Thesis (MSc)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: The ability of biofilms to resist antimicrobial treatment, when planktonic microbes cannot, is of not only fundamental scientific interest, but also a concern in industrial and medical fields. The inability to control biofouling of water distribution networks and products, as well as recurrent infections of implanted medical devices, is not only costly, but also potentially lethal. Several mechanisms whereby biofilms are able to evade antibiotic and biocidal agents have been proposed and investigated, but no universally relevant characteristic has been identified. . Initial investigation, involving BacLightTh ! LIVEIDEAD viability probes, epifluorescence microscopy and image analysis into the ability of natural biofilm and planktonic populations, .cultured in situ in a cooling tower, to survive treatment with a commercial biocide was not conclusive. Subsequent laboratory experimentation with a bacterial isolate from the cooling tower water revealed that the ability of attached biofilms to resist antimicrobial treatment exceeded that of planktonic cells shed from the biofilm. The reduced ability of suspended cells to survive antimicrobial treatment was not statistically significant, compared to that of the biofilm (P = 0.05). This is in contrast to the wealth of literature published on the subject of biofilm antimicrobial resistance The dilution rate in the flowcells in which biofilms were cultivated was more than 100 times higher than the maximum specific growth rate of the test organism. Nevertheless, there was typically more than I x 108 cells/ml in the effluent, suggesting that a metabolically active, rapidly dividing layer of cells existed at the biofilm bulk-liquid interface, from where daughter cells continuously detached. Treatment with an antimicrobial agent resulted in a significant reduction in the viability and number of cells detached from the biofilm, suggesting that this metabolically active layer of the biofilm was more sensitive to antimicrobial treatment, possibly due to a higher specific growth rate. Antimicrobial resistance was shown to be affected by the growth rate for planktonic bacterial populations, with an increased ability to survive, correlated with a decrease in specific growth rate. This supports the contention that growth rate plays a role in the susceptibility of the active layer. The bacterial cells in the layers closest to the attachment surface of the biofilm has frequently been shown to be slow growing, due to nutrient and oxygen limitation, while the outer biofilm layer is more susceptible to unfavourable environmental conditions. It is possible that such differentiation, which results in a responsive outer biofilm layer, provides a mechanism for the protection of the cells in the deeper layers, and thus survival over time. The results presented here support several hypotheses put forth in literature to account for the increased resistance of biofilms towards antimicrobial agents. Future work will include an investigation into changes in the patterns of gene expression when a bacteria becomes attached to a surface, upon subsequent release from the biofilm, and the influence this has on the ability to resist antimicrobial treatment. / AFRIKAANSE OPSOMMING: Die vermoë van aangehegte mikrobes, in teenstelling met vrydrywende mikroorganismes, om behandeling met antimikrobiese middels te oorleef, is nie net van belang vanuit 'n fundamenteel wetenskaplike oogpunt nie, maar ook betekenisvol vir die industriële en mediese velde. Die beheer van bio-bevuiling van waterverspreidingsnetwerke en produkte, sowel as herhaalde infeksies van mediese inplantings, is nie net van kostebelang nie, maar ook potensieël lewensgevaarlik. Verskeie meganismes wat biofilms in staat stelom antimikrobiese behandeling te oorleef, IS voorgestel en ondersoek, maar geen alomteenwoordige eienskap is tot dusver geïdentifiseer nie. Aanvanklike ondersoeke na die vermoë van natuurlike biofilms en planktoniese 'gemeenskappe, om biosiedbehandeling in situ in 'n lugversorgingskoeltoring se