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

The identification of Legionella non-pneumophila species (non-Lp) in clinical and environmental samples is based on the mip 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 rpoB gene is proposed to obtain a more reliable identification of 135 Legionella environmental isolates. All isolates were sequenced for the mip and rpoB genes, and the results were compared to study the discriminatory power of the proposed rpoB scheme. Complete concordance between the mip and rpoB 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 rpoB gene, inter- and intraspecies variation intervals were calculated based on a pairwise identity matrix that was built using the entire rpoB 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-Lp isolates. The results suggest that using both the mip and rpoB 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 mip and rpoB 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 Legionella species subtypes.