Acinetobacter baumannii is an opportunistic pathogen which has been implicated in a variety of nosocomial infections among immunocompromised patients worldwide. Recently, Multi-drug resistant (MDR) isolates of A. baumannii have been isolated from military personnel returning from service in Iraq and Afghanistan. Antibiotic resistance of A. baumannii has limited the number of active antibacterial, making very difficult to treat these types of infections. This work investigated the role of Resistance-Nodulation-cell Division (RND) efflux pumps in the antibiotic resistance mechanism of A. baumannii. Expression of six different RND pumps was analyzed in clinical isolates of A. baumannii. A novel RND family pump, AdeMNO, was found to be present in a majority of isolates. The adeMNO operon was cloned, sequenced, and characterized using the single copy gene expression system in an efflux sensitized surrogate Pseudomonas aeruginosa strain. Antibiotics, trimethoprim, chloramphenicol, and clindamicin were identified as the substrates of this pump. In order to understand the mechanisms of regulation of adeMNO operon, a putative regulator belonging to the lysR-family was identified, cloned, and sequenced from the upstream region of the operon. Promoter regions of the adeMNO operon were also sequenced from various clinical isolates and sequence polymorphisms identified that could be implicated in the regulation of adeMNO expression. / UOIT

Carbapenem resistance in Acinetobacter baumannii is a growing public health concern and represents a serious problem for treatment of the infection. Several carbapenem-hydrolysing &beta / -lactamases have been identified from A. baumannii so far. In this study carbapenem resistance in A.baumannii strains recovered from intensive care units of Gulhane Military Medical Academy, Turkey, were investigated via multiplex PCR and with parallel phenotypic tests. From June 2006 to January 2010, 138 clinical A. baumannii isolates were collected. Identification and antimicrobial susceptibility tests of the isolates were performed. The MICs of imipenem and meropenem were determined by using E-test method. Carbapenem resistant A. baumannii strains were included for further study. Firstly, the presence of carbapenemases were determined. The presence of Metallo-beta-lactamase (MBL) were also investigated. Detection of the four groups of OXA carbapenemases (OXA- 23, OXA-24, OXA-51 and OXA-58) was carried out using a multiplex PCR assay. Sequence analyses were performed. Non-duplicate, multidrug resistant 61 clinical A. baumannii isolates were found to be resistant to imipenem and meropenem. In the 61 isolates, the MIC50 of imipenem and meropenem were 16 and &gt / 32 / MIC90 were 192 and &gt / 32 respectively. Modified Hodge Tests (MHT) were positive for all 61 A. baumannii strains. None of these isolates showed MBL activity. As determined through multiplex PCR, all of the 61 isolates had blaOXA-51 genes, 50 isolates had blaOXA-23, and 11 isolated had blaOXA-58 genes. Alleles encoding OXA-24-like enzymes were not detected in any isolates. This study indicated that the clinical isolates in our region contained blaOXA-51, blaOXA-23, and blaOXA-58 resistance genes. However, blaOXA-24 gene was either absent or occur in very low frequency.

Tez (Uzmanlık) - Süleyman Demirel Üniversitesi, Tıp Fakültesi, Mikrobiyoloji ve Klinik Mikrobiyoloji Anabilim Dalı, 2008. / Kaynakça var.

