Acinetobacter baumannii is a Gram-negative, non-fermenting bacillus that has developed into an important nosocomial pathogen, affecting millions of patients worldwide. The widespread ease of transmission, and ability to become multidrug resistant are some of the characteristics that, at the present time, have developed this bacterium into one of the most significant nosocomial pathogens today. The special ability it exhibits in developing resistance to a wide variety of known antimicrobial agents also helped make this a pathogen of profound importance in modern day medical microbiology. Carbapenems are used as a last resort for treating patients infected with resistant or multi-drug resistant (MDR) Acinetobacter baumannii. Hospitals have long served as reservoirs for the transmission of pathogenic bacteria, and this has become a problem in Kuwait. Unfortunately, very little research has been devoted exclusively to investigating Acinetobacter baumannii prevalence, resistance and pathogenicity in Kuwaiti Hospitals. Research on the local population in Kuwaiti Hospitals is important and beneficial to physicians, to help better diagnose and treat the infections, and prevent any outbreaks from spreading. Aim: This study aimed to examine the resistance and identify the genotypic changes in the organism as it spreads through Mubarak Al-Kabeer Hospital. Methods: A total of 88 Acinetobacter baumannii samples were collected from the Mubarak Al- Kabeer Hospital, over a three year period, 2006-2008, and they were identified phenotypically, by Vitek-2 systems, and then genotypically by PCR amplification of blaOXA-51-like gene. The resistance to the carbapenems: imipenem and meropenem, was identified by use of the Minimal Inhibitory Concentration (MIC) test. Pulsed field gel electrophoresis (PFGE) was used to type the strains and classify them into clonal groups. Identification of the blaOXA-51-like gene types of each of the isolates was done via gene sequencing. Results: All 88 isolates were identified as Acinetobacter baumannii by Vitek-2 system and were shown to carry a blaOXA-51-like gene. Resistance to Imipenem was found in 31.8% of the isolates, whereas resistance to meropenem was found in 23.8% of the isolates. Overall carbapenem resistance was observed in 55.7% of the total isolates, with a slight increase in resistance of isolated over the 3 years of collection. In all, there were 10 different blaOXA-51-like genes identified. The sequences of these genes suggested there was some degree of real-time evolution of the blaOXA-51-like genes during the study period. There were four main clonal clusters. There were three main European clones (blaOXA-66, blaOXA-69, and blaOXA-71) plus a new clone with blaOXA-51-like genes with sequences clustered around the blaOXA-98 gene. Conclusion: This study has shown four major clones were found in the hospital during the study period, three of the clones were closely associated with those found in Europe and elsewhere in the world, and one new clone, containing a blaOXA-98-like gene that appears to be more prevalent in this part of Asia. The gradual increase in resistance to carbapenems over the study period warrants further attention and study of this resilient bacterium.

Acinetobacter baumannii (Ab) é um dos principais agentes de infecção hospitalar, principalmente em Unidades de Terapia Intensiva (UTI). A principal característica da bactéria é a sua resistência intrínseca a diversos antimicrobianos, o que favorece sua persistência no ambiente hospitalar, causando infecções de difícil controle e tratamento. Nos esquemas terapêuticos, os antibióticos carbapenêmicos são os fármacos de escolha, porém nos últimos anos a resistência a estas drogas tem aumentado drasticamente em função da emergência e disseminação de cepas produtora de carbapenemases [i.e., oxacilinases (OXA) e metalo-beta-lactamases (MβL)]. O objetivo do presente trabalho foi avaliar a produção de enzimas carbapenemases do tipo OXA e MβLs, em 36 amostras multirresistentes de A. baumannii, previamente triadas, provenientes de 8 hospitais brasileiros, durante 2004/2008. A caracterização fenotípica e genotípica dos isolados foi realizada por meio da determinação de CIM, hidrólise