Thesis (M.Sc. (Microbiology))--Mahidol University, 2006. / LICL has E-Thesis 0014 ; please contact computer services.

Die Inzidenz invasiver H. influenzae-Infektionen in Deutschland steigt seit Jahren an. Die akkurate Identifizierung und Resistenztestung dieses Erregers sind von großer klinischer und epidemiologischer Bedeutung. Daher wurden im Rahmen der vorliegenden Promotionsarbeit umfangreiche Untersuchungen zur Diagnostik und zur Epidemiologie von Antibiotikaresistenzen bei H. influenzae durchgeführt. Es konnte gezeigt werden, dass die in der Routinediagnostik mittlerweile weit verbreitete MALDI-TOF-MS-Diagnostik durch das VITEK MS IVD nur eingeschränkt zur sicheren Unterscheidung von H. influenzae und H. haemolyticus einsetzbar ist. H. influenzae-Isolate erkannte das System mit einer Genauigkeit von 100 %. Bei H. haemolyticus-Isolaten wurden dagegen 42 % der untersuchten Stämme fälschlicherweise als H. influenzae erkannt. Dieser Fragestellung wurde mit der bisher umfangreichsten molekularbiologisch charakterisierten Studienpopulation beider Bakterienspezies nachgegangen. Die kalkulierte antibiotische Therapie einer Sepsis oder Meningitis erfolgt häufig mit Carbapenemen, die leitliniengerechte Therapie invasiver H. influenzae-Infektionen mit Drittgenerations-Cephalosporinen. Imipenem und Cefotaxim gehören zu den Hauptvertretern dieser Gruppen. Bezüglich der Antibiotikaresistenztestung wurde erstmalig für H. influenzae herausgefunden, dass die routinemäßig verwendete Gradientenagardiffusion (GAD) bei der Testung von Cefotaxim im Vergleich zum Goldstandard Bouillon-Mikrodilution gleichwertig und bei Imipenem sogar sensitiver in der Detektion von Heteroresistenzen ist. Die Epidemiologie dieser Resistenzen wurde in dieser Arbeit erstmalig für Deutschland systematisch erfasst, indem alle verfügbaren invasiven Isolate gemeldeter H. influenzae-Infektionen der Jahre 2016 (Imipenem) beziehungsweise 2016-2019 (Cefotaxim) untersucht wurden. Es wurde eine hohe Prävalenz einer Imipenem-Resistenz von 13,5 % festgestellt. Die Prävalenz einer Cefotaxim-Resistenz lag bei 0,9 %. Zur molekularen Typisierung wurde bei den Imipenem-resistenten Isolaten eine Multilocus-Sequenztypisierung, bei den Cefotaxim-resistenten Stämmen eine Sequenzierung des vollständigen Genoms durchgeführt. Hierbei wurde eine hohe genetische Diversität der Stämme festgestellt, was die Schlussfolgerung zulässt, dass resistente Mutanten sporadisch entstehen. Die Untersuchung möglicher spatio-temporaler Cluster führte zum Nachweis einer sehr selten vorkommenden Übertragung eines Imipenem-resistenten Stamms. Durch die Sequenzierung von Resistenzgenen wurde die Epidemiologie und Relevanz bekannter Aminosäuresubstitutionen beleuchtet. Unter anderem wurde für die PBP3-Substitutionen L389F und Y557H eine hochsignifikante Korrelation mit dem Auftreten von Cefotaxim-Resistenzen nachgewiesen. Die gewonnenen Genomdaten bieten die Grundlage für die Forschung an weiteren Antibiotikaresistenzdeterminanten von H. influenzae. / Haemophilus influenzae is a fastidious, facultative anaerobic, Gram-negative bacillus that colonizes the respiratory tract and can cause respiratory and invasive infection such as meningitis and sepsis. Invasive H. influenzae infections are potentially life-threatening and incidence rates have been increasing for years. Therefore, fast and accurate diagnostics, reliable testing of antibiotic resistance and a successful antibiotic treatment is of great importance. Therefore, the objective of the first part of this thesis was to evaluate the diagnostic and discriminative potential of the MALDI-TOF-MS system VITEK® MS regarding H. influenzae and H. haemolyticus. H. influenzae can cause invasive infections, whereas H. haemolyticus is mostly apathogenic. The system showed excellent accuracy for the identification of