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Biochemical characterization of new variants of extended-spectrum ²-lactamases (ESBL) identified at São Paulo, SP, Brazil

Grant number: 13/07943-8
Support type:Scholarships abroad - Research
Effective date (Start): August 05, 2013
Effective date (End): December 04, 2013
Field of knowledge:Biological Sciences - Biochemistry - Enzymology
Principal researcher:Milena Dropa
Grantee:Milena Dropa
Host: Pablo Power
Home Institution: Faculdade de Saúde Pública (FSP). Universidade de São Paulo (USP). São Paulo , SP, Brazil
Research place: Universidad de Buenos Aires (UBA), Argentina  


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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Scientific publications
(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)
DROPA, MILENA; GHIGLIONE, BARBARA; MATTE, MARIA HELENA; BALSALOBRE, LIVIA CARMINATO; LINCOPAN, NILTON; MATTE, GLAVUR ROGERIO; GUTKIND, GABRIEL; POWER, PABLO. Molecular and Biochemical Characterization of CTX-M-131, a Natural Asp240Gly Variant Derived from CTX-M-2, Produced by a Providencia rettgeri Clinical Strain in Sao Paulo, Brazil. Antimicrobial Agents and Chemotherapy, v. 59, n. 3, p. 1815-1817, MAR 2015. Web of Science Citations: 3.

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