Orientador: Pasqual Barretti / Resumo: Peritonite por bacilos Gram-negativos não fermentadores (BGNNF) é complicação importante da diálise peritoneal (DP), com curso clínico grave e elevada taxa de falência do método. Fatores associados à virulência, resistência antimicrobiana, formação de biofilme, entre outros, têm sido relatados, mas o limitado conjunto de evidências não permite concluir sobre os fatores responsáveis pelo pior curso clínico dessas infecções. O objetivo deste trabalho foi avaliar a influência das características microbiológicas, das condições clínicas do paciente e do tratamento na evolução de peritonites por BGNNF, ocorridas num único centro, em período de 18 anos. A sensibilidade in vitro aos antimicrobianos, produção de biofilme, além da análise do perfil clonal das bactérias pela técnica de eletroforese em gel de campo pulsado foram realizadas em todos os isolados bacterianos. Foram pesquisados genotipicamente, em isolados de Pseudomonas aeruginosa, a presença de marcadores de virulência (alginato, exoenzima S, fosfolipases C, exotoxina A, protesase alcalina, elastase e ramnolipídeos). Associações entre as características microbiológicas do paciente e tratamento com a taxa de resolução da peritonite foram estudadas. A espécie mais frequente foi Pseudomonas aeruginosa (45,59%), seguida por isolados do complexo Acinetobacter baumannii (17,65%). O estudo dos fatores de virulência da Pseudomonas aeruginosa revelou a presença de fatores de virulência em 100% dos casos, exceto exoenzima S (58,33%)... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Peritonitis due to non-fermentative Gram-negative bacilli (NFGNB) is a serious complication of peritoneal dialysis (PD), with a severe clinical course and high technique failure rate. Factors as bacterial virulence, antimicrobial resistance, biofilm formation, among others, have been reported, but the limited amount of evidence does not allow to conclude on the factors responsible for the worst clinical course of these infections. The objective of this study was to evaluate the influence of the microbiological characteristics, patients conditions, and treatment on evolution of peritonitis episodes at a single center in an 18 - year period. In vitro susceptibility, biofilm production, and clonal profile analysis of bacteria by pulsed-field gel electrophoresis (PFGE) were performed in all isolates. The presence of virulence markers (alginate, exoenzyme S, phospholipases C, exotoxin A, alkaline protease, elastase, and ramnolipids) was genotyped in bacterial isolates of Pseudomonas aeruginosa. From the data referring to the patient and causal agent, associations between the microbiological, patient characteristics, and treatment on the resolution rate of peritonitis were analyzed. The most frequent species was Pseudomonas aeruginosa (45.59%), followed by Acinetobacter baumannii complex (17.65%). The study of the virulence factors of Pseudomonas aeruginosa revealed the presence of virulence factors in 100% of the cases, except for exonzyme S (58.33%) and hemolytic phospholipase C ... (Complete abstract click electronic access below) / Mestre

Orientador: Mara Correa Lelles Nogueira / Banca: Beatriz Meurer Moreira / Banca: Marina Baquerizo Martinez / Banca: Suzana Margareth Ajeji Lobo / Banca: Fátima Pereira de Souza / Resumo: O gênero Acinetobacter compreende bacilos Gram-negativos ubíquos na natureza e suas principais espécies, A. calcoaceticus, A. baumannii, A. pittii e A. nosocomialis, foram agrupadas no complexo Acinetobacter baumannii-Acinetobacter calcoaceticus (complexo ACB) por apresentarem alta similaridade genética e por serem de difícil diferenciação por métodos bioquímicos. As infecções mais causadas pelo complexo ACB são pneumonias e bacteremias e, para o tratamento destas, são muito utilizados os beta-lactâmicos, aminoglicosídeos e polimixinas. Entretanto, a resistência aos beta-lactâmicos e aminoglicosídeos tem aumentado em Acinetobacter spp., principalmente, devido à produção de enzimas chamadas beta-lactamases e enzimas modificadoras de aminoglicosídeos (EMAs). Neste contexto, os objetivos do estudo foram avaliar o perfil de susceptibilidade aos antimicrobianos, investigar genes de carbapenemases (blaOXA-23like, blaOXA-51like, blaOXA-58like, blaOXA24-40like, blaOXA-143like, blaOXA-48like, blaKPC, blaGES,blaVIM, blaIMPe blaSPM), e de EMAs [aac(3)-Ia, aac(3')-II, aaac(6')-Ih, aph(3')-VI, ant(2')-Ia, aph(3')-Ia e aac(6')-Ib] por PCR e determinar a similaridade genéticados isolados de A. baumannii por REP-PCR. Cem isolados do complexo ACB resistentes aos carbapenêmicos provenientes de pacientes admitidos em unidades de terapia intensiva (UTIs) do Hospital de Base de São José do Rio Preto (HB) foram encaminhados ao laboratório de Microbiologia da Faculdade de Medicina de São José do Rio Preto (FAMERP), onde foram realizadas a extração de DNA, a identificação da espécie, a investigação dos genes e a tipagem molecular por REP-PCR. Todos os isolados apresentaram fenótipo de multirresistência e foram identificados como pertencentes à espécie A. baumannii. Os genes blaOXA-23like e blaOXA-51like foram detectados em 100% dos isolados, e, em todos, ISAba1 estava localizado "upstream" ao gene blaOXA-23like... / Abstract: The Acinetobacter genus comprises ubiquitous Gram-negative bacilli and its main species, A. calcoaceticus, A. baumannii, A. pittii and A. nosocomialis were grouped in the the Acinetobacter baumannii - Acinetobacter calcoaceticus Complex (ACB Complex) due to their high genetic similarity and for their difficult differentiation by biochemical methods. Most infections caused by ACB complex are pneumonia and bacteremias, and, to treat these, beta-lactams, aminoglycosides and polymyxins are widely used. Nevertheless, beta-lactams and aminoglycosides resistance has been