Acinetobacter baumannii (Ab) é um dos principais agentes de infecção hospitalar, principalmente em Unidades de Terapia Intensiva (UTI). A principal característica da bactéria é a sua resistência intrínseca a diversos antimicrobianos, o que favorece sua persistência no ambiente hospitalar, causando infecções de difícil controle e tratamento. Nos esquemas terapêuticos, os antibióticos carbapenêmicos são os fármacos de escolha, porém nos últimos anos a resistência a estas drogas tem aumentado drasticamente em função da emergência e disseminação de cepas produtora de carbapenemases [i.e., oxacilinases (OXA) e metalo-beta-lactamases (MβL)]. O objetivo do presente trabalho foi avaliar a produção de enzimas carbapenemases do tipo OXA e MβLs, em 36 amostras multirresistentes de A. baumannii, previamente triadas, provenientes de 8 hospitais brasileiros, durante 2004/2008. A caracterização fenotípica e genotípica dos isolados foi realizada por meio da determinação de CIM, hidrólise enzimática e PCR para pesquisa dos genes blaOXA e blaMβL, assim como seqüências de inserção (ISAba-1, ISAba-3) responsáveis pela mobilização dos determinantes de resistência do tipo blaOXA. Finalmente a análise da diversidade genética foi realizada por ERIC-PCR com análise de clusters por coeficiente de Dice. Todos os 36 isolados apresentaram 100% de resistência para imipenem (CIM90 > 64 µg/mL), meropenem, ceftazidima, ciprofloxacina e iperacilina/tazobactam, enquanto que os antibióticos com maior atividade in vitro foram a ampicilina/sulbactam (61,2%) > tobramicina (61,1%) > gentamicina (47,3%) > amicacina (28%). Em todos os isolados foi confirmada a presença intrínseca dos genes blaOXA-51 e ISAba-1, não apresentando colinearidade entre eles, enquanto que 41,6% dos isolados carregaram a combinação dos genes ISAba-1/blaOXA-23 (Genbank accession FJ628170), um isolado (2,7%) carregou a combinação de genes ISAba3/blaOXA-58/ISAba3 (Genbank accession FJ492877) e dois isolados (5,5%) clonalmente relacionados, carregaram o gene blaOXA-72 (Genbank accession FJ969387). Surpreendentemente, em um centro hospitalar foi documentada a presença de um surto de infecção por Ab produtor de OXA-23. Finalmente a tipagem epidemiológica dos isolados revelou a presença de 13 clusters, sendo que 8 diferentes clusters carregavam o gene blaOXA-23. Em resumo, nossos resultados confirmam a disseminação de cepas produtoras de OXA-23 associadas com ISAba-1 no Brasil, assim como a presença intrínseca do gene blaOXA-51 em Ab. Por outro lado, este é o primeiro relato de isolados carbapenem resistentes carregando os genes blaOXA-58 e blaOXA-72 em hospitais brasileiros, os quais aparentemente surgiram em 2004 e 2008, respectivamente. / Acinetobacter baumannii (Ab) is a leading cause of hospital infection, mainly in intensive care units. The main characteristic of the bacterium is its intrinsic resistance to diverse antimicrobial agents, which contribute to persistence in hospital environments causing infections of difficult control and treatment. In therapeutic schedules, carbapenems are choice antibiotics, however in recent years the resistance to these drugs has increased drastically in function of the emergency and dissemination of carbapenemase-producing isolates [i.e., oxacilinases (OXA) and metalo-beta-lactamases (MβLs)]. The aim of this work was to evaluate the production of OXA- and MβL-like carbapenemases, in 36 isolates previously screened as multidrug-resistant (MDR) A. baumannii, recovered from 8 Brazilian hospitals, during 2004/2008. Phenotypic and genotypic characterization of MDR Ab was carried out using CIM determination, enzymatic hydrolysis and PCR for screening of the blaOXA- and blaMβL-like genes, and insertion sequences (ISAba-1, ISAba-3) responsible for mobilization of blamOXA-like gene cassettes. Analysis of genetic relationship was carried out by ERIC-PCR with analysis of clusters for Dice`s coefficient. All of the 36 isolates showed 