water te oorleef, was onbeslis. Die eksperimentele metodes het gebruik gemaak van BacLight™ LIVE/DEAD lewensvatbaarheidkleurstof, epifluoressensie-mikroskopie en beeldanalise. Daaropvolgende ondersoeke met 'n bakteriese isolaat vanuit die koeltoring het daarop gedui dat biofilms beter in staat is om antimikrobiese behandeling te oorleef as selle wat vrygelaat word vanuit die biofilm. Die afname in the lewensvatbaarheid van vrydrywende selle, na afloop van biosiedbehandeling, was nie statisties beduidend in vergelyking met die van die biofilm nie (P = 0.05). Die bevinding is in teenstelling met wat algemeen aanvaar word in die literatuur. Die verdunningstempo waaronder die biofilms in die vloeiselle gekweek is, was meer as 100- voudig hoër as die maksimum spesifieke groeitempo van die toetsorganisme. Ten spyte hiervan was daar tipies meer as 1 x 108 selle/ml in die uitvloeisel teenwoordig. Dit dui op 'n metabolies aktiewe, vinnig verdelende laag selle in die boonste laag van die biofilm, naaste aan die vloeistof fase, waarvandaan dogterselle voortdurend vrygestel word. Behandeling met die antimikrobiese agent het 'n beduidende afname in die lewensvatbaarheid en aantal dogterselle tot gevolg gehad, wat lei tot die gevolgtrekking dat die metabolies aktiewe laag van die biofilm meer sensitief is vir antimikrobiese behandeling, moontlik weens 'n hoër spesifieke groeitempo. Daar is verder bewys dat die vermoë om die werking van die antimikrobiese middel teen te staan, afhanklik is van die spesifieke groeitempo van planktoniese populasies. 'n Afname in groeitempo word geassosieer met 'n toename in oorlewing na antimikrobiese behandeling, wat die voorstel dat die groeitempo van die aktiewe laag 'n rol speel in die vatbaarheid daarvan, ondersteun. Dit is bekend dat die metaboliese aktiwiteit van bakteriese selle nader aan die aanhegtingsoppervlak van die biofilm verlaag is, weens 'n afname in diffusie van suurstof en nutriente in daardie deel van die biofilm. Dit is moontlik dat hierdie differensiasie, wat lei tot die vatbaarheid van die buitenste laag van die biofilm vir ongunstige omgewingstoestande, 'n oorlewingsmeganisme daarstel wat die onderliggende selle beskerm. Die resultate wat hier voorgelê word, ondersteun verskeie hipoteses wat die verhoogde weerstandbiedendheid van biofilms teen antimikrobiese middels beskryf. Toekomstige werk sluit ondersoeke in na veranderende patrone van geenuitdrukking wat plaasvind wanneer 'n bakterie in aanraking kom met 'n oppervlak, vasheg en ook weer vrygestel word, asook die invloed hiervan op die vermoë om antimikrobiese behandeling te oorleef.

published_or_final_version / Ecology and Biodiversity / Master / Master of Philosophy

Mini-dissertation submitted in partial compliance with the requirements for the Master's Degree in Technology: Homoeopathy, Durban Institute of Technology, 2003. / The purpose of this study was to determine the efficacy of Alepidea amatymbica tincture and homoeopathic dilutions to the 1st and 6th decimal potency as compared to ethanol (negative control) in the in vitro inhibition of Escherichea coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus and Bacillus cereus in terms of the disc diffusion test. Vancomycin and gentamicin were included in the study as positive controls in order to account for plate-ta-plate variations in the sensitivity of the bacterial strains to antimicrobial substances. Antimicrobial activity was expressed as the ratio of the inhibition zone (mm) produced by the test substance and the inhibition zone (mm) produced by the two combined antibiotic discs. For this study 20 Mueller-Hinton agar plates were assigned to each bacterial species and were inoculated with their respective bacteria. Four dry filter paper discs and two antibiotic discs were placed equidistantly on each agar plate. The filter paper discs had been previously impregnated with one of the test substances or the negative control using a triple impregnation technique utilizing a micro-pipette. The plates were then incubated at 37\xB0 C. The diameters of the zones of inhibition were measured at 18 hours, 24 hours and 48 hours. Data was analysed by means of the Statistical Package for Social Sciences (SPSS). Statistical methods utilized were Friedmans' test, Mann-Whitney U test, and Kruskall - Wallis Non-Parametric Analysis of Variance by Rank test. / M

