Orientador: Noeme Sousa Rocha / Resumo: As doenças de transmissão alimentar (DTA) por bactérias constituem um problema de saúde pública no mundo. O objetivo do trabalho foi identificar por métodos microbiológicos e moleculares a presença de Campylobacter spp., Salmonella spp. e Escherichia coli em frangos de Botucatu-SP, Brasil. Sessenta (60) amostras de frangos resfriados de diferentes marcas foram coletadas aleatoriamente em diferentes estabelecimentos localizados tanto na periferia, quanto no centro da cidade de Botucatu, SP, no período de julho a outubro de 2015. Posteriormente, Campylobacter spp, Salmonella spp e E. coli foram isolados através de métodos microbiológicos convencionais. A confirmação das bactérias isoladas se realizou por provas bioquímicas e PCR. Além disso, as provas para determinar a resistência antimicrobiana e a existência de gene integron classe 1 foram realizadas. Os resultados demostraram a presença de três das principais bactérias que causam doenças de origem alimentar em carcaças de frango provenientes de supermercados e casas de carne de Botucatu. A prevalência de Campylobacter, Salmonella, e E. coli foi 38,3%, 13,3% e 60%, respectivamente. A resistência antimicrobiana dos isolados foi elevada e presença do gene íntegron classe 1 foi identificada em alguns agentes patogénicos. / Abstract: The transmission of Foodborne Diseases (FBD) by bacteria constitutes a public health problem in the world. The objective of this study was to identify by microbiological and molecular methods the presence of Campylobacter spp., Salmonella spp. and Escherichia coli in broiler chickens at Botucatu, SP, Brazil. Sixty samples of chilled chickens of different brands were randomly collected from supermarkets and meat houses located in both the periphery and the center of the city, in the period from July to October 2015. Later, Campylobacter spp., Salmonella spp., and E. coli were isolated by conventional microbiological methods. Confirmation of the isolated bacteria was performed by biochemical and PCR tests. In addition, proofs to determine antimicrobial resistance and the existence of class 1 integron gene were carried out. Results confirmed the presence of the three assessed bacteria in poultry carcasses from selected establishments. The prevalence was 38.3%, 13.3% and 60% to Campylobacter, Salmonella and E. coli, respectively. Antimicrobial resistance of the isolates was high and the presence of integron class 1 gene has been identified in some pathogens. / Doutor

Orientador: Raphael Lucio Andreatti Filho / Banca: Edna Teresa de Lima / Banca: Noeme Sousa Rocha / Banca: Julio Lopes Sequeira / Banca: Marcelo Vasconcelos Meireles / Resumo: Neste trabalho foram analisadas 100 cepas de Salmonella Enteritidis (SE) isoladas de material avícola, visando à detecção dos genes de virulência spvC e invA e resistência a antimicrobianos integron classe 1. Comparando-se com a possível expressão dos fatores de virulência para sobrevivência em condições impróprias de temperatura, pH e concentração de nutrientes e o teste de inibição em placa, respectivamente. Também a capacidade de transferência horizontal do gene integron classe 1 foi avaliada em SE. Das cepas analisadas, duas apresentaram os genes spvC e invA, simultaneamente, com uma provável expressão destes sendo verificada no crescimento com pH 10,0 ou temperatura de 25ºC. Porém em relação à concentração de nutriente, ambas as cepas não cresceram na menor concentração (0,5%). Não houve relação direta entre a presença do gene integron classe 1 com a multiresistência de SE aos 14 antimicrobianos testados, já que 80% das cepas pesquisadas foram resistentes a até três antimicrobianos e não apresentaram o referido gene. Entretanto, a transferência horizontal desse gene e da resistência antimicrobiana foi realizada in vitro, de um Escherichia coli para uma Salmonella Enteritidis, demonstrando a capacidade de disseminação do gene presente em integron classe 1. / Abstract: In this work, 100 strains of Salmonella Enteritidis (SE) isolated from avian material were studied, aiming to detect the genes of virulence spvC and invA, as well as the antimicrobial resistance type 1 