Les métallo-β-lactamases sont des enzymes qui confèrent aux bactéries qui les synthétisent une résistance aux antibiotiques. La classe B représente les β-lactamases, dans lesquelles le site actif contenant un ou deux atomes de Zn favoriserait l'hydrolyse des antibiotiques. La dégradation des antibiotiques par les enzymes bactériennes est un mécanisme majeur de résistance. L’objectif de ce travail de thèse est de mettre en œuvre des outils de modélisation fondés sur des méthodes de mécanique quantique en vue de déterminer les structures de métallo-β-lactamases avec des inihibiteurs, étape nécessaire pour comprendre ultérieurement quels sont les mécanismes de réaction favorisant la dégradation d’un inhibiteur par des métallo-β-lactamases et fournir des informations qui serviront à mieux interpréter les phénomènes biologiques.Nous avons tout d’abord déterminé les géométries et la stabilité de complexes de coordinations métalliques de systèmes modèles contenant du Zn, comme dans les sites métalliques des métallo-β-lactamases, ou du Cu, complexés à des histidines coordonnées par les Nπ ou Nτ, afin de voir s’il y a une préférence géométrique pour l’une ou l’autre des deux coordinations et voir l’influence de ces différentes coordinations possibles sur les paramètres géométriques au niveau du site métallique. Enfin la présence d’eau et l’influence du solvant aqueux a été étudiée. Grâce à ces méthodes de chimie quantique fondées sur la théorie de la fonctionnelle de la densité, nous avons montré comment ces méthodes permettent d’avoir des informations structurales sur la symétrie adoptée par les centres métalliques, du Zn2+ et du Cu2+. Cette étude structurale nous a permis de mettre en évidence des différences structurales entre ces deux ions métalliques et de déterminer les spectres vibrationnels. Ces investigations nous ont permis de mettre en évidence la nature des liaisons métal-ligand grâce à des approches topologiques. Nous avons montré que ces études préliminaires nous ont permis de choisir la meilleure méthode de calculs DFT pour étudier des centres à zinc dans des structures de β-lactamases.Pour compléter l’étude de structures de métallo-β-lactamases, nous avons déterminé la structure de l’enzyme native L1 (β-lactamase) qui a permis de reproduire les paramètres géométriques des structures expérimentales de L1. Nous avons montré que l’approche combinant des études quantiques et classiques (QM/MM) permet de reproduire avec une très bonne confiance les paramètres structuraux de sites actifs de l’enzyme L1.Enfin, nous avons déterminé les structures de certains sites actifs de la famille B3 des Métallo-β lactamases (Enzyme L1) pour comparer les affinités de différentes familles d’inhibiteurs synthétisées à l’IBM de Montpellier (Institut des Biomolécules de Montpellier) et prédire la structure possible de L1 avec différents inhibiteurs par des méthodes QM/MM pour voir si cette stratégie pourra être appliquée à d’autres inhibiteurs pour des métallo-β-lactamases. / Metallo-β-lactamases are enzymes that give the bacteria that synthesize them antibiotic resistance. B Class represents the beta-lactamases, wherein one or two Zn atom(s) promote(s) β-lactams (antibiotics) hydrolysis. The major resistance mechanism is the degradation of the β-lactams by bacterial enzymes called β-lactamases. One major approach to overcome this resistance deals with combination therapy in which a β-lactam drug is given along with a β-lactamase inhibitor, which protects the former from inactivation. The objective of this thesis is to implement modeling tools based on quantum mechanical methods to determine metallo-β-lactamase structures with inhibitors, a step necessary to understand at a later stage the mechanisms of response to the degradation of the inhibitor by β-lactamases and to provide information that will serve to better interpret biological phenomena.We have first determined the geometries and the stability of metal coordination complexes of model systems containing Zn, as in the metallo-β-lactamase metal sites, or Cu, complexed to histidines coordinated by Nπ or Nτ, in order to see if there is a geometric preference for one or the other of the two coordination’s and to see the influence of these different possible coordination’s on the geometrical parameters at the metallic site. Finally, the presence of water and the influence of the aqueous solvent were studied. Using these methods of quantum chemistry based on the density functional theory, we have shown how these methods provide structural information on the symmetry adopted by the metallic centers of Zn2 + and Cu2 +. This structural study allows us to demonstrate structural differences between these two metal ions and to determine the vibrational spectra. These investigations were able to demonstrate the nature of the metal-ligand bonds through topological approaches. We have shown that these preliminary studies have conducted us to choose the best method of DFT calculations for studying zinc centers in β-lactamase structures.To complete the study of metallo-β-lactamase structures, we have determined the structure of the native enzyme L1 (β-lactamase) which permitted to reproduce the geometric parameters of the experimental structures of L1. We have shown that the combination of quantum and classical approaches (QM/MM) allows to reproduce with very good confidence the structural parameters of the L1 enzyme active sites.Finally, we have determined the structures of certain active sites in the B3 family of Metallo-β lactamases (Enzyme L1) to compare the affinities of different families synthesized at IBM in Montpellier (Institute of Biomolecules of Montpellier) and to predict the possible structure of L1 with different inhibitors by QM / MM methods to see if this strategy can be applied to other inhibitors for metallo-β-lactamases.

