TCC - Bacharelado em Ciências Biológicas (Sede)
URI permanente para esta coleçãohttps://arandu.ufrpe.br/handle/123456789/412
Navegar
Item Análise da Influência do número de cópias na expressão do gene blaKPC(2021-03-05) Oliveira, Michelly Maria Pereira e; Almeida, Anna Carolina Soares; http://lattes.cnpq.br/4153747599532886Enterobacteria has stood out in clinical practice by detecting enzymatic mechanisms that confer resistance to antimicrobials, especially Klebsiella pneumoniae carbapenemase (KPC) due to its rapid worldwide dissemination caused by the location of the blaKPC gene in large transferable plasmids and transposons. The expression of the blaKPC gene can be affected by intrinsic characteristics of the host that hosts it or the number of copies. An unusual phenotype was observed in clinical isolates of P. stuartii and M. morganii and their respective transformants, where E. coli cells that received plasmids that carried the blaKPC gene, showed MIC values, for some beta- lactams, higher than in clinical isolates. The objective of this work was to analyze the number of copies of the blaKPC gene, relating them to their respective MICs. The genomic DNA of the bacterial isolates was extracted from the PromegaTM WizardTM Genomic DNA Purification kit according to the manufacturer's guidelines, and was then quantified by spectrophotometry using the NanoDropTM Lite equipment from Thermo Fisher Scientific. The endogenous control genes were chosen from the literature after a robust search, after which they were analyzed in silico. Using databases, software and other tools, primers were designed. To carry out absolute quantification, an estimate was made based on a proportion of the relative quantitative relationship between target and endogenous control, using the formula 2-ΔCq. For the experiments, the SteponeTM Real-time PCR System (Applied Biosystems) and the SYBR Green PCR Master Mix (Applied Biosystems) were used. The designed primers underwent a validation step to evaluate their efficiency (tufMm - 93,019, tufPs - 90,228 and tufKp - 103,474, both with R2 close to 1). The data generated by the absolute qPCR experiments were associated with the different phenotypic profiles observed and from the tests it was determined that the number of copies of the blaKPC gene for transforming isolates and clinical isolates does not vary significantly, pointing out that another mechanism must be interfering in the expression of the blaKPC gene, directly influencing resistance levels.Item Análise da suceptibilidade aos antimicrobianos em Pseudomonas spp. isoladas de produtos saneantes(2020-02-03) Vitor, Mizânia Cabral de Almeida; Almeida, Anna Carolina Soares; Souza, Paula Mariana Salgueiro de; http://lattes.cnpq.br/6281410502740086; http://lattes.cnpq.br/4891800920829895Item Análise de mecanismos de resistência a polimixina em isolados de Klebsiella pneumoniae(2021-12-17) Andrade, Rauana Lins de; Almeida, Anna Carolina Soares; Santos, Bárbara Nazly Rodrigues; http://lattes.cnpq.br/4795529090461229; http://lattes.cnpq.br/4891800920829895; http://lattes.cnpq.br/8836137133949593Infections for Klebsiella pneumoniae Carbapenemase-producing have been a public health problem leading to increased patient morbidity and mortality, which has led to the reintroduction of a previously discontinued antimicrobial for human use, polymyxins. The raise in resistance to polymyxins has made treatment even more difficult, which is worrying due to the high worldwide dissemination of these strains.This study aimed to perform a genetic analysis of two-component systems involved in polymyxin resistance in Klebsiella pneumoniae isolates. Sixteen multiresistant K. pneumoniae isolates were studied. The clonal relationship was performed from the investigation of repeated extragenic palindromic sequences (REP). The genes of the two-component enzyme regulatory systems (pmrA, pmrB, phoP, phoQ) were amplified by polymerase chain reaction (PCR). The identification of mutations was performed by DNA sequencing with comparative analysis using the MGH 78578 strain as reference. It was possible to distinguish the presence of four groups with clonal relationship of the same species with a variation of 2 to 5 unshared bands, indicating a pattern of similarity between all bacteria in the study. All isolates had more than one mutation in the coding regions of the genes studied, the prevalence was of mutations classified as silent in pmrA and phoP. However, Frameshift Nonsense