New Insight regarding Non- Species Identification: Comparison between the Traditional Gene Classification Scheme and a Newly Proposed Scheme Targeting the Gene.

The identification of non- species (non-) in clinical and environmental samples is based on the gene, although several studies suggest its limitations and the need to expand the classification scheme to include other genes. In this study, the development of a new classification scheme targeting the gene is proposed to obtain a more reliable identification of 135 environmental isolates. All isolates were sequenced for the and genes, and the results were compared to study the discriminatory power of the proposed scheme. Complete concordance between the and results based on genomic percent identity was found for 121/135 (89.6%) isolates; in contrast, discordance was found for 14/135 (10.4%) isolates. Additionally, due to the lack of reference values for the gene, inter- and intraspecies variation intervals were calculated based on a pairwise identity matrix that was built using the entire gene (∼4,107 bp) and a partial region (329 bp) to better evaluate the genomic identity obtained. The interspecies variation interval found here (4.9% to 26.7%) was then proposed as a useful sequence-based classification scheme for the identification of unknown non- isolates. The results suggest that using both the and genes makes it possible to correctly discriminate between several species, allowing possible new species to be identified, as confirmed by preliminary whole-genome sequencing analyses performed on our isolates. Therefore, starting from a valid and reliable identification approach, the simultaneous use of and associated with other genes, as it occurs with the sequence-based typing (SBT) scheme developed for Legionella pneumophila, could support the development of multilocus sequence typing to improve the knowledge and discovery of species subtypes. spp. are a widely spread bacteria that cause a fatal form of pneumonia. While traditional laboratory techniques have provided valuable systems for Legionella pneumophila identification, the amplification of the gene has been recognized as the only useful tool for non- species identification both in clinical and environmental samples. Several studies focused on the gene classification scheme showed its limitations and the need to improve the classification scheme, including other genes. Our study provides significant advantages on identification, providing a reproducible new gene classification scheme that seems to be more accurate than gene sequencing, bringing out greater genetic variation on species. In addition, the combined use of both the and genes allowed us to identify presumed new species, improving epidemiological investigations and acquiring new understanding on fields.