increasing in Acinetobacter spp., mainly, due to the enzymes production called beta-lactamases and aminoglycosides modifying enzymes (AMEs). Thus, the study objectives were to evaluate the antimicrobial susceptibility profile, investigate the carbapenemases gene (blaOXA-23like, blaOXA-51like, blaOXA-58like, blaOXA24-40like, blaOXA-143like, blaOXA-48like, blaKPC, blaGES, blaVIM, blaIMPand blaSPM), the AMEs genes [aac(3)-Ia, aac(3)-II, aac(6')-Ih, aph(3')-VI, ant(2')-Ia, aph(3')-Ia and aac(6')-Ib], and to determine the genetic similarity by REP-PCR. One hundred carbapenem resistant isolates of ACB complex were sent to the Microbiology Laboratory of the Faculdade de Medicina de São José do Rio Preto, where DNA extraction, species identification, PCRs and molecular typing by REP-PCR were carried out. All of the isolates showed multidrug resistance phenotype and were identified as A. baumannii. The genes blaOXA-23like and blaOXA-51like were present in 100% of the isolates andISAba1 was "upstream" to the blaOXA-23likegene. The AMES genes detected were aph(3')-VI (55%), aac(6')-Ib (46%), aac(3)-Ia (30%) and aph(3')-Ia (24%). The REP-PCR typing generated five groups (A, B, C, D and E) with 20 clusters. Of these, many were endemic clusters, because they were isolated during all the collection period and in all the HB's ICUs. Furthermore, the horizontal transmission ... / Doutor

Les Acinetobacter sont des bactéries opportunistes impliquées dans les infections nosocomiales.Le but de ce travail était d’étudier leur épidémiologie et écologie au Liban.Tout d’abord, nous avons analysé 119 souches d’A.baumannii isolées de plusieurs hôpitaux. 76.5 % étaient résistantes aux carbapénèmes et le gène OXA-23 était le plus fréquemment trouvé. Le typage par Multilocus sequence typing a montré que le clone international II était majoritairement détecté. L’électrophorèse en champ pulsé a révélé que 72.6% des souches appartenant au ST2 ont été classées dans un même cluster qui semble être prédominant à Beirut et Tripoli. Ensuite, les réservoirs extrahospitaliers ont été investigués sur 2361 prélèvements collectés au Liban. Au total, 171 souches ont été isolées dans l’environnement, les produits alimentaires ainsi que chez l’homme et les animaux. La majorité de ces souches, globalement sensibles aux antibiotiques, était des Acinetobacter non baumannii, Seuls 15 A.baumannii, de 14 STs différents dont 10 nouveaux ont été isolés. Enfin, nous avons conduit une étude taxonomique approfondie sur plusieurs souches d’Acinetobacter non identifiées au rang d’espèce et retrouvées dans notre étude. Nous avons ainsi caractérisé une nouvelle espèce, nommée « Acinetobacter lebanonensis ».Ce travail a montré que le Liban était un pays à forte endémie d’A.baumannii résistants aux carbapénèmes. Nous n’avons toutefois pas mis en évidence de lien entre les souches cliniques et extrahospitalières, les clones correspondants étant globalement différents. D’autres études sont nécessaires pour élucider l’origine des souches multi-résistantes émergeant dans les hôpitaux. / Acinetobacter spp are opportunistic bacteria widely involved in nosocomial infections. The aim of this work was to study the epidemiology and the ecology of these bacteria in Lebanon. First, we have analyzed 119 clinical strains of A.baumannii. 76.5% of them were resistant to carbapenems and the production of OXA-23 was the main mechanism. Multi-locus sequence typing revealed the predominance of international clone II. Pulsed field gel electrophoresis showed that 72.6% of strains belonging to ST2 were classified in the same cluster which appeared to be predominant in Beirut and Tripoli. On the other hands, Acinetobacter reservoirs were investigated on 2361 samples collected in Lebanon. A total number of 171 strains have been isolated in the environment, food, humans and animals. The majority of these strains was identified as non baumannii Acinetobacter and was susceptible to antibiotics. Besides, typing of A.baumannii revealed the presence of 14 STs including 10 new ones. Finally, we have described a novel species called “Acinetobacter lebanonensis” by conducting a taxonomic study on several strains isolated in Lebanon and other countries. Although the data may be limited, this work has shown the endemic situation of carbapenem resistant A.baumannii circulating in the Lebanese hospitals while the extra hospital ones were different. However, further studies are needed to elucidate the origin of these emerging multidrug resistant strains.

This study focuses on the question of the epidemiology of resistance to antibiotics in Acinetobacter baumannii, which is nowadays one of the most problematic bacterial patho- gens associated with failing antimicrobial therapy. Its aim was to define population-genetic properties, epidemiology and the nature of multidrug resistance for a sample of the current population of A. baumannii from Czechia. A total of 55 isolates were collected in eight medi- cal facilities in central Bohemia from October 2016 to May 2018. The isolates were assessed for their identity at the species, clonal and strain levels as well as resistance phenotype and genotype; they were classified into five clonal groups, each of which encompassed isolates that were likely to be epidemiologically related. The 55 isolates studied belonged, nearly exclusively, to global clone ECII, with 53 % of them forming a genetically relatively homoge- neous group characterized by extensive resistance to antibiotics (susceptible only to col- istin), the presence of genes encoding ArmA a OXA-23 (resistance to all aminoglycosides and carbapenems) and spread in all locations. The in-depth epidemiological analysis of isolates from the city of Příbram and its vicinity indicated the regional spread of two strains, one of which belonged to the...

