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Masters Degrees (Microbiology and Infection Control)

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    Molecular characterization of the tetM gene in clinical isolates of neisseria gonorrhoeae in KwaZulu-Natal, South Africa.
    (2017) Rambaran, Santhuri.; Moodley, Prashini.
    Abstract available in PDF file.
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    Detection of carbapenem-resistant Enterobacteriaceae amongst neonates in a regional hospital in KwaZulu-Natal : screening for carbapenemase production and MIC correlation.
    (2014) Govender, Kathleen.; Moodley, Prashini.
    Carbapenemases are the primary cause for the increase in carbapenem resistance in Gram- negative Enterobacteriaceae. These enzymes are β-lactamases and have the ability to hydrolyse almost all β-lactam antibiotics thereby inactivating carbapenems that are used for the treatment of severe nosocomial infections. Multiple CPE outbreaks and epidemics have been reported in several hospitals in South Africa since the year 2011. This resulted in an increase in the morbidity and mortality rates and are slowly disseminating globally among more vulnerable individuals including neonates. Therefore, the aim of the study was to determine appropriate techniques for the rapid detection of carbapenem-resistant Enterobacteriaceae (CRE) (including CPE) isolated from neonates from King Edward VIII Hospital as well as to determine the molecular mechanisms conferring carbapenemase production in this subset of isolates. A total of 94 Klebsiella pneumoniae and 41 Enterobacter cloacae samples were isolated in this study. Among these species 10 % (9/ 94) and 39 % (16/ 41) of K. pneumoniae and E .cloacae respectively, were resistant to the carbapenems based on the Kirby-Bauer susceptibility tests, microbroth-dilution and E-tests. However, screening for carbapenemase production using chromogenic agar (Brilliance™ CRE agar and ChromID® CARBA agar), Modified Hodge test and amoxycillin-clavulanate double disc synergy test did not correlate with these resistance patterns and exhibited false positive results possibly due to the presence of extended spectrum beta-lactamase (ESBL) production by these organisms. Due to such discrepancies in the phenotypic results, further detection for the presence of carbapenemases was performed using multiplex real-time PCR assays. This revealed the presence of the blaOXA-48 gene in only 1 K. pneumoniae isolate. Further molecular characterisation will be required to determine if alternate mechanisms of resistance are present in the resistant isolates detected in this study.
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    Morphology, membrane characterization and detection of a bacterium associated with ratoon stunting disease of sugarcane.
    (1984) Pillay, Dorsamy.; Roth, G.; Oellermann, Rolf Alfred.
    Ratoon stunting disease (RSD) of sugarcane was first recognized in 1944 in Queensland, Australia (Steindl, 1961). The disease occurs worldwide and causes significant yield losses, especially during drought. RSD produces no external symptoms except a non-specific stunting (Steindl, 1961). RSD, which was first recorded 1n South Africa in 1953 (Anon., 1960), causes a greater overall loss in yield than any other sugarcane disease in South Africa. Yields of sugarcane are reduced by 20% to 40% and the harvest of affected fields declines progressively with successive ratoons (Anon., 1980b). A virus was originally thought to cause RSD, but in 1973, a coryneform bacterium was implicated as the causal agent (Gillaspie et al., 1973; Teakle et al., 1973). In 1980, our laboratory reported the successful isolation and culture of a coryneform bacterium associated with RSD of sugarcane and was indicated to be the causal agent (Nayiager et al., 1980). The lack of a rapid diagnostic technique applicable to mass screening of sugarcane has hindered progress in the control of the disease. There are two types of commonly used diagnostic tests. One test depends on the evaluation of internal stalk symptoms which may require from two to twenty six weeks to develop (Gillaspie et al., 1966; Matsuoka, 1971; Schexnayder, 1960; Singh, 1969). However, these symptoms are not always present in RSD affected plants and similar symptoms can sometimes result from other causes (Steindl, 1961). The other test involves establishing the presence of the coryneform bacterium associated with diseased plants. The bacterium is visible under high magnification by phase-contrast microscopy (Gillaspie et al., 1973) or by electron microscopy (Teakle et al., 1973). Although identification by the latter methods requires little time, the technology involved severely limits the number of samples that can be examined. Recently, serological techniques have been used (Brlansky et al., 1982; Damann et al., 1977; Davis et al., 1980; Gillaspie, 1978b; Gillaspie et al., 1979; Harris and Gillaspie, 1978) but their success has been limited. Besides problems with diagnosis of the disease, the precise morphology and taxonomy of the causal organism is unclarified. The objectives of this research programme were, firstly, to characterize the cultured intact bacterium and its constituent membranes both ultrastructurally and immunologically, and secondly, to evaluate various immunological methods for detection of the bacterium. This study should contribute to enhancing the taxonomic status of the bacterium and to the use of a rapid diagnostic technique applicable to mass screening of sugarcane. Such a technique should eventually contribute to effective control of RSD.
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    In vitro culture and isoenzyme analysis of giardia lamblia.
    (1999) Kwitshana, Zilungile L.; Jackson, Terry F. H. G.; Sturm, Adriaan Willem.
