Made available in DSpace on 2016-06-02T19:55:33Z (GMT). No. of bitstreams: 1 4561.pdf: 2229870 bytes, checksum: e51008e96773b0184eb28a316cf86d57 (MD5) Previous issue date: 2012-04-27 / Financiadora de Estudos e Projetos / Penicillin G acylase (PGA, E.C.3.5.1.11) from Escherichia coli is an enzyme of great industrial importance, widely used for the hydrolysis of penicillin G, producing the 6-aminopenicillanic acid (6-APA), which is a key molecule for the synthesis of semi-synthetic penicillins. Among them, amoxicillin has a broad spectrum of activity against a variety of bacteriological infections. Industrially, amoxicillin is produced by chemical processes, which require drastic reaction conditions, several steps of protection and deprotection of reactive groups in order to prevent non-selective hydrolytic reactions, use of organochloride solvents with non-recyclable waste generation, which are toxic and harmful to the environment. The enzymatic synthesis is a more attractive alternative from the environmental point of view and economic. The tendency of the pharmaceutical industry is the development of enzymatic methods to produce these β-lactam semi-synthetic antibiotics, including amoxicillin. Nevertheless, a major obstacle to its industrial implementation is the limited yield, as a consequence of undesirable hydrolytic side-reactions, which lead to the formation of the by-product (p-hydroxyphenylglycine, POHPG) throughout the course of the reaction. This drawback can be partially avoided by reducing the water activity (aw) in the medium. For this purpose, ionic liquids (ILs) have emerged as an alternative to conventional organic media due to their high thermal and chemical stability, non-flammability, easy recycling, and negligible vapor pressure. Within this context, this work researched the development of an integrated green process for the recovery, reuse and recycle of the by-product (POHPG) of the kinetically controlled enzymatic synthesis of amoxicillin, employing PGA immobilized on Sepabeads® in a totally aqueous medium reaction (sodium phosphate buffer 100 mM, pH 6.5), and assessed the catalytic activity of this biocatalyst in the presence of different ILs as cosolvents for these synthetic reactions, in terms of selectivity (synthesis/hydrolysis, S/H ratio) and conversion of the substrate 6-aminopenicillanic acid (6-APA). The recovery of the by-product (POHPG) of the kinetically controlled enzymatic synthesis of amoxicillin in a totally aqueous reaction medium was done efficiently, achieving a final purity of 99% for the POHPG, which was successfully reused for the production of the substrate p-hydroxyphenylglycine ethyl ester (POHPGEE), achieving a conversion of 93%. Then, POHPGEE was recycled to the reactor (without any further purification) for another batch of enzymatic synthesis of amoxicillin, following the characteristic profile that is expected for these synthetic reactions. This integrated green process generated sodium chloride (NaCl) as waste, which is an inert and harmless salt. Moreover, the assessment of the use of ILs as cosolvents for the reactions of kinetically controlled enzymatic synthesis of amoxicillin presented promising results. An increase of 400% in the selectivity was observed for the reactions carried out in the presence of 1-butyl-3-methylimidazolium hexafluorophosphate (BMI.PF6), as cosolvent at a concentration of 75% (VIL/VWATER) in relation to the standard reaction performed in totally aqueous medium. Similarly, this figure reached more than 350% for reactions conducted in 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide (BMI.NTf2) at the same volume fraction, while for 1-butyl-3-methylimidazolium tetrafluoroborate (BMI.BF4) there was only a slight increase in selectivity (about 57%). The highest conversion of 6-APA was achieved using BMI.NTf2 as cosolvent at a concentration of 71% (VIL/VWATER), representing an increase of more than 36% compared to standard aqueous reaction. No deactivation of the enzyme was observed after the reactions in any of the ILs, and the physical integrity of the biocatalyst particles was entirely maintained. The results of this work collaborated for the advance in the study of the enzymatic synthesis of semi-synthetic penicillins through the use of technologies more green . / Penicilina G acilase (PGA, E.C.3.5.1.11) de Escherichia coli é uma enzima de grande importância industrial, amplamente utilizada para a hidrólise de penicilina G, produzindo o ácido 6-aminopenicilânico (6-APA), que é uma molécula chave para a síntese de penicilinas semi-sintéticas, dentre elas, a amoxicilina, que possui um amplo espectro de ação contra uma variedade de infecções bacteriológicas. Industrialmente, a amoxicilina é produzida por meio de processos químicos, os quais requerem condições drásticas de reação, diversos passos de proteção e desproteção de grupos reativos para impedir reações hidrolíticas não seletivas, utilização de solventes organoclorados com geração de resíduos não recicláveis, que são tóxicos e nocivos ao meio ambiente. A síntese enzimática é uma alternativa mais interessante do ponto de vista ambiental e econômico. A tendência da indústria farmacêutica é o desenvolvimento de métodos enzimáticos para a produção destes antibióticos β-lactâmicos semi-sintéticos, incluindo a amoxicilina. Entretanto, um