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Clostridium difficile causes pseudomembranous colitis in humans and a similar ileocecitis in hamsters. This organism can colonize the intestines after antibiotic therapy disrupts the normal intestinal microflora. Once established in the intestines, the organism causes disease by producing two toxins, designated toxin A and toxin B. Only toxin A is active on intestinal epithelium, thus toxin A is the cause of the initial tissue damage in the intestines. In order for a toxin to affect a cell, it must first bind to the cell. Toxin A has been shown to bind to Galα1- 3Galβ 1-4GIcNAc on the intestinal epithelium of hamsters. I provide evidence that toxin A can use this trisaccharide as a functional receptor on cell lines, and that the expression of the carbohydrate receptor increases the sensitivity of the cells to toxin A. Furthermore, the intestinal epithelium of infant hamsters bound less toxin A at 37C than did the adult tissue, and infants are less sensitive to the disease caused by C. difficile than are adults. This provides further evidence that the activity of toxin A is increased by the binding of the toxin to Galα1-3Galβ1- 4GlcNAc. Even though Galα1-3Galβ 1-4GlcNAc was a receptor for toxin A on animal cells, it probably is not a receptor for toxin A in humans, because people do not normally express this carbohydrate. Instead, I found that toxin A bound to the carbohydrate antigens designated I, X, and Y, which are present on the intestinal epithelium of humans. These carbohydrates could be receptors for toxin A. The possible significance of these receptors is discussed. / Ph. D.

Clostridium sordellii cause gas gangrene in man and animals, and more recently it has been implicated as a causal agent of diarrhea and enterotoxemia in domestic animals. This organism was once believed to cause pseudomembranous colitis (PMC) in humans, however, Clostridium difficile, not C. sordellii, was found to be the causative agent of this disease. It is now known that C. difficile produces two toxins, designated A and B, that are implicated in the pathogenesis of the disease. C. sordellii produces two toxins, designated HT (Hemorrhagic Toxin) and LT (Lethal Toxin), that are similar to toxins A and B of C. difficile. The goal of my research was to purify and characterize the two toxins of C. sordellii, and compare their properties to those of C. difficile. Toxin HT was purified from C. sordellii (VPI strain 9048) culture filtrate by ultrafiltration through an XM-100 membrane filter and immunoaffinity chromatography using a monoclonal antibody to toxin A of C. difficile as the ligand. Toxin LT was purified to 80% homogeneity by ultrafiltration on an XM-100 membrane filter and ion-exchange chromatography. Toxin HT migrated as a major band with molecular weight of 525,000 and a minor band at 450,000 on non-denaturing PAGE. By SDS-PAGE the molecular weight was estimated at 300,000. Isoelectric focusing indicated a pI of 6.1. Like toxin A, toxin HT was cytotoxic to cultured cells, lethal for mice, and elicited an accumulation of hemorrhagic fluid in rabbit ileal loops. Toxin LT exhibited properties similar to toxin B, although LT was about a 1000-fold less cytotoxic than toxin B. By SDS-PAGE the molecular weight was estimated at 260,000. Immunodiffusion analysis revealed a reaction of partial identity between these toxins and their amino-terminal sequences were very similar. Toxins HT and LT of C. sordellii have retained remarkable immunological similarities as well as physicochemical and biological properties with toxins A and B of Q. difficult however the toxins are not identical. / Ph. D.