Made available in DSpace on 2015-03-26T13:51:59Z (GMT). No. of bitstreams: 1 01 - capa_abstract.pdf: 112846 bytes, checksum: f260bcc7fb2e9aa227782fd9e02a5c86 (MD5) Previous issue date: 2006-10-04 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / The diversity of resistance mechanisms to antimicrobial compounds was investigated in E. coli strains isolated from poultry. All 30 isolates were resistant to ampicillin and 26 of them were resistant to at least two antibiotics. Multi-resistance profiles were confirmed, including intermediate levels. Forty percent of the strains presented four resistance markers and 93% of the isolates were resistant to tetracycline, an antibiotic commonly used in poultry farms. The β-lactamase diversity among the strains was probed against seven compounds. Among strains bearing the same resistance profile for other groups of antimicrobial drugs, it was possible to observe different β-lactamases activity against the tested substrates. This fact suggests either a pool of resistance genes or regulatory differences. The isolate that displayed confirmed activity against six of the β-lactamic antibiotics bears a large molecular mass plasmid that confers the AmpR phenotype. A fragment of this plasmid with approximately 5 kb was subcloned in the pCCR9 vector which is commonly used to detect β-lactamase genes. The amino acid deduced from nucleotide sequencing displayed 100% of identity to TEM-1, a class A serine β-lactamase. Next to the gene encoding TEM-1 was found putative transposon related to the Tn3 family, albeit with some differences in the order and direction of transcription of the genes. / A diversidade dos mecanismos de resistência a antimicrobianos foi investigada em Escherichia coli, originadas de frangos de corte. Dos 30 isolados, todos resistentes à ampicilina, 26 apresentaram mais de uma marca de resistência. Foram confirmados modelos de multirresistência, incluindo níveis intermediários de resistência. Em 40% dos casos, ocorreram quatro marcas e 93% dos isolados resistiram à ação de tetraciclina, um promotor de crescimento usual na agropecuária. A diversidade de β-lactamases entre os isolados foi demonstrada pela ação direta contra sete β-lactâmicos. Os isolados com mesmo modelo de resistência possuíam, sob mesmas condições, espectro de ação e de atividade diferentes, mostrando que há amplo pool de genes de resistência, ou diferenças regulatórias nessas bactérias. O isolado com maior espectro de ação, confirmado sobre seis β-lactâmicos, contém um plasmídeo de alta massa molecular, que confere resistência a ampicilina. Um fragmento deste plasmídeo, com aproximadamente 5 kb, foi clonado em pCCR9, vetor usado para detecção de genes de resistência a β-lactâmicos. As análises das seqüências obtidas revelaram 100 % de identidade com TEM-1, uma serina β-lactamase da classe A. Próximo ao gene codificador da TEM-1, foi encontrado um transposon putativo relacionado com os da família Tn3, porém com particularidades na ordem e direção de transcrição dos genes componentes.

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2016-08-18T14:20:32Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) 00 TESE PAULA Doc Final VD-30-03-2016 Com Ficha cat.pdf: 3292123 bytes, checksum: 3a9cccec1214620cbdc7f55f0d159fe1 (MD5) / Made available in DSpace on 2016-08-18T14:20:32Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) 00 TESE PAULA Doc Final VD-30-03-2016 Com Ficha cat.pdf: 3292123 bytes, checksum: 3a9cccec1214620cbdc7f55f0d159fe1 (MD5) Previous issue date: 2015-12-16 / CAPEs / Pseudomonas aeruginosa, Klebsiella spp. e Acinetobacter spp. estão entre as cinco principais causas de infecções relacionadas à assistência à saúde (IRAS), e estão associadas a um elevado índice de mortalidade em pacientes oncológicos. Diante da vulnerabilidade destes pacientes e da crescente incidência de patógenos multidroga resistentes (MDR), o presente trabalho teve por objetivo realiza um estudo epidemiológico