Navegando por Palavras-chave "Resistência Bacteriana"
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- ItemSomente MetadadadosCaracterização fenotípica e molecular de Staphylococcus aureus isolados de infecções de corrente sanguínea adquiridas em ambiente hospitalar em estudo multicêntrico - Projeto SCOPE Brasil(Universidade Federal de São Paulo (UNIFESP), 2020-10-29) Lorenzete, Katia Daliria [UNIFESP]; Pignatari, Antonio Carlos Campos [UNIFESP]; Universidade Federal de São PauloBloodstream infections (BSIs) rank as a major problem not only in Brazil but also in developed countries. Staphylococcus aureus ranks as the five most prevalent agents causing BSI in healthcare settings. In the present study we evaluated the epidemiological analysis and performed phenotypic and molecular tests of Staphylococcus aureus isolated from a National Surveillance Study in Brazil - BrSCOPE. We studied 191 episodes of BSI caused by Staphylococcus aureus and we performed phenotypic and molecular tests for the characterization of resistance mechanisms to oxacillin. We also carried out molecular chracterization of SCCmec and MLST of the isolates. Most of the patients were men with median age of 56 years old. Pediatric population accounted for 12.5% of the cases. Central venous catheter, urinary catheter, and mechanical ventilation were the most predisposing factors observed and malignancy and renal diseases were the underlying conditions most observed in this population. The crude mortality was 35.6%. For patients who needed ICU support this rate was 45.3% and 27.8% for those who did not. From the 191 isolates of Staphylococcus aureus, 83 (43.4%) were oxacillin resistant. The crude mortality for patients who had BSI caused by MRSA was 46.9% (39) while those with MSSA 28.0% (28)(p=0,002) We performed the molecular characterization of the SCCmec of 44 isolates oxacillin resistant. We observed the prevalence of the SCCmec type III (43%) followed by the SCCMEC type II (27%) and IV (18%). Only one isolate was nontypable according the protocol used and belonged to the ST 239.
- ItemSomente MetadadadosDesenvolvimento e avaliação de duas técnicas para estudo de sinergia de combinações de antimicrobianos frente à Pseudomonas aeruginosa multirresistente(Universidade Federal de São Paulo (UNIFESP), 2021) Santos, Gerlan Da Rocha [UNIFESP]; Kiffer, Carlos Roberto Veiga [UNIFESP]; Universidade Federal de São PauloThe Metallo-β-lactamases (MβLs) enzymes identified in Pseudomonas aeruginosa are increasing sources of resistance, causing hydrolysis to almost all classes of β-lactams, except aztreonam. In addition, MβL-producing P. aeruginosa pose a significant threat to Brazilian health system, especially the Sao Paulo metallo-β-lactamase enzyme (SPM-1). In the presente study, we performed in vitro tests of drug combinations for MβL- and EsβL- (Extended-spectrum β-lactamase) producing P. aeruginosa in order to evaluate their synergistic activity. Initially, the presence of blaSPM-1, blaIMP, blaVIM, blaCTX-M e blaGES-1 genes was confirmed by the polymerase chain reaction (PCR) technique. The minimum inhibitory concentrations (MICs) for some antibiotics and isolates were determined by the broth microdilution or agar dilution method. Then epsilometer crossing tests were performed. In order to interpret the epsilometer crossing test results, the Fraction inhibitory concentration index (FICI) was calculated and the combinations were defined as: synergy (≤0,5), additive (0,5-1), no effect (1-4), and antagonism (≥4). Two of the best performing combinations were also studied by time-kill assay: ceftolozane/tazobactam-aztreonam (C/T-ATM) and ceftolozane/tazobactam-fosfomycin (C/T-FOS). A 88.8% synergy (24/27) was detected for C/T-FOS and 14.8% (4/27) for C/T-ATM by epsilometer crossing test. In comparison, C/T-FOS combination presented synergistic activity in one (1/6) by the time-kill assay. The C/T-ATM combination showed no activity against the six isolates tested by TK assay. In the context of increased resistance to carbapenems among P. aeruginosa, techniques that aim to analyze the synergistic effect and combinations of antimicrobials must studied and the findings may help identifying new treatment options.
