Antibiotic resistance is an ongoing global medical crisis and we are in great need of new antibacterial agents to combat rapidly emerging resistant pathogens. Tigecycline is one of few drugs that have been introduced into medicine during the last two decades. It is a broad-spectrum third generation tetracycline that is active against multidrug-resistant bacteria that cause complicated infections. In this thesis I examined the development of tigecycline resistance in Escherichia coli and associated in vitro and in vivo fitness effects. Selections of spontaneous E. coli mutants revealed relatively high accumulation rates of changes in the multidrug efflux system AcrAB-TolC regulation network and in heptose biosynthesis and transport pathways important for lipopolysaccharide (LPS) synthesis. Both groups of mutations led to reduced susceptibility to tigecycline and slower growth compared to the wild-type bacteria. Additional in vitro fitness assays and in vivo competitions showed that LPS mutants were less fit than efflux mutants, providing a possible explanation for why up-regulation of multidrug efflux pumps is the main tigecycline resistance mechanism reported in clinical isolates. Tigecycline was designed to evade the two most common tetracycline resistance mechanisms conferred by Tet proteins, efflux and ribosomal protection. However, tigecycline is a substrate for the tetracycline modifying enzyme Tet(X). Screening of Tet protein mutant libraries showed that it is possible to select Tet mutants with minimal inhibitory concentrations of tigecycline that reach clinically relevant levels. Mutations in Tet proteins that permitted a better protection from tigecycline frequently exhibited reduced activity against earlier generations of tetracyclines, except for the Tet(X) enzyme mutants, which were better at inactivating all tested tetracyclines. This is particularly worrisome because different variants of Tet(X) have recently spread to multidrug-resistant pathogens through horizontal gene transfer. Therefore, Tet(X) mutants with improved activity threaten the medical future of tetracyclines. Multidrug resistance is easily disseminated through horizontally spreading conjugative plasmids. pUUH239.2 is an example of a successful conjugative plasmid that caused the first clonal outbreak of extended spectrum β-lactamase-producing Klebsiella pneumoniae in Scandinavia. This plasmid was formed after rearrangements between two different plasmid backbones and it carries resistance genes to multiple antibiotic classes, heavy metals, and detergents.

Master of Science / Department of Clinical Sciences / David E. Anderson / Drug delivery systems for time release of recombinant human bone morphogenetic protein-2 (rhBMP-2) and antibiotics in orthopedic surgeries continue to be developed. Recently, a biodegradable novel polymeric matrix has been developed for this purpose. We hypothesized that impregnation of the matrix with rhBMP-2 would enhance bone healing. The objectives of the study were to characterize elution of rhBMP-2 and two antimicrobials (tigecycline, tobramycin) from the matrix, and bone response to the matrix in the presence or absence of rhBMP-2 and antimicrobials. In vitro elution of tigecycline, tobramycin, and rhBMP-2 from the matrix was investigated. Drug concentration in media were measured on days 1-6, 8, 10, 13, 15, 17, 21, 25, 28, and 30 using high pressure liquid chromatography/mass spectrometry/mass spectrometry (HPLC/MS/MS; antimicrobials) and ELISA (rhBMP-2). In vivo testing was done using a unicortical defect created into each tibia of twenty adult goats. Animals were randomly assigned to one of 5 groups: 1) control (untreated defect); 2) matrix; 3) matrix+ antimicrobials (tigecycline+tobramycin); 4) matrix+rhBMP-2; and 5) matrix+antimicrobials+rhBMP-2. Plasma concentration of tigecycline and tobramycin and serum concentration of rhBMP-2 were measured by the above techniques on days 1-7, 9, 11, 13, 15, 17, 22, 26, and 30. Bone response was assessed on days 0, 14, and 30 using radiographic scoring and dual energy X-ray absorptiometry (bone mineral density [BMD]). After euthanasia on day 30, histomorphologic analyses of the bone defects were done. Categorical variables were analyzed using a generalized linear model, and continuous variables using an ANOVA with P < 0.05 considered significant. In vitro elution was characterized by a rapid release on day 1 followed by a slow release until day 30 for both antimicrobials and rhBMP-2. Plasma antimicrobial concentrations showed continued release throughout the study period. Serum rhBMP-2 concentration, radiographic scores and BMD were not significantly different between groups. Periosteal and endosteal reaction surface areas were significantly greater surrounding the defects in group 4 (matrix+rhBMP-2). There was no significant difference between the groups for the percent of bone filling the defect. The matrix served as an appropriate antimicrobial and rhBMP-2 delivery system and successfully stimulated bone production when rhBMP-2 was present.