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Bactérias Gram-negativas não fermentadoras são frequentemente encontradas em águas superficiais, sendo muitas vezes carregadoras de múltipla resistência. O presente trabalho teve como objetivo principal analisar a participação de Pseudomonas sp. e Acinetobacter sp. na manutenção da resistência a antimicrobianos em quatro pontos na laguna de Tramandaí e se a presença deles poderia contribuir para a sua permanência. As amostras de água superficial foram coletadas em quatro pontos de coleta na laguna e submetidas a tratamento com os antimicrobianos: ácido nalidíxico, ceftazidima, imipenem e tetraciclina na concentração de 20mg/L. Em cada ponto de coleta uma das alíquotas não foi suplementada com os mesmos sendo utilizada como controle. Os isolados de Pseudomonas sp. foram identificados por provas bioquímicas e MALDI-TOF MS, enquanto que os isolados de Acinetobacter sp. somente por provas bioquímicas. O perfil de suscetibilidade de ambos foi avaliado pela técnica de disco-difusão e a produção de ESBL pela técnica de disco combinado. Os pontos 3 e 4 foram os que exibiram maior número de isolados resistentes. Os maiores percentuais de resistência estiveram associados às amostras submetidas ao tratamento com antimicrobianos. Em todos os pontos de coleta foram encontrados isolados de P. aeruginosa e Acinetobacter sp. multirresistentes. Ambas amostras (com e sem tratamento) exibiram diferentes padrões de resistência nos diferentes pontos de coleta e tanto isolados de P. aeruginosa como de Acinetobacter sp. exibiram isolados produtores de ESBL. A presença de P. aeruginosa e Acinetobacter sp. multirresistentes na Laguna de Tramandaí atenta para o risco de disseminação de resistência neste ambiente e que o mesmo pode estar atuando como reservatório de resistência. / Non-fermenting Gram-negative bacteria are often found in surface water, and are often carriers of multiple resistance to antimicrobials. The present work had as main objective to analyze the role of Pseudomonas sp. and Acinetobacter sp. in maintaining antimicrobial resistance at four points in the Tramandaí lagoon. The surface water samples were collected at four sampling points in the lagoon and treated with the antimicrobials: nalidixic acid, ceftazidime, imipenem and tetracycline at a concentration of 20mg/L. At each collection point, one of the aliquots was not supplemented with the antimicrobial and were used as the control for the treatment. The isolates of Pseudomonas sp. were identified by biochemical tests and MALDITOF MS, whereas the isolates of Acinetobacter sp. were identified only by biochemical tests. The susceptibility profile of both was evaluated by the disc diffusion method and the ESBL production by the combined disk method. Sampling points 3 and 4 showed the highest number of resistant isolates. The highest percentages of resistance were associated with the samples that were submitted to antimicrobial treatment. In all sampling points, multiresistant P. aeruginosa and Acinetobacter were isolated. Both samples (with and without treatment) showed different resistance patterns within the sampling points. P. aeruginosa and Acinetobacter sp. isolates exhibited ESBL producers. The presence of multiresistant P. aeruginosa and Acinetobacter sp. in the Tramandaí Lagoon attempted to the risk of spreading resistance in the aquatic environment, since it can act as a reservoir of resistance.

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O gênero Acinetobacter tem emergido como um importante patógeno nosocomial que apresenta, não apenas resistência intrínseca a muitos antimicrobianos, como também uma grande habilidade de adquirir novos mecanismos de resistência. É de grande importância o conhecimento da epidemiologia local dos isolados para que se possa estabelecer o melhor tratamento a ser adotado e as medidas de controle epidemiológico mais adequadas para evitar a disseminação destes microrganismos. O objetivo deste trabalho foi de comparar isolados de Acinetobacter spp. provenientes de amostras clínicas e de efluente hospitalar quanto ao perfil de suscetibilidade a antimicrobianos e a presença de genes de resistência aos carbapenêmicos, assim como determinar a relação clonal destes isolados. Para isso isolados clínicos e amostras de efluente hospitalar de cinco hospitais em Porto Alegre, RS, Brasil foram coletadas. Os isolados bacterianos foram identificados como pertencentes ao gênero Acinetobacter através da amplificação do rDNA16S. O perfil de suscetibilidade a antimicrobianos foi avaliado pela técnica de disco-difusão e a presença dos genes blaIMP, blaVIM, blaSPM, blaOXA-23, blaOXA-51 foi analisada por PCR em todos os isolados resistentes a carbapenêmicos. A relação clonal dos isolados foi avaliada pela amplificação de seqüências repetitivas do genoma (ERIC-PCR) e análise de macrorestrição do genoma (PFGE). Foram analisados 577 isolados, sendo 274 clínicos e 303 de efluente hospitalar. Foram encontrados 68% de isolados clínicos e 30% de isolados de efluente multirresistentes. Não foram identificados isolados com genes para metalo-b-lactamases, mas 61,2% dos isolados clínicos e três isolados de efluente hospitalar apresentaram o gene blaOXA-23. O gene blaOXA-51 foi identificado em 84% dos isolados clínicos e 56% de efluente. As técnicas de ERIC-PCR e PFGE indicaram uma disseminação de isolados clones entre os