Resumo

Matrix-assisted laser desorption-ionization time of flight mass spectrometry (MALDI/TOF-MS) is a rapid and accurate method for identification of bacteria. The application for this technique has been recently introduced for the diagnosis of bovine subclinical mastitis and the objective of this work is to optimize the methodology for rapid and high accuracy for protein identification of mastitis pathogens by comparing direct colony smear with preparatory extraction. To prepare the samples for MS, the strains isolated from milk were thawed and cultured for 24h in a brain heart infusion agar. The direct colony analysis involves the application of bacterial colonies from growth plates directly onto the MALDI steel plate as a thin film and allowed to dry at room temperature. Subsequently 1 µL of formic acid solution 70% was applied onto the colony and allowed to dry again. The volume of 1 µL of α-cyano-4-hydroxycinnamic acid matrix was applied onto the colony and allowed to dry before data acquisition. For bacterial extraction, 900µl of 100% ethanol was added in pellet of bacteria, vortexed, and centrifuged (13,000g for 2 min). The supernatant was decanted, and the pellet was dried at room temperature. A solution of 70% formic acid was added proportionally to the size of pellet to lyse bacterial cells and release the inner-cell proteins, predominantly the ribosomal proteins that produce diagnostic ions in MALDI fingerprinting. Obtained spectra were analyzed with MALDI Biotyper 3.0 software (Bruker Daltonik). In this work, 47 strains were compared using the two sample preparation approaches. Using the direct colony smear on plate with formic acid, analysis of 14 samples (7 Corynebacterium bovis; 3 S. chromogenes; 2 S. aureus; 1 S. hominis; 1 S. epidermidis) showed secure genus identification and probable species identification (score value – sv≥2.00); 22 samples resulted in probable genus identification (sv=1.7to1.99) and 11 samples had no reliable identification (sv<1.7). Through the extraction method, for 28 samples (15 Corynebacterium bovis; 3 S. chromogenes; 2 Corynebacterium xerosis; 2 S. epidermidis; 2 S. aureus; 1 S. hominis; 1 Corynebacterium sp.; 1 Paenibacillus lactis; 1 Brevibacillus laterosporus) there were secure genus identification and probable species identification (sv≥2.00); 13 samples showed probable genus identification (sv=1.7to1.99) and 6 samples had no reliable identification (sv<1.7). In conclusion, even though faster, the formic acid extraction directly on plate method was ineffective to generate good quality mass spectra of gram positive rods such as Corynebacterium sp. However, results obtained from the extraction method showed equivalent results compared to direct colony smear for bacteria from cocci genus.