Made available in DSpace on 2015-07-22T20:50:06Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-09-29 / Centro de Integração Empresa Escola (CIEE) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Fundação de Apoio e Desenvolvimento ao Ensino, Pesquisa e Extensão Universitária no Acre (FUNDAPE) / Nesta dissertação são apresentados dois estudos envolvendo isolados clínicos de Acinetobacter baumannii que apresentaram mecanismos enzimáticos de resistência aos carbapenêmicos, a produção de carbapenemases do tipo OXA. O primeiro estudo avalia a relação genética de amostras de A. baumannii multirresistentes produtoras de OXA-23 provenientes de distintas cidades brasileiras. 91 amostras clínicas de A. baumannii foram isoladas em 17 centros médicos localizados nas cidades de São Paulo (SP), Rio de Janeiro (RJ), Belo Horizonte (MG), Porto Alegre (RS), Blumenau (SC), Curitiba (PR), São Luiz (MA) e Salvador (BA). Nesta coleção observamos altas taxas de resistência aos carbapenêmicos (91.2%) e presença do gene blaOXA-23-like em 83,5% dos isolados. O elemento de insersão ISAba1 à montante ao gene blaOXA-23 foi detectado em todos os 76 isolados de A. baumannii produtores de OXA-23. Nove grupos de genótipos foram observados entre os 76 isolados produtores de OXA-23. Nossos achados sugerem que o gene blaOXA-23 presente nestes isolados estava localizado DNA cromossomal. Três principais grupos (A, B e D) foram observados circulando em seis cidades das regiões sudeste e sul do Brasil, sendo que o genótipo predominante (A) apresentou relação clonal àquele primeiramente descrito na cidade de Curitiba, em 2003. Em contrapartida foi encontrado um único genótipo de A. baumannii produtor de OXA-23 na cidade de São Luís. Embora existam estudos brasileiros prévios reportando a disseminação de clones de A. baumannii pordutores de OXA-23 localmente, nenhum estudo brasileiro demonstrou antes, i) a análise comparativa dos genótipos originários de diferentes e distantes cidades brasileiras, ii) a provável localização do gene blaOXA-23 e iii) a presença do elemento ISAba1 à montante ao gene blaOXA-23,o que pode ter levado ao alto grau de resistência aos carbapenens observado. Desta maneira, o estudo demonstra a circulação de clones de A. baumannii produtores de OXA-23 no Brasil e enfatiza que medidas de controle de infecção são urgentemente necessárias para reduzir tanto a disseminação de isolados multirresistenes quanto o número de infecções causadas por este patógeno. O segundo estudo trata de um relato de caso de um paciente internado em um hospital da cidade de São Paulo, que apresentou uma infecção por A. baumannii produtor de OXA-72. O isolado em questão, A 30235, apresentou resistência a todos os antimicrobianos testados, com excessão à ampicilina/sulbactam tendo apresentado redução da sensibilidade a esta associação. Foi realizada com sucesso a transformação do plasmídeo presente no isolado A30235 na cepa de A. baumannii ATCC 19606. A confirmação da presença do gene blaOXA-72 no transformante evidenciou a localização plasmidial deste determinante de resistência. O plasmídio contendo o gene blaOXA-72 apresentou um peso molecular de aproximadamente 86 Kb. Neste estudo identificamos pela primeira vez no Brasil, um isolado clínico de A. baumannii produtor de OXA-72. A presença deste determinante de resistência codificado por um gene localizado em um elemento genético móvel demonstra a crescente diversidade de carbapenemase do tipo OXA no Brasil com potencial de disseminação. Medidas de controles adequadas deverão ser tomadas para evitar a disseminação de isolados produtores de OXA-72 entre os hospitais brasileiros, o que tem ocorrido com os isolados de A. baumannii produtor de OXA-23. / In this dissertation we present two studies involving carbapenem-resistant Acinetobacter baumannii clinical isolates due to the production of carbapenems modifying enzymes, the OXA-type carbapenemases. The first study aimed to determine the genetic relationship of multi-drug-resistant A. baumannii producing OXA-23 that was isolated in distinct Brazilian cities. A total of 91 A. baumannii clinical isolates were recovered from 17 medical centers located at eight cities, namely São Paulo (SP), Rio de Janeiro (RJ), Belo Horizonte (MG), Porto Alegre (RS), Blumenau (SC), Curitiba (PR), São Luiz (MA) and Salvador (BA). In this collection we observed high rates of carbapenems resistance (91.2%). Also, the blaOXA- 23-like gene was present in 83.5% of isolates. The insertion sequence ISAba1 was positioned upstream the blaOXA-23 gene in all OXA-23-producing A. baumannii identified. Nine clusters were observed among OXA-23 producers. Our fidings suggest that the blaOXA-23 gene was probably chromosomally-located in all isolates studied. Three clusters (A, B and D) were found in six cities from southeast and southern reagions of Brazil. In addition, the predominant cluster (A) was clonally related to that first described in Curitiba, in2003. In contrast, a single cluster of A. baumannii producing OXA-23 was found in São Luís city. Although there were previous reports regarding the spread of OXA-23-producing A. baumannii in Brazil the following features had not yet been assessed: