Abstract New rapid methods for sensitive and specific detection of pneumococci are not only needed to improve the diagnosis of pneumococcal disease but are also essential for vaccine and carriage studies. The purpose of this study was to develop sensitive PCR methods for the detection and quantification of S. pneumoniae and to study the applicability of these methods to detecting pneumococci in clinical samples. A previously described PCR method was first developed further by introducing a Europium-labelled hybridisation probe for the detection of amplification products. The hybridisation method was easy to use and improved the specificity of the PCR assay. The developed PCR assay was established as a sensitive method for detecting pneumococcal DNA when the presence of pneumococcal DNA in over 2500 middle ear fluid (MEF) samples of children with acute otitis media (AOM) was studied by using the method. Pneumococcal findings increased by 76% when using PCR detection in addition to culture, compared to using culture alone. However, the PCR-positive, culture-negative AOM events represented a less severe type of disease compared to the culture-positive events. A positive PCR finding seems to indicate the presence of viable, although often non-culturable pneumococci within the middle ear cleft. To be able to rapidly detect and quantify the initial numbers of pneumococcal genome copies in clinical samples, a real-time PCR method for the detection and quantification of pneumococcal DNA was developed. In real-time PCR, amplification and detection of amplification products occur simultaneously, which makes it possible to monitor the phase of the reaction at a particular stage or continuously. The method developed here was applied to the analysis of MEF samples and to investigating the nasopharyngeal carriage of pneumococcus. The sensitivities of bacterial culture and real-time PCR in detecting pneumococci were also compared. The real-time PCR assay was found to be rapid and sensitive and to provide information about the differences between the numbers of bacteria in samples. However, the quantitative results were shown to be dependent on the DNA extraction method applied. The real-time PCR method developed appears to be a good aid in research where an accurate and sensitive pneumococcal diagnosis is needed.

Magister Scientiae (Medical Bioscience) - MSc(MBS) / Staphylococcus aureus is a Gram-positive coccus belonging to the Stapylococcaeae family. S. aureus causes a wide range of infections that range from skin infections to lifethreatening infections such as pneumonia and endocarditis and is the major cause of hospital and community-acquired infections. Despite antibiotics being available for the treatment of S.aureus infections, resistance to a number of antibiotics has developed over the years due to their improper and continuous use. S. aureus develops resistance to various drugs via different mechanisms, one of which is the extrusion of the antibiotics through efflux pumps that play a role in its acquisition of multidrug resistance. The ability of methicillin-resistant S.aureus to develop resistance to a variety of antibiotics is causing global concern as treatment options are being limited. Various antimicrobial studies carried out on purified plant-based flavonoids have shown that flavonoids enhance the antibacterial effect of antibiotics. This study analysed antibacterial effects of the antibiotics; tetracycline, ampicillin, methicillin and vancomycin and three flavonoids; chrysin, naringenin and 7-hydroxyflavone, against methicillin-sensitive ATCC 25923 (MSSA) and methicillin-resistant ATCC 33591 (MRSA) S. aureus strains, using the Kirby-Bauer disk diffusion and microtitre microdilution assays. In the Kirby- Bauer assay, the antibiotics demonstrated inhibitory effects on the growth of MSSA ATCC 25923. However MRSA ATCC 33591 was only susceptible to vancomycin, with minimal inhibition zones observed with ampicillin. The flavonoids did not enhance or reduce the antibacterial activities of the antibiotics as the zones of inhibition sizes remained unchanged in the combination studies. Microtitre assay results revealed that naringenin enhanced the antibacterial activities of the antibiotics tetracycline and ampicillin, against MSSA ATCC 25923 and MRSA 33591. This was evident as calculated synergistic ratios by the Abbot formula showed that naringenin had an additive effect. The presence of the efflux pump genes in MSSA ATCC 25923 and MRSA ATCC 33591 was compared using polymerase chain reaction (PCR). The mepA and gyrA genes were identified in both strains whereas sepA was identified in MRSA ATCC 33591. The presence of efflux pump genes in both MSSA ATCC 25923 and MRSA ATCC 33591 also confirmed that the presence or absence of the genes may contribute to antibiotic resistance. The presence of sepA in the MRSA and not the MSSA confirmed that this gene plays a role in conferring drug resistance.

