Characterisation of the wild type AmpC b-lactamase CHE
Abstract number: 1734_20
Fernea A., Mercuri P., Arlet G., Galleni M.
Objectives: AmpC-CHE is a chromosomal class C b-lactamase produced by Enterobactercloacae3. The most remarkable feature of this enzyme is a 6 amino acid deletion (SKVALA, position 289298) corresponding to a deletion of 18 nucleotides in the gene. Furthermore the alignments with other AmpC b-lactamase sequences shows that the 6 amino acid sequence is conserved in the AmpC b-lactamases of Citrobacter freundii and in the plasmid-mediated b-lactamases derived from. Our study is focused on the determination of the influence of this deletion by producing, purifying and undertaking kinetic characterisation of wild type AmpC-CHE.
Methods: AmpC-CHE was isolated from a culture of Enterobactercloacae. The bacteria were grown at 37°C in BHI media in the presence of amoxicillin 20 mg/mL. At an OD of 1.2 at 600 nm, cefoxitin (5 mg/mL final concentration) was added to the media and the culture was incubated for 4 additional hours. The bacteria were collected by centrifugation, resuspended in 15 mM sodium cacodylate pH 6 (buffer A) and lysed by sonication. The crude extract was loaded on a SP sepharose equilibrated in buffer A. The b-lactamase was eluted with a linear salt gradient. The active fractions were collected, dialysed against 10 mM sodium phosphate buffer pH 6.8 (buffer B) and loaded on a hydroxyapatite column equilibrated in buffer B. The AmpC enzyme was recovered by elution of the column with a linear gradient (10400 mM sodium phosphate pH 6.8). The N-terminal sequence and the exact molecular mass of CHE were determined.
Finally, the kinetic profile of the enzyme was characterised.
Results: The purification yield was estimated at 10%. The N-terminal sequence of AmpC was in good agreement with the predicted cleavage site of the pre-b-lactamase (TPSVE-). The mass of the mature form was also in good agreement with the molecular mass determined by mass spectrometry (38725 Da). The activity of AmpC-CHE was measured against nitrocefin, cephalothin, benzylpenicillin, cefotaxime, cefepime and cefpirome. The enzyme exhibited a broad spectrum of activity where benzylpenicillin and nitrocefin were the best substrates.
Conclusion: No major difference in the catalytic properties could be found when compared with the other enzymes of class C b-lactamases. The impact of the deletion on the stability of the enzyme is in progress.
|Session name:||European Society of Clinical Microbiology and Infectious Diseases|
|Location:||ICC, Munich, Germany|
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