H. influenzae isolates, as 100 % of the 236 isolates were correctly identified. When testing 50 H. haemolyticus strains, however, the system showed significant limitations, since 42 % of these strains were misidentified as H. influenzae. According to the current German guidelines for the treatment of sepsis and meningitis, treatment of invasive H. influenzae infections is carried out using carbapenems, such as imipenem, or third-generation cephalosporins, such as cefotaxime. Therefore, the prevalence of antibiotic resistances to these substances was investigated and possible resistance mechanisms were examined. The two antibiotic susceptibility testing methods Gradient agar diffusion (GAD) and broth microdilution (BMD) were compared. As a result, for the determination of the cefotaxime MIC, the two methods showed an excellent correlation, whereas for imipenem there were significant differences in the measured MIC values. Since strains tested by GAD often showed double or fuzzy inhibition zones, heteroresistances may be more apparent using this method and GAD may be more sensitive at detecting imipenem resistance. The prevalence of imipenem resistance was determined for the year 2016. The analysis of 474 different invasive isolates showed a high prevalence of 5.5 %. If including all resistant isolates according to GAD, the prevalence would be even as high as 13.5 %. MLST was performed on all isolates to investigate the genetic relationship. As a result, however, some sequence types were observed more frequently, it revealed a significant diversity. Both the analysis of ftsI and acrR showed previously described amino acid substitutions. Cefotaxime resistance was investigated for all 2432 invasive H. influenzae for the years 2016-2019. The low prevalence of 0.9 % shows that cefotaxime is still well suited for the treatment of invasive H. influenzae infections. For the investigation of the genetic relationship and possible causes of cefotaxime resistance whole genome sequencing (WGS) was performed on all resistant isolates. The strains showed high genetic diversity and the geographic analysis also showed that the resistant strains were evenly spread throughout the population in Germany. This led to the conclusion that cefotaxime resistance is more likely caused by sporadic mutation events rather than by specific clones spreading in certain areas. The analysis of the ftsI gene showed that the amino acid substitutions L389F and Y557H are significantly associated with elevated cefotaxime MICs. This dissertation provides comprehensive data regarding diagnostics, antimicrobial susceptibility testing and the epidemiology of antibiotic resistances of invasive H. influenzae. It could be shown that the VITEK MS IVD, although established in the routine diagnostics, can only be used to a limited extent for reliably differentiating H. influenzae and H. haemolyticus isolates. Regarding antibiotic susceptibility testing, it was found that GAD showed similar results compared to the gold standard BMD when testing cefotaxime. When testing imipenem, this method was even more sensitive in detecting heteroresistances compared to the gold standard. For the first time, the epidemiology of antibiotic resistance against cefotaxime and imipenem was carried out using a large set of precisely defined invasive H. influenzae isolates to obtain representative data for the prevalence of imipenem and cefotaxime resistance in Germany. By investigating amino acid substitutions in the ftsI and acrR gene the epidemiology and relevance of these substitutions could be shown. A well-founded statement on the relationship of the resistant strains could be made using the state-of-the-art typing methods MLST and whole genome sequencing. The genome data also offers the possibility of examining other genes of these strains in more detail.