diferentes hospitais analisados, assim como uma similaridade genética entre isolados de efluente hospitalar e isolados clínicos, indicando a disseminação dos últimos através do efluente. / The genus Acinetobacter has emerged as an important nosocomial pathogen that presents not only intrinsic resistance to many antibiotics, but also a great ability to acquire new resistance mechanisms. It is very important to study local epidemiology of bacterial isolates so the best treatment and the most appropriate epidemic control measurements to prevent the spread of these microorganisms can be determined. The aim of this study was to compare isolates of Acinetobacter spp. from clinical specimens and hospital wastewater on their antibiotic susceptibility profile and the presence of resistance genes to carbapenems, as well as to determine the clonal relationship of these isolates. Clinical isolates and hospital wastewater samples from five hospitals in Porto Alegre, RS, Brazil were collected. The isolates were identified as belonging to the genus Acinetobacter by amplification of the rDNA16S. The susceptibility profile was determined by the disk-diffusion method and the presence of the blaIMP, blaVIM, blaSPM, blaOXA-23, blaOXA-51 genes were screened by PCR. The clonal relationship of the isolates was evaluated by amplification of repetitive sequences from the genome (ERIC-PCR) and macrorestriction analysis of the genome (PFGE). We analyzed 577 isolates, where 274 were of clinical origin and 303 from the hospital wastewater samples. We found that 68% of the clinical isolates and 30% of the wastewater isolates were multiresistant. None of the isolates presented genes of metallo-β-lactamase, in other hand, 61.2% of the clinical isolates and three from the hospital wastewater had the gene blaOXA-23. The gene blaOXA-51 was identified in 84% of the clinical isolates and in 56% of the hospital wastewater ones. The PFGE and ERIC-PCR analysis showed a possible dissemination of clones strains among the hospitals studied, and a genetic similarity among the hospital sewage and clinical isolates, indicating their dissemination through the wastewater.

Acinetobacter sp. apresenta altos níveis de resistência intrínseca a muitos antimicrobianos que pode estar relacionada à perda ou à diminuição da expressão de proteínas de membrana externa (OMPs). O presente trabalho teve como objetivos: identificar as espécies e analisar o perfil fenotípico de OMPs de 19 isolados multirresistentes de Acinetobacter sp. (16 de origem clínica e 3 de efluente hospitalar) e seus revertentes cultivados na presença e ausência de imipenem (IMP) e ceftazidima (CAZ), para verificar possível alteração no perfil das OMPs diante destas condições; detectar fenotipicamente sistema de efluxo e avaliar os dados de outros mecanismos de resistência encontrados em trabalhos anteriores com os isolados em estudo. Os isolados foram identificados através da amplificação por reação em cadeia da polimerase (PCR) e sequenciamento dos genes 16S rRNA e rpoB, para verificar qual é mais discriminatório. Para a detecção de bomba de efluxo foi comparada a concentração inibitória mínima (CIM) de IMP e CAZ na presença e ausência de carbonil-cianeto-m-clorofenilhidrazona (CCCP). A extração de OMPs foi realizada conforme Laemmli (1970), com padronização e posterior análise por eletroforese em gel de poliacrilamida dodecil sulfato de sódio (SDS-PAGE). O gene rpoB demonstrou ser mais discriminatório que o gene 16S rRNA, identificando os isolados com 99 a 100% de similaridade com Acinetobacter baumanni. Foi observada alteração no perfil de OMPs em quatro isolados sendo que em três destes, a perda da expressão de proteínas de 34-35-kDa e 53-kDa pôde ser associada à resistência ao IMP. Maioria dos isolados apresentaram mais de um mecanismo de resistência antimicrobiana. / Acinetobacter sp. shows high levels of intrinsic resistance to many antimicrobials which can be associated with the loss or reduced expression of outer membrane proteins (OMPs). This study aimed to: identify the species and analyze the OMPs profile of 19 multirresistant strains of Acinetobacter sp. (16 of clinical origin and 3 of hospital wastewater) and its revertants grown in the presence and absence of imipenem (IMP) and ceftazidime (CAZ) to verify possible changes in the profile of OMPs on these conditions; phenotypically detect efflux system and evaluate data from other resistance mechanisms found in previous studies with the isolates under study. The isolates were identified through amplification by polymerase chain reaction (PCR) and sequencing of 16S rRNA and rpoB genes to verify which is more discriminatory. For detection of efflux pump was compared the minimal inhibitory concentration (MIC) of IMP and CAZ in the presence and absence of carbonyl-cyanide-m-chlorophenylhydrazone (CCCP). The extraction of OMPs was performed according to Laemmli (1970), with standardization and subsequent analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The rpoB gene proved to be more discriminating than the 16S rRNA gene, identifying the isolates with 99 to 100% similarity to Acinetobacter baumannii. It was observed changes in OMPs profile in four strains and in three of these, the loss of expression of proteins of 34-35 kDa and 53 kDa, could be associated with resistance to IMP. Most isolates showed more than one mechanism of antimicrobial resistance.