dos principais impedimentos para a sua implementação industrial é o rendimento limitado, em decorrência de reações laterais de hidrólise indesejáveis, que levam à formação do subproduto (p-hidroxifenilglicina, POHFG) durante todo o andamento da reação. Este inconveniente pode ser parcialmente evitado reduzindo a atividade da água (aw) no meio. Para esta finalidade, os líquidos iônicos (LIs) surgiram como uma alternativa aos meios orgânicos convencionais, devido à sua elevada estabilidade térmica e química, não inflamabilidade, fácil reciclagem e pressão de vapor desprezível. Neste contexto, este trabalho pesquisou o desenvolvimento de um processo integrado verde para a recuperação, reutilização e reciclo do subproduto (POHFG) da síntese enzimática cineticamente controlada de amoxicilina, empregando PGA imobilizada em Sepabeads® em um meio de reação totalmente aquoso (tampão fosfato de sódio 100 mM, pH 6,5), e avaliou a atividade catalítica deste biocatalisador na presença de diferentes LIs como cossolventes para estas reações sintéticas, em termos de seletividade (síntese/hidrólise, relação S/H) e conversão do substrato ácido 6-aminopenicilânico (6-APA). A recuperação do subproduto (POHFG) da síntese enzimática cineticamente controlada de amoxicilina em meio totalmente aquoso foi realizada eficientemente, atingindo uma pureza final de 99% para a POHFG, a qual foi reutilizada com sucesso para a produção do substrato éster etílico da p-hidroxifenilglicina (EEPOHFG), atingindo uma conversão de 93%. Em seguida, o EEPOHFG foi reciclado ao reator (sem qualquer purificação adicional) para outra batelada de síntese enzimática de amoxicilina, seguindo o perfil característico que é esperado para estas reações sintéticas. Este processo integrado verde gerou como resíduo o sal cloreto de sódio (NaCl) que é inerte e inofensivo. Além disso, a avaliação da utilização de LIs como cossolventes para as reações de síntese enzimática cineticamente controlada de amoxicilina apresentou resultados promissores. Um acréscimo de 400% na seletividade foi observado para as reações realizadas na presença de hexafluorfosfato de 1-butil-3-metilimidazólio (BMI.PF6), como cossolvente na concentração de 75% (VLI/VÁGUA) em relação à reação padrão realizada em meio totalmente aquoso. De maneira similar, este número alcançou mais do que 350% para as reações conduzidas em bis(trifluormetilsulfonil)imida de 1-butil-3-metilimidazólio (BMI.NTf2) na mesma fração volumétrica, enquanto que para tetrafluorborato de 1-butil-3-metilimidazólio (BMI.BF4) houve apenas um ligeiro aumento na seletividade (cerca de 57%). A mais elevada conversão de 6-APA foi obtida empregando BMI.NTf2 como cossolvente na concentração de 71% (VLI/VÁGUA), representando um aumento de mais do que 36% em comparação à reação padrão aquosa. Nenhuma desativação da enzima foi observada após as reações em qualquer um dos LIs, e a integridade física das partículas do biocatalisador foi integralmente mantida. Os resultados deste trabalho colaboraram para o avanço no estudo da síntese enzimática de penicilinas semi-sintéticas através do emprego de tecnologias mais verdes .

A penicilina G é um dos antibióticos mais importantes. Além de possuir um baixo preço e comprovada eficácia de tratamento, mostra inúmeras possibilidades para a redução da morbidade e mortalidade por doenças infecciosas em todo o mundo. Como eventualmente este medicamento causa sequelas no parênquima renal e estruturas associadas, e sendo que a secreção da rede tubular renal contribui para a excreção da penicilina G, onde cerca de 60% do antibiótico é eliminado pela urina, nos propomos a fazer um estudo das principais alterações que possam ocorrer na pelve renal de ratos normais e ratos induzidos à diabetes. Este projeto tem o propósito de descrever e analisar as fibras colágenas, musculares lisas e elásticas da pelve renal de ratos wistar observando alterações estruturais e ultraestruturais dos grupos experimentais quando comparados ao grupo controle com relação ao uso da penicilina G. Os ratos foram divididos em 4 grupos, ratos Wistar normais (N); ratos Wistar tratados com penicilina G (NP); ratos Wistar induzidos à diabetes (D); ratos Wistar diabéticos com penicilina G (DP). Os ratos dos grupos D e DP foram induzidos ao diabetes por aloxano. A região da pelve renal com representação das fibras foi coletada e reduzida em pequenos fragmentos. Os cortes obtidos foram utilizados para Microscopia Eletrônica de Transmissão e corados pelos seguintes métodos para Microscopia Óptica: Hematoxilina Férrica para evidenciação de fibras elásticas; Resorcina fucsina para evidenciação de fibras elásticas e elaunínicas; Resorcina fucsina após oxidação com solução aquosa a 1% de oxona para evidenciação de fibras elásticas, elaunínicas e oxitalânicas; Azan para evidenciação do componente colágeno e muscular lisa; Picrosírius para observação do componente colágeno (especificamente tipo I e III); e Hematoxilina e Eosina, para evidenciação do componente celular. A análise microscópica e a histomorfometria mostraram que a Penicilina G altera os componentes fibrosos da pelve renal, fazendo com que as áreas de fibras musculares lisas e de colágeno tipo III fossem aumentadas e as fibras elásticas maduras diminuídas (neste caso, apenas entre N e NP). O Diabetes mellitus mostrou-se como uma doença metabólica também