A total of 27 Listeria isolates that could not be classified to the species level were obtained from soil samples from different locations in the contiguous United States and an agricultural water sample from New York. Whole-genome sequence-based average nucleotide identity blast (ANIb) showed that the 27 isolates form five distinct clusters; for each cluster, all draft genomes showed ANI values of <95 % similarity to each other and any currently described Listeria species, indicating that each cluster represents a novel species. Of the five novel species, three cluster with the Listeria sensu stricto clade and two cluster with sensu lato. One of the novel sensu stricto species, designated L. cossartiae sp. nov., contains two subclusters with an average ANI similarity of 94.9%, which were designated as subspecies. The proposed three novel sensu stricto species (including two subspecies) are Listeria farberi sp. nov. (type strain FSL L7-0091T=CCUG 74668T=LMG 31917T; maximum ANI 91.9 % to L. innocua ), Listeria immobilis sp. nov. (type strain FSL L7-1519T=CCUG 74666T=LMG 31920T; maximum ANI 87.4 % to L. ivanovii subsp. londoniensis ) and Listeria cossartiae sp. nov. [subsp. cossartiae (type strain FSL L7-1447T=CCUG 74667T=LMG 31919T; maximum ANI 93.4 % to L. marthii ) and subsp. cayugensis (type strain FSL L7-0993T=CCUG 74670T=LMG 31918T; maximum ANI 94.7 % to L. marthii ). The two proposed novel sensu lato species are Listeria portnoyi sp. nov. (type strain FSL L7-1582T=CCUG 74671T=LMG 31921T; maximum ANI value of 88.9 % to L. cornellensis and 89.2 % to L. newyorkensis ) and Listeria rustica sp. nov. (type strain FSL W9-0585T=CCUG 74665T=LMG 31922T; maximum ANI value of 88.7 % to L. cornellensis and 88.9 % to L . newyorkensis ). L. immobilis is the first sensu stricto species isolated to date that is non-motile. All five of the novel species are non-haemolytic and negative for phosphatidylinositol-specific phospholipase C activity; the draft genomes lack the virulence genes found in Listeria pathogenicity island 1 (LIPI-1), and the internalin genes inlA and inlB, indicating that they are non-pathogenic.

No abstract

Reference methods developed for L. monocytogenes are commonly used for Listeria spp. detection. Improved method performance data are needed, since the genus Listeria has expanded from 6 to 26 species and now includes several Listeria sensu lato species, which can show phenotypes distinct from Listeria sensu stricto . Here, we evaluated growth of 19 Listeria spp., including 12 recently described sensu lato species, using the media specified by (i) the U.S. Food and Drug Administration (FDA) Bacteriological Analytical Manual , (ii) the U.S. Department of Agriculture (USDA) Microbiology Laboratory Guidebook , and (iii) the International Organization for Standardization (ISO). The FDA enrichment procedure allowed all species to grow to detectable levels (≥ 4 log 10 ), yielded the highest mean growth (7.58 log 10 ), and was the only procedure where no sensu lato species yielded significantly higher bacterial growth than a sensu stricto species. With the USDA or ISO enrichment procedures several sensu lato species yielded significantly higher bacterial growth than either L. seeligeri or L. ivanovii , suggesting that these two sensu stricto species could be outgrown by sensu lato species. On selective and differential agars, L. seeligeri, L. ivanovii, and L. grayi yielded atypical colony morphologies and/or showed inhibited growth (which may lead to incorrect classification of a sample as negative), while several newly described sensu lato species grew well and showed typical morphologies. Overall, our study shows that the ability to detect different Listeria spp. can be impacted by the specific broth and selective and differential agars used. Our data will aid with selection of media and detection methods for environmental Listeria monitoring programs and facilitate selection of methods that are most likely to detect the targeted Listeria groups (e.g., Listeria sensu stricto, which appear to be the most appropriate index organisms for the pathogen L. monocytogenes ).

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.