Submitted by MIRIAN MARCOLAN DE MELLO null (marcolanmirian@yahoo.com.br) on 2016-02-16T08:13:42Z No. of bitstreams: 1 TESE Doutorado Mirian14_02_2016REPOSITÓRIO.pdf: 1649527 bytes, checksum: c26e5ed45e88f8a3889873caa917015a (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-02-17T13:45:10Z (GMT) No. of bitstreams: 1 mello_mm_dr_sjc.pdf: 1649527 bytes, checksum: c26e5ed45e88f8a3889873caa917015a (MD5) / Made available in DSpace on 2016-02-17T13:45:10Z (GMT). No. of bitstreams: 1 mello_mm_dr_sjc.pdf: 1649527 bytes, checksum: c26e5ed45e88f8a3889873caa917015a (MD5) Previous issue date: 2015-12-14 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / 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 8 meses. Entre os isolados de A. baumannii, 58% demonstraram resistência aos antibióticos imipenem e meropenem por meio de teste de difusão em disco. A seguir 3 isolados clínicos sensíveis e 18 isolados resistentes a esses antibióticos foram selecionados para o presente estudo. O valor de CIM para os isolados sensíveis variou de ˂ 0,5 a 1µg/mL e para os isolados resistentes de 64 a >128µg/mL. A PDT in vitro reduziu o número de células de A. baumannii em todos os isolados testados, mas o percentual de redução foi dependente dos isolados analisados. Além disso, verificou-se nos testes in vivo, que o tratamento com PDT, antibióticos (Imipenem e Meropenem) e associação de PDT+Antibióticos resultaram na sobrevivência das lagartas de G. mellonella, porém sem efeito sinérgico. Conclui-se que a PDT teve ação antimicrobiana contra isolados clínicos de A. baumannii sensíveis e resistentes aos carbapenêmicos, mas não apresentou efeito sinérgico quando associada com antibióticos. / Due to the rapid growth of microorganisms resistant to antibiotics and the limited development of new antimicrobial agents, infections by Gram-negative 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 antibiotics were selected for this study. The MIC value for sensitive isolates ranged from 0.5 to ˂ 1μg / mL and resistant isolates from 64 to> 128μg / mL. The PDT in vitro reduced the number of A. baumannii cells in all isolates tested, but the percentage of reduction was dependent on the analyzed isolates. Furthermore, it was found in in vivo tests, treatment with PDT, antibiotic (Imipenem and Meropenem) and PDT + Antibiotics association resulted in the survival of G. mellonella caterpillars, but no synergistic effect. It was concluded that PDT has antimicrobial activity against clinical isolates of A. baumannii sensitive and resistant to carbapenems, but it had no synergistic effect when combined with antibiotics.

Submitted by Ana Cláudia Moro Lima dos Santos (anna.moro@hotmail.com) on 2018-05-07T20:15:18Z No. of bitstreams: 1 Dissertaçãofinal.pdf: 1989003 bytes, checksum: c02493762a88023038e4fc08eea12f1c (MD5) / Approved for entry into archive by Sulamita Selma C Colnago null (sulamita@btu.unesp.br) on 2018-05-09T16:37:26Z (GMT) No. of bitstreams: 1 santos_acml_me_bot.pdf: 1989003 bytes, checksum: c02493762a88023038e4fc08eea12f1c (MD5) / Made available in DSpace on 2018-05-09T16:37:26Z (GMT). No. of bitstreams: 1 santos_acml_me_bot.pdf: 1989003 bytes, checksum: c02493762a88023038e4fc08eea12f1c (MD5) Previous issue date: 2018-02-26 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Peritonite por bacilos Gram-negativos não fermentadores (BGNNF) é complicação importante da diálise peritoneal (DP), com curso clínico grave e elevada taxa de falência do método. Fatores associados à virulência, resistência antimicrobiana, formação de biofilme, entre outros, têm sido relatados, mas o limitado conjunto de evidências não permite concluir sobre os fatores responsáveis pelo pior curso clínico dessas infecções. O objetivo deste trabalho foi avaliar a influência das características microbiológicas, das condições clínicas do paciente e do tratamento na evolução de peritonites por BGNNF, ocorridas num único centro, em período de 18 anos. A sensibilidade in vitro aos antimicrobianos, produção de biofilme, além da análise do perfil clonal das bactérias pela técnica de eletroforese em gel de campo pulsado foram realizadas em todos os isolados bacterianos. Foram pesquisados genotipicamente, em isolados de Pseudomonas aeruginosa, a presença de marcadores de virulência (alginato, exoenzima S, fosfolipases C, exotoxina A, protesase alcalina, elastase e ramnolipídeos). Associações entre as características microbiológicas do paciente e tratamento com a taxa de resolução da peritonite foram estudadas. A espécie mais frequente foi Pseudomonas aeruginosa (45,59%), seguida por isolados do complexo Acinetobacter baumannii (17,65%). O estudo dos fatores de virulência da Pseudomonas aeruginosa revelou a presença de fatores de virulência em 100% dos casos, exceto exoenzima S (58,33%) e fosfolipase C não hemolítica (87,5%). Houve elevada proporção de BGNNF resistentes aos antimicrobianos testados, em particular à amicacina (36,73%) e à ciprofloxacina (44,9%), sendo que a sensibilidade aos betalactâmicos esteve acima de 70%. Observou-se elevada proporção de isolados produtores de biofilme (73,08%). Os resultados da tipagem por PFGE revelaram um perfil policlonal para a maioria dos isolados, entretanto para isolados do complexo Acinetobacter baumannii a análise revelou um cluster, entre 2000-2008, com perfil de multiresistência aos antimicrobianos, sugerindo fonte hospitalar. A evolução dos episódios mostrou reduzida taxa de cura (35,29%). A sensibilidade à amicacina e cefepime, se associaram de modo independente à maior chance de cura, enquanto a presença concomitante de infecção do óstio de saída do cateter de DP foi preditor independente de não resolução do episódio. Não se observaram associações