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No. of bitstreams: 2 Tese- Cláudia Castelo Branco Artiaga Kobayashi - 2013.pdf: 3424505 bytes, checksum: 8f6814112115a98fa01748f80463bf8c (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2013-04-12 / Carbapenemase-producing Klebsiella pneumoniae is an enterobacteria, commonly associated with resistance to multiple antibiotics. The presence of multidrug resistance phenotype is an important challenge in the epidemiological point of view, regarding the therapeutic management and infection control. A retrospective observational study was conducted in a tertiary-care hospital in Goiânia, Brazil, in the period of 2006 to 2011 to investigate the carbapenemases phenotypes, the in vitro activity of tigecycline and polymyxin B and determine the risk factors associated with infection or colonization by carbapenemase-producing K. pneumoniae. The serine-carbapenemase production was detected by modified Hodge test and combined disk test with boronic acid; metallo-β-lactamases - MβL by double-disk synergy test with imipenem and EDTA; extended spectrum β-lactamases - ESBL by combined disk with cefotaxime and ceftazidime with and without clavulanate, and plasmid AmpC type by combined disk with boronic acid and cefoxitin. The in vitro activity of tigecycline and polymyxin B against the enzyme-producing isolates was assessed using the broth microdilution method and for not β-lactam antimicrobials by disk diffusion and semi-automated system. The association between independent risk factors and colonization or infection by carbapenemase-producing K. pneumoniae was determined by logistic regression analysis. The carbapenem resistance was found in 8.06% of all isolates K. pneumoniae and 6.90% were carbapenemase producers. Among carbapenemases producers isolates, 77.78% were KPC type β-lactamase, 37.04% demonstrated the simultaneous production of plasmid AmpC β-lactamase and KPC, 29.63% associated with MβL or ESBL and KPC, 11.11% MβL/ESBL/KPC and 7.41% MβL/ESBL/KPC/plasmid AmpC. High percentages of not β-lactams antimicrobial resistance were observed, ranging from 61.10% - 74.70% to fluoroquinolones and 52.10% - 54.30% to aminoglycosides. Polymyxin B showed limited activity against the carbapenemase-producing K. pneumoniae (35.70%) and tigecycline was the only antimicrobial that inhibited 75.0% of these multidrug-resistant strains. The independent risk factors for K. pneumoniae carbapenemase producers were prolonged hospitalization (p = 0.031), previous use of carbapenems (p = 0.041) and exposure to more than three antimicrobials (p = 0.034). The detection of multidrug- resistant carbapenemase-producing isolates and the knowledge of different resistance mechanisms can result in significant impact on the treatment outcomes and strategies for infection control and prevention. / Klebsiella pneumoniae produtora de carbapenemase é uma enterobactéria, comumente associada à resistência aos múltiplos antimicrobianos. A presença deste fenótipo de multirresistência consiste em um importante desafio do ponto de vista epidemiológico, quanto ao manejo terapêutico e ao controle de infecção. Um estudo observacional retrospectivo foi conduzido em um hospital público terciário em Goiânia, Go, Brasil, no período entre 2006 e 2011, para investigar os fenótipos de carbapenemases, a atividade in vitro da tigeciclina e polimixina B e determinar os fatores de risco associados à infecção ou colonização por K. pneumoniae produtora de carbapenemase. A presença de serina carbapenemase foi detectada por meio do teste de Hodge modificado e disco combinado com ácido borônico; metalo-β-lactamases – MβL por sinergismo de disco duplo com imipenem e EDTA; β-lactamases de espectro estendido – ESBL, através do disco combinado com cefotaxima e ceftazidima com e sem ácido clavulânico e AmpC plasmidial por disco combinado com ácido borônico e cefoxitina. A atividade in vitro