MALDI-TOF MS Analysis of Burkholderia Pseudomallei and Closely Related Species Isolated from Soils and Water in Khon Kaen, Thailand.

Melioidosis is caused by Burkholderia pseudomallei (Bp) acquired from the environment. Conventional identifi-cation methods for environmental Bp are challenging due to the presence of closely related species. Matrix -assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is accurate for bacte-rial identification, but has been little used to identify Bp from environmental samples. This study aims to evaluate MALDI-TOF MS for the identification of Bp and closely related species isolated from environmental samples in Thailand using whole-genome sequencing (WGS) as the gold standard, including determining the best sample preparation method for this purpose. We identified Bp (n = 22), Burkholderia spp. (n = 28), and other bacterial species (n = 32) using WGS. MALDI-TOF analysis of all Bp isolates yielded results consistent with WGS. A decision-tree algorithm identified 16 important variable peaks, using the protein extraction method (PEM), demonstrating distinct MALDI-TOF profiles for the three categories (Bp, Burkholderia spp. and "other bacterial species"). Three biomarker peaks (4060, 5196, and 6553 Da) could discriminate Bp from other Burkholderia and closely related species with 100% sensitivity and specificity. Hence, the MALDI-TOF technique has shown its potential as a species discriminatory tool, providing results comparable to WGS for classification and surveillance of environmental Bp.