sensitive and efficient ultra high performance liquid chromatography electrospray ionization tandem mass spectrometry method was developed and validated for the rapid determination of 16 bioactive constituents in different parts of the fruit of M. fragrans and its marketed polyherbal formulations using a polarity switching technique. Chromatographic separation was achieved on an Aquity UPLC BEH C18 column in 9.4 min. Quantitative analysis was performed using multiple reaction monitoring mode with continuous polarity switching in a single analysis. The developed method was found to be accurate with overall recovery in the range from 95.95 to 102.07% (RSD ≤ 1.91%), precise (RSD ≤ 1.98%), and linear (r(2) ≥ 0.9992) over the concentration range of 0.1-200 ng/mL. Quantitative analysis indicated that the total content of the 16 bioactive constituents was highest in the mace of M. fragrans. Thus, this rapid and sensitive method could be utilized as a promising reference method for the quality control of M. fragrans and its marketed herbal formulations/food products.