Analysis of immune responses in Bartonella bacilliformis carriers are needed to understand acquisition of immunity to Carrion’s disease and may allow identifying biomarkers associated with bacterial infection and disease phases. Serum samples from 144 healthy subjects from 5 villages in the North of Peru collected in 2014 were analyzed. Four villages had a Carrion’s disease outbreak in 2013, and the other is a traditionally endemic area. Thirty cytokines, chemokines and growth factors were determined in sera by fluorescent bead-based quantitative suspension array technology, and analyzed in relation to available data on bacteremia quantified by RT-PCR, and IgM and IgG levels measured by ELISA against B. bacilliformis lysates. The presence of bacteremia was associated with low concentrations of HGF (p = 0.005), IL-15 (p = 0.002), IL-6 (p = 0.05), IP-10 (p = 0.008), MIG (p = 0.03) and MIP-1α (p = 0.03). In multi-marker analysis, the same and further TH1-related and pro-inflammatory biomarkers were inversely associated with infection, whereas angiogenic chemokines and IL-10 were positively associated. Only EGF and eotaxin showed a moderate positive correlation with bacteremia. IgM seropositivity, which reflects a recent acute infection, was associated with lower levels of eotaxin (p = 0.05), IL-6 (p = 0.001), and VEGF (p = 0.03). Only GM-CSF and IL-10 concentrations were positively associated with higher levels of IgM (p = 0.01 and p = 0.007). Additionally, IgG seropositivity and levels were associated with high levels of angiogenic markers VEGF (p = 0.047) and eotaxin (p = 0.006), respectively. Our findings suggest that B. bacilliformis infection causes immunosuppression, led in part by overproduction of IL-10. This immunosuppression probably contributes to the chronicity of asymptomatic infections favoring B. bacilliformis persistence in the host, allowing the subsequent transmission to the vector. In addition, angiogenic markers associated with bacteremia and IgG levels may be related to the induction of endothelial cell proliferation in cutaneous lesions during chronic infections, being possible candidate biomarkers of asymptomatic infections.

Abstract The cytochrome P450 (CYP) enzyme system in human lung is an essential component in the pulmonary carcinogenicity of several inhaled xenobiotic compounds. The aim of this study was to elucidate the expression and regulation of xenobiotic-metabolizing CYP enzymes in human lung. To evaluate which of the two is a better surrogate cell model for lung tissue, the expression patterns of CYP enzymes in alveolar macrophages and peripheral blood lymphocytes were clarified by reverse transcriptase-polymerase chain reaction (RT-PCR) and compared to the expression in lung tissue. The pattern of CYP expression in alveolar macrophages was found to closely resemble the expression pattern in human lung tissue, while the pattern in lymphocytes was markedly different. The expression of CYP2B6, CYP2C, CYP3A5, and CYP4B1 mRNAs in alveolar macrophages was demonstrated for the first time. To facilitate mechanistic studies on human pulmonary CYP induction, the A549 lung adenocarcinoma cell line was characterized by RT-PCR, and the CYP expression pattern was found to compare reasonably well to human lung epithelial cells. The induction of CYP1A1 by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) behaved as predicted, and CYP1B1 and CYP3A5 were also inducible by TCDD and dexamethasone, respectively. TCDD elevated the level of CYP1A1 mRNA (56-fold), while the induction of CYP1B1 mRNA was more modest (2.5-fold). The tyrosine kinase inhibitor genistein and the protein kinase C inhibitor staurosporine blocked CYP1A1 induction by TCDD, but did not affect CYP1B1 induction. The serine/threonine protein phosphatase inhibitor calyculin A and okadaic acid enhanced CYP1B1 induction slightly, but did not alter CYP1A1 induction. The expression of CYP3A forms in human pulmonary tissues was studied with RT-PCR and immunohistochemistry, and both methods established CYP3A5 as the main CYP3A form. CYP3A4 was expressed in only about 20% of the cases. In A549 cells, CYP3A5 was induced about 4-fold by the glucocorticoids budesonide, beclomethasone dipropionate, and dexamethasone. Maximal induction was achieved by concentrations as low as ~100 nM, suggesting that CYP3A5 could be induced in vivo in patients using inhaled glucocorticoids. However, there were no differences in CYP3A5 expression in alveolar macrophages in current glucocorticoid users, ex-users, and non-users. Cigarette smoking had a marked decreasing effect on CYP3A5 levels in alveolar macrophages. The presence and possible induction of CYP3A5 by glucocorticoids in human lung could have consequences for the maintenance of physiological steroid hormone balance in lung and the individual susceptibility to lung cancer of patients using glucocorticoids.

