Doctoral Degrees (Microbiology)

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Binary interactions between bacteria and Candida albicans
(Stellenbosch : Stellenbosch University, 2017-03) Benade, Eliska; Botha, Alfred; Mouton, Marnel; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
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.
Community-level analysis of the microbiology in constructed wetlands treating distillery effluent
(Stellenbosch : University of Stellenbosch, 2006-04) Du Plessis, Keith R. (Keith Roland); Wolfaardt, Gideon M.; Botha, Alfred; University of Stellenbosch. Faculty of Science. Dept. of Microbiology.
ENGLISH ABSTRACT: Constructed wetlands have been widely used in the treatment of industrial and domestic wastewater to reduce biological and chemical oxygen demand (BOD and COD), to remove nitrate and enteric viruses as well as to generally improve water quality. Distillery wastewater has a complex character due to high concentrations of sugars, lignins, hemicellulose, dextrins, resins, polyphenols and organic acids, leading to a high COD that may exceed 100 000 mg/L. The potential application for the treatment of distillery wastewater by means of constructed wetlands is relatively unexplored. In 1999 a study was initiated at Distell Goudini distillery, Western Cape, South Africa, to explore the possibility of using constructed wetlands to treat distillery wastewater. It was found that constructed wetlands do have the ability to treat distillery wastewater providing that the influent COD does not exceed 15 000 mg/L for extended periods and the correct substrate material is used. The present study expanded on the above-mentioned study and specifically aimed to provide information on the microbiological controls in wetland systems in an applied sense that may contribute to improved treatment efficiency. Furthermore, this project aimed to contribute to our fundamental understanding of the microbial ecology of constructed wetlands used for the treatment of distillery wastewater. This study revealed that a highly dynamic microbial composition exists within wetlands. Furthermore it was found that wetlands can efficiently remove COD even though a low degree of similarity exists between microbial communities in various zones of the same wetland and those between different wetlands, as well as low similarity between communities sampled from the same zone over time. This demonstrates that it will be difficult to define the ‘ideal’ degradative community in terms of microbiological criteria and serves as a reminder that various indicators should be considered for monitoring system health. Furthermore the shifts in microbial community composition illustrate the ability of microbial communities to adapt to changes in the environment without compromising their functional efficacy. When studying the attached microbial communities within wetland systems it was found that different morphotypes are detected at certain stages of biofilm development while some organisms are present at most phases of biofilm formation. Measurement of CO2 production and dissolved organic carbon (DOC) removal in laboratory scale columns showed that grazing protists had a notable effect on overall microbial activity and that organic loading influenced these predator-prey interactions. Interestingly, increased clogging of pores occurred in the presence of protists, resulting in reduced flow through the porous matrix. Terminalrestriction fragment length polymorphism (T-RFLP) analysis of biofilms on gravel in experimental wetlands indicated that the presence of protists and algae had an effect on the microbial community composition. Scanning electron microscopy (SEM) showed that the presence of algae also had an influence on biofilm structure suggesting that the algae provided labile nutrients that were utilized by the bacterial and yeast members of the community. Finally, augmentation with a commercial mixture or microbial populations isolated from distillery effluent demonstrated that the concentration at which supplements are applied influence degradative efficiency.
