Diarrhoeal disease is a major cause of mortality in children in developing countries. It also remains a serious problem among all age groups throughout the world. Whereas studies to determine the epidemiological and aetiological factors of diarrhoeal disease have been reported for other parts of South Africa and the world, as yet no information is available for the Eastern Cape. Therefore this study was undertaken to determine the factors for this area. Enteropathogens were compared for the different ages in the various population groups and, where possible, seasonal and geographical differences were emphasised. A total of 7 278 faecal samples were examined by six laboratories in the Eastern Cape during the period November 1988 to October 1990. Data was recorded noting the age, sex and population group of the patients. The towns selected were Port Elizabeth, Uitenhage, Cradock, Grahamstown and their surrounding areas. The isolation rates for the pathogens studied in the various population groups were compared to those reported in similar studies in other countries. The seasonal incidences of the various selected pathogens were compared with those reported from elsewhere in South Africa. It was thought that the higher temperature of summer may influence the finding in the White population group, while rainfall would play a greater role for the Coloured and Black populations. The geographical distribution of the pathogens emphasised the difference in living conditions between the different population groups. For example a generally higher infestation rate of Helminths occurred in rural areas and in the groups living under poorer conditions. The low isolation rates for certain bacteria and the large percentage of samples from which no pathogens were isolated indicate the need for further research. However, the finding should be valuable for determining Public Health priorities and in the management of outbreaks of diarrhoeal disease.

Le contrôle de la qualité microbiologique de l’eau constitue une problématique majeure en termes sanitaire et économique. L’efficacité des traitements appliqués est actuellement vérifiée par des méthodes standards basées sur la mise en culture des microorganismes. Cependant, leur mise en œuvre ne permet d’obtenir des résultats ne présentant qu’une vision partielle de la population microbienne présente dans un échantillon et ce dans un délai qui n’est pas compatible avec le niveau de réactivité requis dans le domaine de la gestion de l’eau.Les présents travaux visaient à démontrer que l’application de la cytométrie en flux pour le suivi microbiologique au sein de filières de production d’eau potable et de circuits de tours aéroréfrigérantes représente une approche alternative aux méthodes réglementaires et classiques utilisées dans ce domaine. Une attention particulière a été portée sur le fait d’être en mesure de quantifier les cellules totales et viables et d’être capable d’identifier l’impact de différents traitements chimiques et/ou physiques (ozone, UV, biocides oxydants) au niveau de la cellule microbienne. La démarche de calibration et l’utilisation conjointe de différents marqueurs fluorescents incluant le SYBR Green II, l’iodure de propidium et le Chemchrome V6 a permis d’atteindre ce double objectif. Par ailleurs, 3 systèmes de cytométrie en flux différents ont été utilisés selon les études, comprenant le FACSCaliburTM, le FACSCantoTM et l’ACCURITM C6.Au regard des résultats obtenus, il apparait que la cytométrie en flux représenterait un atout dans le domaine du diagnostic rapide d’installations de par sa simplicité, son délai de résultat court (1 heure) et le niveau d’information apporté sur la population microbienne (impact sur la membrane, le matériel génétique ou le métabolisme). / The water microbiological quality control is a major issue in health and economic terms. The effectiveness of treatments applied is currently tested using culture-based standard methods. However, their application leads to a partial view of the microbial population present in a sample and within a period that is not compatible with the required responsiveness concerning water management.The present work aims to demonstrate that the application of flow cytometry for microbiological monitoring of drinking water treatment plants and cooling tower circuits represents an alternative approach to regulatory and conventional methods used in this field. Particular attention was paid to being able to quantify total and viable cells and be able to identify the impact of different chemical and/or physical treatment (ozone, UV, oxidizing biocides) on a microbial cell. The calibration process and the joint use of various fluorescent dyes including SYBR Green II, propidium iodide and ChemChrome V6 have achieved this double objective. Furthermore, three different flow cytometric systems have been used according to the studies comprising the FACSCaliburTM, FACSCantoTM and ACCURITM C6.Given the results, flow cytometry appears as an asset in the field of rapid diagnostic for treatment efficiency due to its ease of use, short time to result (1 hour) and the information provided related to the microbial population (impact on membrane, genetic material or metabolism).