capaz de alterar a morfologia da pelve, fazendo com que a área de fibras musculares lisas aumentasse, a área de colágeno tipo I e a quantidade de fibras elásticas maduras e elaunínicas diminuísse e as oxitalânicas aumentassem, além de um notável aumento na quantidade de mitocôndrias. Podemos inferir que a antibioticoterapia feita pela penicilina G e o diabetes, provocam diferenças estruturais e ultraestruturais na pelve renal dos ratos Wistar, principalmente na organização dos componentes fibrosos elástico, muscular e colágeno. / Penicillin G is the most important antibiotics. Besides having a low cost and proven effectiveness of treatment, it shows great possibilities for reducing morbidity and mortality from infectious diseases worldwide. As this medicine may cause sequelae in the renal parenchyma and associated structures, and since the net renal tubular secretion contributes to the excretion of penicillin G, where about 60% of the antibiotic is eliminated in urine, this study aims to investigate the main structural and ultrastructural changes occurring in the kidney of normal and diabetes rats. Thus, this project aims to describe and analyze the collagen fibers, smooth muscle and elastic fibers of the renal pelvis of Wistar rats, comparing control and penicillin G-treated animals. The animals were divided into 4 groups, normal rats (N), Wistar rats treated with penicillin G (NP); rats induced diabetes (D), diabetic Wistar rats with penicillin G (DP). The diabetes was induced in groups D and DP by alloxan. The fibrotic region of the renal pelvis was collected and reduced into small fragments. The sections were used for the transmission electron microscopy and stained by the following methods for optic microscopic: Iron Hematoxylin for disclosure of elastic fibers; Resorcin fuchsin for disclosure of elastic and elauninic fibers; Resorcin fuchsin after oxidation with 1% aqueous solution of oxone for disclosure of elastic, elauninic and oxytalan fibers; Azan evidencing the collagen and smooth muscle components; Picrosirius for observation of the collagen component (specifically type I and III); and Hematoxylin and Eosin, to show the cellular component. Microscopic and histomorphometry analysis showed that penicillin G alters the fibrous components of the renal pelvis, increasing areas of smooth muscle fibers and collagen type III deposition and decreasing mature elastic fibers (in this case, only between N and NP). Diabetes mellitus proved to be a metabolic disease also able to alter the morphology of the pelvis, leading to the augmentation of smooth muscle fiber area. Moreover, the area of type I collagen and the amount of mature elastic and elauninic fibers were diminished, while oxytalan fibers increased, together with a remarkable increase in the number of mitochondria. We can infer that the antibiotic therapy made by penicillin G and the diabetes, cause structural and ultrastructural differences in the renal pelvis of rats, mainly in the organization of elastic fiber, muscular and collagen components.

Abstract The purpose of the present study was to gain more insight into the blood oxygen level-dependent (BOLD)-contrast functional MRI (fMRI) in the brain and its connection to EEG, both in global and local scales of their temporal and spatial relations. BOLD signal changes were studied during hyperventilation (HV) induced EEG reactivity of intermittent rhythmic delta activity (IRDA). The BOLD signal in gray matter decreased 30% more in subjects with IRDA (N = 4) than in controls (N = 4), during the first two minutes of HV. This difference disappeared during IRDA in EEG. BOLD signal changes may provide additional information about dynamic hemodynamic changes relative to HV induced EEG reactivity. BOLD signal changes were investigated during sudden deepening of thiopental anesthesia into EEG burst-suppression level in pigs (N = 5). Positive (6–8%) or negative (-3– -8%) group average BOLD signal changes correlated to the thiopental bolus injection were seen. Positive and negative responses covered 1.6% and 2.3% of the brain voxels, respectively. BOLD signal changes in brain are associated with sudden deepening of thiopental anesthesia into EEG burst-suppression level, but they are spatially inconsistent and scarce. Somatosensory BOLD response was studied in brain before and after globally induced methotrexate (MTX) exposition in pigs (N = 4). After the MTX exposure, reduced (from 2–4% to 0–1%) or negative (-2% to -3%) BOLD responses were detected. Somatosensory BOLD-contrast response shows a slight difference in brain before and after globally induced MTX exposition. An experimental epilepsy model for development of simultaneous EEG and BOLD-contrast fMRI in the localization of epilepsy was developed and tested. Dynamic penicillin induced local epilepsy was applied in deep isoflurane anesthesia in pigs (N = 6). Relatively high (10–20%) and localized BOLD signal increase was found. The dynamic penicillin induced focal epilepsy model in deep isoflurane anesthesia with simultaneous EEG and BOLD-contrast fMRI is feasible for the development of these methods for localization of epileptic focus or foci. In conclusion, with careful experimental design and analysis, BOLD-contrast fMRI with EEG provides a potential tool for monitoring and localising functional changes in the brain.

