Acinetobacter baumannii est un pathogène opportuniste qui prend une importance clinique croissante du fait de l’acquisition de multi-résistance. Nous avons étudié chez A. baumannii les caractéristiques et la régulation des intégrons de classe 1 (IM1) qui sont des systèmes génétiques favorisant l’acquisition, l’expression et la dissémination des gènes de résistance aux antibiotiques. Nous avons montré qu’il existe une prédominance des promoteurs des cassettes Pc fort in vivo dans une collection d’isolats cliniques et d’environnement hospitalier et in silico dans les IM1 chez A. baumannii. Nous avons aussi montré que l’expression des Pc chez A. baumannii est 4 fois plus faible que chez E. coli, quel que soit le variant de Pc. Deux explications sont possibles pour la sélection des Pc forts chez A. baumannii : (i) la nécessité d’avoir un niveau d’expression suffisant en clinique pour survivre à la pression de sélection antibiotique et (ii) la nécessité d’une régulation de l’expression de l’intégrase, représentant un coût biologique important. En effet, A. baumannii ne possède pas le système de répression par LexA existant chez E. coli. Nos résultats ouvrent le champ de l’étude de la régulation des IM1 chez A. baumannii et ainsi l’identification de nouvelles voies d’action pour lutter contre l’antibio-résistance / Acinetobacter baumannii is an opportunistic pathogen of increasing clinical importance due to the acquisition of multi-resistance. We studied in A. baumannii the characteristics and regulation of class 1 integrons (IM1), which are genetic systems that favor the acquisition, expression and dissemination of antibiotic resistance genes. We have shown that there is a predominance of strong Pc cassette promoters, in vivo, in a collection of clinical and hospital environment isolates, and in silico, from A. baumannii IM1 published in NCBI. We have also shown that the expression of Pc in A. baumannii is 4-fold lower than in E. coli, regardless of the Pc variant. Explanations that can be raised for the selection of strong Pc in A. baumannii are: (i) the need for a sufficient level of antibiotic resistance expression to survive the selection pressure in clinical environment; and (ii) the need for regulation of the integrase expression, which is of significant biological cost. Indeed A. baumannii does not have the LexA repression system existing in E. coli. Our results open the field of the study of IM1 regulation in A. baumannii and thus the identification of new pathways to fight antibiotic resistance.

Introdução: O gênero Acinetobacter sp apresenta considerável heterogeneidade possuindo inúmeras espécies. Atualmente, 23 espécies já foram nomeadas e nove outras espécies já foram descritas. Quatro destas espécies possuem contextos clínicos e epidemiológicos diferentes, no entanto são agrupadas em um complexo denominado Acinetobacter calcoaceticus-Acinetobacter baumannii (ABC) devido à sua similaridade genética e fenotípica. Diversos testes para diferenciação do complexo já foram descritos, porém a maioria não pode ser realizado na rotina laboratorial, pois são caros e laboriosos. Objetivos: Avaliar um método rápido e viável na rotina laboratorial, capaz de diferenciar as espécies do complexo ABC; Determinar a prevalência das diferentes espécies do complexo ABC; Avaliar o perfil de suscetibilidade aos antimicrobianos nas diferentes espécies. Métodos: Foram analisadas 118 amostras de dois hospitais de Porto Alegre-RS através do método Multiplex PCR para o gene gyrB e posteriormente confirmadas pelo padrão-ouro: sequenciamento do 16S-23S ITS. O perfil de suscetibilidade foi realizado através de microdiluição em caldo. Resultados: Das 118 amostras identificadas inicialmente como Acinetobacter sp., a grande maioria dos isolados (106 -89.9%) foram identificados como A. baumannii; mas doze isolados foram identificados como sendo das demais espécies do complexo ABC: 6 (5.1%) A. nosocomialis, 5 (4.2%) A. pittii, e 1 (0.8%) A. genoespécie 10, através da técnica de Multiplex PCR. Todos os resultados foram confirmados por sequenciamento. A. baumannii apresentou um elevado nível (72,6%) de resistência ao imipenem em comparação com as outras espécies seguido da espécie A. nosocomialis que apresentou metade de seus isolados resistentes. Todas as espécies apresentaram baixos índices (inferior a 7,5%) de resistência à Polimixina B e Tigeciclina. Conclusão: O Multiplex PCR para o gene gyrB apresentou resultados fidedignos quando comparados ao padrão-ouro, demonstrando, assim, ser um método confiável para a identificação das espécies do complexo ABC. Outras espécies, além de A. baumannii, ABC podem apresentar percentuais significativos de resistência ao imipenem. / Background: Introduction: The genus Acinetobacter sp presents considerable heterogeneity possessing numerous species. Currently, 23 species have been named and nine other species have been described. Four of these species have different clinical and epidemiological contexts, but are grouped in a complex called Acinetobacter calcoaceticus-Acinetobacter baumannii (ABC) due to their genetic and phenotypic similarity. Several tests for differentiation of the complex have been described, but most can not be performed routinely in the laboratory, they are expensive and laborious. Objectives: To evaluate a fast and feasible in routine laboratory able to differentiate the species of the complex ABC; determine the prevalence of different species of the complex ABC; evaluate the antimicrobial susceptibility profile of the different species. Methods: We analyzed 118 samples from two hospitals in Porto Alegre-RS by the method of Multiplex PCR for gene gyrB and subsequently confirmed by the gold standard: sequencing the 16S-23S ITS. The susceptibility profile was performed by microdilution. Results: Of the 118 samples initially identified as Acinetobacter sp. The great majority of isolates (106 -89.9%) were identified as A. baumannii, but twelve isolates were identified as being from other species of the complex ABC: 6 (5.1%) A. nosocomialis, 5 (4.2%) A. pittii, and 1 (0.8%) A. genospécie 10 by Multiplex PCR technique. All results were confirmed by sequencing. A. baumannii showed a high level (72.6%) of imipenem resistance in comparison with the other species followed by the species A. nosocomialis showed that half of his resistant isolates. All species showed low levels (less than 7.5%) of resistance to Polymyxin B and Tigecycline. Conclusion: Multiplex PCR for gene gyrB results presented totally reliable when compared to the gold standard, demonstrating thus be a safe method for the laboratory. Other species besides A. baumannii, ABC may have significant percentages of resistance to imipenem.