100% resistance to imipenem (CIM90 > 64 µg/mL), meropenem, ceftazidime, ciprofloxacin and piperacillin/tazobactam, whereas antibiotics exhibiting a best in vitro activity were ampicillin/sulbactam (61.2%) > tobramycin (61.1%) > gentamicin (47.3%) > amikacin (28%). Presence of blaOXA-51 and ISAba-1 genes was confirmed in all isolates, not presenting collinearity between them, whereas 41.6% isolates carried the ISAba-1/blaOXA-23 gene array (Genbank accession FJ628170). One Ab isolate harbored the ISAba-3/blaOXA-58/ISAba-3 gene array (2.7%) (Genbank accession FJ492877) and 5.5% of Ab isolates harbored the blaOXA-72 gene (Genbank accession FJ969387). Surprisingly, an outbreak of infection with MDR Ab producing OXA-23 enzyme was documented. Finally the ERIC-PCR typing revealed the presence of 13 clusters, of which 8 different clusters carried the blaOXA-23 gene. In summary, our results confirm the dissemination of OXA-23-producing Ab isolates associated with the ISAba-1 gene, in Brazil, as well as the intrinsic presence of the blaOXA-51 gene cassette. On the other hand, this is the first report of carbapenem-resistant Ab isolates harboring genes blaOXA-58 and blaOXA-72 recovered in Brazilian hospitals, which most likely emerged in 2004 and 2008, respectively.

Acinetobacter baumannii est un pathogène nosocomial qui induit principalement des infections du système respiratoire ou urinaire, et des septicémies chez les patients immunodéprimés. L'émergence de souches multi résistantes aux antibiotiques et l'augmentation de nombreuses d'infections par A. baumannii fait de ce pathogène un enjeu majeur de santé publique. De plus aujourd'hui émerge des souches hypervirulentes. Nous nous sommes intéressés à différentes souches afin de caractériser le phénotype hypervirulent de ces souches. L'étude du système de sécrétion de type VI montre la complexité des mécanismes de virulence d'A. baumannii et sa régulation dépendante des souches. Dans un second temps l'étude des souches cliniques hypervirulentes et nous avons mis en évidence deux nouveaux potentiels mécanismes de virulence : une phase de réplication intracellulaire et une limitation de la réponse immunitaire. Ces mécanismes peuvent expliquer la virulence accrue de ces souches chez l'homme. L'étude nous montre également qu'A. baumannii est un pathogène complexe et qu'on son étude à l'heure actuelle nécessité l'emploi de souche représentative des souches infectant les patients / A. baumannii is an hospital acquired pathogen which causes mainly ventilator associated infection, urinary tract infection and bacteraemia. Last years Multi Drug Resistant strains increase and nosocomial infection cause by A. baumannii also which led him as a serious health care problem. We compare different strains in propose to find phenotype that can explain hypervirulent strain emergence. We studied type six secretion and showed that the complexity of A. baumannii virulence mechanism. Indeed type six secretion system regulation is strain dependant. Secondary we study hypervirulent strain and showed that intracellular stage exists and there is intracellular replication. Also hypervirulent strain induces less immune response. Those two mechanisms can explain A. baumannii hypervirulent phenotype

Orientador: Juliana Campos Junqueira / Banca: Antonio Olavo Cardoso Jorge / Banca: Fernanda Malagutti Tomé / Banca: Renato Araújo Prates / Banca: Raduan Hage / Resumo: Devido ao rápido aumento dos micro-organismos resistentes aos antibióticos e ao desenvolvimento limitado de novos agentes antimicrobianos, as infecções por bactérias Gram-negativas estão se tornando um desafio para os profissionais da saúde e uma ameaça para a saúde pública internacional. O objetivo desse estudo foi avaliar o efeito sinérgico dos antibióticos convencionais associados a terapia fotodinâmica antimicrobiana (PDT) no controle de Acinetobacter baumannii. Para realização desse trabalho, foram obtidos isolados