Obligat anaerob wachsende Bakterien sind an einer Vielzahl von Infektionen beteiligt. Dabei ist Bacteroides fragilis einer der wichtigsten opportunistischen Erreger unter den Anaerobiern. Bei Verdacht auf eine Infektion durch obligate Anaerobier muss nach Materialentnahme für die mikrobiologische Diagnostik unverzüglich eine kalkulierte Therapie eingeleitet werden. Oft ist eine chirurgische Therapie notwendig, die ebenso wie eine adäquate Antibiotikatherapie entscheidend für den Verlauf der Erkrankung ist. Wichtige Substanzen für eine Therapie bei Infektionen mit Beteiligung von B. fragilis sind Clindamycin, Imipenem, Metronidazol und Piperacillin/Tazobactam. Um Aussagen zur in vitro Wirksamkeit dieser Antibiotika gegenüber obligaten Anaerobiern treffen zu können, wurden in der vorliegenden Arbeit die Aktivitäten von verschiedenen Konzentrationen des jeweiligen Antibiotikums auf das Wachstum von sensiblen und resistenten B. fragilis Stämmen mittels Absterbekinetik untersucht. In Abhängigkeit von der zuvor ermittelten minimalen Hemmkonzentration des jeweiligen Antibiotikums wurden die Stämme in 2 Gruppen eingeteilt. Die erste Gruppe umfasst alle Stämme mit einer MHK ≤ 8 µg/ml. In der zweiten Gruppe sind die Stämme mit einer MHK > 8 µg/ml zusammengefasst. Die einzelnen Stämme wurden mit einem Vielfachen der minimalen Hemmkonzentration (MHK) beziehungsweise einem Vielfachen der im menschlichen Blutplasma maximal erreichbaren Konzentration (Cmax) des jeweiligen Antibiotikums inkubiert und die Bakterienkonzentration zu definierten Zeitpunkten ermittelt. Dadurch können sowohl die Wirksamkeit unterschiedlicher Antibiotikakonzentrationen als auch verschiedene Antibiotikaklassen miteinander verglichen und Aussagen zu Empfehlungen für kalkulierte Therapien getroffen werden.

Made available in DSpace on 2015-07-22T20:50:21Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-07-27 / P. aeruginosa é um importante microorganismo em infecções de corrente sanguínea. O tratamento da P. aeruginosa resistente aos carbapenêmicos é um desafio, já que as drogas mais utilizadas para este fim, as polimixinas, tem uma ação inferior aos carbapenêmicos. No entanto, o uso combinado de polimixinas com carbapenêmicos tem demonstrado sinergismo em estudos in vitro. Objetivos: Avaliar a resposta ao tratamento com polimixina B versus polimixina B e imipenem em pacientes com infecção da corrente sanguínea associada a assistência à saúde, primária ou secundária, causada por Pseudomonas aeruginosa resistente aos carbapenêmicos e identificar os fatores associados à mortalidade. Métodos: Realizamos um estudo tipo coorte retrospectivo, com pacientes internados no Hospital São Paulo - UNIFESP, no período de 01 de janeiro de 2000 à 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 a diversos fatores associados à letalidade hospitalar e relacionada a infecção. Foi realizada pesquisa de metalobetalactamase nas amostras de P. aeruginosa que puderam ser recuperadas, por técnica de biologia molecular. Resultados: Foram estudadas 69 infecções de corrente sanguínea por P. aeruginosa resistente aos carbapenêmicos, das quais 35 foram tratadas com monoterapia com polimixina B e 34 com terapia combinada. O óbito relacionado a infecção, definido como aquele que ocorreu nos primeiros 14 dias do diagnóstico da infecção da corrente sanguínea, foi de 42,8% nos pacientes que utilizaram monoterapia e 44,1% naqueles que utilizaram terapia combinada (p=0,917). Foram fatores associados à mortalidade durante a internação hospitalar, o uso prévio de glicopeptídeos (OR 10,71, IC 1,20-95,34, p=0,033) e o escore de Charlson no momento da internação (OR 1,9, IC 1,22-2,94, p=0,004). Foram variáveis associadas à mortalidade relacionada à infecção, a presença de choque séptico (OR 9,99, IC 1,81- 55,22, p=0,006) e uso de nutrição parenteral (OR 7,45, IC 1,23-45,24, p=0,029). Foi encontrada uma alta taxa de mortalidade. A terapia combinada não modificou a evolução dos pacientes. A mortalidade hospitalar no grupo com monoterapia foi de 77,1% entre os pacientes que receberam monoterapia e 79,4% nos que receberam terapia combinada (p=0,819). Em pacientes tratados com terapia combinada, quando analisada a presença ou não de metalobetalactamase, não houve diferença estatisticamente significativa quanto aos desfechos. Conclusões: Nesta população, foram fatores independentes para o óbito durante a internação o uso prévio de glicopeptídeos e a pontuação no score de Charlson e, para o óbito relacionado à infecção, a presença de choque séptico