Introdução: O gênero Acinetobacter sp apresenta considerável heterogeneidade possuindo inúmeras espécies. Atualmente, 23 espécies já foram nomeadas e nove outras espécies já foram descritas. Quatro destas espécies possuem contextos clínicos e epidemiológicos diferentes, no entanto são agrupadas em um complexo denominado Acinetobacter calcoaceticus-Acinetobacter baumannii (ABC) devido à sua similaridade genética e fenotípica. Diversos testes para diferenciação do complexo já foram descritos, porém a maioria não pode ser realizado na rotina laboratorial, pois são caros e laboriosos. Objetivos: Avaliar um método rápido e viável na rotina laboratorial, capaz de diferenciar as espécies do complexo ABC; Determinar a prevalência das diferentes espécies do complexo ABC; Avaliar o perfil de suscetibilidade aos antimicrobianos nas diferentes espécies. Métodos: Foram analisadas 118 amostras de dois hospitais de Porto Alegre-RS através do método Multiplex PCR para o gene gyrB e posteriormente confirmadas pelo padrão-ouro: sequenciamento do 16S-23S ITS. O perfil de suscetibilidade foi realizado através de microdiluição em caldo. Resultados: Das 118 amostras identificadas inicialmente como Acinetobacter sp., a grande maioria dos isolados (106 -89.9%) foram identificados como A. baumannii; mas doze isolados foram identificados como sendo das demais espécies do complexo ABC: 6 (5.1%) A. nosocomialis, 5 (4.2%) A. pittii, e 1 (0.8%) A. genoespécie 10, através da técnica de Multiplex PCR. Todos os resultados foram confirmados por sequenciamento. A. baumannii apresentou um elevado nível (72,6%) de resistência ao imipenem em comparação com as outras espécies seguido da espécie A. nosocomialis que apresentou metade de seus isolados resistentes. Todas as espécies apresentaram baixos índices (inferior a 7,5%) de resistência à Polimixina B e Tigeciclina. Conclusão: O Multiplex PCR para o gene gyrB apresentou resultados fidedignos quando comparados ao padrão-ouro, demonstrando, assim, ser um método confiável para a identificação das espécies do complexo ABC. Outras espécies, além de A. baumannii, ABC podem apresentar percentuais significativos de resistência ao imipenem. / Background: Introduction: The genus Acinetobacter sp presents considerable heterogeneity possessing numerous species. Currently, 23 species have been named and nine other species have been described. Four of these species have different clinical and epidemiological contexts, but are grouped in a complex called Acinetobacter calcoaceticus-Acinetobacter baumannii (ABC) due to their genetic and phenotypic similarity. Several tests for differentiation of the complex have been described, but most can not be performed routinely in the laboratory, they are expensive and laborious. Objectives: To evaluate a fast and feasible in routine laboratory able to differentiate the species of the complex ABC; determine the prevalence of different species of the complex ABC; evaluate the antimicrobial susceptibility profile of the different species. Methods: We analyzed 118 samples from two hospitals in Porto Alegre-RS by the method of Multiplex PCR for gene gyrB and subsequently confirmed by the gold standard: sequencing the 16S-23S ITS. The susceptibility profile was performed by microdilution. Results: Of the 118 samples initially identified as Acinetobacter sp. The great majority of isolates (106 -89.9%) were identified as A. baumannii, but twelve isolates were identified as being from other species of the complex ABC: 6 (5.1%) A. nosocomialis, 5 (4.2%) A. pittii, and 1 (0.8%) A. genospécie 10 by Multiplex PCR technique. All results were confirmed by sequencing. A. baumannii showed a high level (72.6%) of imipenem resistance in comparison with the other species followed by the species A. nosocomialis showed that half of his resistant isolates. All species showed low levels (less than 7.5%) of resistance to Polymyxin B and Tigecycline. Conclusion: Multiplex PCR for gene gyrB results presented totally reliable when compared to the gold standard, demonstrating thus be a safe method for the laboratory. Other species besides A. baumannii, ABC may have significant percentages of resistance to imipenem.

