The incidence of CF related diabetes is on the rise as patient life expectancy continues to improve. Sugars elevated in diabetics include glucose, fructose, and mannose. These sugars, in addition to mannitol (recently approved as an inhaled osmolyte) are the basis for this study, aimed at assessing the impact these clinically relevant sugars have on virulence in Burkholderia multivorans. B. multivorans is a member of the Burkholderia cepacia complex (Bcc), and is the most frequent cause of Bcc infection in CF patients. Using an exopolysaccharide-deficient knockout in macrophage and Galleria mellonella infection models, biofilm formation, and adhesion assays, this study has identified exopolysaccharide-dependent and -independent phenotypes. Sequencing of B. multivorans C1576, a CF outbreak isolate, identified three putative adhesins in clinical isolate C1576 but not present in the sequenced environmental strain ATCC17616. Mannitol promoted adhesion and enhanced expression of these adhesins. This study characterised these adhesins and assessed the distribution within other clinical and environmental isolates of B. multivorans and the Bcc. Additionally, transcriptomic profiling of B. multivorans assessed the sugar response and EPS regulation during growth on clinically relevant sugars. Where possible, links were made between phenotypic studies and transcriptome data. B. multivorans EPS derived from fructose and mannitol was subjected to composition analysis using mass spectrometry, and assessed for biological activity. Still relevant to CF related diabetes, the ability of some members of the Bcc to bind insulin was assessed. Results indicated that a minority of strains bound insulin. Furthermore, by using flow cytometry cell sorting and fluorescence microscopy, results also showed only a small number of cells within a given population that bound insulin. In all, this study has added to the knowledge base of B. multivorans but more work is needed to fully understand virulence strategies exploited by this CF pathogen.

Submitted by Mario BC (mario@bc.ufrpe.br) on 2017-03-10T13:49:44Z No. of bitstreams: 1 Maria Jacyelle dos Santos Muniz.pdf: 928440 bytes, checksum: 0a22b8f42ff3831d58110079cdfe6edd (MD5) / Made available in DSpace on 2017-03-10T13:49:44Z (GMT). No. of bitstreams: 1 Maria Jacyelle dos Santos Muniz.pdf: 928440 bytes, checksum: 0a22b8f42ff3831d58110079cdfe6edd (MD5) Previous issue date: 2016-03-16 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Bacteria Plant Growth Promoting (BPGP) has been studied as environmental remediation, as many agricultural soils are contaminated by residue of pesticides. Among these bacteria are Burkholderia and Pseudomonas, which have the ability to grow in different environments, having the ability to conserve the environment through their activity to biodegrade polluting compounds. Thus, the objective of this study was to evaluate the potential of tolerance and degradation of bacteria Burkholderia sp. UAGC867 and Pseudomonas sp. UAGC97, in relation to the cypermethrin and methomyl insecticides, used to control Spodoptera frugiperda; the respirometric rate ground when influenced with the cypermethrin insecticide and bacterial strains; the effect of cypermethrin on the indole acetic acid production by BPGP and the influence of bacterial inoculation with cypermethrin in corn seeds. For the tolerance test inrelation to cypermethrin and methomyl insecticide , the strains UAGC867 and UAGC97 were cultivated in mineral liquid medium plus glucose and each treatment received 25-50-100 and 200 mg L-1 of each insecticide. The treatment control was free insecticide. Tha degradation test of insecticides by bacteria followed the same methodology, differing only in the absence of glucose treatments. The influence of bacterial inoculum and concentrations different of cypermethrin (0-25-50-100-200 mg L-1) in the soil, the experiment was conducted in microcosms installed in a greenhouse, and later collected 100 g ground and transferred to pots in order to evaluate the respiration rate of the soil in the laboratory. It was also evaluated the production of indole acetic acid (IAA) by PBPG, through the influence of cypermethrin insecticide in the field concentration. Finally, the strains Burkholderia sp. UAGC867 and Pseudomonas sp. UAGC97 were cultured in the presence and absence of cypermethrin (100 mg L-1) to evaluate the germination and growth of corn seed. The two bacteria evaluated were able to express growth in the presence of pesticides concentrations, demonstrating their tolerance for the same. Burkholderia sp. UAGC867 was able to degrade all methomyl