Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-09-27T14:21:32Z No. of bitstreams: 1 osmarnascimentosilva.pdf: 5346948 bytes, checksum: c6c4bb9eae4172e04c4553f57d4e27d0 (MD5) / Approved for entry into archive by Diamantino Mayra (mayra.diamantino@ufjf.edu.br) on 2016-09-27T15:15:36Z (GMT) No. of bitstreams: 1 osmarnascimentosilva.pdf: 5346948 bytes, checksum: c6c4bb9eae4172e04c4553f57d4e27d0 (MD5) / Made available in DSpace on 2016-09-27T15:15:36Z (GMT). No. of bitstreams: 1 osmarnascimentosilva.pdf: 5346948 bytes, checksum: c6c4bb9eae4172e04c4553f57d4e27d0 (MD5) Previous issue date: 2015-04-28 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Com o aumento na incidência de infecções resistentes a múltiplos antibióticos, existe hoje um grande interesse pelos peptídeos antimicrobianos (PAMs) como modelos para a produção de novos antibióticos. Os PAMs são mediadores multifuncionais da resposta imune inata, com atividade antibacteriana direta. O uso de PAMs como agentes terapêuticos tem algumas limitações, como a estabilidade, a toxicidade e alta massa molecular. Apesar dessas limitações, eles apresentam propriedades compensatórias, como imunomodulatória e antitumoral bem como a capacidade de inibir β-lactamases. O desenho racional de PAMs tem sido usado para gerar análogos com atividade melhorada. No presente trabalho avaliamos a atividade antibacteriana in vitro e in vivo da clavanina A e através da modificação incremental criamos dois análogos dos peptídeos mostoparano-L e clavanina A (clavanina-MO e mastoparanoMO),além disso, utilizamos o desenho racional de peptídeos para a criação de dois inibidores de β-lactamase (dBLIPs 1 e 2). A clavanina A mostrou se eficiente na eliminação de S. aureus em um modelo de infecção de ferida e impediu o início da sepse e, assim, reduziu a mortalidade de camundongos infectados em um modelo de infecção bacteriana sistêmica. A clavanina-MO e mastoparano-MO impediram o crescimento de bactérias planctônicas e levaram à erradicação de biofilmes bacterianos maduros. Os peptídeos modificados mostram-se promissores como agentes terapêuticos contra infecções bacterianas sistêmicas e biofilmes causadas por uma variedade de bactérias. dBLIP-1 e dBLIP-2 em combinação com antibióticos convencionais foram eficazes na eliminação de Escherichia coli e Staphylococcus aureus que expressam β-lactamases em um modelo murino de infecção sistêmica. dBLIPs 1 e 2 fornecem pistas para superar a resistência à base de β-lactamase. / With the increased incidence of multiple antibiotic resistant infections, there is huge interesting in antimicrobial peptides (AMPs) as templates to produce novel antibiotics. The AMPs are multifunctional mediators of innate immune response with direct antibacterial activities. Nevertheless, the use of AMPs as therapeutic agents has certain limitations such as stability, toxicity and high molecular mass. Despite such limitations, they show additional properties such as antitumor and immunomodulatory as well as the ability to inhibit β-lactamases. Furthermore, the rational AMPs design has been used to produce analogues with improved activity. In the present study, we utilized the rational AMPs design for generation of two β-lactamase inhibitors (dBLIPs 1 and 2) and through two incremental modification created analogues of clavanin A and mostoparan-L (clavanin-MO and mastoparan-MO respectively) peptides. Both inhibitors in combination with conventional antibiotics were effective for control of Staphylococcus aureus and Escherichia coli expressing β-lactamase in a murine model of systemic infection. dBLIPs 1 and 2 provide clues to overcome resistance to β-lactamase base. The clavanin-MO and mastoparan-MO prevented the growth of planktonic bacteria, leading to the mature biofilm eradication of pathogenic Gram-negative and -positive. The clavanina-MO and mastoparan-MO are promising therapeutic agents against systemic infections and bacterial biofilms caused by a wide bacterial variety.