e molecular de infecções ocasionadas por P. aeruginosa, Klebsiella spp. e Acinetobacter spp. em pacientes com neoplasias sólidas, internados em um hospital de Recife-PE. Para tal, foi realizado um estudo descritivo de corte transversal dos dados clínicos, microbiológicos e hospitalares e os genes de resistência blaSPM-1, blaIMP, blaVIM, blaKPC, blaTEM e blaCTX-M, foram investigados por meio da reação em cadeia da polimerase. Também foi realizada tipagem molecular dos isolados MDR utilizando a técnica Enterobacterial Repetitive Intergenic Consensus-based PCR (ERIC-PCR). Entre 2012 e 2014, foram obtidos 169 isolados, sendo 58 P. aeruginosa, 36 Acinetobacter spp. e 75 Klebsiella spp.. A frequência de patógenos MDR (69,6%, IC95% 61,5% - 76,9%) foi significativamente maior que os isolados não MDR (30,4%, IC95% 23,1% - 38,5%). Dentre as bactérias resistentes aos carbapenêmicos, o gene blaSPM-1 foi detectado em P. aeruginosa (35,5%) e Acinetobacter spp. (3,8%), e o gene blaKPC detectado apenas em P. aeruginosa (25,8%). Já entre os isolados resistentes às cefalosporinas de terceira e quarta geração, o gene blaTEM estava presente em Acinetobacter spp. (25,9%) e Klebsiella spp. (30,6%) e o gene blaCTX-M em 58,3% das Klebsiella spp.. Também foi observado que o câncer de pulmão e os pacientes do sexo masculino foram predominantes na amostra e que o uso prévio de antimicrobianos durante o internamento e até três meses antes do isolamento do patógeno descrito neste estudo, apresentaram associação com o desenvolvimento de infecções por patógenos MDR. Assim, os resultados encontrados podem contribuir com epidemiologia molecular dos genes de resistência presentes no ambiente hospitalar, além de ressaltar a importância da monitorização contínua das IRAS em pacientes com câncer internados por longos períodos, a fim de melhorar os cuidados prestados a estes pacientes. / Pseudomonas aeruginosa, Klebsiella spp. and Acinetobacter spp. are among the five leading causes of Healthcare-associated Infections (HAI) in cancer patients, and those associated with a high mortality rate. Because of the vulnerability of patients and the increasing incidence of multidrug-resistant pathogens (MDR), this study aimed to realize an epidemiological and molecular study of infections caused by P. aeruginosa, Klebsiella spp. and Acinetobacter spp. in patients with solid tumors, admitted to a Recife-PE hospital. Therefore, we conducted a descriptive cross-sectional study of clinical, microbiological and hospital, and the resistance genes blaSPM-1, blaIMP, blaVIM, blaKPC, blaTEM and blaCTX-M, were investigated by polymerase chain reaction. It was also performed molecular typing of MDR isolates using Enterobacterial Repetitive Intergenic Consensus-based PCR (ERIC-PCR). Between 2012 and 2014, were obtained 169 isolates, 58 P. aeruginosa, 36 Acinetobacter spp. and 75 Klebsiella spp.. The frequency of MDR pathogens (69.6% 95% 61.5% - 76.9%) was significantly higher than non-MDR isolates (30.4% 95% 23.1% - 38.5%). Among the carbapenem-resistant bacteria, the blaSPM-1 gene was detected in P. aeruginosa (35.5%) and Acinetobacter spp. (3.8%), and blaKPC gene detected only in P. aeruginosa (25.8%). Among the resistant isolates to the third and fourth generation cephalosporins, the blaTEM gene was present in Acinetobacter spp. (25.9%) and Klebsiella spp. (30.6%) and blaCTX-M gene in 58.3% of the Klebsiella spp.. It was also noted that lung cancer and male patients were predominant in the sample and the previous use of antimicrobials during hospitalization and antibiotic use up to three months before the pathogen isolation described in this study were associated with the development of pathogen infections MDR. Thus, the results can contribute to molecular epidemiology of resistance genes present in the hospital, and underline the importance of continuous monitoring of HAI in cancer patients hospitalized for long periods aiming improve the care provided to these patients.