e o uso de nutrição parenteral. A presença de metalo-beta-lactamase não influiu no desfecho. / Pseudomonas aeruginosa is an important pathogen causing nosocomial bloodstream infections. The treatment of carbapenem-resistant P. aeruginosa is a challenge as the drugs used for this purpose, the polymyxins, have lower activity compared with carbapenems. However, it has been suggested that polymyxins have the ability to make gram-negative bacteria more susceptible to other antibiotics. As carbapenems are considered the main drugs against P. aeruginosa, it would be interesting to demonstrate the efficacy of this combination in vivo, targeting a more effective therapy. Objectives: To evaluate the response of the treatment with polymyxin B versus polymyxin B and carbapenem in patients with nosocomial bloodstream infection caused by carbapenenresistant Pseudomonas aeruginosa and identify factors associated with mortality among those patients Methods: A retrospective cohort study was performed at Hospital São Paulo - UNIFESP, from January 1st, 2000 to December 31, 2009. The identification of patients was done through data collection from the blood cultures report. Patients were initially divided into deaths and survivors, and assessed for exposure to various factors potentially associated with in-hospital mortality and infection-related mortality. Presence of metalobetalactamase was tested trough PCR technique. Results: We studied 69 bloodstream infections caused by carbapenems-resistant P. aeruginosa. Thirthy-five were treated with polymyxin B monotherapy and 34 with combined therapy. In- hospital mortality was 77.1% and 79.4% in the monotherapy group and combined therapy group, respectively (p = 0.819). The infection-related mortality was 42.8% among patients who received monotherapy, and 44.1% in those receiving combined therapy (p = 0.917). Factors associated with mortality were previous use of glycopeptides (OR 10.71, CI95% 1.20 to 95.34, p = 0.033) and Charlson score (OR 1.9, CI95% 1, 22 to 2.94, p = 0.004). Infection-related mortality was associated with the presence of septic shock (OR 9.99, CI95% 1.81 to 55.22, p = 0.006) and parenteral nutrition (OR 7.45, CI95% 1.23 - 45.24, p = 0.029). No statistically significant difference was found between patients with MBLharboring and non-MBL-harboring strains treated with combined therapy. Conclusions: No difference was found between the monotherapy and combined therapy group regarding mortality. Independent factors related to in- hospital mortality were prior use of glycopeptides and the Charlson score. Presence of septic shock and use of parenteral nutrition were independently associated with infection-related mortality. / TEDE / BV UNIFESP: Teses e dissertações

Obligat anaerob wachsende Bakterien sind an einer Vielzahl von Infektionen beteiligt. Dabei ist Bacteroides fragilis einer der wichtigsten opportunistischen Erreger unter den Anaerobiern. Bei Verdacht auf eine Infektion durch obligate Anaerobier muss nach Materialentnahme für die mikrobiologische Diagnostik unverzüglich eine kalkulierte Therapie eingeleitet werden. Oft ist eine chirurgische Therapie notwendig, die ebenso wie eine adäquate Antibiotikatherapie entscheidend für den Verlauf der Erkrankung ist. Wichtige Substanzen für eine Therapie bei Infektionen mit Beteiligung von B. fragilis sind Clindamycin, Imipenem, Metronidazol und Piperacillin/Tazobactam. Um Aussagen zur in vitro Wirksamkeit dieser Antibiotika gegenüber obligaten Anaerobiern treffen zu können, wurden in der vorliegenden Arbeit die Aktivitäten von verschiedenen Konzentrationen des jeweiligen Antibiotikums auf das Wachstum von sensiblen und resistenten B. fragilis Stämmen mittels Absterbekinetik untersucht. In Abhängigkeit von der zuvor ermittelten minimalen Hemmkonzentration des jeweiligen Antibiotikums wurden die Stämme in 2 Gruppen eingeteilt. Die erste Gruppe umfasst alle Stämme mit einer MHK ≤ 8 µg/ml. In der zweiten Gruppe sind die Stämme mit einer MHK > 8 µg/ml zusammengefasst. Die einzelnen Stämme wurden mit einem Vielfachen der minimalen Hemmkonzentration (MHK) beziehungsweise einem Vielfachen der im menschlichen Blutplasma maximal erreichbaren Konzentration (Cmax) des jeweiligen Antibiotikums inkubiert und die Bakterienkonzentration zu definierten Zeitpunkten ermittelt. Dadurch können sowohl die Wirksamkeit unterschiedlicher Antibiotikakonzentrationen als auch verschiedene Antibiotikaklassen miteinander verglichen und Aussagen zu Empfehlungen für kalkulierte Therapien getroffen werden.