concentrations, and 25 mg L-1 of the insecticide cypermethrin. Pseudomonas sp. UAGC97 exhibited degradation only in 25 mg L-1 methomyl insecticide. There carbon dioxide captured oscillations throughout the experiment, however, was not observed between treatments unclustering by principal component analysis. The IAA synthesis by BPGP was not inhibited by the insecticide cypermethrin, however, there was prominent production by Pseudomonas sp. UAGC97 on the seventh day of evaluation. There was no statistical difference between treatments evaluated in the germination speed index and the germination percentage. The assessments of plant growth promotion revealed that the cultivation of Burkholderia sp. UAGC867 in the presence of 100 mg L-1 cypermethrin stimulated the growth of shoots of corn seedlings, in addition to presenting significant results in fresh and shoot dry. The evaluated bacteria exhibit promoting characteristics of plant growth, with potential for more exploration of their interaction with the plant. / O uso de Bactérias Promotoras de Crescimentos em Plantas (BPCP) vem sendo estudado quanto a remediação ambiental, visto que muitos dos solos agrícolas estão contaminados pelo resíduo dos pesticidas. Dentre tais bactérias, estão os gêneros Burkholderia e Pseudomonas, que possuem a capacidade de crescer em variados ambientes, possuindo habilidade de conservar o meio ambiente, através de sua atividade em biodegradar compostos poluentes. Sendo assim, o objetivo do presente trabalho foi avaliar o potencial de tolerância e degradação das bactérias Burkholderia sp. UAGC867 e Pseudomonas sp. UAGC97, quanto aos inseticidas cipermetrina e metomil utilizados no controle da Spodoptera frugiperda; avaliar a taxa respirométrica do solo quando influenciada com o inseticida cipermetrina e as linhagens bacterianas; avaliar a influência da cipermetrina sobre a produção do ácido indol acético pelas BPCP e avaliar a influência da inoculação bacteriana, cultivada com cipermetrina, em sementes de milho. Para o teste de tolerância aos inseticidas cipermetrina e metomil pelas bactérias Burkholderia sp. UAGC867 e Pseudomonas sp. UAGC97, foi utilizado o meio mínimo mineral liquido modificado acrescido de glicose, onde cada tratamento recebeu 25-50-100 e 200 mg L-1 de cada inseticida, além do tratamento controle, insento de inseticida. Para o teste de degradação dos inseticidas cipermetrina e metomil pelas bactérias avaliadas, seguiu-se a mesma metodologia, diferindo apenas da ausência da glicose nos tratamentos. Para o teste da influência do inóculo bacteriano e das diferentes concentrações de cipermetrina (0-25-50-100-200 mg L-1) no solo, o experimento foi conduzido em microcosmos instalados em casa de vegetação, sendo posteriormente coletados 100g do solo e transferidos para potes afim de avaliar a taxa respiratória do solo, em laboratório. Também foi avaliado a produção do ácido indol acético (AIA) pelas BPCP, através da influência do inseticida cipermetrina na concetração de campo. E por fim, as bactérias Burkholderia sp. UAGC867 e Pseudomonas sp. UAGC97, foram cultivadas em meio na presença e na ausência do cipermetrina (100 mg L-1) afim de serem utilizados na avaliação de germinação e desenvolvimento do milho, sendo inculadas em sementes. As duas bactérias avaliadas conseguiram expressar crescimento na presença das concentrações dos inseticidas, demonstrando sua tolerância quanto ao mesmo. A Burkholderia sp. UAGC867 degradou o metomil em todas as concentrações, e mostrou degradação em 25 mg L-1, do inseticida cipermetrina,pelo método avaliado. Já a Pseudomonas sp. UAGC97 exibiu degradação apenas na concentração 25 mg L-1 do inseticida metomil. Houve oscilações de dióxido de carbono capturado entre os tratamentos avaliados, no entanto, não foi observado desagrupamenteo entre os tratamentos através da análise de componentes principais. A síntese do AIA produzido pelas BPCP, não foi inibido pelo inseticida cipermetrina, contudo, houve destaque da produção de AIA pela Pseudomonas sp. UAGC97 no sétimo dia de avaliação. Não houve diferença estatística entre os tratamentos avaliados no índice de velocidade de germinação e na percentagem de geraminação, contudo, nas avaliações de promocação de crescimento vegetal observou que o cultivo da Burkholderia sp. UAGC867 na presença de 100 mg L-1 cipermetrina estimulou o crescimento da parte aérea das plântulas de milho, além de apresentar resultados significativos na massa fresca e seca da parte aérea. As bactérias avaliadas exibem características de promoção de crescimento vegetal, apresentando potencial para exploração mais minunciosa da sua interação com a planta.