entre fatores de virulência, produção de biofilme e características do paciente e tratamento com o desfecho dos episódios. Em conclusão, peritonites em DP, por BGNNF, são infecções com reduzida taxa de cura; a resistência bacteriana é fator associado à menor chance de resolução e peritonite por bactérias do gênero Acinetobacter spp. podem representar infecção grave, potencialmente de origem hospitalar, o que deve fazer redobrar os cuidados quanto ao seu manejo clínico. / Peritonitis due to non-fermentative Gram-negative bacilli (NFGNB) is a serious complication of peritoneal dialysis (PD), with a severe clinical course and high technique failure rate. Factors as bacterial virulence, antimicrobial resistance, biofilm formation, among others, have been reported, but the limited amount of evidence does not allow to conclude on the factors responsible for the worst clinical course of these infections. The objective of this study was to evaluate the influence of the microbiological characteristics, patients conditions, and treatment on evolution of peritonitis episodes at a single center in an 18 - year period. In vitro susceptibility, biofilm production, and clonal profile analysis of bacteria by pulsed-field gel electrophoresis (PFGE) were performed in all isolates. The presence of virulence markers (alginate, exoenzyme S, phospholipases C, exotoxin A, alkaline protease, elastase, and ramnolipids) was genotyped in bacterial isolates of Pseudomonas aeruginosa. From the data referring to the patient and causal agent, associations between the microbiological, patient characteristics, and treatment on the resolution rate of peritonitis were analyzed. The most frequent species was Pseudomonas aeruginosa (45.59%), followed by Acinetobacter baumannii complex (17.65%). The study of the virulence factors of Pseudomonas aeruginosa revealed the presence of virulence factors in 100% of the cases, except for exonzyme S (58.33%) and hemolytic phospholipase C (87.5%). There was a high proportion of antimicrobial resistant, in particular to amikacin (36.73%) and ciprofloxacin (44.9%), with sensitivity to betalactam above 70%. A high (73.08%) proportion of biofilm producing isolates was observed. The results of the PFGE typing revealed a polyclonal profile for most of the isolates; however, for the Acinetobacter baumannii complex species the analysis revealed a cluster at interval from 2000 to 2008, with antimicrobial multi resistance profile, suggest that peritonitis by this agent had a hospital source. The evolution of the episodes showed a reduced resolution rate (35.29%). The susceptibility to amikacin and to cefepime were independently associated with a higher odds of resolution, while the concomitant presence of PD catheter exit site infection was an independent predictor of non-resolution. In conclusion, peritonitis due to NFGNB in PD are infections with reduced resolution rate; bacterial resistance is an independent predictor of lower odds of resolution. Peritonitis by Acinetobacter spp. can represent serious / 64736017.2.0000.5411

Over time, “superbugs,” or bacteria that have become resistant to antibiotics, have become a great concern in modern medicine. Viable alternates are currently being looked into as effective and safe ways to prevent or treat infections caused by these superbugs. One such method is through the utilization of the second messenger molecule cyclic-di-GMP (c-di-GMP) that has been shown to regulate phenotypes within other bacteria that may control surface colonization in Acinetobacter baumannii. Through a series of experiments, the active enzymes that create c-di-GMP - diguanylate cyclases - and break down c-di- GMP - phosphodiesterases - have been inactivated in mutants to test phenotypes including biofilm formation, motility, antibiotic resistance, and desiccation survival. The research’s objective is to show that manipulation of c-di-GMP within the multi-drug resistant strain of Acinetobacter baumannii may serve as a means to control this bacteria.

Bibliography / Thesis (MMed (Pathology. Medical Microbiology))--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: Acinetobacter baumannii has emerged as one of the most troublesome nosocomial pathogens globally. This organism causes infections that are often extremely difficult to treat because of the widespread resistance to the major antibiotic groups. Colonization or infection with multidrugresistant A. baumannii is associated with the following risk factors: prolonged hospital stay, admission to an intensive care unit (ICU), mechanical ventilation, and exposure to broad spectrum antibiotics, recent surgery, invasive procedures, and severe underlying disease. A. baumannii has been isolated as part of the skin flora, mostly in moist regions such as axillae, groin and toe webs. It has also been isolated from the oral cavity and respiratory tract of healthy adults. Debilitated hospitalized patients have a high rate of colonization, especially during nosocomial Acinetobacter outbreaks. This organism is an opportunistic pathogen as it contains few virulence factors. Clinical manifestations of A. baumannii include nosocomial pneumonia, nosocomial bloodstream infections, traumatic battlefield and other wound infections, urinary tract infections, and post-neurological surgery meningitis. Fulminant community-acquired pneumonia has recently been reported, indicating that this organism can be highly pathogenic. The number of multidrug-resistant A. baumannii strains has been increasing worldwide in the past few years. Therefore the selection of empirical antibiotic treatment is very challenging. Antibiotic combinations are used mostly as empirical therapy in critically ill patients. One