- ItemAcesso aberto (Open Access)Determinação da estrutura tridimensional por difração de raios-X da Brazilian Klebisiella Carbapenemase- BKC-1(Universidade Federal de São Paulo (UNIFESP), 2019-07-25) Silva, Paola Jacque De Souza Nascimento Da [UNIFESP]; Oliveira, Vitor Marcelo Silveira Bueno Brandao De [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)Background: The emergence and spread of antimicrobial resistance is one of the biggest threats to global health, particularly among the pathogens of medical significance, as the Gram-negative bacilli and members of the family Enterobacteriaceae, also species Klebsiella pneumoniae. The mechanism of resistance of these pathogens is the production of β-lactamases. These enzymes degrade antimicrobial containing a β-lactam ring. Even though well described the process by which the extended spectrum β-lactamase promote catalysis of antimicrobials β-lactam antibiotics, the mode by which the carbapenemases degrade the carbapenems are not yet fully established. Methods: The aim of this study was to study the structure and the mechanisms of action of a new class A carbapenemase of the Ambler classification scheme, BKC-1, identified in clinical specimes of Klebsiella pneumoniae resistant to carbapenems, isolated from two hospitals located in São Paulo, Brazil. In order to achieve the goal proposed we use recombinant enzyme and the gene blaBKC-1 was cloned and expressed in Escherichia coli BL21 (DE3) derived strains grown to 20° for 16 hours. The conditions of extraction and purification of the recombinant enzyme have been optimized. Different osmotic shock protocols for protein extraction of periplasmic space were realized, the protein fraction of the periplasmic content was treated with ammonium sulphate solution, 85% saturated, to remove some contaminants and allow the reduction of volume of material. Purification of the recombinant protein was performed by the hydrophobic interaction chromatography, ion-exchange and molecular exclusion. The purified protein was stored in 10 mm Tris pH 8.0 to -20°C. For the tests of crystallization was adopted the sitting drop vapor diffusion technique, single crystals were obtained of the BKC, which were submitted to x-ray diffraction. The mutagenesis by PCR-driven overlap extension was used to evaluate the participation of the most conserved amino acid residues in the catalytic activity and mechanism of action of the enzyme. Were produced and sequenced six mutants. The mutant pET-BKC-1-S92A was expressed following the same protocol for protein extraction and purification for the BKC-WT. Was performed several co-cristalização tests for this mutant complexed with different ligands. The best crystals were selected and diffracted x-ray on Laboratório Nacional de Luz Síncrotron, in Campinas-SP. Results: 3D structure of BKC-1 showed similarity to other β- xxi lactamases of the class A, consisting of two domains. The absence of the Cys238 residue and the insertion of the amino acid residue Tyr in the handle (Ω loop) suggest that a greater degree of flexibility of this protein conformational changes occur allows that can explain the carbapenemase activity. The data sets obtained from recombinant protein cocristalização with mutation in the residue Ser92Ala complexed with benzylpenicillin presented low densities, while for the other complexes, were not observed electronic densities which were characteristics of these substances.