da tigeciclina e polimixina B foi avaliada através do método de microdiluição em caldo e dos antimicrobianos não β-lactâmicos por disco-difusão e sistema semiautomatizado. A associação entre fatores de risco independentes e colonização ou infecção foi determinada através de regressão logística. A resistência aos carbapenêmicos foi verificada em 8,06% do total de K. pneumoniae e a atividade de carbapenemase em 6,90%. Dentre os isolados produtores de carbapenemases, 77,78% foram considerados β-lactamase tipo KPC, 37,04% demonstraram a produção simultânea de AmpC plasmidial e KPC, 29,63% associação com MβL ou ESBL e KPC, 11,11% MβL/ESBL/KPC e 7,41% MβL/ESBL/KPC/AmpC plasmidial. Altas taxas de resistência aos antimicrobianos não β-lactâmicos foram observadas, variando de 61,10% - 74,70% para fluoroquinolonas e 52,10% - 54,30% para aminoglicosídeos. A polimixina B apresentou baixa atividade in vitro contra estes isolados (35,70%) e a tigeciclina foi o único antimicrobiano que inibiu 75,0% dos mesmos. Os fatores de risco independentes para K. pneumoniae produtora de carbapenemase foram a internação prévia prolongada (p = 0,031), uso prévio de carbapenêmicos (p = 0,041) e exposição a mais de três antimicrobianos (p = 0,034). A detecção de isolados produtores de carbapenemase multirresistentes e o conhecimento dos diferentes mecanismos de resistência podem resultar em significante impacto nos resultados terapêuticos e estratégias de controle de infecção e prevenção.

Early administration of broad-spectrum antibiotics is the first goal in sepsis treatment. Besides from bacteriostatic/bactericidal effects, some antibiotics may also modify the host´s response to infection. The novel antibiotic tigecycline may exert such properties; however, this property has not been evaluated in large-animal trials. We compared tigecycline with doxycycline and placebo in relation to anti-inflammatory, circulatory and organ dysfunction effects in a sterile pig model of sepsis. Doxycycline, but not tigecycline, reduced the inflammatory response as manifested by tumor necrosis factor alpha levels in plasma. Tigecycline, however, had a stabilizing effect on the circulation not exerted by doxycycline or placebo. To achieve rapid restoration of the circulating blood volume - another major goal in sepsis treatment - fluid bolus administration of is some-times practiced. In addition to crystalloids, albumin-containing solutions are suggested. Yet, some animal-experimental data suggests that rapid bolus administration of albumin reduces albumin’s plasma-expanding effect. We compared a rapid intravenous bolus of radiolabeled albumin with a slow infusion in a sterile pig model of sepsis. Rapid bolus of administration did not reduce plasma levels of albumin following administration and did not increase the amount of albumin that left the circulation. Inadequate oxygen delivery (DO2) by the circulation to the tissues may cause increased plasma lactate, which is the most striking effect of sepsis on the metabolism. However, experimental data and clinical trials refute this link, instead, suggesting other mechanisms, including impaired oxygen extraction, mitochondrial dysfunction and accelerated aerobic glycolysis. We investigated the impact of DO2, oxygen consumption (VO2), hemodynamic parameters and inflammatory response on plasma lactate and organ dysfunction in two experimental sepsis models. In the most severe cases of shock, with DO2, there was an increase in plasma lactate, but without a decrease in VO2, invalidating the assumption that the increase in lactate is due to anaerobic metabolism. To identify critical steps in the sepsis-induced increase in lactate, we inhibited the major energy-producing step in the electron transport chain (ETC). The combination of sepsis and ETC inhibition led to a cellular energy crisis. This finding suggests that early sepsis induces a partial mitochondrial dysfunction.

