Genetic diversity of <i>Legionella</i> species in culture-negative clinical and environmental specimens by sequencing the 23S-5S ribosomal intergenic spacer region

Abstract The diagnosis of Legionnaires’ disease (LD) caused by Legionella non- pneumophila species is likely to increase with broader use of PCR targeting Legionella spp. In this context, accurate species identification in PCR-positive but culture-negative samples is essential to improve understanding of disease epidemiology and to support source attribution. Here, we presented a validated and user-friendly bioinformatic pipeline compatible with next-generation sequencing (NGS) for analyzing the hypervariable 23S-5S region, paired with a curated database encompassing all described Legionella species as of January 2026. Parameters were optimized for sensitivity and specificity using both strains and culture-positive clinical and environmental samples. We then applied the pipeline retrospectively to 92 culture-negative PCR-positive samples collected from 2023 to 2025. Legionella species were successfully assigned in 60% (55/92) of tested samples and revealed a high diversity. Co-infections were detected in clinical samples, including combinations of L. pneumophila with L. longbeachae or L. bozemanii , while environmental samples contained up to six different species. These results demonstrate that 23S-5S amplicon NGS enables species-level identification in the absence of cultured isolates, improving surveillance of non-pneumophila Legionella cases. The proposed pipeline, implemented in QIIME2 and accompanied by a publicly available database, provides a practical framework for routine molecular monitoring and outbreak investigation. Author Notes All supporting data have been provided within the article or through supplementary data files. Four supplementary tables are available with the online version of this article. Impact statement Legionnaires’ disease caused by non- pneumophila Legionella species remains under-characterized because species identification is often impossible when cultures are negative. We developed and validated a next-generation sequencing approach targeting the Legionella 23S-5S region, coupled with a curated reference database and a user-friendly bioinformatic pipeline, enabling species-level identification directly from PCR-positive samples. Application to clinical and environmental specimens revealed substantial Legionella diversity in environmental samples and detected mixed-species infections that would otherwise have been missed. This open and reproducible framework strengthens molecular surveillance and outbreak investigations in the era of culture-independent diagnostics, as well as our understanding of Legionella diversity. Data summary All isolates whole-genome sequences used in this work are available in Table S1, available in the online version of this article. Amplicon-specific information can be found in Table S2, available in the online version of this article.