Introdução: O gênero Acinetobacter sp apresenta considerável heterogeneidade possuindo inúmeras espécies. Atualmente, 23 espécies já foram nomeadas e nove outras espécies já foram descritas. Quatro destas espécies possuem contextos clínicos e epidemiológicos diferentes, no entanto são agrupadas em um complexo denominado Acinetobacter calcoaceticus-Acinetobacter baumannii (ABC) devido à sua similaridade genética e fenotípica. Diversos testes para diferenciação do complexo já foram descritos, porém a maioria não pode ser realizado na rotina laboratorial, pois são caros e laboriosos. Objetivos: Avaliar um método rápido e viável na rotina laboratorial, capaz de diferenciar as espécies do complexo ABC; Determinar a prevalência das diferentes espécies do complexo ABC; Avaliar o perfil de suscetibilidade aos antimicrobianos nas diferentes espécies. Métodos: Foram analisadas 118 amostras de dois hospitais de Porto Alegre-RS através do método Multiplex PCR para o gene gyrB e posteriormente confirmadas pelo padrão-ouro: sequenciamento do 16S-23S ITS. O perfil de suscetibilidade foi realizado através de microdiluição em caldo. Resultados: Das 118 amostras identificadas inicialmente como Acinetobacter sp., a grande maioria dos isolados (106 -89.9%) foram identificados como A. baumannii; mas doze isolados foram identificados como sendo das demais espécies do complexo ABC: 6 (5.1%) A. nosocomialis, 5 (4.2%) A. pittii, e 1 (0.8%) A. genoespécie 10, através da técnica de Multiplex PCR. Todos os resultados foram confirmados por sequenciamento. A. baumannii apresentou um elevado nível (72,6%) de resistência ao imipenem em comparação com as outras espécies seguido da espécie A. nosocomialis que apresentou metade de seus isolados resistentes. Todas as espécies apresentaram baixos índices (inferior a 7,5%) de resistência à Polimixina B e Tigeciclina. Conclusão: O Multiplex PCR para o gene gyrB apresentou resultados fidedignos quando comparados ao padrão-ouro, demonstrando, assim, ser um método confiável para a identificação das espécies do complexo ABC. Outras espécies, além de A. baumannii, ABC podem apresentar percentuais significativos de resistência ao imipenem. / Background: Introduction: The genus Acinetobacter sp presents considerable heterogeneity possessing numerous species. Currently, 23 species have been named and nine other species have been described. Four of these species have different clinical and epidemiological contexts, but are grouped in a complex called Acinetobacter calcoaceticus-Acinetobacter baumannii (ABC) due to their genetic and phenotypic similarity. Several tests for differentiation of the complex have been described, but most can not be performed routinely in the laboratory, they are expensive and laborious. Objectives: To evaluate a fast and feasible in routine laboratory able to differentiate the species of the complex ABC; determine the prevalence of different species of the complex ABC; evaluate the antimicrobial susceptibility profile of the different species. Methods: We analyzed 118 samples from two hospitals in Porto Alegre-RS by the method of Multiplex PCR for gene gyrB and subsequently confirmed by the gold standard: sequencing the 16S-23S ITS. The susceptibility profile was performed by microdilution. Results: Of the 118 samples initially identified as Acinetobacter sp. The great majority of isolates (106 -89.9%) were identified as A. baumannii, but twelve isolates were identified as being from other species of the complex ABC: 6 (5.1%) A. nosocomialis, 5 (4.2%) A. pittii, and 1 (0.8%) A. genospécie 10 by Multiplex PCR technique. All results were confirmed by sequencing. A. baumannii showed a high level (72.6%) of imipenem resistance in comparison with the other species followed by the species A. nosocomialis showed that half of his resistant isolates. All species showed low levels (less than 7.5%) of resistance to Polymyxin B and Tigecycline. Conclusion: Multiplex PCR for gene gyrB results presented totally reliable when compared to the gold standard, demonstrating thus be a safe method for the laboratory. Other species besides A. baumannii, ABC may have significant percentages of resistance to imipenem.