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A sepse após a injúria térmica é a maior causa de morbidade e mortalidade em pacientes queimados, uma vez que profundas alterações ocorrem na farmacocinética de agentes antimicrobianos. Investigaram-se trinta e um pacientes, portadores de sepse documentada e apresentando lesões ativas; utilizou-se o tratamento empírico conforme seguem os regimes de dose: 1 g, 12/12 h para a vancomicina, 1 g, 6/6 h para o imipenem e 2 g, 8/8 h para o cefepime. Sete coletas seriadas de sangue foram realizadas através de cateter venoso (2 mL/cada); o plasma foi obtido pela centrifugação e armazenado no congelador (-80o C) até o ensaio. A concentração plasmática dos antimicrobianos foi determinada simultaneamente pela aplicação do método bioanalítico desenvolvido no estudo. O método de cromatografia líquida de alta eficiência demonstrou boa linearidade, precisão e exatidão para a determinação simultânea da vancomicina, cefepime e imipenem plasmáticos; a plicação desse método bioanalítico permitiu o monitoramento plasmático terapêutico e o estudo farmacocinético. Com base nos resultados obtidos de concentração plasmática versus tempo, aplicou-se a modelagem para investigar a farmacocinética desses agentes antimicrobianos nos pacientes queimados. Os parâmetros cinéticos foram estimados com base no modelo aberto de um compartimento pela aplicação do programa PK Solutions v. 2.0; a estatística foi realizada pela utilização do programa GraphPad Prism v. 4.0. Com base na farmacocinética alterada, as concentrações obtidas para a vancomicina e imipenem se mostraram abaixo dos valores recomendados para atingir eficácia; por outro lado, as concentrações obtidas para o cefepime se mostraram dentro da faixa recomendada para atingir eficácia, uma vez que não se registrou alteração da farmacocinética deste antimicrobiano nos pacientes queimados. Desta forma, o monitoramento plasmático terapeutico se mostrou importante, permitindo o ajuste de dose para a vancomicina e para o imipenem, uma vez que a concentração minima efetiva (CME) não foi atingida para ambos pela utilização do regime de dose empírica nos pacientes queimados. Por outro lado, o monitoramento do cefepime plasmático também se mostrou de relevância, uma vez que os pacientes queimados com longa permanência na terapia intensiva podem apresentar disfunção renal em alguma fase da internação; consequentemente, a individualização de dose será recomendada para esses pacientes. Adicionalmente, investigou-se a disposição cinética da vancomicina em nove pacientes queimados após duas diferentes intervenções cirúrgicas. Comparou-se a farmacocinética da vancomicina pós-desbridamento versus pos-enxerto com base no monitoramento plasmático após o regime de dose empírica (1 g, 12/12 h). Após multiplas infusões, o vale da vancomicina plasmática foi obtido pela coleta de sangue imediatamente antes da infusão subsequente e está relacionado ao acúmulo no estado de equilíbrio. Em conseqüência da depuração aumentada e meia-vida biológica reduzida pós-desbridamento comparado ao pós-enxerto, registrou-se para a vancomicina vale abaixo da concentração efetiva mínima nos pacientes queimados. Finalmente, os resultados obtidos no presente estudo permitem concluir que a farmacocinética da vancomicina e do imipenem está alterada nos pacientes queimados com sepse, e recomenda-se o monitoramento das concentrações plasmáticas para garantir a eficácia de forma a previnir a emergência bacteriana. / Sepsis after thermal injury is the major cause of morbidity and mortality in burn patients, once deep changes on the pharmacokinetics of antimicrobials agents are expected. Thirty one burn patients were investigated, all of them had documented sepsis and presented active lesions; they were treated with empirical dose regimen as follows: 1 g, 12/12 h for vancomycin, 1 g, 6/6 h for imipenem and 2 g, 8/8 h for cefepime. A serial of seven blood samples were collected from the venous catheter (2 mL/each); plasma was obtained by centrifugation and storaged in an ultra-low freezer (-80o C) until assay. Drug plasma concentration was determined simultaneously by application of a bioanalytical method described previously. High performance liquid chromatographic method showed good linearity, precision and accuracy for vancomycin, cefepime and imipenem plasma measurements; its application permitted therapeutic drug monitoring and pharmacokinetic studies. Pharmacokinetic modeling was applied to data