Salmonella species is the etiologic agent of salmonellosis, which is a zoonotic infection that is characterized by diarrhea and systemic infection. Contaminated foods are usually the vehicles of Salmonella transmission along the food supply chain. Asymptomatic food production animals and effluents also contribute to contamination of meat. Antimicrobials have contributed significantly to treatment of salmonellosis. However, uncontrolled antimicrobial use is among the causes of antibiotic resistance, which results in treatment failure. The aim of this research study was to determine the extent of Salmonella spp contamination during the cattle slaughtering process in South African rural abattoirs (n = 23), water and cattle feaces. In addition, the aim was to determine antimicrobial resistance profiles of the Salmonella spp isolates. The specific objectives were: i) to establish the occurrence and distribution of Salmonella spp on cattle carcasses, hides, and intestinal contents and environmental samples using classical microbiology and molecular techniques; ii) to determine the Salmonella serovars using serotyping; and iii) to determine antimicrobial resistance patterns and multidrug resistance among the Salmonella isolates using the Kirby-Bauer disc diffusion method. Materials and Classical microbiology techniques were used to analyze cattle faeces (n = 400), hides (n = 67), intestinal contents (n = 62), carcass sponges (n = 100), and water from the abattoirs (n = 75) for the presence of Salmonella spp. Further confirmation of the Salmonella isolates was done using Polymerase Chain Reaction whereby the invA gene was targeted. A total of 92 Salmonella spp isolates were recuperated. The 92 Salmonella were serotyped as described in the White-Kauffmann-Le Minor scheme. The 92 Salmonella spp isolates were further subjected to antimicrobial susceptibility examination towards the following antimicrobials: ampicillin (10μg), cefotaxine (30μg), kanamycin (30μg), oxytetracycline (30μg), and enrofloxacin (5μg) by using the Kirby-Bauer disk diffusion procedure. All the isolates carried the invA genes. The average Salmonella spp occurrence on carcasses, hides, and intestinal contents was 35.37% (n = 81). Eleven of the faecal samples (2.75%) tested positive for Salmonella spp. The Salmonella serovar that occurred more frequently was S. Enteritidis. Different serovars that were recognized on carcasses were not automatically found on the hides and intestinal contents. The incompatible frequency of the different Salmonella serovars on carcasses, intestinal contents and hides means that in addition to carriage on hides and in intestinal contents, new external causes that did not form part of this study also play a vital role concerning carcass contamination. Most Salmonella spp (n = 66; 71.7%) isolates were resistant to a minimum of one antimicrobial with main resistance detected towards oxytetracycline (51.90%). This emphasizes on the call for wise antimicrobial use at some stage in animal production and strict sanitation for the duration of slaughtering. Briefly, cattle slaughtered in South African rural abattoirs harboured different types of Salmonella serovars that were resistant to antimicrobials, which could be a public health risk and danger. The outcome should support policymakers with determining the effectiveness of existing sanitary measures during cattle slaughtering in rural abattoirs, which is vital from socio-economic, public health, and epidemiological perspectives. / College of Agriculture and Environmental Sciences

M.Tech. / RNA interference (RNAi) is a gene silencing mechanism that uses short RNA duplexes to block gene expression. This mechanism has been widely explored to determine functions of genes. Furthermore, this phenomenon has been used to silence unwanted genes such as viral genes. RNAi has been successfully employed in non-mammalian organisms such as plants, where long dsRNAs (more than 30 bp) have been used without inducing non-specific effects. However, in mammalian cells, cytoplasmic dsRNAs of more than 30 bp trigger non-specific induction of many genes, which may result from the activation of dsRNA-dependent protein kinase (PKR) and 2’,5’-oligoadenylate synthetase (2’,5’-OAS), via the interferon response pathway. In this study, we describe a novel and simple strategy to overcome nonspecific effects induced by longer RNA duplexes. This strategy uses sodium bisulphite which is a mutagen that deaminates cytosine residue to uracil residues in order to introduce mutations in the sense strand of the duplex. Introduction of these mutations results in the formation of G:U pairings between the sense and antisense strands of the long hairpin RNA. RNA duplexes with mismatches have been shown to be able to prevent interferon induction in mammalian