O desenvolvimento de adesivos bioativos mostra-se como uma alternativa interessante para agregar benefícios aos novos materiais. O objetivo do estudo foi sintetizar nanopartículas de prata (NAg) e de hidroxiapatita (NHA), avaliar sua influencia no efeito antibacteriano, citotoxicidade de um adesivo com NAg e sua resistência de união à dentina associado ao uso de NHA. As nanopartículas foram caracterizadas por fluorescência de raio x por reflexão total (TRXF), difratometria de raio x (DRX), microscopia eletrônica de transmissão (TEM) e espalhamento de luz visível (DLS). O sistema Scotchbond Multi-Purpose (SBMP) foi modificado com adição de 0,05% e 0,1% de NAg no primer e no bond. Molares humanos foram restaurados com o sistema adesivo modificado após o pre-tratamento da dentina com suspensão aquosa de NHA a 0,5% e 1% para realização do ensaio de resistência de união imediata (24h) e após 1 ano de envelhecimento. A interface adesiva foi caracterizada por Microscopia Confocal Raman (MCR). Para mensurar o efeito antibacteriano (S.mutans) do adesivo com NAg foi realizado o ensaio de difusão em ágar com template e contagem de Unidades Formadoras de Colônias (UFC). A citotoxicidade foi avaliada com uso de células tronco da polpa dentária (DPSCs) em contato com adesivos não polimerizados e polimerizados em diferentes concentrações de meio condicionado (0% 25%, 75% e 100%). Foram produzidas NAg esféricas, com estrutura cúbica de face centrada, e com 16 nm de diâmetro médio. As NHA exibiram estrutura prismática com tamanho médio de 79,4 nm. O adesivo com NAg e o tratamento da dentina com NHA não afetaram a resistência de união imediata ou após 1 ano de envelhecimento, e as interfaces adesivas mostraram-se íntegras. Os adesivos modificados por NAg exibiram citotoxicidade similar ao adesivo controle e maior efeito antibacteriano. Os adesivos com adição de NAg são promissores quanto à obtenção de um material bioativo antimicrobiano que não altera a resistência de união ou a biocompatibilidade. / The development of bioactive adhesives is shown an interesting alternative to add benefits to the new materials. The aim of the study was to synthesize silver nanoparticles (NAg) and hydroxyapatite (NHA) and to evaluate their influence on the bond strength, antimicrobial effect, and biomaterials cytotoxicity. The nanoparticles were characterized by x-ray fluorescence by total reflection (TRXF), x-ray diffractometry (XRD), transmission electron microscopy (TEM) and visible light scattering (DLS). The Scotchbond Multi-Purpose system (SBMP) was modified with 0.05% and 0.1% silver in the primer and bond. Human molars were restored with the modified adhesive after pre-treatment of dentin with 0.5% and 1% NHA for the immediate bond strength test (24h), and after 1 year of aging. The adhesive interface was characterized by Confocal Raman Microscopy (MCR). To measure the antibacterial effect (S.mutans) of the adhesive with NAg, the agar diffusion assay with template was performed and counting of Colony Forming Units (UFC). In order to evaluate the cytotoxicity, dental pulp stem cells (DPSCs) were used in contact with unpolymerized and polymerized adhesives in different concentrations of conditioned medium (0% 25%, 75% and 100%). Spherical NAg, with a cubic face centered structure, and 16 nm in diameter were produced. The NHA exhibited a prismatic structure with approximately 79.4 nm. The adhesive with NAg and the treatment of dentin with NHA did not affect the bond strength immediately or after 1 year of aging, and the adhesive interfaces were shown to be intact. NAg-modified adhesives exhibited cytotoxicity similar to the control adhesive and higher antibacterial effect. The adhesives with NAg addition are promising in obtaining a bioactive antimicrobial material that does not alter the bond strength or the biocompatibility.