integron genes using the polymerase chain reaction (PCR), comparing it with the possible expression of the virulence factors to survive under inappropriate conditions of temperature, pH and nutrient concentration, and the plaque inhibition assay, respectively. Capacity of horizontal transfer of type 1 0,integron gene was also evaluated in SE. Two of the analyzed strains showed spvC and InvA genes simultaneously, with a probable expression verified through growing in pH 10.0 or 25°C temperature. However, regarding to nutrient concentration, the aforementioned strains did not grow at the lowest concentration (0.5%). There was no direct relation between type 1 integron gene and the multiresistance of SE to the 14 tested antibiotics, because 80% of the strains did not showed the gen, but was resistant till three antibiotics. However, horizontal transfer of this gene was performed in vitro, showing the capacity of dissemination of the type 1 integron gene between bacteria. / Doutor

Les intégrons de multirésistance sont des plateformes génétiques permettant aux bactéries de s’adapter à des pressions antibiotiques . Ils leur permettent de capturer et d’exprimer de s gènes de résistance sous forme de cassettes. La capture et le réarrangement des cassettes sont réalisés par une intégrase dont l’expression est régulée par la réponse SOS chez E. coli . L’expression des cassettes est quant à elle assurée par le promoteur Pc. Les travaux présentés dans ce manuscrit visent à préciser deux aspects liés à la dynamique d’évolution des intégrons . L’étude du coût biologique des intégrons a montré que ceux - ci sont des structures génétiques très peu coûteuses pour E. coli . Ce faible coût est notamment lié à la répression du gène de l’intégrase par la réponse SOS. Dans ces conditions de répression, le coût d’un intégron dépend de l’expression du réseau de cassettes et de son contenu. Ainsi ce coût augmente avec la force du promoteur Pc et le nombre de cassettes dans le réseau. D’autre part, le coût lié à la nature des cassettes est variable. L’étude de la dynamique d’évolution du promoteur Pc visait à vérifier l’hypothèse selon laquelle des pressions antibiotiques auraient conduit à l’émergence de promoteurs forts à partir d’un variant ancestral faible. L’évolution d’une souche de E. coli , contenant un intégron plasmidique portant un variant faible d e Pc, a été réalisée en chemostat sur 200 générations . L’analyse des populations évoluées par deep - sequencing n’a pas permis de mettre en évidence l’émergence de variants forts de Pc . Néanmoins, l ’ étude de ce s populations évoluées révèle une part majoritaire d’évolution chromosomique. Dans ces conditions, l’ absence d’évolution du Pc pourrait atteste r soit d’une réalité biologique ou soit d’un protocole expérimental d’évolution à optimiser . / Resistance integrons are genetic platforms able to catch and express resistance genes embedded within gene cassettes. Capture and reshuffling of gene cassettes are mediated by the integrase whose expression is regulated by the SOS response in E. coli. Gene cassettes are then expressed from the Pc promoter.This work aims to clarify the evolution dynamic of integrons.In a first part, the fitness cost of class 1 integron was assessed in E. coli. Results reveal that integrons are low cost structures and that their cost is reduced by the SOS-mediated repression system. While repressed, the cost of an integron mostly depends on cassettes array expression. The cost of an integron therefore increases with Pc strength and the number of cassettes in the array. Furthermore, different cassettes exhibit different costs.In a second part, the evolution dynamic of Pc promoter was assessed in response to antibiotic pressures. An E. coli strain, carrying a plasmidic integron with a weak Pc promoter, was propagated in chemostat for 200 generations. The deep-sequencing of evolved populations did not reveal any mutations in the promoter region. On the other hand, evolved bacteria presented evidence of chromosomal adaptation. In these conditions, the lack of evolution within the Pc region could reveal either a biological reality or an experimental protocol to optimize.