The use of reporter gene fusions to assess cellular processes such as protein targeting and regulation of transcription or translation is established technology in archaeal, bacterial, and eukaryal genetics. Fluorescent proteins or enzymes resulting in chromogenic substrate turnover, like β-galactosidase, have been particularly useful for microscopic and screening purposes. However, application of such methodology is of limited use for strictly anaerobic organisms due to the requirement of molecular oxygen for chromophore formation or color development. We have developed β-lactamase from Escherichia coli (encoded by bla) in conjunction with the chromogenic substrate nitrocefin into a reporter system usable under anaerobic conditions for the methanogenic archaeon Methanosarcina acetivorans. By using a signal peptide of a putative flagellin from M. acetivorans and different catabolic promoters, we could demonstrate growth substrate-dependent secretion of β-lactamase, facilitating its use in colony screening on agar plates. Furthermore, a series of fusions comprised of a constitutive promoter and sequences encoding variants of the synthetic tetracycline-responsive riboswitch (tc-RS) was created to characterize its influence on translation initiation in M. acetivorans. One tc-RS variant resulted in more than 11-fold tetracycline-dependent regulation of bla expression, which is in the range of regulation by naturally occurring riboswitches. Thus, tc-RS fusions represent the first solely cis-active, that is, factor-independent system for controlled gene expression in Archaea.

No description available.

Trafficking of GPCRs is a dynamic process that is tightly regulated and sometimes defective in human diseases. Therefore it is important to develop new methods to allow simple and quantitative measurement of surface expression of membrane proteins. Here we describe the development and validation of a new assay for quantification of cell surface expression of GPCRs using β-lactamase as a reporter. For this assay we N-terminally fused β-lactamase (βlac) to the β2-adrenergic receptor (β2AR) and GABA b R1 (GBR1). The results obtained by the βlac assay are quantitatively and qualitatively similar to well established ELISA when measuring agonist induced internalization of β2AR. We also show that measurement of GBR1 surface expression with GBR2 co-expression is quantitatively identical between the βlac and ELISA. In conclusion, our results show that our newly developed βlac assay is quantitatively similar while being less expensive, more robust and higher throughput compared to an ELISA.

Trafficking of GPCRs is a dynamic process that is tightly regulated and sometimes defective in human diseases. Therefore it is important to develop new methods to allow simple and quantitative measurement of surface expression of membrane proteins. Here we describe the development and validation of a new assay for quantification of cell surface expression of GPCRs using β-lactamase as a reporter. For this assay we N-terminally fused β-lactamase (βlac) to the β2-adrenergic receptor (β2AR) and GABA b R1 (GBR1). The results obtained by the βlac assay are quantitatively and qualitatively similar to well established ELISA when measuring agonist induced internalization of β2AR. We also show that measurement of GBR1 surface expression with GBR2 co-expression is quantitatively identical between the βlac and ELISA. In conclusion, our results show that our newly developed βlac assay is quantitatively similar while being less expensive, more robust and higher throughput compared to an ELISA.