clínicos de A. baumannii do Laboratório de Análises Clínicas Valeclin da cidade de São José dos Campos/SP, identificados pelo método de bioquimismo e submetidos ao teste de difusão em disco para verificar a sensibilidade antimicrobiana. Os isolados selecionados foram transferidos para o ICT/UNESP, onde foi realizado testes para determinação da Concentração Inibitória Mínima aos antibióticos Imipenem e Meropenem seguindo as normas da CLSI. Cepas sensíveis e resistentes aos antibióticos foram avaliadas quanto a sensibilidade in vitro à terapia fotodinâmica antimicrobiana. Além disso, foram testados os efeitos dos antibióticos convencionais, da PDT e da terapia combinada de antibióticos e PDT nas infecções experimentais induzidas em G. mellonella por isolados clínicos de A. baumannii resistentes aos antibióticos. Os resultados das terapias na infecção experimental foram avaliados por meio da curva de sobrevivência das lagartas de G. mellonella. Os dados dos testes in vitro foram submetidos à Análise de Variância e teste de Tukey. Os dados obtidos na curva de sobrevivência de G. mellonella foram analisados pelo método de Log-rank. Em todos os testes, foi considerado nível de significância de 5%. Nos resultados desse estudo, observou-se que o Laboratório Valeclin identificou 1,54% de amostras positivas para A. baumannii entre as 13.715 amostras clínicas analisadas em um período de... / Abstract: Due to the rapid growth of microorganisms resistant to antibiotics and the limited development of new antimicrobial agents, infections by Gramnegative bacteria are becoming a challenge for health professionals and a threat to international public health. The aim of this study was to evaluate the synergistic effect of conventional antibiotics associated with antimicrobial photodynamic therapy (PDT) in control of Acinetobacter baumannii. In order to conduct this project were obtained clinical isolates of A. baumannii at the Clinical Laboratory Valeclin situated in the city of São José dos Campos / SP, identified by bioquimismo method and submitted to disk diffusion test to verify the antimicrobial sensitivity. The selected isolates were transferred to the ICT / UNESP, which were conducted tests to determine the Minimum Inhibitory Concentration to Imipenem and Meropenem antibiotics following the rules of the CLSI. Sensitive and resistant strains to antibiotics were evaluated in vitro sensitivity to antimicrobial photodynamic therapy. Besides, the effects of conventional antibiotics, and combined PDT, and PDT of antibiotics in experimental infections induced in G. mellonella by clinical isolates of A. baumannii resistant to antibiotic therapy were tested. The results of therapies in experimental infection were evaluated by survival curve of worms G. mellonella. Data from in vitro tests were submitted to ANOVA and Tukey test. The data obtained in G. mellonella survival curve were analyzed by log-rank method. In all tests it was considered 5% significance level. The results of this study, it was observed that the Valeclin Laboratory identified 1.54% of positive samples for A. baumannii between the 13,715 clinical specimens analyzed in a period of 8 months. Among the isolates of A. baumannii, 58% were resistant to antibiotic imipenem and meropenem by disk diffusion test. Next, 3 isolates clinical sensitive and 18 isolates resistant to those ... / Doutor