Les intégrons sont des plateformes génétiques bactériennes de capture et d'expression de gènes. Les intégrons cliniques sont les principaux responsables de la forte augmentation récente des bactéries multirésistantes aux antibiotiques. Cependant, dans l’environnement, ils ne représentent qu’une part minime de l’importante diversité des intégrons. Ainsi, les objectifs de cette thèse étaient (i) d’évaluer l’étendue de la diversité des intégrons dans l’environnement, (ii) de comprendre les phénomènes responsables de la structuration du pool d’intégrons dans le compartiment sédimentaire d’un milieu estuarien anthropisé et (iii) de rechercher l’existence potentielle d’intégrons indicateurs du niveau de contamination chimique.Le suivi des intégrons de classes 1, 2 et 3 et des populations d’E. coli dans des sédiments du bassin versant de la Risle impactés par des sources de contamination fécale bien caractérisées ont montré qu’en étiage, les souches d’E. coli d’origine humaine se disséminaient sur de courtes distances. Les intégrons de classe 1 se disséminent sur des distances un peu plus importantes et se maintiennent dans les 12 premiers centimètres du compartiment sédimentaire au moins.Une méthode a été développée permettant, pour la première fois, l’analyse de la diversité des intégrons via séquençage haut-débit. L’application de cette méthode sur une carotte sédimentaire de 4,8 m de profondeur prélevée dans l’estuaire fluvial de la Seine a permis de mettre en évidence plusieurs milliers de classes d’intégrons dont de nombreuses intégrases encore jamais répertoriées. La diversité des intégrons chute fortement avec la profondeur. Les intégrons de classe 1, majoritaires dans les sédiments de surface, ont une abondance qui chute fortement avec la profondeur cependant ils répondent plutôt positivement à la contamination chimique renforçant l’idée de leur utilisation comme proxy de pollutions anthropiques récentes. Trois classes d’intégrons dominent dans la vase consolidée représentant 38% des séquences obtenues dans la carotte sédimentaire mais répondant plutôt négativement à la contamination chimique. Enfin, la structure du pool d’intégrons est fortement corrélée à celle de la communauté bactérienne mais semble en partie indépendante de la communauté bactérienne dans deux des fractions sédimentaires profondes avec la dominance d’une nouvelle classe d’intégrons qui semble sélectionnée par les HAP. / Integrons are bacterial genetic platforms allowing acquisition and expression of genes. Clinical integrons play a major role in the strong increase of antibiotic multi-resistant bacteria recently observed. However, in the environment, they represent only a tiny fraction of the large integron diversity. Therefore, the aims of this thesis were (i) estimating the extent of the integron diversity in the environment, (ii) understanding phenomena responsible for integron pool structure in anthropized estuarine sediments and (iii) looking for integrons potentially proxy of chemical pollution level. The research of class 1, 2 and 3 integrons and the analysis of E. coli populations in sediments from the Risle drainage basin impacted by well-characterized fecal contamination sources show that E. coli strains with human origins were spread on short distances during low water level periods. However, class 1 integrons are spread on slightly longer distances and remain present in the 12 first centimeters of sediments at least. A methodology was developed allowing, for the first time, the analysis of integron diversity by high-throughput sequencing. In this way, the analysis of a 4.8 meter core sediment from the fluvial Seine estuary highlighted several thousands integron classes including many new integrases absent from data bases. Integron diversity decreases along with depth. Class 1 integrons are the majority integrons in surface sediments but their abundance strongly decreases in deep sediments. Class 1 integron abundance rather responds positively to chemical pollutions accentuating the idea that class 1 integrons could be used as proxy of recent anthropogenic pollutions. In the sediment core, three integron classes outshine the whole dataset: they represent 38% of all the sequences from the sediment core. However, abundances of these three majority integron classes rather respond negatively to chemical pollution levels. Integron pool structure is highly correlated to bacterial community diversity but seems to be partially independent to bacterial community diversity within two deep fractions from the sediment core: in these two sediment fractions, a new integron class outshines the rest of integron classes and seems to be specific to these two sediment fractions. Furthermore, this new integron class seems to be selected by PAH.

