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

Authors: STONE, NATHAN - Northern Arizona University; HALL, CARINA - Northern Arizona University; ORTIZ, MARIELISA - Animal And Plant Health Inspection Service (APHIS); HUTTON, SHELBY - Northern Arizona University; SANTANA-POPPER, ELLA - Northern Arizona University; CELONA, KIMBERLY - Northern Arizona University; WILLIAMSON, CHARLES - Northern Arizona University; BRATSCH, NICOLE - Northern Arizona University; FERNANDES, LUIS - Butantan Institute; BUSCH, JOSEPH - Northern Arizona University; PEARSON, TAMILA - Northern Arizona University; RIVERA-GARCIA, SARAI - Animal And Plant Health Inspection Service (APHIS); SOLTERO, FRED - Animal And Plant Health Inspection Service (APHIS); GALLOWAY, RENEE - Centers For Disease Control And Prevention (CDC) - United States; WEINER, ZACHARY - Centers For Disease Control And Prevention (CDC) - United States; HOFFMASTER, ALEX - Centers For Disease Control And Prevention (CDC) - United States; SAHL, JASON - Northern Arizona University; Nally, Jarlath; WAGNER, DAVID - Northern Arizona University

Submitted to: PLOS Neglected Tropical Diseases
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/4/2022
Publication Date: 5/18/2022
Citation: Stone, N.E., Hall, C.M., Ortiz, M., Hutton, S., Santana-Popper, E., Celona, K.R., Williamson, C.H., Bratsch, N., Fernandes, L.G., Busch, J.D., Pearson, T., Rivera-Garcia, S., Soltero, F., Galloway, R., Weiner, Z., Hoffmaster, A.R., Sahl, J.E., Nally, J.E., Wagner, D.M. 2022. Divergent lineages of pathogenic Leptospira species are widespread and persisting in the environment in Puerto Rico, USA. PLOS Neglected Tropical Diseases. 16(5). e0009959. https://doi.org/10.1371/journal.pntd.0009959.
DOI: https://doi.org/10.1371/journal.pntd.0009959

Interpretive Summary: Leptospirosis is a common zoonotic disease worldwide, but more prevalent in the tropics. It is spread more readily during severe weather events, a likely result of flooding, which distributes soil and water contaminated with Leptospira spp., the disease agents. Human cases increased following the 2017 hurricanes that ravaged Puerto Rico (Maria and Irma), prompting environmental sampling of soil and water to assess the presence, abundance, and persistence of pathogenic leptospires in these environments. The goal was to better understand these potential reservoirs of human and animal disease. Divergent and novel groups of pathogenic Leptospira were abundant and widespread in soil and water in Puerto Rico and persisted in these environments for >1 year. However, most groups that we identified have not previously been described from humans and/or other animals, so the disease potential of these novel organisms is unknown. The results of this study reveal a tremendous amount of previously uncharacterized Leptospira diversity in soil and water in Puerto Rico, which could cryptically contribute to disease. The description and characterization of these novel types improves our understanding of the genus Leptospira, and will aid in the development of improved diagnostics and preventative tools to advance public health outcomes.

Technical Abstract: 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 potential environmental sources of leptospirosis are rare. Furthermore, understanding of environmental Leptospira that contain pathogenic genes and may be 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 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 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 whole genome sequenced six saprophytic and two pathogenic Leptospira isolates; the latter represent a novel pathogenic Leptospira species that likely belongs 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. Several persisted for >1 year in soil and this reservoir could contaminate water. This work increases understanding of environmental Leptospira and should improve leptospirosis surveillance and diagnostics.