published_or_final_version / Pathology / Master / Master of Philosophy

Stroke is one of the most common causes of disability and death worldwide. The most commonly experienced stroke in the clinical setting is focal ischaemia in which the middle cerebral artery (MCA) is occluded and leads to a complex series of various pathophysiological pathways that ultimately lead to neuronal cell death. Several studies have been conducted on various therapeutic agents in the search for a neuroprotective drug and various animal models have been used to carry out this research. While theanine, a component of green tea and minocycline, a tetracycline antibiotic, have been shown to possess some neuroprotective properties, the mechanisms by which these two agents carry out these effects still remains unclear. The objectives of this study were to investigate the mechanisms by which these drugs carry out these neuroprotective effects and their neuroprotective ability in a MCA occlusion model of focal ischaemia. Ischaemia leads to oxidative stress due to the imbalance of free radicals and the endogenous antioxidant defence system. An antioxidant assay using the stable 2, 2-diphenyl-1-picrylhydrazyl (DPPH●) radical was used to assess the antiradical properties of each drug. It was found that minocycline showed superior antioxidant activity in vitro when compared to theanine. Further studies on the drugs‟ ability to attenuate the Fenton reaction (in which iron catalyses the formation of reactive species) were elucidated using electrochemical analysis, UV/VIS studies, ferrozine and ferritin assays. It was found that minocycline, in contrast to theanine, was able to bind to iron ions and thus potentially prevent the participation of iron in metal catalysed radical reaction. The antioxidant activity of both drugs was further investigated by assessing their effect on cyanide-induced superoxide generation and quinolinic acid (QA)-induced lipid peroxidation (LP). Experimental evidence shows that both drugs had no significant effect on the generation of superoxide in vitro and that there was a significant decrease in LP for minocycline in vitro and theanine in vivo. The metal binding and antioxidant properties were postulated to be a possible mechanism through which these agents reduced lipid peroxidation. A study was conducted to determine the effects of the drugs on the biosynthesis of the neurotoxin, QA and it was found that minocycline increases the levels of holoenzyme activity of tryptophan-2, 3-dioxygenase (TDO) in vitro and that theanine reduces the levels of the same enzyme in vivo after treatment for 10 days. TDO is the enzyme that converts tryptophan to other products that enable enzymatic activity to change it to QA. Minocycline was thought to bring about this effect as it has been shown from preceding experimental studies that it is an effective reducing agent. Theanine on the other hand is hypothesised to bring about a reduction in holoenzyme activity by changing the binding of tryptophan to the enzyme or affecting the radicals that participate in the enzymatic degradation of tryptophan. A focal ischaemic model of stroke was induced by occluding the MCA. Histological examination of the hippocampus post -ischaemia shows a reduction in the size of the infarct after pre-treatment with minocycline only. A further study into the effects of the drugs on the generation of superoxide and on the levels of the endogenous glutathione after a stroke was carried out. Pre-treatment of the animals with either theanine or minocycline showed no significant effects on the generation of the radical species or of the endogenous antioxidant which ruled out these as a mechanism of neuroprotection of both drugs, post-ischaemia.The findings of this study provide novel information on the possible mechanisms by which both theanine and minocycline act to bring about neuroprotection. In particular in this study, pre-treatment with minocycline has shown promise in the focal ischaemic model of stroke.

The use of minocycline for the treatment of a broad range of systemic infections and for severe acne has been associated with vestibular side effects. The severity of side effects may lead to poor adherence to therapy by patients. The use of sustained release formulations of minocycline that display slow dissolution of minocycline following administration may be beneficial in reducing the incidence and severity of side effects. Therefore, sustained release capsule dosage forms containing 100 mg minocycline (base) were manufactured and assessed for use as sustained release oral dosage forms of minocycline. Minocycline sustained release capsules were manufactured based on matrix technologies using hydroxypropylmethyl cellulose (HPMC) and Compritol® as release retarding polymers. The rate and extent of minocycline release from the capsules was evaluated using USP Apparatus 1 and samples were analysed using a validated High Performance Liquid Chromatographic (HPLC) method with ultraviolet (UV) detection. Differences in the rate and extent of minocycline release from formulations manufactured using HPMC or Compritol® were influenced by the concentration of polymer used in the formulations. The rate and extent of minocycline release was faster and greater when low concentrations of polymer were used in formulations. The effect of different excipients on the release pattern(s) of minocycline and particularly their potential to optimise minocycline release from experimental formulations was investigated. The use of diluents such as lactose and microcrystalline cellulose (MCC) revealed that lactose facilitated minocycline release when HPMC was used as the polymer matrix. In contrast, the use of lactose as diluent resulted in slower release of minocycline from Compritol® based formulations. The addition of sodium starch glycolate to HPMC based formulations resulted in slower release of minocycline than when no sodium starch glycolate was used. Compritol® based formulations were observed to release minocycline faster following addition of sodium starch glycolate and Poloxamer 188 to experimental formulations. In vitro dissolution profiles were compared to a target or reference profile using the difference and similarity factors, ƒ1 and ƒ2 , and a one way analysis of variance (ANOVA). In addition, the mechanism of minocycline release was elucidated following fitting of dissolution data to the Korsmeyer-Peppas, Higuchi and Zero order models. Minocycline release kinetics were best described by the Korsmeyer-Peppas model and the values of the release exponent, n (italics), revealed that drug release was a result of the combined effects of minocycline diffusion through matrices and erosion of the matrices. These in vitro dissolution profiles were better fit to the Higuchi model than to the Zero order model. Two formulations that displayed a fit to the Zero order model were identified for further studies as potential dosage forms for sustained release minocycline.