Introdução: O Clostridium difficile é um bacilo Gram-positivo, anaeróbio estrito, formador de esporos, que produz toxinas que podem causar diarreia, colite pseudomembranosa, dilatação do cólon, sepse e até morte. Nos últimos anos o quadro clínico e epidemiológico das infecções por Clostridium difficile tem se modificado e as limitações das opções terapêuticas tornaram-se mais evidentes. Objetivo Primário: Comparar as metodologias de disco-difusão e ágar-diluição na detecção de sensibilidade/resistência de isolados de Clostridium difficile. Objetivos Secundários: Avaliar prospectivamente o perfil de sensibilidade/resistência de isolados clínicos hospitalares de Clostridium difficile provenientes de seis hospitais terciários da cidade de São Paulo e fornecer evidências para fundamentar o diagnóstico e o tratamento empírico das diarreias causadas por Clostridium difficile. Métodos: utilizamos os métodos de disco-difusão e ágar-diluição, de acordo com os critérios estabelecidos pelo CLSI e EUCAST. Resultados: Os coeficientes de correlação observados entre os diâmetros dos halos de inibição e Concentração Inibitória Mínima foram abaixo do esperado tornando inviável o método de disco-difusão para determinação de sensibilidade aos antimicrobianos nitazoxanida, teicoplanina e tigeciclina. Todas as 50 cepas deste estudo foram sensíveis ao metronidazol (MIC50 foi de 1 ?g/mL a MIC90 foi de 2 ug/mL). Para o método de disco-difusão, sugerimos que halos de inibição >= 33mm possam ser interpretados como sensíveis. Devido à moderada correlação, significância estatística e distribuição de halos de inibição das amostras próximos aos valores encontrados utilizando a cepa ATTC, sugere-se a utilização do método de disco-difusão para vancomicina, onde halos com diâmetro >= 22mm possam ser considerados como sensíveis pelo método. Para o moxifloxacino houve uma boa correlação entre as duas metodologias: discodifusão e de ágar-diluição (O coeficiente de Pearson foi de -0,84, e o valor de p foi menor que 0,00001), sugerindo que halos de inibição >= 18mm possam ser interpretados como sensíveis pela metodologia de disco-difusão. A nitazoxanida foi à droga que mostrou melhor atividade in vitro (MIC50 foi 0,06 ?g/mL e a MIC90 de 0,12 ug/mL). Por se mostrar uma droga com potente atividade in vitro (MIC50 e a MIC90 foi de 0,12 ug/mL), a tigeciclina poderia ser mais uma opção terapêutica em infecções por Clostridium difficile, dependendo de mais estudos para avaliar sua real eficácia clínica e segurança. Conclusão: Os resultados verificados neste estudo indicam a necessidade de mais estudos in vitro e clínicos para definir os limites de sensibilidade/resistência para a teicoplanina e a nitazoxanida, pois faltam critérios de interpretação tanto para disco-difusão quanto para ágar-diluição. Os resultados deste trabalho in vitro confirmaram a utilidade do metronidazol como uma droga eficaz no tratamento de infecção por Clostridium difficile. A nitazoxanida foi à droga que mostrou melhor atividade in vitro por método dilucional. Sugerimos a utilização do método de disco-difusão para: metronidazol, vancomicina e moxifloxacino. Os resultados desse trabalho sugerem que halos de inibição para metronidazol ( >= 33mm), moxifloxacino ( >= 18mm) e vancomicina ( >= 22mm) poderiam ser considerados como sensíveis pelo método de disco-difusão. O método de ágardiluição é um método de boa acurácia, porém trabalhoso para ser executado na rotina laboratorial / Introduction: Clostridium difficile is a Gram-positive bacillus, strictly anaerobic, spore-forming, which produces toxins that can cause diarrhea, colitis pseudomembranous, colon expansion, sepsis and even death. In recent years the clinical and epidemiological picture of infection by Clostridium difficile has been modified and limitations of therapeutic options have become more evident. Primary Objective: Comparing the methods of disk diffusion and agar dilution in the detection sensitivity/resistance isolates of Clostridium difficile. Secondary Objectives: Prospectively evaluate the profile of sensitivity/resistance of hospital clinical isolates of Clostridium difficile from six tertiary hospitals in São Paulo city and provide evidence to support the diagnosis and empirical treatment of diarrhea caused by Clostridium difficile. Methods: We use the disk diffusion method and agar dilution method, according to the established criteria by CLSI and EUCAST. Results: The observed correlation coefficients between the