The genus Acinetobacter currently contains 34 species, the vast majority of which are not regularly implicated in causing infection. However, incidences of hospitalacquired infection with Acinetobacter species are increasing, mainly due to the rise in the number of infections caused by the species Acinetobacter baumannii in immunocompromised patients particularly in intensive care units (ICUs). Due to high levels of resistance in A. baumannii to many classes of antibiotic, the carbapenems have been portrayed as the ‘drugs of choice’ for treating infections with this species. However, the activity of the carbapenems against A. baumannii has come under threat with the identification of four groups of class D β-lactamases carried by members of the species. Of these, the OXA-51-like enzymes have been suggested to be ubiquitous and intrinsic enzymes within A. baumannii. This presents the worrying scenario of the potential for all A. baumannii to become carbapenem resistant, leaving few treatment options available for this species. This project aimed to investigate the epidemiological spread of the OXA-51-like β-lactamases, examine the diversity within these enzymes, and whether this diversity may have implications for their ability to confer resistance to the carbapenems. A functional map showing the amino-acid similarities between the OXA-51-like enzymes demonstrated that the enzymes fall into distinct closely-related groups, with notable clusters surrounding OXA-66, OXA-69 and OXA-98. PCR and sequencing analysis of a geographically diverse group of 64 A. baumannii isolates demonstrated that isolates forming specific sequence groups (SGs) as defined by Turton et al (2007) also contained the same or closely related blaOXA-51-like gene. Higher minimum inhibitory concentrations (MICs) of carbapenems were found in association with acquired carbapenemases, or with the presence of ISAba1 upstream of the blaOXA-51- like gene. Pulsed-field gel electrophoresis (PFGE) analysis of the isolates did not demonstrate relatedness between isolates which formed the same sequence group. Multilocus sequence typing (MLST) of a subset of 44 isolates grouped isolates more consistently with the SGs and blaOXA-51-like alleles, indicating that PFGE is unreliable for use with A. baumannii unless studying a short time period, and that blaOXA-51-like alleles are a good epidemiological marker. Mutation studies using meropenem with five A. baumannii isolates encoding different OXA-51-like enzymes, while resulting in an increase in meropenem MICs of between 8- and 128-fold, did not result in a nucleotide substitution in the blaOXA- 51-like genes or a change in the upstream region of the genes in any isolate suggesting that the carbapenems may not be producing a strong selective pressure on the blaOXA- 51-like genes. Analysis of πN/πS ratios for the blaOXA-51-like genes, MLST genes and the TEM, SHV and CTX-M β-lactamase families showed the blaOXA-51-like genes to be under less positive selection than these other β-lactamases, though under less purifying selection than the MLST genes. Phylogenetic analysis of the MLST genes and the blaOXA-51-like genes indicates that the blaOXA-51-like genes have been evolving within A. baumannii since its speciation, and that different groups of blaOXA-51-like genes have been evolving at different rates corresponding to different rates of evolution within their parent lineages. Structural modelling studies based upon the published crystal structure for OXA-40 indicated that amino-acid variation at particular sites in the OXA-51-like enzymes are likely to have an effect of enzyme function. Alterations at amino-acid position 167 change the shape of the entrance to the active site which may affect hydrolysis by accommodating the antibiotic differently, or may affect the substrate profile of the enzyme. The substitution of glutamine for proline at position 194 may significantly alter the shape of the enzyme thereby affecting substrate hydrolysis. This project found that specific groups of blaOXA-51-like genes are associated with specific A. baumannii lineages and that these genes could serve as convenient epidemiological markers. Most of the diversity within the OXA-51-like enzymes is due to their continued evolution within A. baumannii since the species’ emergence. However, certain amino-acid changes may play a role in altering the rate of hydrolysis or substrate profile of these enzymes.