enzimática e PCR para pesquisa dos genes blaOXA e blaMβL, assim como seqüências de inserção (ISAba-1, ISAba-3) responsáveis pela mobilização dos determinantes de resistência do tipo blaOXA. Finalmente a análise da diversidade genética foi realizada por ERIC-PCR com análise de clusters por coeficiente de Dice. Todos os 36 isolados apresentaram 100% de resistência para imipenem (CIM90 > 64 µg/mL), meropenem, ceftazidima, ciprofloxacina e iperacilina/tazobactam, enquanto que os antibióticos com maior atividade in vitro foram a ampicilina/sulbactam (61,2%) > tobramicina (61,1%) > gentamicina (47,3%) > amicacina (28%). Em todos os isolados foi confirmada a presença intrínseca dos genes blaOXA-51 e ISAba-1, não apresentando colinearidade entre eles, enquanto que 41,6% dos isolados carregaram a combinação dos genes ISAba-1/blaOXA-23 (Genbank accession FJ628170), um isolado (2,7%) carregou a combinação de genes ISAba3/blaOXA-58/ISAba3 (Genbank accession FJ492877) e dois isolados (5,5%) clonalmente relacionados, carregaram o gene blaOXA-72 (Genbank accession FJ969387). Surpreendentemente, em um centro hospitalar foi documentada a presença de um surto de infecção por Ab produtor de OXA-23. Finalmente a tipagem epidemiológica dos isolados revelou a presença de 13 clusters, sendo que 8 diferentes clusters carregavam o gene blaOXA-23. Em resumo, nossos resultados confirmam a disseminação de cepas produtoras de OXA-23 associadas com ISAba-1 no Brasil, assim como a presença intrínseca do gene blaOXA-51 em Ab. Por outro lado, este é o primeiro relato de isolados carbapenem resistentes carregando os genes blaOXA-58 e blaOXA-72 em hospitais brasileiros, os quais aparentemente surgiram em 2004 e 2008, respectivamente. / Acinetobacter baumannii (Ab) is a leading cause of hospital infection, mainly in intensive care units. The main characteristic of the bacterium is its intrinsic resistance to diverse antimicrobial agents, which contribute to persistence in hospital environments causing infections of difficult control and treatment. In therapeutic schedules, carbapenems are choice antibiotics, however in recent years the resistance to these drugs has increased drastically in function of the emergency and dissemination of carbapenemase-producing isolates [i.e., oxacilinases (OXA) and metalo-beta-lactamases (MβLs)]. The aim of this work was to evaluate the production of OXA- and MβL-like carbapenemases, in 36 isolates previously screened as multidrug-resistant (MDR) A. baumannii, recovered from 8 Brazilian hospitals, during 2004/2008. Phenotypic and genotypic characterization of MDR Ab was carried out using CIM determination, enzymatic hydrolysis and PCR for screening of the blaOXA- and blaMβL-like genes, and insertion sequences (ISAba-1, ISAba-3) responsible for mobilization of blamOXA-like gene cassettes. Analysis of genetic relationship was carried out by ERIC-PCR with analysis of clusters for Dice`s coefficient. All of the 36 isolates showed 100% resistance to imipenem (CIM90 > 64 µg/mL), meropenem, ceftazidime, ciprofloxacin and piperacillin/tazobactam, whereas antibiotics exhibiting a best in vitro activity were ampicillin/sulbactam (61.2%) > tobramycin (61.1%) > gentamicin (47.3%) > amikacin (28%). Presence of blaOXA-51 and ISAba-1 genes was confirmed in all isolates, not presenting collinearity between them, whereas 41.6% isolates carried the ISAba-1/blaOXA-23 gene array (Genbank accession FJ628170). One Ab isolate harbored the ISAba-3/blaOXA-58/ISAba-3 gene array (2.7%) (Genbank accession FJ492877) and 5.5% of Ab isolates harbored the blaOXA-72 gene (Genbank accession FJ969387). Surprisingly, an outbreak of infection with MDR Ab producing OXA-23 enzyme was documented. Finally the ERIC-PCR typing revealed the presence of 13 clusters, of which 8 different clusters carried the blaOXA-23 gene. In summary, our results confirm the dissemination of OXA-23-producing Ab isolates associated with the ISAba-1 gene, in Brazil, as well as the intrinsic presence of the blaOXA-51 gene cassette. On the other hand, this is the first report of carbapenem-resistant Ab isolates harboring genes blaOXA-58 and blaOXA-72 recovered in Brazilian hospitals, which most likely emerged in 2004 and 2008, respectively.

