A dissertation submitted to the Faculty of Medicine, University of the Witwatersrand, Johannesburg, for the degree of Master of Science in Medicine. / Analysis of organisms involved in hospital infections has shown that Gram-positive bacteria have assumed an increasingly important role. Examples that have been recognised as important pathogens are staphylococci , enterococci, streptococci, Corynebacterium jeikeium and Leuconostoc species. Methicillin resistance in staphylococci has become a major problem in certain hospitals. Viridans streptococci continue to be the most frequent cause of native valve endocarditis. Leuconostoc species are being increasingly isolated from blood cuIture specimens. strains of Gram-positive bacteria have become resistant to specific antibiotics; e.g. staphylococci to methicillin, enterococci to ampicillin, and viridans streptococci to penicillin. JK corynebacteria are sensitive only to vancomycin and resistant to other antimicrobials normally used for treating infection caused by Gram-positive bacteria. In this study various combinations of antimicrobials against 35 clinical isolates of Gram-positive bacteria obtained from three hospitals in the Johannesburg area (Johannesburg, Hillbrow, and Baragwanath) from 1987- 1988 were investigated. The MIC / MBC results conformed to others described in worldwide studies. Results when different methodologies for determining synergy were used, varied. This emphasizes the need for standardization, especially with regard to the time-kill studies. Most antimicrobial combinations demonstrated tested against Leuconostoc species synergy using the checkerboard method, but these results were not confirmed by time-kill procedures, which showed mainly indifference. Synergy was also obtained when gentamicin plus ciprofloxacin was combined Corynebacterium jeikeium. Because of increasing resistance and the fact that Gram- positive bacteria cause serious infections, various and new combinations of antimicrobials need to be tested before treating these infections. Parts of this dissertation have been presented at the following congresses: 10th Annual Congress of the Society of Medical. Laboratory Technologists of South Africal Sun city 1989 75th Anniversary Congress of Pathology Johannesburg 1990 11th Annual Congress of the Society of Medical Laboratory Technologists of South Africa, Durban 1991 / Andrew Chakane 2019

No description available.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Tetrodecamycin (1) is a novel α-(γ-hydroxyacyl) tetronic acid based polyketide antibiotic isolated from the culture broth of Streptomyces nashvillensis MJ885-mF8 by Takeuchi et al. in 1994. Compound 1 shows potent inhibitory activity against Gram-positive bacteria including Bacillus anthracis and methicillin resistant Staphylococcus aureus (MRSA). It was proposed that an Aldol reaction of trans-decalin core 2 and tetronic acid derivative 3 followed by a face selective epoxidation and a subsequent epoxide-opening reaction would deliver the 6,6,7,5-skeleton of tetrodecamycm (1). To investigate this proposal, the silyl enol ether 5 was prepared from cycloheptene 4 in 7 steps. An unusual domino silyl enol ether reaction sequence was observed when the silyl enol ether 5 was submitted to a Diels-Alder reaction. It afforded cycloadduct 6, which was converted to the key intermediate 2 after another 3 steps (Scheme 1). Concurrently, double functionalisation of simple cyclic silyl enol ethers was investigated. Because of some difficulties in reproducing good overall yields to the cycloadduct 6, a second synthetic route was proposed. It was envisaged that a palladium-catalysed oxidative cyclisation or an organoselenium-mediated cyclisation reaction of compound 8 would construct the 6,6,7,5- skeleton 7, which would be converted to tetrodecamycin (1) via dihydroxylation followed by an introduction of the exo-methylene group. The intramolecular Diels-Alder reaction of trienal 11 afforded trans-decalin 10, which was converted to β-keto ester 9 in 2 steps. A Dieckmann-type cyclisation of 9 afforded compound 8 in good yield. However, so far transformation to compound 7 has not been achieved (Scheme 2).

Several bacteria were isolated from the bottom sediments of Tyume River and investigated for bioflocculant production potentials. Kaolin clay suspension (4 g/l) was used to measure the flocculating activity and three of the positive isolates were identified by 16S rRNA gene nucleotide sequence analyses and the sequences deposited in GenBank as Streptomyces sp Gansen (accession number HQ537129), Brachybacterium sp UFH (accession number HQ537131.), and Cellulomonas sp Bola (accession number HQ537132). Streptomyces sp Gansen exhibited its maximum flocculating activity using lactose (85% activity), peptone (76.3% activity), Ca2+ as sole sources of carbon, nitrogen and cations respectively, and at a neutral pH of 7.0, while, the bioflocculant produced by Brachybacterium sp UFH with glucose, urea and Ca2+ as carbon, nitrogen and cations sources yielded 82% and 97% flocculation activity respectively at a neutral pH. Also, glucose (73.2% activity), ammonium chloride (78.2% activity) and Ca2+ resulted in optimal production of bioflocculant by Cellulomonas sp Bola, also at a neutral pH. Chemical analysis confirmed that bioflocculant produced by Streptomyces Gansen is a polysaccharide while Brachybacterium sp UFH and Cellulomonas sp Bola produces a glycoprotein compound. This freshwater actinomycetes appears to have a tremendous potential as sou rces of new bioflocculants.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Thesis (M. Med. Sc.)--University of Hong Kong, 2007.