    Giardia lamblia, an enteric protozoan parasite, infects a large number of individuals worldwide. In South Africa prevalences ranging between 4 and 63% are documented, however, the impact of giardiasis is underreseached in this country. Giardia infections vary from asymptomatic carriage or a self-limiting acute symptomatic illness to chronic, debilitating malabsorption syndrome. The factors responsible for development of symptomatic versus asymptomatic infection are poorly understood. It is believed by some that host factors determine the clinical outcome of infection. On the other hand, the possibility of the existence of pathogenic and non-pathogenic strains (a situation akin to Entamoeba spp.) remains to be explored. One requirement for investigation of the potential contribution of strain differences to pathogenecity of infection is establishment of laboratory cultures of different strains isolated from symptomatic and asymptomatic patients. The present study was undertaken to develop and modify existing methods for: (i) establishment of laboratory cultures of Giardia trophozoites from excystation of faecal cysts, (ii) long-term maintenance and cryopreservation of the cultures and (iii) preliminary characterisation methodology. One thousand and twenty-three stool specimens were collected from day care centres, hospital wards and Hlabisa hospital laboratory. A further 6246 were retrieved from the Microbiology Laboratory at King Edward VIII Hospital and screened by direct wet preparation. Giardia was detected by light microscopy following formol-ether concentration (127 of 1023 samples) or direct examination of wet preparations (78 of 6246 samples). Cysts were purified from the positive specimens by sucrose gradient separation. Viability was assessed by a dye-exclusion method (eosin). Three in vitro excystation techniques were employed in an attempt to obtain trophozoites for initiation and establishment of viable cultures thereof. Culture conditions were optimised using two reference strains of Giardia, WB & H7 (obtained from the National Institutes of Health, USA). The percentage excystation ranged between 0-42% with all the in vitro methods of excystment. Excysted trophozoites remained viable in TYI-S-33 culture medium for periods ranging between 12-72 hours or up to 9 days, and gradually died, hence viable trophozoite cultures could not be established. Some culture initiates (overall 65%) were lost through overwhelming bacterial and!or fungal contaminants. An animal model was subsequently set up in which C57BL/6 and Praomys (Mastomys) coucha mice were used for in vivo excystation experiments. 1-3 day old suckling mice were intragastrically injected with 10,5 -cysts/ ml in 0,1 ml distilled water. Trophozoites were retrieved from the stomachs of infected mice 7-10 days after inoculation and cultivated in TYI-S-33 medium. Six local isolates were axenised using the in vivo excystation method. They have been maintained for more than 15 months in culture after stabilates and Iysates of confluent growths had been cryopreserved in Liquid Nitrogen. Successful (100%) retrieval of the cryopreserved cultures has been achieved. Seven isoenzyme electrophoresis systems have been set up and optimised. Reproducible results were obtained in six of the enzymes. Some differences in banding patterns of the enzymes were demonstrated.
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    Evaluation of laboratory methods for susceptibility testing of staphylococcus aureus.
    (1988) Jansen van Rensburg, Hermanus Christoffel.
    The susceptibility of 80 StaphyIococcus aureus isolated to oxacillin was investigated using microtitre, agar dilution and Stokes' disc diffusion methods. There was a bimodal distribution of the isolates according to the oxacillin minimum inhibitory concentration (MIC) values. For the sensitive isolates, the agar dilution method generally gave lower MIC values than the microtitre method, while for the resistant isolates the agar dilution method gave comparable to slightly lower MIC values than the microtitre method. The Stokes disc diffusion method yielding the best results when performed on Mueller-Hinton agar incubated at 30°C for 18 hours; however local strains grew poorly when incubated at 30 C for 18 hours. The next best medium which provided clear disc diffusion results plus good growth was Mueller-Hinton agar incubated at 35°C for 18 hours, on which 10 % of the sensitive isolates appeared intermediate in susceptibility, and none resistant, while all the resistant isolates (microtitre MIC >8mg/1) appeared resistant. Oxacillin resistance among strains of Staphylococcus aureus tested by Stokes' disc diffusion method correlated best with gentamicin resistance, and less often with tetracycline resistance. Therefore gentamicin- or tetracycline-resistance may indicate oxacillin resistance in Staphylococcus aureus.
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    Cloning of the endomannanase from Scopulariopsis candida LMK008 and evaluation of its effect on the digestibility on animal feed.
    (2012) Gareeb, Ashant Pravin.; Govinden, Roshini.; Setati, Mathebata Evodia.
    Present within the biodiverse hypersaline environment are a wide variety of halotolerant filamentous fungi. Many of these phytopathogens are capable of hydrolysing plant cell wall polysaccharides such as hemicellulose which are comprised of mannans and heteromannans which are polymers of the mannose sugars. Endoacting hydrolytic enzymes such as endo-β-1,4-mannanases are secreted into the extracellular environment and are involved in the catalysis of the random hydrolysis of β-1,4-mannosidic linkages within the backbone of mannan, galactomannan, glucomannan, and galactoglucomannan. Poultry are monogastric animals that are unable to efficiently digest high-fibre and mannan rich feeds such as soybean meals and this results in decreased or depressed animal performance. The use of feeds supplemented with β-mannanases has been shown to enhance the feeding value of mannan-based meals. In the current study, the degradation of β-mannan polysaccharides present in poultry feed by halotolerant Scopulariopsis candida LMK008 β-mannanase was investigated. SDS-PAGE, Native-PAGE in conjunction with zymogram analysis was used to assess the molecular weight of the endomannanases. At least three isozymes were detected: two of 56 kDa (pI 3.5 and 6.7) and one of 28 kDa. Anion exchange chromatography was used to purify the 28 kDa isozyme. Three mannan-based substrates, viz., locust bean gum, guar gum and soybean flour, were used to evaluate the hydrolysis capability of the crude as well as the purified β-mannanase via the release of reducing sugars and was detected using the DNS assay. The β-mannanase exhibited low activity with pure guar gum but high activity with locust bean gum galactomannan and soybean flour mannan. The hydrolysis activities of the crude and purified enzyme were then tested further on mannan-based soybean meals. In general it was found that more reducing sugars were released from the grower feed than the starter and layer feeds. Another common hydrolysis pattern observed in all feed types was that after prolonged incubation of 24 h there was a decrease in the amount of reducing sugars released which could be attributed to the presence of naturally-occurring microorganisms in the feed sample which metabolised the simple sugars resulting from the enzymatic hydrolysis of the mannan components in the feed samples. This was confirmed by standard plate count assays. The results obtained are encouraging and the purified β-mannanase could be applied as an industrial feed additive within the animal feed industry, however, further testing of the enzyme in situ is needed in order to prove its applicability. The cloning of the endomannanase has to date proven unsuccessful despite numerous techniques being employed and further research is also needed to accomplish this task.
