Evaluation of Two Protein Extraction Protocols Based on Freezing and Mechanical Disruption for Identifying Nontuberculous Mycobacteria by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry from Liquid and Solid Cultures.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has proved to be a useful diagnostic method for identifying conventional bacteria. In the case of mycobacteria, a good protein extraction protocol is essential in order to obtain reliable identification results. To date, no such protocol has been definitively established. The aim of this study was to compare the manufacturer's recommended protein extraction protocol (protocol A) with two novel protocols (protocols B and C), which apply different freezing temperatures and mechanical disruption times using an automatic tissue homogenizer. A total of 302 clinical isolates, comprising 41 nontuberculous mycobacteria (NTM) species, were grown in parallel on solid and liquid media and analyzed: 174 isolates were slow-growing mycobacteria (SGM) and 128 isolates were rapid-growing mycobacteria (RGM). Overall, MALDI-TOF MS identified a higher number of NTM isolates from solid than from liquid media, especially with protocol C (83.4 and 68.2%, respectively; P < 0.05). From solid media, this protein extraction method identified 57.9 and 3.9% more isolates than protocols A ( P < 0.001) and B ( P < 0.05), respectively. In the case of liquid media, protocol C identified 49.7 and 6.3% more isolates than protocols A and B, respectively ( P < 0.001). With regard to the growth rate, MALDI-TOF MS identified more RGM isolates than SGM isolates in all of the protocols studied. In conclusion, the application of freezing and automatic tissue homogenizer improved protein extraction of NTM and boosted identification rates. Consequently, MALDI-TOF MS, which is a cheap and simple method, could be a helpful tool for identifying NTM species in clinical laboratories.