An external ecological niche for Candida albicans within reducing, oxygen-limited zones of wetlands and riverbanks

Stone, Wendy (2011-03)

Thesis (MSc)--Stellenbosch University, 2011.

Thesis

ENGLISH ABSTRACT: The ascomycetous yeast, Candida albicans, has been almost exclusively studied in a clinical context, due to the medical risk and costs associated with the yeast. Most environmental research into the external survival of this opportunistic pathogen has been concerned with short-term, severe pollution challenges. However, a study of literature indicates that the habitat characteristics of the oxygenlimited zones in wetlands and riverbanks are comparable to those of the gastrointestinal source of sewage-borne C. albicans. Interestingly, these are the external, environmental regions to which sewage-borne C. albicans is often exposed. In addition, oxygen-limitation is the predominant parameter in stimulating conjugation of C. albicans. Based on these observations, this study aimed to assess polluted river bank and wetland environments in the Western Cape of South Africa as potential habitats to accommodate a niche for C. albicans, particularly comparing the presence of this yeast in oxygen-limited, plant debris-rich zones and aerobic, clear, flowing zones. The second objective was to employ in vitro microcosm studies to investigate the survival and growth of C. albicans in various microhabitats similar to those comprising the oxygen-limited zones of wetlands. These included the rhizosphere of wetland flora, various soil and mud types and decomposing plant debris. The final objective was to establish the presence of sufficient nutrient and energy sources within this environment for the growth of C. albicans. In particular, cellulosic substrates and mono- and disaccharides released by the natural degradation of wetland plant debris were investigated as potential energy sources for this human commensal in the wetland environment. These study objectives combined to demonstrate the potential of such an oxygen-limited, plant debris-rich environment as a niche for C. albicans external to its human host. Both semi-quantitative culturing techniques and quantitative Real-Time PCR demonstrated the improved survival of C. albicans in oxygen-limited, plant debris-rich zones in wetland and river bank environments, in comparison to aerobic, clear subsurface water zones in the same environments. These zones were compared in the Plankenburg and Diep Rivers, situated in the Western Cape of South Africa. Correlations between coliform concentrations and total yeast concentrations were demonstrated in each of the different river zones, with higher pollution levels characteristic of the dry season. Candida albicans numbers in flowing water (zone W), rock-filtered (zone R) and plant-filtered water (zone P) were compared during the progress of the rainy and dry seasons. No C. albicans was observed in clear, flowing water throughout the analysis. Early in the rainy season, both rock-filtered (aerobic, poor in plant debris) and plant-filtered (oxygen-limited, rich in plant debris) water demonstrated C. albicans numbers at approximately equivalent levels of 10²-10³ cells/100 mL. However, as the rainy season progressed and total yeast and coliform numbers in all zones of the rivers dropped to negligible levels, C. albicans could no longer be detected in aerobic, rock-filtered zones; but its numbers remained at constant levels in oxygen-limited, plant-filtered zones. This suggests that oxygen-limited wetland and river bank zones rich in plant matter, analogous to the human gastrointestinal tract, may provide an ideal habitat in which C. albicans could establish a niche external to its host. The survival of this yeast in the various microhabitats that comprise this anaerobic, reducing wetland environment was evaluated with in vitro microcosms. The rhizosphere of wetland plants had no influence on C. albicans growth and survival in comparison to bulk soil away from the plant, and wetland mud microbiota was demonstrated to be inhibitory to its survival. However, decaying plant debris was shown to increase the survival of the yeast in this inhibitory mud environment. Candida albicans was shown to compete well saprophytically in anaerobic plant debris microcosms. In addition, the tendency of C. albicans to associate with plant matter in an aquatic environment was demonstrated by inoculating the yeast in water containing Hydrilla, a submerged macrophyte found in South African aquatic environments. Plate and liquid analyses, as well as an ANKOM NDF analysis, indicated unequivocally that the C. albicans strains evaluated in this work were unable to utilise the complex carbohydrates of the wetland habitat, including cellulose and fibre. However, HPLC, along with GCMS, demonstrated the anaerobic assimilation by C. albicans of monosaccharides released by natural lignocellulose degradation of wetland plant matter. An analysis of total nitrogen by digestion in a nitrogen analyser, as well as evaluation of ammonium, nitrate and nitrite in a KCL extract, also showed that C. albicans assimilates nitrogenous compounds released by the decomposition of wetland plant matter. This decay process occurs constantly in wetland and river bank habitats. It may therefore provide energy and nutrients for C. albicans, particularly in the anaerobic zones where conjugation may possibly occur and a niche may be established, as indicated by the results obtained for the Plankenburg and Diep Rivers.

