| Summary: | This study developed and validated a method, based on the coupling of Fourier-transform infrared spectroscopy (FT-IR) and machine learning, for the automated serotyping of Legionella pneumophila serogroup 1, Legionella pneumophila serogroups 2-15 as well as their successful discrimination from Legionella non-pneumophila. As Legionella presents significant intra- and inter-species heterogeneities, careful data validation strategies were applied to minimize late-stage performance variations of the method across a large microbial population. A total of 244 isolates were analyzed. In details, the method was validated with a multi-centric approach with isolates from Italian thermal and drinking water (n = 82) as well as with samples from German, Italian, French, and British collections (n = 162). Specifically, robustness of the method was verified over the time-span of 1 year with multiple operators and two different FT-IR instruments located in Italy and Germany. Moreover, different production procedures for the solid culture medium (in-house or commercial) and different culture conditions (with and without 2.5% CO2) were tested. The method achieved an overall accuracy of 100, 98.5, and 93.9% on the Italian test set of Legionella, an independent batch of Legionella from multiple European culture collections, and an extra set of rare Legionella non-pneumophila, respectively.
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