Thesis (PhD)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: The global rise in microbial resistance, ranging from the agricultural industry to the medical sector, has created the urgent need for novel or supplementary antibiotics. Antimicrobial peptides or “nature’s antibiotics” may be the answer to this major problem. In this study a group of antimicrobial peptides, cyclic decapeptides named tyrocidines, produced by the soil bacterium Bacillus aneurinolyticus, was investigated for their antifungal activity, possible mode of antifungal action and potential applications. The study illustrated that the tyrocidines have significant antifungal activity against a range of phytopathogens, including Fusarium solani and Botrytis cinerea, as well as the human pathogen Candida albicans. The activity of the tyrocidines is influenced by the identity of both the target organism and the media environment. Further evidence was obtained in support of the hypothesis that the tyrocidines are extremely sensitive to their environmental conditions and that they tend to self-assemble to form oligomers. The assessment of a small tyrocidine library and analogues, comprised of eight peptides, revealed no overt structure-activity relationships against fungal pathogens, except for the importance of a tyrosine residue. This indicated an important role for the conserved sequence of the tyrocidines, NQYVOLfP, together with the tendency of the tyrocidines to oligomerise into higher-order active structures in their antifungal activity. The tyrocidines were found to be membrane active toward the fungal pathogens. However, supporting evidence was also obtained for additional mode(s) of antifungal action for the tyrocidines which inter alia induces morphological abnormalities in filamentous fungal target cells. Furthermore, the results also indicated that the membrane activity of the tyrocidines may be influenced by additional factors to that of the composition of the target cell membrane, for instance components of the fungal cell wall. This investigation also indicated the significant potential of the tyrocidines to be developed for the commercial sector. The potent activity of the tyrocidines against agronomically important phytopathogens (significantly higher than the commercial fungicide bifonazole) together with their relative salt stability bodes well for their development as bio-fungicides for the agricultural sector. The tyrocidines also exhibited an overt sinergistic effect on the in vitro candidacidal activity of two key antifungal drugs, caspofungin and amphotericin B. Furthermore, tyrocidine A and caspofungin exhibited synergistic activity in vivo which had a significant positive effect on the survival of C. albicans infected Caenorhabditis elegans. Latter results highlighted their potential to serve as candidates for combinatorial treatment in the medical industry. / AFRIKAANSE OPSOMMING: Die globale verskynsel van mikrobiese weerstand, wat strek vanaf die landbou sektor tot in die mediese bedryf, het ’n dringende behoefte vir die ontwikkeling van nuwe antmikrobiese middels geskep. Antimikrobiese peptiede of “die natuur se antibiotika”, kan moontlik die antwoord op hierdie ernstige problem wees. Tydens hierdie studie is ‘n groep sikliese antimikrobiese peptiede, naamlik die tirosidiene wat deur die grondbakterium Bacillus aneurinolyticus geproduseer word, vir hulle antifungiese aktiwiteit, hulle moontlike meganisme(s) van antifungiese werking en hulle potensiёle aanwendings bestudeer. Hierdie studie het getoon dat die tirosidiene uitsonderlike antifungiese aktiwiteit teen ‘n reeks fitopatogene, insluitend Fusarium solani en Botrytis cinerea, asook teen die mens patogeen Candida albicans het. Die aktiwiteit van die tirosidiene is deur beide die identiteit van die teikenorganisme sowel as die mediumomgewing beїnvloed. Daar is ook verdere bewyse verkry wat die hipotese dat tirosidiene uiters sensitief is tot hulle omgewing en dat hulle neig om te oligomeriseer, ondersteun. Die studie van die klein tirosidien-biblioteek, saamgestel uit agt tirosidiene en analoё, het geen ooglopende struktuur-aktiwiteit verwantskappe opgelewer nie, behalwe vir die oёnskynlike invloed van die tirosien-residu. Laasgenoemde het die belangrikheid van die gekonserveerde aminosuurvolgorde van die tirosidiene, NQYVOLfP, asook die neiging van tirosidiene om hoё-orde aktiewe strukture te vorm deur self-verpakking, beklemtoon. Tydens die studie is daar gevind dat die tirosidiene membraan-aktiewiteit toon teenoor fungiese patogene. Daar is egter ook goeie bewyse vir alternatiewe meganisme(s) van antifungiese