i) the comparative analysis of OXA-23 producers genotypes originating in distinct and distant Brazilian cities, ii) the genetic location of the blaOXA-23 gene and iii) the presence of ISAba1 upstream blaOXA-23 probably resulting in high degree of carbapenem resistance. Thus, our study demonstrates that the clonal dissemination of OXA-23- producing A. baumannii had occurred in Brazil. These findings emphasize that infection control measures are urgently needed to reduce both the spread of multidrug resistantstrains and the number of infections caused by this pathogen. The second study refers to a case report involving a hospitalized patient that presented an wound infection due to OXA-72-producing A. baumannii. The referred clinical isolate, A 30235, was resistant to most antibiotics tested and showed reduced susctptibility to ampicillin/sulbactam. Successful transformation assays using A. baumannii ATCC 19606 as the recipient strain revealed the plasmid location of the blaOXA-72 gene. This plasmid showed molecular weight of about 86 Kb. We identified for the first time in Brazil, an A. baumannii clinical isolate producing OXA-72. The presence of this resistance determinant encoded by a gene located in a mobile genetic elemet, points out to an increasing diversity of OXA-type carbapenemase in Brazil with potential spread. Appropriate control measures should be taken to prevent the spread of OXA-72 producers among Brazilian hospitals, which it we have experienced with OXA-23- producing-A. baumannii in this country. / TEDE / BV UNIFESP: Teses e dissertações

Hospital acquired pneumonia is a major problem in the nosocomial environment worldwide. The rise in the number and level of antibiotic resistant strains of bacteria means that conventional therapies are no longer as effective as they once were. Many of the main causative organisms are Gram-negative rods, such as Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Haemophilus influenzae and one that has become a greater problem in the last twenty years Acinetobacter genospecies 13 TU. Lipopolysaccharide (LPS) is a molecule that is found on the cell surface of all Gram-negative organisms. LPS is a vital part of the outer membrane of Gram-negative bacteria and is a major factor in these organisms’ ability to cause serious infection and disease. While many Gram-negative organisms, such as E. coli and Klebsiella pneumoniae, are well characterised, other species that have become potential nosocomial pathogens more recently, such as Acinetobacter genospecies 13 TU, are much less well characterised. It is unknown as to how widespread exposure to Acinetobacter genospecies 13 TU is in a healthy population. Also, little is also about pathogenesis of Acinetobacter genospecies 13 TU such as the capacity for induction of cytokines by the Acinetobacter genospecies 13 TU LPS LPS was extracted with the aqueous phenol method and re-purified by Voegel’s method from eight strains of Acinetobacter genospecies 13 TU, four strains of Haemophilus influenzae, two strains of Pseudomonas aeruginosa, two strains of Klebsiella pneumoniae and two strains of E. coli. These LPSs were used in enzyme linked immunosorbant assays (ELISAs) with serum taken from 475 blood donors from the Southeast Scotland Blood Transfusion Service. The results from the ELISAs were averaged for each individual blood donor across all the species tested. These averaged results were compared across the species. LPS from two strains of each species, ten in all, were used to challenge the THP-1 human monocytic cell line and the mRNA was extracted and used in quantitative polymerase chain reactions to measure cytokine induction. It was seen that exposure to Acinetobacter genospecies 13 TU LPS is about as widespread in a healthy population from Southeast Scotland as exposure to Pseudomonas aeruginosa LPS and somewhat similar to Klebsiella pneumoniae LPS. Antibodies to E. coli LPS and Haemophilus influenzae LPS were similarly widespread in a healthy population from Southeast Scotland. These last two were much more widely spread than the other organisms tested. Some individuals seem to produce antibodies at high levels to all of the LPSs tested. It may be possible to use serum from these individuals to make a hyper-immune immunoglobulin preparation to be used in the immunotherapy of hospital associated pneumonia. The LPS from one of the strains of Acinetobacter genospecies 13 TU was able to induce similar levels of cytokine production as Klebsiella pneumoniae and Pseudomonas aeruginosa. It was able to induce higher levels of cytokine production over a greater number of cytokines than both Haemophilus influenzae and E. coli LPS. LPS from the other strain of Acinetobacter genospecies 13 TU tested induced lower levels of cytokines compared to the other strain. These levels were lower than those developed by Haemophilus influenzae and E. coli LPS as well as those induced by the LPSs from Klebsiella pneumoniae and Pseudomonas aeruginosa. It seems that there is a range of different levels of cytokine production induced by Acinetobacter genospecies 13 TU LPS with some strains inducing high levels and others inducing low levels of cytokines.