The introduction of antibiotics in healthcare is one of the most important medical achievements with regard to reducing human morbidity and mortality. However, bacterial pathogens have acquired antibiotic resistance at an increasing rate, and due to a high prevalence of resistance to some antibiotics they can no longer be used therapeutically. The antibiotic mecillinam, which inhibits the penicillin-binding protein PBP2, however, is an exception since mecillinam resistance (MecR) prevalence has remained low. This is particularly interesting since laboratory experiments have shown that bacteria can rapidly acquire MecR mutations by a multitude of different types of mutations. In this thesis, I examined mechanisms and dynamics of mecillinam resistance in clinical and laboratory isolates of Escherichia coli. Only one type of MecR mutations (cysB) was found in the clinical strains, even though laboratory experiments demonstrate that more than 100 genes can confer resistance Fitness assays showed that cysB mutants have higher fitness than most other MecR mutants, which is likely to contribute to their dominance in clinical settings. To determine if the mecillinam resistant strains could compensate for their fitness cost, six different MecR mutants (cysB, mrdA, spoT, ppa, aspS and ubiE) were evolved for 200-400 generations. All evolved mutants showed increased fitness, but the compensation was associated with loss of resistance in the majority of cases. This will also contribute to the rarity of clinical MecR isolates with chromosomal resistance mutations. How MecR is mediated by cysB mutations was previously unclear, but in this thesis I propose and test a model for the mechanism of resistance. Thus, inactivation of CysB results in cellular depletion of cysteine that triggers an oxidative stress response. The response alters the intracellular levels of 450 proteins, and MecR is achieved by the increase of two of these, the LpoB and PBP1B proteins, which rescue the cells with a mecillinam-inhibited PBP2. Mecillinam is used for UTI treatments and to investigate mecillinam resistance in a more host-like milieu, MecR strains were grown in urine and resistance was examined. Interestingly, this study showed that neither laboratory, nor clinical cysB mutants are resistant in urine, most likely because the cysteine present in the urine phenotypically reverts the bacteria to susceptibility. These findings suggest that mecillinam can be used to treat also those clinical strains that are identified as MecR in standard laboratory tests, and that testing of mecillinam susceptibility in the laboratory ought to be performed in media that mimics urine to obtain clinically relevant results. In summary, the work described in this thesis has increased ourgeneral knowledge of mecillinam resistance and its evolution. Hopefully this knowledge can be put to good use in clinical settings to reduce the negative impact of antibiotic resistance.

A Febre Reumática, complicação tardia de uma infecção de orofaringe causada pelo Streptococcus pyogenes (estreptococo -hemolítico do grupo A de Lancefield), tem como conseqüência a Cardiopatia Reumática, explicada pelo mimetismo molecular entre proteínas cardíacas humanas e a associação de proteínas e carboidratos da membrana do S. pyogenes. A profilaxia secundária com a PGB 1.200.000 UI IM propõe-se a evitar novos surtos, sendo administrada em intervalos de vinte e um dias nos países com alto índice de estreptococcia. A lesão valvar predispõe à Endocardite Infecciosa, que resulta de bacteriemias causadas por focos infecciosos de origem bucal em cerca de 40% dos casos. Os Streptococcus Viridans constituem o grupo mais comumente encontrado nas Endocardites Infecciosas, em especial os Streptococcus sanguinis e Streptococcus oralis. O efeito do uso crônico da PGB não foi estudado com especificidade para essa microbiota. Assim, foi avaliada, qualitativa e quantitativamente, a microbiota bucal de 100 pacientes, aos 7 e 21 dias, após profilaxia secundária para a Febre Reumática com a PGB 1.200.000 UI IM e comparada com a de 100 pacientes portadores de doença arterial coronariana sem antecedentes de Febre Reumática. As espécies avaliadas foram divididas em S. sanguinis, S. oralis e outras espécies de Streptococcus Viridans Foram coletadas amostras de saliva pela mastigação de goma de parafina e transportadas em meio VMGA II S. As culturas foram semeadas em ágar Columbia CNA com 5% de sangue desfibrinado puro de carneiro com acréscimo de penicilina G. e incubadas a 35ºC em estufa de CO2 por 72 horas. As colônias sugestivas de Streptococcus foram submetidas a testes bioquímicos para confirmação de gênero e espécie. A concentração inibitória mínima foi determinada pelo método Etest e interpretada segundo os padrões do Clinical and Laboratory Standards Institute. Não houve diferença quanto à presença do S. sanguinis nos grupos estudados (P=0,40). O S. oralis prevaleceu aos 7 dias de PGB em relação ao grupo controle (P=0,01). Quanto à identificação de outras espécies, houve maior número de cepas nos pacientes do grupo controle quando comparados aos do grupo de estudo aos 7 e 21 dias de PGB (P<0,001). Os números de UFC/ml de S. sanguinis, S. oralis e de outras espécies foram comparados entre os grupos e não houve diferença entre eles (P=0,96; P=0,60 e P=0,77; respectivamente). Quanto às CIM do S. sanguinis e do S. oralis, não houve diferença entre os grupos (P=0,79 e P=0,13; respectivamente). Todos os testes estatísticos foram realizados em um nível de