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    Cationic liposome mediated transfection with/without a targeting component.
    (2005) Singh, Ashika.; Naidoo, Richard.; Singh, Moganavelli.
    The transfer and expression of genes in cells is an important technique for basic research and gene therapy of human disease. A model for gene therapy has been investigated making use of a transfection complex consisting of three components, the DNA i.e. the gene to be transferred and expressed; a gene delivery vehicle viz. a cationic liposome and a cell specific targeting ligand, asialoorosomucoid (AOM). Cationic liposomes are positively charged liposomes that have been prepared from synthetic lipids and have been shown to complex or bind to DNA via electrostatic attraction. They have shown potential as an efficient non-viral gene delivery vehicle in human gene therapy. In this investigation, a novel cationic liposome consisting of 3B [N -(N',N'-dimethylaminopropane)carbamoyl] cholesterol (Chol-T), dioleoylphosphatidylethanolamine (DOPE) and biotinylcholesteryl formylhydrazide was prepared and assessed as a mediator of DNA delivery in a mammalian cell culture system viz. the HepG2 cell line. The cationic liposome was synthesised and characterised by electron microscopy. Foreign DNA may be specifically delivered to target cells by a carrier system which makes use of the recognition of the asialoglycoprotein AOM by cognate receptors on the HepG2 cell plasma membrane. The positively charged AOM was biotinylated and due to this biotinylation, binds streptavidin which contains specific binding sites for biotin. The cationic liposome itself contains biotin residues in its bi-Iayer which in turn binds streptavidin resulting in a ternary complex. Further, due to the DNA binding capability of the cationic liposome, a transfection complex is produced consisting of the three components. The experiments were based on the following concepts: (i) Hepatocytes possess a unique receptor that binds to and internalises galactose-terminal asialoglycoproteins by receptor mediated endocytosis. (ii) Due to electrostatic attraction, DNA binds to cationic liposomes forming soluble complexes. (iii) Through the biotin-streptavidin reaction, the biotinylated AOM is attached to the cationic liposome containing biotin forming complexes enabling targeted delivery of the DNA. (iv) DNA containing the pGL3 gene for the luciferase enzyme was used and following transfection experiments, the luciferase assay was performed to ensure successful transfection. The complexes were tested on the hepatocellular carcinoma cell line, HepG2, which possess the asialoglycoprotein receptor. Transfection studies were conducted using a transient expression system, the luciferase assay system. Some degree of success in the transfection of HepG2 cells was observed. Results obtained in this study suggest that transfection using our targeted transfection complex consisting of cationic liposomes and cell specific targeting ligands does in fact transfect cells by receptor mediation.
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    Microbiological aspects of enterococci isolated at King Edward VIII Hospital, Durban.
    (1999) Pillay, Nithianandhi.; Sturm, Adriaan Willem.; Peer, Abdool Kader Cassim.; Bhamjee, Ahmed.
    The increasing frequency of enterococci as a major cause of nosocomial infections and the transmission of these organisms amongst hospital patients demands a greater awareness of the Enterococcus. Therapy of enterococcal infections is complicated by the pathogens continually changing resistance patterns to many broad-spectrum antibiotics. In addition, the ability of enterococci to cause serious invasive infections including endocarditis and septicaemia with associated high mortality rates; prompted this study which was aimed at identifying the biological properties of enterococci isolated from blood cultures of patients admitted at King Edward VIII hospital, Durban. Enterococci were identified to species level by the API 20 Strep system which identified 68% and a conventional biochemical system of Facklam and Collins which identified 100% of the isolates.The emergence of beta-Iactamase producing enterococci in other countries encouraged the testing of all isolates for this enzyme. All were beta-Iactamase negative. The reported false susceptibility for aminoglycosides and cephalosporins with blood enriched media encouraged the testing of these antibiotics with and without the supplementation of 5% lysed blood. The results showed that an average false susceptibility of 55 % occurred for gentamicin and 35% for tobramycin and netilmicin. The cephalosporins affected, cefotaxime and cefuroxime showed a false susceptibility of 28% and 17% respectively. The choice of treatment for serious enterococcal infections is a syllergistic combination of a beta-Iactam antibiotic plus an aminoglycoside for enterococci with intrinsic low-level resistance. The development of high-level aminoglycoside resistance, MIC 22000,ug/ml results in loss of synergism. This study showed that 26.4 % of enterococcal isolates displayed high level aminoglycoside resistance i.e. to gentamicin and streptomycin. Time-kill study showed reduced killing rate for these organisms for the beta-Iactams and glycopeptides with low-level gentamicin resistance. The results confirmed that a cell-wall active agent combined with gentamicin can be successfully used for enterococcal therapy if the organism has intrinsic low-level resistance to this amino glycoside. Pulsed-field gel electrophoresis (PFGE) carried out on a selected number of Enterococcus faecalis and Enterococcus faecium with high-level aminoglycoside resistance showed a variability in the restriction endonucelase digestion patterns. This suggests independent development of high-level gentamicin resistance and not clonal expression. The ease and reliability with which enterococcal isolates may be typed using this technique to compare different strains represent a significant advance.
