Binary interactions between bacteria and Candida albicans

Thesis (PhD)--Stellenbosch University, 2017.ENGLISH ABSTRACT: The primary habitat of the ascomycetous yeast Candida albicans is thought to be the mammalian gastrointestinal (GI) tract. This opportunistic pathogen however, was also found to be capable of sustainable growth in the anaerobic zones of fecal contaminated rivers. Although it is known that bacteria may impact the survival of C. albicans under aerobic conditions, the effect of an anaerobic environment on these bacteria/yeast interactions has never been explored. Therefore, using both aerobic and anaerobic liquid co-cultures, binary interactions between C. albicans and a number of environmental bacterial isolates were studied at 26ºC. The bacteria represented Aeromonas hydrophila, Bacillus cereus, Bacillus subtilis, Clostridium, Enterobacter, Klebsiella pneumoniae, Kluyvera ascorbata and Serratia marcescens. Using plate counts it was found that bacterial growth inhibits the yeast’s growth under aerobic conditions; however, the inhibitory effect was ameliorated under anaerobic conditions. Bacterial enzymes capable of degrading yeast cell walls, including chitinases and mannanases, were produced by the majority of the bacteria. In contrast, except for A. hydrophila, these enzymes were not detected in anaerobic bacterial cultures, nor was the antimicrobial compound, prodigiosin found in anaerobic cultures of S. marcescens. Crude extracellular enzyme preparations from K. pneumoniae and S. marcescens had no effect on the yeast’s growth, but were found to enhance the toxicity of prodigiosin towards the yeast, especially in combination with mannanases. The above-mentioned in vitro methods were also used to study binary interactions at 37ºC between C. albicans and selected GI bacteria. The latter included Bacteroides fragilis, Bacteroides vulgatus, Clostridium perfringens, Escherichia coli, K. pneumoniae, Lactobacillus acidophilus and Lactobacillus plantarum. Using live/dead yeast viability staining in combination with epifluorescence microscopy it was demonstrated that the aerobic filamentous phase of C. albicans, which is usually induced at 37ºC, was either killed or inhibited by the bacteria; however, the bacteria had no effect on anaerobic yeast growth. Mannanase containing crude enzyme preparations from K. pneumoniae and B. fragilis also had no effect on anaerobically cultured yeast cells. Also, the enzyme preparations did not enhance the antagonistic effect of antimicrobials, such as Amphotericin B and prodigiosin, towards the yeast. Generally, bacterial growth was unaffected by the presence of C. albicans, neither at 26 or 37ºC, nor under aerobic or anaerobic conditions. Under anaerobic conditions at 37ºC however, the presence of either C. albicans or the model ascomycete Saccharomyces cerevisiae, significantly increased the numbers of mannan utilizing B. fragilis and B. vulgatus. Conversely, this increase was not as pronounced in the presence of engineered S. cerevisiae strains with compromised cell wall mannan layers. Treating monocultures of the two Bacteroides species with intact dead yeast cells also resulted in significant increases in bacterial numbers, while removal of the cell wall mannan layers from these dead yeasts resulted in no increase in Bacteroides numbers. These findings indicated that mannan utilizing Bacteroides strains may form commensalistic interactions with C. albicans. Overall, our findings suggest that the symbioses between C. albicans and bacteria may differ depending on the presence or absence of oxygen.AFRIKAANSE OPSOMMING: Die soogdier se spysverteringskanaal word beskou as die primêre habitat van die askomisete gis, Candida albicans. Hierdie opportunistiese patogeen was egter ook al gevind om volhoubare groei in die anaërobiese sones van fekaal besmette riviere te handhaaf. Alhoewel dit bekend is dat bakterieë die oorlewing van C. albicans onder aerobiese toestande kan beïvloed, is die uitwerking van 'n anaërobiese omgewing op hierdie bakterieë/gis interaksies nog onbekend. Dus is beide aërobiese en anaërobiese vloeibare mede-kulture gebruik om binêre interaksies by 26ºC tussen C. albicans en 'n aantal omgewings-bakteriële isolate te bestudeer. Die bakterieë verteenwoordig Aeromonas hydrophila, Bacillus cereus, Bacillus subtilis, Clostridium, Enterobacter, Klebsiella pneumoniae, Kluyvera ascorbata en Serratia marcescens. Deur van plaattellings gebruik te maak is dit gevind dat bakteriële groei die gis groei onder aerobiese toestande inhibeer, maar nie onder anaerobiese toestande nie. Bakteriële ensieme wat instaat is om gis selwande aftebreek, insluitend chitienases en mannanases, was geproduseer deur die meerderheid van die bakterieë. In teenstelling, behalwe vir A. hydrophila, was hierdie ensieme nie gevind in anaërobiese bakteriële kulture nie, en die antimikrobiese verbinding, prodigiosin was ook nie gevind in anaërobiese kulture van S. marcescens. Ru ekstrasellulêre ensiem voorbereidings van K. pneumoniae en S. marcescens het geen effek op gis groei gehad nie, maar dit het wel die toksisiteit van prodigiosin teenoor die gis vermeerder, veral in kombinasie met mannanases. Bogenoemde in vitro metodes is ook gebruik om binêre interaksies by 37ºC tussen C. albicans en geselekteerde spysverteringskanaal bakterieë te bestudeer. Laasgenoemde sluit in, Bacteroides fragilis, Bacteroides vulgatus, Clostridium perfringens, Escherichia coli, K. pneumoniae, Lactobacillus acidophilus en Lactobacillus plantarum. Die gebruik van lewendige/dooie gis-lewensvatbaarheidskleuring in kombinasie met epi-fluoressensie mikroskopie, het getoon dat die aerobiese filamentagtige fase van C. albicans, wat gewoonlik by 37ºC geïnduseer word, doodgemaak of geïnhibeer word deur die bakterieë. In teenstelling hiermee, het die bakterieë geen uitwerking op anaërobiese gis groei getoon nie. Ru-ensiem voorbereidings van K. pneumoniae en B. fragilis wat mannanase bevat, het ook geen uitwerking op anaërobies gekweekte gisselle gehad nie. Die ensiem voorbereidings het ook nie die antagonistiese effek van antimikrobiese middels, soos Amfoterisien B en prodigiosin, teenoor die gis verbeter nie. Oor die algemeen was bakteriële groei nie geaffekteer deur die teenwoordigheid van C. albicans nie, nie by 26 of 37ºC of onder aerobiese of anaerobiese toestande nie. Alhoewel, onder anaerobiese toestande by 37ºC in die teenwoordigheid van beide C. albicans of die model askomisete gis, Saccharomyces cerevisiae, was die groei van beide B. fragilis en B. vulgatus, wat mannaan kan benut, aansienlik verhoog. Hierdie toename was egter nie so beslis in die teenwoordigheid van S. cerevisiae stamme met verswakte mannaan lae nie. Die behandeling van mono-kulture van die twee Bacteroides stamme met dooie gisselle het ook gelei tot aansienlike verhogings in bakteriële getalle, maar die verwydering van die mannaan lae van hierdie dooie giste het geen verhoging in Bacteroides getalle tot gevolg gehad nie. Hierdie bevindinge het aangedui dat Bacteroides stamme wat van mannaan gebruik kan maak, moontlik kommensalistiese interaksies met C. albicans kan vorm. In geheel dui ons bevindinge daarop dat die simbiose tussen C. albicans en bakterieë kan verskil afhangende van die teenwoordigheid of afwesigheid van suurstof