The occurrence of Legionella species other than Legionella pneumophila in clinical and environmental samples in Denmark identified by mip gene sequencing and matrix-assisted laser desorption ionization time-of-flight mass spectrometry

In Denmark, several laboratories use PCR as a routine diagnostic method for Legionnaires' disease, and almost all PCR-positive samples are investigated by culture. From 1993 to 2010, isolates of Legionella species other than Legionella pneumophila were obtained from respiratory samples from 33 patients, and from 1997 to 2010, 42 isolates of Legionella non-pneumophila species were obtained and saved from water samples from 39 different sites in Denmark. Macrophage infectivity potentiator gene (mip) sequencing was used as a reference method to identify the Legionella non-pneumophila species. Only one of the 75 isolates did not meet the acceptance criterion of a similarity of ≥98% to sequences in the database. The species distribution between clinical and environmental isolates varied. For the former, four species were detected, with Legionella bozemanae and Legionella micdadei predominating (both 44%). For the latter, eight species were detected, with Legionella anisa predominating (52%). The distribution among the Danish clinical isolates was different from the general distribution both in Europe and outside Europe, where L. bozemanae and Legionella longbeachae are the most commonly found clinical Legionella non-pneumophila species. The 75 isolates were also investigated by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS): 64 were correctly identified, with a score of ≥2.0; eight had a score of <2.0, but only two of these were wrongly identified; and three gave no results with MALDI-TOF MS. Both mip sequencing and MALDI-TOF MS are robust methods for Legionella species identification.