A deeper understanding of the urease enzyme in the opportunistic yeast Cryptococcus neoformans
(Stellenbosch : Stellenbosch University, 2020-12) Toplis, Barbra-Ann; Botha, Alfred; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
ENGLISH ABSTRACT: Urease enzyme production is a well-studied virulence mechanism of the opportunistic pathogen Cryptococcus neoformans. It is believed that the main function of urease is to provide these yeasts with a readily assimilable nitrogen source in the form of ammonia, via the hydrolysis of exogenous urea. The latter is encountered by cryptococci both within the human host, as well as in the natural environment. Although largely unexplored in C. neoformans, urease can also hydrolyse urea that originates endogenously from metabolic reactions within the yeast cell, such as those involved in polyamine biosynthesis. The overall aim of this study was therefore to determine whether cryptococcal urease is linked to metabolic pathways related to the yeast’s endogenous urea production. Thus, throughout the study, biochemical analyses were conducted on wild-type, urease-deficient and urease-reconstituted strains of C. neoformans H99. To investigate cryptococcal physiology at mammalian body temperature, all experiments were carried out at 37 °C in addition to 26 °C. The results revealed that urease plays a role in cryptococcal growth and compared to nutrient-rich conditions and a temperature of 26 °C, urease enzyme activity was highest under nutrient-limited conditions at 37 °C. The latter findings were obtained in the absence of exogenous urea, which together with the observation of elevated urea levels in crude extracts of urease-deficient cells, indicated that urea was produced endogenously by C. neoformans. In addition, it was found that arginase enzyme activity, a potential source of intracellular urea, was highest in cells of the urease- deficient mutant. The latter finding was a suggested explanation for the elevated urea levels that were observed for this strain. Additional aspects linked to arginase metabolism were also explored and it was found that compared to the wild-type, urease-deficient cells differed with respect to agmatinase activity, polyamine biosynthesis and intracellular levels of proline and reactive oxygen species. Very importantly, the urease-negative strain also showed differences in melanin production, which provided the first evidence that urease may affect the functioning of another cryptococcal virulence mechanism. Overall, the results provided strong evidence that the urease enzyme of C. neoformans is linked to vital metabolic pathways within the yeast cell, of which many are known to be involved in polyamine metabolism. Thus, using a polyamine-free growth medium, cryptococcal polyamine biosynthesis was subsequently explored further. Mass spectrometry analysis showed that agmatine and spermidine are the major extra- and intracellular polyamines of C. neoformans at both 26 and 37 °C. Cadaverine and putrescine were identified as minor polyamines and spermine was undetected. Temperature was found to influence the results obtained for all three strains as significant differences were detected in the relative percentages of extra- and intracellular polyamines between 26 and 37 °C. Strain differences in polyamine metabolism were observed at both tested temperatures where, compared to the wild- type, relative percentages of extracellular putrescine and spermidine were found to be significantly lower and agmatine significantly higher in cultures of the urease-deficient mutant. The inverse was true for intracellular spermidine and agmatine. All three strains were more susceptible to treatment with the polyamine inhibitor cyclohexylamine compared to DL-α-difluoromethylornithine and all inhibitory effects were more pronounced at 37 °C than at 26 °C. At both tested temperatures, the urease-deficient mutant was less susceptible to cyclohexylamine compared to the wild-type. Growth inhibition by cyclohexylamine and DL-α-difluoromethylornithine was reduced with spermidine and putrescine supplementation, respectively. Taken together, the results provided novel insight into polyamine metabolism by C. neoformans, highlighting for the first time the involvement of urease in biogenic amine production. Based on our findings, cryptococcal urease plays a complex cellular role that extends far beyond the use of exogenous urea as a nitrogen source.
The effect of different soil yeasts on the growth and physiology of lupin and wheat
(Stellenbosch : Stellenbosch University, 2018-03) Moller, Leandra; Botha, Alfred; Valentine, Alexander J.; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
ENGLISH ABSTRACT: Leguminous plants are often used in break-crop and crop rotation systems to fix atmospheric di-nitrogen (N2), thereby reducing the need for nitrogen (N) fertiliser input during cereal production. In these systems, wheat (Triticum aestivum L.) yield increases following blue lupin (Lupinus angustifolius L.) cultivation. Yields can also be increased by plant growth promoting microorganisms such as yeasts. These plant growth promoting yeasts (PGPY) enhance seed germination, produce plant growth promoting (PGP) factors, and partake in beneficial tripartite and quadripartite symbioses with their host plant and its root symbionts. Despite the importance of blue lupin in agriculture, it was unclear how tripartite and quadripartite interactions involving PGPY affect this legume’s biological N2 fixation (BNF) and growth. In addition, the effect of a single PGPY on germination and growth of plants used in break-crop or crop rotation systems, such as blue lupin and wheat, was unknown. To address these aspects, the impact of a rhizosphere yeast and mycorrhizal fungi on growth and nutrition of nodulated blue lupin was firstly evaluated. The ability of this rhizosphere yeast to affect a break-crop or crop rotation system was subsequently assessed by studying the yeast’s influence on the germination and developmental physiology of blue lupin and wheat. Papiliotrema laurentii (syn. Cryptococcus laurentii) was isolated from the rhizosphere of blue lupin. Nodulated blue lupin seedlings, treated with either P. laurentii CAB 91 (PL), or mycorrhizal fungi (MF), or with both symbionts (PLMF), were then cultivated in a glasshouse, after which the plants’ biomass parameters, symbiotic colonisation, and mineral nutrition were analysed. The PGP traits of PL were subsequently evaluated by comparing them to that of two other rhizosphere yeasts, i.e. Hannaella zeae CAB 1119 (HZ) and Saitozyma podzolica CAB 1199 (SP). The yeasts were used to coat seeds of blue lupin and wheat, where after their influence on the germination of these seeds was assessed under controlled conditions. The cold test was used to evaluate the influence of the three yeast strains on the vigour of both plants. To determine the effect of HZ, PL, and SP on growth and photosynthesis of nodulated blue lupin and wheat, plants were cultivated under glass house conditions. It was found that the MF treatment had no effect on blue lupin seedlings under glass house conditions. Improved growth of PL treated seedlings was underpinned by increased BNF efficiency, while greater nodulation and efficient growth on N resources supported the increased biomass of PLMF treated plants. Subsequent trials with the three rhizosphere yeasts mentioned above, indicated that of these three yeasts only PL can be used to coat the seeds of both blue lupin and wheat, since this yeast increased the germination of blue lupin and the vigour of wheat. Additionally, under glass house conditions PL promoted higher relative growth rates during the early developmental stages of both plants. The latter coincided with enhanced photosynthetic metabolism and water relations. These findings indicate that PL may potentially serve as an efficient bio-fertiliser of blue lupin and wheat to benefit breakcrop and crop rotation systems.
