Análise da Influência do número de cópias na expressão do gene blaKPC

Abstract

Enterobacteria 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.