rationale for the use of combination therapy is to achieve synergy between agents. The checkerboard and time-kill methods are two traditional methods that have been used for synergy testing. These methods are labor intensive, cumbersome, costly, and time consuming. The E-test overlay method is a modification of the E-test method to determine synergy between the different antibiotics. This method is easy to perform, flexible and time efficient. The aim of this study was to assess the in vitro activity of different combinations of colistin, rifampicin, imipenem, and tobramycin against selected clinical strains of A. baumannii using the checkerboard and the E-test synergy methods. The MICs obtained with the E-test and broth microdilution method were compared. The results of the disk diffusion for imipenem and tobramycin as tested in the routine microbiology laboratory were presented for comparison. Overall good reproducibility was obtained with all three methods of sensitivity testing. The agreement of MICs between the broth dilution and E-test methods was good with not more than two dilution differences in MIC values for all isolates, except one in which the rifampicin E-test MIC differed with three dilutions from the MIC obtained with the microdilution method. However, the categorical agreement between the methods for rifampicin was poor. Although MICs did not differ with more than two dilutions in most cases, many major errors occurred because the MICs clustered around the breakpoints. The combinations of colistin + rifampicin, colistin + imipenem, colistin + tobramycin, rifampicin + tobramycin, and imipenem + tobramycin all showed indifferent or additive results by the E-test method. No results indicating synergy were obtained for all the above-mentioned combinations. There was one result indicating antagonistic effect for the combination of colistin + tobramycin. The results of the checkerboard method showed results indicating synergy in four of the six isolates for which the combination of colistin and rifampicin was tested. The other two isolates showed indifferent/additive results. All the other combinations showed indifferent/additive results for all isolates except isolate 30 (col + tob) and isolate 25 (rif + tob) which showed synergism. No antagonistic results were observed by the checkerboard method. When the results obtained with the E-test and checkerboard methods were compared, it was noted that for most antibiotic combinations an indifferent/additive result was obtained. However, for the colistin + rifampicin combination, the checkerboard method showed synergism for 4 of 6 isolates, whereas the E-test method showed indifference and an additive result in one. For the rifampicin + tobramycin, and colistin + tobramycin combinations, synergism was also shown with the checkerboard method in one isolate for each combination. The E-test method however showed an indifferent and additive result respectively. . The E-test method was found to be a rapid, reproducible, easy-to-perform, and flexible method to determine synergistic antibiotic activity. This study was however limited by low numbers of isolates. This might explain why no synergistic results were obtained with the E-test method and few synergistic results with the checkerboard method. Genotypic analysis using pulse-field gel electrophoresis (PFGE) may be considered in future studies to determine relatedness of the isolates which will facilitate the selection of different strains for synergy testing. Furthermore, clinical studies are needed to establish whether in vitro synergy testing is useful in the clinical setting and whether the results of synergy testing will have any bearing on the clinical outcome of patients infected with multidrug resistant A. baumannii. / AFRIKAANSE OPSOMMING: Acinetobacter baumannii het wêreldwyd as een van die mees problematiese nosokomiale patogene verskyn. Hierdie organisme veroorsaak infeksies wat dikwels baie moeilik is om te behandel weens wydverspreide weerstandigheid teen major antibiotikagroepe. Kolonisasie of infeksie met multi-weerstandige A. baumannii word geassosieer met die volgende riskofaktore: verlengde hospitaalverblyf, toelating tot ‘n intensiewe sorgeenheid (ICU), meganiese ventilasie, blootstelling aan breëspektrum antibiotika, onlangse chirurgie, indringende prosedures en ernstige onderliggende siekte. A. baumannii kan deel vorm van die normale velflora, veral in die axillae, inguinale area en tussen die tone. Dit is ook al vanuit die mondholte en die respiratoriese traktus van gesonde volwassenes geïsoleer. Verswakte gehospitaliseerde pasiënte word veral gekoloniseer gedurende nosokomiale Acinetobacter uitbrake. Hierdie organisme is ‘n opportunistiese patogeen en bevat min virulensie faktore. Kliniese manifestasies van A. baumannii sluit nosokomiale pneumonie, nosokomiale bloedstroom infeksies, troumatiese slagveld- en ander wondinfeksies, urienweginfeksies en meningitis wat volg op neurologiese chirurgie in. Fulminerende gemeenskapsverworwe pneumonie is onlangs beskryf en dui aan dat hierdie organisme hoogs patogenies kan wees. Die aantal multi-weerstandige A. baumannii stamme het wêreldwyd toegeneem oor die laaste paar jare. Daarom is die seleksie van empiriese antibiotiese behandeling ‘n uitdaging. Antibiotika kombinasies word meestal as empiriese behandeling in ernstige siek pasiënte gebruik. Die beginsel hiervan is om sinergistiese werking tussen agente te verkry. Die “checkerboard” en “time-kill” metodes is twee tradisionele metodes van sinergisme toetsing. Hierdie metodes is werksintensief, duur en tydrowend. Die E-toets sinergisme metode is gebaseer op die E-toets metode. Hierdie metode is maklik, buigbaar en tydseffektief. Die