Acinetobacter baumannii (Ab) é um dos principais agentes de infecção hospitalar, principalmente em Unidades de Terapia Intensiva (UTI). A principal característica da bactéria é a sua resistência intrínseca a diversos antimicrobianos, o que favorece sua persistência no ambiente hospitalar, causando infecções de difícil controle e tratamento. Nos esquemas terapêuticos, os antibióticos carbapenêmicos são os fármacos de escolha, porém nos últimos anos a resistência a estas drogas tem aumentado drasticamente em função da emergência e disseminação de cepas produtora de carbapenemases [i.e., oxacilinases (OXA) e metalo-beta-lactamases (MβL)]. O objetivo do presente trabalho foi avaliar a produção de enzimas carbapenemases do tipo OXA e MβLs, em 36 amostras multirresistentes de A. baumannii, previamente triadas, provenientes de 8 hospitais brasileiros, durante 2004/2008. A caracterização fenotípica e genotípica dos isolados foi realizada por meio da determinação de CIM, hidrólise enzimática e PCR para pesquisa dos genes blaOXA e blaMβL, assim como seqüências de inserção (ISAba-1, ISAba-3) responsáveis pela mobilização dos determinantes de resistência do tipo blaOXA. Finalmente a análise da diversidade genética foi realizada por ERIC-PCR com análise de clusters por coeficiente de Dice. Todos os 36 isolados apresentaram 100% de resistência para imipenem (CIM90 > 64 µg/mL), meropenem, ceftazidima, ciprofloxacina e iperacilina/tazobactam, enquanto que os antibióticos com maior atividade in vitro foram a ampicilina/sulbactam (61,2%) > tobramicina (61,1%) > gentamicina (47,3%) > amicacina (28%). Em todos os isolados foi confirmada a presença intrínseca dos genes blaOXA-51 e ISAba-1, não apresentando colinearidade entre eles, enquanto que 41,6% dos isolados carregaram a combinação dos genes ISAba-1/blaOXA-23 (Genbank accession FJ628170), um isolado (2,7%) carregou a combinação de genes ISAba3/blaOXA-58/ISAba3 (Genbank accession FJ492877) e dois isolados (5,5%) clonalmente relacionados, carregaram o gene blaOXA-72 (Genbank accession FJ969387). Surpreendentemente, em um centro hospitalar foi documentada a presença de um surto de infecção por Ab produtor de OXA-23. Finalmente a tipagem epidemiológica dos isolados revelou a presença de 13 clusters, sendo que 8 diferentes clusters carregavam o gene blaOXA-23. Em resumo, nossos resultados confirmam a disseminação de cepas produtoras de OXA-23 associadas com ISAba-1 no Brasil, assim como a presença intrínseca do gene blaOXA-51 em Ab. Por outro lado, este é o primeiro relato de isolados carbapenem resistentes carregando os genes blaOXA-58 e blaOXA-72 em hospitais brasileiros, os quais aparentemente surgiram em 2004 e 2008, respectivamente. / Acinetobacter baumannii (Ab) is a leading cause of hospital infection, mainly in intensive care units. The main characteristic of the bacterium is its intrinsic resistance to diverse antimicrobial agents, which contribute to persistence in hospital environments causing infections of difficult control and treatment. In therapeutic schedules, carbapenems are choice antibiotics, however in recent years the resistance to these drugs has increased drastically in function of the emergency and dissemination of carbapenemase-producing isolates [i.e., oxacilinases (OXA) and metalo-beta-lactamases (MβLs)]. The aim of this work was to evaluate the production of OXA- and MβL-like carbapenemases, in 36 isolates previously screened as multidrug-resistant (MDR) A. baumannii, recovered from 8 Brazilian hospitals, during 2004/2008. Phenotypic and genotypic characterization of MDR Ab was carried out using CIM determination, enzymatic hydrolysis and PCR for screening of the blaOXA- and blaMβL-like genes, and insertion sequences (ISAba-1, ISAba-3) responsible for mobilization of blamOXA-like gene cassettes. Analysis of genetic relationship was carried out by ERIC-PCR with analysis of clusters for Dice`s coefficient. All of the 36 isolates showed 100% resistance to imipenem (CIM90 > 64 µg/mL), meropenem, ceftazidime, ciprofloxacin and piperacillin/tazobactam, whereas antibiotics exhibiting a best in vitro activity were ampicillin/sulbactam (61.2%) > tobramycin (61.1%) > gentamicin (47.3%) > amikacin (28%). Presence of blaOXA-51 and ISAba-1 genes was confirmed in all isolates, not presenting collinearity between them, whereas 41.6% isolates carried the ISAba-1/blaOXA-23 gene array (Genbank accession FJ628170). One Ab isolate harbored the ISAba-3/blaOXA-58/ISAba-3 gene array (2.7%) (Genbank accession FJ492877) and 5.5% of Ab isolates harbored the blaOXA-72 gene (Genbank accession FJ969387). Surprisingly, an outbreak of infection with MDR Ab producing OXA-23 enzyme was documented. Finally the ERIC-PCR typing revealed the presence of 13 clusters, of which 8 different clusters carried the blaOXA-23 gene. In summary, our results confirm the dissemination of OXA-23-producing Ab isolates associated with the ISAba-1 gene, in Brazil, as well as the intrinsic presence of the blaOXA-51 gene cassette. On the other hand, this is the first report of carbapenem-resistant Ab isolates harboring genes blaOXA-58 and blaOXA-72 recovered in Brazilian hospitals, which most likely emerged in 2004 and 2008, respectively.