Insights into the lignocellulosic physiology of the yeast pathogen Cryptococcus neoformans var. grubii
(Stellenbosch : Stellenbosch University, 2014-04) Botes, Angela; Botha, Alfred; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
ENGLISH ABSTRACT: Cryptococcus neoformans (Sanfelice) Vuillemin is an opportunistic pathogen that causes cryptococcal meningitis, predominantly in immuno-compromised individuals, particularly in those suffering from human immuno virus (HIV) or acquired immuno-deficiency syndrome (AIDS). This basidiomycetous yeast species is sub-divided into two main varieties, C. neoformans var. neoformans (serotype D) and C. neoformans var. grubii (serotype A), as well as a rare hybrid variety, C. neoformans (serotype AD). The global incidence of cryptococcosis among AIDS sufferers is approximately 30 % with 90 % of these cases being attributed to serotype A. Cryptococcus neoformans has been isolated from numerous environmental sources including guano, soil, and particularly decaying wood and tree hollows. Infection occurs when particles originating from these environments are inhaled. The ecological niche of C. neoformans was thought to be avian guano; however, recent findings indicate that the true ecological niche may rather be woody material. Representatives of this species, particularly C. neoformans var. grubii, were found to grow on agar plates containing carboxymethyl cellulose as carbon source; however, little is known about its ability to degrade hemi-cellulose. As such, the overall aim of this project was to study the interactions of C. neoformans var. grubii ATCC H99 with cellulosic and hemi-cellulosic materials. Growth studies revealed that C. neoformans var. grubii ATCC H99 was capable of utilizing carboxymethyl cellulose, glucomannan and galactomannan as sole carbon sources. This yeast also assimilated simple degradation products of lignocellulose such as L-arabinose, D-galactose, D-glucose, D-mannose, L-rhamnose and D-xylose. D-Mannose and D-glucose resulted in the highest maximum specific growth rates. Screening the genome of C. neoformans var. grubii ATCC H99 resulted in the identification of three putative cellulases, specifically an endo-glucanase (EC7) and two cellulases (CC1 and CC6). Evaluation of the deduced amino acid sequences indicated that all three enzymes belong to glycoside hydrolase family 5 (GHF5). Phylogenetic analyses revealed that the three enzymes grouped in distinct clades with other GHF5 members. Automated homology modeling of the three-dimensional structure revealed that CC1 and CC6 displayed the classical (α/β)8 TIM barrel fold associated with GHF5. Modeling of EC7 did not produce the classic GHF5 structure, suggesting that this enzyme may be classed in a separate GHF. Green fluorescent protein (GFP) tagging confirmed that CC6 forms part of the cryptococcal secretome. Real-time quantitative PCR (qPCR) analyses indicated that the three proteins responded differently on a transcriptional level in the presence of various carbohydrates. Cellulase CC6 displayed the most dynamic expression profile, indicating up-regulation in the presence of mannose, galactose and cellobiose. The use of Acacia mearnsii debris and the aqueous extract thereof also resulted in a significant up-regulation of all three enzymes. This confirms previous findings that the woody phyloplane is a natural habitat of C. neoformans var. grubii. In the presence of mucin, the transcription of CC6 was up-regulated. Similar to laccase and urease, CC6 may aid the survival of C. neoformans within the human respiratory system. Understanding the carbohydrate metabolic regulatory system and its impact on virulence would increase our overall knowledge of this pathogen‘s survival capabilities and infection strategies.
