Diverse lineages of pathogenic Leptospira species are widespread in the environment in Puerto Rico, USA

Abstract

Background: Leptospirosis, caused by Leptospira bacteria, is a common zoonosis worldwide, especially in the tropics. Reservoir species and risk factors have been identified but surveys for environmental sources are rare. Furthermore, understanding of environmental Leptospira containing virulence associated genes and possibly capable of causing disease is incomplete, which may convolute leptospirosis 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 DNA from potentially pathogenic Leptospira 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 within the pathogenic P1 group. 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 these lineages is unknown but several were consistently detected for >1 year in soil, which could contaminate water. This work increases understanding of environmental Leptospira diversity and should improve leptospirosis surveillance and diagnostics.

Fig 1. Map of Puerto Rico indicating sites where environmental samples were collected for the detection of pathogenic Leptospira spp. DNA from 2018–2020.

Black dots represent paired water and soil sites (n = 22); 20 samples were collected from each site (10 soil and 10 water). Yellow triangles indicate sites where pathogenic Leptospira DNA was detected (15/22 sites). One site in San Juan (site 16, red circle) was serially sampled three times over a 14-month period to assess persistence of pathogenic Leptospira in soil. This map was created using ArcGIS software by Esri. ArcGIS and ArcMap are the intellectual property of Esri and are used herein under license. Copyright Esri. All rights reserved. For more information about Esri software, please visit www.esri.com. Basemap: http://goto.arcgisonline.com/maps/Canvas/World_Light_Gray_Base.

Fig 2. Genetically diverse and previously undescribed lineages of pathogenic Leptospira are widespread in Puerto Rico.

A) Maximum likelihood phylogeny constructed using a 202bp segment of the lipL32 gene, including sequences obtained from soil and water samples collected in this study (blue text) and those available in public databases (black text). Nine major genetic clades are represented, seven of which were present in Puerto Rico, including four not previously described. Clades are labelled 1 through 9 on the tree and color coded. B) Sites where each major clade was collected, with colors within the pie charts corresponding to those in the phylogeny; superscript indicates if soil (S) and/or water (W) samples were positive at each site. Fifteen positive sites are included on this map. This map was created using ArcGIS software by Esri. ArcGIS and ArcMap are the intellectual property of Esri and are used herein under license. Copyright Esri. All rights reserved. For more information about Esri software, please visit www.esri.com. Basemap: http://goto.arcgisonline.com/maps/Canvas/World_Light_Gray_Base.

Fig 3. Site 16 in Caño Martín Peña, San Juan, Puerto Rico where sampling was conducted at three separate timepoints to assess environmental persistence of pathogenic Leptospira in soil near the edge of the water.

A) Maximum likelihood phylogeny of all lipL32 sequences obtained from this soil site over the course of three sampling periods (color coded by date of sampling period); two of five clades detected at this site were identified at all three timepoints, suggesting environmental persistence. Clade colors correspond to Fig 2. Two isolates representing a novel pathogenic Leptospira species were obtained from soil sample 27 (red asterisk). B) Photo of exact location where samples 21–30 were collected and the isolates were obtained. C) Transect where 40 soil samples were collected over three sampling periods. Circles are color coded to indicate sampling period as in the phylogeny (panel A) and the red asterisk denotes the location where the Leptospira isolates originated. D) Location of site 16 in Caño Martín Peña. The maps in panels B and C were created using ArcGIS software by Esri. ArcGIS and ArcMap are the intellectual property of Esri and are used herein under license. Copyright Esri. All rights reserved. For more information about Esri software, please visit www.esri.com. Basemap for panel C: Community, https://www.arcgis.com/home/item.html?id=273bf8d5c8ac400183fc24e109d20bcf. Basemap for panel D: World Imagery (WGS84), https://www.arcgis.com/home/item.html?id=898f58f2ee824b3c97bae0698563a4b3.

Fig 4. Whole genome dendrogram of 802 known pathogenic, intermediate, and saprophytic Leptospira spp. isolates, including six saprophytic and two pathogenic isolates obtained in this study from a single soil sample from site 16.

A) Pairwise genomic distance dendrogram contextualizing the relationship among all known saprophytic (S1 and S2), intermediate (P2), and pathogenic (P1) Leptospira spp. reveals the pathogenic isolates LGVF01 and LGVF02 represent two genotypes of the same previously undescribed pathogenic species, whereas the saprophytes belong to three known Leptospira spp. B) Detailed view of the S1 clade that contains four saprophytic isolates obtained during this study. This maximum likelihood phylogeny reveals significant diversity among isolates that fall within the L. bandrabouensis clade. C) Detailed view of the P1 clade that contains two pathogenic isolates obtained during this study. This maximum likelihood phylogeny reveals two genotypes of a novel pathogenic Leptospira spp. isolated from soil in Puerto Rico.