Acinetobacter baumannii is an opportunistic pathogen and can cause severe disease in immune-suppressed and/or injured patients. It is an extreme-drug resistant bacterium with the ability to form biofilms thereby significantly increasing resistance to treatment. Because of the extreme drug resistance and relatively unknown immunological profile of A. baumannii new treatment options are needed. A. baumannii has been reported to express a Biofilm Associated Protein (BAP); a high molecular weight protein composed of multiple repeat modules and thought to be surface exposed on planktonic bacterium and upregulated in biofilm. While it is unknown if BAP has any role in in vivo infection of humans, the repeats of BAP proteins are thought to function in intercellular adhesion to support the mature biofilm and thus represent potential targets for immunotherapeutic intervention. Herein my thesis is aimed at trying to verify that BAP is surface exposed, upregulated in biofilm and to prove a role for BAP in pathogenesis, as well as investigating A. baumannii interactions with components of the innate immune system in vitro. Consensus synthetic peptides corresponding to the major internal repeats of BAP were designed and conjugated to carrier proteins and recombinant proteins were manufactured to correspond to the non-repetitive N and C terminals of the protein for murine immunization and assay development. Serum from immunized mice was collected and analyzed in ELISA and western immunoblot to determine reactivity with planktonic and biofilm whole organism. Anti-serum to whole bacteria was also tested in opsonisation assays to determine direct killing ability of serum on bacteria in vitro. Anti-serum to whole bacteria showed direct killing of the organism in vitro when in high concentrations (diluted 1/10), relative to pre-immune serum, but was less effective in lower concentrations (diluted 1/50). Despite generating antibody reagents to multiple domains and epitopes spanning the published BAP sequence, we were unable to confirm that BAP is expressed by A. baumannii as reported by others. However, if BAP is indeed expressed in A. baumannii our DNA and immunochemical data collectively suggest that BAP is potentially mosaic in this pathogen.

Acinetobacter baumannii is an opportunistic pathogen and can cause severe disease in immune-suppressed and/or injured patients. It is an extreme-drug resistant bacterium with the ability to form biofilms thereby significantly increasing resistance to treatment. Because of the extreme drug resistance and relatively unknown immunological profile of A. baumannii new treatment options are needed. A. baumannii has been reported to express a Biofilm Associated Protein (BAP); a high molecular weight protein composed of multiple repeat modules and thought to be surface exposed on planktonic bacterium and upregulated in biofilm. While it is unknown if BAP has any role in in vivo infection of humans, the repeats of BAP proteins are thought to function in intercellular adhesion to support the mature biofilm and thus represent potential targets for immunotherapeutic intervention. Herein my thesis is aimed at trying to verify that BAP is surface exposed, upregulated in biofilm and to prove a role for BAP in pathogenesis, as well as investigating A. baumannii interactions with components of the innate immune system in vitro. Consensus synthetic peptides corresponding to the major internal repeats of BAP were designed and conjugated to carrier proteins and recombinant proteins were manufactured to correspond to the non-repetitive N and C terminals of the protein for murine immunization and assay development. Serum from immunized mice was collected and analyzed in ELISA and western immunoblot to determine reactivity with planktonic and biofilm whole organism. Anti-serum to whole bacteria was also tested in opsonisation assays to determine direct killing ability of serum on bacteria in vitro. Anti-serum to whole bacteria showed direct killing of the organism in vitro when in high concentrations (diluted 1/10), relative to pre-immune serum, but was less effective in lower concentrations (diluted 1/50). Despite generating antibody reagents to multiple domains and epitopes spanning the published BAP sequence, we were unable to confirm that BAP is expressed by A. baumannii as reported by others. However, if BAP is indeed expressed in A. baumannii our DNA and immunochemical data collectively suggest that BAP is potentially mosaic in this pathogen.