The loss of rice plants (Oryza sativa L.) due to damage caused by abiotic stresses (water deficit and allelopathy) are recurrent, the objective was to evaluate the behavior of rice plants through agronomic and morphological changes under different layers of water in the soil (LAS) of 100%, 70%, 50% and 30% of field capacity (CC), and to identify the anatomical and physiological changes in plant in LAS 100% and 50% of CC, Induced by PGPR (Burkholderia pyrrocinia BRM-32113 and Pseudomonas fluorescens BRM-32111), that indicate the mitigating effect of the damages caused by the water deficit. And in plantings with residue of rice plants in the soil was to identify and understand the effect of the application of rhizobacteria on highland rice plants in consecutive plantations. Test one, The PGPR were subjected to temperature abiotic stresses (30, 35, 40 oC), salinity (0.5%, 7.5%), and water deficit (0, -0.2, - 0.4, -0.6, -0.8, -1.0, -1.2 Mpa) simulated by PEG with 10 replicates and evaluated the growth. Test two, the rice plants inoculated with: BRM-32111, BRM-32113 and control, were submitted the LAS of: 100, 70, 50 and 30% of CC, with three pots per treatment and five plants per pot. were evaluated: evapotranspirated water and water potential (ψam), biomass and length of plant and root, leaf area and relative chlorophyll content. Test three, plants Control, inoculated with BRM-32111 and BRM-32113, submitted to 100% and 50% of CC, and maintained for 28 days, and evaluated growth, physiology and anatomy. Test four, were used four treatments consisting of seed rice inoculated with P. fluorescens BRM-32111 in soil with residue, B. pyrrocinia BRM-32113 in soil with residue, plants control in soil with residue (CR)and plants control on soil without residue (SR) roots of plants rice (residues of Allelochemicals), the essay were in (DIC), and results submitted to ANOVA, Duncan's test (p <0.05). Test one, It was verified that PGPR were tolerant to abiotic stresses of: salinity (80%), temperature (96%) and dry (96%), test two, plants have reached the critical LAS of up to 63% of CC, plants with BRM-32111 had the effect of reducing the damage caused by the water deficit, In biomass 30%, root length 88% in LAS of 30% CC when compared to control plants. Test three, The seeds with BRM-32111 differed from the control in germination, and with BRM 32113 differed from the control in IVG. The plants inoculated with BRM 32111 and BRM32113 had a smaller reduction in root diameter, number of protoxilema pores, cortex thickness, and increase in density of stomata, and increase in carbon assimilation rate (A), efficiency of water use (EUA), carboxylation efficiency rubisco (A / Ci), higher accumulation of chlorophyll a, proline and reduction in the concentration of malonic aldehyde (MDA), in relation to control plants submitted to the same LAS. Test four, the growth of seedlings and plants control rice (CR) was negatively affected by allelopathic compounds. In sowing with residue, plants inoculated with rhizobacteria P. fluorescens BRM-32111 and B. pyrrocinia BRM-32113 induced an increase of 88% in biomass, 3% in the leaf area, 40% and 67% in length and root biomass, respectively, 21% in chlorophyll a, 50% in A, and 63% in the EUA compared to CR control plants CR. These results, evidences that the rhizobacteria are tolerant to the temperature stresses, salinity and osmotic pressure and help to alleviate the harmful effect caused by the water deficit, and increase tolerance of rice plants to stress with allelochemicals in upland rice. / A perda na produtividade de plantas de arroz (Oryza sativa L.) devido aos danos causados por estresses abioticos (déficit hídrico e alelopatia) são recorrentes, o objetico foi avaliar o comportamento de plantas de arroz através das alterações agronômicas e morfológicas sob diferentes lâminas de água no solo (LAS) de 100%, 70 %, 50% e 30 % da capacidade de campo (CC), e identificar as modificações anatômicas e fisiológicos em planta em LAS de 100 % e 50 % da CC induzidas por PGPR (Burkholderia pyrrocinia BRM-32113 e Pseudomonas fluorescens BRM-32111), que indiquem o efeito mitigador dos danos causados pelo déficit hídrico. E em plantios com resíduo de plantas do arroz no solo foi identificar e compreender o efeito da aplicação de rizobactérias em plantas de arroz de terras altas em plantios consecutivos. No primeiro ensaio, as PGPR foram submetidas aos estresses abióticos de temperatura (30, 35 e 40 oC), salinidade (0.5 % e 7.5%) e déficit hídrico (0, -0.2, -0.4, -0.6, -0.8, -1.0 e -1.2 Mpa) simulado por PEG com 10 repetições e avaliado o crescimento. No segundo ensaio, as plantas