significância de 5%. Concluiu-se que o S. oralis prevaleceu aos 7 dias de PGB 1.200.000 UI IM; os Streptococcus Viridans de outras espécies prevaleceram no grupo controle; o número de UFC/mL de saliva não diferiu nos grupos estudados, a susceptibilidade dos S. sanguinis e S. oralis à penicilina G não foi alterada pela ação da PGB 1.200.000 UI IM a cada 21 dias e, por fim, a PGB não provocou reações de hipersensibilidade em nenhum paciente do estudo / Rheumatic fever is the result of a Streptococcus pyogenes (group A -hemolytic Streptococcus) infection of the upper respiratory tract. Rheumatic heart disease is a rheumatic fever consequence and is elucidated by the molecular mimicry between human cardiac proteins and group A streptococcal proteins and carbohydrates association. The secondary prophylaxis with 1,200,000 U BPG every three weeks is used for prevention of recurrent rheumatic fever in developing countries. Valvar defects are a risk for infective endocarditis which is resulted of bacteriemia caused for oral infectious focuses in 40% of cases. Viridans streptococci are the predominant group recovered in infective endocarditis, specially Streptococcus sanguinis and Streptococcus oralis. The effect of chronic BPG wasnt studied with specificity to these pathogens yet. Therefore, the oral microbiota was evaluated, qualitatively and quantitatively, at 7 and 21 days after secondary prophylaxis with BPG to rheumatic fever (study group), in a hundred patients and in comparison to another hundred patients with coronary heart disease who never acquired rheumatic fever (control group). The species evaluated were divided in S. sanguinis, S. oralis and another Streptococcus species. It was collected samples of chewing-stimulated saliva (1ml) and transported in VMGA II S medium. The samples were cultured in pure and with penicillin G 5% sheep blood Columbia ágar (CNA), incubated for 72 hours in an atmosphere containing 5% CO2 at 35ºC. The strains that were suggestive to Streptococcus were identified by biochemical tests to confirm bacteria species and genus. Minimal inhibitory concentration was determined by Etest method and interpreted in accordance to Clinical and Laboratory Standards Institute. The results showed that there was no difference in S. sanguinis presence in all groups (P=0.40). S. oralis prevailed in 7 days BPG group in comparison to control group (P=0.01). The control group showed the highest number of others species in comparison to 7 and 21 days BPG (P<0.001). CFU/ml numbers of S. sanguinis, S. oralis and other species strains were compared in 7 and 21 days BPG to control group and there was no difference among themselves (P=0.96, P=0.60 and P=0.77; respectively). There was no difference in S. sanguinis and S. oralis MICs among the study and control groups (P=0.79 and P=0.13). All statistic tests were done at 5% significance level. It was concluded that S. oralis prevailed in 7 days BPG group in comparison to control group; other species of Viridans streptococci prevailed in control group. The number of CFU/mL did not differ in both studied groups; the penicillin susceptibility of S. sanguinis and S. oralis did not change by BPG every three weeks and, by the end, it was not observed hypersensitivity reactions to penicillin in neither of the patients of this study

Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

BACKGROUND AND OBJECTIVES: Streptococcus agalactiae (Group B Streptococcus, GBS) is the leading cause of neonatal infections and deaths in human. It can also cause infections in pregnant women and non-pregnant adults. Penicillin and ampicillin are antibiotics of choice for the treatment of GBS infections. Erythromycin and clindamycin are used as alternative therapy in penicillin allergic patients, however resistance to these agents has been increasingly observed. This present study was undertaken to determine the colonization rate of GBS, susceptibility profile and the mechanism of antibiotic resistance in pregnant women and their babies at Dr. George Mukhari Academic Hospital in Pretoria. METHODS: Rectal and vaginal swabs were collected from pregnant women; ear and umbilical swabs from newborns over an 11 month period. Samples were cultured on selective media (CNA agar and Todd-Hewitt broth) and GBS positively identified using morphological and biochemical tests including Gram staining, hemolytic activity, catalase test, bile esculin, CAMP test and Latex agglutination test. The susceptibility testing was done using the Kirby-Bauer and E-test methods. The D-test method was used to determine the inducible clindamycin resistance. Multiplex PCR with were used to detect different genes coding for resistance. RESULTS: Out of the 413 patients evaluated, 128 (30.9%) were positive with GBS. All isolates were sensitive to penicillin and ampicillin. Erythromycin and clindamycin resistance was 21.1% and 17.2% respectively; of which 69% harbouring constitutive MLBB, 17.4% inducible MLSB. The alteration of ribosomal target encoded by ermB genes was the commonest mechanism of resistance observed in 55% of isolates, 38% of isolates had both ermB and linB genes and efflux pump mediated by mefA genes was detected in one of isolates. Conclusion: This study reaffirms the appropriateness of penicillin as the antibiotic of choice for treating GBS infection. However it raises the challenges of resistance to the macrolides and lincosamides. More GBS treatment options for penicillin allergic patients need to be researched. / Health Studies / M.Sc. (Life Sciences (Microbiology))

