| Grant number: | 13/07943-8 |
| Support Opportunities: | Scholarships abroad - Research |
| Start date: | August 05, 2013 |
| End date: | December 04, 2013 |
| Field of knowledge: | Biological Sciences - Biochemistry - Enzymology |
| Principal Investigator: | Milena Dropa |
| Grantee: | Milena Dropa |
| Host Investigator: | Pablo Power |
| Host Institution: | Faculdade de Saúde Pública (FSP). Universidade de São Paulo (USP). São Paulo , SP, Brazil |
| Institution abroad: | Universidad de Buenos Aires (UBA), Argentina |
Abstract Introduction. Bacterial resistance is a worldwide public health problem, which is facilitated by the selective pressure of antimicrobials use in clinical practice and other activities. Extended-spectrum ²-lactamases (ESBL) are one of the most prevalent mechanism of resistance in Gram negatives all over the world, and due to the frequency of point mutations that occur in these enzymes, there is a great likelihood for the emergence of new enzymatic variants, what can lead to new ²-lactam resistance profiles. Justification. The finding of two new ESBL variants in nosocomial strains from a recent study shows that, in addition to the worldwide issue on the spread of resistance genes, the antimicrobial selective pressure applied on bacteria can enable the emergence of enzymes containing wider catalytic activities. Therefore, the biochemical properties of the new enzymatic variants need to be characterized properly. Objective. To characterize biochemically the new ESBL variants TEM-197 and CTX-M-131. Material and Methods. The new enzymes encoding genes, which were identified in a previous study, will be cloned into an expression system, and the clones will be subjected to minimal inhibitory concentration measures using Etest, enzyme extraction by freezing (-20ºC) and thawing (20ºC) method, and enzyme purification in a ionic exchange column connected to a purifier. The enzymes active fractions will be detected by an iodometric system, and the samples purification degree will be evaluated by SDS-PAGE. Isoelectric focusing will be determined by bidimensional polyacrilamide gel electrophoresis, the hydrolitic activity will be measured by spectrophotometry using nitrocefin as a substrate, and the molecular mass will be assessed by mass spectrometry-HPLC. Kinetic parameters - Km and kcat constants, as well as the kcat/Km relation (catalytic efficiency), will be measured for each b-lactam compound, by the absorbance variation in spectrophotometer. Expected Results. Biochemical characterization of the new enzymes is expected to provide technical and epidemiologic information for the scientific community, as well as we expect that the methodology applied herein can be brought to Brazil in the future. (AU) | |
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