Acinetobacter baumannii is an opportunistic pathogen and can cause severe disease in immune-suppressed and/or injured patients. It is an extreme-drug resistant bacterium with the ability to form biofilms thereby significantly increasing resistance to treatment. Because of the extreme drug resistance and relatively unknown immunological profile of A. baumannii new treatment options are needed. A. baumannii has been reported to express a Biofilm Associated Protein (BAP); a high molecular weight protein composed of multiple repeat modules and thought to be surface exposed on planktonic bacterium and upregulated in biofilm. While it is unknown if BAP has any role in in vivo infection of humans, the repeats of BAP proteins are thought to function in intercellular adhesion to support the mature biofilm and thus represent potential targets for immunotherapeutic intervention. Herein my thesis is aimed at trying to verify that BAP is surface exposed, upregulated in biofilm and to prove a role for BAP in pathogenesis, as well as investigating A. baumannii interactions with components of the innate immune system in vitro. Consensus synthetic peptides corresponding to the major internal repeats of BAP were designed and conjugated to carrier proteins and recombinant proteins were manufactured to correspond to the non-repetitive N and C terminals of the protein for murine immunization and assay development. Serum from immunized mice was collected and analyzed in ELISA and western immunoblot to determine reactivity with planktonic and biofilm whole organism. Anti-serum to whole bacteria was also tested in opsonisation assays to determine direct killing ability of serum on bacteria in vitro. Anti-serum to whole bacteria showed direct killing of the organism in vitro when in high concentrations (diluted 1/10), relative to pre-immune serum, but was less effective in lower concentrations (diluted 1/50). Despite generating antibody reagents to multiple domains and epitopes spanning the published BAP sequence, we were unable to confirm that BAP is expressed by A. baumannii as reported by others. However, if BAP is indeed expressed in A. baumannii our DNA and immunochemical data collectively suggest that BAP is potentially mosaic in this pathogen.

Acinetobacter baumannii is an opportunistic pathogen and can cause severe disease in immune-suppressed and/or injured patients. It is an extreme-drug resistant bacterium with the ability to form biofilms thereby significantly increasing resistance to treatment. Because of the extreme drug resistance and relatively unknown immunological profile of A. baumannii new treatment options are needed. A. baumannii has been reported to express a Biofilm Associated Protein (BAP); a high molecular weight protein composed of multiple repeat modules and thought to be surface exposed on planktonic bacterium and upregulated in biofilm. While it is unknown if BAP has any role in in vivo infection of humans, the repeats of BAP proteins are thought to function in intercellular adhesion to support the mature biofilm and thus represent potential targets for immunotherapeutic intervention. Herein my thesis is aimed at trying to verify that BAP is surface exposed, upregulated in biofilm and to prove a role for BAP in pathogenesis, as well as investigating A. baumannii interactions with components of the innate immune system in vitro. Consensus synthetic peptides corresponding to the major internal repeats of BAP were designed and conjugated to carrier proteins and recombinant proteins were manufactured to correspond to the non-repetitive N and C terminals of the protein for murine immunization and assay development. Serum from immunized mice was collected and analyzed in ELISA and western immunoblot to determine reactivity with planktonic and biofilm whole organism. Anti-serum to whole bacteria was also tested in opsonisation assays to determine direct killing ability of serum on bacteria in vitro. Anti-serum to whole bacteria showed direct killing of the organism in vitro when in high concentrations (diluted 1/10), relative to pre-immune serum, but was less effective in lower concentrations (diluted 1/50). Despite generating antibody reagents to multiple domains and epitopes spanning the published BAP sequence, we were unable to confirm that BAP is expressed by A. baumannii as reported by others. However, if BAP is indeed expressed in A. baumannii our DNA and immunochemical data collectively suggest that BAP is potentially mosaic in this pathogen.