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    Microbiology and molecular epidemiology of multiresistant haemophilus influenza type B in Durban, South Africa.
    (1988) Peer, Abdool Kader Cassim.; Van den Ende, Jan.; Smith, Arnold L.; Ward, Joel I.
    Microbiological and molecular epidemiological studies were conducted on 36 multi-resistant Haemophilus influenzae strains, isolated from paediatric patients, over a 26 month period (April 1986 to May 1988). The majority of strains (80,5%) had been isolated from blood and cerebrospinal fluid. More than 80% of isolates tested belonged to biotype II and 90% were of serotype B. Minimal inhibitory concentrations against 6 antibiotics (ampicillin, chloramphenicol, tetracycline, rifampicin, streptomycin and cefotaxime) confirmed the presence of multi-resistant strains. Resistance to rifampicin was confirmed in 6 (16,7%) strains. All strains were susceptible to cefotaxime. Ten transconjugants analysed with respect to their plasmid content were shown to harbour an identical 41 MDa plasmid. Restriction endonuclease digests of these plasmids with Eco R1 and Sst1 revealed almost identical restriction patterns. Outer membrane protein profiles of 19 strains revealed the predominance of one particular subtype. By combining the microbiological and molecular epidemiological findings, it is concluded that one strain of H. influenzae type b is responsible for the nosocomial acquisition of infections amongst paediatric patients. The implifications of these findings are discussed.
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    A study of the prevalence of campylobacter pylori in patients with upper gastrointestinal symptoms, and an evaluation of various laboratory methods to detect its presence.
    (1988) Miller, N. M.; Van den Ende, Jan.
    Antral mucosal biopsies were examined microbiologically and histologically for the presence of Campylobacter pylori in 224 patients with upper gastrointestinal symptoms. One hundred and eighty three (83%) patients were found to harbour Campylobacter pylori in their gastric mucosa. Campylobacter pylori was strongly associated with the presence of histological gastritis (93%) and was detected in only 10% of 30 patients whose gastric biopsies showed normal histology. Endoscopically diagnosed duodenal lesions were more strongly associated with the presence of Campylobacter pylori than were gastric lesions (p<0.001). A variety of laboratory methods were evaluated to determine the sensitivity and specificity to detect the presence of Campylobacter pylori. Histology was the most sensitive and specific method to detect the presence of Campylobacter pylori. Although culture was highly specific, it was less sensitive than histology in detecting Campylobacter pylori in gastric antral mucosal specimens. The "conventional" gastric urease assay, although specific, needs be performed under controlled conditions (37°C) for optimal results. The "one-minute" urease assay was more sensitive than the "conventional" gastric urease assays and was highly specific. ELISA to detect specific-IgG antibodies to Campylobacter pylori was a moderately sensitive non-invasive method to detect Campylobacter pylori infection, but was non-specific.
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    Neutrophil cytoplasmic antibodies : their clinical associations and an improved method for their detection.
    (1993) Duursma, June.; Pudifin, Dennis James.
    The test for antineutrophil cytoplasmic antibodies (ANCA) was introduced into the author's laboratory in 1987. An improved indirect immunofluorescent method was developed, using a system which allows 16 instead of one serum sample to be screened on each microscope slide. The known disease associations of ANCA that have been explored include systemic vasculitis, renal limited vasculitis, chronic inflammatory bowel disease and HIV disease. In general the findings are similar to those which are emerging from other centres and confirm the value not only of the positivity but also the relevance of the intracellular disposition of the neutrophil cytoplasmic fluorescence in diagnosis. In this study 85% of patients with Wegener's granulomatosis were found to have C-ANCA. C, P and X-ANCA staining patterns were found in 57% of patients with ulcerative colitis. Forty one per cent of patients with symptomatic HIV have ANCA. Certain histological features such as neutrophil and vascular damage in invasive amoebiasis, and the established lytic effect of amoebae on neutrophils prompted the investigation of the possibility that ANCA may be generated in this disease. Seventy eight amoebiasis sera were screened and 98,70/0 gave a positive ANCA test with a pattern of fluorescence resembling that found in Wegener's granulomatosis. An ELISA test for specificity confirmed that, as in Wegener's granulomatosis, this amoebiasis-associated ANCA had proteinase 3 specificity. Of practical clinical importance is the fact that both HIV and amoebiasis are associated with a high level of ANCA positivity. These findings will need to be considered when ANCA tests are used in clinical decision making in an area where HIV disease and amoebiasis are endemic. A large number of normal volunteer blood donors have been tested and the false positivity rate of 0,5% confirms the specificity of the test.
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    Spread of multi drug resistant tuberculosis (MDR) including extensively drug resistant turberculosis (XDR TB), in rural KwaZulu-Natal.
    (2011) Ramtahal, Melissa Afton.; Moodley, Prashini.
