Subtyping of Listeria monocytogenes at the haplotype level by Fourier transform infrared (FT-IR) spectroscopy and multivariate statistical analysis

Listeria monocytogenes is a widespread foodborne pathogen that represents a major concern with respect to food safety. Rapid identification of this bacterium at a subspecies level is important to trace back an outbreak and improve risk-based inspection programs. A method for subtyping L. monocytogenes at the serotype and haplotype levels was developed using Fourier transform infrared (FT-IR) reflectance microscopy. Thirty strains of L. monocytogenes belonging to four different PCR serotypes (1/2a, 1/2b, 4b, and 4c) that had previously been characterized by Multilocus genotyping (MLGT) and Pulsed field gel electrophoresis (PFGE) assays were used in this study. The FT-IR based identification and classification was compared to the known MLGT and PFGE subtyping of the L. monocytogenes. Canonical variate analysis (CVA) of the spectra resulted in 96.6% correct identification of L. monocytogenes at the serotype level. Hierarchical cluster analysis (HCA) and CVA of the spectra showed 91.7% correct identification of strains at the haplotype level consistent with their MLGT groupings. FT-IR spectra of strains were also differentiated correctly in accordance with their PFGE haplotyping. Additionally, by using HCA of FT-IR spectra, each bacterium was differentiated at the strain level. Starting from a pure culture, this method enabled classification of L. monocytogenes at the serotype, haplotype, and/or strain level within 18 h, which is faster and potentially less expensive than the molecular methods and previous FT-IR methods. This is the first report of the identification of L. monocytogenes at the haplotype level using FT-IR.