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / In recent years, the isolation of bacteria producing β-lactamases has caused concern around the world, due to the fact these enzymes hydrolysis the ring β-lactam antimicrobials used in the main clinic. This aim of this study was asses the prevalence metallo-β-lactamases (MbL) in isolates of Pseudomonas aeruginosa and Acinetobacter baumannii obtained from patients admitted at the University Hospital of Santa Maria (HUSM). The profile of susceptibility for all isolates was evaluated by the disk diffusion method standardized by CLSI. The antimicrobial disks were distributed in a way that allows the identification of strains producers of AmpC and ESBL. For the identification of the producers of MbL the test of disk approximation with EDTA 0.1 M, EDTA 0,5M and acid 2-mercaptopropionic were performed. Isolates that did not have any of the mechanisms of resistance search were classified as multiresistant (MDR). The minimum inhibitory concentration (MIC) for ceftazidima, imipenem and polymyxin B was assessed by broth method microdilution for all isolated, according to CLSI. From January to June 2006, were obtained 32 isolates the P.aeruginosa and 41 the A. baumannii, the those 17 (23.29%) were β-lactamase AmpC-type producers, 11 (15.07%) were MbL producers, and 45 (61,64%) were classified as MDR. All strains producing MbL were Pseudomonas aeruginosa. The sensitivity of the isolates according to the CIM for antimicrobial evaluated were: 90,28% for polymyxin B, 36,11% for imipenem and 18% for ceftazidima. There was a high prevalence of MDR isolates and producers of β-lactamase-type AmpC and MbL in HUSM, this is extremely worrying once there is limiting therapy available. This situation becomes even more worrying with the find of isolates resistant the polymyxin B, witch is one of the last options of treatment for MDR isolates and producers of MbL. The detection of microorganisms is extremely important for the committees of infection hospital with the goal of preventing outbreaks, as well as guide the medical team on the conduct therapy, since there are few effective antimicrobial clinically for these pathogens and no prospects for development the new antimicrobial in the near future. / Nos últimos anos, o isolamento de bactérias produtoras de β-lactamases tem causado preocupação em todo o mundo, devido ao fato dessas enzimas hidrolisarem o anel β- lactâmico dos principais antimicrobianos utilizados na clínica. Este trabalho teve por objetivo avaliar a prevalência de metalo-β-lactamases (MbL) em isolados de Pseudomonas aeruginosa e Acinetobacter baumannii obtidos de pacientes atendidos no Hospital Universitário de Santa Maria (HUSM). O perfil de sensibilidade para todos os isolados foi avaliado pelo método de disco difusão padronizado pelo CLSI. Os discos de antimicrobianos utilizados foram distribuídos de forma que permitisse a identificação dos isolados produtores de AmpC e ESBL. Para a identificação dos produtores de MbL utilizou-se o teste de disco aproximação com os seguintes agentes quelantes: EDTA 0,1M, EDTA 0,5 M e ácido 2-mercaptopropiônico. Os isolados que não possuíam nenhum dos mecanismos de resistência pesquisados foram classificados como multirresistentes (MDR). A concentração inibitória mínima (CIM) para ceftazidima, imipenem e polimixina B foi avaliada pelo método de microdiluição em caldo para todos os isolados, de acordo com o CLSI. Durante o período de janeiro a junho de 2006 foram obtidos 32 isolados de P.aeruginosa e 41 de A. baumannii, destes 17 (23,29%) foram produtores de β-lactamase do tipo AmpC, 11 (15,07%) foram produtores de MbL e 45 (61,64%) foram classificados como MDR. Todas as cepas produtoras de MbL foram de Pseudomonas aeruginosa. A sensibilidade dos isolados de acordo com a CIM para os antimicrobianos avaliados foram as seguintes: 90,28% para polimixina B, 36,11% imipenem e 18% ceftazidima. Observou-se uma alta prevalência de isolados MDR no HUSM, além de isolados produtores de β-lactamase do tipo AmpC e MbL, o que é extremamente preocupante devido limitar a terapia a poucos antimicrobianos. Esta situação torna-se ainda mais preocupante com a detecção de isolados resistentes a polimixina B, a qual é uma das últimas opções de tratamento para infecções causadas por isolados de P. aeruginosa e Acinetobacter baumannii MDR e produtores de MbL. A detecção desses microrganismos é de grande importância para as comissões de controle de infecção hospitalar com o objetivo de prevenir surtos, bem como orientar a equipe médica sobre a conduta terapêutica, uma vez que há poucos antimicrobianos efetivos clinicamente para esses patógenos e as perspectivas para o desenvolvimento de novos antimicrobianos em um futuro próximo são mínimas.