INTRODUÇÃO: Cepas de Pseudomonas aeruginosa (PA) produtoras de Metalo-b- lactamase (MBL) tem sido reportadas como sendo uma importante causa de infecções nosocomiais assim como um grande problema terapêutico mundial. No complexo HC-FMUSP, o maior hospital universitário do Brasil, a prevalência de PA resistente aos carbapenêmicos também se apresenta de forma crítica. No entanto, a prevalência de MBL em nossa instituição e a padronização para detecção fenotípica deste mecanismo de resistência ainda não foram estabelecidos. OBJETIVOS: Determinar a prevalência de MBL em cepas de PA resistentes ao imipenem; comparar métodos fenotípicos e moleculares na detecção de MBL; avaliar a clonalidade dos isolados produtores de MBL e determinar o perfil de suscetibilidade de todos os isolados incluídos no estudo. MÉTODOS: Foi realizado um estudo retrospectivo com 69 cepas de PA resistentes ao imipenem isoladas de hemoculturas de pacientes do HC-FMUSP no ano de 2005. Os isolados foram submetidos ao teste de suscetibilidade pelo método de microdiluição e Etest® para colistina. Estes foram testados para a produção de MBL pelos métodos fenotípicos de Disco Aproximação (DA), Etest® MBL e Teste de Hodge Modificado (THM). A Reação em Cadeia da Polimerase (PCR) foi realizada para detecção dos seguintes genes: (blaSPM-1, blaIMP-1, blaIMP-2, blaVIM-1 e blaVIM-2). A clonalidade dos isolados produtores de MBL foi avaliada por Eletroforese em Campo Pulsado (PFGE). RESULTADOS: Cinqüenta e três cepas (76.8%) foram positivas pelos métodos de DA e Etest® MBL, e 19 cepas (27.5%) foram positivas pelo THM. Vinte e um isolados (30.4%) demonstraram o gene blaMBL por PCR, sendo que 17 (81%) foram positivos para o gene blaSPM-1 e 4 (19%) para o gene blaVIM-2. Os genes blaIMP-1, blaIMP-2 e blaVIM-1 não foram detectados. O inibidor ácido 2-mercaptoacético (MAA) e o THM mostram a melhor concordância com a PCR, com índices de kappa variando de 0.81 a 0.86 e 0.79, respectivamente O ácido etilenodiaminotetracético (EDTA), demonstrou alta sensibilidade (100%), baixa especificidade (33.3%), e pobre concordância com a PCR. Entre os isolados positivos para o gene blaSPM-1, 5 foram indistinguíveis, 11 foram estreitamente relacionados e 1 possivelmente relacionado. Entre os isolados positivos para o gene blaVIM-2, 2 foram indistinguíveis, 1 foi estreitamente relacionado e 1 diferente. Entre os isolados produtores de MBL, a colistina e o aztreonam foram as drogas mais ativas com 90.5% e 85.7% de sensibilidade, respectivamente. CONCLUSÃO: Este é o primeiro relato de PA produtora de MBL no HC-FMUSP, reforçando a epidemiologia brasileira onde a enzima SPM-1 é a mais prevalente entre isolados de PA. Os métodos de DA com o inibidor MAA e o THM mostraram-se boas opções para detecção fenotípica de MBL em laboratórios de microbiologia. Os produtores de MBL demonstraram fenótipo multiresistente com um padrão clonal, enfatizando a necessidade de práticas de controle de infecções em nossa instituição / INTRODUCTION: Pseudomonas aeruginosa (PA) strains Metallo-b-lactamase (MBL) producing have been reported as an important cause of nosocomial infection, also becoming a critical therapeutic problem worldwide. At HC-FMUSP complex, the largest teaching hospital in Brazil, the prevalence of PA resistant to carbapenems is also presented as a critical problem. However, MBL prevalence and the standardization for phenotypical detection of this resistance mechanism still had not been established. PURPOSE: To determine MBL prevalence in PA resistant to imipenem; to compare phenotypic and molecular methods to detect MBL; to evaluate the clonality of the MBL producers strains and to determine the susceptibility profile of all isolates included in this study. METHODS: A retrospective study was carried analyzing 69 PA strains resistant to imipenem isolated from blood cultures of patients at HC-FMUSP during 2005. They were submitted to the susceptibility profile test by microdilution method and Etest® to colistin. The isolates were also tested for MBL production by phenotypic methods like Double Disk Synergy (DDS), Etest® MBL, Modified Hodge Test (MHT). The Polimerase Chain Reaction (PCR) was used to detect the following genes: (blaSPM-1, blaIMP-1, blaIMP-2, blaVIM-1 and blaVIM-2). The clonality of the MBL producers was evaluated by Pulsed Field Gel Electrophoresis (PFGE). RESULTS: Fifty-three isolates (76.8%) had positive results with DDS and Etest® MBL, and 19 isolates (27.5%) were positive by MHT. Twenty-one isolates (30.4%) had a blaMBL gene by PCR, being 17 (81%) positive for blaSPM-1 and 4 (19%) for blaVIM-2. The blaIMP-1, blaIMP-2 and blaVIM-1 genes had not been detected. Mercaptoacetic acid (MAA) inhibitor and MHT showed the best agreement with PCR, with kappa value ranging from 0.81 to 0.86 and 0.79, respectively. Etilenodiaminotetracetic acid (EDTA) inhibitor showed high sensibility (100%), low specificity (33.3%), and poor agreement with PCR. Among isolates producing blaSPM-1 gene, 5 were indistinguishable, 11 were closely related and 1 was possibly related. Among isolates producing blaVIM-2 gene, 2 were indistinguishable, 1 closely related and 1 was different. Among MBL-producing strains, colistin and aztreonam were the most active drugs with 90.5% and 85.7% of sensitivity, respectively. CONCLUSION: This is the first report of PA MBL producers at HCFMUSP, reinforcing the Brazilian epidemiology where SPM-1 enzyme is the most prevalent among PA isolates. The DDS method with MAA inhibitor and MHT had the best agreement with PCR, showing themselves as good options for MBL phenotypical detection in microbiology laboratories. The MBL producers had shown multiresistant phenotype with a clonal standard, reinforcing the necessity of infection control practices in our institution