BACKGROUND AND OBJECTIVES: Streptococcus agalactiae (Group B Streptococcus, GBS) is the leading cause of neonatal infections and deaths in human. It can also cause infections in pregnant women and non-pregnant adults. Penicillin and ampicillin are antibiotics of choice for the treatment of GBS infections. Erythromycin and clindamycin are used as alternative therapy in penicillin allergic patients, however resistance to these agents has been increasingly observed. This present study was undertaken to determine the colonization rate of GBS, susceptibility profile and the mechanism of antibiotic resistance in pregnant women and their babies at Dr. George Mukhari Academic Hospital in Pretoria. METHODS: Rectal and vaginal swabs were collected from pregnant women; ear and umbilical swabs from newborns over an 11 month period. Samples were cultured on selective media (CNA agar and Todd-Hewitt broth) and GBS positively identified using morphological and biochemical tests including Gram staining, hemolytic activity, catalase test, bile esculin, CAMP test and Latex agglutination test. The susceptibility testing was done using the Kirby-Bauer and E-test methods. The D-test method was used to determine the inducible clindamycin resistance. Multiplex PCR with were used to detect different genes coding for resistance. RESULTS: Out of the 413 patients evaluated, 128 (30.9%) were positive with GBS. All isolates were sensitive to penicillin and ampicillin. Erythromycin and clindamycin resistance was 21.1% and 17.2% respectively; of which 69% harbouring constitutive MLBB, 17.4% inducible MLSB. The alteration of ribosomal target encoded by ermB genes was the commonest mechanism of resistance observed in 55% of isolates, 38% of isolates had both ermB and linB genes and efflux pump mediated by mefA genes was detected in one of isolates. Conclusion: This study reaffirms the appropriateness of penicillin as the antibiotic of choice for treating GBS infection. However it raises the challenges of resistance to the macrolides and lincosamides. More GBS treatment options for penicillin allergic patients need to be researched. / Health Studies / M. Sc. (Life Sciences (Microbiology))

Made available in DSpace on 2016-06-02T19:02:43Z (GMT). No. of bitstreams: 1 5706.pdf: 7869131 bytes, checksum: 3a35e736b3418ca357ef4fc2e657c0af (MD5) Previous issue date: 2013-10-16 / Universidade Federal de Minas Gerais / Enzymes are potent catalysts, but operationally fragile, expensive and soluble. Industrial applications of enzymes, often, are possible only using immobilized enzyme. Nowadays, various studies have been performed aiming to immobilize enzymes onto magnetic carriers, which allow the selective recovery of the derivative by applying an external magnetic field even in complex reaction media containing other suspended solids. There are many studies using magnetic carriers in enzymes immobilization procedures, however there are no commercially available enzymes immobilized onto magnetic materials. In these studies usually are used carriers with not ideal characteristics for applications in industrial processes. The present study aimed to develop new magnetic carriers and methods for immobilization of enzymes in these carriers, penicillin G acylase (PGA) and cellulases have been used as model enzymes. The thesis was divided into five parts, in the first part (Chapter 1) the state-of-art is presented. The second part (Chapter 2) describes the synthesis of magnetic carriers robust, cheap and with good characteristics for applications in bioprocesses. For this purpose were tested the synthesis of silica magnetic microparticles (SMMps) in water-in-oil micro-emulsion using sodium silicate as silica source and superparamagnetic iron oxide nanoparticles as magnetic core. Materials with good magnetic properties, high surface area and mesoporous structure were obtained. SMMps structure was characterized, it was possible to control the final structure of the material according to the synthesis conditions. In the third part of this study (Chapter 3) was evaluated a new concept in enzymes immobilization using magnetic materials. Magnetic tags were co-aggregated with PGA and cross-linked with glutaraldehyde, producing magnetic cross-linked enzymes aggregates (M-CLEAs). Several reaction conditions were tested producing M-CLEAs with different characteristics and strong response to external magnetic fields. Derivatives with good recovered activity and increased thermal and methanol 50% (v/v) stabilities were obtained. M-CLEAs presented superior performance, in comparison with the free enzyme, in penicillin G hydrolysis experiments, being reused for three reaction cycles without loss of activity. In the fourth part of this study (Chapter 4) the immobilization of the Trichoderma reesei cellulolytic complex onto 17 carriers using 60 different immobilization conditions was evaluated. Covalent methods to cellulases immobilization resulted in total loss of the enzymatic activity. The immobilization by adsorption allowed preserving a portion of the enzymatic activity, however, the enzyme was desorbed from the carrier with the increase in the ionic strength. The best results were achieved for adsorption in MANAE-agarose followed by cross-linking with glutaraldehyde. Hydrolysis experiments using insoluble substrates showed that it is possible to hydrolyze such substrates even using immobilized enzyme onto porous carriers. The derivative was reused for ten reaction cycles (hydrolysis of filter