Extended-spectrum β-lactamases (ESBLs) constitute a major problem given their broad substrate specificity and ability to hydrolyse many of the extended-spectrum third-generation cephalosporins currently in use in hospital settings. Guiana extended-spectrum-type (GES-1 – GES-9) ESBL enzymes have mainly been found in Pseudomonas aeruginosa (P. aeruginosa) and only at a limited number of geographical sites, mainly France, Greece and South Africa. Detection of GES-type ESBL-producing P. aeruginosa isolates in the clinical microbiology laboratory using conventional methods is problematic with molecular methods yielding better results. The aim of this study was to utilise various molecular techniques to determine the prevalence of GES-type ESBLs, characterise their genetic determinants and determine their clonal relatedness. The study further aimed to apply a sequence-selective, competitive PNA-based multiplex PCR in real-time for the identification and differentiation of GES-type enzymes. The prevalence of GES-type ESBLs was determined successfully through DNA sequencing. An increase in GES-2 prevalence since 2000 was noted which emphasised the importance of constant surveillance to monitor antibiotic determinants, their spread and overall prevalence. The knowledge on prevalence could be used in turn to monitor the efficacy of infection control measures and antibiotic regimens. Repeated sequencing confirmed the presence of blaGES-5 in P. aeruginosa isolates. As far as could be established, this study reported the first occurrence of GES-5 in South Africa and was the second description of GES-5 in P. aeruginosa. Application of a sequence-specific, competitive PNA-based multiplex PCR in real-time utilising SYBR Green was not suitable for the identification and differentiation of the blaGES genes. Although the method achieved different melting temperatures for the bla<GES genes tested, these temperatures were not suitable for accurate differentiation. Melting temperatures obtained for the same blaGES gene varied and those for different genes overlapped. An approach exploiting the high temperature shift caused by the PNA-probe rather than its competitive nature might be more successful. Random amplified polymorphic DNA typing has been described as a fast and simple method with high discriminatory power for the typing of P. aeruginosa and was thus used to determine the clonal relatedness of the bla<GES positive P. aeruginosa isolates. The occurrence of identical or similar P. aeruginosa isolates producing ESBLs in a single hospital setting emphasised the importance of constant surveillance. The study further identified identical P. aeruginosa clones that occurred in different hospitals indicating spread from a common external reservoir into these hospitals. The occurrence of highly drug-resistant P. aeruginosa in the environment has serious implications in a country with an ever increasing immune-compromised population. These finding were of concern since they demonstrated that acquired GES ESBLs can rapidly emerge and become a major cause of broad-spectrum β-lactam resistance among nosocomial pathogens. The information obtained in this study should be used to create awareness of the potential ESBL problem threatening current antimicrobial regimens in South Africa. / Dissertation (MSc (Medical Microbiology))--University of Pretoria, 2008. / Medical Microbiology / unrestricted

SYNTHESIS AND BILOGICAL SIGNIFICANCE OF 1,2,4-OXADIAXOLIDIN-5-ONE Chimdi kalu, Austin Miller and Dr. Abbas G. Shilabin Department of Chemistry, East Tennessee State University, Johnson City, TN 37614. ABSRTACT Due to the challenge posed by microbial resistance to broad spectrum of antibiotics, there has been a great need to synthesize of a novel compound which has a different mechanism of action on microbial activity. 1,2,4-oxadiaxolidin-5-One constitute an important class of compound with tremendous potential as pharmaceutical and otherwise biologically relevance substance due to the fact that its five member ring is a configurationally stable building block. This unit is found in other compound like alkaloids, with vast medical application. This study describes the synthesis of 1,2,4-oxadiaxolidin-5-one in two-step procedure using nitroethane and benzaldehyde as starting materials to produce nitrone, which in turn undergoes 1,3- dipolar cycloaddition with phenyl isocyanate to give 1,2,4-oxadiaxolidin-5-one. The product was characterized using proton NMR and GC-MS. There is an ongoing investigation on the summary of some important inhibitory activity against class A β-lactamase by 1,2,4-oxadiaxolidin-5-one heterocyclic core structure to provide effective antimicrobial β-lactamase inhibitors, hence, solving the problem of microbial resistance to currently used antimicrobial drugs.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21958号 / 医博第4500号 / 新制||医||1037(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 中川 一路, 教授 松原 和夫, 教授 西渕 光昭 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Gene expression of porin and beta-lactamases genes during the beta-lactam antibiotic treatment and effect of inoculum size on Klebsiella pneumoniae clinical isolates ABSTRACT In recent years, Klebsiella pneumoniae has been increasingly reported to be one of the most important nosocomial pathogens, and it is usually resistant to many antibiotics. In this work, we focused on the expression of the AmpC group β- lactamase DHA-1 and its negative regulator AmpR, as well as the porins OmpK35 and OmpK36 and on effect of inoculum. We used well-characterized Klebsiella pneumoniae strains in this study. Plasmids obtained from these strains were also transformed into different wild-type Klebsiella pneumoniae strains, which were typed by pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). Gene expression analysis was performed by RT-PCR using specific primers and TaqMan probes. In most strains, expression was dependent on the presence of an inducer. The highly resistant strain showed a different expression pattern, but the expression of blaDHA-1 remained inducible by cefoxitin. Different regulation was also observed in the transformants. Based on our data, we suggest that the previously described regulatory pathway for AmpC is not generally suitable, and we propose that there are more...