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.

Submitted by Ray Andra Pinheiro (andraray304@gmail.com) on 2018-08-27T14:16:46Z No. of bitstreams: 1 MARCILENE.pdf: 494238 bytes, checksum: a4d7c74268dd986254a649368e4766af (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2018-08-27T15:52:38Z (GMT) No. of bitstreams: 1 MARCILENE.pdf: 494238 bytes, checksum: a4d7c74268dd986254a649368e4766af (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2018-08-27T15:56:03Z (GMT) No. of bitstreams: 1 MARCILENE.pdf: 494238 bytes, checksum: a4d7c74268dd986254a649368e4766af (MD5) / Made available in DSpace on 2018-08-27T15:56:03Z (GMT). No. of bitstreams: 1 MARCILENE.pdf: 494238 bytes, checksum: a4d7c74268dd986254a649368e4766af (MD5) Previous issue date: 2013-09-19 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Acinetobacter baumannii is the most common species of the isolated genus from clinical specimens and hospital environment, often affecting patients in intensive care units ( ICUs ). It is a bacterium which seems to have a propensity to develop antimicrobial resistance extremely fast. Practices as the quite higher use of antibiotics in patient in ICUs contribute to develop the A. Baumannii resistance. It is getting importance because it is related to nasocomiais infections in units where patients are using invasive procedures, immunoimpaired, elderly or treated with broad-spectrum antibiotics. For the reasons above, it was decided to do this study in patients hospitalized at Santa Casa de Misericordia do Pará (FSCMP) to verify the cases of infeccion by Acinetobacter baumannii related to health care epidemiology isolayting clinical specimens with emphasis on sensitivity profile in a period from 2007 to 2011. Having such results, 48.39% of cases of bloodstream infection as the site of infection by A. Baumannii, followed by infection of the urinary tract and trachea, 12.9% each, with the largest number of cases in adult and neonatal ICUs. The A.baumannii showed significant resistance to cephalosporins 3rd and 4th generation, with troubling resistance to carbapenems, with annual growth from 6.25% to 30%. Concluding that there was a change in resistance profile of A. Baumannii in five years, and evidenced a rich field of research regarding the epidemiology of Acinetobacter baumannii, showing the necessity of new approaches to aspects such as clinical findings, duration of hospitalization, previous antibiotic use, and others, as a contribution to the clinical intervention against nosocomial infections caused by the under study agent. / O Acinetobacter baumannii é a espécie mais comum do gênero isolado de amostras clínicas e de ambiente hospitalar, frequentemente afetando pacientes em unidades de tratamento intensivo (UTIs). É uma bactéria que parece ter uma propensão para desenvolvimento de resistência antimicrobiana extremamente rápida. Práticas como, o uso significativamente alto de antimicrobianos por paciente em UTIs contribuem para o desenvolvimento da resistência da A. Baumannii.. Ganhando importância por estar relacionado a infecções nasocomiais em pacientes em uso de procedimentos invasivos além de pacientes imunocomprometidos, idosos e tratados com antibióticos de amplo espectro. Foi realizado estudo em pacientes internados na Fundação Santa Casa de Misericórdia do Pará (FSCMP) para se verificar a epidemiologia dos casos de infecção relacionada à assistência à saúde por Acinetobacter baumannii isolado de espécimes clínicos com ênfase no perfil de sensibilidade num período de 2007 a 2011. Do total de 63 casos de IRAS por A. baumannii, 48,39% representaram infecção de corrente sanguínea, seguidos pela infecção de trato urinário e infecção de foco pulmonar, com 12,9% cada, sendo um maior número de casos oriundos das UTIs adulto e neonatal. O A.baumannii demonstrou alta taxa de resistência, com destaque para Cefatzidima (100%), Cefepime (52,38%), Piperacilina-Tazobactam (52,46%), com emergente resistência para os carbapenêmicos, com progressão anual de 6,25% a 30%. Conclusão: A vigilância de 5 anos do perfil de suscetibilidade demonstrou um rápido aumento das cepas multirresistentes de A. baumannii, particularmente nos dois últimos anos do estudo. O monitoramento do padrão de resistência poderá ajudar na otimização da terapia empírica destas infecções, sendo necessários esforços nas medidas intervencionais de controle de infecção na instituição.

