MALDI-TOF mass spectrometry proteomic based identification of clinical bacterial isolates.

Background & objectives: Pathogenic bacteria often cause life threatening infections especially in immunocompromised individuals. Therefore, rapid and reliable species identification is essential for a successful treatment and disease management. We evaluated a rapid, proteomic based technique for identification of clinical bacterial isolates by protein profiling using matrix-assisted laser desorption-ionization time - of - flight mass spectrometry (MALDI-TOF MS). Methods: Freshly grown bacterial isolates were selected from culture plates. Ethanol/formic acid extraction procedure was carried out, followed by charging of MALDI target plate with the extract and overlaying with α-cyano-4 hydroxy-cinnamic acid matrix solution. Identification was performed using the MALDI BioTyper 1.1, software for microbial identification (Bruker Daltonik GmbH, Bremen, Germany). Results: A comparative analysis of 82 clinical bacterial isolates using MALDI -TOF MS and conventional techniques was carried out. Amongst the clinical isolates, the accuracy at the species level for clinical isolates was 98.78%. One out of 82 isolates was not in accordance with the conventional assays because MALDI-TOF MS established it as Streptococcus pneumoniae and conventional methods as Streptococcus viridans. Interpretation & conclusions: MALDI - TOF MS was found to be an accurate, rapid, cost-effective and robust system for identification of clinical bacterial isolates. This innovative approach holds promise for earlier therapeutic intervention leading to better patient care.

Group III PLA2 from the scorpion, Mesobuthus tamulus: cloning and recombinant expression in E. coli

Phospholipases A2 (PLA2) are enzymes that specifically hydrolyze the sn-2 fatty acid acyl bond of phospholipids, producing a free fatty acid and a lyso-phospholipid. We report the cloning and expression of a secretory phospholipase A2 (sPLA2) from Mesobuthus tamulus, Indian red scorpion. The nucleotide sequence codes for a 167 residue enzyme. The open reading frame codes for a 31 amino acid signal peptide followed by a mature portion of the protein. The primary structure shows the calcium binding motif, catalytic residues, 8 highly-conserved cysteines and C-terminal extension which classify it as a group III PLA2. The entire transcript was expressed in Escherichia coli and was purified by metal affinity chromatography under denaturing conditions. The protein was refolded by serial dilutions in the refolding buffer to its active form. Hemolytic assays indicate that the protein adopts a functional conformation. The functional requisites such as optimum pH of 8 and calcium dependency are shown. This report provides a simple but robust methodology for recombinant expression of toxic proteins.