obtained based on drug plasma concentrations versus time, to investigate those antimicrobial agents in burn patients. Estimated kinetic parameters were based on the one compartment open model by application the software PK Solutions v. 2.0; statistics was performed by using the software GraphPad Prism v. 4.0. Based on altered pharmacokinetics, obtained plasma concentrations to reach drug efficacy were below the recommended values for vancomycin and imipenem; on the other hand, cefepime plasma concentrations to reach drug efficacy were in the recommended range, once its pharmacokinetics didnt change in burn patients. Then, therapeutic plasma monitoring was cost-effective permitting dose adjustment for vancomycin and imipenem, once the minimum effective concentration (MEC) wasnt reached for both antimicrobial agents by using the empirical dose regimen for burn patients. On the other hand, cefepime plasma monitoring was also cost-effective, since burn patients long term therapy can present renal dysfunction at the minimum one period in the intensive care unit; consequently, dose adjustment could be required for them. Additionally, vancomycin kinetic disposition was investigated in nine burn patients after two different surgical interventions. Vancomycin pharmacokinetics post-debridement versus post-skin grafting procedure was compared based on drug plasma monitoring by using the empirical dose regimen (1 g, 12/12 h). Trough vancomycin plasma level after multiple infusions, obtained by blood collection before de next dose, is related to drug accumulation at the steady state level; then, trough below the minimum effective concentration (MEC) were obtained after both surgical procedures performed in burn patients. Meanwhile, increased plasma clearance and reduced biological half-life were obtained after debridement compared skin grafting procedure. Finally, data obtained in the present study permit to conclude that the pharmacokinetics is altered for vancomycin and imipenem in burn patients with sepsis, and drug plasma monitoring is recommended to guarantee drug efficacy and to prevent the bacterial emergency.

Após o surgimento e disseminação das β-lactamases de amplo espectro em membros da família Enterobacteriaceae, os antibióticos carbapenêmicos (imipenem, meropenemeertapenem) têm sido considerados a terapia de escolha devido à estabilidade apresentada contra estas enzimas. A desvantagem destes antibióticos é a sua capacidade de induzir resistência aos β-lactâmicos e a outros antibióticos quimicamente não relacionados. O imipenem tem favorecido a indução de cefalosporinases cromossômicas (AmpC) e também tem sido relacionado, in vivo, com a seleção de mecanismos intrínsecos de resistência, contribuindo com o perfil multi -droga resistente (MDR). Esse perfil é freqüentemente associado à diminuição da permeabilidade por alteração na síntese de porinas em conjunto com um aumento da atividade de bombas de efluxo, as quais não permitem o estabelecimento de uma concentração ativa do antibiótico no interior da célula bacteriana. O presente trabalho teve como objetivo avaliar o estabelecimento do perfil MDR em enterobactérias provenientes de isolados clínicos em função da exposição a diferentes concentrações de imipenemin vitro. A seleção do grupo das amostras estudadas foi feito por meio da determinação do perfil de sensibilidade dos isolados, tipagem molecular e ensaio de hidrólise de Imipenem. Nos isolados selecionados para a indução foi realizada numa etapa inicial (etapa basal) a análise de porinas de membrana externa por SDS-PAGE e o estudo de genes codificadores de β-lactamases pela técnica de PCR. O estudo do estabelecimento do perfil MDR foi feito por meio de passagens sucessivas das amostras em meio contendo concentrações sub-inibitórias de imipenem seguido de análise fenotípica (CIM e acúmulo do antibiótico intracelular e SDS-PAGE), e a análise da expressão gênica de genes associados a permeabilidade de membrana (ompC, ompF eAcrA) e genes reguladores(marA e ompR). Após a indução com o imipenem, 77% dos isolados induzidos aumentaram a CIM para os carbapenêmicos, mudando assim o perfil de resistência observado na etapa basal Também foi afetado o perfil de resistência para outros antibióticos não relacionados a β-lactámicos, porém numa percentagem menor. Com relação à alteração da permeabilidade, a perda de porina foi observada apenas para um isolado, no entanto a diminuição na expressão gênica de Omp36 foi significativa desde o começo