Interactions between gut microbiota and the central nervous system, with emphasis on quorum sensing between commensal lactic acid bacteria and human cells
(Stellenbosch : Stellenbosch University, 2023-03) Dicks, Leon Milner Theodore; Botha, Alfred; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
ENGLISH ABSTRACT: The human gut hosts close to 4 trillion microorganisms, which is nearly equivalent to the estimated 3.0×1013 human cells in a 70 kg body. Although the composition of gut microbiota changes with age, variation in diet, medication, hormone levels, stress and other environmental factors, a core group of autochthonous bacteria, between 400 and 500 species, are always present. More than 90% of the gut microbiome is represented by Proteobacteria, Actinobacteria, Bacteroidetes and Firmicutes, with the latter in the majority. Fusobacteria and Verrucomicrobia make up the remaining 10% of the gut microbiome. The human gut microbiome supersedes the number of cells in our bodies ten-fold. Since lactic acid bacteria (LAB) are the predominant gut microbiota, it is safe to conclude that changes in this group will affect the entire microbiome, ultimately leading to adjustments in the behaviour of intestinal epithelial cells (IECs). Changes in the immune system and quorum sensing (QS) signals instigated by an altering gut environment trigger a cascade of hormonal and neurological reactions. Activation of Toll-like receptors, for instance, induce strong immune and inflammatory reactions, but at the same time stimulate the secretion of hormones such as 5-hydroxytryptamine (5-HT, or serotonin), glucagon-like peptide-1 (GLP-1), peptide tyrosine tyrosine (PYY), glucose-dependent insulinotropic peptide (GIP), cholecystokinin (CCK), ghrelin, leptin, pancreatic polypeptide (PP), oxyntomodulin and neurotensin. Serotonin act as neurotransmitter but also regulates diverse functions such as platelet aggregation, bone development, immune response, cardiac function and gut homeostasis, and control enteric motor and secretory reflex. Gut bacteria also synthesize, or regulate, the production of serotonin and other neurotransmitters such as glutamine (Glu), gamma-amino butyric acid (GABA), dopamine (DA), norepinephrine and histamine. These molecules communicate with the central nervous system (CNS) via afferent fibers in the Vagus nerve (VN), autonomic sympathetic and parasympathetic nervous systems, but also the hypothalamic-pituitary-adrenal axis (HPA). Intermediate compounds such as short-chain fatty acids (SCFAs), tryptophan and secondary bile acids produced by gut bacteria also communicate with the CNS. Signals received from the brain are sent back to entero-epithelial cells (EECs) via the HPA and efferent VN fibers to complete the circle of communication referred to as the gut-brain axis (GBA). The modulation, development, and renewal of neurons in the enteric nervous system (ENS) are controlled by gut microbiota, especially those with the ability to produce and metabolize hormones. Minor activation of the ENS and VN results in drastic changes in the production of neurotransmitters, which also affects digestion, intestinal permeability, gastric motility, and immune regulation. GABA, in addition to other metabolites, play an important role in anti-inflammatory responses and help alleviate psychiatric symptoms stemming from inflammation. Treatment of schizophrenic and bipolar patients with probiotics alleviated symptoms associated with irritable bowel disease (IBD), and autistic children benefitted from probiotic treatment. Obsessive compulsive disorder (OCD)-like behavior could also be controlled by treatment with LAB. Inter- and intra-species signalling systems have been well studied, but far less is known about interkingdom quorum sensing (QS), especially between gut bacteria and intestinal epithelial cells (IECs). Although the auto-inducer 3 (AI-3)/epinephrine (Epi)/norepinephrine (NE) QS signalling system described for pathogenic Escherichia coli, Salmonella typhimurium and Ctirobacter rodentium are widely used by Gram-negative pathogenic bacteria, not all species have receptors that recognize these signals. Instead, they have developed “broad-range” “solo” LuxR-type receptors such as SdiA (a LuxR homolog) and QscR to improve their communication abilities. Despite our knowledge on QS, the effect of these signalling molecules on the CNS is ill-researched. Several QS peptides (QSPs) have the ability to diffuse through the intestinal mucosa and enter the circulatory system, from where they may penetrate the blood-brain barrier (BBB). It may be that LAB communicate with the CNS using small linear or cyclized oligopeptides (QS peptides, QSPs) of 5 to 17 amino acids long, as reported for other Gram-positive bacteria. In our own research we have shown that bacteriocins can indeed transverse epithelial (Caco-2) and endothelial (HUVECs) monolayers without changing the integrity of the membranes and with no toxic effect. Once in the blood stream, bacteriocins may cross the BBB, similar to that reported for the heptapeptide PapRIV produced by Bacillus. Our understanding of exactly how gut microorganisms control cognitive behavior, mood, and neuropsychiatric disorders remains limited. However, the more we discover about the gut microbiome, QS, neurotransmitters and the GBA, the greater the chance of developing novel therapeutics, probiotics and psychobiotics to treat gastro-intestinal disorders such as inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS), but also improve cognitive functions and prevent or treat mental disorders. This calls for in-depth deciphering of the complex, everchanging network between cells and neurons. Research on the quenching of QS signals need to be prioritised. We need to understand how quorum quenching (QQ) therapy will affect beneficial gut microbiota. Biomarkers need to be developed to identify differences in the gut microbiome of individuals suffering from psychological disorders. Interactions between drugs used in treatment and gut microbiota need to be studied in greater depth. We need to understand the effect psychiatric medication may have on the composition of the gut microbiome. Are intestinal microbiota able to metabolise these drugs? Studies should include multi-omics of gut and oral microbiota to have a better understanding of the mutual interplay between phyla. Will it be possible to develop probiotics to treat dysbiosis and neuropsychiatric abnormalities?
