Novel Streptomycete antimicrobial compounds: activity against pathogens from domestic rainwater harvesting systems

Durrell, Kim Angeline (2016-03)

Thesis (MSc)--Stellenbosch University, 2016.

Thesis

ENGLISH ABSTRACT: Due to increased urbanisation, frequent droughts and poor water sources in rural communities, the scarcity of water has become a major concern. Water is an essential part of life – in order to meet the ever growing demand for water, it is imperative to search for alternative water sources. Domestic rainwater harvesting (DRWH) is considered a potential source of water and is commonly used in most parts of the world. However, the microbial quality of harvested rainwater poses a serious health risk to the consumer. Pathogenic bacteria such as Staphylococcus spp., Salmonella spp., Pseudomonas aeruginosa and Klebsiella spp., amongst others, have been associated with rainwater harvesting systems and the poor quality of the water is mainly attributed to faecal contamination caused by mammals, birds and reptiles. Generally, these pathogenic strains are highly virulent and are often resistant to common antimicrobials utilised for the treatment of diseases caused by these pathogens. An increase in drug-resistant infectious diseases presents a significant challenge to antimicrobial therapies. Urgent developments of novel antimicrobial agents are required to treat a wide spectrum of targets which include bacteria, fungi, viruses and parasites. Actinomycetes are a group of soil microorganisms known for the production of bio-active compounds. Streptomyces pharetrae CZA14T is one such actinomycete which was first described in 2005. Preliminary tests revealed that the strain has the ability to produce bio-active compounds. Subsequently, the genome of CZA14T was sequenced and submitted to an online secondary metabolite prediction software, antiSMASH, to predict secondary metabolite biosynthetic gene clusters (smBGCs) which then govern the expression of the compounds of interest. Fifty-five smBGCs encoding for secondary metabolites of interest were predicted by antiSMASH, of which, 23 were non-ribosomal peptide synthetases (NRPSs), seven were lantipeptide/lassopeptides/bacteriocins, eight were polyketide synthases (PKSs) and the remaining 17 biosynthetic gene clusters include genes for siderophores, terpenes, butyrolactones, ectoines, melanin and genes labelled as “other”. In the current study, previously identified optimal production media and fermentation conditions for maximum antimicrobial production by CZA14T were scaled-up from 0.5 L, 1 L and 2 L baffled flasks, to fermentation in a 3 L airlift reactor (ALR) and a 5 L continuous stirred tank reactor (CSTR). Solvent extractions were performed on both the supernatant and mycelia to test for extra- and intra-cellular production of bio-active compounds. Fermentation samples were also collected from the ALR at days 3, 6, 9 and 12, and used for molecular studies for which RNA extraction was performed and cDNA was synthesised and sequenced. Using molecular techniques, it was determined that CZA14T possess the genes for the expression of a Type-II PKS – curamycin A. Sequence information indicated that the Type II PKS involved in the production of curamycin A was expressed under the fermentation conditions used in the ALR. This Type-II PKS corresponded to the predicted Type-II PKS from contig 196, cluster 24 of the CZA14T genome. Solvent extracts were analysed using Thin Layer Chromatography (TLC) and the inhibitory efficacy of the crude and partially purified extracts were determined using filter disk diffusion and bioautography assays against a range of ATCC clinical test strains and environmental DRWH system isolates. Compounds separated by TLC showed bands with similar Rf values when visualised under UV light throughout all fermentation processes, but the focus of the study was mostly on the extracts prepared from the fermentation in the ALR. Compounds produced by CZA14T exhibited a broad spectrum of bio-activity against all the test strains used in this study, displaying both anti-bacterial and anti-fungal activity. Compounds produced by CZA14T in the ALR displayed various chemical and physicochemical characteristics as determined by different staining methods of the developed TLC plates and UV/Vis spectroscopy of the biologically active compounds. The crude compounds were partially purified using silica column chromatography and the fractions collected were analysed by liquid chromatography-mass spectrometry (LC-MS). Furthermore, the ALR fermentation broth was subjected to ammonium sulphate precipitation, dialysis and organic solvent extraction. This yielded a set of bio-active compounds with activity against various ATCC strains and DRWH system isolates. This sample was also subjected to LC-MS analyses and results showed the presence of a mass (1374.8 m/z) that corresponds to that of curamycin A, which serves as additional confirmation that the smBGC that encodes for curamycin A, is expressed under the fermentation conditions used in the ALR. Future studies will focus on the purification and structural elucidation of the bio-active compounds produced by CZA14T, including curamycin A.