werking, wat ondermeer tot morfologiese abnormaliteite in filamentagtige fungi-teikenselle lei, vir die tirosidiene verkry. Die resultate het verder ook daarop gewys dat die membraan-aktiwiteit van die tirosidiene ook deur ander faktore, soos deur komponente van die fungiese selwand, en nie net deur die samestelling van die fungiese membraan beїnvloed word nie. Hierdie ondersoek het ook die aansienlike potensiaal van die tirosidiene vir kommersiёle ontwikkeling en gebruik uitgelig. Die merkwaardige aktiwiteit van die tirosidiene teen fitopatogene van agronomiese belang (wat selfs beter as diè van die kommersiёle swamdoder bifonazole was) tesame met die relatiewe sout stabiliteit van die tirosidiene, is belowende tekens om die tirosidiene as bio-swamdoders vir die landbou sektor te ontwikkel. Die tirosidiene het ook ‘n uitgesproke sinergistiese effek op die in vitro candidasidiese aktiwiteit van twee sleutel antifungiese middels, caspofungin en amphotericin B, getoon. Verder is daar in vivo sinergistiese aktiwiteit gewys deur die kombinasie van tirosidien A en caspofungin wat ’n beduidende positiewe effek op die oorlewing van C. albicans geïnfekteerde Caenorhabditis elegans gehad het. Laasgenoemde dui op die potensiaal van die tirosidiene om in die mediese bedryf as kandidate vir kombinasie-behandeling te dien.

Candida species were discovered more than a century ago as a causative organism of oral thrush. In HIV patients, the presence of oral candidiasis has been shown to be the earliest opportunistic infection. Candidiasis lesions associated with HIV infections are primarily a reflection of the specific change of the host's immune response caused by the virus. Studies of AIDS all over the world show that 58-81% of all patients contract a fungal infection at some time during the primordial stage or after developing AIDS and 10-20% have died as a direct consequence of fungal infections. Twenty four South African medicinal plants were screened using a modification of the NCCSL broth microdilution antifungal test against Candida albicans standard strain ATCC 10231 and two clinical isolates from a 5-month- old baby and an adult. This assay was performed in order to find a traditional remedy to treat oral candidiasis. Of all the screened plants Allium sativum L., Glycyrrhiza glabra L., Polygala myrtifolia L. and Tulbaghia violacea L. aqueous extracts were found to have the best activity. Allium sativum and Tulbaghia violacea aqueous bulb extracts had MIC values of 0.56 mgml-1 and 3.25 mgml-1 respectively, whilst Polygala myrtifolia leaf extracts and Glycyrrhiza glabra rhizome extracts had MIC values of 1.56 mgml-1 and 3.25 mgml-1 respectively when tested against the isolate from a 5-month-old baby, which was the most susceptible of the isolates used. All the extracts had higher MIC values against the standard strain (ATTC 10231), which was the least susceptible to the extracts used. Stability testing was performed on fresh aqueous extracts of A. sativum, G. glabra, T. violacea and P. myrtifolia stored at 4°C, 23°C and 33°C over a period of one week, to determine the stability of the extracts in solution. All A. sativum extracts maintained stability for three days in solution, whilst T. violacea extracts remained stable for only two days in solution. TLC fingerprinting of A. sativum and T. violacea extracts indicated the presence of the known antibacterial and antifungal compound allicin. The activity of allicin and other active compounds was observed by using the bioautographic assay, which was performed on these extracts. P. myrtifolia and G. glabra extracts lost stability 24 hours after preparation at all tested temperatures. However, it was clear with the four plant extracts tested that storage of solutions at higher temperatures reduced their activity and stability. The unpleasant taste and smell of A. sativum and G. glabra could however not be masked, since the intake of these two extracts would result in HIV patients being recognised. These two plants where therefore not considered for further investigation. G. glabra and P. myrtifolia are both saponin containing plants. These could be the active constituents responsible for the anticandidal action. G. glabra is known for its biological activity as an antibacterial agent, whilst other Polygala species have been reported to possess antifungal saponins. Although P. myrtifolia and G. glabra are not stable for more than 24 hours, they do not have an unpleasant smell or taste. These plants are therefore further investigated for use as oral mouthwash in clinics and homes. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2003.

