Submitted by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2018-04-19T15:07:55Z No. of bitstreams: 4 Dissertação Parcial (Cap. 1-4) - Luma C. Loureiro.pdf: 1497392 bytes, checksum: 13f91a02fd24dc31022afc14786f874e (MD5) Reprodução Não Autorizada.pdf: 47716 bytes, checksum: 0353d988c60b584cfc9978721c498a11 (MD5) Dissertação Parcial (Cap. 7) - Luma C. Loureiro.pdf: 260521 bytes, checksum: 479ddd5a004985a4fea7ef881128da94 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2018-04-19T15:08:50Z (GMT) No. of bitstreams: 4 Dissertação Parcial (Cap. 1-4) - Luma C. Loureiro.pdf: 1497392 bytes, checksum: 13f91a02fd24dc31022afc14786f874e (MD5) Reprodução Não Autorizada.pdf: 47716 bytes, checksum: 0353d988c60b584cfc9978721c498a11 (MD5) Dissertação Parcial (Cap. 7) - Luma C. Loureiro.pdf: 260521 bytes, checksum: 479ddd5a004985a4fea7ef881128da94 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-04-19T15:08:50Z (GMT). No. of bitstreams: 4 Dissertação Parcial (Cap. 1-4) - Luma C. Loureiro.pdf: 1497392 bytes, checksum: 13f91a02fd24dc31022afc14786f874e (MD5) Reprodução Não Autorizada.pdf: 47716 bytes, checksum: 0353d988c60b584cfc9978721c498a11 (MD5) Dissertação Parcial (Cap. 7) - Luma C. Loureiro.pdf: 260521 bytes, checksum: 479ddd5a004985a4fea7ef881128da94 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-06-27 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Gram-negative bacteria, such as Klebsiella pneumoniae, have LPS in their membranes, a component that triggers inflammatory process, by cells of the host immune system such as macrophages (MA), produced cytokines, chemokines and lipid mediators. Inflammation is important in preventing microorganisms, but the exacerbation can cause serious tissue damage. Indeed, MA are also important in the control of infections, phagocytosis and death of microorganisms. Membrane lipids have been the subjects of studies to development of new pharmacological therapies (Membrane Lipid Therapy - MLT). On this way, lipid of surfactant liquid (LS) were target to development pulmonary MLT. Among the most abundant phospholipids in LS, we found POPC, a phosphatidylcholine (PC), the oxidized phospholipids product, such as PaldoPC. In this work, our aim was to evaluate the effects of LS-derived PCs on modulation of polarization (M1 and M2), inflammatory and phagocytic activity of macrophages against K. pneumoniae. Our results demonstrated that POPC and PaldoPC increase NO production when M1 are stimulated with LPS. Indeed, the treatment with PC increased the production of inflammatory cytokines and chemokines in the M0 and M1 profile, post LPS-stimulated. The increment on inflammatory mediators production by MA treated with PC, correlated with the increased on gene expression of TLR2, TLR4, and MYD88. The treatment with POPC increased phagocytosis of K. pneumoniae, corroborating with the increased production of PGD2. However, PaldoPC inhibited the phagocytosis and increased the production of PGE2. Our data suggest that PC did not have inflammatory effect direct on MA, but increased the MA response against LPS, increasing expression of innate immune receptors, and modulation of prostaglandins production; influenced the pulmonary microenvironment immune response. / Bactérias Gram-negativas, como Klebsiella pneumoniae, possuem LPS em suas membranas, componente responsável por desencadear processo inflamatório por células do sistema imune como os macrófagos (MA), produzindo citocinas, quimiocinas e mediadores lipídicos. A inflamação é importante no combate aos microrganismos, mas a exacerbação deste evento pode causar sérios prejuízos ao tecido. Da mesma forma, os MA são importantes no controle de infecções, atuando na fagocitose e morte de microrganismos. Os lipídios de membrana têm sido alvo de estudos para o desenvolvimento de novas terapias farmacológicas (Terapia de Lipídios de Membrana - TLM). Neste sentindo, os lipídios do líquido surfactante (LS) são alvos para desenvolvimento de TLM pulmonar. Dentre os fosfolipídios mais abundantes do LS, encontramos o POPC, uma fosfatidilcolina (PC), e também seus produtos oxidados, como PaldoPC. Neste trabalho, nosso objetivo foi avaliar os efeitos das PC derivadas do LS na modulação da polarização (M1 e M2), atividade inflamatória e fagocítica de MA contra K. pneumoniae. Nossos resultados demonstram que POPC e PaldoPC aumentaram a produção de NO nos M1 estimulados com LPS. Da mesma forma, o tratamento com as PC aumentaram a produção de citocinas e quimiocinas inflamatórias no perfil M0 e M1 pós-estimulados com LPS. O aumento da produção de mediadores inflamatórios nos MA tratados com PC, correlacionou com o aumento da expressão gênica de TLR2, TLR4, e MYD88. Além disso, o tratamento com POPC aumentou a fagocitose de K. pneumoniae, corroborando com o aumento produção de PGD2. No entanto, o tratamento com PaldoPC inibiu a fagocitose, e aumentou a produção de PGE2. Nossos dados sugerem que PC não tem efeito inflamatório direto sobre MA, mas, potencializa a resposta de MA com LPS, aumentando a expressão de receptores da imunidade inata e modulando a produção de prostaglandinas; influenciando a resposta imune do microambiente pulmonar.