Submitted in partial fulfillment of the requirements for the Degree of Master of Technology: Biotechnology, Durban Institute of Technology, 2000 / M

It was the purpose of the present investigation to determine if sensitivity to other antibiotics can likewise be affected by subjecting S. aureus to heparin contact. It is of special interest in this problem to determine whether heparin in some manner affects the combining process of penicillin with the cells of several strains of S. aureus.

Diarrhea is a global concern that the United Nations Children’s Fund (UNICEF) and the World Health Organization (WHO), have confirmed to be the second major cause of death in children under the age of five. Major bacterial pathogens that cause diarrhea include Escherichia coli, Salmonella species, Shigella species and Staphylococcus aureus. Antibiotic therapy is recommended depending on the severity and presentation of the disease; however, the appearance of antibiotic-resistant bacteria is an emerging global threat to the ability to treat these bacterial infections. This situation could be overcome by the discovery of new natural antibiotics. Plants have been a source of medicine for centuries and have been used to treat diseases including diarrhea. This makes plants a natural potential target to study for their antibiotic properties. The objective of this study was to determine the antibiotic properties of medicinal plants against known pathogens that cause bacterial diarrhea. Three medicinal plants, Cassia abbreviata, Kigelia africana, and Geranium incanum were investigated for their antimicrobial properties against these strains of microorganisms: American Type Culture Collection (ATTC) and Clinical Strains (CS). The plant materials were ground into powder, which was then dissolved in methanol, acetone and distilled water to extract the active compounds. The plant extracts were then used to (i) determine their antibiotic activity, (ii) determine the minimum inhibitory concentration (MICs), (iii) analyze the thin layer chromatography (TLC) fingerprints, and (iv) analyze the autobiography assay. The results obtained in this study met the aim and objectives of this study. The antimicrobial activities of the selected plants were obtained as discussed in Chapter 2 and 3. These results indicated that the traditional plants could be used as antimicrobials. In the screening assays, the test microorganisms were inhibited by the plant extracts, when they were subjected to plant extracts. This was performed on Mueller Hinton agar as sensitivity testing, which revealed clear zones of inhibition. The MIC values for each plant extract were established which ranged from 0.101 to 13.3 mg/dl. The TLC analysis revealed the spots which contained the active compounds which inhibited the bacterial growth. A bioautography assay was performed on the TLC plates, which exposed the exact spots containing the active compound inhibiting the bacteria. These results are clearly consistent with what former scientists have observed. Detailed explanations on the results are in Chapter 3 and 4 of this paper. It is important to note that all the procedures performed in this study were in vitro assays. Some effective in vitro assay activity may not always result in the same effective in vivo activity, because some active compounds may be metabolized and degraded into inactive metabolites. For this reason, the in vitro results obtained in this study, may not reflect the true effectiveness of the compounds in in vivo trials. It is therefore advised that future scientists should take a step further in analyzing the plant extracts through in vivo assays. Further testing and study on these plants at an advanced molecular level will be beneficial in the medical fields in the search for new antibiotics to treat infectious diseases. Purification and further analysis of their products can be helpful in the production of pure natural medicines. This will discover the active ingredients and compounds responsible for inhibition of the microorganisms. This will make the compounds potential candidates for a scientific validation and analysis for future scientists to bring a new dawn in the fight against infectious diseases.

