Identification of Legionella anisa, Legionella longbeachae and Legionella pneumophila using MALDI-TOF MS: A method validation study with environmental isolates

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) allows species-level microbial identification quickly and cost-effectively. The aim of this study was to validate MALDI-TOF MS for the identification of Legionella anisa, Legionella longbeachae and Legionella pneumophila and to examine whether incubation time or sample preparation method affect identification results. Environmental Legionella strains and ATCC reference strains were used in the study. The strains underwent 16S rRNA sequencing and latex agglutination and fluorescence tests to which MALDI-TOF MS results were compared. The strains, incubated for 24 and 48 h, were analysed with MALDI-TOF MS using direct transfer and extraction methods. Following performance characteristics were calculated from the results: sensitivity, specificity, relative accuracy, predictive values, repeatability and reproducibility. Species-level identification was achieved for 63 out of the 69 environmental and reference strains regardless of incubation time or preparation method. MALDI-TOF MS proved to be very specific (100 %) and sensitive (94.6-100 %) to the Legionella species analysed. With the extraction method, incubation time had a statistically significant, but species-dependent effect on the interpretative criteria, i.e. score values, in all species studied. No significant difference in any of the species' score values was found between the direct transfer and extraction methods with 24 h incubation. MALDI-TOF MS score values showed high repeatability (std 0.01-0.23) and reproducibility (std 0.02-0.18) for all Legionella species analysed. Based on the results, MALDI-TOF MS suits well for the analysis of environmental Legionella strains already after 24 h of incubation and using direct transfer method.