Made available in DSpace on 2015-07-22T20:50:13Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-04-27 / Acinetobacter baumannii é um dos principais agentes causadores de infecção associada à assistência a saúde, e, portanto, a melhor compreensão dos fatores associados à letalidade e fatores de risco para aquisição de Acinetobacter baumannii multirresistentes em pacientes com infecção de corrente sanguínea, bem como dados a respeito da escolha terapêutica, são necessários. Objetivos: Analisar os fatores associados a letalidade em pacientes com infecção de corrente sanguínea (ICS) por Acinetobacter baumannii resistente à carbapenêmicos e avaliar o impacto da terapêutica dos pacientes tratados com polimixina B ou ampicilina-sulbactam. Método: Trata-se de um estudo tipo coorte retrospectivo realizado no Hospital São Paulo durante o período de 01 de janeiro de 2000 a 31 de dezembro de 2009. A identificação dos pacientes foi realizada através de levantamento de dados da ficha de notificação de hemoculturas com posterior revisão dos prontuários. Os pacientes foram inicialmente divididos em óbitos e sobreviventes e avaliados quanto a exposição à diversos fatores potencialmente associados à letalidade hospitalar, posteriormente aqueles que foram tratados com os antimicrobianos em questão foram analisados em relação à letalidade em 14 dias. Resultados: Foi identificado um total de 150 episódios de infecção de corrente sanguínea por Acinetobacter baumannii resistente a carbapenêmicos; o sexo masculino foi predominante com 90 pacientes (60%) e a mediana de idade foi 62 anos, quanto a abordagem terapêutica, 60 pacientes (40%) foram tratados com polimixina B, 28 (18,6%) com ampicilinasulbactam. A gravidade avaliada pelo APACHE II foi maior ou igual a 15 em cerca de 70% dos casos. Com relação aos óbitos, houve 116 óbitos que correspondeu a 77% da população estudada e a mediana do tempo decorrido da internação até o óbito foi 35 dias. A análise multivariada identificou o uso de hemodiálise (OR = 3,32, IC 95% 1,03-10,68, p = 0,044) presença de choque séptico (OR = 3,75, IC 95% 1,09 - 12,87, p = 0,036), infecção polimicrobiana (OR = 5,50, IC 95% 1,12 – 26,92, p = 0,036) e idade mais elevada (OR = 1,04, IC 95% 1,02 - 1,07, p = 0,003) como fatores de risco independentes para mortalidade nesta população. O tratamento com polimixina B ou ampicilina-sulbactam (OR = 0,07, IC 95% 0,02 - 0,33, p = 0,001) foi um fator de proteção. Ao analisarmos especificamente os 88 pacientes que utilizaram polimixina B ou ampicilina-sulbactam, as únicas variáveis que foram preditores de risco para mortalidade em 14 dias foram: idade elevada (OR = 1,04, IC 95% 1,01 - 1,07, p = 0,010) e tratamento com polimixina B (OR = 5,56, IC 95% 1,87 - 16,49, p = 0,002); enquanto a nutrição parenteral (OR = 0,21, IC 95% 0,06 - 0,70, p = 0,011) foi fator de proteção para este grupo. Conclusões: As ICS por A. baumannii resistente aos carbapenêmicos são potencialmente graves e tem como fatores independentes de mortalidade o uso de hemodiálise, a presença de choque séptico, infecção polimicrobiana e idade mais elevada. O tratamento com polimixina B foi fator relacionado à maior mortalidade em comparação com ampicilina-sulbactam. O uso de nutrição parenteral foi relacionado à menor mortalidade. / Acinetobacter baumannii is a leading cause of healthcare-associated infections. A better understanding of risk factors for acquisition and factors associated with mortality among patients with bloodstream infection caused by multidrug-resistant Acinetobacter baumannii, as well as, the appropriate therapy for these infections is needed. Objectives: The aim of this study is to analyze the factors associated with mortality in patients with bloodstream infection (BSI) caused by carbapenem-resistant Acinetobacter baumannii (CRAB) and to assess the impact of therapy among patients treated with polymyxin B and ampicillin-sulbactam for at least 48 hours. Method: A retrospective cohort study