Trillions of microbes line the gastrointestinal tract to form the gut microbiome, a symbiotic organ whose supportive functions include energy production, immune homeostasis, and defense against pathogens. Disturbances to gut microbial composition, in turn, drive the pathogenesis of various metabolic, inflammatory, and carcinogenic diseases. Much effort has been dedicated to elucidating environmental triggers of gut dysbiosis, not the least of which is the consumption of medications. Antibiotics eradicate keystone commensals and enhance pathogenic behaviors of persisting pathobionts, whose resistance mechanisms can have off-target effects on human physiology and treatment response. Recent evidence indicates that the spectrum of antimicrobial compounds that disturb the gut microbiome extends far beyond traditional antibiotics, and includes commonly prescribed cardiovascular, neuropsychiatric, metabolic, and cancer medications. Although the capacity of non-antibiotic pharmaceuticals to induce gut dysbiosis is well appreciated, their impact on gut microbial function has not been studied systematically. Bacterial multi-omic profiling offers a cost-effective, high-throughput approach to understanding bacterial genetic responses to chemical perturbations, and how these functional changes might reciprocally impact relevant human phenotypes. Our laboratory, which houses a personal strain biobank of over 30,000 gut bacterial isolates spanning over 400 taxa, has established scalable pipelines for bacterial genomic and transcriptomic profiling that are readily applicable to diverse non-model gut microbes. We applied these methodologies to healthy fecal samples and bacterial isolates to elucidate strain-level responses to common pharmaceuticals with known gut microbiome associations. We first performed a gut microbiota transcriptomic screen of 19 representative fecal isolates against 20 top-prescribed orally delivered medications. Computational analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed induction of pathways associated with metabolism and multidrug resistance, including upregulation of efflux machinery by lipid-lowering drugs, antidepressants and cardiovascular medications. We discovered many bacterial responses with clinical significance, which we computationally validated using clinical metagenomic datasets. Most importantly, we showed that statin-mediated overexpression of the AcrAB-TolC efflux pump generates collateral toxicity in dietary retinol and secondary bile acids, resulting in depletion of pump-containing Bacteroidales species from patient microbiomes. We next performed the first comprehensive screen for antimicrobial activity in cancer drugs by exposing three healthy fecal samples to a panel of 41 first-line cancer therapeutics. Using 16S-genomic profiling, we identified several members of the targeted kinase inhibitor (TKI) class that induced gut dysbiosis, including first-line hepatocellular carcinoma (HCC) treatment sorafenib. We profiled natural bacterial isolates exposed to different TKI HCC treatments, and again observed transcriptional induction of conserved multidrug efflux pumps. Adaptive evolution assays identified Resistance-Nodulation-Division (RND) efflux pumps as effectors of TKI resistance. Remarkably, we demonstrated that acquired TKI resistance in evolved Bacteroidales lineages generated strain-specific cross-resistances and collateral sensitivities to several unrelated antibiotics. Collectively, our work demonstrates the importance of profiling xenobiotic impacts on the gut microbial resistome, as bacterial adaptations to pharmaceutical toxicities can feed back onto microbiome communities and the human host to affect health outcomes.