    Mycobacterium tuberculosis (MTB) is an airborne pathogen that is easily transmitted from person to person. An intact immune system prevents the organism from causing disease in most individuals. In South Africa, the prevalence of human immunodeficiency virus (HIV) has reached astronomical levels and is now fuelling the tuberculosis (TB) epidemic. Drug resistant MTB strains combined with a weakened host immune system is a lethal combination. Multi-drug resistant (MDR) including extensively drug resistant (XDR) tuberculosis is on the increase, with Tugela Ferry in KwaZulu-Natal South Africa, reporting the largest cluster of XDR cases in the world. It is unknown whether a single clone of the drug resistant strain is circulating in this area or whether there are multiple strains at play. Using 2 complementary genotyping methods, we showed that the MDR strains present are the result of clonal spread associated with the F28 family, as well as de novo resistance which manifests as unique patterns. The XDR epidemic in Tugela Ferry is the result of clonal spread of a strain belonging to the F15/LAM4/KZN family.
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    Characterization of 1, 2-DCA degrading Ancylobacter aquaticus strains isolated in South Africa.
    (2011) Pillay, Thiloshini.; Pillay, Basil Joseph.; Olaniran, Ademola Olufolahan.
    1,2-Dichloroethane (1,2-DCA), a highly toxic and recalcitrant compound, is produced anthropogenically in larger quantities than any other chlorinated compound. It is regarded as a mutagen and carcinogen, thus making it a priority target molecule for biological degradation. In addition, the intermediates of 1,2-DCA degradation are highly reactive and toxic, due to the electrophilic nature of the carbonyl groups in these compounds. Aerobic biodegradation of 1,2-DCA, resulting in complete mineralization, has previously been reported in Xanthobacter autotrophicus GJ10 and some Ancylobacter aquaticus strains. X. autotrophicus GJ10 has been found to possess chloroacetaldehyde (CAA) dehydrogenase and haloacid (HA) dehalogenase enzymes, both of which play a crucial role in 1,2-DCA degradation. Five strains of Ancylobacter aquaticus capable of utilizing 1,2-DCA as a sole carbon and energy source have recently been isolated in our laboratory. The degradation potential and specific dehalogenase activities of these bacterial isolates against 1,2-DCA and other halogenated compounds as a carbon source were investigated and compared to previously characterized organisms, viz., X. autotrophicus GJ10 and Ancylobacter aquaticus strains AD25 and AD27. Furthermore, this study proposed to detect the presence of the CAA dehydrogenase (aldB) and HA dehalogenase (dhlB) encoding genes in these isolates. Growth of all strains in the presence of 1,2-DCA as a carbon source was monitored over an 84 h period, in minimal medium supplemented with either vitamins or yeast extract. Dehalogenase activities were measured colorimetrically by monitoring halide release by crude cell extracts of the isolates. In order to detect the presence of dhlB and aldB genes, genomic DNA of the isolates was digested with individual restriction endonucleases, viz., EcoRI, PstI, HindIII and BamHI, and then subjected to Southern hybridization experiments. All isolates demonstrated significant growth rates in both vitamin and yeast extract supplemented media, with the former having a greater overall growth effect. Ancylobacter aquaticus DH5 demonstrated the highest growth rate of 0.147.h-1 in the presence of vitamins while Ancylobacter aquaticus DH12 displayed the highest growth rate of 0.118.h-1 with yeast extract. Optimum haloalkane dehalogenase activities of these bacterial isolates were confirmed at pH 8, similar to the activity in X. autotrophicus GJ10, while haloaciddehalogenase activity had a broader pH range. Hydrolytic dehalogenase activity of the bacterial isolates using a range of halogenated aliphatic compounds was also determined. Results demonstrated a wide substrate range with activity being observed on 1,3- dibromopropane, 1,2-dibromoethane and 1,3-dichoropropene, for all isolates. Southern Hybridization experiments confirmed the presence of both aldB and dhlB genes in X. autotrophicus GJ10. The dhlB probe produced a positive signal for an EcoRI fragment in Ancylobacter aquaticus DH12 while the aldB probe hybridized and produced a single positive signal on similar sized PstI fragments for all organisms except A. aquaticus AD25 which produced two positive signals. The results in this study demonstrate the potential application of the newly isolated strains of Ancylobacter aquaticus. in future bioremediation strategies. The detection of the genes involved in 1,2-DCA degradation further support the use of these isolates and/or their enzymes for the degradation of 1,2- DCA as well as other halogenated compounds. Future work need to determine sequence similarity of these genes detected in A. aquaticus strains to the genes in Xanthobacter autotrophicus GJ10 and other previously reported genes. It may also be important to investigate the activity of the enzymes under various environmental conditions and to determine enzyme structure and the catalytic sites, so as to gain knowledge of their degradation potential on site. Characterization of enzymes at both the molecular and protein levels may be necessary and beneficial for implementation in strategies involving bioremediation for the biological degradation of a wide range of halogenated aliphatic hydrocarbons.
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    Response of endothelial cells to exposure to Chlamydia trachomatis, biovar LGV.
    (2011) Seipone, Ikanyeng Dolly.; Sturm, Adriaan Willem.