de arroz inoculadas com: BRM-32111, BRM-32113 e controle, foram submetidas as LAS de: 100, 70, 50 e 30 % da CC, com três vasos por tratamento e cinco plantas por vaso. Foram avaliados: água evapotranspirada e potencial hídrico (ψam), biomassa e comprimento da planta e raiz, área foliar, teor relativo de clorofila. No terceiro ensaio, foram usadas plantas controle, inoculadas com BRM-32111 e BRM-32113, submetidas a 100% e 50% da CC, e mantido até aos 28 dias, e avaliado crescimento, fisiologia e anatomia. No quarto ensaio, foram utilizados quatro tratamentos constituídos de sementes de arroz inoculados com P. fluorescens BRM32111 em solo com resíduo, com B. pyrrocinia BRM-32113 em solo com resíduo e plantas controle em solo com resíduo (CR) e plantas controle em solo sem resíduo (SR) de raízes de plantas de arroz (resíduos de Aleloquímicos), todos os ensaios foram em DIC, e os resultados submetidos ANOVA, teste de Duncan (p < 0.05). No primeiro ensaio, verificou - se que as PGPR foram tolerantes aos estresses abióticos de: salinidade (80%), temperatura (96%) e seca (96%), no segundo ensaio, as plantas atingiram a LAS crítica de até 63 % da CC, as plantas com BRM-32111 tiveram os efeitos amenizador aos danos causado pelo déficit hídrico, na biomassa 30%, comprimento da raiz 88% em LAS de 30% de CC quando comparados a plantas controle. No terceiro ensaio, as sementes com BRM-32111 diferiram do controle na germinação, e com BRM 32113 diferiram do controle no IVG. As plantas inoculadas com BRM 32111 e BRM-32113 tiveram menor redução no diâmetro radicular, número de poros de protoxilema, espessura do córtex, e aumento na densidade de estômatos, e aumento em taxa de assimilação de carbono (A), eficiência do uso da água (EUA) e eficiência de carboxilação da rubisco (A / Ci), maior acumulo de clorofila a, prolina e redução na concentração de aldeído malônico (MDA), em relação a plantas controle submetidas a mesma LAS. No quarto ensaio, o crescimento de plântulas e plantas de arroz controle (CR) foi afetado negativamente pelos compostos alelopáticos. Em semeio com resíduo, plantas inoculadas com as rizobactérias P. fluorescens BRM-32111 e B. pyrrocinia BRM-32113 induziram aumento em 88% na biomassa, 3% na área foliar, 40% e 67% no comprimento e biomassa radicular, respectivamente, 21% na clorofila a, 50% na A, e 63% no EUA comparado as plantas controle CR. Estes resultados, evidenciam que as rizobactérias são tolerantes aos estresses de temperatura, salinidade e pressão osmótica e auxiliam na amenização do efeito danosos causados pelo déficit hídrico, e aumentam a tolerância de plantas de arroz ao estresse com aleloquímicos no arroz de terras altas

Melioidosis is a neglected tropical disease that continues unabated in many countries, particularly in Southeast Asia. There is no vaccine and antimicrobial treatment is expensive and complicated. Virulence models are important tools used to investigate genes involved in pathogenesis. Galleria mellonella is the larvae of the wax worm moth that has been used to model various infections. Based on previous studies, we attempted to establish an infection model using Burkholderia pseudomallei and Burkholderia thailandensis, a related species which is avirulent in humans. Injections of various forms of these species (fresh and frozen) were used to develop Kaplan-Meier plots. We also tested Burkholderia cepacia, Burkholderia vietnamiensis, Burkholeria ambifaria, and Burkholderia multivorans to understand how they affect the larvae. We found that larvae injected with B. pseudomallei and B. thailandensis did not accurately model the respective infections these species cause in humans, while the other non-virulent species did not produce disease, as expected. We conclude that G. mellonella is not an appropriate infection model for B. pseudomallei and B. thailandensis. Healthcare-associated infections (HAI) are on the rise, and place a heavy burden on our healthcare system each year. Disinfectants used in healthcare settings can reduce HAIs, but first must be evaluated for proper efficacy. To date there are few statistical models that are useful in comparing disinfectant test methods. We conducted a head-to-head comparison of two common test methods, suspension and surface, using Clostridium difficile spores as the test organism. A novel statistical method was developed to evaluate which test method better predicted disinfectant performance. An activated disinfectant that gradually lost activity over time was used in these evaluations. Results showed that the suspension test method was less variable, and was a better predictor of disinfectant efficacy over time.