Thesis (M. Med. Sc.)--University of Hong Kong, 2008. / Includes bibliographical references (p. 23-34)

The Type VII secretion system (T7SS) is linked to virulence and long-term pathogenesis in a broad range of Gram-positive bacteria, including the human commensal and pathogen Staphylococcus aureus. The Type VIIb secretion system (T7SSb) is responsible for the export of small toxic proteins, which induce antibacterial immune responses and mediate bacterial persistence in the host. In addition, it is also involved in bacterial competition. The T7SSb requires several proteins to build up the secretion machinery. This work focuses on the structural and functional investigation of the motor ATPase EssC and the putative pore forming, multi-pass membrane component EsaA. Both proteins are indispensable for substrate secretion. EssC belongs to the FtsK/SpoIIIE ATPase family and is conserved among the T7SSs. It contains three C-terminal, cytosolic ATPase domains, designated as EssC- D1, -D2 and -D3, whereby EssC-D3 is the most distal one. In this thesis, I am presenting the crystal structure of the EssC-D3 at 1.7 Å resolution. As the deletion of EssC-D3 abrogates substrate export, I have demonstrated that this domain comprises a hydrophobic, surface-exposed pocket, which is required for substrate secretion. More specifically, I have identified two amino acids involved in the secretion process. In addition, my results indicate that not only EssC-D3 is important for substrate interaction but also EssC-D2 and/or EssC-D1. Unlike in the related Yuk T7SSb of Bacillus subtilis, the ATPase activity of D3 domain contributes to substrate secretion. Mutation of the modified Walker B motif in EssC-D3 diminishes substrate secretion completely. The membrane protein EsaA encompasses an extracellular segment spanning through the cell wall of S. aureus. I was able to reveal that this part folds into a stable domain, which was crystallized and diffracted up to 4 Å. The first attempts to dissolve the structure failed due to a lack of homologues structures. Therefore, crystals for single-wavelength anomalous dispersion, containing selenomethionyl-substitutes, were produced and the structure solution is still in progress. Preliminary experiments addressing the function of the extracellular domain indicate an important role in substrate secretion and bacterial competition. / Das Typ VII Sekretionssystem (T7SS) ist wichtig für Virulenz und Langzeit- Pathogenität von Gram-positiven Bakterien. Zu diesen gehört auch Staphylococcus aureus, bekannt als Kommensal und Pathogen im Menschen. Das Typ VIIb Sekretionssystem (T7SSb) exportiert kleine, toxische Proteine, die antibakterielle Immunantworten auslösen und für bakterielle Persistenz verantwortlich sind. Außerdem ist es an dem Konkurrenzkampf zwischen Bakterien beteiligt. Das System benötigt verschiedene Komponenten, um eine Sekretion zu ermöglichen. Diese Doktorarbeit konzentriert sich auf zwei dieser Proteine, die ATPase EssC und das Membranprotein EsaA. Beide Komponenten sind unentbehrlich für eine vollständige Funktionalität. EssC gehört zu der Familie der FtsK/SpoIIIE ATPasen und ist evolutionär in allen T7SSs erhalten. EssC besitzt drei C-terminale, zytosolische ATPase Domänen, bezeichnet als EssC-D1, -D2 und D3, wobei EssC-D3 C-terminal gelegen ist. In dieser Arbeit präsentiere ich die Kristallstruktur der ATPase Domäne EssC-D3, aufgelöst bis zu 1.7 Å. Die Domäne ist unabdingbar für die Sekretion. Durch die Strukturauflösung wurde eine hydrophobe, Oberflächen-exponierte Substrat- Bindetasche bestimmt, die eine essenzielle Rolle für den Export der toxischen Substrate einnimmt. Durch dieses Projekt konnten zwei Aminosäuren in dieser Tasche bestimmt werden, die für den Prozess der Substratsekretion wichtig sind. Weiterhin wurde bewiesen, dass nicht nur EssC-D3, sondern auch die ATPase Domäne EssC-D2 und/oder EssC-D1 mit den Substraten interagieren kann. Im Gegensatz zu dem verwandten T7SSb in Bacillus subtilis, verfügt EssC-D3 über ATPase Aktivität und ermöglicht dadurch den Substratexport. Das Membranprotein EsaA besitzt einen extrazellulären Abschnitt, der sich durch die Zellwand von S. aureus erstreckt. Dieser extrazelluläre Part besteht aus einer stabilen Domäne, welche kristallisiert werden konnte und bis zu 4 Å diffraktiert. Aufgrund von fehlenden homologen Strukturen konnte die Struktur der Domäne noch nicht bestimmt werden. Für die Phasenbestimmung, die wichtig für die Strukturauflösung ist, wurden Kristalle mit Selenomethionyl-Substituten hergestellt. Die Strukturauflösung ist noch nicht beendet. Erste Experimente bezüglich der extrazellulären Domäne zeigen, dass diese ebenfalls wichtig für die Substratsekretion und zusätzlich am Konkurrenzkampf zwischen Bakterien beteiligt ist.

published_or_final_version / abstract / toc / Microbiology / Master / Master of Philosophy