Made available in DSpace on 2014-06-12T18:30:31Z (GMT). No. of bitstreams: 2 arquivo3565_1.pdf: 5205665 bytes, checksum: d3f5d67863a34c85659602984a49cb65 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2009 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Klebsiella pneumoniae é uma enterobactéria responsável por uma alta incidência de infecções hospitalares, causadas geralmente por linhagens portadoras de multirresistência e produtoras de -lactamases de amplo espectro (ESBL). Este trabalho teve como objetivo determinar os genes envolvidos na formação de beta-lactamases clássicas e ESBLs em isolados de K. pneumoniae de Recife-PE, Brasil, assim como investigar o efeito ultra-estrutural causado por diferentes concentrações de antibióticos beta-lactâmicos, em isolados de K. pneumoniae produtores de diferentes tipos de betalactamases. Foram analisados 52 isolados de K. pneumoniae quanto a presença dos genes blaSHVe blaTEM, originados da microbiota normal e de infecções na comunidade e hospitalares e para a investigação do gene blaCTX-M foram utilizados 19 isolados clínicos de K. pneumoniae que apresentaram resistência a cefalosporinas de terceira geração ou ao aztreonam. Os MICs dos isolados foram determinados frente aos antibióticos ceftazidima, cefotaxima e aztreonam. Dois isolados de infecção nosocomial de K. pneumoniae foram submetidos a diferentes Sub-MICs de ceftazidima, cefotaxima e aztreonam para a análise pela Microscopia Eletrônica de Transmissão e de Varredura (MET e MEV). O gene blaSHV foi detectado em 16 isolados hospitalares, 4 da comunidade e 9 da microbiota, enquanto o gene blaTEM foi detectado em 18 isolados hospitalares, 4 da microbiota e 2 da comunidade. Através do sequenciamento de DNA, o gene blaCTX-M2 foi detectado em três isolados resistentes tanto a ceftazidima quanto a cefotaxima. Adicionalmente, foi encontrada uma nova variante SHV em um dos isolados e a presença de duas variantes anteriormente relatadas a SHV-28 e a SHV-108. As análises dos 2 isolados de K. pneumoniae pela MET e MEV mostraram grandes alterações morfológicas e ultra-estruturais dos isolados, quando submetidos aos diferentes Sub-MICs de ceftazidima e cefotaxima. *Nossos resultados mostram que isolados de K. pneumoniae portadores de diferentes β-lactamases e resistentes a diferentes antimicrobianos podem sofrer alterações ultra-estruturais significativas quando submetidos a Sub-MICs da droga, o que poderia possibilitar uma melhor atuação do sistema imune de um paciente infectado com esta espécie bacteriana

In healthcare settings and beyond, the ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) among other pathogens exchange antibiotic resistance and virulence factors and emerge as new infectious clones. According to the Saudi General Authority for Statistics (stats.gov.sa), Saudi Arabia is a country where more than 27 million pilgrims meet in annual continual mass-gathering events. This massive influx of people could introduce novel pathogens to the community that could not necessarily be detected with traditional culture-dependent clinical microbiological tests. Conventional clinical microbiology and environmental pathogen detection methods have had many limitations and narrow search scope. These methods can only target known and culturable pathogens. Over the past decade, applications of next-generation sequencing (NGS) and bioinformatics tools have revolutionized the way pathogens are detected and their relevant phenotypes such as clonal types, antibiotic resistance are predicted to aid in clinical decision making as additional practice to traditional clinical microbiology-based testing protocols. The aim of this study was to apply whole-genome sequencing (WGS) and bioinformatic analysis tools on clinical samples and bacterial isolates in order to pave the way for transforming current clinical microbiology practices in a tertiary referral hospital in Jeddah, Saudi Arabia. My attempt to utilize