inhibitions diameter zone of the and Minimum Inhibitory Concentration were under expectations impeding the disk diffusion method for determining sensitivity to nitazoxanide antimicrobial, teicoplanin and tigecycline. All 50 strains of this study were sensitive to metronidazole (MIC50 was 1 Ug/ml to MIC90 was 2 ug/ml). For the method disk diffusion, we suggest that inhibition zones >= 33mm can be interpreted as sensitive. Due to the moderate correlation, statistical significance and distribution of zones of inhibition on samples of the next found values using the strain ATTC, we suggest using the disk diffusion method for vancomycin where halos diameter >= 22mm can be considered as sensitive by the method. There was a good correlation to moxifloxacin between the two methodologies: disk diffusion and agar dilution (Pearson\'s coefficient was -0.84 , and the \"p\" value was less than 0.00001), suggesting that inhibition zones >= 18mm can be interpreted as sensitive by disk diffusion method. Nitazoxanide was the drug that showed a better performance in vitro activity (MIC50 was 0.06 ?g/ml and MIC90 0.12 ug/ml). For a drug that shows potent activity in vitro (MIC50 and MIC90 was 0.12 ug/ml), the tigecycline could be a therapeutic option in infection by Clostridium difficile, depending on further studies to evaluate their real clinical efficacy and security. Conclusion: Obtained results in this study indicate the need for further studies in vitro and clinicians to define the limits of sensitivity/resistance to teicoplanin and nitazoxanide, so there is no interpretation criteria for both disk diffusion and for agar dilution. Results of this work in vitro study confirmed the utility of metronidazole as an effective drug in the treatment of infection by Clostridium difficile. Nitazoxanide was the drug that showed better performance in vitro by dilutional method. We suggest the use of disk diffusion method: metronidazole, vancomycin and moxifloxacin. This work suggest that inhibition zones for metronidazole ( >= 33mm), moxifloxacin ( >= 18mm) and vancomycin ( >= 22mm) could be considered as sensitive by disk diffusion method. The agar dilution method is a method to be accurate, but laborious to run in the laboratory routine

Introdução: O Clostridium difficile é um bacilo Gram-positivo, anaeróbio estrito, formador de esporos, que produz toxinas que podem causar diarreia, colite pseudomembranosa, dilatação do cólon, sepse e até morte. Nos últimos anos o quadro clínico e epidemiológico das infecções por Clostridium difficile tem se modificado e as limitações das opções terapêuticas tornaram-se mais evidentes. Objetivo Primário: Comparar as metodologias de disco-difusão e ágar-diluição na detecção de sensibilidade/resistência de isolados de Clostridium difficile. Objetivos Secundários: Avaliar prospectivamente o perfil de sensibilidade/resistência de isolados clínicos hospitalares de Clostridium difficile provenientes de seis hospitais terciários da cidade de São Paulo e fornecer evidências para fundamentar o diagnóstico e o tratamento empírico das diarreias causadas por Clostridium difficile. Métodos: utilizamos os métodos de disco-difusão e ágar-diluição, de acordo com os critérios estabelecidos pelo CLSI e EUCAST. Resultados: Os coeficientes de correlação observados entre os diâmetros dos halos de inibição e Concentração Inibitória Mínima foram abaixo do esperado tornando inviável o método de disco-difusão para determinação de sensibilidade aos antimicrobianos nitazoxanida, teicoplanina e tigeciclina. Todas as 50 cepas deste estudo foram sensíveis ao metronidazol (MIC50 foi de 1 ?g/mL a MIC90 foi de 2 ug/mL). Para o método de disco-difusão, sugerimos que halos de inibição >= 33mm possam ser interpretados como sensíveis. Devido à moderada correlação, significância estatística e distribuição de halos de inibição das amostras próximos aos valores encontrados utilizando a cepa ATTC, sugere-se a utilização do método de disco-difusão para vancomicina, onde halos com diâmetro >= 22mm possam ser considerados como sensíveis pelo método. Para o moxifloxacino houve uma boa correlação entre as duas metodologias: discodifusão e de ágar-diluição (O coeficiente de Pearson foi de -0,84, e o valor de p foi menor que 0,00001), sugerindo que halos de inibição >= 18mm possam ser interpretados como sensíveis pela metodologia de disco-difusão. A nitazoxanida foi à droga que mostrou melhor atividade in vitro (MIC50 foi 0,06 ?g/mL e a MIC90 de 0,12 