Antimicrobial peptides (AMPs) are found throughout the animal kingdom and act as a natural defence against a broad spectrum of pathogens. These peptides are toxic to invading organisms without acting on host cells, so are of interest for their potential to act as potent new drugs against pathogenic organisms. AMPs traverse the cell wall and predominantly target the plasma membrane, resulting in destabilisation, leakage of intracellular components and cell death. In this thesis the mode of action of several AMPs was investigated. The role of the cell wall was studied and found to mediate peptide binding, the inhibition of certain cell wall components also increased peptide action, subsequent internalisation events were observed with varying localisation patterns and the effect of several genes that alter cell susceptibility to AMP were examined. Several Candida albicans mutants, each deficient in cell wall protein mannosylation, were tested in relation to their susceptibility to AMPs. It was discovered that cells lacking or deficient in the phosphomannan fraction, with a concomitant reduction in surface negative charge, correlated with reduced susceptibility to AMP action. To ascertain whether peptide binds to negatively charged phosphate, the effect of exogenous glucosamine 6-phosphate (but not glucosamine hydrochloride) was studied demonstrating that peptide efficacy was reduced due to the presence of exogenous phosphate. More specifically, sequestration of the truncated cationic AMP dermaseptin S3 (DsS3(1-16)) was reduced in these phosphomannan deficient mutants. Microscopy analysis of fluorescein tagged DsS3(1-16) also revealed the differential localisation patterns of this AMP: transiently binding to the plasma membrane, localisation to the vacuole or diffuse distribution throughout the cytoplasm. It is proposed that for these cationic AMPs to exert their full antifungal action they must first bind to the negatively charged phosphate. The echinocandins are a relatively new class of antifungal that function by inhibiting 1,3-β glucan synthase resulting in reduced 1,3-β glucan in the cell wall. As AMPs have to traverse the cell wall it was postulated that cells lacking this fraction would display increased AMP binding to the membrane. Clinical isolate strains of Candida and Cryptococcus spp. were acquired to test their susceptibility to AMP and echinocandin combinations. Comparing the fractional inhibitory concentration index (FICI) (supported by viable cell counts and on a solid surface using disc diffusion assays) synergy was observed between caspofungin, anidulafungin and several AMPs in vitro. In vitro toxicity assays revealed no increase in haemolytic or cytotoxic action on combination. These synergistic combinations could provide a novel treatment against fungal pathogens. The final area of study was based upon work that identified genes whose expression altered cell susceptibility to AMPs. Three genes were selected for investigation that upon deletion increased the action of DsS3(1-16) or magainin 2 on S. cerevisiae. Results from growth analysis, peptide sequestration and cell viability counts confirmed that deletion of HAL5, LDB7 or IMP2’ did increase susceptibility. Additionally, deletion of HAL5 increased the probability of cell depolarisation upon peptide exposure. Expression of GFP-tagged Imp2’ also increased when cells were exposed to DsS3(1-16). It was concluded that deletion of HAL5 increases depolarisation due to insufficient potassium efflux, leading to ion leakage and cell death facilitated by AMP action. Double strand break repair and DNA protection are probably compromised upon deletion of LDB7 and IMP2’, increasing the inhibitory action of DsS3(1-16) that has previously been shown to bind to DNA.

M.Sc.(Nanoscience) / The synthesis and application of gold nanoparticles (AuNPs) has been intensively studied worldwide. However, the toxicity of these nanoparticles is still a concern. We considered that various physiochemical methods used to synthesize AuNPs are energy driven, costly and require the use of harmful chemicals. Thus, this makes them not environmentally-friendly. The aim of this study was therefore to synthesize AuNPs via a greener route using Aspalathus linearis tea leaves. The AuNPs were used to coat eight commercial antifungal discs (i.e. amphotericin B, fluconazole, clotrimazole, econazole, flucytosine, ketoconazole, miconazole and nystatin) against four Aspergillus spp. for enhanced antifungal activity. The aqueous extract of A. linearis was characterized by high performance liquid chromatography and liquid chromatography–mass spectroscopy. The AuNPs were characterized using ultravioletvisible (UV-vis) spectroscopy, dynamic light scattering, nanoparticle tracking analysis, Fourier transforms infrared spectroscopy (FTIR), high-resolution transmission electron microscopy and X-ray diffraction. The toxicity of the synthesized AuNPs was studied by 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay and xCELLigence test on HepG-2 cell lines and results revealed very little to no toxicity of the AuNPs. The pristine antifungal and AuNPs coated antifungal discs were characterized by FTIR, scanning electron microscopy (SEM) and antifungal activity performed using the disc diffusion method. A strong resonance peak was observed at 529 nm of the AuNPs measured using UV-vis spectroscopy. Average size of AuNPs was ~44±1 nm and demonstrated excellent in-vitro stability under various solutions (5% NaCl, phosphate buffered saline) at varying pH levels. The SEM images revealed that the AuNPs were attached onto the coated antifungal discs when compared with the pristine antifungal discs. Antifungal results indicated that AuNPs significantly (p<0.001) enhanced the antifungal activity of the coated antifungal discs against the tested fungi when compared to the pristine antifungal discs. The AuNPs coated econazole disc exhibited the greatest (broad spectrum) activity than other antifungal agents tested. In conclusion, A. linearis can be used as a reducing agent in the synthesis of stable AuNPs. Furthermore, the AuNPs coated antifungal discs demonstrated considerable antifungal activity over the pristine antifungal discs...