paper) saving more than 90% of its activity. Finally, in Chapter 5, the T. reesei cellulolytic complex was immobilized by adsorption onto SMMp activated with amino groups followed by glutaraldehyde cross-linking achieving good results in terms of recovered activity. / Enzimas são potentes catalisadores, porém frágeis operacionalmente, caras e solúveis. Aplicações industriais desses catalisadores, muitas vezes, são possíveis apenas com o uso de enzima imobilizada. Estudos indicam que o uso de suportes magnéticos para imobilizar enzimas pode permitir a recuperação seletiva do derivado através da aplicação de um campo magnético externo mesmo em meios complexos contendo outros sólidos em suspensão. Apesar de existirem muitos estudos empregando suportes magnéticos para imobilização de enzimas, não existem enzimas imobilizadas em materiais magnéticos disponíveis comercialmente. Nestes estudos geralmente são utilizados suportes magnéticos com características não ideais para aplicações em bioprocessos. O presente estudo teve como principal objetivo o desenvolvimento de novos suportes magnéticos e métodos para imobilização de enzimas nestes suportes, a enzima penicilina G acilase (PGA) e celulases foram utilizadas como modelo. O estudo foi dividido em cinco partes, no Capítulo 1 é apresentada uma introdução indicando o estado da arte. O Capítulo 2 apresenta o preparo de novos suportes magnéticos robustos, baratos e com características ótimas para aplicações em bioprocessos. Nesta etapa foi testada a síntese de micro-partículas magnéticas de sílica (SMMps) em micro-emulsão água-em-óleo, empregando silicato de sódio como fonte de sílica e nanopartículas superparamagnéticas de óxido de ferro como núcleo magnético. Os materiais obtidos apresentaram excelentes propriedades magnéticas, alta área de superfície e estrutura mesoporosa. A partir da caracterização físico-química e morfológica das SMMps foi possível controlar a estrutura final do material de acordo com as condições de síntese. No Capítulo 3 foi avaliado um novo conceito em imobilização de enzimas empregando materiais magnéticos. Neste estudo etiquetas magnéticas foram co-agregadas com PGA e entrecruzadas com glutaraldeído, gerando agregados enzimáticos entrecruzados com propriedades magnéticas (M-CLEAs). Várias condições reacionais foram testadas rendendo M-CLEAs com diferentes características e com resposta robusta a campos magnéticos externos. Derivados imobilizados com boa atividade recuperada e incremento na estabilidade térmica e frente a metanol 50% (v/v) foram obtidos. M-CLEAs apresentaram desempenho superior ao observado para a enzima livre em experimentos de hidrólise de penicilina G, sendo reutilizados por três ciclos reacionais sem perda de atividade. No Capítulo 4 foi avaliada a imobilização do complexo celulolítico de Trichoderma reesei em 17 suportes, empregando 60 diferentes condições de imobilização. Os experimentos de imobilização realizados empregando técnicas de imobilização por união covalente ocasionaram perda total de atividade enquanto métodos de imobilização por adsorção permitiram conservar boa atividade enzimática, porém a enzima dessorveu do suporte com o aumento na força iônica do meio. Os melhores resultados foram alcançados para adsorção em MANAE-agarose seguido de entrecruzamento com glutaraldeído. Experimentos de hidrólise de substratos insolúveis mostraram que é possível hidrolisar este tipo de substrato mesmo com enzima imobilizada em suportes porosos. O derivado foi reutilizado por dez ciclos (hidrólise de papel filtro) conservando mais de 90% de sua atividade. Por fim, no Capítulo 5, o complexo celulolítico de T. reesei foi imobilizado por adsorção em SMMp ativado com grupos amino seguido de entrecruzamento com glutaraldeído apresentando bons resultados em termos de atividade recuperada.

Made available in DSpace on 2016-06-02T19:56:40Z (GMT). No. of bitstreams: 1 3187.pdf: 3659896 bytes, checksum: 975ac91a3eb67a4347c326de8f22bf8e (MD5) Previous issue date: 2010-03-31 / Universidade Federal de Minas Gerais / This master's thesis project aims at studying the dynamic optimization of the operation of a bench scale (up to 5L) automated, agitated and aerated bioreactor, where the semi-continuous cultivation of recombinant Pichia pastoris is run. This yeast was cloned using the PGK1 promoter, which precludes the use of methanol as inducer, expressing constitutively the enzyme penicillin G Acylase (PGA) from Bacillus megaterium. While the group of molecular biology of DEQUFSCar is working on cloning the PGA, d P. pastoris expressing the enzyme &#61665;- amylase from Bacillus subtilis was cultivated. This clone, provided by prof. Fernando Torres, UnB, uses the same construction and, therefore, its kinetics of growth and production should be very similar to the PGA s. Cultivation of recombinant Pichia pastoris was performed in flasks (skaker) using standard culture medium, aiming at obtaining kinetic data, which are the starting point for the escalation to a benchtop bioreactor. Following that, tests were performed in a 5L bioreactor in batch and fed batch operation modes. With the bioreactor data , kinetic parameters of growth, to be further used in the simulations, were estimated, using a hybrid algorithm (which combines the global method Simulated Annealing, with the local one Levenberg- Marquardt). This algorithm, is implemented in Matlab and available in the software library of Ladabio (Laboratory of Development and Automation of Bioprocesses ). From these data, models of microbial