Background . Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry accurately identifies both selected bacteria and bacteria in select clinical situations. It has not been evaluated for routine use in the clinic.

Methods . We prospectively analyzed routine MALDI-TOF mass spectrometry identification in parallel with conventional phenotypic identification of bacteria regardless of phylum or source of isolation. Discrepancies were resolved by 16S ribosomal RNA and rpo B gene sequence-based molecular identification. Colonies (4 spots per isolate directly deposited on the MALDI-TOF plate) were analyzed using an Autoflex II Bruker Daltonik mass spectrometer. Peptidic spectra were compared with the Bruker BioTyper database, version 2.0, and the identification score was noted. Delays and costs of identification were measured.

Results . Of 1660 bacterial isolates analyzed, 95.4% were correctly identified by MALDI-TOF mass spectrometry; 84.1% were identified at the species level, and 11.3% were identified at the genus level. In most cases, absence of identification (2.8% of isolates) and erroneous identification (1.7% of isolates) were due to improper database entries. Accurate MALDI-TOF mass spectrometry identification was significantly correlated with having 10 reference spectra in the database (P=.01). The mean time required for MALDI-TOF mass spectrometry identification of 1 isolate was 6 minutes for an estimated 22%–32% cost of current methods of identification.

Conclusions . MALDI-TOF mass spectrometry is a cost-effective, accurate method for routine identification of bacterial isolates in <1 h using a database comprising ⩾10 reference spectra per bacterial species and a ⩾1.9 identification score (Brucker system). It may replace Gram staining and biochemical identification in the near future.