Diarrhoea, particularly infectious diarrhoea, in children below five years of age is recognised as one of the leading cause of morbidity and mortality throughout the world. This is especially true in residential areas of developing countries where there is substandard sanitation and overcrowding which are reservoirs for farther transmission. Shigellosis is endemic in developing countries and in Sub-Saharan Africa, including South Africa, a region where unique geographic, economic, political, sociocultural, and personal factors interact to create distinctive continuing challenges to prevention and control. Our study was undertaken to establish baseline information on incidences of Shigella, its serotype and resistant pattern of isolates from human faeces from residents of Johannesburg, South Africa. All stools received from January to April 2013 from the private health care system were cultured on standard media for isolation of Shigella and confirmed by standard biochemical reactions and serological method. Antibiotic sensitivity test was determined by the agar diffusion method. 11009 stool samples were assayed from patients aged between 22 days to 94 years with a 110 Shigella isolates yield, of which 47 (43%) were S. flexneri, 61 (55%) S. sonnei and 1 (1%) of S. dysentriae and S. boydii respectively. Majority of patients 76 (69%) were children between < 1 to 5 years old followed by those between 6 to 10 years 13(12%). Of the four species isolated from children of up to 10 years old, S. sonnie was confirmed in 52 cases (59%) and S. flexneri in 36 cases (41%). A total of 53 (48%) males and 57 (52%) females were infected. However, a hundred per cent susceptibility to ciprofloxacin and ceftriaxone but high levels of resistance to Co-trimoxazole (83%), tetracycline (72%), and ampicillin (26%) was noted. From the 110 isolates, 96 (87%) were resistant to one or more drugs while 14 (13%) were fully susceptible. These results show that S. sonnei followed by S. flexneri as predominating aetiology of shigellosis and Ceftriaxone and ciprofloxacin as effective drugs against all four Shigella species. / Health Studies / M. Sc. (Life Sciences)

Diarrhoea, particularly infectious diarrhoea, in children below five years of age is recognised as one of the leading cause of morbidity and mortality throughout the world. This is especially true in residential areas of developing countries where there is substandard sanitation and overcrowding which are reservoirs for farther transmission. Shigellosis is endemic in developing countries and in Sub-Saharan Africa, including South Africa, a region where unique geographic, economic, political, sociocultural, and personal factors interact to create distinctive continuing challenges to prevention and control. Our study was undertaken to establish baseline information on incidences of Shigella, its serotype and resistant pattern of isolates from human faeces from residents of Johannesburg, South Africa. All stools received from January to April 2013 from the private health care system were cultured on standard media for isolation of Shigella and confirmed by standard biochemical reactions and serological method. Antibiotic sensitivity test was determined by the agar diffusion method. 11009 stool samples were assayed from patients aged between 22 days to 94 years with a 110 Shigella isolates yield, of which 47 (43%) were S. flexneri, 61 (55%) S. sonnei and 1 (1%) of S. dysentriae and S. boydii respectively. Majority of patients 76 (69%) were children between < 1 to 5 years old followed by those between 6 to 10 years 13(12%). Of the four species isolated from children of up to 10 years old, S. sonnie was confirmed in 52 cases (59%) and S. flexneri in 36 cases (41%). A total of 53 (48%) males and 57 (52%) females were infected. However, a hundred per cent susceptibility to ciprofloxacin and ceftriaxone but high levels of resistance to Co-trimoxazole (83%), tetracycline (72%), and ampicillin (26%) was noted. From the 110 isolates, 96 (87%) were resistant to one or more drugs while 14 (13%) were fully susceptible. These results show that S. sonnei followed by S. flexneri as predominating aetiology of shigellosis and Ceftriaxone and ciprofloxacin as effective drugs against all four Shigella species. / Health Studies / M. Sc. (Life Sciences)