- ItemSomente MetadadadosObtenção, caracterização e reconstituição da protease PgtE de Salmonella enterica sorovar Typhimurium em lipossomos: um estudo do mecanismo de resistência aos peptídeos antimicrobianos(Universidade Federal de São Paulo (UNIFESP), 2020-09-24) Silva, Pamela Jacque De Souza Nascimento Da [UNIFESP]; Perez, Katia Regina [UNIFESP]; Universidade Federal de São PauloMultidrug-resistant gram-negative bacteria represents one of the most prominent public health problems worldwide. Omptin family of the Outer membrane protein, such as PgtE from Salmonella spp. Enterica serovar Typhimurium, are able to cleave a variety of protein substrates, including antimicrobial peptides that have shown promise in the treatment of multi-resistant bacteria. Objective: to obtain and characterize the PAM-resistant proteoliposomes of S. typhi from the incorporation of the recombinant PgtE protein in liposomes. Methods: In this work, we describe how to use recombinant PgtE in its soluble and active form by cloning the gene encoding native PgtE S. typhi in the pET 28a (+) expression vector. The recombinant proteins expression was conducted in the hosted type E. coli BL21 (Dε3) under moderate conditions to avoid protein aggregation in inclusion bodies. It also describes the rational development of the protocols used for the extraction of proteins from the membrane through solubilization with the surfactant SDS. After protein solubilization, establish a convenient purification protocol to obtain a protein with a high degree of homogeneity and proteolytic activity used with SDS and CHAPS surfactant. Determined as kinetic constants (kcat, KM and kcat/KM) against a peptide substrate with intramolecular suppression of fluorescence in order to analyze the specific activity of the recombinant protein used in its solubilized and purified form. To use the proteoliposomes, we perform the incorporation of the recombinant protein in the DPPC or DPPC:DPPG 1:1 liposomes using the co-solubilization method that uses the BioBeads® resin. And it characterizes these proteoliposomes that are conducted by biophysical approaches of calorimetry through DSC and fluorescence to evaluate how changes in the incorporation in the thermodynamic characteristics of the membranes, as well as in the recovery of enzymatic activity when a substance is reconstituted. Results: The use of surfactant was important to obtain recombinant PgtE in its active soluble form after being removed from the lipid bilayer. The optimization of the solubilization and purification protocol using the SDS and CHAPS surfactants allowed obtaining the soluble and active protein with the highest possible yield and a high degree of homogeneity. The incorporation by the method of co-solubilization of lipids, protein and detergent with subsequent selective adsorption of the detergent using the Biobeads® resin was a methodology for obtaining the DPPC and DPPC proteoliposomes: effective DPPG, since we reached values between 87,1 and 90,7% xix protein incorporation yield. The DSC assays showed a more significant change in the profile of the membrane lipids phase transition thermogram, greater changes in the ∆H and in the cooperativity of the lipids of proteoliposomes made up of DPPC: DPPG. Kinetic assays suggest that negative charge on the membrane may influence PgtE activity. Conclusion: The strategies adopted in all stages, from obtaining the protein and purification to the characterization of the proteoliposome system, allowed to study the proteolytic mode of action of PgtE in a less complex and non-pathogenic system and opportunity to allowing the study of candidate peptides to treatment of infections by multi-resistant bacteria as an alternative to treatment using antibiotics.
- ItemSomente MetadadadosPerfil de consumo de antibacterianos e correlação com multirresistência de bactérias isoladas em unidades críticas de um hospital de ensino de São Paulo(Universidade Federal de São Paulo (UNIFESP), 2021) Rattes, Alysson Leandro Ribeiro [UNIFESP]; Burattini, Marcelo Nascimento [UNIFESP]; Universidade Federal de São PauloThe evolution of multi resistance bacteria, resulting from selection pressure related to the inappropriate use of antibiotics, remains an alarming issue. OBJECTIVES: To analyze temporal trends in antibiotic consumption and its correlation with the development of antimicrobial resistance in first bacterial isolates of blood cultures from ten consecutive years. METHODS: observational, retrospective, single-center study carried out in a Brazilian tertiary university hospital. The correlation of temporal trends of antibiotic use (DDD/100 patient-day) with the rate of resistant isolates from selected bacteria first blood cultures were analyzed over ten years. RESULTS Cephalosporins were the most used class of antibiotics and presented the highest resistance profiles, with ceftriaxone being the most used antibiotic. However, there were different patterns of time trends relating the use of antibiotics and the development of bacterial resistance. There was an increase in resistance to A. baumannii (amikacin, ceftazidime, imipenem and piperacillin-tazobactam) and K. pneumoniae (cephalosporins, carbapenems, piperacillin-tazobactam, polymyxin, Tigecycline). K. pneumoniae showed a significant positive correlation between consumption and resistance for imipenem, meropenem, piperacillintazobactam, polymyxin, tigecycline and A. baumannii with ampicillin-sulbactam. CONCLUSION: There were significant correlations between antibiotic use and development of resistance, but with different evolution patterns related to the different bacteria and antibiotics analyzed which may imply different genetic mechanisms involved for Acinetobacter baumannii and Klebsiella pneumoniae.