Mycobacterium tuberculosis et Mycobacterium abscessus produisent les β-lactamases BlaC et BlaMab qui contribuent à la résistance intrinsèque de ces bactéries aux β-lactamines. Notre objectif est de caractériser l’inhibition de ces β-lactamases par l’avibactam et le clavulanate pour contribuer au développement de nouveaux traitements. Nous avons déterminé le profil de substrat et d’inhibition de BlaMab ainsi que sa structure cristalline, révélant trois différences majeures avec BlaC. BlaMab a une activité supérieure à celle de BlaC pour toutes les β-lactamines sauf la céfoxitine qui est utilisée pour les infections dues à M. abscessus. BlaC est inhibée irréversiblement par le clavulanate et inefficacement par l’avibactam alors que BlaMab présente le comportement inverse impliquant une hydrolyse du clavulanate et une inhibition très rapide par l’avibactam. La structure de BlaMab diffère de celle de BlaC principalement par le remplacement du motif conservé SDN par SDG. L’introduction de SDG dans BlaMab et de SDN dans BlaC a montré que cette différence détermine le profil d’inhibition des β-lactamases. Une seule mutation peut donc entraîner l’émergence d’une résistance aux combinaisons d’une β-lactamine avec le clavulanate ou l’avibactam mais pas avec les deux inhibiteurs. L’avibactam et le clavulanate offrent donc des alternatives thérapeutiques en cas de résistance à l’un des inhibiteurs. Nous nous sommes également intéressés aux β-lactamines partenaires du clavulanate, pour le traitement de la tuberculose et montrer que la structure des carbapénèmes pouvait être optimisée pour améliorer l’inactivation des cibles et diminuer l’hydrolyse par BlaC. / Mycobacterium tuberculosis and Mycobacterium abscessus produce the β-lactamases BlaC and BlaMab that contribute to the intrinsic resistance of those bacteria to β-lactams. Our objective was to characterize the inhibition of these β-lactamases by avibactam and clavulanate in order to contribute to the development of new treatments. We have determined the inhibition and substrate profiles of BlaMab, as well as its crystal structure, revealing three major differences with BlaC. BlaMab is more active than BlaC with respect to hydrolysis of all β-lactams except cefoxitin, which is used for the treatment of infections due to M. abscessus. BlaC is inhibited irreversibly by clavulanate and inefficiently by avibactam. In contrast, BlaMab shows the opposite behavior involving hydrolysis of clavulanate and a rapid inhibition by avibactam. Structurally BlaC differs from BlaMab mainly by the replacement of the conserved motif SDN by SDG. The introduction of SDG in BlaMab and of SDN in BlaC revealed that this difference determines the inhibition profile of the β-lactamases. A single mutation can therefore lead to the emergence of resistance to the association of β-lactam with clavulanate or avibactam, but not to both associations. Thus, avibactam and clavulanate offer therapeutic alternatives in case of resistance to one of the two inhibitors. We have also investigated the β-lactam partners of clavulanate for the treatment of tuberculosis and showed that the structure of carbapenems could be optimized to enhance the inactivation of the targets and to reduce hydrolysis by BlaC.

Submitted by Priscila Nascimento (pnascimento@icict.fiocruz.br) on 2013-03-22T18:57:42Z No. of bitstreams: 1 Bruno_Gabriel_N_Andrade.pdf: 1653673 bytes, checksum: 187a641b69c50d36212eb943747940e5 (MD5) / Made available in DSpace on 2013-03-22T18:57:42Z (GMT). No. of bitstreams: 1 Bruno_Gabriel_N_Andrade.pdf: 1653673 bytes, checksum: 187a641b69c50d36212eb943747940e5 (MD5) Previous issue date: 2012 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil. / As Metallo-β-Lactamases (Mβls) são uma família de enzimas cuja importância clínica reside na sua capacidade de hidrolisar praticamente todos os antibióticos da classe dos β-Lactâmicos, incluindo as carbapenemas, que são os compostos mais poderosos utilizados atualmente, a única exceção são os monobactâmicos. Estas enzimas são classificadas em três subclasses, B1, B2 e B3, com base nas suas identidades em nível de sequência e perfil de atividade. As enzimas das subclasses B1 e B3, caracterizam-se por ter amplo espectro de ação enquanto que, as da subclasse B2, são carbapenemases estritas. O conjunto de genes associados à resistência das bactérias aos antimicrobianos, presentes em um determinado ambiente, é conhecido como “Resistoma”. Evidências caracterizam a microbiota de ambientes naturais como fonte e/ou reservatório destes genes e o resistoma ambiental como reservatório original das Mβls. Nosso estudo tem como objetivo buscar em projetos de metagenomas marinhos públicos evidências da existência de um resistoma neste bioma. Buscamos, in silico, por Mβls putativas similares àquelas encontradas em bactérias presentes em ambientes clínicos. Os projetos de metagenomas marinhos públicos foram obtidos no banco de armazenamento do Cyberinfrastructure for Advanced Microbial Research (CAMERA), buscas por Metallo-β-Lactamases foram feitas com o software HMMer V3, empregando um limiar de similaridade baseado em um e-value de 10-20, utilizando os alelos de Metallo-β-Lactamases conhecidos para a construção de perfis HMM, empregados como sondas. Para determinar a relação com as Metallo-β-Lactamases clínicas, uma segunda busca foi feita com os resultados do HMMer contra o banco de dados de proteínas não redundantes (NR) utilizando o programa Blastp do pacote Blast+. Motivos conservados característicos das Mβls foram identificados visualmente para