WGS as a tool on pathogenic strains in this study combined with the clinical data has resulted in discovering a silent outbreak of an emerging hypervirulent strain of Klebsiella pneumoniae (Chapter 2). Analysis of the strains antimicrobial profiles genetically has yielded the first characterization of a misidentified Klebsiella quasipneumoniae harboring plasmid-mediated carbapenemases of Klebsiella pneumoniae carbapenemases (KPC) (Chapter 3). Similarly, I was able to study mobile colistin resistance genes in the isolates and identify a novel occurrence of mcr-1 and mcr-8 (Chapter 4). I applied clinical metagenomic protocol on an intestinal biopsy of an inflammatory bowel disease patient with Crohn’s disease, where I identified an association of three co-occurring and an actively replicating non-tuberculosis mycobacteria (Chapter 5). The deployment of whole-genome sequencing and metagenomic in infectious disease surveillance and diagnostics could prove beneficial in limiting epidemics and detect transmission patterns of antimicrobial-resistant genes.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第21819号 / 農博第2332号 / 新制||農||1067(附属図書館) / 学位論文||H31||N5191(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 三上 文三, 教授 植田 充美, 教授 宮川 恒 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

This master thesis deals with analysis of structural variation of genome and with methods of its sequencing across all generations. Subsequently it contains a description of copy number variation and methods of its detection. The experimental part focuses on algorithm proposal for CNV detection according analysis and testing of uneven coverage in genome, variable representation of GC content and distance of sequence reads. Finally, the algorithm for detecting copy number variation is tested on genomic data of bacteria Klebsiella pneumoniae.

Orientador: Cristiane Angélica Ottoni / Resumo: A utilização de nanopartículas de prata (AgNP) biológicas nos dias atuais é uma alternativa promissora frente as obtidas por via sintética (VS), uma vez que, não geram resíduos em seu processo de síntese e possuem superfície revestida por proteínas que viabilizam sua incorporação em diversos compostos comerciais, sendo os fármacos os de maior destaque. O efeito do descarte de AgNP sintéticas no meio ambiente é atualmente fonte de investigação de diversos grupos de pesquisa, entretanto, são restritas as informações associadas as AgNP biológicas. Diante deste contexto, o presente estudo utilizou a AgNP IBCLP20 biossintetizada pelo fungo Aspergillus niger para avaliar a sua ação antimicrobiana e efeito tóxico em ambiente aquático dulcícula utilizando organismos pertencentes a diferentes níveis tróficos. Nos ensaios antimicrobianos a AgNP IBCLP20 apresentou excelente ação antibacteriana em uma faixa de concentração de 5 a 100 µg/mL e ação antifúngica na faixa de concentração de 20 a 100 µg/mL. A densidade celular da microalga Chrorella vulgaris, quando exposta a AgNP IBCLP20 e ao sal precursor AgNO3, na maior concentração analisada (100 uM/mL) e após 96 horas de incubação, apresentou uma taxa de redução de 34,4% e 85,71%, respectivamente. Nas mesmas condições supracitadas, só foi detectada viabilidade celular da microalga quando exposta a AgNP biológica (65,38%). A letalidade média em D. silimis causada pelas AgNP IBCLP20 foi estimada a uma concentração de 4,06 μg/L (2,29 -6,42).... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The use of biological silver nanoparticles (AgNP) today is a promising alternative compared to those obtained synthetically (VS), since they do not generate residues in their synthesis process and have a surface coated with proteins that enable their incorporation in several commercial compounds, with drugs being the most prominent. The effect of discarding synthetic AgNP in the environment is currently a source of investigation for several research groups, however, information associated with biological AgNP is restricted. In this context, the present study used the AgNP IBCLP20 biosynthesized by the fungus Aspergillus niger to evaluate its antimicrobial action and toxic effect in aquatic dulcicle environment using organisms belonging to different trophic levels. In antimicrobial tests, AgNP IBCLP20 showed excellent antibacterial action in a concentration range of 5 to 100 µg / mL and antifungal action in the concentration range of 20 to 100 µg / mL. The cell density of the microalgae Chrorella vulgaris, when exposed to AgNP IBCLP20 and the precursor salt AgNO3, in the highest concentration analyzed (100 uM / mL) and after 96 hours of incubation, showed a reduction rate of 34.4% and 85.71 %, respectively. Under the same conditions mentioned above, cell viability of the microalgae was only detected when exposed to biological AgNP (65.38%). The average lethality in D. silimis caused by AgNP IBCLP20 was estimated at a concentration of 4.06μg/L (2.29 -6.42). , The physiological ... (Complete abstract click electronic access below) / Mestre