growth and of production were developed, following a classic approach (unstructured, non-segregated). Computer simulations using different feeding strategies and employing these models allowed mapping the dynamics of the system. From this information, optimal control strategies were proposed to define optimal feeding profiles. Cellular concentrations of 5.4 g/L (dry weight) were reached in shaker (20h of cultivation, when glucose is exhausted), expressing 218 U/mL of &#61665;-amylase, compared to 11.4 g/L (dry weight) that were achieved in cultures in a bioreactor in batch simple (10h of cultivation, when glucose is exhausted), expressing 156 U/mL of &#61665;-amylase In fed-batch cultures, cell concentrations of up to 45 g/L were achieved, expressing up to 260 U/mL of &#61665;- amylase, with a productivity of 5.2 U/mL/ h. In fed-batch cultures of P. pastoris expressing PGA, cell concentrations of up to 35 g/L were achieved. Enzyme activity was not detected in the culture broth due to the effect of glycosylation. Immunodetection reaction confirmed the expression of the recombinant enzyme. Four specific growth rate equations were adjusted, with different types of inhibition by one product, detected at significant levels by liquid chromatography highperformance, but not yet identified. This metabolite was added as an inhibitor in kinetic models, using the peak areas, normalized as a pseudoconcentration. The best fit to the experimental data were the Monod kinetic model with non-competitive inhibition. Typical values obtained for the maximum specific growth and glucose/ cell conversion factor in bioreactor were &#61676;max=0,24 h-1 and YX/S = 0,48. Algorithm for optimal control in open loop was developed and successfully implemented, providing a robust profiles of great power, whose validation is proposed as a continuation of this work. / Este mestrado se propoe a estudar a otimizacao dinamica de biorreator automatizado, tipo tanque agitado e aerado, em escala de bancada (ate 5L), onde se processa o cultivo semi-continuo de Pichia pastoris recombinante. Essa levedura foi clonada pelo grupo do prof. Fernando Torres, da UnB, utilizando o promotor PGK1, que dispensa a utilizacao de metanol como indutor, expressando constitutivamente a enzima &#61665;-amilase de Bacillus subtilis. Durante a execucao deste mestrado, a enzima penicilina G acilase (PGA) de Bacillus megaterium esta sendo clonada pelo grupo de biologia molecular do DEQ-UFSCar usando a mesma construcao e, portanto, a cinetica de crescimento e producao da PGA heterologa devera ser muito semelhante as da &#61665;-amilase, utilizada como estudo de caso para otimizacao do bioprocesso. Cultivos de Pichia pastoris recombinante foram realizados em frascos agitados, utilizando meio de cultivo padrao, objetivando o levantamento de dados cineticos, ponto de partida para o escalonamento em biorreator de bancada. Posteriormente, foram realizados ensaios em biorreator de 5L, em batelada e batelada alimentada. Com os dados obtidos nos cultivos em biorreator, e utilizando algoritmo hibrido para estimativa de parametros (que combina o metodo global Simulated Annealing, com o local de Levenberg-Marquardt), implementado em MatLab e disponivel no LaDABio (Laboratorio de Desenvolvimento e Automacao de Bioprocessos), foram ajustados parametros cineticos de crescimento, para serem utilizados nas simulacoes dos cultivos em biorreator. A partir dai, foi desenvolvido modelo de crescimento microbiano e de producao, utilizando um enfoque classico (modelo nao-estruturado, nao-segregado) para descrever o sistema. Com isso, torna-se possivel realizar simulacoes em computador usando diferentes estrategias de alimentacao, para mapear a dinamica do sistema. A seguir, foram desenvolvidos algoritmos de controle otimo em malha aberta para definicao de estrategias de alimentacao. Concentracoes celulares de 5,4 g/L (massa seca) foram alcancadas em cultivos em camara rotatoria (20h de cultivo, quando se esgota a glicose), expressando 218 U/mL de &#61665;-amilase, comparado com 11,4 g/L(massa seca) que foram atingidos em cultivos em biorreator em bateladas simples (10h de cultivo, quando se esgota a glicose), expressando 156 U/mL de &#61665;-amilase. Em cultivos em batelada alimentada concentracoes celulares de ate 45 g/L foram atingidas, expressando ate 260 U/mL de &#61665;-amilase, com uma produtividade de 5,2 U/mL/h. Em cultivo em batelada alimentada de P. pastoris expressando PGA, concentracoes celulares de ate 35 g/L foram atingidas. Nao foi detectada atividade enzimatica no caldo de cultivo devido ao efeito da glicosilacao. Reacao de imunodeteccao confirmou a expressao da enzima recombinante. Foram ajustadas quatro equacoes de velocidade especifica de crescimento, com diferentes tipos de inibicao por um produto, detectado em niveis importantes por cromatografia liquida de alto desempenho, mas ainda nao identificado. Esse metabolito foi inserido como inibidor nos modelos cineticos, utilizando as areas dos picos, normalizadas, como uma pseudoconcentracao. Os melhores ajustes aos dados experimentais foram com modelo cinetico de Monod com inibicao nao-competitiva. Valores tipicos obtidos para a velocidade especifica maxima de crescimento e de fator de conversao glicose/celula em biorreator foram &#61676;max = 0,24 h-1 e YX/S = 0,48. Algoritmo de controle otimo em malha aberta foi desenvolvido e implementado com sucesso, prevendo de forma robusta perfis otimos de alimentacao, cuja validacao fica proposta como continuidade deste trabalho.