and Missense mutations were identified in pmrB and phoQ genes, which led to alterations in the amino acid chain and production of a non-functional protein. Nucleotide alterations in the coding regions of the TCS regulatory genes (phoPQ and pmrAB) and the compromise of the protein sequence are considered the most relevant mechanisms regarding the mediation of resistance to polymyxins.Item Análise de mecanismos de resistência em bactérias clínicas oriundas de hospitais de Pernambuco(2021-02-17) Silva, Jonas de Melo Silvestre da; Almeida, Anna Carolina Soares; http://lattes.cnpq.br/4891800920829895; http://lattes.cnpq.br/9044602995334190The spread of resistant bacteria or superbugs has been considered a catastrophic threat to the health of the population and represents one of the main challenges in the area of health worldwide. This work's main objective is to identify genetic and molecular determinants, resistance mechanisms, and the clonal relationship between twenty-two isolates obtained from two hospitals in Pernambuco. Isolates of: Klebsiella pneumoniae, Klebsiella ozaenae, Escherichia coli, and Enterobacter spp were included, all of which showed a phenotypic profile of multidrug resistance (MDR). Isolates from the University Hospital of the Federal University of Vale do São Francisco had a higher resistance profile compared to isolates from the University Hospital Oswaldo Cruz located in Recife, mainly to aminoglycosides and 3rd and 4th generation cephalosporins. Using the polymerase chain reaction (PCR) technique, it was possible to detect at least one of the B-lactam resistance genes tested in all isolates, except for 2 isolates that did not present any of the genes evaluated. The blaCTX-M gene was the most prevalent found in this study. And despite half of the samples having a resistance profile to carbapenems, the blaKPC gene was the least detected. In addition, clonal relationship analysis using the REP-PCR technique revealed a possible endemicity of a single clonal type in the Intensive Care Unit at Hospital Universitário Oswaldo Cruz located in Recife. At the University Hospital of the Federal University of Vale do São Francisco, the establishment of two clonal groups that have been disseminated for at least 3 months was identified. The presence of multiresistant bacteria in hospital units reinforces the need for strategies to contain infections and spread these pathogens, especially in ICUs.Item Análise in silicode iniciadores de genes referência utilizados como normalizadores em estudos utilizando qPCR para avaliação da expressão de isolados de Klebsiella pneumoniae(2019) Fonseca, Bárbara Schneyder Oliveira Pereira da; Almeida, Anna Carolina Soares; Nascimento, Crisvânia Pedrosa dos Santos; http://lattes.cnpq.br/9308656350291661; http://lattes.cnpq.br/4891800920829895; http://lattes.cnpq.br/0924501124844316Klebsiella pneumoniaeis a pathogenic bacterium considered to be an "urgent threat to human health"becausethe number of antibiotic-resistant bactéria is increasing, especially those considered to be the last line for its treatment, such as colistin.Forthis it is necessary to understand their mechanisms of resistance to know the best forms of treatment and to develop new drugs to treat infections. For this purpose, real-time quantitative PCR in relative expression studies has become one of the most effective tools to understand bacterial functioning at the transcriptional level, but for the results to be reliable and real, it is necessary to perform the normalization step, which among the possible the most common is through the useof reference genes. However, the choice of the genes to be used as normalizers among the genes pointed out in the literature has been controversial and, in many cases, with little reliability. This difficulty would be eliminated if there were a robust database for various types of studies for species other than humans and rats. Thus, there was a need to evaluate amongthe bacterial gene expression studies using qPCR, as normalizing genes and the primers used to amplify them.In a literature review, available in Pubmed, the 16Sand rpoBgenes were most commonly used as normalizers. Through in silico analysis after literature analysis it was possible to observe that the sequences of shared primer pairs were only smaller series within the ideal standard and the best ones in the future usedin later experiments, among others only one referring to one of the genes most commonly used, the 16S.