Introdução: A pressão de colonização (PC) representa a estimativa da probabilidade de transmissão cruzada de microrganismos entre pacientes dentro de uma unidade ou hospital. Pouco se sabe sobre a influência das bactérias Gram negativas na pressão de colonização comparativamente às Gram positivas. Ainda assim, alguns estudos realizados com pacientes que adquiriram Acinetobacter baumannii multirresistente (ACMR) em Centros de Terapia Intensiva (CTIs) demonstram a PC como fator de risco associado à aquisição desta bactéria. Além da PC e de outros fatores de risco tradicionais, como presença de comorbidades, procedimentos invasivos e antibioticoterapia prévia, a própria colonização por ACMR representa um fator de risco associado ao desenvolvimento de infecção. A identificação de pacientes portadores de ACMR pode servir tanto como marcador para o desenvolvimento de infecções clínicas subsequentes como para identificar potenciais transmissores desta bactéria a outros pacientes. No entanto, a implantação de estratégias de vigilância sistemática ainda apresenta limitações como a baixa sensibilidade dos métodos de rastreamento e a determinação do melhor sítio anatômico a ser pesquisado. Nosso estudo investigou o papel da PC e outros fatores preditores para aquisição de (ACMR) e também estimou a sensibilidade das culturas de rastreamento e a prevalência de ACMR entre pacientes adultos internados em Centros de Terapia Intensiva (CTI) com baixa endemicidade. Métodos: Estudo realizado com os pacientes internados no CTI adulto de hospital universitário terciário, no período de junho de 2009 a maio de 2012. Para estimar a sensibilidade dos rastreamentos e prevalência de ACMR foi realizado estudo transversal incluindo todos os pacientes adultos consecutivos, admitidos no CTI durante o período, submetidos pelo menos, a um rastreamento semanal para ACMR. As amostras foram coletadas através de swabs, de pelo menos três sítios anatômicos: orofaringe, pele e região perianal. No caso de pacientes em ventilação mecânica, o sítio orofaringe era substituído pela aspiração de secreção traqueal e se houvesse ferida operatória e/ou cateter, era coletado um swab de ferida operatória e um swab do sítio de inserção do cateter. Para estimar a sensibilidade foram selecionados os pacientes que, além de submetidos pelo menos a um rastreamento semanal, apresentaram uma cultura positiva para ACMR proveniente de amostra clínica recente, obtida até 10 dias antes ou até 7 dias depois da coleta dos swabs para rastreamento. Para investigar a PC e fatores preditores para aquisição de ACMR foi realizada coorte retrospectiva incluindo-se todos os pacientes adultos, maiores de 18 anos, consecutivamente admitidos no CTI durante o período, submetidos pelo menos a uma oportunidade de rastreamento semanal para pesquisa de ACMR durante o período da internação e sem diagnóstico prévio de colonização/infecção por ACMR no momento da admissão. Pacientes em que ACMR foi isolado nas primeiras 48 horas de internação ou com tempo de permanência no CTI igual ou inferior a 48 horas foram excluídos do estudo. Para este delineamento, considerada somente a primeira internação de cada paciente durante o período avaliado. A PC foi estimada em nível individual como a proporção mensal de pacientes-dia identificados como portadores de ACMR no CTI X 100 no mês em que ocorreu a aquisição de ACMR, óbito ou alta da unidade. Além da PC, fatores como a presença de comorbidades, idade, sexo, escore APACHE II, cirurgia, uso de cateter vascular central, sonda vesical de demora, ventilação mecânica, antibioticoterapia com carbapenêmicos e/ou quinolonas e tempo de permanência no CTI foram investigados como preditores durante a hospitalização mediante análise univariável e multivariável. Resultados: Durante o período do estudo, 2561 pacientes foram admitidos no CTI e 1706 (66,6%) foram rastreados para pesquisa de ACMR. Foram realizados 14.638 rastreamentos ao total, dos quais 192 foram positivos (1,3%), identificados em 118 pacientes. A prevalência de pacientes colonizados ou infectados por ACMR no CTI foi de 9,5% (163/1706 pacientes), considerando isolados obtidos de amostras de rastreamento e amostras clínicas. A prevalência de pacientes colonizados ou infectados estimada somente por amostras de rastreamento foi de 6,9% (118/1706 pacientes) e a prevalência de pacientes colonizados foi 3,7% (64/1706 pacientes). Os sítios que apresentaram maior sensibilidade foram: aspirado traqueal (41,5%), orofaringe (40%) e ferida operatória (37,5%). A menor sensibilidade constatada foi quanto