a eliminação de falsos positivos, análises filogenéticas foram feitas utilizando o programa MEGA v5. Identificamos, em sítios de vários oceanos e mares, sequências apresentando Mβls putativas, apresentando similaridade de sequência estatisticamente relevante com Mβls clínicas, como: VIM, CFIA, BLAB, SPM, CPHA, GOB, L1, FEZ1 e CAU-1. Algumas destas Mβls putativas apresentaram identidade de sequência com suas similares clínicas, as Mβls VIM e SPM. Nossa análise mostrou que a Mβl GOB apresenta uma grande abundância e dispersão. Assim, apresentamos as primeiras evidências da existência de um resistoma marinho, caracterizado pela presença de sequências das três subclasses de Mβls e com ampla distribuição pelo meio marinho. / The Metallo-β-lactamases (Mβls) are a family of enzymes with high impact in the clinic due to their ability to hydrolyze virtually all antibiotics belonging to the β-lactams group, excluding the monobactams. These enzymes are classified into three subclasses, B1, B2 and B3, based on their sequence similarity and the activity profile. The enzymes from subclass B1 and B3 present a broad spectrum of activity and the subclass B2 are strict carbapenemases. The set of genes associated with bacteria antimicrobial resistance present in an environment is known as “Resistome”. Studies have presented evidences characterizing the microbiota of natural environments as the source and/or reservoir of resistance genes and the environmental resistome as the original reservoir of Mβls. Our study aims to search publicly available marine metagenome projects for evidences of a marine resistome. We performed an in silico analysis to reveal putative Mβls that are similar to those found in the clinical setting. The marine metagenomic were downloaded from the Cyberinfrastructure for Advanced Microbial Research (CAMERA), and screened for Metallo-β-lactamases with the HMMer V3 software, with similarity threshold of an e-value of 10-20, using the known Metallo-β- lactamases alleles to build HMM profiles, and used them as queries. In order to determine the relationship between these two groups, the results were queried against the NCBI non-redundant protein database using the Blastp software with the same e-value cutoff. The Mβl motifs were visually identified to eliminate false positives, and phylogenetic analysis were made using the MEGA v5 software. Putative sequences showing identity with clinical Mβls, such as: VIM, CFIA, BLAB, SPM, CPHA, GOB, L1, FEZ1 and CAU-1 were found in samples from distinct oceans. Our analysis showed that the Mβl GOB is variable and dispersed in various sites of the oceans. Thus, we could map and bring evidences of the marine resistome based on the presence of the three subclasses of Mβls distributed in the ocean.

Escherichia coli and Klebsiella pneumoniae cause a wide range of infections. Multidrug-resistance strains carrying extended-spectrum β-lactamases (ESBLs) has become a growing problem worldwide. The CTX-M type ESBLs has emerged distinctly, especially in Escherichia coli and Klebsiella pneumoniae. CTX-M type has been associated with many outbreaks of infections both in the hospitals and community. CTX-M-15 is now identified as the most predominantly distributed CTX-M enzyme. Clonal outbreaks of CTX-M-15 producing Enterobacteriaceae have been described in many countries including the United Kingdom, and Escherichia coli is the most commonly involved species. A total of 100 isolates were received in 2010 from London St George’s hospital, England, 50 Escherichia coli, 17 Klebsiella spp, 9 Enterobacter spp, 13 Proteus spp, 6 Lactose fermenting coliforms, 2 Pantoea spp, one Serratia marcescens, one Morganella morganii, and one Hafnia alvei. The antimicrobial susceptibility results showed that 5 Escherichia coli and one Klebsiella pneumoniae isolates were found to be resistance to cefotaxime, ceftazidime, ceftriaxone, cefotaxime, ciprofloxacin, and gentamicin, making them multi-drug resistant bacteria. None of the isolates showed resistance to imipenem, ertapenem, or morepenem, thus making carbapenems the drug of choice for the treatment of these infections due to multi-resistant isolates. The overall frequency of CTX-M-15 type ESBL-producers detected in this study was 6 (6%) most of them 5/6 (83%) were from Escherichia coli and one was (17%) Klebsiella pneumoniae isolates. The 6 CTX-M-positive isolates were typed by PFGE, only two strains of Escherichia coli showed more than 85% similarity, owing to clonal homology for both strains. The rest strains showed less than 85% similarity. S1 nuclease plasmid profiles were obtained for ESBL-producers isolates. A total of one to three plasmids per isolate, ranging from approximately 78.0 to 152.0 kb, were observed. The plasmids from most isolates were assigned to be IncFA and IncFB replicons. Analysis of phylogenetic groups showed group A and group B2. The method of phylogenetic classification of exteraintestinal pathogenic Escherichia coli depends on examine and combination of two preserved genes (chuaA and yjaA) and the DNA fragment TSP. Primer walking and PCR experiments were used for the genetic environment studies which showed 5 different genetic constructions for the described blaCTX-M-15 genes. Conjugation studies were used to detect the transferability of the plasmids harbouring the reported blaCTX-M-15 genes. Three isolates were found transferable by conjugation. In conclusion, this study reports the presence of hospital highly resistant blaCTX-M-15 in St George’s hospital. The spread of blaCTX-M-15 is probably due to horizontal gene transfer harbouring ISEcp1 and the conjugative properties of plasmids carrying blaCTX-M-15.