was conducted at São Paulo Hospital from January 1, 2000 to December 31, 2009. The identification of patients was done through data collection from the blood culture reporting and subsequent chart review. Patients were initially divided into those who died and those who survived and evaluated against exposure to various factors potentially associated with hospital mortality. Subsequently, patients treated with the polymyxin B and ampicillin/sulbactam were analyzed concerning to 14-day mortality. Results: A total of 150 episodes of bloodstream infection caused by carbapenem-resistant Acinetobacter baumannii was identified: male sex was predominant in 90 patients (60%), the median age was 62 years, 60 patients (40 %) were treated with polymyxin B and 28 (18.6%) were treated with ampicillin-sulbactam. The APACHE II score was greater than or equal to 15, in about 70% of cases. Regarding to deaths, there were 116 deaths corresponding to 77% of the population and the median time elapsed from hospital admission to death was 35 days. Multivariate analysis identified the use of hemodialysis (OD = 3.32, p = 0.044, CI 1.03 to 10.68), presence of septic shock (OR = 3.75, p = 0.036, CI 1.09 to 12, 87), polymicrobial infection (OD = 5.50, p = 0.036, CI 1.12 to 26.92) and elderly age (OD = 1.04, p = 0.003, CI 1.02 to 1.07) as independent risk factors for mortality in this population. Treatment with polymyxin B and ampicillin-sulbactam (OD = 0.07, p = 0.001, CI 0.02 to 0.33) was a protective factor. By analyzing specifically the 88 patients who received polymyxin B or ampicillin-sulbactam, the only variables found as predictors of 14-day mortality were: elderly age (OD = 1.04, p = 0.010, CI 1.01 to 1, 07) and polymyxin B treatment (OD = 5.56, p = 0.002, CI 1.87 to 16.49), while parenteral nutrition (OD = 0.21, p = 0.011, CI 0.06 to 0, 70) was a protective factor for this group. Conclusions: Bloodstream infections caused by carbapenem-resistant A. baumannii are a potentially serious infection. The independent factors associated with mortality were: use of hemodialysis, presence of septic shock, polymicrobial infection and elderly age. The treatment with polymyxin B was related to a greater mortality when compared to ampicillin-sulbactam. The use of early parenteral nutrition was a protective factor. / TEDE / BV UNIFESP: Teses e dissertações

Este trabalho recebeu a Menção Honrosa no Prêmio Capes de Teses 2014 / Made available in DSpace on 2014-09-09T12:30:32Z (GMT). No. of bitstreams: 2 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) 16.pdf: 6304996 bytes, checksum: 71c341f48f8e1ace48b22de15c0ccb89 (MD5) Previous issue date: 2013 / Acinetobacter baumannii é um patógeno oportunista com crescente importância em infecções relacionadas a assistência em saúde. No Brasil é particularmente problemático devido à sua alta prevalência e multirresistência, com as carbapenemases tipo OXA representando o principal mecanismo responsável por esta resistência. Esse trabalho teve como objetivo determinar a diversidade genética, caracterizar fenotípica e molecularmente a resistência a antimicrobianos e avaliar fatores de virulência em Acinetobacter baumannii isolados de 10 hospitais públicos e privados no Rio de Janeiro no período de 2005 a 2007. Durante este período foram estudados 141 isolados de A. baumannii coletados de pacientes internados em UTIs ou enfermarias cirúrgicas. A identificação da espécie foi confirmada através da detecção do gene intrínseco blaOXA-51.Os 110 isolados de A. baumannii resistentes ao imipenem apresentaram perfil de multirresistência com taxas superiores a 98,2% para 8 dos 10 antimicrobianos testados e 98(87,3%) produziram a carbapenemase OXA-23. Não houve produto de amplificação para os genes blaOXA-24, blaOXA-58, blaIMP, blaVIM e Int1 pela técnica de PCR. As drogas com atividade in vitro foram a polimixina B e tigeciclina. Observou-se uma diversidade clonal, com a presença de 5 genótipos tipados por PFGE, com prevalência