Interactions of the lignicolous fungus Coniochaeta pulveracea
(Stellenbosch : Stellenbosch University, 2015-12) Van Heerden, Andrea; Botha, Alfred ; Van Zyl, Willem Heber ; Cruywagen, C. W.; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
ENGLISH ABSTRACT: Coniochaeta pulveracea is a microcyclic dimorphic lignicolous fungus that has mostly been isolated from the woody cauloplane. Since the majority of studies conducted on this fungus were taxonomic investigations that focused on macro- and microscopical characterization little is known about their eco-physiology. The latter include morphogenesis as a result of changing environmental conditions, as well as interactions with other fungi, such as yeasts, also occurring on the woody cauloplane. As such, the overall aim of this project was to study the role of Coniochaeta pulveracea’s physiology and morphogenesis in relation to its interactions on the woody cauloplane, including symbioses with yeasts originating from this environment. A representative of the microcyclic fungus C. pulveracea was isolated from a decaying Acacia tree and subsequently identified using morphology, as well as sequence analysis of the D1/D2 (600 – 650 bp) region of the large subunit ribosomal deoxyribonucleic acid gene complex. Plate assays for extracellular hydrolytic enzyme activity indicated that this C. pulveracea isolate, as well as representatives of its closest relatives including Coniochaeta boothii, Coniochaeta rhopalochaeta, and Coniochaeta subcorticalis, were able to produce cellulase and xylanase. All representatives were also able to degrade birchwood toothpicks during a 50-day incubation period at 30°C. To test the ability of these fungi and their enzymes to release simple sugars from complex cellulosic substrates, filtrates obtained from liquid cultures of Coniochaeta, cultivated on carboxymethyl cellulose (CMC) as sole carbon source, were analyzed using high-performance liquid chromatography analysis. Consequently, the presence of mono- and disaccharides such as glucose and cellobiose was confirmed in these culture filtrates. Two subsequent experiments were conducted to determine whether these simple sugars released from woody material by filamentous growth of Coniochaeta may enhance growth of yeasts previously isolated from wood degrading fungi or decaying wood. In the first experiment, representatives of Coniochaeta were co-cultured with selected yeasts suspended in agar plates containing birchwood toothpicks, followed by examination of plates for colony formation. Results indicated that Coniochaeta growth on the toothpicks enhanced growth of nearby yeast colonies in the agar plates. In the second experiment, representatives of selected yeasts and Coniochaeta species were co-cultured on CMC and xylan containing plates where after yeast colony formation was recorded on the plates. Saccharomyces cerevisiae strains, engineered to utilize specific wood degradation products, i.e., cellobiose or xylose, as sole carbon source were used as positive controls. While it was found that cellobiose released from CMC was assimilated by the yeasts, no evidence could be obtained that xylose released from xylan was used as carbon source by the yeasts. These ambiguous results could be ascribed to secretion of nutritious metabolic end products, other than the products of fungal xylanases. Since the above-mentioned interactions were studied whilst the Coniochaeta strains were mostly in their filamentous growth stage, microcycle conidiation of C. pulveracea strains and representatives of the closely related species, C. boothii and C. subcorticalis was also studied under different environmental conditions. The strains were found to exhibit hyphal growth on solid substrates and underwent a dimorphic switch to produce microcycle conidiation upon transfer to a liquid medium which differed in physico-chemical composition compared to the original solid medium. Factors that were found to contribute to this dimorphic switch were temperature, pH and the presence of complex nitrogen sources such as casamino acids and peptone in the medium. However, C. pulveracea showed intraspecific differences with regard to its response to changes in the physico-chemical environment. The interactions of microcyclic Coniochaeta strains with selected yeasts, such as representatives of Meyerozyma guilliermondii and Cryptococcus neoformans, were also studied in complex liquid media and it was found that, depending on medium composition, the microcyclic Coniochaeta exerted different effects on the different yeasts strains. In some co-cultures a positive effect on yeast growth was observed, while in other cases microcyclic Coniochaeta inhibited yeast growth.