doel van hierdie studie was om die in vitro aktiwiteit tussen verskillende antibiotika kombinasies van colistin, rifampisien, imipenem, en tobramisien teen geselekteerde kliniese A. baumannii isolate te toets met die “checkerboard” en E-toets sinergisme toetsing metodes. Die minimum inhibitoriese konsentrasies (MIKs) verkry met die E-toets en “broth microdilution” metode is ook vergelyk. Die resultate van die skyfie diffusie metode (die metode wat in die roetiene mikrobiologie laboratorium gebruik word) vir imipenem en tobramisien word ook verskaf vir vergelyking van die resultate van verskillende sensitiwiteitsmetodes. In oorsig is goeie herhaalbaarheid van resultate verkry met al drie metodes van sensitiwiteitstoetsing. Die ooreenstemming van MIKs tussen die “broth dilution” en E-toets metodes was goed en resultate het met nie meer as twee verdunnings in MIK waardes verskil nie. Daar is een uitsondering waar die rifampisien E-toets MIK waarde met drie verdunnings van die MIK waarde verkry met die “microdilution” metode verskil. Die ooreenstemming tussen die sensitiwiteitskategorie resultate tussen die twee metodes was egter swak vir rifampisien. Alhoewel die MIKs in die meeste gevalle met nie meer as twee verdunnings in waarde verskil het nie, was daar baie major foute aangetoon omdat die MIKs rondom die breekpunte geval het. Die kombinasies van colistin + rifampisien, colistin + imipenem, colistin + tobramisien, rifampisien + tobramisien, en imipenem + tobramisien het oorwegend slegs matige interaksie met die E-toets metode getoon. Geen sinergisme is verkry met enige van die antibiotika kombinasies met hierdie metode nie. Daar was egter een resultaat wat antagonisme getoon het vir die kombinasie van colistin + tobramycin. Die resultate van die “checkerboard” metode het sinergisme getoon in vier van die ses isolate wat vir die kombinasie van colistin en rifampisien getoets was. Die ander twee isolate het slegs matige interaksie getoon. Al die ander kombinasies het ook slegs matige interaksie getoon, behalwe in isolaat 30 (col + tob) en isolaat 25 (rif + tob) waar die spesifieke kombinasies sinergisme getoon het. Geen antagonisme is waargeneem met die “checkerboard” metode nie. Met vergelyking van die E-toets en “checkerboard” metodes, is dit opmerklik dat vir die meeste van die antibiotika kombinasies slegs matige interaksie verkry is. Vir die colistin + rifampisien kombinasie toon die “checkerboard” metode egter sinergisme vir 4 uit 6 isolate, terwyl die E-toets metode slegs matige interaksie toon. Vir rifampisien + tobramisien, en colistin + tobramisien kombinasies is sinergisme getoon met die “checkerboard” metode in een isolaat vir elke kombinasie. Die E-toets metode het slegs matige interaksie getoon. Die E-toets sinergisme metode was vinnig, herhaalbaar en maklik om uit te voer. Hierdie studie word egter beperk deur lae getalle van isolate. Dit mag verklaar waarom geen sinergistiese resultate met die E-toets metode verkry is nie en die min sinergistiese resultate met die “checkerboard” metode. Genotipiese analiese met “pulse-field gel electrophoresis” mag in aanmerking geneem word in toekomstige studies om die verwantskap tussen isolate te bepaal wat die seleksie van verskillende stamme vir sinergisme toetsing sal vergemaklik. Verder, kliniese studies is nodig om te bepaal of in vitro sinergisme toetsing van waarde is en of die resultate van sinergisme toetsing ‘n rol speel in die kliniese uitkoms van pasënte geïnfekteer met multiweerstandige A. baumannii. / The National Health Laboratory Serivice

Submitted by Genilton Cardenes (genilton_cardenes@outlook.com) on 2018-10-31T16:11:12Z No. of bitstreams: 3 Avaliação do potencial de Acinetobacter junii SB132 na degradação de hidrocarbonetos do diesel.pdf: 3349884 bytes, checksum: 7e54351525a269ee4bacb489c4b8ee09 (MD5) Ata de defesa de dissertação.pdf: 313772 bytes, checksum: c312101e5d7eadad598267ac8a263549 (MD5) Carta de encaminhamento BDTD e termo de autorização para publicação.pdf: 776943 bytes, checksum: f8362a56a57c08b24e6f2f4266042eb4 (MD5) / Rejected by PPGBIOTEC Biotecnologia (ppg_biotec.ufam@yahoo.com.br), reason: O arquivo da Dissertação está faltando a ficha catalográfica. on 2018-11-06T20:40:25Z (GMT) / Submitted by Genilton Cardenes (genilton_cardenes@outlook.com) on 2018-11-08T19:53:33Z No. of bitstreams: 3 Ata de defesa de dissertação.pdf: 313772 bytes, checksum: c312101e5d7eadad598267ac8a263549 (MD5) Carta de encaminhamento BDTD e termo de autorização para publicação.pdf: 776943 bytes, checksum: f8362a56a57c08b24e6f2f4266042eb4 (MD5) Avaliação do potencial de Acinetobacter junii SB132 na degradação de hidrocarbonetos do diesel.pdf: 2974599 bytes, checksum: 772fb4ba6951912ffcf1ffe6fc6dfc4e (MD5) / Approved for entry into archive by PPGBIOTEC Biotecnologia (ppg_biotec.ufam@yahoo.com.br) on 2018-11-12T19:16:22Z (GMT) No. of bitstreams: 3 Ata de defesa de dissertação.pdf: 313772 bytes, checksum: c312101e5d7eadad598267ac8a263549 (MD5) Carta de encaminhamento BDTD e termo de autorização para publicação.pdf: 776943 bytes, checksum: f8362a56a57c08b24e6f2f4266042eb4 (MD5) Avaliação do potencial de Acinetobacter junii SB132 na degradação de hidrocarbonetos do diesel.pdf: 2974599 bytes, checksum: 772fb4ba6951912ffcf1ffe6fc6dfc4e (MD5) / Rejected by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br), reason: * A carta inserida deve ser a "Carta de Encaminhamento para o Autodepósito" disponível em http://biblioteca.ufam.edu.br/servicos/teses-e-dissertacoes * O Termo de Autorização é dispensável. A menos que a Tese/Dissertação seja Confidencial e/ou passível de patente, conforme orientações em http://biblioteca.ufam.edu.br/servicos/teses-e-dissertacoes Dúvidas? ddbc@ufam.edu.br