Submitted by Pedro Barros (pedro.silvabarros@ufpe.br) on 2018-07-04T22:20:31Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) TESE Carmelita de Lima Bezerra Cavalcanti.pdf: 1587660 bytes, checksum: eade0c1462dbaba07af02453d8fb4173 (MD5) / Made available in DSpace on 2018-07-04T22:20:31Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) TESE Carmelita de Lima Bezerra Cavalcanti.pdf: 1587660 bytes, checksum: eade0c1462dbaba07af02453d8fb4173 (MD5) Previous issue date: 2017-02-21 / Apesar do grande envolvimento de isolados de Acinetobacter baumannii em Infecções Relacionadas à Assistência à Saúde (IRAS) e do seu alto índice de multidroga-resistência, levando a poucas opções terapêuticas em casos de infecções causadas por esta espécie bacteriana, há muito a ser descoberto sobre os mecanismos de virulência e resistência desta espécie. Portanto, este trabalho teve como objetivo estabelecer o perfil clonal, de resistência e virulência de isolados multidroga-resistentes (MDRs) de A. baumannii obtidos em hospitais de Recife-PE, determinando a prevalência de genes de resistência e virulência e a capacidade de expressão de genes de virulência após submissão dos isolados a diferentes antimicrobianos utilizados na prática clínica. Para determinar a prevalência dos genes de resistência e virulência foram utilizados 37 isolados do complexo A. baumannii provenientes de dois hospitais públicos de Recife – PE. A análise da expressão dos genes de virulência foi realizada utilizando três isolados MDR selecionados, além da ATCC 19606 de A. baumannii submetidos in vitro a colistina, meropenem e associação destes antimicrobianos. Os isolados foram avaliados quanto à confirmação da espécie através da detecção do gene blaOXA-51-like e da técnica de MALDI-TOF. A tipagem molecular desses isolados foi realizada através da técnica de PFGE utilizando a enzima de restrição Apa1. A detecção e sequência dos genes de resistência, blaOXA-51-like, blaOXA-23-like, blaOXA-143-like, blaIMP, blaVIM, blaKPC, o elemento de inserção, ISAba1, e dos genes de virulência, basC, ompA, pilA e csuE foi realizada através de PCR e sequenciamento gênico. Todos os isolados pertenciam à espécie A. baumannii, distribuídos em 07 padrões de PFGE, com três isolados apresentando 100% de similaridade, os quais foram obtidos nos dois hospitais públicos do estudo, sugerindo uma disseminação inter-hospitalar. Todos os isolados apresentaram o gene de resistência blaOXA-51-like e a maioria possuía o gene ISAba1, além desses, também apresentavam o gene blaOXA-143-like ou blaOXA-23-ike. Estes resultados demonstram uma ampla resistência dos isolados aos carbapenêmicos, além de outras classes de antimicrobianos. Os dados são preocupantes quanto à disseminação clonal desses genes entre os isolados obtidos nos hospitais analisados. Todos os isolados do estudo apresentaram os genes de virulência basC, ompA, pilA e csuE, com exceção de um isolado que não apresentou o gene csuE. Também pode-se demonstrar uma tendência ao aumento da expressão dos genes de virulência csuE, bfmS e baeS após tratamento in vitro com meropenem, colistina e associação destes antimicrobianos, reforçando a necessidade de vigilância quanto ao tratamento de IRAS causadas por A. baumannii MDR, mesmo em uso associado de antimicrobianos. / Due to the increasing resistance of clinical isolates of A. baumannii to antimicrobial and the consequent decrease of effective therapeutic options, this study aimed to establish the clonal profile, resistance and virulence of A. baumannii multidrug-resistant isolates (MDRs) obtained in hospitals in Recife-PE, determining the prevalence of resistance and virulence genes and the in vitro influence of different antibiotics used in clinical practice, alone and in combination, on bacterial growth and expression of these genes. To perform the first stage of this study 37 isolates of A. baumannii complex, most of them MDR. Analysis of virulence gene expression was performed using three MDR isolates beyond ATCC 19606 from A. baumannii submitted in vitro to colistin, meropenem and association of these antimicrobials. The isolates were evaluated for confirmation of the species through detection of the gene for intrinsic β-lactamase, blaOXA-51-like, and the Matrix-Assisted Laser Desorption / Ionization (MALDI-TOF) technique. The clonal profile of these isolates was determined by Pulsed-field Gel Electrophoresis (PFGE) using Apa1 enzyme. Detection and sequence of blaOXA-51-like, blaOXA-23-like, blaOXA-143-like, blaIMP, blaVIM and blaKPC genes the insertion element, ISAba1, and the virulence genes, basC, ompA, pilA and csuE were performed using Polymerase Chain Reaction (PCR) and sequencing. All isolates belonged to the A. baumannii species, distributed in 07 PFGE patterns, three isolates showed 100% similarity, which were obtained in two different hospitals of the study, suggesting an interhospital spread. Most isolates had the blaOXA-51-like and ISAba1 resistance genes, and either blaOXA-143-like or blaOXA-23-like gene. These results demonstrated high level resistance to carbapenems and other classes of antimicrobials, corroborating the MDR profile. This data alert for the spread of these genes among isolates in this hospitals. All isolates showed basC, ompA, pilA and csuE virulence genes, except for one isolate that did not show the csuE gene. Expression of csuE, bfmS e baeS virulence genes increased after in vitro submission to meropenem, colistin and the associated use may be demonstrated, reinforcing the need for surveillance for treating infections caused by MDR A. baumannii infections, even in associated antimicrobial use.