Microbial degradation of mycotoxins
(Stellenbosch : Stellenbosch University, 2007-04) Alberts, Johanna Francina; Van Zyl, Willem Heber; Botha, Alfred; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
ENGLISH ABSTRACT: Aflatoxins are mycotoxins predominantly produced by the filamentous fungi Aspergillus flavus and Aspergillus parasiticus. Aflatoxin B1 (AFB1), the most abundant aflatoxin, is highly mutagenic, toxic, carcinogenic and teratogenic to humans and animals and is particularly correlated with the incidence of hepatocellular carcinoma in parts of Africa, China and South East Asia. In this regard aflatoxin is classified as a type I human carcinogen by the International Agency for Research on Cancer. Furthermore, aflatoxin contamination of food and feed is responsible for extensive economic losses due to loss of crops and farm animals. In spite of regulations regarding acceptable levels of aflatoxin in food, aflatoxin contamination remains a serious worldwide problem, especially in developing countries where it occurs predominantly in dietary staples. Inactivation of aflatoxin by physical and chemical methods has not yet proved to be effective and economic. However, biological detoxification offers an attractive alternative for eliminating toxins as well as safe-guarding the desired quality of food and feed. In this study, the biological degradation of AFB1 by bacteria and fungi was investigated. Several bacteria, including Rhodococcus spp., as well as white rot fungi have the potential to degrade a wide range of polycyclic hydrocarbon compounds due to the large repertoire of enzymes they produce and therefore the ability of some of these microorganisms to degrade AFB1 was investigated. Effective degradation of AFB1 by intracellular extracts of Mycobacterium fluoranthenivorans sp. nov. DSM 44556T, Nocardia corynebacterioides DSM 20151 and N. corynebacterioides DSM 12676 was demonstrated. Furthermore, AFB1 was effectively degraded by liquid cultures as well as intra- and extracellular extracts of Rhodococcus erythropolis DSM 14303. Significant (P<0.001) reduction in AFB1 was observed following treatment with R. erythropolis extracellular extracts with only 33.20% residual AFB1 after 72 h. Results indicated that the degradation by R. erythropolis DSM 14303 is enzymatic and that the enzymes are constitutively produced. The degradation of AFB1 when treated with R. erythropolis DSM 14303 extracellular extract coincided with a total loss of mutagenicity. In addition, treatment of AFB1 with culture fractions containing recombinant 2,3-dihydroxybiphenyl dioxygenase, which was produced through extracellular expression of the bphC1 gene of R. erythropolis DSM 14303 in Escherichia coli BL21, resulted in significant (P<0.0001) degradation (49.32%) and reduced mutagenic potency (42.47%) of the molecule. Significant (P<0.0001-0.05) degradation of AFB1 was obtained following treatment with culture extracts containing laccase enzyme produced by white rot fungi (17.10- 76.00%), purified fungal laccase from Trametes versicolor (1 U/ml, 87.34%) as well as with recombinant laccase produced by Aspergillus niger (118 U/L, 55.00%). Furthermore, treatment of AFB1 with purified fungal laccase enzyme (1 U/ml) resulted in loss of the mutagenic potency of the molecule. The decrease in the fluorescence and mutagenic properties of AFB1 following treatment with the microbial preparations imply changes to the furofuran- and/or lactone rings of the molecule. The current study contributes towards developing genetic engineered microbial strains which could be applied as an important bio-control measure. Such strains could exhibit multifunctional technological properties including degradation of AFB1, to significantly improve the quality, safety and acceptability of food.