The predominant tool for adaptation in Gram-negative bacteria is a genetic system called integron. It rearranges gene cassettes, promoting multiple antibiotic resistances, a recognized major global health threat. It is based on a unique recombination process involving a Tyrosine recombinase – called integrase IntI – and folded single-stranded DNA hairpins – called attC sites. Four recombinases and two attC sites form a macromolecular synaptic complex, which is key to the entire recombination process and the focus of our study. The bottom strand of all attC sites shows highest recombination in vivo, however, it still varies greatly and the underlying reason is unknown. We hypothesize that the difference in recombination efficiency arises from the variable mechanical stability of the synaptic complex, which in turn is affected by the attC site. Here, we established an optical tweezers force-spectroscopy assay that allows us to probe the synaptic complex stability for different DNA substrates and protein variants. We discovered a strong correlation between recombination efficiency and the mechanical stability of the synapse, indicating a regulatory mechanism from the DNA sequence to the quaternary complex structure stability. We have discovered protein residues interacting with the DNA in trans, within the synaptic complex, which reduces its stability. Furthermore, we discovered that the C-terminal helix, a conserved structural feature of tyrosine recombinases plays a key role in the stabilization of the tetramer assembly on the DNA, which upon mutation significantly destabilized the synaptic complex. Expanding upon this new understanding of synapse stability regulation we developed a novel approach for destabilizing the synaptic complex, potentially reducing the recombination efficiency. We designed α-helix mimicking peptides that would compete with the C-terminal tail of the integrase, block the interlocking interaction, and lead to synaptic complex destabilization. We have observed a prominent destabilizing effect on the synaptic complex already at 10 µM peptide concentration. Overall, our findings reveal new regulatory mechanisms in the recombination efficiency of the bacterial integron and provide first data for the active synapse destabilization mechanism. This novel understanding of the regulatory role the synaptic complex plays in the recombination efficiency of the integron system introduces a new approach to reduce the spread of antibiotic resistance among bacteria. / Das vorherrschende Anpassungsmittel bei gramnegativen Bakterien ist ein genetisches System, das Integron genannt wird. Es ordnet Genkassetten neu an und fördert so multiple Antibiotikaresistenzen, die eine globale Gesundheitsbedrohung darstellen. Es basiert auf einem einzigartigen Rekombinationsprozess, an dem eine Tyrosin-Rekombinase - Integrase IntI genannt - und gefaltete einzelsträngige DNA-Hairpins - attC-Stellen genannt - beteiligt sind. Vier Rekombinasen und zwei attC-Stellen bilden einen makromolekularen synaptischen Komplex, der für den gesamten Rekombinationsprozess entscheidend ist und im Mittelpunkt unserer Forschung steht. Der untere Strang aller attC-Stellen weist in vivo die höchste Rekombinationsrate auf, die jedoch aus unbekannten Grund stark variier. Wir vermuten, dass der Unterschied in der Rekombinationsrate auf die unterschiedliche mechanische Stabilität des synaptischen Komplexes zurückzuführen ist, die wiederum von der attC-Stelle beeinflusst wird. Hier haben wir einen Test mittels Kraft-spektroskopie mit einer optischen Pinzette entwickelt, mit dem wir die Stabilität des synaptischen Komplexes