Submitted by Pedro Barros (pedro.silvabarros@ufpe.br) on 2018-07-04T22:20:31Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) TESE Carmelita de Lima Bezerra Cavalcanti.pdf: 1587660 bytes, checksum: eade0c1462dbaba07af02453d8fb4173 (MD5) / Made available in DSpace on 2018-07-04T22:20:31Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) TESE Carmelita de Lima Bezerra Cavalcanti.pdf: 1587660 bytes, checksum: eade0c1462dbaba07af02453d8fb4173 (MD5) Previous issue date: 2017-02-21 / Apesar do grande envolvimento de isolados de Acinetobacter baumannii em Infecções Relacionadas à Assistência à Saúde (IRAS) e do seu alto índice de multidroga-resistência, levando a poucas opções terapêuticas em casos de infecções causadas por esta espécie bacteriana, há muito a ser descoberto sobre os mecanismos de virulência e resistência desta espécie. Portanto, este trabalho teve como objetivo estabelecer o perfil clonal, de resistência e virulência de isolados multidroga-resistentes (MDRs) de A. baumannii obtidos em hospitais de Recife-PE, determinando a prevalência de genes de resistência e virulência e a capacidade de expressão de genes de virulência após submissão dos isolados a diferentes antimicrobianos utilizados na prática clínica. Para determinar a prevalência dos genes de resistência e virulência foram utilizados 37 isolados do complexo A. baumannii provenientes de dois hospitais públicos de Recife – PE. A análise da expressão dos genes de virulência foi realizada utilizando três isolados MDR selecionados, além da ATCC 19606 de A. baumannii submetidos in vitro a colistina, meropenem e associação destes antimicrobianos. Os isolados foram avaliados quanto à confirmação da espécie através da detecção do gene blaOXA-51-like e da técnica de MALDI-TOF. A tipagem molecular desses isolados foi realizada através da técnica de PFGE utilizando a enzima de restrição Apa1. A detecção e sequência dos genes de resistência, blaOXA-51-like, blaOXA-23-like, blaOXA-143-like, blaIMP, blaVIM, blaKPC, o elemento de inserção, ISAba1, e dos genes de virulência, basC, ompA, pilA e csuE foi realizada através de PCR e sequenciamento gênico. Todos os isolados pertenciam à espécie A. baumannii, distribuídos em 07 padrões de PFGE, com três isolados apresentando 100% de similaridade, os quais foram obtidos nos dois hospitais públicos do estudo, sugerindo uma disseminação inter-hospitalar. Todos os isolados apresentaram o gene de resistência blaOXA-51-like e a maioria possuía o gene ISAba1, além desses, também apresentavam o gene blaOXA-143-like ou blaOXA-23-ike. Estes resultados demonstram uma ampla resistência dos isolados aos carbapenêmicos, além de outras classes de antimicrobianos. Os dados são preocupantes quanto à disseminação clonal desses genes entre os isolados obtidos nos hospitais analisados. Todos os isolados do estudo apresentaram os genes de virulência basC, ompA, pilA e csuE, com exceção de um isolado que não apresentou o gene csuE. Também pode-se demonstrar uma tendência ao aumento da expressão dos genes de virulência csuE, bfmS e baeS após tratamento in vitro com meropenem, colistina e associação destes antimicrobianos, reforçando a necessidade de vigilância quanto ao tratamento de IRAS causadas por A. baumannii MDR, mesmo em uso associado de antimicrobianos. / Due to the increasing resistance of clinical isolates of A. baumannii to antimicrobial and the consequent decrease of effective therapeutic options, this study aimed to establish the clonal profile, resistance and virulence of A. baumannii multidrug-resistant isolates (MDRs) obtained in hospitals in Recife-PE, determining the prevalence of resistance and virulence genes and the in vitro influence of different antibiotics used in clinical practice, alone and in combination, on bacterial growth and expression of these genes. To perform the first stage of this study 37 isolates of A. baumannii complex, most of them MDR. Analysis of virulence gene expression was performed using three MDR isolates beyond ATCC 19606 from A. baumannii submitted in vitro to colistin, meropenem and association of these antimicrobials. The isolates were evaluated for confirmation of the species through detection of the gene for intrinsic β-lactamase, blaOXA-51-like, and the Matrix-Assisted Laser Desorption / Ionization (MALDI-TOF) technique. The clonal profile of these isolates was determined by Pulsed-field Gel Electrophoresis (PFGE) using Apa1 enzyme. Detection and sequence of blaOXA-51-like, blaOXA-23-like, blaOXA-143-like, blaIMP, blaVIM and blaKPC genes the insertion element, ISAba1, and the virulence genes, basC, ompA, pilA and csuE were performed using Polymerase Chain Reaction (PCR) and sequencing. All isolates belonged to the A. baumannii species, distributed in 07 PFGE patterns, three isolates showed 100% similarity, which were obtained in two different hospitals of the study, suggesting an interhospital spread. Most isolates had the blaOXA-51-like and ISAba1 resistance genes, and either blaOXA-143-like or blaOXA-23-like gene. These results demonstrated high level resistance to carbapenems and other classes of antimicrobials, corroborating the MDR profile. This data alert for the spread of these genes among isolates in this hospitals. All isolates showed basC, ompA, pilA and csuE virulence genes, except for one isolate that did not show the csuE gene. Expression of csuE, bfmS e baeS virulence genes increased after in vitro submission to meropenem, colistin and the associated use may be demonstrated, reinforcing the need for surveillance for treating infections caused by MDR A. baumannii infections, even in associated antimicrobial use.