Molecular characterisation of Cryptococcus neoformans and Cryptococcus gattii present in tree hollows, their antifungal susceptibility, and chemical factors influencing their growth on woody debris
(Stellenbosch : Stellenbosch University, 2020-03) Vreulink, Jo-Marie; Botha, Alfred; Vismer, Hester; Boekhout, Teun; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
ENGLISH ABSTRACT: Members of the Cryptococcus neoformans/Cryptococcus gattii species complex causes cryptococcosis; a disease that is a major burden among HIV/AIDS patients in South Africa. Infection is likely obtained via inhalation of basidiospores from the environment. Basidiospores are produced during opposite-sex mating that occurs between MATα and MATa strains or through same-sex mating. Although it is well established that the habitats of pathogenic cryptococci can serve as infection sources, little was known about the prevalence of environmental pathogenic cryptococci and their relation to clinical isolates in South Africa. It was suggested that environmental factors could affect mating type occurrence, yet nothing was known about this aspect. Furthermore, it was unestablished if South African environmental isolates presented different antifungal susceptibilities than clinical isolates. To address these facets, pathogenic cryptococci were first isolated from the South African environment. Following molecular characterisation, the epidemiological relationship between the environmental and clinical isolates was investigated. Subsequently, the effect of chemical factors on the growth of strains found to colonise trees differentially was examined. Lastly, the antifungal susceptibility of the environmental isolates was compared to that of clinical isolates. Pathogenic cryptococci were isolated from woody debris samples collected on three sampling dates from trees situated in a public park in Cape Town (PPCT) and on one sampling date from trees located on a privately-owned game farm in the Northern Cape (GFNC), whereafter the isolates were molecularly characterised. Since trees were not permanently colonised by C. gattii sensu stricto MATa, it was estimated whether chemical factors in the wood could have affected the occurrence of C. gattii s.s. MATa. A positive correlation was detected between the numbers of C. gattii s.s. MATa and the ammonium-nitrogen (NH₄-N) concentration. Subsequently, the effect of low and high ammonium chloride (NH₄Cl) concentrations on the growth of C. gattii s.s. MATa and MATα was investigated in woody microcosms. Antifungal susceptibility differences between environmental and clinical isolates were evaluated by determining the amphotericin B (AmB) and fluconazole (FLU) susceptibility of the isolates. The pathogenic cryptococci isolated from the PPCT trees were predominantly C. gattii s.s. ST230 followed by C. neoformans s.s. genotype AFLP1/VNI ST23. Since ST23 was previously isolated from infected patients, the PPCT trees might be an infection source. Growth studies revealed that C. gattii s.s. MATa isolates were able to compete better with MATα isolates in microcosms supplemented with a high NH₄Cl concentration, which provided a possible explanation for the absence of MATa in PPCT trees after the first sampling date. All of the environmental isolates were more susceptible to AmB than clinical isolates. In contrast, environmental C. neoformans s.s. genotype AFLP/VNI and C. gattii s.s. isolates were less susceptible to FLU than corresponding clinical isolates, indicating that FLU resistance can develop among environmental isolates. Furthermore, temporal variation in C. gattii s.s. ST230 isolates’ FLU susceptibility was potentially linked to variations in iron, NH₄-N and sodium concentrations within their original woody habitat. This study indicates that environmental factors can affect the numbers and antifungal susceptibility of pathogenic cryptococci in the South African environment.
Rhizofiltration of urban effluent : microbial ecology and conceptual treatment mechanisms
(Stellenbosch : Stellenbosch University, 2016-03) Postma, Ferdinand; Botha, Alfred; Mouton, Marnel; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
ENGLISH ABSTRACT: Polluted urban runoff is a challenge that is globally met by governing bodies employing best management practices (BMPs). One such BMP is rhizofiltration, a novel type of phytoremediation BMP designed to mimic riparian ecology, with the goal of rapidly filtering large volumes of urban runoff before it enters rivers. The physical, chemical and biological mechanisms behind pollutant removal within a rhizofiltration system however, are still largely unknown. The overall aim of this study was therefore to assess the ability of a pilot scale rhizofiltration system to reduce concentrations of the physico-chemical pollutants ammonium, chemical oxygen demand (COD), nitrate, phosphate, sulphate and suspended solids, as well as microbial indicators of faecal pollution, in simulated urban runoff. The faecal indicators included coliphages, faecal coliforms, potentially pathogenic yeasts (PPY) and Salmonella and/or Shigella. To achieve this study’s aim a conceptual model was first constructed to identify potential bacterial mechanisms of pollution removal and to estimate the effect of physico-chemical conditions on microbial communities within the rhizofilter medium. Then, the overall performance of the filter was measured with regard to its bioregeneration and sorption capacity for the abovementioned pollutants. Sorption equilibrium, for most of the pollutants in the simulated runoff percolating through the filter, was reached within 45 minutes. Partial