Metabolomic profiling of antibiofilm compounds from fungal endophytes derived from Scottish seaweeds

Student thesis: Doctoral Thesis

Abstract

In the search for a new antimicrobial bioactive compound from natural source against both biofilm-forming Staphylococcus aureus and Pseudomonas aeruginosa, endophytic fungi associated with seaweeds are an interesting source of bioactive secondary metabolites. Twenty-four endophytes were isolated from eight different Scottish seaweeds namely; Ascophyllum nodosum, Laminaria hyperborean, Ulva intestinalis, Ulva lactuca, Himanthalia elongata, Fucus vesiculosis, Fucus serratus, and Fucus spiralis. Endophytes responsible for producing bioactive compounds were identified using internal transcribed spacer (ITS) gene sequencing. Depending on their biological activity and chemical profiles, three endophytic fungi, namely; Dendryphiella salina (D. salina), Hypoxylon rubiginosum (H. rubiginosum), and Mariannaea elegans (M. elegance) were identified and subjected for media optimisation studies on five liquid media and four solid media in three different incubation periods. For liquid media; this included: malt extract broth with and without sea salt, Wickersham liquid media with and without sea salt and lastly, marine broth. While for the solid media, rice and oat, both with and without sea salt were used. The chemical profile for each extract was monitored using Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS) and nuclear magnetic resonance spectroscopy (NMR). Antimicrobial activity was monitored using both AlamarBlue® and planktonic assays. The appropriate medium and the incubation period were chosen for a scale-up of the selected endophytic fungus according to the following factors: 1) higher yield, 2) improved antimicrobial activity, and 3) diverse chemical profile. Based on these factors, D. salina was chosen for scale-up and isolation work. D. salina was grown on malt extract with salt and oat without salt. The scaled-up extracts of D. salina were subjected to liquid-liquid partitioning followed by fractionation using either medium pressure flash chromatography or flash chromatographic technique. The first fractionation of the scaled-up extracts was tested against both biofilm-forming S. aureus and P. aeruginosa and analysed using 1H NMR and LC-HRMS. The HRMS data was processed using MZmine followed by dereplication using an in-house method then subjected to orthogonal partial least square-discriminant analysis (OPLS-DA). OPLS-DA results were used determine the antimicrobial active secondary metabolites. As a result, eight compounds were isolated from the bioactive fractions of D. salina. D. salina grown on malt extract broth yielded linoleic acid, orsellinic acid, orcinol and 2,5-dihydroxy-3-(hydroxymethyl)benzoic acid. While D. salina inoculated on oat solid media afforded the acetonide and glycerylglycoside derivatives of linoleic acid along with the peptide turnagainolide A. All isolated compounds, except for linoleic acid and its glycerol congener, displayed antibiofilm activity with MBEC (Minimum Biofilm Eradication Concentration) values between 45 and 360 µM concentrations against biofilm-forming bacteria S. aureus and P. aeruginosa. F10-2 (turnagainolide A) and F10-8 (gingerglycolipid B stereoisomer) displayed the highest potency with MIC and MBEC values between 45 and 75 µM concentrations. Conclusion: Endophytic D. salina derived from Scottish seaweed L. hyperborea grown on malt extract broth and oat solid media showed the ability to produce compounds with antimicrobial bioactivity against biofilm-forming bacteria. In addition, three new antimicrobial natural products: 2,5-dihydroxy-3-(hydroxymethyl)benzoic acid along with an acetonide and glycerylglycoside derivatives of linoleic acid were isolated from D. salina extracts. Thus, marine endophytic fungi are promising source of bioactive compounds.
Date of Award6 Oct 2021
Original languageEnglish
Awarding Institution
  • University Of Strathclyde
SponsorsUniversity of Strathclyde
SupervisorRuangelie Edrada-Ebel (Supervisor) & Katherine Duncan (Supervisor)

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