<p>Vanlig tarmbakterie som Escherichia coli (E. coli) kan bland annat orsaka infektioner i bukhålan och urinvägsinfektioner. Infektioner orsakade av bakterien har ofta behandlats med betalaktamantibiotika, som penicilliner och cefalosporiner, vilket har resulterat i selektion av antibiotikaresistenta bakterier. Extended-Spectrum Beta-Lactamases (ESBL) är enzymer som hydrolyserar 3:e generationens cefalosporiner som cefotaxim, ceftazidim och ceftriaxon, men kan även bryta ner penicilliner, monobaktamer och övriga cefalosporiner. ESBL förekommer främst hos E. coli och Klebsiella pneumoniae. I början utgjordes ESBL-enzymerna av TEM och SHV, men idag är CTX-M den vanligaste ESBL-varianten. CTX-M genen finns i över 50 olika varianter, vilka delas in i fem subgrupper (CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9 och CTX-M-25). Syfte med arbetet var att utföra en genotypisk bestämning av ESBL-producerande E. coli, som isolerats i Kronobergs län 2009, med hjälp av realtids-PCR (Polymerase Chain Reaction). För detektion av CTX-M användes multiplex-realtids-PCR med vilken de fem olika grupperna kunde detekteras under samma PCR-analys. Resultatet visade att den vanligaste ESBL-varianten bland ESBL-producerande E. coli i Kronobergs län 2009 var CTX-M-1 (88 positiva av 123), men ett stort antal var positiva både för CTX-M-1 och TEM (41 isolat). Den minst förekommande varianten visade sig vara SHV (2 isolat). En ökad antibiotikaresistens kan leda till svårigheter att behandla vissa vanliga infektioner som urinvägsinfektioner och infektioner efter bukoperationer. Med tanke på att E. coli är en vanlig tarmbakterie är det viktigt att motverka uppkomst och spridning av ESBL. En genotypisk karaktärisering av förekommande ESBL-stammar i Kronoberg är därför en viktig del av övervakningen<em>.</em></p>

Šio tyrimo tikslas palyginti diskų difuzijos ir automatizuotą metodus, nustatančius Enterobacteriaceae šeimos bakterijų plataus veikimo &#946;-laktamazių (PVBLazių) fenotipą ir įvertinti PVBLazių paplitimą Vilniaus universiteto ligoninės Santariškių klinikose 2008-2010 metais. Tyrimų analizei buvo pasirinkta 58 Enterobacteriaceae šeimos bakterijos, išskirtos 2009-2011 metais ligoniams, besigydžiusiems Vilniaus Universiteto ligoninės Santariškių klinikose. Bakterijos ištirtos naudojant fenotipinius PVBL nustatymo metodus: automatizuotą Phoenix sistemą (Becton Dickenson, Sparks, MD, JAV), kombinuotą diskų difuzijos metodą ir dvigubos difuzijos metodą. Automatizuota Phoenix sistema tyrime laikyta kaip patvirtinantis metodas identifikuojant bakterijas ir nustatant PVBLazių gamybos fenotipą. Tiriamąją grupę sudarė šios bakterijų rūšys: K. pneumoniae, E.coli, Enterobacter spp., Proteus spp., Serratia spp., Morganella spp. ir Providencia spp.. Ištyrus 58 bakterijas patvirtinačiu metodu nustatyta, kad 31 iš jų gamina plataus veikimo beta-laktamazes. Atliktas palyginamasis diskų difuzijos metodų įvertinimas ir nustatytas jautrumas, specifiškumas, teigiama prognostinė vertė (PPV) ir neigiama prognostinė vertė (NPV). Įvertinus atliktų tyrimų duomenis nustatyta, kad kombinuoto diskų difuzijos metodo jautrumas - 100%, specifiškumas - 100%, PPV - 100%, NPV - 100%. Dvigubos difuzijos metodo jautrumas - 100%, specifiškumas – 88,9%, PPV – 91,2%, NPV - 100%. Gauti tyrimų rezultatai rodo, jog... [toliau žr. visą tekstą] / The aim of this study is to compare disk diffusion and automated methods for determining the Enterobacteriaceae extended spectrum-&#946;-lactamases (ESBL) phenotype and to assess the prevalence of (ESBL) at University Hospital Clinics in 2008-2010. For the study were chosen 58 bacteria from the Enterobacteriaceae family that were isolated in 2009-2011 in patients that were treated at Vilnius University Hospital Clinic. The bacteria studied using phenotypic methods ESBL: Phoenix automated system (Becton Dickenson, Sparks, MD, USA) combined disc diffusion method and the double-diffusion method. Automated system of Phoenix for the study was considered as a method of confirming the identification of bacteria and determination of (ESBL) production phenotype. The studied group