    Although both are caused by Chlamydia trachomatis, Lymphogranuloma Venereum (LGV) presents differently from the infections caused by Oculogenital (OG) strains. The endothelium of blood and lymph vessels allows passage of cells to the site of infection. Endothelial cells also secrete chemokines and cell adhesion molecules which act as attractants and binding sites for various cellular immune components. Since LGV biovar affect the lymphoid tissue we studied the effect of C. trachomatis on endothelial cells. Human umbilical vein endothelial cells (HUVEC) were infected with C. trachomatis LGV serovars L1, L2, L3 and the OG strain E at multiplicity of infection (MOI) of 1 and incubated for 24 hours. Stimulation of Interleukin-8 (IL-8) and monocyte chemokine protein-1 (MCP-1) chemokines and the intercellular adhesion molecule -1 (ICAM-1) were quantified by enzyme linked immunosorbent assays (ELISA). Transendothelial migration of neutrophils and monocytes was carried out in transwells. The lactate dehydrogenase (LDH) release assay was used to measure cell necrosis. Apoptotic cell death was analysed using the BioVisionTM CaspGLOW Fluorescein Caspase Staining Kit and DeadEndTM Colorimetric Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) system with the C. trachomatis Culture Confirmation kit as a counter stain. All Chlamydia trachomatis serovars (L1, L2, L3 and E) successfully infected and replicated in HUVEC after 24 hours of infection. Only L3 stimulated significantly higher production of IL-8, MCP-1 and ICAM-1 by HUVEC as compared to the negative control and mock-infected cells. However, the remaining LGV serovars (L1 and L2) and the OG serovar E showed no significant difference in the stimulation of IL-8, MCP-1 and ICAM-1 when compared to the controls. Comparison of LGV and OG serovars showed no significant difference between these two biovars in inducing production of IL-8 and MCP-1, but L3 stimulated ICAM-1 at a significantly higher level than E. There was no significant difference in the number of migrated neutrophils between untreated HUVEC, mock infected HUVEC and HUVEC infected with Chlamydia serovars. L2 and L3 had significally higher amount of migrated monocytes than the controls with L3 being the highest. L3 was the only serovar that had a significant level of cell death by necrosis. Apototic cells were observed in both uninfected and infected HUVEC which is due to normal cell turn over. None of the infected cells showed TUNEL positive nuclei. It can be concluded that L3 is more virulent than the other serovars during the first 24 hours of infection. Infection with C. trachomatis serovars does not seem to cause any cell death by apoptosis 24 hours post infection. The only cell death that occurs is by necrosis and only on serovar L3 infected cells.
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    Biodeterioration of aluminium hot roll mill emulsions.
    (1998) Ramsden, Peter John.; Wallis, Frederick Michael.
    An in-depth study of the biodeterioration of the Hulett Aluminium hot roll mill emulsion, Prosol, was conducted. Samples of the emulsion in use at the hot roll mill were taken from various areas of the emulsion reticulation system in order to identify regions of highest microbial contamination. Standard plate count techniques and diagnostic procedures were employed to quantify and identify the microorganisms in these samples. In some of the highly contaminated areas of the emulsion reticulation system, microorganisms exceeded lxlO'CFUml'1 emulsion. A range of bacteria was identified which included members of the genera: Bacillus; Pseudomonas; Escherichia; Enterobacter; Sporosarcina; Micrococcus; Aeromonas; Chromobacterium and Desulfovibrio. Various fungi, including several yeasts, were also isolated and some of the filamentous spore-forming types were identified zsAspergillus spp.; Penicillium spp. and a Cladosporium sp. A visual scale was developed to assess the growth rate of the isolated microorganisms on a range of specific media containing various emulsion components as carbon and energy source. Although the results obtained by using this scale were not conclusive, a few biodegradable components were nonetheless identified. It was found that mixed cultures of the above microorganisms had a greater biodeteriorative effect on the emulsion than did any of the pure cultures when applied separately. This suggested complex microbial interactions were involved in the breakdown of the emulsion. A laboratory-scale model system representative of the Hulett Aluminium hot roll mill was designed and constructed to carry out a series of tests on unprotected and biocide-treated emulsions. A range of biocide concentrations were tested from which the minimum biocide inhibitory concentration was calculated. It was shown that microorganisms exposed to sublethal doses of the biocide Busan (active component glutaraldehyde) over a prolonged period of time, exhibited greater levels of tolerance and resistance to the biocide than did those microorganisms not previously exposed. It was deduced that less frequent, shock doses of biocide are more effective in the control and eradication of emulsion degrading microorganisms than are frequent, low level doses of the same biocide. In addition to the biocide studies, three imported so-called 'biostable' emulsions were evaluated as possible replacements for the susceptible Prosol. Of these three imported emulsions, two viz. HRF3 and Houghton Biostable were shown to be more resistant than Prosol to biodeterioration. After assessing the current hot roll mill management practices, a number of recommendations were made, including: the improvement of plant hygiene; education of the mill workers; improvement of emulsion monitoring; improvement of down-time management and improvement of biocide dosing regimes. Recommendations are also made for minimizing potential microbial growth in the new hot roll mill currently under construction at the Hulett Aluminium processing plant at Pietermaritzburg, South Africa.
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    Effects of management practices on soil organic matter content, soil microbial activity and diversity in the KwaZulu-Natal midlands.
    (2002) Nsabimana, Donat.; Wallis, Frederick Michael.
