Application of FT-IR spectroscopy using the IR Biotyper® for Leptospira: protocol optimization and first spectral insights

This study aimed to optimize a Fourier Transform Infrared (FT-IR) based protocol using the IR Biotyper® system for Leptospira strains and to provide the first spectral insights supporting its feasibility for serogroup and serovar differentiation. Reference Leptospira strains were cultured in Ellinghausen–McCullough–Johnson–Harris medium. A sample preparation workflow was optimized to accommodate liquid cultures, including chemical and thermal inactivation protocols to ensure biosafety. FT-IR spectra were acquired using the IR Biotyper® spectrometer and analyzed by Principal Component Analysis (PCA) in the 1300–800 cm⁻¹ spectral region. Whole Genome Sequencing (WGS) was used as the reference approach for taxonomic confirmation. Identification and serovar differentiation from genomic data were performed using the jSpecies, KmerFinder, and Pathogenwatch platforms. The sample preparation methods tested produced spectra suitable for analysis while ensuring biosafety. Spectral acquisition showed good reproducibility for most serovars across technical and biological replicates. Analysis of WGS performed using the jSpecies platform successfully differentiated the two major human-pathogenic serovars, Icterohaemorrhagiae and Copenhageni, whereas other bioinformatics tools showed limitations. PCA of FT-IR spectra generated by the IR Biotyper® demonstrated promising discriminatory potential among the serovars and serogroups evaluated, including closely related serovars such as Icterohaemorrhagiae and Copenhageni and serovars within the Australis and Sejroe serogroups. The IR Biotyper offers a rapid FT-IR approach for Leptospira typing. Under the experimental conditions evaluated, the method allowed differentiation of some serovars. Although preliminary, these findings suggest that FT-IR may represent a promising complementary tool for epidemiological surveillance of leptospirosis.