für verschiedene DNA-Substrate und Proteinvarianten untersuchen können. Wir stellten eine starke Korrelation zwischen der Rekombinationsrate und der mechanischen Stabilität der Synapse fest, was auf einen Regulationsmechanismus zwischen der DNA-Sequenz und der Stabilität der quaternären Komplexstruktur hinweist. Wir haben Proteinreste entdeckt, die innerhalb des synaptischen Komplexes mit der DNA in trans interagieren, was zu einer Verringerung dessen Stabilität führt. Darüber hinaus stellten wir fest, dass die C-terminale Helix, ein konserviertes Strukturmerkmal von Tyrosin-Rekombinasen, eine Schlüsselrolle bei der Stabilisierung des Tetramer-Aufbaus an der DNA spielt, die bei Mutation den synaptischen Komplex erheblich destabilisiert. Auf der Grundlage dieses neuen Verständnisses der Regulierung der Synapsenstabilität haben wir einen neuen Ansatz zur Destabilisierung des synaptischen Komplexes entwickelt, der die Effizienz der Rekombination verringern könnte. Wir entwarfen α-Helix-nachahmende Peptide, die mit dem C-terminalen Ende der Integrase konkurrieren, die Interlocking-Interaktion blockieren und zur Destabilisierung des synaptischen Komplexes führen. Wir haben eine deutliche destabilisierende Wirkung auf den synaptischen Komplex bereits bei einer Peptidkonzentration von 10 µM beobachtet. Insgesamt zeigen unsere Ergebnisse neue Regulationsmechanismen für die Rekombinationsleistung des bakteriellen Integrons auf und liefern erste Daten für den Mechanismus der aktiven Destabilisierung der Synapse. Dieses neue Verständnis der regulatorischen Rolle, die der synaptische Komplex bei der Rekombinationseffizienz des Integronsystems spielt, eröffnet einen neuen Ansatz zur Verringerung der Verbreitung von Antibiotikaresistenzen unter Bakterien.

P. aeruginosa e espécies de Acinetobacter são causas comuns de diversas infecções em pacientes hospitalizados, principalmente nos internados em centros de tratamento intensivo. Além disso, esses microrganismos se destacam por apresentarem resistência, intrínseca e adquirida, a várias classes de antibióticos, conferindo à bactéria fenótipos de multirresistência e panresistência. O objetivo deste estudo foi avaliar a participação de integrons (elementos genéticos que carreiam genes de resistência), de genes codificadores de metalo--lactamases, da perda de porinas (canais protéicos da membrana externa), e da atividade de efluxo aumentada, como determinantes do fenótipo de multirresistência e panresistência. Foram estudadas 147 P. aeruginosa e 57 Acinetobacter spp. isolados de pacientes hospitalizados no Hospital Universitário da Universidade Federal de Juiz de Fora, no período de 2003 a 2006. O perfil de sensibilidade destes isolados foi determinado por disco de difusão e utilizado para classificá-las como multirresistentes (MDR) e não multirresistentes (n-MDR). A variabilidade clonal dos isolados foi investigada por PFGE. Os isolados pertencentes aos grupos MDR e n-MDR foram investigados quanto a presença de integrons de classe 1, 2 e 3, por PCR e análise de RFLP. Os cassetes gênicos contidos nestes integrons, assim como genes codificadores de carbapenemases (ex. IMP, VIM e SPM), foram detectados por PCR e identificados por seqüenciamento. Avaliação da expressão gênica de bombas de efluxo (mexB, mexY, mexD e adeB) e de porina (OprD) foi conduzida por real-time RT-PCR. Os dados apresentados para os isolados do grupo MDR foram comparados àqueles do grupo n-MDR e a associação entre os determinantes de resistência e o fenótipo MDR foi calculada estatisticamente. Fenótipo de multiresistência foi observado em 42,2% e 84,2% das P. aeruginosa e Acinetobacter