cells. According to the obtained results, long hairpins RNA with and without mismatches were unable to inhibit the expression of the target region, which was the U5 region of the HIV-1 subtype C LTR. The U5 region of the LTR is actively involved in the reverse transcription of HIV-1. Therefore silencing of this region would have led to the inhibition or reverse transcription blockage. Furthermore, data showed that the interferon response was induced when using these long hairpin RNA duplexes. Due to the sensitivity of mammalian cells, the action of sodium bisulphite could have stimulated certain genes of the interferon pathway. Even though hairpins constructed in this study were unable to prevent the induction of the interferon response pathway and also could not silence the target, this strategy of using sodium bisulphite has a great potential as shown by its ability to induce changes in cytosine residues and leaving other nucleotides unchanged.

M.Sc. / LPS, as a pathogen associated molecular pattern (PAMP) molecule can interact with eukaryotic host cells. Interaction occurs by either direct contact or due to the release of micelles containing LPS from bacterial cell surfaces. LPS activates innate host defence systems in both invertebrate and vertebrate animal/insect cells via analogous pathways, where the lipid A component,is responsible for the activities. LPS from several plant pathogens have been shown to activate a number of defence-related responses in plants. Initial concentration studies and cell viability assays were conducted to assess isonitrosoacetophenone (INAP) and LPS as elicitors of defensive responses in tobacco (Nicotiana tabacum cv. Samsun) cell suspensions. The effective concentrations were found to be 100vM INAP and 100μg/ml LPS. RNA was isolated, quantified and analysed to confirm the quality of the starting material for differential display analysis. The DDRT-PCR technique was successfully applied in order to obtain comparative "displays" of PCR amplicons derived from three sub-divided mRNA pools (i.e. each of the three different anchor primers, per treatment). Significant differences in the profiles of control, INAP and LPS treated cells were observed, indicating that the eliciting agents had prominent effects on cellular homeostasis, resulting in an altered gene expression profile. DDRT-PCR can be technically challenging at a number of steps. Modifications were incorporated to initially obtain differentially expressed transcripts (DETs), as well as reamplify the DETs. 223 Putative DETs were isolated from denaturing polyacrylamide sequencing gels. 172 Putative DETs were re-amplified, of which 126 appeared as good candidates for further analysis. Finally, 96 putative DETs were chosen for reverse Northern analysis. DDRT-PCR has been reported to be plagued by false positives. Reverse Northern analysis confirms the presence of the putative DET from the subdivided RNA pool, as well as affirming the differential expression, compared between the control and inducer blots. 26 DETs were selected for cloning, of which 16 were sequenced. Homologies between the DETs and known sequences were determined using BLASTN and BLASTX alignments, DNAssist software, as well as MIPS alignments to the Arabidopsis genome. Five of the DETs were assigned putative functions in plant signal perception, transduction and the defence response, based on their respective sequence homologies to sequences involved in innate immunity. It is proposed that the DET, HAP3-15, represents the plant equivalent of a component of the innate immunity pathway in mammals and Drosophila. It is further proposed that HAP3-15 represents a S-Receptor kinase protein (SRK), with a defensive role in distinguishing self from potential pathogens. Therefore, as a SRK, HAP3-15 would function as a transmembrane receptor able to conduct an external signal through the membrane to the cytoplasm as a form of signal perception. Subsequently HAP3-15 could ii play a role in phosphorylation cascades through the kinase domain and, consequently, be responsible for signal transduction. In addition, LPS would then represent the ligand creating the signal perceived by the SRK, HAP3-15, with oligosaccharide binding ability. HAP3-15 was also identified as a true positive by the INAP probe in reverse Northerns, implying that both the biological and chemical inducers used, activated the same receptor kinase. Whether the same signalling pathway was followed during the phosphorylation cascades has not been determined. Further analysis will require Northern blots in a time study to investigate the kinetics of induction. In addition, longer sequence information for each of the five DETs needs to be obtained to identify the corresponding genes in order to investigate their roles in innate immunity in plants.