Traditional bacterial disease therapies utilize compounds that ultimately kill the target bacteria but it exerts a strong selective pressure on the bacteria to develop multi-drug resistance mutants. The increasing occurrence of resistance in common pathogens has highlighted the need to identify new anti-microbials that target the control of bacterial pathogenicity in a non-extermination manner to reduce the incidence of bacteria resistance. One new strategy exploits the discrete signaling molecules that regulate the various bacterial signaling pathways, which are responsible for the expression of pathogenicity traits. Halogenated furanones (fimbrolides) from the marine red alga, Delisea pulchra have been shown to interfere with the key signaling pathway present in Gram-negative bacteria by competitively displacing the cognate signaling molecule from the transcription protein. This project focused on the design and synthesis of 1,5-dihydropyrrol-2-ones, a new class of fimbrolide derivatives capable of displaying strong antagonistic properties of the fimbrolides. Primary synthetic methodologies examined include the halolactamization of allenamides and the direct lactone-lactam transformation. No doubt, both methodologies yielded the lactam ring, the former failed to introduce the crucial C-5 bromomethylene group essential for bioactivity. A facile high yielding two-step lactone-lactam transformation method was developed and using this method, a wide range of substituted 5-bromomethyl- and 5-dibromomethylene-1,5-dihydropyrrol-2-ones were synthesized. Furthermore, a new class of tricyclic crown-ether type compounds with no literature precedent were discovered. To vary the diversity of the compounds, a related class of compounds, 5,6-dihydroindol-2-ones, were examined. A general versatile method for the synthesis of 7-substituted 5,6-dihydroindol-2-ones was developed. The synthetic strategy proceeds via the established Suzuki-Miyaura cross-coupling reaction of halogenated dihydroindol-2-ones with arylboronic acids/esters. The Suzuki methodology was found to be reliable in furnishing a wide range of 7-substituted products in high yields. A preliminary molecular modeling approach was used to assist in the design of new anti-microbials via the ligand-docking analyses of the TraR and LasR protein. A positive correlation was observed between the docking scores and biological activity and the methodology was further developed into an initial screening tool to filter potential active and non-active compounds. The newly synthesized compounds were analysed for their efficacy in reducing the expression of the Green Fluorescent Protein (GFP) in the presence of natural AHL signaling molecules in an AHL-monitor strain, indicative of the inhibition of bacterial phenotype expression. The dihydropyrrol-2-one class of compounds showed significant biological activity and this highlighted their potential for further development.

The pharmacokinetics of two antimicrobials were investigated in alpacas. Six healthy alpacas were each administered a single dose of 10 mg/kg of oxytetracycline by IV injection and IM injection. In addition, a single dose of 20 mg/kg of florfenicol by IV administration was given to alpacas in a separate study. The pharmacokinetic parameters of oxytetracycline and florfenicol in alpacas were compared to the results previously obtained in llamas. There were significant differences between llamas and alpacas in several of oxytetracycline pharmacokinetic parameters but there were no significant differences in all of florfenicol pharmacokinetic parameters in these two animals. It can be concluded that llamas and alpacas have different oxytetracycline disposition kinetics while they have similar disposition kinetics of florfenicol. The pharmacokinetics of clorsulon, a narrow-spectrum anthelmintic agent, was investigated in llamas following oral administration at a single dose of 14 mg/kg. The plasma levels of clorsulon produced by this dose was lower than the values reported in the clorsulon pharmacokinetic studies carried out in sheep and goats following oral administration at a single dose of 7 mg/kg This suggests the entire dose of clorsulon is not absorbed in llamas. Since the differential equations describing one-compartment system with first-order input and two-compartment system after IV administration with nonlinear elimination kinetics cannot be solved, there is no mathematical expression for the AUC for drugs following these models. The AUC values calculated from the proposed preliminary AUC equations for drugs following these models were compared to the AUC calculated using the trapezoidal rule method based on computer-generated data using the fourth-order Runge-Kutta method. Except for a few exceptions, the predicted AUC from the proposed equations matched the values calculated from the theoretically generated data. / Graduation date: 2004

Thesis (Ph. D.)--University of Pretoria, 2002. / Includes bibliographical references.

