Discovery of novel anti-Candida albicans compounds from environmental bacteria

Date
2023-12
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Publisher
Stellenbosch : Stellenbosch University
Abstract
ENGLISH ABSTRACT: The opportunistic fungal pathogen, Candida albicans (C. albicans), has the ability to colonize different epithelial tissues (e.g. vaginal, intestinal and oral tissues) causing moderate to severe and fatal infections. The fatal infections are due to fungal cells that enter the bloodstream and circulate after being dispersed from their resistant biofilms. Organisms that have adapted to form biofilms are associated with improved tolerance to drugs, which was also found for some drugs in this study. A medium-throughput multiplex assay system (MPAS) has been developed for the in-parallel study of several fungal life-cycle stages of different strains of C. albicans. MPAS was designed to use conventional 96-well plates and 96-pin lids in four different assay layouts. In MPAS, the metabolic activity measurements were used to determine the effect of antimicrobial compounds in assay targeting planktonic cell susceptibility, biofilm prevention, biofilm eradication, as well as susceptibility of cells released from biofilms. MPAS detection limits were further optimized by testing the activity of antimicrobial culture extracts from known bacterial producers on the different C. albicans life stages. The MPAS data showed which producer extracts were more active against certain strains of C. albicans, as well as highlighted which fungal forms are more sensitive to certain culture extracts. This led to the successful application of MPAS to identify active culture extracts of unknown soil microbes against planktonic and biofilm forms of C. albicans for further characterization. Ultra-performance liquid chromatography linked to high resolution tandem mass spectrometry (HR UPLC-MSᵉ) was used to identify and characterize known antifungal lipopeptides (such as surfactins, iturins and fengycins), rhamnolipids, as well as active small quorum sensing compounds in the most active culture extracts. The overall success of the MPAS together with HR UPLC-MSᵉ was confirmed by the successful identification of known and unknown compounds, from soil sample bacteria, with activity against C. albicans. The study showed the potential of the medium throughput MPAS together with HR UPLC-MSᵉ to discover and characterize potential antibiofilm and antifungal compounds for further development.
AFRIKAANSE OPSOMMING: Die opportunistiese swampatogeen, Candida albicans (C. albicans), het die vermoë om verskillende epiteelweefsels (bv. vaginale, derm- en mondweefsel) te koloniseer, wat matige tot ernstige en dodelike infeksies veroorsaak. Die dodelike infeksies is te wyte aan swamselle wat in die bloedstroom sirkuleer nadat hulle van hul weerstandbiedende biofilms vrygestel is. Organismes wat aanpas om biofilms te vorm, word geassosieër met verbeterde verdraagsaamheid teenoor medikamente, wat ook in hierdie studie vir sommige middels gevind is. ‘n Medium-deurset multipleks essaï sisteem (MPAS) is vir die in-parallel studie van verskeie swam-lewensiklus-stadia van verskillende stamme van C. albicans ontwikkel. MPAS is ontwerp om gebruik te maak van konvensionele 96-putplate en 96-pendeksels in vier verskillende toets-uitlegte. In MPAS is die metaboliese aktiwiteitsmetings gebruik om die effek van antimikrobiese verbindings te bepaal in essaïs wat planktoniese sel vatbaarheid, biofilm voorkoming, biofilm uitwissing, sowel as vatbaarheid van selle vrygestel uit biofilms teiken. MPAS deteksielimiete is verder geoptimiseer deur die aktiwiteit van antimikrobiese kultuurekstrakte van bekende bakteriële produsente, op die verskillende C. albicans lewenstadia te bepaal. Die MPAS data het getoon watter produsent-ekstrakte meer aktief was teen sekere stamme van C. albicans, asook uitgelig watter swamvorme meer sensitief is vir sekere kultuurekstrakte. Dit het gelei tot die suksesvolle toepassing van MPAS om aktiewe kultuurekstrakte van onbekende grondmikrobes teen planktoniese en biofilmvorme van C. albicans te identifiseer vir verdere karakterisering. Ultradoeltreffendheid vloeistofchromtografie gekoppel met hoë resolusie tandem massaspektrometrie (HR UPLC-MSᵉ) is gebruik om bekende antifungiese lipopeptiede (soos surfaktiene, ituriene en fengisien), ramnolipiede, asook aktiewe klein kworumwaarnemingsverbindings, te identifeer en te karakteriseer in die mees aktiewe kultuurekstakte. Die algehele sukses van die MPAS saam HR UPLC-MSᵉ is bevestig deur die suksesvolle identifikasie van bekende en onbekende verbindings, vanaf grondmonsterbakterieë, met aktiwiteit teen C. albicans. Die studie het die potensiaal van die mediuim deurvloei MPAS tesame met HR UPLC-MSᵉ gewys om potensiële antibiofilm en antifungiese verbindings te ontdek en te karakteriseer vir verdere ontwikkelling.
Description
Thesis (PhD)--Stellenbosch University, 2023.
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