>Magister Scientiae - MSc / Genetic variability in genes encoding drug metabolizing enzymes, transporters and targets are known to be the main factors of inter-individual differences in therapeutic outcome. Genetic factors are estimated to be responsible for about 15-30% of inter-individual variation in drug disposition and response. Single-nucleotide polymorphisms (SNPs) are the most prevalent class of genetic variation that could explain the variability in drug efficacy and undesired side effects for patients. The aims of this study were to develop and evaluate the performance of robust and high throughput techniques for genotyping ten polymorphisms related to anticancer drugs and ten polymorphisms related to cholesterol lowering drugs. SNaPshot minisequencing and high resolution melt analysis (HRM) genotyping panels were developed, optimized, and their performances were evaluated and compared. SNaPshot minisequencing systems were developed and successfully optimized for the genotyping of ten SNPs associated with anticancer drug therapy, and ten SNPs associated with cholesterol lowering drugs. These systems were used to genotype the selected SNPs in 130 healthy Cape Admixed participants residing in Cape Town, South Africa. Population genetics data obtained for the studied SNPs were analysed using several statistical analysis software tools. Important population genetic parameters were calculated. Among others, allelic and genotypic frequencies were determined and compared with other populations in the world. High resolution melt analysis (HRM) genotyping panels were developed, optimized and their performance were evaluated and compared to the SNaPshot assays. HRM was explored as an alternative inexpensive and rapid methodology to genotype five SNPs related to anticancer therapy and five SNPs related to cholesterol lowering therapy (statins). Unlike the SNaPshot assays, rigorous optimization was required for the detection heterozygous genotypes via HRM. Both assays were validated using direct sequencing and compared to each other. The HRM system is a closed tube, cheap and (theoretically) rapid method for identifying genetic variations. HRM was however found to be more time consuming, needed further optimization, primer redesigning and more evaluation. The developed genotyping systems could be further validated using clinical samples from patients. This could help in optimizing drug therapy for cancer and cholesterol treatment.

