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Listeria spp. are widely distributed environmental bacteria associated with human foodborne illness. The ability to detect and characterize Listeria strains in the natural environment will contribute to improved understanding of transmission routes of contamination. The current standard for surveillance and outbreak source attribution is whole-genome sequencing (WGS) of Listeria monocytogenes clinical isolates. Recently, metagenomic sequencing has also been explored as a tool for the detection of Listeria spp. in environmental samples. This study evaluated soil samples from four locations across altitudes ranging from 1,500 to 4,500 ft in the Nantahala National Forest in North Carolina, USA. Forty-two Listeria isolates were cultured and sequenced, and 12 metagenomes of soil bacterial communities were generated. These isolates comprised 14 distinct strains from five species, including Listeria cossartiae subsp. cayugensis ( n = 8; n represents the number of distinct strains), L. monocytogenes ( n = 3), " Listeria swaminathanii " (Lsw) ( n = 1), Listeria marthii ( n = 1), and Listeria booriae ( n = 1). Most strains ( n = 13) were isolated from lower altitudes (1,500 or 2,500 ft), while the L. swaminathanii strain was isolated from both higher (4,500 ft) and lower (1,500 ft) altitudes. Metagenomic analysis of soil described a reduction in both bacterial community diversity and relative abundance of Listeria spp. as the altitude increased. Soil pH and cation exchange capacity were positively correlated ( P < 0.05) with the abundance of Listeria spp. as detected by metagenomics. By integrating culture-independent metagenomics with culture-based WGS, this study advances current knowledge regarding distribution of Listeria spp. in the natural environment and suggests the potential for future use of culture-independent methods in tracking the transmission of foodborne pathogens., Importance: As a foodborne pathogen, Listeria continues to cause numerous illnesses in humans and animals. Studying the diversity and distribution of Listeria in soil is crucial for understanding potential sources of contamination and developing effective strategies to prevent foodborne outbreaks of listeriosis. Additionally, examining the ecological niches and survival mechanisms of Listeria in natural habitats provides insights into its persistence and adaptability, informing risk assessments and public health interventions. This research contributes to a broader understanding of microbial ecology and the factors influencing foodborne pathogen emergence, ultimately enhancing food safety and protecting public health. Moreover, using a metagenomic approach provides a detailed understanding of the soil microbial ecosystems, leading to more effective monitoring and control of foodborne pathogens. This study also highlights the potential for integrating metagenomics into routine surveillance systems for food safety in the near future.

Listeria monocytogenes, a foodborne pathogen, and other Listeria spp. are present in natural environments. Isolating and characterizing strains from natural reservoirs can provide insight into the prevalence and diversity of Listeria spp. in these environments, elucidate their contribution to contamination of agricultural and food processing environments and food products, and lead to the discovery of novel species. In this study, we evaluated the diversity of Listeria spp. isolated from soil in a small region of the Great Smoky Mountains National Park, the most biodiverse national park in the U.S. National Park system. Of the 17 Listeria isolates recovered, whole-genome sequencing revealed that 14 were distinct strains. The strains represented a diversity of Listeria species (L. monocytogenes [ n  = 9], L. cossartiae subsp. cossartiae [ n  = 1], L. marthii [ n  = 1], L. booriae [ n  = 1], and a potentially novel Listeria sp. [ n  = 2]), as well as a diversity of sequence types based on multilocus sequence typing (MLST) and core genome MLST, including many novel designations. The isolates were not closely related (≥99.99% average nucleotide identity) to any isolates in public databases (NCBI, PATRIC), which also indicated novelty. The Listeria samples isolated in this study were collected from high-elevation sites near a creek that ultimately leads to the Mississippi River; thus, Listeria present in this natural environment could potentially travel downstream to a large region that includes portions of nine southeastern and midwestern U.S. states. This study provides insight into the diversity of Listeria spp. in the Great Smoky Mountains and indicates that this environment is a reservoir of novel Listeria spp. IMPORTANCE Listeria monocytogenes is a foodborne pathogen that can cause serious systemic illness that, although rare, usually results in hospitalization and has a relatively high mortality rate compared to other foodborne pathogens. Identification of novel and diverse Listeria spp. can provide insights into the genomic evolution, ecology, and evolution and variance of pathogenicity of this genus, especially in natural environments. Comparing L. monocytogenes and Listeria spp. isolates from natural environments, such as those recovered in this study, to contamination and/or outbreak strains may provide more information about the original natural sources of these strains and the pathways and mechanisms that lead to contamination of food products and agricultural or food processing environments.