    The objective of this study was to investigate the effects of land use and management practice on the soil organic matter content and the size, activity and diversity of the microbial biomass. These effects were investigated using samples taken from the top (0-10 cm) layer of the soils from long-term agricultural managements including natural grassland, maize under conventional (maize CT), maize under zero tillage (maize ZT), annual ryegrass, Eucalyptus, Pinus, and permanent kikuyu pasture. The natural grassland was used as a control since records indicated that no agricultural activity had ever been exerted on the soil. The measurements used to investigate these effects included soil organic C, total N, soil pH, microbial biomass C, basal respiration rate, microbial quotient, metabolic quotient, dehydrogenase activity, fluorescein diacetate (FDA) hydrolysis, arginine ammonification rate, arylsulphatase activity and acid and alkaline phosphatase activities. The microbial functional diversity was measured using the Biolog Ecoplate and catabolic response profiles methods. Soil organic Cand total Nwere lowest under maize CT, followed by maize ZT and annual ryegrass and were higher under natural grassland, Eucalyptus and Pinus plantations while permanent kikuyu pasture had the highest values. The other analyses, namely microbial biomass C, basal respiration rate, FDA hydrolysis, arginine ammonification rate and arylsulphatase activity also followed the same pattern. Annual cultivation was responsible for a decrease in microbial biomass C, basal respiration rate and enzyme activity, principally because there was an appreciable decrease in soil organic matter content. Conversely, permanent pasture, Eucalyptus and Pinus plantations increased appreciably the amount of organic C and consequently, promoted the size and activity of the microbial biomass in the soils. The principle component scores showed that management practices affected the microbial functional diversity because different treatments were found in separate zones of the principle component spaces. The regression analysis showed that the variation in the PC1 and PC2 scores was correlated with the variation in soil organic C, exchangeable acidity, extractable P and exchangeable K and Mg. In addition, richness, evenness, Shannon, and Simpson diversity indices showed that any management practice affects the dynamics of soil microbial diversity.
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    The assessment of humoral immunity in the vaginal mucosa of pregnant and non-pregnant women.
    (2003) Omar, Momeen.; Sturm, Adriaan Willem.
    Mucosal surfaces are prominent in the gastrointestinal, urogenital, and respiratory tracts and provide portals of entry for pathogens. The mucosal immune system consists of molecules, cells, and organised lymphoid structures intended to provide immunity to pathogens that impinge upon mucosal surfaces. The aim of this study was to assess humoral immunity in the vaginal mucosa and compare this immune response to a systemic response. The use of commercially available tampons provided a self-administered, pain free method for the collection of vaginal secretions. To standardise specimens, a total protein determination was performed on vaginal secretions and on sera. All subjects were screened for sexually transmitted infections (STIs) using conventional and deoxyribonucleic acid (DNA) amplification tests. Immunoglobulin levels in vaginal secretions and in sera were quantitated using a quantitative sandwich enzyme- linked- immunosorbent assay (ELISA). The immunoglobulin levels quantitated were analysed on the basis of pregnancy status and the presence or absence of an STI. Immunoglobulin results for serum showed a significant increase in IgG and IgA in women with an STI regardless of pregnancy (p< 0.001). This study showed a decrease in vaginal IgG and IgA in women with an STI. Non-pregnant women with an STI had significantly lower levels of IgG and IgA in the cervico-vaginal secretions as compared to the controls (p=0.002 and p=0.0002 respectively). This was also observed in pregnant women (p= 0.03 and p< 0.001 respectively). IgM levels were mostly too low to be detectable but showed a tendency to increase in vaginal secretions of women with an STI. Pregnancy did not have an effect on immunoglobulin levels except for IgA. The effects observed were due to the presence of an STI. All the STI pathogens studied displayed a similar effect on immunoglobulin levels. Bacterial vaginosis, however, appears to exert an effect specifically on lowering IgG (p=0.008) in vaginal fluid and increasing IgG levels (p=0.008) in serum. Once a more complete understanding of the mechanisms associated with the host defence of the vaginal mucosa is obtained, specific immunotherapeutic strategies can be developed. A greater knowledge of host defence factors specific to the vagina will provide insights into understanding susceptibility to opportunistic infections and STIs.
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    Cryptosporidium and cryptosporidiosis.
    (1990) Moodley, Dhayendre.; Jackson, Terry F. H. G.