Submitted by Tiótrefis Fernandes (ppgssea@ufam.edu.br) on 2017-09-19T15:01:02Z No. of bitstreams: 4 Carta orientador Marcilene Maria de Souza Viana.pdf: 67670 bytes, checksum: c77de740b0ec45224e57fedbd9bb8212 (MD5) Pre Textuais.pdf: 144156 bytes, checksum: 303fc10d5636659a54a366f20b368dbb (MD5) Textuais Finais.pdf: 614930 bytes, checksum: 09b99dac0f864d42c18c34b81b661c28 (MD5) termo ma 001.jpg: 1164946 bytes, checksum: 72cb9c00fc7cf57ac3fa3f057419f594 (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2017-09-20T14:08:34Z (GMT) No. of bitstreams: 4 Carta orientador Marcilene Maria de Souza Viana.pdf: 67670 bytes, checksum: c77de740b0ec45224e57fedbd9bb8212 (MD5) Pre Textuais.pdf: 144156 bytes, checksum: 303fc10d5636659a54a366f20b368dbb (MD5) Textuais Finais.pdf: 614930 bytes, checksum: 09b99dac0f864d42c18c34b81b661c28 (MD5) termo ma 001.jpg: 1164946 bytes, checksum: 72cb9c00fc7cf57ac3fa3f057419f594 (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2017-09-20T14:23:54Z (GMT) No. of bitstreams: 4 Carta orientador Marcilene Maria de Souza Viana.pdf: 67670 bytes, checksum: c77de740b0ec45224e57fedbd9bb8212 (MD5) Pre Textuais.pdf: 144156 bytes, checksum: 303fc10d5636659a54a366f20b368dbb (MD5) Textuais Finais.pdf: 614930 bytes, checksum: 09b99dac0f864d42c18c34b81b661c28 (MD5) termo ma 001.jpg: 1164946 bytes, checksum: 72cb9c00fc7cf57ac3fa3f057419f594 (MD5) / Made available in DSpace on 2017-09-20T14:23:54Z (GMT). No. of bitstreams: 4 Carta orientador Marcilene Maria de Souza Viana.pdf: 67670 bytes, checksum: c77de740b0ec45224e57fedbd9bb8212 (MD5) Pre Textuais.pdf: 144156 bytes, checksum: 303fc10d5636659a54a366f20b368dbb (MD5) Textuais Finais.pdf: 614930 bytes, checksum: 09b99dac0f864d42c18c34b81b661c28 (MD5) termo ma 001.jpg: 1164946 bytes, checksum: 72cb9c00fc7cf57ac3fa3f057419f594 (MD5) Previous issue date: 2013-09-18 / 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.

Acinetobacter baumannii is an important nosocomial pathogen, frequently associated with morbidity and mortality in immunocompromised patients due to the immuno-ablative treatments, neutropenia and prolonged hospitalization. The ability of A. baumannii to survive in the healthcare setting makes it a frequent problematic pathogen in cancer centres. Much of the interest in A. baumannii has been attributed to its remarkable rapid acquisition of resistance mechanisms A. baumannii is an excellent example of genetic plasticity, with its ability to acquire and express resistance in plasmids and chromosome particularly to carbapenems The aim of this thesis is to look at the molecular epidemiology and resistance mechanisms of 34 non-duplicate A. baumannii in two cancer centres in Cairo, Egypt. Initial sequencing of the ubiquitous blaOXA-51-like gene revealed a large diversity within the strains, with eight different genes identified: blaOXA-64, blaOXA-65, blaOXA-66, blaOXA-69, blaOXA-71, blaOXA-78, blaOXA-94, blaOXA-89/100. Typing with Pulsed-field Gel Electrophoresis (PFGE) showed an overall similarity at only 28.69% between the isolates, with variation in pattern for isolates with similar blaOXA-51-like genes. Typing with Multilocus Sequence Typing (MLST) identified 6 new Sequence Types: ST408 - ST414, in addition to ST331 and ST108 which have been previously found in other regions of the world. All three OXA-type carbapenemases: blaOXA23, blaOXA40 and blaOXA58, responsible for conferring carbapenem resistance were found in the collection studied. Insertion sequences ISAba1, ISAba2 and ISAba3 have been found to upregulate the expression of blaOXA genes. ISAba1 was found upstream of blaOXA23 in 18 strains in this collection The first report of ISAba2 was identified upstream of a blaOXA-51-like gene in this collection. Additionally, ISAba3 was bracketing the blaOXA58 genes, and two isolates harboured hybrid promoters with IS1006 and IS1008 interrupting the upstream ISAba3 sequence. Resistance to Ceftazidime was mediated by Extended-spectrum β-lactamase (ESBL) genes belonging to PER-like group: blaPER-1, blaPER-7 and the first report of blaPER-3 gene and its genetic environment in A. baumannii. In conclusion, this study shows the diversity exhibited by A. baumannii in Egypt. The various resistance mechanisms illustrate the ability of A. baumannii in acquiring and expressing resistance genes, either on plasmids or in the chromosome. Furthermore, the results indicate an urgent need to strict infection control policies and surveillance of antimicrobial use in Egyptian hospitals.

