Whole-Genome Sequencing and Metagenomics Reveals Diversity and Prevalence of Soil Listeria spp. in the Nantahala National Forest

Background. Listeria spp. are widely distributed environmental bacteria associated with human foodborne illness. Whole-genome sequencing (WGS) of Listeria monocytogenes clinical isolates is the current standard for surveillance and identifying outbreaks. WGS of Listeria isolates can assist in understanding the diversity of Listeria species. The capability of precise detection and characterization of Listeria spp. strains in the natural environment is important for identifying transmission routes that can lead to food contamination and human illness. Recently, metagenomic sequencing has emerged as a promising tool for the direct detection of Listeria spp. in environmental samples. The objective of this study was to use a practical hybrid approach that can provide valuable information on the occurrence of Listeria spp. in soil environments in the southern Appalachian region of the United States. Results. This study involved soil sampling from four locations across varying altitudes in the Nantahala National Forest. Forty-two Listeria spp. isolates and 12 metagenomes of soil bacterial communities were obtained. Analysis of the isolates genomes revealed 14 distinct strains, representing a diversity of Listeria species. The predominant species isolated was L. cossartiae subsp. cayugensis (n = 8), with the other species including L. monocytogenes (n = 3), ‘ L. swaminathanii ’ (n = 1), L. marthii (n = 1), and L. booriae (n = 1). Most distinct strains (n = 13) were isolated from lower altitudes (1,500 or 2,500 ft), while the ‘ L. swaminathanii ’ strain was isolated from two different altitudes. Metagenomic analysis demonstrated a decline in both bacterial community diversity and relative abundance of Listeria spp. with increasing altitude. Of the soil factors assessed, pH and cation exchange capacity were significantly positively correlated with the abundance of Listeria spp. in soil as detected by metagenomics. Conclusions. By integrating culture-independent metagenomics with culture-based WGS, this study provides insights into the diversity and prevalence of Listeria spp. in the Nantahala National Forest. These findings contribute to the current knowledge on the distribution of Listeria spp. in the natural environment and offer a potential culture-independent method for tracking the transmission of foodborne pathogens.