Recent work has shown that microorganisms are surprisingly like animals in having sophisticated behaviours such as cooperation, communication, and recognition, as well as many kinds of symbioses. Here we show first that the social amoeba Dictyostelium discoideum has a primitive farming symbiosis that includes dispersal and prudent harvesting of the crop. About one-third of wild-collected clones engage in husbandry of bacteria. Instead of consuming all bacteria in their patch, they stop feeding early and incorporate bacteria into their fruiting bodies. They then carry bacteria during spore dispersal and can seed a new food crop, which is a major advantage if edible bacteria are lacking at the new site. However, if they arrive at sites already containing appropriate bacteria, the costs of early feeding cessation are not compensated, which may account for the dichotomous nature of this farming symbiosis. We also observed farmer Dictyostelium discoideum clones carry bacteria that they do not use as food. We hypothesized that these bacteria may play a defensive role against other D. discoideum clones. In our second study, we investigated the impact of these bacteria-carrying farmers on non-farming D. discoideum clones. We found that the presence of farming clones reduces spore production in non-farmers. Furthermore, this effect increases with frequency of farming clones, demonstrating the vulnerability of non-farming clones to farmers though in this experiment we had not separated the effects of the farmer clone and the bacteria they carry. In our third study we exposed non-farmers to a filtered supernatant from the most common non-food carried bacterium, Burkholderia xenovorans . This supernatant is likely to carry whatever the bacteria are producing. We treated Dictyostelium clones at the beginning of the social stage and found that the supernatant enhanced spore production of farming clones and hurt spore production of non-farming clones. This study shows that the effects of the bacteria can be restricted to a filtered supernatant alone. This discovery of symbiosis of D. discoideum with bacteria, and its impact on social interactions among D. discoideum clones will provide a fertile ground for further experiments on the evolution of sociality.

El problema que trae aparejado la falta de un diagnóstico certero y rápido de los patógenos respiratorios en pacientes fibroquísticos tiene un fuerte impacto en el tratamiento antimicrobiano, en el control de infecciones y principalmente en la calidad de vida y sobrevida de estos pacientes. En el caso particular de los organismos del complejo Burkholderia cepacia, que colonizan a estos pacientes, son ampliamente reconocidas las dificultades y limitaciones de los métodos bioquímicos tradicionales para su identificación. En los últimos años se han puesto muchos esfuerzos en desarrollar nuevos métodos de identificación rápidos, sencillos y de bajo costo que permitan la diferenciación e identificación inequívoca de bacilos no fermentadores, pertenecientes y no pertenecientes al complejo Burkholderia cepacia. Este trabajo de tesis fue concebido con la idea de avanzar en el conocimiento general sobre las técnicas de identificación, las características genotípicas y fenotípicas de organismos del complejo B. cepacia y a su vez resolver problemas de diagnóstico del sector hospitalario y epidemiológicos de nuestro país.

