Abstract
Legionella spp. are widespread bacteria in aquatic environments with a growing impact on human health. Between the 61 species, Legionella pneumophila is the most prevalent in human diseases; on the contrary, Legionella non-pneumophila species are less detected in clinical diagnosis or during environmental surveillance due to their slow growth in culture and the absence of specific and rapid diagnostic/analytical tools. Reliable and rapid isolate identification is essential to estimate the source of infection, to undertake containment measures, and to determine clinical treatment. Matrix-assisted laser desorption ionization–time-of-flight mass spectrometry (MALDI–TOF MS), since its introduction into the routine diagnostics of laboratories, represents a widely accepted method for the identification of different bacteria species, described in a few studies on the Legionella clinical and environmental surveillance. The focus of this study was the improvement of MALDI–TOF MS on Legionella non-pneumophila species collected during Legionella nosocomial and community surveillance. Comparative analysis with cultural and mip-gene sequencing results was performed. Moreover, a phylogenetic analysis was carried out to estimate the correlations amongst isolates. MALDI–TOF MS achieved correct species-level identification for 45.0% of the isolates belonging to the Legionella anisa, Legionella rubrilucens, Legionella feeleii, and Legionella jordanis species, displaying a high concordance with the mip-gene sequencing results. In contrast, less reliable identification was found for the remaining 55.0% of the isolates, corresponding to the samples belonging to species not yet included in the database. The phylogenetic analysis showed relevant differences inside the species, regruped in three main clades; among the Legionella anisa clade, a subclade with a divergence of 3.3% from the main clade was observed. Moreover, one isolate, identified as Legionella quinlivanii, displayed a divergence of 3.8% from the corresponding reference strain. However, these findings require supplementary investigation. The results encourage the implementation of MALDI–TOF MS in routine diagnostics and environmental Legionella surveillance, as it displays a reliable and faster identification at the species level, as well as the potential to identify species that are not yet included in the database. Moreover, phylogenetic analysis is a relevant approach to correlate the isolates and to track their spread, especially in unconventional reservoirs, where Legionella prevention is still underestimated.
Highlights
Legionella spp. are Gram-negative bacteria ubiquitous in natural fresh water such as rivers, lakes, and thermal springs
Applying the cut-offs recommended by the manufacturer, the MALDI Biotyper system identified at the genus level 90/202 (44.5%) isolates
The role of Legionella in human respiratory infections emphasizes the importance of its surveillance in conventional and unconventional artificial environments that could represent a reservoir of infections (Szewzyk et al, 2000)
Summary
Legionella spp. are Gram-negative bacteria ubiquitous in natural fresh water such as rivers, lakes, and thermal springs. They can be found in moist soil and mud in which the association with amoebae, protozoa, and biofilms plays a key role in the life cycle of bacteria (Rowbotham, 1980; Fields, 1996; Diederen, 2008). The ability of Legionella to replicate inside protozoa cells allows bacteria to infect human cells, such as alveolar macrophages, causing the disease in humans known as Legionellosis—an aggressive form of pneumonia—and the Pontiac fever, a febrile and generally benign non-pulmonary disease form (Newton et al, 2010; Mercante and Winchell, 2015; Cunha et al, 2016)
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