dos genótipos A (71,8%) e B(22,7%), presente em 7 e 5 hospitais, respectivamente. Dentre os 96 isolados produtores de OXA-23, fori selecionada uma cepa representativa de cada genótipo que foram submetidos a métodos de tipagem baseados em sequenciamento... / Acinetobacter baumannii is an opportunistic pathogen with increasing importance in hospital infections. In Brazil, it is particularly problematic because of its high prevalence and multidrug resistance, with the OXA-type carbapenemases representing the main mechanism responsible for such resistance. This study aimed to determine the genetic diversity, as well as phenotypic and molecular characterization of antimicrobial resistance and evaluation of virulence factors in Acinetobacter baumannii isolated from 10 public and private hospitals in Rio de Janeiro from 2005 to 2007. During this period 141 isolates of A. baumannii collected from patients admitted to ICUs or surgical wards have been studied. Species identification has been confirmed by detection of gene intrinsic blaOXA-51. The 110 isolates of A. baumannii resistant to imipenem showed multidrug resistance profile with rates above 98.2% for 8 of the 10 antibiotics tested and 98 (87.3%) produced the carbapenemase OXA-23. There was no amplification product for genes blaOXA-24, blaOXA-58, blaIMP, blaVIM and Int1 by PCR. The drugs with in vitro activity were polymyxin B and tigecycline. There was a clonal diversity, the presence of five genotypes typed by PFGE, with a prevalence of genotypes A (71.8%) and B (22.7%), present in 7 and 5 hospitals, respectively. Among the 96 strains producing OXA-23, one representative for each genotype that were subjected to typing methods based on sequencing was selected. By MLST (related to the database Oxford) four new STs were detected: ST131, ST132, ST133 and ST134 and when using the MLST-IP (developed by Institut Pasteur) the ST79, ST15 and two new allelic profiles were detected. Four SGs (SG1, SG4 and two new profiles) have been identified, allowing the association of 70% of isolates with the European Clone II. Sequencing of the blaOXA-51-like gene revealed the presence of blaOXA-66, blaOXA-95 and blaOXA-132. The blaOXA-23 gene was consistently found associated with the transposon Tn2006 and was chromosomally encoded in all isolates. Of 31 isolates susceptible to imipenem, 5 showed the blaOXA-23 gene and the insertion sequence ISAba1. However, the association of this sequence to the gene blaOXA-23 was detected only in isolates resistant to imipenem used as controls. The ISAba4 insertion sequence was not found in any isolated. These isolates were susceptible to imipenem and meropenem, with MICs ε 4 mg / mL for both antimicrobials. These isolates producing blaOXA-23 have been grouped into four distinct genotypes (B, C, G and I). Most isolates included in this study had plasmid (83.7%), with profiles ranging from 1 to 4. In search of biofilm production by colorimetric assay using crystal violet it was possible to observe the production of biofilm in 96.5% of isolates. Two representative isolates of the prevalent genotypes were evaluated for the degree of association at monolayer of A-549 cells by flow cytometry, and fluorescence optical microscopy. The adhesion was observed in all methodologies, with low rates of association for genotype A (11.9%) and genotype B (9.7%). The search for quorum-sensing carried out with representative isolates of the same prevalent genotypes were negative for the production of AHL inducer using Agrobacterium tumefaciens strain NT1. While the extract of the supernatant of the positive control of P. aeruginosa PAO1 and the reference standard AHL-C8 and AHL -C12 were positive. Data from this study are relevant to public health because they allow the knowledge of the molecular epidemiology of this species, as well as some of the virulence factors, which have been rarely described in Brazil, thus enhancing the monitoring and implementing control measures.