No description available.

Genetic typing of Klebsiella pneumoniae is used for epidemiological referencing. In the clinical setting it can be useful in outbreak investigations, understanding transmission and managing hospital infections. Multi-drug resistant bacteria exist and proliferate either due to natural selection of clonal lineages or the transfer of mobile genetic elements, sometimes in response to antibiotic-use selective pressure. Pulsed-field gel electrophoresis (PFGE) is highly discriminatory and the gold standard typing method for the characterisation of K. pneumoniae isolates. The aim of the study was to genetically characterise K. pneumoniae isolates by PFGE and multilocus sequence typing (MLST). One hundred unrepeated ESBL-producing K. pneumoniae isolates were collected from the National Health Laboratory Service (NHLS). The PFGE was performed on a Rotaphor VI system (Biometra, Germany). Clonal representatives were further characterised by MLST. All the strains were typeable by PFGE using XbaI, which discerned multiple pulsotypes and MLST identified 10 different STs including a novel sequence type, ST1632. The diverse pulsotypes of K. pneumoniae isolates are not suggestive of clonal spread of particular strains. The MLST results further confirmed the variability among isolates tested and elucidated several STs, some of which have been identified internationally and often associated with carbapenem-resistance. Data on K. pneumoniae STs is still limited in the South African clinical setting, although the close monitoring of resistance profiles and characterisation of isolates is imperative for outbreak analysis, identification of prominent STs in clinical settings as compared to international counterparts and surveillance of expanding resistance. / Dissertation (MSc (Medical Microbiology))--University of Pretoria, 2015. / Medical Microbiology / MSc (Medical Microbiology) / Unrestricted

Bacteriophages (phages) are the smallest biological entity on the planet. They provide vast amounts of valuable knowledge to biologists. Phage genomes are relatively simple compared to the organisms they infect (prokaryotes) and yet continually point to the complexity surrounding molecular- and microbiological mechanisms of life. By studying phages we can learn of the systems of gene expression, protein interaction and DNA organization. Phages are useful not only from an academic perspective, but may also have useful clinical applications. In the face of the rise of antibiotic-resistant bacterial “super pathogens”, scientists and researchers turn to phages as alternative treatments to these types of infections. Phages are capable of infecting and killing even the deadliest of bacterial pathogens, such as carbapenem-resistant Enterobacteriaceae (CRE) or Bacillus anthracis, and may prove increasingly useful in the future for combatting harmful pathogens. This thesis looks at several aspects of phage biology—from the underlying genetics contributing to phage virulence, to the clinical application of phage therapy to treat infections. First, a look at CRE-Klebsiella pneumoniae isolates and phages capable of infecting some strains may reveal a potential therapeutic approach in the future. Additionally, genomic analysis reveals interesting features that may explain aspects of phage virulence and evolutionary history. Then, a collection of genetically diverse phages is used in infection assays on pathogenic strains of Bacillus anthracis to establish the first-reported phages capable of infecting these strains. Finally, the process of preparing phage samples for therapeutic application is explored in-depth to conclude with discussion of clinical application. During the course of these projects over 25 phages were isolated, as many phage genomes were assembled and annotated, resulting in the preparation of two genome announcements and near-completion of two publishable first-author papers (chapters II and III). In addition, participation in a variety of collaborative efforts may lead to a handful of co-author papers and on various topics, including phage biology and application.