spp. estudadas. Nenhum isolado bacteriano apresentou fenótipo panresistente. Em 65 (44,2%) dos isolados de P. aeruginosa, foram detectados integrons de classe 1. Esses elementos apresentaram relação estatisticamente significativa com fenótipos MDR em P. aeruginosa. Entretanto, a maioria desses integrons não carreava nenhum cassete gênico (43/65) ou continham apenas cassetes gênicos de resistência a aminoglicosídeos (19/65). Entre os isolados de Acinetobacter spp., 11 (17,5%) apresentaram integrons de classe 1 e 30 (47,6%) integrons de classe 2. Apenas os últimos foram estatisticamente associados com fenótipos MDR. A pesquisa de metalo--lactamase (MBL) revelou a produção de enzimas SPM em 24 isolados de P. aeruginosa. Os estudos de expresão gênica demonstraram que, entre os sistemas de efluxo mais relatados para P. aeruginosa, MexXY-OprM foi o que mostrou maior diferença entre o nível de expressão dos grupos MDR e n-MDR, sugerindo que este sistema de efluxo desempenha importante papel no fenótipo MDR. Diminuição, em média de 66,4%, da produçãode OprD também foi um padrão encontrado nos isolados MDRem relação aos n-MDR. Dois grupos clonais de P. aeruginosa e dois de Acinetobacter spp. foram predominantes e tiveram relação com presença de integrons, produção de SPM-1 e com fenótipo MDR. Portanto, esse fenótipo pode ser consequência de acúmulo de determinantes de resistência em clones específicos. / The non-fermenting pathogenic bacteria Pseudomonas aeruginosa and Acinetobacter spp. are important causes of nosocomial infections. Theses species are often associated with a multidrug resistance (MDR) phenotype, due to intrinsic and acquired resistance genes. Some determinants of resistance, such as integrons, carbapenemases, overexpression of efflux systems and porins loss may be associated with the MDR phenotype. The aim of this study was to evaluate the association of non-MDR and MDR phenotypes in P. aeruginosa and Acinetobacter spp. to the presence of integrons and carbapenemases encoding genes, the overexpression of mexY, mexB, mexD and adeB genes and loss of the outer membrane protein, OprD. These resistance determinants were evaluated in 147 P. aeruginosa and 57 Acinetobacter spp., isolated from in-patients of University Hospital of UFJF. Isolates with different PFGE and non-susceptibility profiles were grouped according to MDR or non-MDR phenotypes. PCR and real-time RT-PCR were used to investigate the presence of class 1, 2 and 3 integrons and carbapenemase encoding genes and the expression of mexY, mexB, mexD and adeB efflux pumps and OprD porin, respectively. Class 1 integrons were one of the most common genetic elements present in MDR P. aeruginosa (44,2%), but the phenotype could not be attributed to these elements, since they showed empty (43/65) or only aminoglycoside gene cassettes (19/65). Class 2 integrons were the most common genetic elements in MDR Acinetobacter spp., and this association was statistically significant. SPM encoding gene was the only carbapenemase gene found in P. aeruginosa and, predominantly, in the PFGE cluster A. Expression of MexXY-OprM determined by real-time RT-PCR was the highest variable between MDR and non-MDR P. aeruginosa isolates (almost 10-fold). Reduction of 66.4% in OprD expression was observed in MDR P. aeruginosa, in comparison with non-MDR ones. It is concluded that the most important genetic determinants in the MDR phenotype of P. aeruginosa were SPM-1 production, followed by MexXY-OprM over expression and diminished production of OprD, while class 2 integrons was the most important genetic determinant of MDR phenotype in A. baumannii.