This thesis aimed to investigate the features and properties of the ground states, transient species and photoproducts involved in the photophysical and photochemical processes for four kinds of drug compounds: lomefloxacin (LF), norfloxacin (NF), tiaprofenic acid (TPA), and flurbiprofen (Fp). The investigation used femtosecond transient absorption (fs-TA), nanosecond transient absorption (ns-TA), UV/Vis absorption spectra (UV/Vis), nanosecond transient resonance Raman (ns-TR2) and nanosecond time-resolved resonance Raman spectroscopy (ns-TR3), as well as density functional theory (DFT) calculations. Although many previous investigations have indicated that photo-defluorination or photo-decarboxylation reactions may account for the phototoxicity for these compounds, detailed information on the mechanisms remains unclear. In this thesis, the photo-defluorination reaction of LF was explored in neutral water at pH 7.2. The fs-TA results revealed that the lowest lying excited singlet state species (S1) partially decayed into the ground state through fluorescence emission and partially underwent cleavage of the carbon-fluorine bond at position 8 to generate into a singlet aryl cation. Subsequently, intersystem crossing (ISC) allowing the transformation from singlet cation to triplet carbene was observed. Finally, a cyclization reaction with the N-ethyl chain took place for the triplet carbene to generate the final product. The mechanism underlying NF phototoxicity involves a photo-defluorination reaction in neutral water (pH=7.2). The fs-TA spectra indicated that the S1 underwent efficient ISC to swiftly transform into lowest excited triplet (T1) The ns-TA gained under nitrogen-saturated condition observed a new transient species produced from T1 that was proposed to be a transient species derived from the photo-defluorination reaction involving a SN2Ar* mechanism. The photo-defluorinated product ultimately experienced an ISC process to produce the final product. The photo-decarboxylation mechanism of TPA was studied in a neutral phosphate buffered solution (PBS). The fs-TA data revealed that S1 went through an efficient ISC to rapidly transform into T1 that then undergoes a photo-decarboxylation reaction to produce a triplet biradical species (denoted as TB3). The ns-TA and ns-TR3 results supplied evidence of the protonation process of TB3 that produces the neutral species (denoted as TBP3) that then decayed through ISC to give rise to the singlet TBP species, which underwent further reaction to make the final product (DTPA). The photo-decarboxylation reaction of Fp was explored in pure acetonitrile (MeCN). The second excited singlet (S2) went through internal conversion (IC) to decay to S1. Intriguingly, three different pathways for S1 decay co-exist. One pathway is fluorescence emission and the second is an ISC process. The third pathway is the homolysis of the carbon α bond reaction that proceeds to generate two radical species, one being a carboxyl species and the other being the residual, denoted as FpR that was liable to be oxidized under an oxygen-saturated condition to yield a new radical species with the addition of one oxygen molecule which is denoted as FOR that then experienced intramolecular hydrogen transfer (IHT) and dehydroxylation (DHO) to produce the final product. / published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

A significant potion of the population in Lesotho relies on traditional medicine to meet its health care requirements. Traditional healers and herbalists were interviewed from Qacha's Nek (Highlands) and Mohale's Hoek (Lowlands) districts in Lesotho on plants used by the Basotho in traditional remedies. Fifteen plants were reported to be used for bacterial infections while thirteen plants were used for diseases associated with inflammation . Plant roots were most often used to make water extracts. Mainly high altitude plants are used with lowland healers obtaining most of their plant material from the highlands, either by collecting them or buying them from highland gatherers. Leaves and roots of plants used to treat bacterial infections were extracted with hexane, methanol and water and the respective extracts screened at 100 mg ml¯¹ for anti-bacterial activity using the disc diffusion bioassay. Seven species displayed very high anti-bacterial activity against both Gram-positive and Gram-negative bacteria. A number of plant extracts had medium inhibitory activity, mostly against Gram-positive bacteria. This activity was mainly found in the root extracts. Six of the thirteen plants screened for anti-inflammatory activity using the cyclooxygenase-1 (COX-1) bioassay had activity above 90%. Hexane and methanol extracts were the most active while water extracts usually had lower activity. Malva parviflora, Eriocephalus punctulatus and Asparagus microraphis exhibited high anti-inflammatory activity from hexane, methanol and water extracts made from leaf and root material. High anti-bacterial activity was also recorded from M. parviflora and E. punctulatus hexane, methanol and water extracts. An investigation on seasonal variation and plant part substitution in medicinal activities for these plants was carried out. Extracts of M. parviflora collected between June 1999 and July 2001 showed variation in anti-bacterial activity. Extracts made from leaves and roots inhibited the growth of both Gram-positive and Gram-negative bacteria. More bacterial strains were inhibited by extracts made from roots collected in cooler months. However, a trend in seasonal activity was not evident for either the roots or leaves because there was no detection of activity in some of the extracts made within the same months or seasons of the adjacent years. Variation in anti-inflammatory was detected for M. parviflora extracts. E. punctulatus leaf extracts did not exhibit any seasonal variation in anti-bacterial activity. Anti-inflammatory activity of E. punctulatus showed seasonal variation with the highest activity noted when material was collected during the cooler months and a decline in activity when collections were made during the warmer months. Hexane, methanol and water extracts made from leaves and roots of A. microraphis did not show any seasonal variation in anti-inflammatory activity. Thus, M. parviflora and E. punctulatus should be collected during the cooler months while A. microraphis can be collected throughout the year. Traditional healers, herbalists and vendors need to be encouraged to use aerial parts in substitution of ground parts which are reported to be highly utilized. Effect of storage on anti-bacterial and anti-inflammatory activities of M. parviflora, E. punctulatus and A. microraphis were monitored. Dried, ground leaf and root material of the three plants was stored in a cold room, at room temperature and in the Botanical Garden where the material was exposed to high and large changes in temperature. Dried hexane and methanol extracts made from leaves and roots of these plants were stored in a cold room and at room temperature. Initially, storage of the plant material under the three storage conditions caused an increase in antibacterial activity of the hexane, methanol and water extracts made from leaf and root material of M. parviflora and E. punctulatus. Storage for a longer period resulted in a decrease in inhibitory activity. TLC fingerprints developed from hexane and methanol extracts made from M. parviflora and E. punctulatus stored in a cold room and at room temperature showed a consistent number and colour of spots during the initial storage period. Prolonged storage resulted in a decline in the number and colour of detected spots. The stored hexane and methanol extracts made from leaves and roots showed a similar trend of increases and decreases in anti-bacterial activity as well as changes in spots with the storage of the extracts. Testing of the effect on anti-inflammatory activity of hexane, methanol and water extracts made from leaves and roots of M. parviflora, E. punctulatus and A. microraphis showed no change in inhibitory activity of hexane extracts obtained from the material and the extracts stored at the three storage conditions. Methanol and water extracts made from leaves exhibited an increase in activity with prolonged storage. Generally, the stability of the inhibitory activity was longer for the stored dried material than the plant extracts. Isolation of biological active compounds from M. parviflora was not successful due to loss in anti-bacterial activity as a result of collection of plant material from a different locality. Anti-inflammatory compounds could not be isolated due to insufficient amount and the synergistic effect of the active compounds . The purified compounds exhibited loss of activity following HPLC purification which then re-appeared upon recombining the fractions. A number of compounds were detected from essential oils of E. punctulatus using GC. Fractions containing these compounds gave positive anti-bacterial activity in the disc-diffusion , bioautographic and MIC bioassays as well as high anti-inflammatory activity with COX-1 and COX-2 anti-inflammatory bioassays. No anti-inflammatory compounds were isolated from A. microraphis. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 2003.