Orientador: Antonio Olavo Cardoso Jorge / Banca: Luciane Dias de Oliveira / Banca: Cristiane Aparecida Pereira / Resumo: O número de espécies bacterianas resistentes aos antimicrobianos tem atingido níveis elevados. Com isso, torna-se necessária a realização de pesquisas que avaliem os efeitos de métodos terapêuticos alternativos. Os objetivos desse trabalho foram avaliar a citotoxicidade do extrato glicólico de Zingiber officinale em macrófagos de camundongos e queranócitos humanos, atividade antimicrobiana sobre biofilmes monotípicos (micro-organismos aeróbios: Candida albicans, Staphylococcus aureus, Streptococcus mutans, Pseudomonas aeruginosa, e anaeróbios: Porphyromonas gingivalis, Porphyromonas endodontalis, Prevotella intermedia) e biofilmes heterotípicos (associação: C. albicans e bactérias aeróbias). Para ação citotóxica, as células foram cultivadas em meio DMEM, semeadas (2 x 104 células/poço) na placa de 96 poços. Após aderência inicial, foi adicionado o extrato em diluição seriada (5 min e 24 h), e realizado o teste de MTT. Para avaliar a ação antimicrobiana, a Concentração Inibitória Mínina (CIM) e Concentração Microbicida Mínima (CMM) foram determinadas segundo as normas do CLSI. Para biofilmes, foram adicionados 100 µL meio de cultura e 100 µL suspensão microbiana (107 UFC/mL) em placas de 96 poços, nos heterotípicos foram utilizados 50 µL de cada micro-organismo. As placas foram incubadas (37ºC), por 48 h (aeróbios) ou por 7 dias (anaeróbios). Aplicou-se o extrato (5 min e 24 h), nas concentrações efetivas pré-determinadas (CMM) e duas superiores, depois os biofilmes foram desa... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The number of bacterial species resistant to antimicrobials has reached high levels. Thus, it is necessary to perform research that evaluates the alternative therapeutic methods, such as ginger. The present study was to evaluate the cytotoxicity of the Zingiber officinale glycolic extract in human and mouse macrophages, antimicrobial activity on monotypic biofilms (aerobic microorganisms: Candida albicans, Staphylococcus aureus, Streptococcus mutans, Pseudomonas aeruginosa, and anaerobes: Porphyromonas gingivalis, Porphyromonas endodontalis, Prevotella intermedia) and heterotypic biofilms (association: C. albicans and aerobic bacteria). To cytotoxic action, the cells have been cultivated in DMEM medium, seeded (2x104 cells/well) in the 96-well plate. After initial adhesion, the extract has been added to serial dilution (5 min and 24 h), and the MTT tests were performed. In order to assess the antimicrobial action, Minimal Inhibitory Concentration (MIC) and Minimum Microbicidal Concentration (MMC) were determined in according to CLSI standards. In the biofilms case, 100 μL culture medium and 100 μL microbial suspension (107 CFU / mL) were added to 96-well plates, 50 μL of each microorganism was used in the heterotypics. The plates were incubated (37°C) for 48 h period (aerobic) or for 7 days (anaerobic). The extract (5 min and 24 h) was applied at the pre-determined effective concentrations (MMC) and two higher concentrations, after which the biofilms were disaggregated and se... (Complete abstract click electronic access below) / Mestre

"Fungal infections have large implications in agriculture and medicine, and there are few interventions available in the form of antifungal agents due to their toxicity to the host. Saccharomyces cerevisiae is an excellent model for pathogenic fungi because it is a well-studied, tractable organism and shares some traits with pathogenic fungi. Like most pathogenic fungi, S. cerevisiae is dimorphic and transitions from the benign yeast form to a filamentous form in which it produces psuedohyphae. Finding novel routes of suppressing dimorphic transition in a model like S. cerevisiae could lead to the discovery of new antifungal agents. Recently, quorum-sensing mechanisms have been under investigation as new avenues for microbial control. Quorum sensing is a signaling phenomenon that is well described in bacteria. It is the regulation of gene expression in response to cell density via the accumulation of small signaling molecules in the immediate environment. Indole-3-acetic acid (IAA) demonstrates some of the criteria for being a quorum-sensing molecule in S. cerevisiae. The purpose of this thesis was to further explore IAA as a quorum-sensing molecule in S. cerevisiae by demonstrating IAA production by the organism in culture. Radio-labeled tryptophan incorporation experiments followed by Thin Layer Chromatograph (TLC) analysis demonstrated that IAA is produced in culture by S. cerevisiae. A screen of a commercially available gene deletion library using the radio-labeled trypophan incorporation assay identified genes implicated in the IAA biosynthetic pathway. Some of these genes are homologous to those in an IAA pathway in the fungus Ustilago maydis. Further investigation of deletion strains of these candidate genes shows that Ald2 and Ald3, two aldehyde dehydrogenases, are involved in IAA production. The double mutant, ald2∆ald3∆, makes less IAA than wild type and is unable to demonstrate haploid invasive growth. This supports the idea that IAA biosynthesis in S. cerevisiae is necessary for morphological transition and that IAA could serve as a quorum-sensing molecule in S. cerevisiae with dimorphic transition as the quorum-sensing phenotype."

Orientador: Tarcisio José de Arruda Paes Junior / Coorientador: Fernando Cristovan / Banca: Lafayette Nogueira Júnior / Banca: João Paulo Barros Machado / Resumo: O objetivo deste trabalho foi sintetizar e caracterizar nanopartículas de sílica recobertas por prata, e avaliar a influência quando incorporadas à resina acrílica quimicamente ativada, ao reembasador macio, e a um glaze, em suas características microbiológicas, microestruturais e mecânicas. Foi confeccionada, pelo método de hidrólise e condensação controlada (método de Stober), uma solução contendo nanopartículas de sílica revestidas por nanopartículas de prata em duas proporções, 10 milimols e 30 milimols, onde as mesmas foram caracterizadas. As nanopartículas de sílica incorporadas com a prata foram analisadas por energia dispersiva de raios-X integrado (EDS), microscopia eletrônica de varredura (MEV), e espalhamento de luz dinâmico (DLS). Para análise microbiológica foram confeccionadas 10 amostras cilíndricas (2mmx10mm), em resina acrílica quimicamente ativada onde as partículas foram incorporadas em duas concentrações: 2.5% e 5% com as duas molaridades diferentes 10 mM e 30 mM. Em outra situação foram confeccionadas 10 amostras cilíndricas (2mmx10mm) de reembasador macio, com concentrações de 2,5% e 5% com as duas molaridades diferentes 10 mM e 30 mM, no terceiro caso as partículas foram acrescentadas a um glaze nas duas concentrações e molaridades e aplicada sob uma amostra de reembasador macio. Uma suspensão de Candida albicans e Estreptococos mutans foi utilizada para análise de concentração inibitória mínima. Amostras retangulares em resina acrílica (n=6) de 30x10x3... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The aim of this work was to synthesize and characterize silver - coated silica nanoparticles, and to evaluate the influence when incorporated into chemically activated acrylic resin, soft reliner, and glaze, in their microbiological, microstructural and mechanical characteristics. A solution containing silica nanoparticles coated by silver nanoparticles in two proportions, 10 millimols and 30 millimols, where they were characterized, was prepared by the hydrolysis and controlled condensation method (Stober method). Silica nanoparticles incorporated with silver were analyzed by integrated X-ray dispersive energy (EDS), scanning electron microscopy (SEM), and dynamic light scattering (DLS).For the microbiological analysis, 10 cylindrical samples (2mmx10mm) were made in chemically activated acrylic resin where the particles were incorporated in two concentrations: 2.5% and 5% with two different molarities of 10 mM and 30 mM. In another situation, 10 cylindrical samples (2mmx10mm) of soft reliner were made, with concentrations of 2.5% and 5% with the two different molarities 10 mM and 30 mM, in the third case the particles were added to a glaze in the two concentrations and molarities and applied under a sample of soft reliner. A suspension of Candida albicans and Streptococcus mutans was used for analysis of minimal inhibitory concentration. The samples were made in acrylic resin (n = 6) of 30x10x3mm and were made to perform the three-point flexural strength test of EMIC (Model ... (Complete abstract click electronic access below) / Mestre