Acinetobacter baumannii is an opportunistic pathogen prevalent in nosocomial infections, most commonly infecting humans with compromised immune systems during their hospital stays. The organism's success in such circumstances has to do with its ability to survive on dry, abiotic surfaces (e.g. catheters, bed railings, and other medical equipment) and its increasingly apparent antibiotic resistance. These factors make A. baumannii a serious problem for healthcare professionals and in public health generally. A. baumannii is paradigmatic and representative of the issues confronting healthcare in the ongoing antibiotic crisis, and many strains are showing multidrug resistant (MDR) phenotypes. Given that the patients infected by A. baumannii tend to be very vulnerable and traditional antibiotic treatment seems to be getting less and less effective, it is imperative to explore alternative treatment options that may lead to better outcomes, especially if their mechanisms are not the same as the traditional antibiotics that exert the selective pressures that have led to the current antibiotic crisis. A small molecule called M64 is known to inhibit a LysR-type transcription regulator (LTTR) important for virulence, but not cell growth or viability, in another opportunistic pathogen, Pseudomonas aeruginosa. The experiments presented here show that M64 was able to rescue mice infected with A. baumannii and to downregulate the expression of important metabolic genes downstream from A. baumannii LTTRs BenM and CatM in vitro while having no effect on bacterial growth. BenM and CatM regulate genes involved in the metabolism of benzoate and catechol respectively, both of which are parts of tryptophan metabolism and are eventually broken down to form acetyl-CoA and succinyl-CoA for energy production in the citric acid cycle. Such a pharmacodynamic profile offers a starting point in the design of alternative treatments of MDR bacterial infections, as successful outcomes are observed without the direct killing of cells in vitro seen in traditional antibiotics. In this case, as catechol metabolism is important for siderophore biosynthesis and thus bacterial virulence, inhibition of the transcription of genes involved in catechol metabolism may be playing a role in the observed rescue of infected mice. Further studies are required to ascertain the nature of the inhibitor's effect, however.

Acinetobacter baumannii é considerado um dos patógenos de maior importância clínica atualmente, sendo responsável por uma variedade de infeções nosocomiais como, bacteremias, infecções no trato urinário, pneumonias associadas a ventilação mecânica, meningites secundárias e infecções em feridas. Desde a última década o tratamento de infecções por Acinetobacter spp. vem sendo dificultado pela emergência de cepas multirresistentes. Nesse contexto, o objetivo deste trabalho foi determinar a dinâmica populacional e as características moleculares envolvidas na resistência de Acinetobacter spp. Foram avaliados isolados de Acinetobacter spp. provenientes de seis hospitais da cidade de Porto Alegre coletados entre janeiro de 2013 e março de 2014. A espécie Acinetobacter baumannii foi identificada pela presença do gene blaOXA-51, além de PCR multiplex para o gene gyrB. Carbapenemases (blaNDM, blaOXA-23-like, blaOXA-24/40-like, blaOXA-58-like e blaOXA-143-like), além dos integrons de classe 1 e 2 foram pesquisados por PCR. Um total de 524 isolados de Acinetobacter spp. foram coletados, e a maioria (487/92,9%) foram identificados como A. baumannii, seguidos pelas espécies A. nosocomialis (9/1,7%), A. pittii (3/0,6%), A. calcoaceticus (3/0,6%) e por fim, 22 (4,2%) isolados não pertenciam ao complexo A. baumannii-calcoaceticus. Quanto ao perfil de sensibilidade, 83% dos isolados demonstraram não ser sensíveis aos carbapenêmicos (imipenem e meropenem). Dos isolados de A. baumannii, 429 (88,1%) continham o gene blaOXA-23, além disso, foram observados dois isolados de A. nosocomialis contendo o gene blaOXA-23. Foi possível identificar dois isolados de A. baumannii produtores de blaOXA24/40 (0,8%) pela primeira vez na região Sul do país. A análise do gene por sequenciamento caracterizou como sendo a variante blaOXA-72, e a tipagem por MLST caracterizou os isolados como pertencentes a ST730 (CC79). Também foram analisados por MLST, isolados produtores de OXA-72 do estado de São Paulo e do Paraná. A análise dos dados demonstrou que estes isolados estão associados aos complexos clonais epidêmicos CC15 e CC79. A pesquisa para o gene blaNDM foi positiva para um isolado de A. pittii, sendo o primeiro isolado de Acinetobacter não baumannii produtor de NDM-1 no Brasil. A análise do contexto genético de blaNDM-1 demonstrou a presença de ISAba125 upstream ao gene, além disso, o gene demonstrou estar localizado no cromossomo da bactéria. Foi observada uma maior prevalência de integrons de classe 2 nos isolados avaliados (134/25,5%), quando comparada aos integrons de classe 1 (72/13,7%). Raros (2/0,4%) isolados apresentaram ambas as classes de integrons. Um total 