ug/mL). Por se mostrar uma droga com potente atividade in vitro (MIC50 e a MIC90 foi de 0,12 ug/mL), a tigeciclina poderia ser mais uma opção terapêutica em infecções por Clostridium difficile, dependendo de mais estudos para avaliar sua real eficácia clínica e segurança. Conclusão: Os resultados verificados neste estudo indicam a necessidade de mais estudos in vitro e clínicos para definir os limites de sensibilidade/resistência para a teicoplanina e a nitazoxanida, pois faltam critérios de interpretação tanto para disco-difusão quanto para ágar-diluição. Os resultados deste trabalho in vitro confirmaram a utilidade do metronidazol como uma droga eficaz no tratamento de infecção por Clostridium difficile. A nitazoxanida foi à droga que mostrou melhor atividade in vitro por método dilucional. Sugerimos a utilização do método de disco-difusão para: metronidazol, vancomicina e moxifloxacino. Os resultados desse trabalho sugerem que halos de inibição para metronidazol ( >= 33mm), moxifloxacino ( >= 18mm) e vancomicina ( >= 22mm) poderiam ser considerados como sensíveis pelo método de disco-difusão. O método de ágardiluição é um método de boa acurácia, porém trabalhoso para ser executado na rotina laboratorial / Introduction: Clostridium difficile is a Gram-positive bacillus, strictly anaerobic, spore-forming, which produces toxins that can cause diarrhea, colitis pseudomembranous, colon expansion, sepsis and even death. In recent years the clinical and epidemiological picture of infection by Clostridium difficile has been modified and limitations of therapeutic options have become more evident. Primary Objective: Comparing the methods of disk diffusion and agar dilution in the detection sensitivity/resistance isolates of Clostridium difficile. Secondary Objectives: Prospectively evaluate the profile of sensitivity/resistance of hospital clinical isolates of Clostridium difficile from six tertiary hospitals in São Paulo city and provide evidence to support the diagnosis and empirical treatment of diarrhea caused by Clostridium difficile. Methods: We use the disk diffusion method and agar dilution method, according to the established criteria by CLSI and EUCAST. Results: The observed correlation coefficients between the inhibitions diameter zone of the and Minimum Inhibitory Concentration were under expectations impeding the disk diffusion method for determining sensitivity to nitazoxanide antimicrobial, teicoplanin and tigecycline. All 50 strains of this study were sensitive to metronidazole (MIC50 was 1 Ug/ml to MIC90 was 2 ug/ml). For the method disk diffusion, we suggest that inhibition zones >= 33mm can be interpreted as sensitive. Due to the moderate correlation, statistical significance and distribution of zones of inhibition on samples of the next found values using the strain ATTC, we suggest using the disk diffusion method for vancomycin where halos diameter >= 22mm can be considered as sensitive by the method. There was a good correlation to moxifloxacin between the two methodologies: disk diffusion and agar dilution (Pearson\'s coefficient was -0.84 , and the \"p\" value was less than 0.00001), suggesting that inhibition zones >= 18mm can be interpreted as sensitive by disk diffusion method. Nitazoxanide was the drug that showed a better performance in vitro activity (MIC50 was 0.06 ?g/ml and MIC90 0.12 ug/ml). For a drug that shows potent activity in vitro (MIC50 and MIC90 was 0.12 ug/ml), the tigecycline could be a therapeutic option in infection by Clostridium difficile, depending on further studies to evaluate their real clinical efficacy and security. Conclusion: Obtained results in this study indicate the need for further studies in vitro and clinicians to define the limits of sensitivity/resistance to teicoplanin and nitazoxanide, so there is no interpretation criteria for both disk diffusion and for agar dilution. Results of this work in vitro study confirmed the utility of metronidazole as an effective drug in the treatment of infection by Clostridium difficile. Nitazoxanide was the drug that showed better performance in vitro by dilutional method. We suggest the use of disk diffusion method: metronidazole, vancomycin and moxifloxacin. This work suggest that inhibition zones for metronidazole ( >= 33mm), moxifloxacin ( >= 18mm) and vancomycin ( >= 22mm) could be considered as sensitive by disk diffusion method. The agar dilution method is a method to be accurate, but laborious to run in the laboratory routine