The n-Hexane, dichloromethane, methanol and aqueous crude extracts of Garcinia kola (Heckel) seeds were screened for their anti-Vibrio activities against 50 Vibrio bacteria isolated from wastewater final effluents. The 50 isolates consisted of different Vibrio species namely V. fluvialis (14), V. vulnificus (12), V. parahaemolyticus (12), V. metschnikovii (3) and 9 others unidentified to the specie level. The n-Hexane, dichloromethane and methanol extracts had activities against 16 (32 percent) of the Vibrio isolates, while the aqueous extracts had activities against 12 (24 percent) all at a screening concentration of 10 mg/ml. The minimum inhibitory concentrations (MICs) were 0.313-0.625 mg/ml, 0.313-0.625 mg/ml, 0.313-2.5 mg/ml and 10 mg/ml for n-Hexane, dichloromethane, methanol and aqueous extracts respectively. Rate of kill studies were carried out against three different Vibrio species namely V. vulnificus (AL042), V. parahaemolyticus (AL049) and V. fluvialis ( AL040) using the n-Hexane, dichloromethane and methanol extracts at 1× to 4 × MICs and 2 hour exposure. About 96.3 percent, 82.2 percent, and 78.1 percent (V. fluvialis AL040); 92.6 percent, 87.8 percent and 68.9 percent (V. parahaemolyticus AL049); and 91.6 percent, 64.4 percent, 60 percent (V. vulnificus AL042) of the bacteria were killed by the crude n-Hexane, dichloromethane and methanol extracts respectively after 2 hour exposure time at 4× MIC. The patterns of activity were bacteriostatic, with the n-Hexane extracts being most effective in activity. We conclude that the Garcinia kola seeds have promise in the treatment and management of infections caused by Vibrio species.

Helicobacter pylori classified as a class 1 carcinogen is a common human pathogen implicated in certain gastrointestinal diseases. Helicobacter pylori infection is acquired mainly in childhood, especially in developing countries. H. pylori infection causes peptic ulcer, duodenitis, gastritis and cancer. The growing resistance of H. pylori to antibiotics used in its treatment as well as other innate limitations of the triple therapy has necessitated a search for alternative treatment from natural sources which could be readily available, less cost effective. The antimicrobial activity of solvents (acetone, ethanol, methanol, chloroform and water) crude extracts of Lippia javanica were investigated against 31 H. pylori strains by the agar well diffusion technique. The minimum inhibitory concentration (MIC) was determined by spectrophotometric analysis at 620 nm using the broth micro dilution method and the rate of kill by broth dilution method. Phytochemical analysis was also performed. H. pylori standard strain NCTC 11638 was included as a positive control. Metronidazole and amoxicillin were used as positive control antibiotics. The ANOVA test was used to analyze the results using SPSS version 17.0. The strains were inhibited by all the extracts with inhibition zones of diameter ranging from 0-36 mm and 0-35 mm for the control antibiotic, clarithromycin. The MIC90 ranged from 0.039- 0.625 mg/mL for acetone; 0.039-1.25mg/mL for methanol, 0.00195-0.313 mg/mL for ethanol; 0.01975-2.5 mg/mL for metronidazole and 0.0048-2.5 mg/mL for amoxicillin. Acetone extract completely inhibited strain PE369C at MIC (0.1 mg/mL) and 2× MIC (0.2 mg/mL) in 18h and at ½× MIC (0.05 mg/mL) in 36h. Strain PE466C was completely inhibited at 4× MIC in 72h. Phytochemical analysis revealed the presence of flavonoids, saponins, tannins, steroids and alkaloids. The results indicate that the extracts of the leaves of L. javanica may contain compounds with anti-H. pylori activity and merits further study to identify the compounds.