Salmonella enterica subsp. enterica serovar Newport ( S . Newport) is a clinically and epidemiologically significant serovar in the United States. It is the second most prevalent clinically isolated Salmonella serovar in the United States, and it can contaminate a wide variety of food products. In this study, we evaluated the population structure of S . Newport clinical isolates obtained by the Tennessee Department of Health during routine surveillance ( n  = 346), along with a diverse set of other global clinical isolates obtained from EnteroBase ( n  = 271). Most of these clinical isolates belonged to established lineages II and III. Additionally, we performed lineage-specific phylogenetic analyses and were able to identify 18 potential epidemiological clusters among the isolates from Tennessee, which represented a greater proportion of Tennessee isolates belonging to putative epidemiological clusters than the proportion of isolates of this serovar that are outbreak related. IMPORTANCE This study provides insight on the genomic diversity of one of the Salmonella serovars that most frequently cause human illness. Specifically, we explored the diversity of human clinical isolates from a localized region (Tennessee) and compared this level of diversity with the global context. Additionally, we showed that a greater proportion of isolates were associated with potential epidemiological clusters (based on genomic relatedness) than historical estimates. We also identified that one potential cluster was predicted to be multidrug resistant. Taken together, these findings provide insight on Salmonella enterica serovar Newport that can impact public health surveillance and responses and serve as a foundational context for the Salmonella research community.

Soil samples collected in the Great Smoky Mountains National Park yielded a Listeria isolate that could not be classified to the species level. Whole-genome sequence-based average nucleotide identity BLAST and in silico DNA-DNA Hybridization analyses confirmed this isolate to be a novel Listeria sensu stricto species with the highest similarity to L. marthii (ANI = 93.9%, isDDH = 55.9%). Additional whole-genome-based analysis using the Genome Taxonomy Database Toolkit further supported delineation as a novel Listeria sensu stricto species, as this tool failed to assign a species identification. Phenotypic and genotypic characterization results indicate that this species is nonpathogenic. Specifically, the novel Listeria species described here is phenotypically (i) nonhemolytic and (ii) negative for phosphatidylinositol-specific phospholipase C activity; the draft genome lacks all virulence genes found in the Listeria pathogenicity islands 1, 2, 3, and 4 as well as the internalin genes inlA and inlB . While the type strain contains an apparently intact catalase gene ( kat ), this strain is phenotypically catalase-negative (an unusual characteristic for Listeria sensu stricto species). Additional analyses identified a nonsynonymous mutation in a conserved codon of kat that is likely linked to the catalase-negative phenotype. Rapid species identification systems, including two biochemical and one matrix-assisted laser desorption/ionization, misidentified this novel species as either L. monocytogenes , L. innocua, or L. marthii . We propose the name L. swaminathanii, and the type strain is FSL L7-0020 T (=ATCC TSD-239 T ). IMPORTANCE L. swaminathanii is a novel sensu stricto species that originated from a US National Park and it will be the first Listeria identified to date without official standing in the nomenclature. Validation was impeded by the National Park's requirements for strain access, ultimately deemed too restrictive by the International Committee on Systematics of Prokaryotes. However, lack of valid status should not detract from the significance of adding a novel species to the Listeria sensu stricto clade. Notably, detection of non -monocytogenes sensu stricto species in a food processing environment indicate conditions that could facilitate the presence of the pathogen L. monocytogenes. If isolated, our data show a potential for L. swaminathanii to be misidentified as another sensu stricto, notably L. monocytogenes. Therefore, developers of Listeria spp. detection and identification methods, who historically only include validly published species in their validation studies, should include L. swaminathanii to ensure accurate results.