P. aeruginosa e espécies de Acinetobacter são causas comuns de diversas infecções em pacientes hospitalizados, principalmente nos internados em centros de tratamento intensivo. Além disso, esses microrganismos se destacam por apresentarem resistência, intrínseca e adquirida, a várias classes de antibióticos, conferindo à bactéria fenótipos de multirresistência e panresistência. O objetivo deste estudo foi avaliar a participação de integrons (elementos genéticos que carreiam genes de resistência), de genes codificadores de metalo--lactamases, da perda de porinas (canais protéicos da membrana externa), e da atividade de efluxo aumentada, como determinantes do fenótipo de multirresistência e panresistência. Foram estudadas 147 P. aeruginosa e 57 Acinetobacter spp. isolados de pacientes hospitalizados no Hospital Universitário da Universidade Federal de Juiz de Fora, no período de 2003 a 2006. O perfil de sensibilidade destes isolados foi determinado por disco de difusão e utilizado para classificá-las como multirresistentes (MDR) e não multirresistentes (n-MDR). A variabilidade clonal dos isolados foi investigada por PFGE. Os isolados pertencentes aos grupos MDR e n-MDR foram investigados quanto a presença de integrons de classe 1, 2 e 3, por PCR e análise de RFLP. Os cassetes gênicos contidos nestes integrons, assim como genes codificadores de carbapenemases (ex. IMP, VIM e SPM), foram detectados por PCR e identificados por seqüenciamento. Avaliação da expressão gênica de bombas de efluxo (mexB, mexY, mexD e adeB) e de porina (OprD) foi conduzida por real-time RT-PCR. Os dados apresentados para os isolados do grupo MDR foram comparados àqueles do grupo n-MDR e a associação entre os determinantes de resistência e o fenótipo MDR foi calculada estatisticamente. Fenótipo de multiresistência foi observado em 42,2% e 84,2% das P. aeruginosa e Acinetobacter spp. estudadas. Nenhum isolado bacteriano apresentou fenótipo panresistente. Em 65 (44,2%) dos isolados de P. aeruginosa, foram detectados integrons de classe 1. Esses elementos apresentaram relação estatisticamente significativa com fenótipos MDR em P. aeruginosa. Entretanto, a maioria desses integrons não carreava nenhum cassete gênico (43/65) ou continham apenas cassetes gênicos de resistência a aminoglicosídeos (19/65). Entre os isolados de Acinetobacter spp., 11 (17,5%) apresentaram integrons de classe 1 e 30 (47,6%) integrons de classe 2. Apenas os últimos foram estatisticamente associados com fenótipos MDR. A pesquisa de metalo--lactamase (MBL) revelou a produção de enzimas SPM em 24 isolados de P. aeruginosa. Os estudos de expresão gênica demonstraram que, entre os sistemas de efluxo mais relatados para P. aeruginosa, MexXY-OprM foi o que mostrou maior diferença entre o nível de expressão dos grupos MDR e n-MDR, sugerindo que este sistema de efluxo desempenha importante papel no fenótipo MDR. Diminuição, em média de 66,4%, da produçãode OprD também foi um padrão encontrado nos isolados MDRem relação aos n-MDR. Dois grupos clonais de P. aeruginosa e dois de Acinetobacter spp. foram predominantes e tiveram relação com presença de integrons, produção de SPM-1 e com fenótipo MDR. Portanto, esse fenótipo pode ser consequência de acúmulo de determinantes de resistência em clones específicos. / The non-fermenting pathogenic bacteria Pseudomonas aeruginosa and Acinetobacter spp. are important causes of nosocomial infections. Theses species are often associated with a multidrug resistance (MDR) phenotype, due to intrinsic and acquired resistance genes. Some determinants of resistance, such as integrons, carbapenemases, overexpression of efflux systems and porins loss may be associated with the MDR phenotype. The aim of this study was to evaluate the association of non-MDR and MDR phenotypes in P. aeruginosa and Acinetobacter spp. to the presence of integrons and carbapenemases encoding genes, the overexpression of mexY, mexB, mexD and adeB genes and loss of the outer membrane protein, OprD. These resistance determinants were evaluated in 147 P. aeruginosa and 57 Acinetobacter spp., isolated from in-patients of University Hospital of UFJF. Isolates with different PFGE and non-susceptibility profiles were grouped according to MDR or non-MDR phenotypes. PCR and real-time RT-PCR were used to investigate the presence of class 1, 2 and 3 integrons and carbapenemase encoding genes and the expression of mexY, mexB, mexD and adeB efflux pumps and OprD porin, respectively. Class 1 integrons were one of the most common genetic elements present in MDR P. aeruginosa (44,2%), but the phenotype could not be attributed to these elements, since they showed empty (43/65) or only aminoglycoside gene cassettes (19/65). Class 2 integrons were the most common genetic elements in MDR Acinetobacter spp., and this association was statistically significant. SPM encoding gene was the only carbapenemase gene found in P. aeruginosa and, predominantly, in the PFGE cluster A. Expression of MexXY-OprM determined by real-time RT-PCR was the highest variable between MDR and non-MDR P. aeruginosa isolates (almost 10-fold). Reduction of 66.4% in OprD expression was observed in MDR P. aeruginosa, in comparison with non-MDR ones. It is concluded that the most important genetic determinants in the MDR phenotype of P. aeruginosa were SPM-1 production, followed by MexXY-OprM over expression and diminished production of OprD, while class 2 integrons was the most important genetic determinant of MDR phenotype in A. baumannii.