South Africa has the highest species diversity of ericaceous plants belonging to the Erica genus. There are over 850 identified species in the Cape Floral Region. The Albany Centre of Endemism (ACOE) is located within this region and is a hotspot of diversity consisting of various plant genera. The success of Erica plants is ubiquitously attributed to mycorrhizal relationships they engage in with a diverse group of fungi. This symbiosis is known as the ericoid mycorrhizal (ERM) association. The overall aim of this study was to establish the diversity of root fungi associated with Erica plants using morphological, molecular and 454 pyrosequencing techniques. Six Erica species were identified using leaf and flower morphology according to taxonomic keys. The identified plants were Erica cerinthoides, Erica demissa, Erica chamissonis, Erica glumiflora, Erica caffra and Erica nemorosa. Roots from sampled plants were stained and examined microscopically to determine their mycorrhizal status. Ericoid mycorrhizal associations together with dark septate endophyte (DSE) structures and hyphae that did not form any specific structure were observed in all the roots. In addition arbuscular mycorrhizal (AM) structures in the form of vesicles were detected in E. glumiflora and E. cerinthoides. In order to identify the culturable fungi associated with the respective hosts, sterilised roots were placed on various culture media for cultivation. Thereafter isolated fungi were morphologically classified into 67 morphotypes. These were mostly sterile and darkly pigmented. Non-sporulating mycelia of variable colouration such as white, cream-yellowish, beige, green and brown were also observed. Further identification was carried out using molecular techniques. DNA was extracted separately from pure cultures and amplified using ITS1 and ITS4 primers in a polymerase chain reaction (PCR). Thereafter sequencing and Basic Local Alignment Search Tool (BLAST) were used to identify the isolates to generic level. The fungi were taxonomically classified into 54 operational taxonomic units and 94 percent were Ascomycetes and Helotiales was the dominant order. Unclassified Helotiales with affinities to fungi currently identified as Epacrid root fungus was common in all hosts. Other isolates that were identified included Oidiodendron, Meliniomyces, Phialocephala, Cadophora, Lachnum, Leohumicola Cryptosporiopsis, Chaetomium, Acremonium and Epicoccum species. Basidiomycetes were represented by two OTUs belonging to the genus Mycena. Four OTUs comprised fungi that had no significant alignments in the reference databases. Direct root DNA extraction together with 454 pyrosequencing was used to detect the diversity of culturable and unculturable fungi associated with the identified hosts. The ITS2 region was targeted for sequencing. Although Ascomycetes remained the dominant phyla, Basidiomycetes were also detected in all host plants. Glomeromycota was present in E. caffra and E. cerinthoides. Helotiales was dominant in all Erica plants with the exception of E. cerinthoides and E. chamissonis which were dominated by the order Chaetothyriales. The OTUs identified to genus level included Epacris pulchella root fungus, Oidiodendron cf. maius, Acremonium implicatum, Leohumicola, Lachnum, Capronia and Mycena species. Culture-based techniques and pyrosequencing detected similar fungal composition comprising Ascomycetes, while, pyrosequencing was able to detect Glomeromycetes and Basidiomycetes.

The polymerase chain reaction (PCR) was investigated as a potential tool in microbial screening for 13-lactam. producing organisms. Optimization of PCR conditions and the addition of acetamide to the PCR reaction allowed for the successful amplification of the isopenicillin N synthetase (lPNS) gene in S. clavuligerus, S. tanashiensis, S. griseus, S. olivaceus, S. lipmanii, and S. chartreusis. PCR was used to produce a radiolabelled probe from S. clavuligerus that was used to detect analogous genes in bacteria and fungi. Southern blot and dot blot analysis using the lPNS probe revealed the presence of IPNS-like sequences in seventeen organisms. Fourteen of these sequences belonged to known 13-lactam. producing organisms; one unidentified soil isolate; and two non-/3-lactam. producing organisms viz. S. venezuelae ATCC 10712 and S. hygroscopicus ATCC 21703. The lPNS gene was also detected in a 13-lactam producer (S. chartreusis) that had lost its ability to produce antibiotic. It would therefore have been overlooked in a conventional antibiotic screening program. The use of PCR, coupled with Southern hybridization and dot blot analysis, increased the sensitivity and specificity of the antibiotic screening procedures and allowed for the investigation of evolutionary relationships between the eukaryotes and the prokaryotes. A preliminary investigation into the potential use of RAPD PCR and protein fmgerprinting as tools for solving discrepancies in streptomycete identification was conducted. A variety of streptomycete species that were chosen as being representative of a number of numerical taxonomic classes were amplified using various RAPD primers. Streptomycetes appear to be genetically diverse organisms as was reflected by their RAPD and protein profiles. The application of PCR in an antibiotic screening program showed great potential as a specific and sensitive tool in the detection of /3-lactam producers and in the elimination of duplicate strains.