Submitted by Tiótrefis Fernandes (ppgssea@ufam.edu.br) on 2017-09-19T15:01:02Z No. of bitstreams: 4 Carta orientador Marcilene Maria de Souza Viana.pdf: 67670 bytes, checksum: c77de740b0ec45224e57fedbd9bb8212 (MD5) Pre Textuais.pdf: 144156 bytes, checksum: 303fc10d5636659a54a366f20b368dbb (MD5) Textuais Finais.pdf: 614930 bytes, checksum: 09b99dac0f864d42c18c34b81b661c28 (MD5) termo ma 001.jpg: 1164946 bytes, checksum: 72cb9c00fc7cf57ac3fa3f057419f594 (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2017-09-20T14:08:34Z (GMT) No. of bitstreams: 4 Carta orientador Marcilene Maria de Souza Viana.pdf: 67670 bytes, checksum: c77de740b0ec45224e57fedbd9bb8212 (MD5) Pre Textuais.pdf: 144156 bytes, checksum: 303fc10d5636659a54a366f20b368dbb (MD5) Textuais Finais.pdf: 614930 bytes, checksum: 09b99dac0f864d42c18c34b81b661c28 (MD5) termo ma 001.jpg: 1164946 bytes, checksum: 72cb9c00fc7cf57ac3fa3f057419f594 (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2017-09-20T14:23:54Z (GMT) No. of bitstreams: 4 Carta orientador Marcilene Maria de Souza Viana.pdf: 67670 bytes, checksum: c77de740b0ec45224e57fedbd9bb8212 (MD5) Pre Textuais.pdf: 144156 bytes, checksum: 303fc10d5636659a54a366f20b368dbb (MD5) Textuais Finais.pdf: 614930 bytes, checksum: 09b99dac0f864d42c18c34b81b661c28 (MD5) termo ma 001.jpg: 1164946 bytes, checksum: 72cb9c00fc7cf57ac3fa3f057419f594 (MD5) / Made available in DSpace on 2017-09-20T14:23:54Z (GMT). No. of bitstreams: 4 Carta orientador Marcilene Maria de Souza Viana.pdf: 67670 bytes, checksum: c77de740b0ec45224e57fedbd9bb8212 (MD5) Pre Textuais.pdf: 144156 bytes, checksum: 303fc10d5636659a54a366f20b368dbb (MD5) Textuais Finais.pdf: 614930 bytes, checksum: 09b99dac0f864d42c18c34b81b661c28 (MD5) termo ma 001.jpg: 1164946 bytes, checksum: 72cb9c00fc7cf57ac3fa3f057419f594 (MD5) Previous issue date: 2013-09-18 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Acinetobacter baumannii is the most common species of the isolated genus from clinical specimens and hospital environment, often affecting patients in intensive care units ( ICUs ). It is a bacterium which seems to have a propensity to develop antimicrobial resistance extremely fast. Practices as the quite higher use of antibiotics in patient in ICUs contribute to develop the A. Baumannii resistance. It is getting importance because it is related to nasocomiais infections in units where patients are using invasive procedures, immunoimpaired, elderly or treated with broad-spectrum antibiotics. For the reasons above, it was decided to do this study in patients hospitalized at Santa Casa de Misericordia do Pará (FSCMP) to verify the cases of infeccion by Acinetobacter baumannii related to health care epidemiology isolayting clinical specimens