ao sítio de inserção de cateter (14,5%). A sensibilidade total do método foi de 60%, considerando a abordagem de rastreamento de múltiplos sítios. Entre os 1706 pacientes rastreados para pesquisa de ACMR e incluídos no estudo transversal, 1375 perfizeram os critérios de elegibilidade para o estudo de coorte. Destes, 75 (5,4%) adquiriram ACMR durante a internação no CTI. A densidade de incidência de ACMR no CTI variou entre zero a 10,6 casos/1000 pacientes-dia e os valores mensais da PC entre zero a 26,8%. Manifestações do trato digestivo (RR = 2,13; IC 1,22 – 3,69), diagnóstico de trauma e complicações relacionadas ao mesmo (RR = 2,39; IC 1,01 – 5,66) e a pressão de colonização (RR = 1,08; IC 1,06 – 1,10) foram identificados como fatores de risco independentes para aquisição de ACMR na análise multivariada. Conclusões: Em nosso estudo, o método utilizado para realizar o rastreamento de portadores de ACMR no CTI apresentou baixa sensibilidade, o que deve certamente subestimar a real prevalência desta bactéria. A pressão de colonização por ACMR esteve associada a risco de aquisição da bactéria em pacientes adultos submetidos à terapia intensiva mesmo num contexto de baixos níveis endêmicos, mesmo considerando-se a presença de outros fatores de risco já tradicionalmente conhecidos. / Introduction: Colonization pressure (CP) can represent an estimation of the probability of cross-transmission of a particular organism within a defined hospital unit or hospital. Few studies have assessed the role of gram-negative bacteria risk of acquisition in CP in comparison to the gram-positive. Some of these studies that are performed on patients who acquired Acinetobacter baumannii multiresistant (MRAB) in intensive care units, have showed that CP is associated at risk to acquire this bacteria. Besides CP and others traditional risk factors like comorbidities, invasive procedures and therapy with antibiotic, colonization by MRAB can be a risk factor to develop an infection. Establishing infection control measures to limit the crosstransmission is recommended when a MRAB carriage is identified and thus reduces the risk of development of clinical infections. The implementation of a screening surveillance policy to MRAB has some limitations including the low sensitivity of the method and the best anatomic site to be screened. The aim of this study is to evaluate the role of CP and other risk factors in the acquisition of multiresitant Acinetobacter baumannii (MRAB), estimate the sensitivity of surveillance cultures and estimate the prevalence of MRAB in adult patients admitted at intensive care unit (ICU) with low endemic levels of the bacteria. Methods: This study was conducted in the ICU at a tertiary hospital from June 2009 to May 2012. A cross-sectional study conducted to estimate the sensitivity of surveillance cultures and the prevalence of MRAB. We included all consecutive adult patients admitted to ICU, who had at least 1weekly surveillance cultures performed during their ICU stay. The samples were collected by swabs at least 3 body sites: pharynx, skin and rectum. In case of mechanical ventilation tracheal aspiration was collected instead of pharynx site. If patient had a surgery wound and/or catheter, a swab of surgical wound and insertion catheter site would be collected. Bacterial cultures of clinical specimens were performed by medical criteria. Patients whom MRAB isolated from a clinical specimen in the preceding 10 days or 7 days after performing surveillance cultures were selected to estimate the sensitivity of surveillance cultures. A retrospective cohort study was conducted to evaluate the role of CP and others risk factors in the acquisition of MRAB. We included all consecutive adult patients admitted to ICU without previous diagnosis of MRAB colonization/infection, who had at least 1 weekly surveillance cultures performed during their stay at ICU. Patients who stayed in the ICU for less than 48 hours, as well as those in whom MRAB was detected within the first 48 hours in the unit, were excluded from the cohort. Only the first ICU admission per patient was included in the analysis. CP was estimated individually as a monthly proportion of the number of patient-days positive to MRAB in ICU X100 in the month of MRAB acquisition, death or discharge. Multivariate analysis to determine risk factors for the MRAB acquisition was performed including additional variables such as patients’ demographic data, comorbidities, APACHE II score, performance of surgery, duration of ICU admission, quinolones or