da indução. A expressão da bomba de efluxoAcrAB foi afetada pela indução com imipenem, aumentando significativamente a expressão de AcrA, enquanto os reguladores estudados, MarA e OmpR tiveram a sua expressão induzida pelo imipenem. Foi possível observar também associação do nível de expressão gênica do regulador MarA com a expressão de AcrA,porém não foi possível observar uma associação estatisticamente significativa deste regulador com o perfil de expressão de OMPs. A indução de OmpR foi associado com um aumento da expressão de RNAm de Omp35, já para Omp36 foi possível observar apenas uma tendência na repressão deste gene. O estudo da resposta destes genes reguladores e determinantes de resistência, em resposta à exposição ao com o imipenem in vitro, permitiu reportar o comportamento molecular da bactéria numa resposta adaptativa no estagio inicial do estabelecimento do fenótipo MDR. A utilização de isolados clínicos com diversos determinantes de resistência permitiu observar a variabilidade nas respostas adaptativas das enterobacterias, o que é fundamental para a compreensão dos mecanismos de adaptação da bactéria e sua contribuição na falha terapêutica. / After emergence and broad dissemination of extended spectrum β-lactamases into the Enterobacteriaceae family, the carbapenemic antibiotics (imipenem, meropenem and ertapenem) have been considered the chosen therapy in the treatment of nosocomial infections by the stability that these antibiotics show to these enzymes. The disadvantage of carbapenems is theirs capacity to induce resistance against β-lactamics and to other chemically unrelated antibiotics. The imipenem has been shown to induce chromosomal cephalosporinases (AmpC) and it was also related, in vivo, with the selection of intrinsic mechanism leading to multi-drug resistance profile (MDR). This profile is usually associated with membrane impermeability due to reduced outer membrane porin synthesis with an incremented activity of efflux pumps, which results in a reduced concentration of antibiotics inside the bacteria. This study aimed to evaluate the establishment of the MDR profile in Enterobacteriaceae from clinical isolates by exposure to different concentrations of imipenem in vitro. The selection of the study group was performed by determination of antibiotic susceptibility profile,molecular typing and hydrolysis assay of imipenem. In the selected isolates submitted to induction, in an initial step (baseline), was performed the outer membrane porin analysis by SDS-PAGE and the gene-specific amplification of B-lactamase enzymes by PCR. The study of the establishment of MDR was performed by progressive passages with subclinical concentrations of imipenem, followed each one by the evaluation of phenotypic profile (MIC, accumulation antibiotic in celland SDS-PAGE) and gene expression analysisof genes related to membrane permeability (ompC, ompF and acrA) and regulatory genes(MarA and ompR). After induction with imipenem, 77 % of the isolates increased the MIC for the carbapenems, changing the resistance profile at the baseline. In a lesser percentage, the resistance profile to other β-lactams-unrelated antibiotics was also affected. Loss of porin was observed only for an isolated, however a significantly decreased Omp36 mRNA expression was observed from the start of induction. The expression of the efflux pump AcrAB ,was also affected by the imipenem induction, significantly increasing the AcrA gene expression, whereas the studied regulatory genes,MarA and OmpR,were induced by the imipenem. It was also possible to observe an association between the expression of the regulator MarA and the expression of AcrA, nevertheless no association was observed between this regulator and OMPs . OmpR induction was associated with an increased Omp35mRNA expression, however only a trend for the repression of Omp36was observed. The study of the response of these regulatory genes and genetic determinants of resistance, in response to the imipenem exposure in vitro, allowed to report the molecular behavior of the bacteria in an adaptive response in the initial stage of the establishment of a MDR phenotype. The use of clinical isolates with diverse resistance determinants allowed observing the variability in adaptive responses in enterobacteria, which is important to understand the adaptive mechanisms of bacteria to this antibiotic, the involvement in the emergence of the MDR profile and its contribution to the treatment failure.