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Doctoral Thesis
DOI
10.11606/T.9.2011.tde-22122011-085102
Document
Author
Full name
Elizabeth Harummyy Takagi
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2011
Supervisor
Committee
Martinez, Marina Baquerizo (President)
Barth, Afonso Luis
Garcia, Doroti de Oliveira
Marques, Elizabeth de Andrade
Sampaio, Jorge Luiz Mello
Title in Portuguese
Caracterização molecular e fenotípica de amostras bacterianas pertencentes ao complexo Acinetobacter calcoaceticus-Acinetobacter baumannii
Keywords in Portuguese
A. 3
A.13TU
Acinetobacter baumannii
Biofilme
Identificação bacteriana
Quorum sensing
Resistência bacteriana
Abstract in Portuguese
Nos últimos 30 anos, Acinetobacter tornou-se um dos patógenos de maior preocupação clínica pela falta de terapias eficazes em virtude do fenótipo de multirresistência frequentemente apresentado. Dentre as espécies do gênero Acinetobacter, A. baumannii, A. genoespécie 3 e A. genoespécie 13TU são as mais comumente encontradas a partir de amostras biológicas. Estas espécies ao lado de A. calcoaceticus constituem o complexo A. calcoaceticus-A. baumannii (ACB). Este estudo propõe um esquema composto de duas PCRs para a identificação das espécies de interesse médico que fazem parte do complexo ACB. O método é simples, rápido e, além de identificar as espécies, permite pesquisar a presença de genes de resistência. Foram identificadas 515 amostras do complexo ACB, isoladas de pacientes no período de janeiro de 2005 a dezembro de 2010. A identificação das espécies do complexo ACB foi realizada por esquema composto de duas reações de PCR. Foram avaliados os perfis de sensibilidade por disco difusão e a pesquisa da presença dos genes blaOXA-23-like, blaOXA-24-like, blaOXA-51-like, blaOXA-58-like, blaIMP, blaVIM, blaSIM, blaSPM e blaGIM foi realizada por PCR utilizando-se iniciadores específicos. No grupo de amostras estudas, 82,5% são A. baumannii (425), 11,5% A. genoespécie 13TU (59) e 6,0% A. genoespécie 3 (30), sendo A. baumannii mais isolado em pacientes internados em UTIs (p=0,0407) e A. genoespécie 13TU mais isolado em pacientes de outros ambientes hospitalares (p=0,0204). A. baumannii apresentou menor sensibilidade a todos os antimicrobianos quando comparado com A. genoespécie 13TU e A. genoespécie. 3 (p<0,05). Foi possível observar ao longo do período estudado o aumento significativo da resistência aos carbapenêmicos e da sensibilidade a gentamicina por A. baumannii entre os isolados de pacientes de UTIs (p<0.05). Nenhum dos genes codificadores para metalo-lactamases foi detectado nas amostras estudadas Dentre os cepas resistentes aos carbapenêmicos (176) o gene blaOXA-23 foi detectado em 81,25% e uma amostra de A. baumannii apresentou o gene codificador para OXA-72. A tipagem molecular foi realizada por RAPD e para os isolados resistentes aos carbapenêmicos também por PFGE. Resultados obtidos por RAPD revelaram menor diversidade entre os isolados de pacientes internados em UTIs. O dendrograma obtido utilizando-se PFGE separou dois clones cujos componentes eram resistentes aos carbapenêmicos, no entanto não apresentavam o gene blaOXA-23-like. A produção de acil-homoserina lactona, autoindutor-2 e autoindutor-3 de três amostras de cada espécie clínica do complexo ACB foi pesquisada utilizando-se bioensaios. Apenas Autoindutor 3 foi detectado por bioensaio e em menor quantidade no meio précondicionados obtido a partir de A. genoespécie 3 quando comparado com A. genoespécie 13TU e A. baumannii (p<0.05). Três cepas de cada espécie clínica do complexo ACB foi avaliada quanto a capacidade de adesão em monocamada de células Hep-2, MRC-5 e NCI-H292, sendo essa última a que revelou diferenças entre as espécies clínicas do complexo ACB. A. baumannii apresentou adesão difusa, A. genoespécie 13 TU adesão com formação de agrupamentos e A. genoespécie 3 não aderiu. Esse mesmo ensaio foi realizado na presença de propanolol e notou-se a diminuição de células aderidas por campo observado. Dez cepas de cada espécie clínica do complexo ACB foram pesquisadas quanto a produção de biofilme por ensaio colorimétrico utilizando cristal violeta e foi possível notar a produção significativa de biofilme por A. baumannii, quando comparado com A. genoespécie 3 (p<0.05). Esse mesmo ensaio na presença de de fentolamina, mostrou a diminuição significativa na produção do biofilme por A. baumannii. A interferência no ensaio de adesão bacteriana e biofilme, na presença de fentolamina ou propanolol, sugerem o envolvimento do autoindutor-3 na regulação desses mecanismos de virulência.