    Cryptosporidium parvum can cause debilitating disease in immunocompetent persons with cholera-like symptoms characterised by self-limiting, profuse diarrhoea; on the other hand asymptomatic infection with this organism frequently occurs. However, in immunocompromised patients, the disease is more severe and is lifethreatening. A pivotal aspect of the present survey was a comparative assessment of four commonly used staining techniques (viz. modified Ziehl-Neelsen, safranin-methylene blue, auramine phenol fluorescence and Sheather's sucrose flotation) for the detection and identification of Cryptosporidium oocysts. The Sheather's flotation method proved to be superior to the other three procedures which were not only less sensitive but also less specific. A modification of the Sheather's flotation technique was developed for use with diarrhoeal stools; this was found to be simple, reliable, costeffective and the least time consuming of the above methods; this was used exclusively in a subsequent survey of the association of Cryptosporidium infection with diarrhoea in hospitalised children. Although previous epidemiological surveys of cryptosporidiosis have been conducted in South Africa standardised methods have not been employed. This initial assessment of diagnostic techniques therefore provided a tool for accurately assessing the importance of Cryptosporidium as a causative organism of diarrhoea. In an extensive study performed on children younger than 10 years old, who were hospitalised with a primary diagnosis of diarrhoea at King Edward VIII Hospital, it was found that 9,0% (111/1229) were passing Cryptosporidium oocysts; this was the second most common enteric pathogen. In 72% (80/111) of patients with Cryptosporidium infections it was the only pathogen. The prevalence of cryptosporidiosis was highest during the months of February, March, April and May; direct correlation between the rainfall in the Durban area and the prevalence of cryptosporidiosis was demonstrated (r = 0,6125). Cryptosporidium infection was more prevalent in the 4-6 month age group (p = 0,001). The fact that Cryptosporidium infections may be symptomatic in some individuals and asymptomatic in others, suggests that strain differences in respect of pathogenic potential may occur. A prerequisite to the investigation of strain differences was to increase parasite numbers; both in vivo and in vitro culture techniques were employed. Culture in chicken embryos failed to increase the parasite population and only limited areas of the chorio-allantoic membranes showed a few developmental stages. Cell cultures proved to be more suitable for Cryptosporidium growth and parasite numbers increased proportionally with duration in culture. Attempts at infecting suckling Balb/c mice were unsuccessful; however experimental infection of immunosuppressed adult rats facilitated the examination of various developmental stages of the parasite. Isoenzyme electrophoresis is an excellent method for demonstrating polymorphism in many species. Of the five enzyme systems that were tested, glucose phosphate isomerase, malic enzyme and phosphoglucose dehydrogenase proved to be the most promising. The electrophoresis of lysates, prepared from oocysts, in an agarose gel system was found to give adequate and reproducible resolution of isoenzyme patterns. Isoenzyme polymorphism could be demonstrated in oocysts harvested from the stools of four children. Such polymorphism has not been described previously and indicates a more extensive study to investigate strain differences, and to correlate these with the clinical histories of infected subjects. This approach may be invaluable in elucidating the pathogenesis of Cryptosporidium infections in man.
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    Detection of drug metabolizing enzyme gene (DMEs) polymorphisms among the Zulu population of South Africa.
    (2007) Makume, Mantha Thandiwe.; Naidoo, Richard.; Naidoo, Kogieleum.; Chelule, Paul Kiprono.
    The ability to metabolise drugs and achieve positive therapeutic outcomes is dependent on both genetic and environmental factors. The focus of this study was to determine the distribution and frequency of clinically relevant DME alleles and to assess the impact of these DME alleles on therapeutic outcomes in a cohort of 50 HIV-TB co-infected Zulu participants. PCR-RFLP was used to generate a genotypic profile of CYPIA2, 2C9, 2C19, 2E1, 3A4, MDR-1 and NAT-2. The distributions of the allelic frequencies were as follows. The CYPIA2 (A) - 50.7%, CYP2C9*2 — 100% and *3 — 56.2%, CYP2C19*2 — 35.4%, CYP2E1 (C2) — 28.4%, CYP3A4*1B (G) — 58.2%, MDR-1 (C3435T) - 16% and NAT-2 slow acetylators — 6.5%. Seventy-three percent of participants had prolonged TB therapy. Within this group, 82.9% of patient displayed wild type and 17.2% variant allele for CYP2E1 gene (p= 0.04) profile. In addition, all the slow acetylators in this study had prolonged TB therapy. In the MDR-1 gene, 87.5% showed wild type allele and 12.5% displayed the variant allele. Unsuccessful TB outcomes were also noted in 22% of this study population. In this group the variant allele was found to be dominant in CYPIA2, CYP3A4 and NAT-2, the opposite was seen in CYP2E1 and MDR-1. It was also interesting to note a similar genetic profile in the group that showed successful TB therapy outcomes. All participants had positive ARV treatment outcomes despite DME genotypic variations. However, 26% of all study participants experienced liver enzyme abnormalities. These findings concur with other studies regarding the ethnic distribution of DME alleles and evidence of an association between ART and TB therapeutic outcomes and DME genotype variation was inconclusive.
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    A microsatellite evaluation of the genetic status of the p27Kip1 and p21Cip1/WAF1 genes in oesophageal cancer.
    (2008) Gaffoor, Zakir.; Naidoo, Richard.
    p21 C/P 1/"El and p 2 7K/P 1 are cyclin-dependant kinase inhibitors that fonn an integral part of the cell cycle process. These proteins function as cell-cycle inhibitors, and are able to induce cell cycle arrest by binding to cyclin complexes at key stages. p21 and p27 have been found to be down-regulated in various cancers. This study investigated aberrations at microsatellite markers linked to the p21 and p27 cell cycle genes, in a large cohort of oesophageal squamous cell carcinomas in South Africa. Fluorescent-based PCR were performed on markers linked to both the p21 and p27. The products were run with a 50-500hp marker on 6% denaturing polyacrylamide gels, on the ALFexpresstm' DNA sequencer. The detection and analysis of PCR products was achieved using the AL F e xp res sT M and Fragment M an a aerTm software programmes. Our findings indicate that markers linked to p27 display infrequent aberrations, with loss of heterozygosity ranging from 19% to 37%, and microsatellite instability at 3% to 7%. However, significant relationships between decreased survival time, and aberrations in markers DI2S391 and Dl2S364, were found to exist. Marker D6S1575 linked to p21 displayed frequent allelic loss at 47%, and was comparable to similar studies on the 6p region Further, LOH-Al in this marker was found to be significantly associated with poorly differentiated tumours. The findings from our study indicate that microsatellite aberrations occur infrequently at the p21 and p27 loci in oesophageal cancer. with the exception of marker D6S1575. In addition,this study clearly demonstrates the accuracy and sensitivity of the technology employed. This is the first microsatellite-based investigation of the p21/p27 gene loci in oesophageal cancer in South Africa, using a fluorescent-based PCR assay.