QepA is a plasmid-mediated efflux pump found in some strains of Escherichia coli, in which it significantly elevates the resistance against quinolones. The protein has similarities with 14-TMS major facilitator superfamily transporters and is situated in the inner membrane of the bacteria. It was acquired by horizontal gene transfer and integrated into a now inactivated class 1 integron, also harbouring several other antibiotic resistance genes such as rmtB and blaTEM-1. QepA alone is not sufficient to raise the resistance level over the clinical breakpoint and is in clinical isolates therefore associated with other quinolone antibiotic resistance genes or quinolone target point mutations. The mechanisms regulating qepA expression are not yet understood. Therefore, in this study the qepA gene was amplified from an E. coli clinical isolate and, together with its upstream promotor sequence, was inserted into the E. coli chromosome. It was shown that qepA gene expression can be induced by exposure to 0.5-fold MIC concentrations of ciprofloxacin, trimethoprim and other DNA damaging antimicrobials. The deletion of a LexA binding site situated after a PcW promotor, which was predicted to drive qepA expression, did not alter this induction behaviour. Nested deletions of up to 200 nts downstream sequence of the PcW promotor, led to the identification of a sequence region required for expression induction. This study showed that qepA expression is induced by environmental factors leading to DNA damage and further identified a previously unknown DNA sequence required for expression regulation.

Antibiotic resistant bacteria are threatening human health, and the resistance is progressingfaster than the development of new antimicrobial compounds. Antibiotic resistant infections cost enormous sums of money and resources, but most importantly human lives. Therefore, early prediction and detection of antibiotic resistance in bacteria are research areas of high priority. The use of analytical instruments such as matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) is a powerful tool for research in antibiotic resistant bacteria, both as quick microbe identification and for other areas in the field.Due to its relatively easy interpretable spectra provided by the soft ionization technique, and ability to ionize macromolecules without compromising sample integrity, it has also been used for biomarker screening for detection of antimicrobial resistance. Although these studies have shown promising results, the area is still progressing and needs further method development and standardized protocols. This study aimed to use MALDI-TOF MS for carbapenemaseassociated screening in Klebsiella oxytoca. The presumed key spectral peaks would derive from presence of the enzyme Verona integron-encoded metallo-β-lactamase, type 1 (VIM-1), and would not be found if VIM-1 were absent. The isolates, carrying the enzyme, used in the study were isolated from wastewater and river water in Örebro, Sweden. Bacteria genus and species was determined by MALDI-TOF identification, whereupon the microbes were tested for antibiotic susceptibility using the disk diffusion method. Carbapenem hydrolysis assay was used to confirm the presence/absence of functional carbapenemase. A genotypic confirmation was performed by polymerase chain reaction and gel electrophoresis. Mass spectra from MALDI-TOF were compared for identification of possible biomarker peaks that could indicate carbapenem resistance. Nine key mass spectral peaks were found that could potentially be used as biomarkers in future studies. The peaks differentiated two groups of Klebsiella oxytocaisolates, one group producing functional carbapenemase and one group that did not, consistent with the aim of this study.

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.