consisted of the following bacterial species: K. pneumoniae, E. coli, Enterobacter spp., Proteus spp., Serratia spp., Morganella spp. and Providencia spp . The examination with the validated method of 58 bacteria showed that 31 of them produce an extended spectrum beta lactamases. A performed comparative disc diffusion method was made and the evaluation of the sensitivity, specificity, positive prognostic value (PPV) and negative prognostic value (NPV) was identified. The evaluation of the data from studies showed that the combined disc diffusion method for sensitivity - 100% and specificity - 100%, PPV - 100%, NPV - 100%. Double-diffusion method for sensitivity - 100% and specificity - 88.9%, PPV - 91.2%... [to full text]

Bactérias Gram-negativas não fermentadoras são frequentemente encontradas em águas superficiais, sendo muitas vezes carregadoras de múltipla resistência. O presente trabalho teve como objetivo principal analisar a participação de Pseudomonas sp. e Acinetobacter sp. na manutenção da resistência a antimicrobianos em quatro pontos na laguna de Tramandaí e se a presença deles poderia contribuir para a sua permanência. As amostras de água superficial foram coletadas em quatro pontos de coleta na laguna e submetidas a tratamento com os antimicrobianos: ácido nalidíxico, ceftazidima, imipenem e tetraciclina na concentração de 20mg/L. Em cada ponto de coleta uma das alíquotas não foi suplementada com os mesmos sendo utilizada como controle. Os isolados de Pseudomonas sp. foram identificados por provas bioquímicas e MALDI-TOF MS, enquanto que os isolados de Acinetobacter sp. somente por provas bioquímicas. O perfil de suscetibilidade de ambos foi avaliado pela técnica de disco-difusão e a produção de ESBL pela técnica de disco combinado. Os pontos 3 e 4 foram os que exibiram maior número de isolados resistentes. Os maiores percentuais de resistência estiveram associados às amostras submetidas ao tratamento com antimicrobianos. Em todos os pontos de coleta foram encontrados isolados de P. aeruginosa e Acinetobacter sp. multirresistentes. Ambas amostras (com e sem tratamento) exibiram diferentes padrões de resistência nos diferentes pontos de coleta e tanto isolados de P. aeruginosa como de Acinetobacter sp. exibiram isolados produtores de ESBL. A presença de P. aeruginosa e Acinetobacter sp. multirresistentes na Laguna de Tramandaí atenta para o risco de disseminação de resistência neste ambiente e que o mesmo pode estar atuando como reservatório de resistência. / Non-fermenting Gram-negative bacteria are often found in surface water, and are often carriers of multiple resistance to antimicrobials. The present work had as main objective to analyze the role of Pseudomonas sp. and Acinetobacter sp. in maintaining antimicrobial resistance at four points in the Tramandaí lagoon. The surface water samples were collected at four sampling points in the lagoon and treated with the antimicrobials: nalidixic acid, ceftazidime, imipenem and tetracycline at a concentration of 20mg/L. At each collection point, one of the aliquots was not supplemented with the antimicrobial and were used as the control for the treatment. The isolates of Pseudomonas sp. were identified by biochemical tests and MALDITOF MS, whereas the isolates of Acinetobacter sp. were identified only by biochemical tests. The susceptibility profile of both was evaluated by the disc diffusion method and the ESBL production by the combined disk method. Sampling points 3 and 4 showed the highest number of resistant isolates. The highest percentages of resistance were associated with the samples that were submitted to antimicrobial treatment. In all sampling points, multiresistant P. aeruginosa and Acinetobacter were isolated. Both samples (with and without treatment) showed different resistance patterns within the sampling points. P. aeruginosa and Acinetobacter sp. isolates exhibited ESBL producers. The presence of multiresistant P. aeruginosa and Acinetobacter sp. in the Tramandaí Lagoon attempted to the risk of spreading resistance in the aquatic environment, since it can act as a reservoir of resistance.