bioregeneration of the filter medium occurred within a week for ammonium, COD, phosphate and sulphate, as well as for the microbial pollutants. Evidence was subsequently obtained supporting the hypothesis that this regeneration is brought about by microbial activity, since metagenomic (16S rDNA high throughput sequencing) and phospholipid fatty acid (PLFA) analyses revealed the presence of viable dynamic microbial populations within the rhizofilter medium. Significant correlations between relative quantities of microbial operational taxonomic units (OTUs) and nutrient concentrations were also uncovered. The rhizofilter plants selected for a microbial community distinct from an unplanted control, however, this did not relate to differences in filter performance. This phenomenon was ascribed to the rapid percolation rate and design of the rhizofilter which maximizes aeration of the filter medium. It was contended that these properties, combined with the composition of the simulated urban runoff, selected for functionally similar organisms. The Actinomycetales were the most abundant bacterial group in both the planted and unplanted filter media. However, the plants appeared to select for Mycobacteriaceae and nitrifiers identified as the Nitrospiraceae. Among the transient OTUs in the filter media were taxa associated with the human gut, including the Campylobacteraceae, Moraxellaceae, Porphyromonadaceae and Prevotellaceae, while the Enterobacteriaceae containing faecal coliforms were below the detection limit of the metagenomic analysis. Strains of four Candida species consistently occurred in the simulated urban runoff. The abundance of these PPY in the influent and effluent of the rhizofilter were affected by physico-chemical factors. Subsequent metagenomic analysis of the fungal community within the filter media revealed a low relative abundance of candidal PPY. In short, the rhizofilter design and wastewater composition selected for copiotrophic aerobic microorganisms capable of mineralizing potentially recalcitrant organic carbon and driving oxidative processes such as nitrification whilst removing human microbial commensialists and pathogens.
The role of nitrogen limitation in cryptococcal virulence and drug tolerance
(Stellenbosch : Stellenbosch University, 2021-12) Bosch, Caylin; Botha, Alfred; Volschenk, H.; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
ENGLISH ABSTRACT: Cryptococcosis, a disease caused by members of the Cryptococcus neoformans/Cryptococcus gattii species complex, has the highest incidence rate among systemic HIV-associated fungal infections and accounts for more than 15% of all AIDS-related deaths globally. The etiological agents of cryptococcosis are widely distributed within the environment with ecological reservoirs including pigeon guano and the woody debris of numerous tree species. Within its natural habitat, C. neoformans must adapt to severe nutrient stresses. In the present work, we delved into the effects of one such environmental stress, namely nitrogen limitation, as it relates to cryptococcal virulence. Moreover, we explored the clinical relevance of these effects by investigating the role of nitrogen stress in cryptococcal susceptibility to two commonly used antifungal drugs, amphotericin B (AmB) and fluconazole (FLU). By culturing C. neoformans and C. gattii in media with different ecologically relevant nitrogen concentrations, we found that low nitrogen conditions enhanced cryptococcal virulence factor production, as well as the levels of the membrane sterol and antifungal target, ergosterol. Evaluation of drug tolerance using time-kill methodology revealed that nitrogen limited cultures had the highest survival percentages in the presence of both AmB and FLU, suggesting that nitrogen concentration may indeed influence drug tolerance. For a deeper understanding of the effects of nitrogen limitation on pathogenic cryptococci, we investigated the transcriptomic response of C. neoformans to low nitrogen concentrations using RNA-sequencing. It was found that nitrogen limited conditions upregulated the expression of antifungal tolerance- related genes, including those involved in ergosterol biosynthetic processes and cell wall integrity. Low nitrogen conditions were also found to modulate the expression of numerous virulence-associated genes, such as CTR4 and CGP1, which encode a copper transporter and a microtubule-associated protein, respectively. Using gene deletion mutants, we demonstrated for the first time that Ctr4 and Cgp1 are functionally associated with cryptococcal adaptation to nitrogen availability, by contributing to cryptococcal growth in low nitrogen conditions, nitrogen source assimilation, oxidative stress tolerance and antifungal susceptibility. Finally, we evaluated the in vivo effects of cryptococcal pre-adaptation to nitrogen limited environments using a Galleria mellonella infection model and by studying cryptococcal- macrophage interactions. We showed that low nitrogen conditions enhanced the virulence of C. neoformans in an invertebrate host and demonstrated that this nutritional stress influences uptake of the fungus by human macrophages. These findings ultimately highlight the importance of isolate origin in the cryptococcal-host interaction. Altogether, the insights gained from our research greatly enhance our understanding of the role of nitrogen availability in cryptococcal pathogenesis and antifungal tolerance, and partially improve our knowledge on how nitrogen influences the survival of these fungal pathogens in natural and host niches.

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