Use of Fourier-Transform Infrared (FTIR) Spectroscopy with IR Biotyper® System for Legionella pneumophila serogroups identification

Legionella spp. are Gram-negative bacteria that inhabit freshwater environments representing a serious risk for human health. Legionella pneumophila (Lp) is the species most frequently responsible for a severe pneumonia, known as Legionnaires’ disease. Lp consists of 15 serogroups (Sgs), usually identified by monoclonal or polyclonal antibodies. Concerning Lp serogrouping, it is well known that phenotyping methods do not have a sufficiently high discriminating power, while genotypic methods although very effective, are expensive and laborious. Recently, mass spectrometry and infrared spectroscopy have proved to be rapid and successful approach for the microbial identification and typing. Different biomolecules (e.g., lipopolysaccharides) adsorb infrared radiation originating a specific microbial fingerprint. The development of a classification system based on the intra-species identification features allows a rapid and reliable typing of strains for diagnostic and epidemiological purposes. The aim of the study was the evaluation of Fourier Transform Infrared Spectroscopy using the IR Biotyper® system (Bruker Daltonik, Germany) for the identification of Lp at serogroup (Sg) level for diagnostic purposes as well as in outbreak events. A large dataset of Lp isolates (n=133) and ATCC reference strains representing the 15 Lp serogroups were included. The discriminatory power of instrument’s classifier, by Principal Component Analysis (PCA) and Linear Discriminant Analysis (LDA) was tested. All isolates were classified as follow: 12/133 (9.0 %) Lp Sg1 and 115/133 (86.5%) as Lp Sg 2-15 (including both ATCC and environmental Lp serogroup). Moreover, a mis-classification for 2/133 (1.5%) isolates of Lp Sg 2-15 returned as Lp Sg1 was observed and 4/133 (3.0%) isolates were not classified. The accuracy of 95.49% and an error rate of 4.51% were calculated. IR Biotyper® is able provide a quick and cost-effective reliable Lp classification with advantages compared to agglutination tests that show ambiguous and unspecific results. Further studies including a larger number of isolates could be useful to implement the classifier obtaining a robust and reliable tool for the routine Lp serogrouping. IR Biotyper® could be a powerful and easy-to-use tool to identify Lp Sgs especially during cluster/outbreak investigations, to trace the source of the infection and promptly adopt preventive and control strategies