Soil Collected in the Great Smoky Mountains National Park Yielded a Novel Listeria sensu stricto Species, L. swaminathanii

Abstract

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). IMPORTANCEL. 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.

Keywords: Listeria sensu stricto; US National Parks; average nucleotide identity; in silico DNA-DNA hybridization; novel species; valid publication.

FIG 1

UPGMA dendrogram based on Average Nucleotide Identity BLAST (ANIb) analysis of 34 reference genomes (consisting of the 30 Listeria species and subspecies type strains described as of June 11, 2021, and one genome representing each of the four L. monocytogenes lineages) and the L. swaminathanii FSL L7-0020^T draft genome. The vertical red dotted line is placed at 95%, representing the species cutoff. The horizontal scale bar indicates ANI percentage similarity.

FIG 2

Maximum Likelihood (ML) phylogenetic tree based on the GTDB-Tk analysis of 120 concatenated protein amino acid sequences of the same 34 reference genomes used for ANIb analysis and the L. swaminathanii draft genome. The phylogeny was inferred using RAxML v8.2.12 (62), and the best fit model for protein evolution, PROTGAMMAILGF, was determined using ProtTest 3.4.2 (70). The values mapped to the nodes represent bootstrap values based on 1,000 replicates; values <70% are not shown. The tree is rooted at the midpoint and includes the outgroup Brochothrix thermosphacta ATCC 11509^T.

FIG 3

Presence/absence of key genes from the L. swaminathanii FSL L7-0020^T draft genome analyses compared to the L. monocytogenes genomes representing each of the four lineages and the type strain. Gray squares indicate the respective operon, loci, or gene (identified at the top of the column) is present, white indicates it is absent. For kat and L. swaminathanii FSL L7-0020^T, the square is not a solid gray color to reflect this gene was detected, but may not be functional (i.e., this strain is catalase-negative). The flagella locus includes 26 genes (Table S1). The nitrate reductase results include genomic analyses for narG, narH, and narI. The nitrite reductase analyses include genomic analyses for (nirB, nirD).