Listeria cossartiae sp. nov., Listeria farberi sp. nov., Listeria immobilis sp. nov., Listeria portnoyi sp. nov. and Listeria rustica sp. nov., isolated from agricultural water and natural environments

Abstract

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 L7-0091^T=CCUG 74668^T=LMG 31917^T; maximum ANI 91.9 % to L. innocua), Listeria immobilis sp. nov. (type strain FSL L7-1519^T=CCUG 74666^T=LMG 31920^T; maximum ANI 87.4 % to L. ivanovii subsp. londoniensis) and Listeria cossartiae sp. nov. [subsp. cossartiae (type strain FSL L7-1447^T=CCUG 74667^T=LMG 31919^T; maximum ANI 93.4 % to L. marthii) and subsp. cayugensis (type strain FSL L7-0993^T=CCUG 74670^T=LMG 31918^T; maximum ANI 94.7 % to L. marthii). The two proposed novel sensu lato species are Listeria portnoyi sp. nov. (type strain FSL L7-1582^T=CCUG 74671^T=LMG 31921^T; maximum ANI value of 88.9 % to L. cornellensis and 89.2 % to L. newyorkensis) and Listeria rustica sp. nov. (type strain FSL W9-0585^T=CCUG 74665^T=LMG 31922^T; 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.

Keywords: ANI; GTDB-Tk; Listeria sensu lato; Listeria sensu stricto; novel species.

Fig. 1.

UPGMA hierarchical cluster dendrogram based on the average nucleotide identity blast (ANIb) analysis of all 27 draft genomes representing the five novel Listeria species and two subspecies proposed here and 28 reference strains representing 21 Listeria species. The vertical bar is placed at 95 corresponding to the proposed 95 % ANIb cut-off for species differentiation [75]. The horizontal scale represents ANI percent similarity. The values placed on nodes represent ANI similarities; for terminal nodes, this value represents the similarity between the two taxa; for internal nodes, the value represents the similarity between the two taxa in the different branches that are most similar to each other. ANI similarities are only shown for nodes that split a given species from one or more other species. The isolates selected for phenotypic characterization are indicated with an ‘*”. The novel species are bolded with type strains identified by a superscript T.

Fig. 2.

Maximum-likelihood consensus phylogeny based on the concatenation of a 120-bacterial protein marker set (bac120) from GTDB-Tk analysis of the 27 draft genomes representing the five novel Listeria species and two subspecies and the same reference set used for ANIb analysis (see Fig. 1). The phylogeny was reconstructed using RAxML v8.2.12 and the PROTGAMMAILGF model. The values on the branches represent bootstrap values based on 1000 replicates; bootstrap values <70 are not shown. The tree is rooted at the midpoint and includes the outgroup Brochothrix thermosphacta ATCC®11509^T. The tree was edited with iTOL v5 [43]. Novel Listeria species are bolded and type strains are identified with a superscript T. The presence/absence of key genes, operons, and loci in the draft genomes were mapped onto the tree using iTOL [43] and indicated with filled/unfilled symbols (filled indicates presence, unfilled indicates absence). Symbols with an ‘*’indicate loci containing diversified genes and consequently only some genes were detected using a blastn search with reference L. monocytogenes genes from the PasteurMLST databases [61, 62]; specifically (i) not all genes in the flagella locus sourced from the cgMLST1748 database (lmo0676 thru lmo0717 [76]; Table S4) were detected in the L. grayi and L. costaricensis genomes, and (ii) not all LIPI-1 genes sourced from the Virulence database were detected in the L. ivanovii and L. seeligeri genomes. Detection of diversified flagella and LIPI-1 genes was achieved via alternative search methods including (i) locating the genes in the NCBI GenBank annotated genomes, or (ii) using more closely related reference genes (e.g. the L. ivanovii prfA gene cluster).

Fig. 3.

Maximum-likelihood phylogeny based on 16S rRNA sequence analysis of the type strains representing the five novel species and two subspecies using mega X. A total of 1078 positions were included in the dataset. The same reference set used for ANIb analysis was included along with Brochothrix thermosphacta ATCC®11509^T for an outgroup. The tree was reconstructed using mega X with 1000 bootstrap replicates and the Kimura two-parameter model [50, 51]; bootstrap values <70 are not shown. Novel Listeria species type strains are bolded and identified with superscript T.