Les chloroéthènes sont les polluants majeurs des eaux souterraines et des nappes phréatiques. De par leur toxicité et leur effet cancérigène, ils représentent une préoccupation majeure pour les autorités publiques et sanitaires. La restauration des sites contaminés est possible par des techniques de dépollution biologique impliquant les microorganismes (bioremédiation microbienne). Cependant, la réussite des traitements dépend à la fois des conditions physicochimiques du site pollué et des capacités de dégradation de la microflore indigène. Ainsi, pour optimiser les processus de décontamination, l’identification et le suivi des différentes populations microbiennes sont indispensables avant et pendant le traitement. Les biopuces ADN fonctionnelles (FGA, Functional Gene Array), outils moléculaires haut débit, sont particulièrement bien adaptées pour des applications en bioremédiation. Leur élaboration nécessite de disposer de logiciels performants pour le design de sondes qui combinent à la fois une forte sensibilité, une très bonne spécificité et un caractère exploratoire, ce dernier étant indispensable pour la détection des séquences connues mais surtout de celles encore jamais décrites au sein d’échantillons environnementaux. Un nouveau logiciel, autorisant la sélection de sondes combinant tous ces critères, a été développé et nommé HiSpOD. Son utilisation pour la construction d’une FGA dédiée aux voies de biodégradation des chloroéthènes a permis d’évaluer l’effet de traitements de biostimulation sur la microflore indigène pour plusieurs sites industriels contaminés. Les données révèlent différentes associations entre microorganismes déhalorespirants qui sont fonction des paramètres environnementaux. / Chlorinated solvents are among the most frequent contaminants found in groundwater and subsurface ecosystems. Because of their high toxicity and carcinogenicity, they represent a serious risk for human health and the environment. Thus, such polluted sites need a rehabilitation treatment. Among remediation solutions, microbial bioremediation represents a less invasive and expensive alternative than physico-chemical treatments. However, the process efficiency greatly depends on the environmental conditions and the microbial populations’ biodegradation capacities. Therefore, bioremediation treatment optimization requires the identification and monitoring of such capacities before and during the treatment. Functional Gene Arrays (FGA), by profiling environmental communities in a flexible and easy-to-use manner, are well adapted for an application in bioremediation. But, constructing efficient microarrays dedicated to microbial ecology requires a probe design step allowing the selection of highly sensitive, specific and explorative oligonucleotides. After a detailed state of the art on probe design strategies suitable for microbial ecology studies, we present new software, called HiSpOD, generating efficient explorative probes for FGA dedicated to environmental applications. Finally, this bioinformatics tool was used to construct a FGA targeting most genes involved in chloroethenes biodegradation pathways which allowed the evaluation of biostimulation treatments conducted on indigenous bacterial populations for several industrial contaminated sites.

It was demonstrated that induction of a major outer protein, HI, was associated with increased resistance to chelators of divalent cations such as EDTA and to the cationic antibiotics polymyxins and aminoglycosides. Outer membrane protein HI was the major cellular protein in cells grown in Mg²⁺-deficient medium (0.02 mM Mg²⁺) and in mutants selected for resistance to polymyxin. Increase in protein HI was associated with decrease in cell envelope Mg²⁺. Induction of protein HI was prevented by supplementation of Mg²⁺-deficient medium with 0.5 mM Mg²⁺, Ca²⁺, Mn²⁺ or Sr²⁺, but not by Zn²⁺, Ba²⁺, or Sn²⁺. Cells grown in Ca²⁺, Mn²⁺ or Zn²⁺ showed enhanced levels of these cations as main major cell envelope associated cation. Only cells grown in the presence of those cations which failed to prevent HI induction were resistant to chelators, polymyxin B and gentamicin. Protein HI overproducing cells also demonstrated altered streptomycin uptake. It was further demonstrated that aminoglycosides could interact with the outer membrane so as to make it more permeable to other substances. Mg²⁺ inhibited aminoglycoside-mediated permeabilization. Both aminoglycosides and polymyxin B could be shown to displace a small amount of Mg²⁺ from the cell envelope. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Since heparin combines with some antibiotics to decrease the toxicity of the antibiotic to the patient, the purpose of this investigation is to determine whether it has any effect upon the development of resistance to antibiotics by Staphylococcus aureus.