Inhibition of Fusarium oxysporum f. sp. cubense race 4 by Burkholderia cepacia was evident when grown on various media (TSA, PDA, PSA, YM, KMB, PPM, NYGA, LA) with different carbon sources and under various pH and temperature conditions. In addition, B. cepacia was able to inhibit several fungal pathogens in vitro. Antagonism of B. cepacia against F. oxysporum f. sp. cubense occured at high levels of Fe³+, which may suggest that antagonism by B. cepacia did not involve siderophore production. Thin layer chromatogram (TLC) examination showed that B. cepacia produced several substances, one of which had similar R[f] value to that described for pyrrolnitrin. Cell-free supernatant of a 4-day culture of 6. cepacia was applied to an Amberlite XAD-2 column and inhibitory activity co-eluted with the 95% methanol (pH 9.5) fraction. The concentrated activated fractions showed inhibitory activity against F. oxysporum f. sp. cubense. A GC-MS chromatogram indicated numerous components in the antifungal extracts. The only compound identified in the Wiley 138 library, was 1,2- Benzenedicarboxylic acid, bis (2-Ethylhexyl) ester. Observations by light microscopy indicated that B. cepacia inhibited spore germination in F. oxysporum f. sp. cubense race 4 and retarded the mycelial growth. The interaction between the endophytic bacterium, B. cepacia and F. oxysporum f. sp. cubense race 4 was investigated with aid of scanning and transmission electron microscopy. This demonstrated that the bacterium was able to colonize the surface of hypha and macrospore of F. oxysporum f. sp. cubense. Mycelial deformation, terminal and/or intercalary swelling were evident. At later stages, hyphae of F. oxysporum f.sp. cubense, colonized by B. cepacia, were found to have collapsed. Further studies in vivo confirmed that B. cepacia colonized the hypha of F. oxysporum f. sp. cubense which had invaded banana roots. TEM observation showed that in the banana plant B. cepacia was closely associated with the healthy banana roots and a matrix was frequently found to be present between the bacterium and the plant surface. In addition, B. cepacia exists mainly in the intercellular space of the banana roots. UV irradiation treatment of B. cepacia resulted in a mutant that had lost inhibitory activity against F. oxysporum f. sp. cubense on TSA agar. Transposon mutagenesis of B. cepacia was performed by Tn5 insertion. Six mutants which had lost or had reduced inhibitory activity against F. oxysporum f. sp. cubense were generated. These mutants showed no inhibitory zones on TSA medium in the presence of the fungus. It was observed that one mutants. cepacia :: Tn5-188 appeared to lose the ability to colonize the fungal hypha, whilst a different mutant B. cepacia ::Tn5 - 217 was still able to colonize the fungal hyphae. TLC analyses showed that there was a decrease in antibiotic production in mutants B. cepacia :: Tn5 - 217 and B. cepacia - UV - 34, compared with the wild type. GC- MS analyses showed that there was no evidence of the peaks at 14.62 minutes, 20.0 minutes and 20.46 minutes in both chromatograms of mutants B. cepacia :: Tn5 -217 and 8. cepacia -UV - 34, compared with the wild type B. cepacia. No PCR products were detected using primers that were developed from sequences within the biosynthetic loci for Phi of P.fluorescens Q2-87(GenBank accession no. U41818) and PCA of P. fluorescens 2-79 (GeneBank no. L48616). Colony hybridization suggested that genomic DNA from B. cepacia could contain both Phi- and PCA probes. It was found that hybridization of genomic DNA digested with Cla-I of B. cepaca with Phl2a probe only occurred at low stringency. A hybridization signal was detected from a Cla-l fragment of approximately 2800bp. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1997.