AFRIKAANSE OPSOMMING: Die askomisete gis Candida albicans is feitlik eksklusief in ‘n kliniese konteks bestudeer weens die mediese risiko en koste daaraan verbonde. Die meeste omgewingsnavorsing op die eksterne oorlewing van hierdie opportunistiese patogeen was toegespits op die uitdagings van ernstige korttermyn besoedeling. ‘n Literatuurstudie toon egter dat die habitat-eienskappe van die suurstof-beperkte sones in vleilande en rivieroewers vergelykbaar is met dié van die gastroïntestinale bron van C. albicans wat in riool gevind word. Interessant genoeg is dit juis hierdie eksterne omgewingsgebiede waaraan C. albicans vanuit riool dikwels blootgestel word. Hierby is suurstof-beperking die vernaamste parameter in die stimulering van konjugasie in C. albicans. Op grond van hierdie waarnemings poog dié studie om besoedelde vleilande en rivieroewers in die Wes-Kaap Provinsie van Suid-Afrika te evalueer as potensiële habitatte wat ‘n nis van C. albicans kan akkommodeer, en veral om die teenwoordigheid van hierdie gis in suurstof-beperkte sones ryk aan plantafval te vergelyk met aerobe, helder, vloeiende sones. Die tweede doelwit was om in vitro mikrokosmos studies te gebruik om die oorlewing en groei van C. albicans in verskillende mikrohabitatte soortgelyk aan suurstof-beperkte sones in vleilande te ondersoek. Dit sluit die risosfeer van vleilandflora in, asook verskillende grond- en moddertipes en ontbindende plantafval. Die laaste doelwit was om die teenwoordigheid van genoegsame voedings- en energiebronne in dié omgewing te bepaal vir die groei van C. albicans. In besonder is sellulose substrate, asook die mono- en di-sakkariede, wat deur die natuurlike afbraak van vleiland plantafval vrygestel word, as potensiële energiebronne van hierdie mens-kommensaal in die vleiland-omgewing ondersoek. Hierdie studiedoelwitte het gesamentlik die potensiaal van so ‘n suurstofbeperkte, plantafvalryke omgewing as ‘n nis vir C. albicans buite die menslike gasheer aangetoon. Beide semi-kwantitatiewe kweektegnieke en kwantitatiewe in-tyd PKR het die verbeterde oorlewing van C. albicans in suurstofbeperkte, plantafvalryke sones in vleiland en rivieroeweromgewings gedemonstreer, in teenstelling met aerobe, helder oppervlakwatersones in dieselfde omgewings. Hierdie sones in die Plankenburg- and Dieprivier in die Wes-Kaap Provinsie, Suid-Afrika, is met mekaar vergelyk. Korrelasies tussen coliform konsentrasies en totale giskonsentrasies is in elk van die verskillende sones in dié riviere gedemonstreer, met hoër vlakke van besoedeling kenmerkend aan die droër seisoen. Candida albicans getalle in vloeiende water (sone W), rots-gefiltreerde (sone R) en plant-gefiltreerde water (sone P) is deur die verloop van die reën- en droë seisoene met mekaar vergelyk. Geen C. albicans is deur die loop van die analises in helder, vloeiende water bespeur nie. Vroeg in die reënseisoen het beide rots-gefiltreerde (aerobe, min plantafval) en plant-gefiltreerde (suurstofbeperk, ryk in plantafval) water vergelykbare vlakke van C. albicans getoon, naamlik 10²-10³ selle/100 mL. Soos die reënseisoen egter verloop het en die totale gis- en coliforme getalle in al die sones van die riviere tot weglaatbare vlakke gedaal het, kon C. albicans egter nie meer in die aerobe, rots-gefiltreerde sones bespeur word nie, hoewel die getalle in suurstofbeperkte, plant-gefiltreerde sones konstant gebly het. Dit dui daarop dat suurstof-beperkte vleiland en rivieroewer sones ryk in plantmateriaal, analoog tot die menslike gastroïntestinale kanaal, die idealke habitat mag bied waarin C. albicans ‘n nis mag vind buite sy gasheer. Die oorlewing van hierdie gis in die verskillende mikrohabitatte wat uit hierdie anaerobe, reduserende vleilandomgewing bestaan, is met in vitro mikrokosmosse geëvalueer. Die risosfeer van vleilandplante het in vergelyking met die grond weg van die plant geen effek op die groei en oorlewing van C. albicans gehad nie, en vleiland modder-mikrobiota is gevind om die oorlewing daarvan te inhibeer. Verrottende plantafval het egter die oorlewingsvlakke van giste in hierdie inhiberende modderomgewing verbeter. Candida albicans kan egter saprofities goed kompeteer in anaerobe plantafval mikrokosmosse. Hierby is die geneigdheid van C. albicans om met plantmateriaal in waterige omgewings te assosieer gedemonstreer deur die gis te innokuleer in water wat Hydrilla, ‘n onderwater makrofiet wat in Suid-Afrikaanse akwatiese omgewings aangetref word, bevat. Plaat en vloeibare analises, asook ‘n ANKOM NDF data-analise, het onteenseglik getoon dat die C. albicans stamme wat in dié werk gebruik is, nie in staat was om die komplekse koolhidrate, insluitende sellulose en vesel, van die vleiland habitat te benut nie. HPLC, saam met GC-MS, toon egter C. albicans se anaerobe assimilasie van monosakkariede wat deur natuurlike lignosellulose afbraak van vleiland plantmateriaal vrygestel is. ’n Totale stikstof analise deur vertering in ’n stikstof analiseerder, en ’n evalueering van ammonium, nitraat en nitriet in ‘n KCl ekstrak, het ook getoon dat C. albicans stikstofverbindings assimileer wat deur die afbraak van vleiland plantmatriaal vrygestel word. Hierdie afbraakproses kom deurlopend in vleiland en rivieroewer habitatte voor en verskaf potensieel energie en voedingstowwe aan C. albicans, spesifiek in die anaerobe sones waar konjugasie moontlik kan plaasvind, en ‘n nis gevestig kan word, soos aangedui deur die Plankenburg- and Dieprivier.

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