with emphasis on sensitivity profile in a period from 2007 to 2011. Having such results, 48.39% of cases of bloodstream infection as the site of infection by A. Baumannii, followed by infection of the urinary tract and trachea, 12.9% each, with the largest number of cases in adult and neonatal ICUs. The A.baumannii showed significant resistance to cephalosporins 3rd and 4th generation, with troubling resistance to carbapenems, with annual growth from 6.25% to 30%. Concluding that there was a change in resistance profile of A. Baumannii in five years, and evidenced a rich field of research regarding the epidemiology of Acinetobacter baumannii, showing the necessity of new approaches to aspects such as clinical findings, duration of hospitalization, previous antibiotic use, and others, as a contribution to the clinical intervention against nosocomial infections caused by the under study agent. / O Acinetobacter baumannii é a espécie mais comum do gênero isolado de amostras clínicas e de ambiente hospitalar, frequentemente afetando pacientes em unidades de tratamento intensivo (UTIs). É uma bactéria que parece ter uma propensão para desenvolvimento de resistência antimicrobiana extremamente rápida. Práticas como, o uso significativamente alto de antimicrobianos por paciente em UTIs contribuem para o desenvolvimento da resistência da A. Baumannii.. Ganhando importância por estar relacionado a infecções nasocomiais em pacientes em uso de procedimentos invasivos além de pacientes imunocomprometidos, idosos e tratados com antibióticos de amplo espectro. Foi realizado estudo em pacientes internados na Fundação Santa Casa de Misericórdia do Pará (FSCMP) para se verificar a epidemiologia dos casos de infecção relacionada à assistência à saúde por Acinetobacter baumannii isolado de espécimes clínicos com ênfase no perfil de sensibilidade num período de 2007 a 2011. Do total de 63 casos de IRAS por A. baumannii, 48,39% representaram infecção de corrente sanguínea, seguidos pela infecção de trato urinário e infecção de foco pulmonar, com 12,9% cada, sendo um maior número de casos oriundos das UTIs adulto e neonatal. O A.baumannii demonstrou alta taxa de resistência, com destaque para Cefatzidima (100%), Cefepime (52,38%), Piperacilina-Tazobactam (52,46%), com emergente resistência para os carbapenêmicos, com progressão anual de 6,25% a 30%. Conclusão: A vigilância de 5 anos do perfil de suscetibilidade demonstrou um rápido aumento das cepas multirresistentes de A. baumannii, particularmente nos dois últimos anos do estudo. O monitoramento do padrão de resistência poderá ajudar na otimização da terapia empírica destas infecções, sendo necessários esforços nas medidas intervencionais de controle de infecção na instituição.