AFRIKAANSE OPSOMMING: Toenemende verstedeliking, gereelde droogtes, swak gehalte water en skaars waterbronne, veral in landelike gebiede, is ‘n bron van kommer wat voorsiening van vars water aanbetref. Water is noodsaaklik vir lewe en om die groeiende vraag na water te voorsien, is dit noodsaaklik om alternatiewe waterbronne te ondersoek. Die opvang van reënwater word as 'n potensiële alternatiewe bron van water beskou en word algemeen in baie dele van die wêreld as ‘n water bron gebruik. Daar is egter ‘n ernstige gesondheidsrisiko verbonde aan die gebruik van versamelde reënwater, veral as dit as drink-water bron gebruik word. Die rede hiervoor is dat patogene soos Staphylococcus spp., Salmonella spp., Pseudomonas aeruginosa en Klebsiella spp. bewys is om teenwoordig te wees in reënwater tenks, en die kontaminasie van die tenks met hierdie patogene word dan algemeen toegeskryf aan fekale besmetting van die opvang area van die tenks deur klein soogdiere, voëls en reptiele. Hierdie patogene is oor die algemeen hoogs virulent en weerstandig teen antimikrobiese middels wat huidiglik gebruik word om siektes te behandel wat deur hierdie organismes veroorsaak word. 'n Toename in antimikrobiese weerstandige infeksies en siektes plaas druk op antimikrobiese terapie vir die suksesvolle behandeling van hierdie infeksies en siektes. Daarom is dit noodsaaklik om nuwe antimikrobiese middels te ondersoek wat effektief sal wees teen ‘n wye spektrum van bakterieë, fungi, virusse en parasiete. Aktinomisete is 'n groep grond mikro-organismes wat bekend is vir die produksie van bio-aktiewe verbindings. Streptomyces pharetrae CZA14T is een so aktinomiseet wat vir die eerste keer in 2005 beskryf is. Voorlopige toetse het getoon dat CZA14T die vermoë het om bio-aktiewe verbindings te produseer. Die genoom volgorde van CZA14T is daarom bepaal. Die genoom is toe gebruik om die teenwoordigheid van sekondêre metaboliet biosintetiese geen klusters (smBGKs) te voorspel, wat beheer uitoefen oor die uitdrukking van bio-aktiewe verbindings. Dit is gedoen deur gebruik te maak van die sagteware AntiSMASH. Vyf-en-vyftig smBGCs is geïdentifiseer deur AntiSMASH, waarvan 23 kodeer vir nie-ribosomale peptied sintetases (NRPSs), sewe kodeer vir lantipeptiede / lassopeptiede / bakteriosiene, agt kodeer vir poliketide sintases (PKSs) en die oorblywende 17 biosintetiese geen klusters kodeer vir siderofore, terpene, butyrolaktone, ektoines, melanien en gene wat as "ander" beskryf is. In die huidige studie is ‘n medium en fermentasie kondisies gebruik wat voorheen bewys is om optimaal te wees vir die produksie van antimikrobiese verbindings deur CZA14T. Die eksperimente is opgegradeer van 0.5 L, 1 L en 2 L gekeepte fles fermentasies na fermentasie in ‘n 3 L “airlift reactor (ALR)” en 5 L aanhoudend gemengde tenk reaktor (CSTR). Ekstraksies is uitgevoer op beide die miselium en supernatant om te toets vir intra- en ekstrasellulêre produksie van bio-aktiewe verbindings. Fermentasie monsters is ook geneem uit die ALR op dag 3, 6, 9 en 12, en daarna gebruik vir molekulêre analises naamlik: RNA ekstraksies, cDNA sintese en cDNA volgorde bepaling. Deur die gebruik van molekulêre tegnieke, is vasgestel dat CZA14T gene besit vir die uitdrukking van 'n Tipe-II PKS kuramisin A. Volgorde inligting het aangedui dat die Tipe-II PKS, onder die gespesifiseerde fermentasie kondisies in die ALR, uitgedruk is. Hierdie Tipe-II PKS het ooreengestem met die voorspelde Tipe-II PKS van kontik 196, kluster 24 van die genoom van CZA14T. Oplosmiddel ekstraksies is ontleed met behulp van dunlaagchromatografie (DLC) en die inhiberende doeltreffendheid van die kru- en gedeeltelike gesuiwerde ekstraksies is bepaal deur gebruik te maak van diffusie filter skyfies en bioautografiese toetse. Die ekstraksies is getoets teen 'n reeks ATCC kliniese isolate en omgewings-isolate wat vanuit reënwater tenks geïsoleer is. Die verbindings wat met behulp van DLC geskei is, het deurentyd dieselfde bande getoon (onder UV-lig gevisualiseer) met ooreenstemmende Rf-waardes vir die fermentasie-tydperk. Die fokus van die studie was egter op die ekstraksies wat vanuit die fermentasie-produk van die ARL voorberei is. Die verbindings wat deur CZA14T geproduseer is, was aktief teen al die toets organismes wat in hierdie studie gebruik is en het dus beide anti-bakteriële en anti-fungi aktiwiteit getoon. Verbindings wat deur CZA14T geproduseer is in die ALR, het verskeie chemiese en fisiese-chemiese eienskappe vertoon, soos bepaal deur verskillende kleuringsmetodes van die ontwikkelde DLC plate en UV / Vis spektroskopie van die biologiese aktiewe verbindings. Die kru-verbindings is gedeeltelik gesuiwer met behulp van silika kolomchromatografie en die versamelde breukdele is ontleed deur vloeistofchromatografie-massaspektrometrie (LC-MS). Daarbenewens is die ALR fermentasie mengsel onderwerp aan ammoniumsulfaat presipitasie, dialise en organise oplosmiddel ekstraksie. ‘n Stel bio-aktiewe verbindings, wat aktiwiteit teen ‘n verskeidenheid ATCC – en reënwater isolate getoon het, is op hierdie manier geïdentifiseer. Hierdie monster is ook blootgestel aan LC-MS analise en resultate het die teenwoordigheid van 'n massa (1374,8 m/z), wat ooreenstem met dié van kuramisin A, getoon. Hierdie resultate dien as addisionele bevestiging dat die smBGC wat kodeer vir kuramisin A uitgedruk word tydens die ALR fermentasie kondisies. Toekomstige studies sal fokus op die suiwering en strukturele ontleding van die bio-aktiewe verbindings wat deur CZA14T geproduseer word, insluitende kuramisin A.

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