Divergent lineages of pathogenic Leptospira species are widespread and persisting in the environment in Puerto Rico, USA

Background: Leptospirosis, caused by Leptospira bacteria, is a common zoonosis worldwide more prevalent in the tropics. Reservoir species and risk factors have been identified but surveys for environmental sources of leptospirosis are rare. Furthermore, understanding of environmental Leptospira containing pathogenic genes and possibly capable of causing disease is incomplete and could result in some pathogenic strains evading detection, thereby convoluting diagnosis, prevention, and epidemiology. Methodology/Principal Findings: We collected environmental samples from 22 sites in Puerto Rico during three sampling periods over 14-months (Dec 2018-Feb 2020); 10 water and 10 soil samples were collected at each site. Samples were screened for pathogenic Leptospira DNA using the lipL32 PCR assay and positive samples were sequenced to assess genetic diversity. One urban site in San Juan was sampled three times over 14 months to assess persistence in soil; live leptospires were obtained during the last sampling period. Isolates were whole genome sequenced and LipL32 expression was assessed in vitro. We detected pathogenic Leptospira DNA at 15/22 sites; both soil and water were positive at 5/15 sites. We recovered lipL32 sequences from 83/86 positive samples (15/15 positive sites) and secY sequences from 32/86 (10/15 sites); multiple genotypes were identified at 12 sites. These sequences revealed significant diversity across samples, including four novel lipL32 phylogenetic clades. Most samples from the serially sampled site were lipL32 positive at each time point. We sequenced the genomes of six saprophytic and two pathogenic Leptospira isolates; the latter represent a novel pathogenic Leptospira species likely belonging to a new serogroup. Conclusions/Significance: Diverse and novel pathogenic Leptospira are widespread in the environment in Puerto Rico. The disease potential of the novel lineages is unknown but several persisted for >1 year in soil, which could contaminate water. This work increases understanding of environmental Leptospira and should improve leptospirosis surveillance and diagnostics.