Orientador: Rosemeire Cristina Linhari Pietro / Banca: Sérgio Ricardo Ambrósio / Banca: Ana Helena Januário / Banca: Jairo Kenupp Bastos / Banca: Tais Maria Bauab / Resumo: Produtos naturais de fungos constituem-se em fontes promissoras de novas moléculas de alta diversidade estrutural e amplo espectro de atividades biológicas, particularmente quando provenientes de organismos que ocupam nichos ecológicos específicos e/ou originários de habitats complexos. O presente trabalho objetivou a prospecção química e biológica de fungos oriundos de associações ecológicas consideradas como altamente promissoras e ainda pouco exploradas para a busca de metabólitos secundários bioativos. Realizou-se isolamento de fungos endofíticos de Piper umbellatum (Piperaceae), o qual resultou em 6 diferentes cepas fúngicas. Estes fungos, assim como duas diferentes espécies de fungos derivados de ambiente marinhos associados à Ascídias foram cultivados em meio líquido e obtidos extratos que foram avaliados em ensaios de atividade antibacteriana e antifúngica. Os extratos provenientes das 6 cepas de fungos endofíticos isoladas, assim como os fungos derivados de ambiente marinho, apresentaram moderada atividade antibacteriana ou antifúngica. De acordo com os resultados destes ensaios de atividade biológica, as duas cepas de fungos derivados de ambiente marinhos associados à ascídias foram selecionadas para cultivos em escala ampliada para obtenção e isolamento de metabólitos secundários bioativos. Cinco substâncias foram isoladas dos extratos dos fungos associados á ascídias e estas substâncias foram submetidas a análises espectroscópicas para elucidação de suas estrutura... (Resumo completo, clicar acesso eletrônico abaixo) / Doutor

Introdução: Dados sobre a relação entre a farmacocinética do fluconazol (FCZ) e anfotericina B (AMB) no líquido cefalorraquidiano (LCR) e a hipertensão intracraniana (HIC) não se encontram disponíveis na literatura. Objetivos: Avaliar a influência da pressão intracraniana na concentração dos antifúngicos AMB e FCZ no LCR de pacientes com meningite criptococócica internados no Hospital de Clínicas de Porto Alegre (HCPA), no período de 1 ano. Métodos: Foram estudados 15 pacientes com meningite criptococócica durante os primeiros 14 dias de tratamento com AmB (1 mg/kg/dia) e FCZ (800 mg/dia). As amostras de LCR foram obtidas por meio de punções lombares de rotina realizadas nos dias 1, 7 e 14 da terapia antifúngica, respectivamente. Os valores das pressões intracranianas de abertura foram obtidos no momento de cada punção lombar. Os níveis de AmB e FCZ no LCR foram medidos pela metodologia de cromatografia líquida de alta performance (HPLC). A concentração inibitória mínima (CIM) para AmB, FCZ, voriconazol (VRZ) e flucitosina (5-FC) de cada isolado de Cryptococcus sp. foi realizada de acordo com o as normas descritas no documento M27-A3, do CLSI, publicado em 2008. Resultados: Entre os 15 pacientes incluídos no estudo, C. gattii foi isolado do LCR de 2 pacientes e C. neoformans foi isolado do LCR de 13 pacientes apresentaram. A condição de imunossupressão encontrada foi a AIDS, seguida de transplante de órgão sólido. Nove pacientes apresentaram cultura negativa de LCR no 14º dia de terapia antifúngica. Os níveis de AmB no LCR foram indetectáveis para a maioria das amostras de LCR durante os 14 dias de terapia antifúngica. Os níveis de FCZ no LCR aumentaram progressivamente do dia 1 ao dia 14 de terapia. Seis pacientes apresentaram HIC no dia 1, com variação da pressão de abertura entre 100 mmH2O e 650 mmH2O no respectivo dia. A pressão intracraniana não interferiu nas concentrações de FCZ no LCR. Não observamos correlação entre a HIC e as concentrações de AMB e FCZ no LCR de acordo com a correlação de Spearman (Spearman p= 0.122). Conclusão: São necessários mais estudos para avaliar o papel da HIC na eficácia terapêutica de diferentes agentes antifúngicos em pacientes com meningite criptococócica. / Introduction: Data considering the