Title in English
Molecular and phenotypic characterization of Acinetobacter calcoaceticus-Acinetobacter baumannii isolates
Keywords in English
A. 13TU
A. 3
Acinetobacter baumannii
Antimicrobial susceptibility
Biofilm
Quorum sensing
Abstract in English
The genus Acinetobacter has emerged as one of the most troublesome pathogens for health care institutions globally. Its clinical significance, especially over the last 15 years, has been driven by its remarkable ability to up regulate or acquire resistance determinants, making it one of the organisms threatening the current antibiotic era. A. baumannii, A. 3 and A. 13TU are the most commonly species found from biological samples. These species beside A. calcoaceticus are very closely related and difficult to distinguish from each other by phenotypic properties. Therefore, it has been proposed to refer to these species as the A.calcoaceticus-A. baumannii complex(ACB). In the period from 2005 to 2009, the most frequent bacterial isolates among the nosocomial infection at the HU-USP was ACB (18%). Due to the frequency with which species are involved in ACB outbreaks of infection in the HU-USP and the emergency clinic because of expression of the phenotype of resistance to several classes of antibiotics, this study aimed to identify and characterize the species of complex ACB by molecular methods, to study their mechanisms of resistance and to characterize the different clones from patients admitted to different hospital areas. Furthermore, the ability to characterize biofilm formation, adhesion to different cell lines as well as the mechanisms of cell-cell communication were analyzed. From the ACB complex, 515 samples were identified, isolated from patients from January 2005 to December 2010. The identification of clinical species of the ACB was performed by molecular methods that were developed and validated for identification of Acinetobacter sp. include two reactions of PCR. The profiles of sensibility were evaluated by disc diffusion and the detection of the presence of genes blaOXA-23-like, blaOXA-24-like, blaOXA-51-like, blaOXA-58-like, blaIMP, blaVIM, blaSIM, blaGIM, and blaSPM were performed using specific primers. Molecular typing was performed by RAPD and isolates resistant to carbapenems also by PFGE. The production of autoinducers of three clinical species complex was sought using bioassays with sensor strains. The ability adhesion was evaluated in monolayer of Hep-2 cells, MRC-5 and NCI-H292E. Ten clinical strains of each species of ACB complex were screened for the production of biofilms by colorimetric assay using crystal violet. Among all the strains studied, 82.5% were A. baumannii (425), 11.5% A.13TU (59) and 6.0% A. 3 (30). A. baumannii strains were more isolated from intensive care unit (ICU) patients (p = 0.0407) and A. 13TU from other patients in different hospital settings (p = 0.0204). A. baumannii showed less sensitivity to all drugs when compared with A. 13TU and A.3 (p <0.05). It was possible to observe during the study period a significant increase in carbapenem resistance and sensitivity to gentamicin by A. baumannii isolates from patients in ICUs (p <0.05). No genes coding for metallo-lactamase was detected in the samples studied. blaOXA-23 gene was detected in 81.25% among the 176 strains resistant to carbapenems. Results obtained by RAPD revealed less diversity among isolates from ICU patients compared to isolates from patients from other hospitals. The dendrogram obtained by PFGE showed less diversity than RAPD It was unable to detect homoserine lactone and autoinducer-2 by bioassay. The survey was positive of autoinducer-3 observed differences in yield among clinical species, smaller amount produced by strains of A. 3 when compared with A. 13TU and A. baumannii (p <0.05). Among the cells studied in adhesion testing, line NCI-H292 showed the greatest power discrimination between adhesion pattern observed and species of the ACB. A. baumannii showed diffuse adherence, A. 13 TU strains showed adhesion clustering and A. 3 did not adhere. This experiment was repeated in the presence of 100 µM of propranolol and it was noted a decrease in cell A. 13TU and A. baumannii adhered per field observed. The biofilm assay showed significantly higher production of biofilms by A.baumannii compared with A. 3 (p <0.05). When the test was conducted in the presence of phentolamine at 100µM, it was observed a significant decrease in the biofilm productions by A. baumannii, which revealing the involvement of the autoinducer-3 in biofilm production. The data obtained suggest that the proposed method of identification is a method for identification of species of medical interest belonging to the ACB complex which could be used in a routine laboratory. The method is simple, fast and beside the identification species, provides data about the resistance genes. Moreover, it revealed that the isolates of A. baumannii are more resistant and OXAbla genes, that was restricted to the ACB complex studied in this work. A. baumannii has also increased capacity for adhesion and biofilm formation, which regulates the expression of the phenotype may be linked to the production of autoinducers-3.
 
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Publishing Date
2012-02-08
 
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