on 2018-11-12T20:52:58Z (GMT) / Submitted by Genilton Cardenes (genilton_cardenes@outlook.com) on 2018-11-13T20:07:30Z No. of bitstreams: 3 Ata de defesa de dissertação.pdf: 313772 bytes, checksum: c312101e5d7eadad598267ac8a263549 (MD5) Avaliação do potencial de Acinetobacter junii SB132 na degradação de hidrocarbonetos do diesel.pdf: 2974599 bytes, checksum: 772fb4ba6951912ffcf1ffe6fc6dfc4e (MD5) Carta de encaminhamento BDTD.pdf: 315084 bytes, checksum: e83e1b1d1918636fcffa71de997b062d (MD5) / Approved for entry into archive by PPGBIOTEC Biotecnologia (ppg_biotec.ufam@yahoo.com.br) on 2018-11-14T18:58:47Z (GMT) No. of bitstreams: 3 Ata de defesa de dissertação.pdf: 313772 bytes, checksum: c312101e5d7eadad598267ac8a263549 (MD5) Avaliação do potencial de Acinetobacter junii SB132 na degradação de hidrocarbonetos do diesel.pdf: 2974599 bytes, checksum: 772fb4ba6951912ffcf1ffe6fc6dfc4e (MD5) Carta de encaminhamento BDTD.pdf: 315084 bytes, checksum: e83e1b1d1918636fcffa71de997b062d (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2018-11-14T20:20:28Z (GMT) No. of bitstreams: 3 Ata de defesa de dissertação.pdf: 313772 bytes, checksum: c312101e5d7eadad598267ac8a263549 (MD5) Avaliação do potencial de Acinetobacter junii SB132 na degradação de hidrocarbonetos do diesel.pdf: 2974599 bytes, checksum: 772fb4ba6951912ffcf1ffe6fc6dfc4e (MD5) Carta de encaminhamento BDTD.pdf: 315084 bytes, checksum: e83e1b1d1918636fcffa71de997b062d (MD5) / Made available in DSpace on 2018-11-14T20:20:28Z (GMT). No. of bitstreams: 3 Ata de defesa de dissertação.pdf: 313772 bytes, checksum: c312101e5d7eadad598267ac8a263549 (MD5) Avaliação do potencial de Acinetobacter junii SB132 na degradação de hidrocarbonetos do diesel.pdf: 2974599 bytes, checksum: 772fb4ba6951912ffcf1ffe6fc6dfc4e (MD5) Carta de encaminhamento BDTD.pdf: 315084 bytes, checksum: e83e1b1d1918636fcffa71de997b062d (MD5) Previous issue date: 2017-10-26 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Industrial exploitation of petroleum as well as the use of its derivatives has been growing due to its importance for society. Petroleum is a complex mixture of several organic compounds, mainly hydrocarbons compounds. The occurrence of contamination of the environment with these components is worrisome because in addition to its difficult degradation, oil requires many stages of processing, from its extraction, transportation, refining to the storage of the derivatives, dramatically increasing its exposure to the environment. An alternative to hydrocarbons degradation is the use of bacteria, by process called biodegradation, that depends ecosystem conditions and the local environment. Thus, bioremediation is a treatment process that uses microorganisms that degrade and transform existing organic pollutants in less complex and generally more easily degradable compounds, which can even reach mineralization. In this study we used the Acinetobacter junii SB132 bacterium previously isolated from aquatic macrophytes of Rio Negro near the city of Manaus (AM). Its hydrocarbon degradation capacity was tested in presence of diesel oil as the only carbon and energy source. In this work, the results obtained by gas chromatography coupled to mass spectrometry (GC-MS) showed that the alkanes of the diesel oil were degraded on average 58% by A. junii SB132 at 30 °C after 4 days of culture. The individual alkanes of diesel oil were degraded between 60 % -87 %. Proteomic study revealed proteins and metabolic pathway of A. junii SB132 involved in the degradation of hydrocarbons, specially alkanes. This study suggests that this degrading bacterial lineage of hydrocarbons has a great potential for bioremediation of the environment contaminated by diesel. / Atualmente, a exploração industrial do petróleo bem como o uso de seus derivados vem crescendo cada vez mais devido à sua importância econômica para a sociedade. O petróleo é uma mistura complexa de vários compostos orgânicos, constituído principalmente por hidrocarbonetos. A ocorrência de contaminação do meio ambiente com estes compostos é agravada, pois, além da sua difícil degradação, o petróleo requer muitas etapas de processamento, desde a sua extração, transporte, refino até a armazenagem dos derivados, aumentando drasticamente a sua exposição ao meio ambiente. Uma alternativa para a degradação de hidrocarbonetos é o uso de bactérias e tal processo, nomeado biodegradação, depende das condições do ecossistema e do meio ambiente local. Com isso, a biorremediação é um processo de tratamento que utiliza microrganismos que degradam e transformam compostos orgânicos poluentes existentes nos ambientes contaminados em compostos menos complexos e geralmente mais facilmente degradáveis, podendo chegar até a sua mineralização. Neste estudo foi utilizada a bactéria Acinetobacter junii SB132 previamente isolada a partir de macrófitas aquáticas do Rio Negro nas proximidades da cidade de Manaus (AM). Sua capacidade de degradação de hidrocarbonetos foi avaliada fornecendo óleo diesel como única fonte de carbono. Os resultados obtidos pela técnica de cromatografia gasosa acoplada à espectrometria de Massas (GC-MS) mostraram que os alcanos do óleo diesel foram degradados em média 58 % por A. junii SB132 após 4 dias de cultivo em meio mínimo a 30 °C. Os alcanos individuais de óleo diesel foram degradados entre 60% -87%. A partir de proteínas extraídas dessa linhagem também foram feitas análises por ESI-MS que identificaram proteínas e rotas metabólicas envolvidas na degradação de hidrocarbonetos como a via de degradação, especialmente de alcanos. Esse estudo sugere que essa linhagem bacteriana possui um grande potencial para biorremediação de ambiente contaminado por diesel.