Acinetobacter baumannii es un cocobacilo aeróbico, Gram negativo y considerado un patógeno nosocomial emergente, que ha desarrollado resistencia a múltiples fármacos. El presente trabajo tuvo como objetivo determinar la resistencia antimicrobiana de cepas de Acinetobacter baumannii aisladas de pacientes internados en el Hospital Nacional Guillermo Almenara Irigoyen y relacionarlo con la presencia de plásmidos, así como determinar la prevalencia de esta especie en dicho nosocomio según el servicio de hospitalización, tipo de muestra, edad, sexo y factores de comorbilidad, mortalidad y estacionalidad. Se identificaron 40 cepas de A. baumannii de origen clínico aisladas de muestras de pacientes hospitalizados del nosocomio Guillermo Almenara Irigoyen durante enero-diciembre del 2012. Las cepas fueron identificadas mediante el sistema automatizado Micro Scan y el método convencional. Se utilizo Agar MacConkey y Agar Leeds Acinetobacter, incubándose a 37 y 44°C respectivamente, en la coloración Gram se observaron cocobacilos Gram negativas, se hicieron pruebas bioquímicas como TSI, citrato y gelatina además de la pruebas de oxidasa y catalasa. Se realizó la sensibilidad antimicrobiana a través del sistema automatizado Micro Scan y el método de difusión en disco. El 100% de las cepas mostraron resistencia a cefalosporina de tercera generación, meropenem, imipenem, ciprofloxacino, ticar/Ac. clavulánico. El 93.5% fueron resistentes a cefepime y sulfametoxazol-trimetoprim, el 95 % a levofloxacino, el 90% a amikacina, el 45% a gentamicina, el 37.5% a tobramicina y el 30% a tetraciclina. Se registraron 12 antibiotipos de resistencia, con una mayor frecuencia en el servicio de Unidad de cuidados Intensivos. Se determinó el perfil plasmídico de las cepas estudiadas, obteniendo 31 perfiles según el número y tamaño de las bandas, que oscila entre 1,240 - 55,459 pb aproximadamente; luego se relacionó con los patrones de resistencia y el origen de aislamiento de la cepa. Además se realizó la curación de las cepas con bromuro de etidio a una concentración del 300 µg/ml. Las cepas que perdieron la resistencia fueron interpretadas como portadoras de resistencia plasmídica a los determinados antibióticos. Además se determinó una prevalencia del 7.42% de total de bacterias gram negativas aisladas, presentando un mayor número de casos en los meses de verano e invierno, con una misma proporción para ambos sexos, la edad promedio de los pacientes infectados fue 62.314±19.745. A.baumannii se aisló principalmente de muestras respiratorias, seguida de hemocultivos y líquidos biológicos provenientes del servicio de UCI, Medicina Interna 1 y Cirugía General 5. Las infecciones se asociaron a pacientes con estados de inmunosupresión debido a procesos quirúrgicos y enfermedades base como cáncer, insuficiencia hepática crónica, insuficiencia renal y EPOC. La tasa de mortalidad asociada a la infección fue del 56.4%. / ---- Acinetobacter baumannii is an aerobic, Gram-negative coccobacillus and is considered an emerging nosocomial pathogen that has developed resistance to multiple drugs. The present study aimed to determine the antimicrobial resistance in Acinetobacter baumannii strains isolated from patients hospitalized in the Guillermo Almenara Irigoyen National Hospital and its relationship with the presence of plasmids, and determine the prevalence of this species in that hospital according to the inpatient service , sample type, age, sex and comorbidity, mortality and seasonality factors. 40 A. baumannii strains of clinical origin, isolated from samples of hospitalized patients at the Guillermo Almenara Irigoyen hospital during January and December 2012, were identified. Strains were identified by the MicroScan Automated System and by the conventional method. MacConkey Agar and Leeds Acinetobacter Agar were used, incubating at 37 and 44ºC, respectively. Gram-negative coccobacillus were observed in the Gram stain. Biochemical test including TSI, citrate and gelatin were performed, in addition to the oxidase and catalase tests. Antimicrobial susceptibility testing was performed using the Micro Scan automated system and the disk diffusion method. 100% of the strains showed resistance to third-generation cephalosporin, meropenem, imipenem, ciprofloxacin, ticar / clavulanic acid. 93.5% were resistant to cefepime and trimethoprim-sulfamethoxazole, 95% to levofloxacin, 90% to amikacin, 45% to gentamicin, 37.5% to tobramycin and 30% to tetracycline. 12 resistance antibiotypes were recorded, with more frequency in the intensive care unit service. The plasmid profile of the studied strains was determined, obtaining 31 profiles according to the number and size of the bands, which ranged from about 1,240– 55,459 bp; these were then associated with the resistance patterns and the isolation origin of the strain. Furthermore, strain curing was performed with ethidium bromide at a concentration of 300 µg/ml. Strains that lost resistance were interpreted as strains carrying plasmidic resistance to certain antibiotics. Moreover, a prevalence of 7.42% of total isolated Gram-negative bacteria was determined, presenting a larger number of cases in the summer and winter, with the same proportion for both sexes; the average age of infected patients was 62.314 ± 19.745. A. baumannii was mainly isolated from respiratory samples, followed by blood cultures and biological fluids samples from the UCI service, Internal Medicine 1 and General Surgery 5. The infections were associated to patients with immunosuppression state caused by surgical procedures and underlying diseases such as cancer, chronic liver failure, kidney failure and EPOC. The mortality rate associated with infection was 56.4%. Keywords: Acinetobacter baumannii, nosocomial infection, antimicrobial resistance plasmids, plasmids healing, hospital prevalence.