relationship between pharmacokinetics of fluconazole (FCZ) and amphotericin B (BPA) in cerebrospinal fluid (CSF) and intracranial hypertension (IH) are not available in the literature. Objectives: To evaluate the influence of intracranial pressure on the concentration of the antifungals AMB and FCZ in the CSF of patients with cryptococcal meningitis admitted at the Hospital de Clínicas de Porto Alegre (HCPA), within a period of one year. Methods: Fifteen patients with cryptococcal meningitis were studied during the first 14 days of treatment with AmB (1 mg / kg / day) and FCZ (800 mg / day). CSF samples were obtained by means of routine lumbar punctures performed on days 1, 7 and 14 of antifungal therapy, respectively. The values of intracranial opening pressures were obtained at the time of each lumbar puncture. The levels of AmB and FCZ in the CSF were measured by high performance liquid chromatography (HPLC) methodology. The minimum inhibitory concentration (MIC) for AmB, FCZ, voriconazole (VRZ) and flucytosine (5-FC) of each isolate of Cryptococcus sp. was performed according to CLSI guideline M27-A3, published in 2008. Results: Among the 15 patients included in the study, C. gattii was isolated from the CSF of 2 patients and C. neoformans was isolated from the CSF of 13 patients presented. The immunosuppressive condition found was AIDS, followed by solid organ transplantation. Nine patients presented negative CSF culture on the 14th day of antifungal therapy. AmB levels in the CSF were undetectable for most of the CSF samples during the 14 days of antifungal therapy. CSF FCZ levels increased progressively from day 1 to day 14 of therapy. Six patients presented IH on day 1, with variation of the opening pressure between 100 mmH2O and 650 mmH2O on the respective day. Intracranial pressure did not interfere with CSF on FCZ concentrations. We did not observe a correlation between IH and the concentrations of AMB and FCZ in the CSF according to the Spearman correlation (Spearman p = 0.122). Conclusion: Further studies are needed to evaluate the role of IH in the therapeutic efficacy of different antifungal agents in patients with cryptococcal meningitis.

Encapsulation is a novel technique that involves the entrapment of materials such as cells, enzymes or chemicals within a semi-permeable matrix and is being explored as a drug delivery system. This project investigated the encapsulation of Streptomyces nodosus in alginate to assess whether this organism can produce the antifungal drug amphotericin B from within the matrix. New methods were developed to immobilise S. nodosus mycelia and spores in alginate capsules, assess bacterial viability and detect ng mL–1 quantities of amphotericin B in culture fluids. When capsules were cultured and cell proliferation was encouraged, organisms formed protrusions on the surface of the capsules. Differentiated branched hyphae that never progressed to sporogenic hyphae were observed on the surface of these structures. Viability was maintained for up to 30 days and low levels of amphotericin B were produced. The emergence of a co-existing free-dwelling population was also observed. Culturing immobilised organisms using conditioned media from an amphotericin deficient S. nodosus strain, augmented the development of the free-dwelling population resulting in the detection of amphotericin B in the culture fluid and full differentiation to sporogenic hyphae. This is the first report of sporulation of S. nodosus in liquid environments and demonstrates that immobilised S. nodosus can produce antibiotics. The sporulation of free-dwelling organisms was also induced using conditioned media and manipulation of quorum size, indicating a solid surface is not required for sporulation. Conditioned media from other Streptomyces spp. induced variable responses including sporulation, pigment formation and antibiotic production, possibly demonstrating communication between species and/or alteration in nutritional status. This new model for the life cycle of S. nodosus will permit the study of developmental pathways, antibiotic production, microbial community structure and inter-species and intra-species signalling. / Doctor of Philosophy (PhD)