Made available in DSpace on 2016-04-28T14:59:00Z (GMT). No. of bitstreams: 1 2008 - Debora Alves Gonzaga da Silva1.pdf: 485199 bytes, checksum: 8ad8d9c6be0e3c6fca263c141e2866c3 (MD5) Previous issue date: 2008-09-26 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / In the objective of advancing the epidemiologic study of the bacterial stain of the tomato, the mechanisms of transmission of Xanthomonas vesicatoria from the plant to the seed and from the seed to the plant have been evaluated. The morphology of seeds and of parts of the seedlings or saplings (tegument, radicles, hypocotyl and cotyledon leaves, and definitive leaves) have also been studied by means of electronic scanning microscopy techniques. The transmission of X. vesicatoria from the plant to the seed has been evaluated upon direct and indirect isolation of parts of ripe fruit (flesh, placenta, placental liquid, water from the first washing of the seeds, entire seeds and ground seeds) in a culture of Agar Nutrient (AN). The fruit were obtained in experimental parcels, conducted at the Horticultural sector of UFRRJ, and inoculated with the phytobacteria through different methods (atomization and injection) combined with different regions of inoculation (flower cluster, flesh, and placenta), during the plant s developing phases (flower, unripened fruit, firm ripe and ripe fruit). The transmission of the phytobacteria from the seeds to the seedlings and saplings was assessed by means of isolating in an AN medium parts of the seedling or sapling (root, tegument, radicles, hypocotyl and cotyledon leaves, and definitive leaves), germinated in different substrata ( germitest paper, sand, and commercialized substrate for saplings). The morphology of seeds and parts of the seedlings was characterized through observations in electron microscope scanning by using samples of fresh seeds (extracted from inoculated fruit), of parts of seedlings or saplings (tegument, radicles, hypocotyl and cotyledon leaves, and definitive leaves) and of seeds inoculated by vacuum procedure at 7, 14, and 21 days after sowing. The treatments applied to the unripened fruit (inoculation by atomization, injection in the placenta and injection in the flesh) were more efficient in the process of transmitting the phytobacteria than the treatment applied to the firm ripe and ripe fruit. It was also observed that the X vesicatoria colonizes the tegument and every part of the seedlings and saplings during the process of germination and emergence. The seed tegument was characterized by an entanglement of trichomes, with a base in the shape of ring and cavity, which may serve as sheltering sites for pathogens, including the X vesicatoria. The process of colonization of tomato seeds by X vesicatoria characterizes itself by the formation of biofilms and fibrils. The presence of stomata colonized by X vesicatoria has been observed in radicles, of seven day old seedlings, and in primary roots originated from the radicles of 20 day old saplings. Few cells of X vesicatoria have been observed on the hypocotyl. The presence of endophytic bacteria forming aggregates with characteristics of an aggressive growth identified as Acinetobacter sp. was detected in many samples of seeds. These same bacteria were detected in many tests of germination and isolation, interfering negatively in the development and recuperation of X vesicatoria in the in vitro tests. / Com o objetivo de se avan?ar no estudo epidemiol?gico da mancha-bacteriana do tomateiro, foram avaliados os mecanismos de transmiss?o de Xanthomonas vesicatoria da planta para a semente e da semente para a planta. Foram feitos, ainda, estudos da morfologia da semente e partes da pl?ntula ou muda (tegumento, rad?cula, hipoc?tilo e folhas cotiledonares e definitivas), atrav?s de t?cnicas de microscopia eletr?nica de varredura. A transmiss?o de X. vesicatoria da planta para a semente foi avaliada a partir de isolamentos diretos e indiretos de partes de frutos maduros (mesocarpo, placenta, l?quido placent?rio, ?gua proveniente da primeira lavagem das sementes, sementes inteiras e sementes trituradas) em meio de cultura Nutriente Agar (NA). Os frutos foram obtidos em parcelas experimentais, conduzidas no setor de Horticultura da UFRRJ, e inoculados com a fitobact?ria por diferentes m?todos (atomiza??o e inje??o) combinados com diferentes regi?es de inocula??o (cacho floral, mesocarpo e placenta), durante as fases de desenvolvimento da planta (flor, frutos verdes, frutos de vez e frutos maduros). A transmiss?o da fitobact?ria das sementes para as pl?ntulas e mudas foi avaliada por isolamentos em meio NA a partir de partes da pl?ntula ou muda (raiz, tegumento, hipoc?tilo, folhas cotiledonares e folhas definitivas), germinadas em diferentes substratos (papel germitest, areia e substrato comercial para mudas). A morfologia das sementes e partes das pl?ntulas foi caracterizada por meio de observa??es em microsc?pio eletr?nico de varredura utilizando-se amostras de sementes frescas (extra?das de frutos inoculados), de partes de pl?ntulas ou mudas (raiz, hipoc?tilo, folha cotiledonar e folha definitiva) e de sementes inoculadas pelo procedimento a v?cuo aos 7, 14 e 21 dias ap?s a semeadura. Os tratamentos aplicados aos frutos verdes (inocula??o por atomiza??o, inje??o na placenta e inje??o no mesocarpo) foram mais eficientes no processo de transmiss?o da fitobact?ria que os tratamentos aplicados aos frutos de vez e maduros. Observou-se, ainda, que X. vesicatoria coloniza o tegumento e todas as partes das pl?ntulas e mudas durante o processo de germina??o e emerg?ncia. O tegumento da semente foi caracterizado por um emaranhado de tricomas, com a base em forma de anel e cavidade, que podem servir como s?tios protetores para pat?genos inclusive X. vesicatoria. O processo de coloniza??o das sementes de tomate por X. vesicatoria se caracteriza pela forma??o de biofilmes e fibrilas. Foram observados em rad?culas, de pl?ntulas com sete dias de idade, e em ra?zes prim?rias, oriundas das rad?culas de mudas com 20 dias de idade, a presen?a de est?matos colonizados por X. vesicatoria. Sobre o hipoc?tilo, foram observadas poucas c?lulas de X. vesicatoria. Em v?rias amostras de sementes foi detectada a presen?a de bact?rias endof?ticas, que formavam agregados com caracter?sticas de crescimento agressivo, identificadas como Acinetobacter sp. Esta mesma bact?ria foi detectada em v?rios testes de germina??o e isolamento interferindo negativamente no desenvolvimento e recupera??o de X. vesicatoria nos testes in vitro.