Orientador: Mara Corrêa Lelles Nogueira / Banca: Alessandra Vidotto / Banca: Fátima Pereira de Souza / Resumo: Acinetobacter baumannii é frequentemente encontrado em infecções hospitalares, ocasionando pneumonia associada à ventilação, bacteremia, infecções de trato urinário e meningite secundária. Nos últimos anos, o uso extensivo de antimicrobianos em hospitais contribuiu para o aumento e emergência de cepas resistentes, incluindo os carbapenêmicos, os principais recomendados no tratamento. Assim, os aminoglicosídeos ganham importância como opção terapêutica. A resistência a estes antimicrobianos é decorrente, principalmente, da produção de enzimas modificadoras de aminoglicosídeos (AMEs) e de enzimas 16S rRNA Metilases. No Brasil, informações sobre o perfil de suscetibilidade e genes que codificam estas enzimas são escassas. Assim, é fundamental a identificação de mecanismos de resistência e reservatórios potenciais, bem como realizar comparação de isolados para controlar a disseminação de A. baumannii no ambiente hospitalar. O presente estudo teve como objetivos avaliar e comparar o perfil de suscetibilidade aos antimicrobianos, genes de enzimas modificadoras de aminoglicosídeos (AMEs), genes de enzimas 16S rRNA Metilases e similaridade genética entre isolados de A. baumannii com importância clínica no Hospital de Base (HB) de São José do Rio Preto, SP. Além disso, estes resultados foram comparados com um estudo anterior realizado no mesmo hospital. Foram avaliados 32 isolados coletados no período entre dezembro de 2013 a maio de 2014. A identificação da espécie foi realizada por metodologias automatizadas e confirmada por Duplex-PCR. Os testes de suscetibilidade aos antimicrobianos foram resultantes de teste automatizado de microdiluição e disco-difusão, para aminoglicosídeos Amicacina (AK), Gentamicina (CN), Tobramicina (TOB) e Canamicina (CAN), de acordo com o CLSI, 2014 e FDA, 2013. Primers específicos e PCRmultiplex foram usados para a detecção dos genes... / Abstract: Acinetobacter baumannii is found in nosocomial infections, causing ventilator-associated pneumonia, bacteremia, urinary tract infections and secondary meningitis. In recent years, the extensive use of antibiotics in hospitals has contributed to the rise and emergence of A. baumannii strains resistant to a wide range of antimicrobials, including carbapenems, main antibiotics recommended on treatment. In this context, aminoglycosides gain importance as therapeutic options. Resistance to aminoglycosides is mainly due to the Aminoglycoside Modifying Enzymes (AMEs) and 16S rRNA Methylases production. In Brazil, high infection rates to carbapenem-resistant A. baumannii are observated, however information about aminoglycoside susceptibility profile and occurance of this genes are scarce. Thus, it is essential to identify resistance mechanisms and potential reservoirs, as well as perform comparison of isolates to control the spread of A. baumannii in the hospital environment. This study aimed to evaluate and compare the antimicrobial susceptibility profile, genes of aminoglycoside modifying enzymes and 16S rRNA Methylases and genetic similarity among A. baumannii isolates with clinical importance at Hospital de Base (HB) in São José do Rio Preto, SP. Additionally, the results were compared to another previous study achieved in the same hospital. 32 isolates collected between December 2013 and May 2014 were evaluated. Specie identification was performed using automated methodology and confirmed by Duplex- PCR. The susceptibility tests were the result of automated and disc-diffusion tests - for aminoglycosides Amikacin (AK), Gentamicin (CN), Tobramycin (TOB) and Kanamycin (K) - according to CLSI, 2014 and FDA, 2013. Specific primers and Multiplex-PCR were used in AMEs' and 16S rRNA Methylases' genes detection and primers REP1-REP2 were used in molecular typing by REP-PCR. All the isolates of this study... / Mestre

Submitted by Erika Demachki (erikademachki@gmail.com) on 2014-10-20T17:09:26Z No. of bitstreams: 2 Dissertação - Cássia Silva de Miranda Godoy - 2012.pdf: 2252636 bytes, checksum: cdb569738a5d271a5bc6ac26e7d4dc4e (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Jaqueline Silva (jtas29@gmail.com) on 2014-10-20T17:39:39Z (GMT) No. of bitstreams: 2 Dissertação - Cássia Silva de Miranda Godoy - 2012.pdf: 2252636 bytes, checksum: cdb569738a5d271a5bc6ac26e7d4dc4e (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2014-10-20T17:39:39Z (GMT). No. of bitstreams: 2 Dissertação - Cássia Silva de Miranda Godoy - 2012.pdf: 2252636 bytes, checksum: cdb569738a5d271a5bc6ac26e7d4dc4e (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2012-03-23 / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / Acinetobacter baumannii (Ab), has an important role in healthcare-associated infections, present a rapid global and emerging multidrug-resistant (MDR) strains, affecting many countries. In Brazil, Ab is responsible for outbreaks infections in intensive care units (ICUs) since 1996, with high rates of antimicrobial resistance. This was a descriptive cohort study of adult infected with Ab during the period of June to December of 2010, that evaluated the clinical and epidemiological profile of infections caused by Ab and analyzed genetically, by pulsed field gel electrophoresis (PFGE), clinical isolates from patients admitted in five ICUs of the Municipality of Goiania. We identified 64 cases of patient infected or colonized with Ab during the study period, 84 samples culture positive for Ab with a global infection rate of 4.8%. Infection incidence at each hospital was as follows: 10 % in ICU-1, 4.3% in ICU-2, 7.8% in ICU-3, 1.3% in ICU-4, and 7.5 % in ICU-5. The mean age of patients was 53.2 years (sd=19) and 59.4% (38) were male. Symptomatic infections occurred in 90.6% of the cases. The most frequent site of infection was pulmonary (53.1%), followed by surgical site (10.9%), and urinary tract (7.8%). The most common underlying diseases were neoplasia (34.4%) and AIDS (17.2%). Most of the patients infected with Ab (98.4%) had received antimicrobial therapy previously. The most frequently used drugs were cephalosporins (71.4%), carbapenems (50.8%), glycopeptides (46.0%), and fluoroquinolones (33.3%). Among the invasive procedures realized prior to Ab infection, intravascular catheters and vesical catheters where the most frequent (93.7%). The culture results from the isolate of each patient revealed that carbapenems resistance was 73.4%, ampicillin-sulbactam 60.9%, amikacin 20.3%, polymyxins 6.2%, tigecycline 3.1%. The overall mortality rate was 79.7% and related mortality rate to Ab infection was 67.2%. PFGE analysis of the isolates from 56 patients demonstrated the dissemination of genetically related clones within the ICU and between the different ICUs, and the identification of a major outbreak of MDR Ab in four out of the five analyzed ICUs involving 12 patients. In conclusion a high rate of antimicrobial resistance was detected as well as a high mortality rate among ICU patients infected with Ab, requiring stronger and more efficient measures to control this agent, as well as urgent measures are needed for the rational utilization of antibiotics in ICUs. / O Acinetobacter baumannii (Ab) tem importante papel nas infecções relacionadas à assistência à saúde e apresenta emergência rápida e global de cepas multidrogarresistentes (MDR), atingindo vários países. No Brasil, o Ab é responsável por surtos de infecções em unidades de terapia intensiva (UTIs) desde 1996, com elevadas taxas de resistência aos antimicrobianos. Esse estudo foi delineado como uma coorte descritiva de pacientes adultos infectados por Ab no período de junho a dezembro de 2010. Avaliou o perfil clínico e epidemiológico das infecções causadas por Ab e analisou geneticamente, por eletroforese em gel de campo pulsado (PFGE), os isolados clínicos destes pacientes em cinco UTIs de Goiânia e Aparecida de Goiânia. Foram identificados 64 casos de pacientes infectados ou colonizados por Ab no período do estudo, 84 amostras de cultura positivas para Ab e taxa global de infecção de 4,8 %. A frequência de infecções por UTIs foi de: 10% na UTI-1; 4,3% na UTI-2; 7,8% na UTI-3 e 1,3% na UTI-4 e 7,5% na UTI-5. A média de idade dos pacientes foi de 53,2 anos (dp=19) sendo 59,4% (38) do sexo masculino. Infecção sintomática ocorreu em 90,6% dos casos. O sítio de infecção mais frequente foi o pulmonar com 53,1%, seguido de infecção do sítio cirúrgico 10,9% e trato urinário 7,8%. As doenças de base mais comuns foram neoplasia (34,4%) e AIDS (17,2%). Dos pacientes com infecção pelo Ab, 98,4% receberam antibacteriano prévio. Os antibacterianos mais usados foram: cefalosporinas 71,4% (45); carbapenêmicos 50,8% (32), glicopeptídeos 46,0% (29) e fluorquinolonas 33,3% (21). Dos procedimentos invasivos realizados previamente à infecção pelo Ab, cateter vascular central e sondagem vesical de demora (93,7%) foram os mais comuns. A letalidade global foi de 79,7% (51/64), e a relacionada às infecções pelo Ab foi de 67,2% (39/58). Avaliando os resultados de cultura positiva, a resistência aos carbapenêmicos foi de 73,4%, à ampicilina/sulbactam de 60,9% (39/64), amicacina de 20,3% (13/64), polimixinas de 6,2% (4/64) e tigeciclina de 3,1% (2/64). A análise por PFGE das isolados bacterianos dos pacientes (56), demonstrou a disseminação clonal de Ab dentro das UTIs, e entre as diferentes UTIs, com identificação de um surto por Ab MDR entre quatro das cinco UTIs investigadas no período do estudo, envolvendo 12 pacientes. Constatamos alto índice de Ab MDR e alta letalidade nas UTIs avaliadas, com necessidade de intensificar o controle deste agente, além de racionalização do uso de antimicrobianos nas UTIs.