carbapenems antibiotic exposure and the use of mechanical ventilation, arterial/central venous catheter and/or urinary catheter. Results: During the study period, 2,561 patients were admitted to ICU and 1706 (66,6%) were screened to MRAB. A total of 14,638 surveillance cultures were performed, 192 were positive (1.3%) of 118 patients. The true prevalence of MRAB colonized or infected patients in ICU was 9.5% (163/1706 patients), while the prevalence estimated by the surveillance cultures was 6.9% (118/1706 patients). The most sensitivity site was tracheal aspirate (41.5%), pharynx (40%) and surgery wound (37.5%). The less sensitivity was obtained by a swab at the catheter insertion site (14.5%). The overall sensitivity of multisite surveillance approach was 60%. Out of the 1706 patients screened to surveillance cultures and included in a crosssectional study, 1375 met inclusion criteria for the cohort study. Of which, 75 (5.45%) acquired MRAB during ICU stay. The incident rates of MRAB ranged from 0 to 10.6 cases/1000 patient-days. CP ranged from 0 to 26.8%. Trauma (RR = 2.39; IC 1.01 – 5.66), gastrointestinal diseases (RR = 2.13; IC 1.22 – 3.69) and the CP (RR = 1.08; IC 1.06 – 1.10) were identified as independent risk factors for acquisition of MRAD at multivariate analysis. Conclusion: In our study, surveillance cultures to screening MRAB carriages showed low sensitivity. Thus, the true prevalence of MRAB in ICU may be underestimated. The CP was associated to the risk of acquisition of MRAB by the patients admitted to ICU despite the low endemic levels and other risk traditional factors included in the multivariate analysis.

Abstract Acinetobacter baumannii is a prevalent nosocomial where infections are typically secondary infections to patients that already have an infection or other source of being immunocompromised. Like many other infectious bacteria, A. baumannii is increasingly considered a multi-drug resistant pathogen. This eliminates the ability to treat A. baumannii infections with traditional antibiotics, hence the need for another method of treating A. baumannii. This research study was designed to find a way to affect the survival of A. baumannii such that it can be applied to a hospital setting to prevent further infections to immunocompromised patients. One mechanism potentially used by A. baumannii to persist on hospital surfaces is through the use of the bacterial second messenger cyclic-di-GMP (c-di-GMP). This nucleotide signal is regulated in response to environmental conditions, and then activates c-di-GMP-binding proteins that induce phenotypic changes. I hypothesized that by deleting these c-di-GMP-binding proteins that it will produce measurable differences in phenotype like biofilm formation, motility, and desiccation survival. Reducing phenotypes such as these may alter A. baumannii’s ability to persist on hospital surfaces, and potentially lead to future surface eradication. A. baumannii encodes two potential c-di-GMP-binding proteins of particular interest, one that contains a sole PilZ domain and another that pairs a PilZ domain with a hydrolase domain. PilZ domains bind c-di-GMP within a conserved binding site, regulating the conformational structure of the protein, and are named for the first studied PilZ domain within the pilus-associated PilZ protein. Pili are used in pilus-mediated motility and surface attachment, and they are A. baumannii’s primary method of motility due to not having flagellum. I hypothesized that by removing these c-di-GMP-binding proteins, I would interrupt the c-di-GMP signaling that might regulate motility. I am testing two A. baumannii strains: 5075, a recent military hospital isolate and 17978, an older lab strain. A notable difference between these two strains is that 5075 demonstrates twitching motility where it utilizes type IV pili, but 17978 demonstrates swarming motility that has unknown mechanisms. Both c-di-GMP-binding proteins were tested for their role in twitching or swarming motility of the respective strains. I found that swarming motility of 17978 is regulated by both c-di-GMP-binding proteins. While I am still generating the deletion strain for the c-di-GMP-binding hydrolase enzyme, the sole PilZ domain protein is also required for twitching motility in the 5075 strain. These results suggest c-di-GMP regulates both forms of motility in A. baumannii. Future plans include determining the role of the c-di-GMP-binding hydrolase enzyme in twitching motility and identifying the role that these proteins play through binding of c-di-GMP.