Lower respiratory tract infectious diseases are a world-wide healthcare burden with bacterial pathogens accounting for a large portion of primary and secondary infections. The human respiratory tract is home to hundreds of species of microbes that comprise the human airway microbiome. These commensals play a crucial role in human health in part by providing colonization resistance against pathogens. In a previous study from the Surette lab it was shown that specific bacterial isolates from the respiratory microbiome inhibits the growth of pathogens aerobically. This included an isolate of Staphylococcus aureus which inhibited the growth of Enterococcus faecium. This activity was further characterized in this thesis and the underlying mechanism was explored through comparative genomics. As well, this observation provided proof-of-concept for a large-scale screen for additional isolates which inhibit pathogen growth. I hypothesized that the respiratory tract microbiota included many other bacteria capable of inhibiting the growth of respiratory tract pathogens in both aerobic and anaerobic environments, and that anaerobic conditions will identify new activities not detected aerobically. To examine and identify potential beneficial bacteria, I have screened ~5000 respiratory tract bacteria from the Surette lab’s airway isolate collection against four pathogens: Streptococcus pneumoniae, Staphylococcus aureus, Pseudomonas aeruginosa and Klebsiella pneumoniae. The respiratory tract commensals were pinned onto the pathogen-lawn and their interaction was expressed as zones of clearing or altered growth phenotypes of the pathogen. The results of the screen showed that anti-pathogen activity was a common feature of respiratory tract commensals. In particular, S. pneumoniae was inhibited by taxonomically diverse members of the microbiota representing three phyla (Proteobacteria, Firmicutes and Actinobacteria). Many of the facultative anaerobes that inhibited S. pneumoniae expressed their activity in anerobic conditions. / Thesis / Master of Science (MSc) / The human respiratory tract harbours commensal and pathogenic bacteria, and the latter cause most of the lower respiratory tract infections. The commensal bacteria help to train the immune system and impede the growth of pathogens through colonization resistance. A previous study by the Surette lab identified bacterial isolates from the respiratory tract that inhibit the growth of select pathogens, among them, a particular strain of Staphylococcus aureus. Based on the results of the earlier study, I hypothesized that the respiratory tract bacteria is a good source of commensals that can inhibit the growth of S. aureus and other respiratory pathogens, such as Streptococcus pneumoniae, Pseudomonas aeruginosa and Klebsiella pneumoniae. To find potential therapeutic bacteria, I screened ~5000 respiratory tract isolates from the Surette lab’s strain collection for the ability to impair growth of target pathogens. Additionally, I further characterized the activity of the previously identified S. aureus strain against various Lactobacillalles strains and used comparative genomics to identify potential biosynthetic genes required for biosynthesis of molecules with antibacterial activity within the genome of S. aureus. The research reported in this thesis demonstrates that many commensal bacteria that live within our airways have the ability to inhibit the growth of bacterial pathogens. This work may provide a new source of antibiotics against respiratory infections and new strategies to reduce susceptibility to infections in vulnerable populations.