Acinetobacter baumannii é um patógeno oportunista, geralmente resistente a muitos antimicrobianos, que causa surtos de infecções hospitalares. Assim, a sua habilidade de formar biofilme pode explicar a capacidade de sobreviver no ambiente hospitalar e em utensílios médicos. O objetivo desse estudo foi avaliar a capacidade de duas cepas clínicas de A. baumannii obtido de hospitais de Porto Alegre, Brasil (abC e abH) e uma cepa controle ATCC 19606 (abA) formar biofilme e realizar a análise transcricional dos genes possivelmente envolvidos na produção e manutenção do biofilme: bap, abaI, ompA, bfmRS, csuAB, pgaA, pilZ, wspR, eal, eagg, IscRSU e csdA. A capacidade de formação de biofilme foi avaliada pelo método cristal violeta em superfície plástica a 25°C e 37°C nos meios LB, LB + 1% glicose, urina pura e LB + 10% sangue de carneiro. A análise transcricional foi feita por real time PCR. A cepas AbH, AbC e AbA foram fortes formadoras de biofileme em LB e LB+glicose à 25 e 37°C. Em LB+sangue, as cepas AbH e AbA foram fortes formadoras e AbC foi fraca formadora de biofilme. Em urina, a cepa AbH foi moderadamente formadora, AbC e AbA foram fracas formadoras de biofilme. Os genes wspR, pgaA, ompA, bap, abaI de AbA, csdA de AbH e o operon IscRSU foram superexpressos em biofilme; os genes eagg de AbH, pilZ e csuAB foram inibidos e os genes bfmRS, eal, eagg de AbA, csdA de AbA e abaI de AbH não possuíram uma variação significativa na expressão durante o biofilme. Portantanto, a regulação positiva dos genes wspR, pgaA, ompA, bap, csdA e do operon IscRSU é um indício de sua importância na formação e manutenção do biofilme por esse patógeno. / Acinetobacter baumannii is an opportunistic pathogen which causes a wide range of nosocomial infections, being usually multirresistent to drugs. Their ability to form biofilm can explain the feature of surviving inside hospital environment and on medical devices. The aim of the present study was to evaluate the ability of two clinically isolated MDR A. baumannii strain, obtained from a general hospital of Porto Alegre, RS, Brazil (AbH and AbC), and the reference ATCC 19606 strain (AbA), to form biofilm and to analyze the relative expression of genes putatively involved in biofilm formation: bap, abaI, ompA, bfmRS, csuAB, pgaA, pilZ, wspR, eal, eagg, IscRSU e csdA. The biofilm formation capability was evaluated by the crystal-violet staining method on plastic surfaces at 25°C and 37°C using the broth LB, LB + 1% glucose, pure urine and LB + 10% sheep blood. The trancritional profiling of the genes was analyzed through real time PCR methodologies. The strains AbH, AbC e AbA were strong biofilm producers in LB e LB+glucose at 25 and 37°C. In the broth LB+blood, the strains AbH and AbA were strong biofilm producers and AbC was week biofilm producer. In urine, the strain AbH was moderated producer, AbC and AbA were week biofilm producers. The genes wspR, pgaA, ompA, bap, abaI from AbA, csdA from AbH and the operon IscRSU were overexpressed in biofilm; the genes eagg de AbH, pilZ e csuAB were suppressed and the genes bfmRS, eal, eagg from AbA, csdA from AbA e abaI from AbH did not vary their expression significantly during the biofilm condition. In conclusion, the factors wspR, pgaA, ompA, bap, csdA and of the operon IscRSU, that were positively regulated, appear to have an important role during the process of biofilm formation by A. baumannii.

A.baumannii é um importante patógeno em infecções nosocomiais principalmente por sua capacidade de se tornar resistente aos antimicrobianos. Surtos de A.baumannii resistente aos carbapenêmicos (ABRC) têm sido descritos em todo mundo. Devido à emergência de resistência aos antimicrobianos e ausência de novas opções de tratamento, as polimixinas reemergiram como opção de terapia contra infecções causadas por A.baumannii. O uso de polimixina é associado a maior mortalidade e menor eficácia comparada a outros antimicrobianos. Alguns estudos in vitro têm avaliado a combinação de polimixina com outros antimicrobianos a fim de aumentar a eficácia dos tratamentos. O objetivo deste estudo foi avaliar o sinergismo entre a polimixina B com outros antimicrobianos em isolados de ABRC, pelo método de Curvas Tempo-Morte bacteriana (Time- Kill Curves). Os isolados foram provenientes de banco de amostras e foram avaliadas as combinações de polimixina B com carbapenêmicos (imipenem e meropenem), tigeciclina, rifampicina, amicacina e ceftazidima. As combinações foram testadas nos tempo 0, 30’, 1,4,12 e 24 h. Sinergismo entre polimixina B foi demonstrado contra todos antimicrobianos para ambos isolados, exceto para ceftazidima e imipenem no isolado 1. Nosso estudo mostrou que tigeciclina, amicacina e rifampicina são agentes mais ativos combinados com polimixina B, sendo assim estes agentes podem apresentar efeito benéfico em combinação com a polimixina no tratamento de ABRC. / A.baumannii is an important pathogen in nosocomial infections primarily for its ability to become resistant to antimicrobials. Outbreaks carbapenem- resistant A.baumannii (CRAB) has been described worldwide. Due to the emergence of antimicrobial resistance and the absence of new treatment options, the polymyxins reemerged as an option therapy against infections caused by A.baumannii. The use of polymyxin is associated with higher mortality and lower effectiveness compared to other antimicrobials. In vitro studies have evaluated the combination of polymyxin with other antimicrobial agents to enhance the effectiveness of the treatments. This study was to evaluate the synergy between polymyxin B with other antimicrobials in isolates from ABRC, by Time-Kill Curves. The isolates were from stool samples and were evaluated combinations of polymyxin B with carbapenems (imipenem and meropenem), tigecycline, rifampin, amikacin and ceftazidime. The combinations were tested at time 0, 30 ', 1,4,12 and 24 h. Synergism between polymyxin B was demonstrated against all antimicrobials for both isolates, except for ceftazidime and imipenem in isolated 2. Our study showed that tigecycline, amikacin and rifampicin more active agents are combined with polymyxin B, and thus these agents may have a beneficial effect in combination with a polymyxin in treating CRAB.