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The Nit1C cluster is a conserved gene cluster of seven genes that confers bacterial growth on cyanide as the sole nitrogen source. Bacteria with this ability are referred to as cyanotrophs. To date, the linkage between Nit1C and cyanotrophy has only been demonstrated for environmental isolates but the cluster also exists in certain medically related bacteria. In this study, a nosocomial isolate, Acinetobacter haemolyticus ATCC 19194, carrying Nit1C also displayed the ability to grow on cyanide. Growth on cyanide was accompanied by the induction of the cluster as was the mere exposure of cells to cyanide. Expression of the cluster was determined by measuring the activity of the nitrilase (NitC) coded for by the cluster and by transcriptional analysis (qRT-PCR). However, a disconnect between nitC message and NitC protein was observed depending on the phase of the growth cycle, the disconnect being related to proteolytic digestion of the NitC protein. Ironically, the cluster was also discovered to be upregulated in the absence of cyanide under nitrogen starvation conditions paralleling biofilm formation. The basis of the genetic linkage to cyanotrophy is not understood but taken together with results showing that nitrogen starvation and biofilm formation are also physiologically associated with Nit1C expression, points to a critical role for the cluster in stress-induced adaptation.

Post-transcriptional regulations (PTRs) have always been considered features of organisms with higher complexity. However recently, the interest toward the post- transcriptional mechanisms in prokaryotes increased. The bacterial proteome is much more complex compared to the genome size, suggesting a tight and articulate regulation of proteins production, extremely important for the bacterial adaptation to an always changing environment. Bacterial PTRs are responsible of modulation of mRNA stability and decay, translation initiation and elongation, modulation of the access of ribosome to the ribosome binding site and control of termination of the transcript. The main actors in the PTRs are small non-coding RNA (responsible of the inhibition of the transcription) and RNA binding proteins (RBPs), which modulate the translation and half-life of the mRNA. RBPs, are particularly of my interest since I wanted to find a possible orthologous of the eukaryotic Elav-like (Elavl) family of proteins in Acinetobacter baumannii. Elav-like proteins are present in all metazoans and are characterized by two highly conserved sequences: RNP-1 (a quite well conserved hexamer) and RNP-2 (a really well conserved octamer) that are responsible of binding to the mRNA. Each species has a different number of Elavl paralogous that is totally independent from the complexity of the organisms, suggesting a more ancient origin. In particular, I focused on the human paralog HuR (human antigen R). HuR is characterized by three RNA Recognition motif (RRM) -domains, is ubiquitously expressed and is mainly localized into the nucleus (where it is responsible of maturation of the mRNA), but under stress stimuli, can shuttle into the cytoplasm where protect the target mRNA from degradation, by binding AU/U rich sequences (ARE sequences). Its high concentration into the cytoplasm can lead to the overexpression of oncogenes and pro-tumorigenic factors. The choice of Acinetobacter baumannii comes from the increasing worldwide concern toward this pathogen that is becoming multidrug resistant. Indeed, in Italy, more the 50% of nosocomial infections are caused by A. baumannii. I found a putative protein (AB-Elavl), composed by a single RRM domain endowed with similar features of the eukaryotic RRM domain as the presence of a quite well conserved RNP-2 and a less conserved RNP-1. I expressed this protein with recombinant tools and confirmed the production of the protein in the host by western blot and mass spectrometry. I evaluated the binding activity of AB-Elavl testing the EC50 and the Kd with different biochemical assays (EMSA, AlphaScreen and HTRF- FRET) toward three different RNA sequences, in order to test the specificity. By X- RAY and NMR, I confirmed the folded structure that can be overlapped to the HuR’s one and the interaction with the probes tested, highlighting the presence of binding, but with different specificity. I also tested some small molecules developed for interfering in the binding of HuR with the target sequence and found a possible compound able to interact with AB-Elavl, by disrupting the binding with the target probe. All these results suggest an ancient origin of the metazoans’ Elavl family of proteins that probably share a common ancestor with AB-Elavl. More studies should be performed to better understand the role of AB-Elavl in A. baumannii as well as in other bacteria. In fact, I found the presence of other ARE sequence-binding proteins also in Pseudomonas aeruginosa. Interesting would be to check the presence of this protein in all the multidrug resistant ESKAPE bacteria.

No description available.