Toshiyuki Masuzawa, Mohd Khairul Nizam Mohd Khalid, Vasantha Kumari Neela, Anissa Amara Korba, Roman Thibeaux, Antony T. Vincent, Olivier Schiettekatte, Ryo Nakao, Eve Bernet, Mathieu Picardeau, Cyrille Goarant, Frédéric J. Veyrier, Nabilah Ismail, Pascale Bourhy, Fairuz Amran, Institut Armand Frappier (INRS-IAF), Institut National de la Recherche Scientifique [Québec] (INRS)-Réseau International des Instituts Pasteur (RIIP), Biologie des Spirochètes / Biology of Spirochetes, Institut Pasteur [Paris], Ecole doctorale Bio Sorbonne Paris Cité (BioSPC) (ED 157), Université Paris Diderot - Paris 7 (UPD7)-Université Paris Descartes - Paris 5 (UPD5), Unité de Recherche et d'Expertise Leptospirose - Leptospirosis Research and Expertise Unit [Nouméa, Nouvelle-Calédonie] (UREL), Institut Pasteur de Nouvelle-Calédonie, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), Universiti Putra Malaysia, Universiti Sains Malaysia (USM), Institute for Medical Research, Chiba Institute of Science (CIS), Hokkaido University [Sapporo, Japan], Laboratory of Parasitology [Sapporo, Japan], Institut Pasteur d'Algérie, Réseau International des Instituts Pasteur (RIIP), This work was supported in part by a donation from Foundation MSD to the ‘PIBnet’ programme of Institut Pasteur, by Public Health France (SPF), by Institut Pasteur through grant PTR 30-2017 and by the Malaysia Ministry of Education through Long-Term Research Grant Scheme (LRGS Phase 2/2014, UPM/700-2/7/LRGS/5526400). This work was part of the PhD thesis of O. S. who received financial support from 'Université Paris Diderot' and 'Sorbonne Paris Cité'. ATV received a Postdoctoral Fellowship from the Natural Sciences and Engineering Research Council of Canada. FJV is a research scholar of the Fonds de Recherche du Québec-Santé., We thank Vincent Enouf and the team of core facility P2M (Institut Pasteur, Mutualized Platform for Microbiology) for genomic sequencing. We also thank Nathalie Armatys, Céline Lorioux, Farida Zinini, Dominique Girault and Marie-Estelle Soupé-Gilbert for technical assistance with the cultures of Leptospira, Sabine Henry, Geoffroy Liegeon, Marie-Estelle Soupé-Gilbert and Emilie Bierque for environmental sampling, Robert Gaultney for animal experiments, and Chantal Bizet from the Collection of Institut Pasteur (CIP) for providing reference strains. We are also grateful to Prof. Aharon Oren for revising the names of novel Leptospira species and Jarlath Nally for the name of L. tipperaryensis., Biologie des Spirochètes, Unité de Recherche et d'Expertise Leptospirose - Leptospirosis Research and Expertise Unit (UREL), Hokkaido University, and Institut Pasteur [Paris] (IP)
The causative agents of leptospirosis are responsible for an emerging zoonotic disease worldwide. One of the major routes of transmission for leptospirosis is the natural environment contaminated with the urine of a wide range of reservoir animals. Soils and surface waters also host a high diversity of non-pathogenic Leptospira and species for which the virulence status is not clearly established. The genus Leptospira is currently divided into 35 species classified into three phylogenetic clusters, which supposedly correlate with the virulence of the bacteria. In this study, a total of 90 Leptospira strains isolated from different environments worldwide including Japan, Malaysia, New Caledonia, Algeria, mainland France, and the island of Mayotte in the Indian Ocean were sequenced. A comparison of average nucleotide identity (ANI) values of genomes of the 90 isolates and representative genomes of known species revealed 30 new Leptospira species. These data also supported the existence of two clades and 4 subclades. To avoid classification that strongly implies assumption on the virulence status of the lineages, we called them P1, P2, S1, S2. One of these subclades has not yet been described and is composed of Leptospira idonii and 4 novel species that are phylogenetically related to the saprophytes. We then investigated genome diversity and evolutionary relationships among members of the genus Leptospira by studying the pangenome and core gene sets. Our data enable the identification of genome features, genes and domains that are important for each subclade, thereby laying the foundation for refining the classification of this complex bacterial genus. We also shed light on atypical genomic features of a group of species that includes the species often associated with human infection, suggesting a specific and ongoing evolution of this group of species that will require more attention. In conclusion, we have uncovered a massive species diversity and revealed a novel subclade in environmental samples collected worldwide and we have redefined the classification of species in the genus. The implication of several new potentially infectious Leptospira species for human and animal health remains to be determined but our data also provide new insights into the emergence of virulence in the pathogenic species., Author summary Leptospirosis which is an emerging zoonotic disease worldwide, is transmitted to humans through contact with soils or surface waters contaminated with the urine of reservoir animals. Species of Leptospira, which include infectious and non-infectious strains, are ubiquitous in the environment. In this study we have sequenced the genomes of strains of Leptospira isolated from several environmental sources worldwide. Comparison of these genomes with other members of the Leptospira genus revealed the existence of 30 novel Leptospira species. A comparative genomic analysis of species of the genus allowed us to identify genes or genome features that are specific of infectious species, providing insights into virulence evolution in these atypical bacteria but also allow refinement of the Leptospira classification.