Leptospira enrichment culture followed by ONT Nanopore sequencing allows better detection of Leptospira presence and diversity in water and soil samples.

BackgroundLeptospirosis, a life-threatening disease in humans and animals, is one of the most widespread global zoonosis. Contaminated soil and water are the major transmission sources in humans and animals. Clusters of disease outbreaks are common during rainy seasons. Methodology/Principal FindingsIn this study, to detect the presence of Leptospira, we applied PCR, direct metagenomic sequencing, and enrichment culture followed by metagenomic sequencing on water and soil samples. Direct sequencing and enrichment cultures followed by PCR or sequencing effectively detected pathogenic and nonpathogenic Leptospira compared to direct PCR and 16S amplification-based metagenomic sequencing in soil or water samples. Among multiple culture media evaluated, Ellinghausen-McCullough-Johnson-Harris (EMJH) media containing antimicrobial agents was superior in recovering and detecting Leptospira from the environmental samples. Our results show that enrichment culture followed by PCR can be used to confirm the presence of pathogenic Leptospira in environmental samples. Metagenomic sequencing on enrichment cultures effectively detects the abundance and diversity of Leptospira spp from environmental samples. Conclusions/SignificanceThe selection of methodology is critical when testing environmental samples for the presence of Leptospira. Selective enrichment culture improves Leptospira detection efficacy by PCR or metagenomic sequencing and can be used successfully to understand the presence and diversity of pathogenic Leptospira during environmental surveillance. Author SummaryLeptospirosis, a life-threatening disease in humans and animals, is one of the most widespread global zoonosis. Contaminated soil and water are major sources of transmission in humans and animals. For this reason, clusters of disease outbreaks are common during the rainy season. In this study, Leptospira enrichment cultures followed by PCR and sequencing detected pathogenic and nonpathogenic Leptospira in soil and water samples. The pathogenic and intermediate groups of Leptospira were more prevalent in soil samples tested. Metagenomic sequencing on enrichment culture is effective in detecting the abundance and diversity of various Leptospira spp. in environmental samples. Soil samples in proximity to water may be an ideal niche for Leptospira growth and survival and may be an appropriate sample of choice for testing.