244 isolados de A. baumannii resistentes aos carbapenêmicos foram submetidos à tipagem por REP-PCR, que demonstrou a presença de 20 grupos clonais entre os isolados analisados. Isolados de diferentes grupos clonais foram selecionados para a tipagem por MLST. Com o objetivo de realizarmos uma análise longitudinal dos clones circulantes desde o primeiro surto de Acinetobacter baumannii resistente aos carbapenêmicos (CRAB) na cidade de Porto Alegre, também foram selecionados para análise por MLST isolados de A. baumannii pertencentes ao nosso banco de dados com diferentes perfis clonais, coletados entre 2007 e 2008. Através da análise dos dados gerados pelo MLST no banco de dados do Instituto Pasteur, foram descritas 13 novas STs: ST883 (CC32), ST884 (CC221), ST885 (CC79), ST886, ST887, ST888, ST889 (CC464), ST892 (CC15), ST899, ST902 (CC1), ST903 (CC79), ST904 (CC15) e ST905 (CC32). A avaliação da dinâmica populacional de A. baumannii nos dois períodos avaliados demonstrou a permanência de isolados pertencentes aos complexos clonais CC15 e CC79 desde o período do primeiro surto de CRAB da cidade de Porto Alegre até o ano de 2014, demonstrando a capacidade desses clones de se manter por longos períodos no ambiente hospitalar. Além disso, estes CCs apresentaram uma elevada prevalência durante o primeiro período avaliado (2007-2008). Este dado que nos permite inferir que o primeiro surto de CRAB produtor de OXA-23 da cidade de Porto Alegre foi relacionado a disseminação de CC15 e CC79. / Acinetobacter baumannii is considered one of the main pathogens of clinical importance currently, being responsible for a wide range of nosocomial infections such as, bacteremias, urinary tract infections, ventilator-associated pneumonia, secondary meningitis and wound infections. Since the last decade the treatment of these infections has been impaired by the emergence of multiresistant strains. In this context, the aim of this study was to determine the population dynamic and the molecular characteristics involved in Acinetobacter spp. resistance. A total of 524 Acinetobacter spp. isolates were collected from six Porto Alegre hospitals from January 2013 to March 2014. A. baumannii species were identified by the presence of blaOXA-51 gene and by the gyrB multiplex PCR. Carbapenemase genes (blaNDM, blaOXA-23-like, blaOXA-24/40-like, blaOXA-58-like and blaOXA-143-like) as well as class 1 and 2 integrons were investigated by PCR. As expected, the majority (487/92.9%) of isolates were identified as A. baumannii, followed by A. nosocomialis (9/1.7%), A. pittii (3/0.6%), and A. calcoaceticus (3/0.6%). A total of 22 (4.2%) isolates were not identified as A. baumannii-calcoaceticus complex. Analysis of the susceptibility profile demonstrated that 83% of the isolates were not susceptible to carbapenems (imipenem e meropenem). Among the A. baumannii isolates, 429/487 (88.1%) presented blaOXA-23 gene. Two A. nosocomialis isolates also presented the blaOXA-23 gene. We also found, for the first time in Southern Brazil, two A. baumannii isolates containing blaOXA24/40 (0.8%). The sequencing of blaOXA24/40 identified the variant blaOXA-72, and MLST analysis characterized both isolates as ST730 (CC79). In addition, OXA-72-producing A. baumannii isolates from the states of São Paulo and Paraná were analyzed by MLST. Data analysis demonstrated that the isolates were associated to the clonal complexes CC15 and CC79. The screening of blaNDM gene was positive for an A. pittii isolate. Therewith, in the present study we described for the first time a Acinetobacter non-baumannii producing NDM-1 in Brazil. Analysis of the genetic environment of blaNDM-1 gene demonstrated the presence of ISAba125 upstream of the gene and that the gene was chromosome-located in A. pittii. It was observed an increased prevalence of class 2 integrons (134/25.5%) compared to class 1 integrons (72/13.7%). Only a few isolates presented both classes (2/0.4%). A total of 244 carbapenem-resistant A. baumannii isolates were typed by REP-PCR, which demonstrated the presence of 20 clonal groups. Isolates belonging to different clonal groups were selected for MLST typing. In order to conduct a longitudinal analysis of circulating clones from the first CRAB outbreak in the city of Porto Alegre, we also selected A. baumannii isolates with different clonal profiles collected between 2007 and 2008. According to MLST database from Pasteur Institute, we identified 13 new STs: ST883 (CC32), ST884 (CC221), ST885 (CC79), ST886, ST887, ST888, ST889 (CC464), ST892 (CC15), ST899, ST902 (CC1), ST903 (CC79), ST904 (CC15) and ST905 (CC32). The analysis of A. baumannii population dynamics in the two periods of the study demonstrated the persistence of the clonal complexes CC15 and CC79 from the first CRAB outbreak in Porto Alegre city, up to the year of 2014. Besides, these CCs presented a high prevalence during the first period (2007-2008) evaluated. These data allow us to infer that the first CRAB OXA-23-producing outbreak in Porto Alegre city was related to the dissemination of CC15 and CC79.