Burkholderia cepacia complex (Bcc) species are opportunistic pathogens in patients with cystic fibrosis (CF), which are able to cause lethal infections. The Bcc are inherently resistant to most classes of antibiotics, which makes successful treatment problematic. Lipid A (also known as endotoxin), the hydrophobic anchor of lipopolysaccaride (LPS), is the bio-active component of LPS. One of several unique characteristics of the lipid A of the Bcc, is the permanent attachment of 4-amino-4-deoxy-L-arabinose (L-Ara4N) to the lipid A molecule. Also, the genes involved in L-Ara4N biosynthesis are necessary for viability in B. cenocepacia. Here we present research on lipid A biosynthesis, modi cation, and transport in the Bcc and highlight promising antimicrobial targets. The synthetic antibiotic CHIR-090 is an inhibitor of LpxC, an enzyme involved in the lipid A biosynthetic pathway. I investigated the activity of CHIR-090 against the Bcc and found that sensitivity to this antibiotic was both species- and strain-specific. CHIR-090 displayed MICs between 0.1 and 12.5 μg/ml against a panel of B. multivorans, the most prevalent Burkholderia species in CF. The species- and strain-specific sensitivity towards CHIR-090 was further explored and a strong correlation was found between the presence of a unique open reading frame, named LpxC2, in resistant species. To address the problem of multiple drug-resistance of the Bcc, we investigated the activity of the pyridoxal 50-phosphate (PLP)-dependent enzyme inhibitor cycloserine (CS) against the Bcc. CS is used as a second line of defense against M. tuberculosis. The activity of the D-enantiomer of CS (DCS) against the Bcc was tested and displayed MICs between 2 and 128 μg/ml and acted bactericidal towards the Bcc. Additionally, DCS inhibition of recombinant ArnB from B. cenocepacia J2315, a PLP-dependent enzyme necessary for viability in the Bcc, was studied. ArnB was inhibited reversibly by DCS. ArnB was further explored as a promising drug-target in the Bcc, but only CS has been identified as an inhibitor so far. In this thesis it was attempted to find the reason why is L-Ara4N modification of lipid A necessary for viability in B. cenocepacia. Therefore, two proteins were characterised, which are involved in lipid A transport: LptA, the periplasmic lipid A binding protein, and LptB, the cytoplasmic ATP-ase. LptA was found to be able to bind both modified and unmodified lipid A in vitro and therefore is not L-Ara4N specific. Furthermore, LptA could bind deep-rough-, rough-, and smooth- LPS, similar to that described for Escherichia coli LptA. The kinetic parameters of LptB were determined in vitro (kcat = 5.71 min-1 and KM = 0.88 mM), and were comparable to E. coli LptB. The ATP-ase activity of LptB was not influenced by the presence of any forms of LPS (modified or non-modified). Therefore, we concluded that both B. cenocepacia J2315 LptA and LptB are not L-Ara4N specific.

Mit einer miniaturisierten Schnellmethode (experimentelles Taxa Profile-Plattensystem, Merlin, Bornheim-Hersel, Deutschland) wurden 570 biochemische Stoffwechselleistungen der 36 Spezies des Genus Burkholderia (B.) untersucht. Die drei verwendeten 384-Loch-Mikrotitratons-Platten sind mit vakuumgetrockneten Substraten beschichtet: Profile A-Platte mit 191 Aminosäuresubstraten, Profile C-Platte mit 191 Kohlenhydratsubstraten und Profile E-Platte mit 188 enzymatischen Substraten. In die Studie wurden 160 Stämme aus insgesamt 36 Spezies, einschliesslich der beiden S 3 Erreger, B. mallei, Verursacher des Rotz und B. pseudomallei, Auslöser des Pseudorotzes (Melioidose), einbezogen. Insgesamt wurden 44 Reaktionsgruppen (Taxons) dargestellt. In fünf Spezies, einschließlich B. mallei und B. pseudomallei, konnten interne homologe Taxons festgestellt werden. Es erfolgte die Erstellung von Prozentwerttabellen der Reaktivität der untersuchten Isolate. Jedes Taxon wurde dann separat ausgewertet. Alle Taxons können eindeutig Spezies zugewiesen werden und ermöglichen so eine Diagnose. Aus den biochemischen Reaktionen wurden 88 Substrate, die eine Differenzierung aller 44 Taxons erlaubt, ausgewählt. Für 14 Taxons konnte eine einzige Schlüsselreaktion beschrieben werden. Mit diesen Substraten wird eine 96-Loch-Mikrotitratons-Platte belegt werden. In Zukunft wird mit diesem optimierten, kostengünstigen Testsystem in jedem Routinelabor innerhalb von 24h die exakte Identifizierung eines unbekannten Burkholderia-Isolates möglich sein. Mit den 160 phänotypisierten Stämmen wurde eine in vitro-Resistenztestung mit 32 antibakteriellen Wirkstoffen mittels einer Mikrodilutionsmethode (GENARS GN4-Platte, Merlin, Bornheim-Hersel, Deutschland) durchgeführt. Die Auswertung erlaubt erstmalig einen vollständigen Überblick über die Resistenzlage des gesamten Genus.

Thematic Paper (M.C.T.M. (Clinical Tropical Medicine))--Mahidol University, 2006.