M.Sc. / An Acinetobacter sp. strain RFB1 isolated in our laboratory has been shown to have the ability to metabolise inorganic cyanide salts, CO 2, and bicarbonate. The enzyme aggregate responsible for the conversion of these substrates, is located extra-cellularly. Resolution of the extra-cellular complex, a crude enzyme filtrate, was attempted in order to characterise the protein responsible for the reduction of CO 2. The crude enzyme filtrate was separated by means of molecular exclusion chromatography and afforded three fractions with molecular masses ranging from 76 000 to 191 000. Analysis by SDS-electrophoresis, showed that the first protein fraction contained more than ten proteins. Certain of these proteins were identified in the second fraction and other proteins in the third protein fraction. This implies that some denaturation already occurred during molecular exclusion separation. The functionali7ation of CO 2 by protein fractions 1 and 3 supports this argument, and, in addition , cyanide ions were only reduced by fractions 1 and 2. Fatty acids, ranging with chainlengths between C5 and C25, were shown to be present and certain fatty acids were unequivocally identified by GC-mass spectroscopy as the products resulting from CO2 functionali7ation and carbon-carbon bond formation. Ferrous ions, in an optimal concentration of 250 gg cm', were necessary and served as an essential ingredient of the reaction mixture. A rather unusual result was, however, that apart from an initial, relatively small uptake of Fe(II), significant amounts of Fe(III) were not formed and the Fe(II) concentration remained approximately constant during the reaction. This implies that the formed Fe(M) is rapidly reduced to Fe(II) again. Spectroscopic measurements, furthermore, strongly suggested the involvement of an iron-sulphur cluster in a cyclic redox process wherein both Fe(II) and Fe(III) are involved. Carefully conducted experiments pointed to light as the outside source of energy. Qualitative similarities with an artificial photosynthetic process, formulated earlier by J-M. Lehlliii, can be drawn and used partly to explain the experimental results.

Pyrimidine nucleotide biosynthesis was studies in Acinetobacter calcoaceticus ADP-1. Pyrimidine auxotrophic mutants were isolated and characterized for this purpose. One such Pyr mutant, strain ADP-1-218 was chosen for further study.

Acinetobacter baumannii is an important nosocomial pathogen, frequently associated with morbidity and mortality in immunocompromised patients due to the immuno-ablative treatments, neutropenia and prolonged hospitalization. The ability of A. baumannii to survive in the healthcare setting makes it a frequent problematic pathogen in cancer centres. Much of the interest in A. baumannii has been attributed to its remarkable rapid acquisition of resistance mechanisms A. baumannii is an excellent example of genetic plasticity, with its ability to acquire and express resistance in plasmids and chromosome particularly to carbapenems The aim of this thesis is to look at the molecular epidemiology and resistance mechanisms of 34 non-duplicate A. baumannii in two cancer centres in Cairo, Egypt. Initial sequencing of the ubiquitous blaOXA-51-like gene revealed a large diversity within the strains, with eight different genes identified: blaOXA-64, blaOXA-65, blaOXA-66, blaOXA-69, blaOXA-71, blaOXA-78, blaOXA-94, blaOXA-89/100. Typing with Pulsed-field Gel Electrophoresis (PFGE) showed an overall similarity at only 28.69% between the isolates, with variation in pattern for isolates with similar blaOXA-51-like genes. Typing with Multilocus Sequence Typing (MLST) identified 6 new Sequence Types: ST408 - ST414, in addition to ST331 and ST108 which have been previously found in other regions of the world. All three OXA-type carbapenemases: blaOXA23, blaOXA40 and blaOXA58, responsible for conferring carbapenem resistance were found in the collection studied. Insertion sequences ISAba1, ISAba2 and ISAba3 have been found to upregulate the expression of blaOXA genes. ISAba1 was found upstream of blaOXA23 in 18 strains in this collection The first report of ISAba2 was identified upstream of a blaOXA-51-like gene in this collection. Additionally, ISAba3 was bracketing the blaOXA58 genes, and two isolates harboured hybrid promoters with IS1006 and IS1008 interrupting the upstream ISAba3 sequence. Resistance to Ceftazidime was mediated by Extended-spectrum β-lactamase (ESBL) genes belonging to PER-like group: blaPER-1, blaPER-7 and the first report of blaPER-3 gene and its genetic environment in A. baumannii. In conclusion, this study shows the diversity exhibited by A. baumannii in Egypt. The various resistance mechanisms illustrate the ability of A. baumannii in acquiring and expressing resistance genes, either on plasmids or in the chromosome. Furthermore, the results indicate an urgent need to strict infection control policies and surveillance of antimicrobial use in Egyptian hospitals.