Phenotypic characterization and analysis of complete genomes of two distinct strains of the proposed species “L. swaminathanii”

Recently, a new Listeria species, “Listeria swaminathanii”, was proposed. Here, we phenotypically and genotypically characterize two additional strains that were previously obtained from soil samples and compare the results to the type strain. Complete genomes for both strains were assembled from hybrid Illumina and Nanopore sequencing reads and annotated. Further genomic analysis including average nucleotide identity (ANI) and detection of mobile genetic elements and genes of interest (e.g., virulence-associated) were conducted. The strains showed 98.7–98.8% ANI with the type strain. The UTK C1-0015 genome contained a partial monocin locus and a plasmid, while the UTK C1-0024 genome contained a full monocin locus and a prophage. Phenotypic characterization consistent with those performed on the proposed type strain was conducted to assess consistency of phenotypes across a greater diversity of the proposed species (n = 3 instead of n = 1). Only a few findings were notably different from those of the type strain, such as catalase activity, glycerol metabolism, starch metabolism, and growth at 41 °C. This study further expands our understanding of this newly proposed sensu stricto Listeria species.

Genomic characterization. Relatedness to other species and taxonomy were assessed using various genomic methods, including ANI, ribosomal multilocus sequence typing (rMLST), and dDDH. Assemblies for all currently described Listeria species type strains and representative strains were downloaded from the RefSeq or GenBank databases on NCBI or the ATCC genome portal, along with the assembly for the "L. swaminathanii" type strain (GCF_014229645.1). PYANI 26 (v0.2.10) was used to calculate ANI between all strains and bactaxR 27 was used to create an ANI dendrogram. The assemblies were also input into the rMLST 28 tool (available on PubMLST) and the Type Strain Genome Server (TYGS) 29 . A whole-genome alignment was performed with the two strains and FSL L7-0020 in Geneious using the progressiveMauve 30 algorithm (Mauve plugin v1.1.3) and visualized with Mauve 30,31 . For the alignment, the FSL L7-0020 assembly contigs reordered relative to the strain genomes and concatenated into a single sequence to form a pseudochromosome using the MCM algorithm in Geneious; the plasmid was also excluded from UTK C1-0015.
Genomes were evaluated for loci associated with antimicrobial resistance, virulence, motility, metal and disinfectants resistance, stress islands, and Listeria genomic islands using ResFinder 32 (v4.1), KmerResistance 33,34 (v2.2), VirulenceFinder 35 (v2.0), and the relevant schemes on Pasteur [36][37][38][39] . Mobile genetic elements (MGEs) were identified and characterized using PlasmidFinder (v.2.0), PLSDB 40 (v.2021_06_23), Phaster 41 , and PhageBoost 42 . BLAST and BLAST Ring Image Generator (BRIG) (v0.95) 43 were used to create a plasmid map for comparison of similar plasmids. Genomic comparison of monocin loci and nucleotide and amino acid identity were determined using BLAST and EasyFig 44  Phenotypic characterization. The phenotypic characterization of UTK C1-0015 and UTK C1-0024 was performed as per the standardized methodology in the FDA Bacteriological Analytical Manual (BAM) Chapter 10 45 and those described by Carlin et al. 10,16 . The following characteristics were assessed: growth at different temperatures (4, 7, 22, 30, 37, and 41 °C), growth under anaerobic conditions, colony morphology on selective and differential agar medium, motility, Gram stain, hemolysis, oxidase and catalase activity, nitrate reduction, and the biochemical tests included in three different commercial test kits (API Listeria, API 20 E, and API 50 CH). For each phenotypic analysis, from frozen stock, strains were streaked for isolation onto Brain Heart Infusion (BHI) agar (BD Biosciences, Franklin Lanes, NJ + Fisher Scientific Agar, Waltham, MA) and incubated aerobically at 30 °C for 24 h. From that plate, isolated colonies were either used directly or to inoculate a BHI broth (BD Biosciences, Franklin Lanes, NJ) tube, followed by aerobic incubation with shaking at 30

Results/discussion
Since 2010, there have been multiple new species added to the Listeria genus, many originally isolated from natural environments 16 . This paper describes the genotypic and phenotypic characterization of two new Listeria isolates obtained from soil samples collected in the Great Smoky Mountains National Park along the North Carolina-Tennessee border 17 . Evaluation of genotypic and phenotypic characteristics of "L. swaminathanii" strains will aid in the characterization of this novel species and contribute to our knowledge of the diversity of Listeria spp. Here, we describe the newly isolated strains, UTK C1-0015 and UTK C1-0024, and compare with the "L. swaminathanii" type strain (FSL L7-0020 T ) and other Listeria spp. Both genomes were able to be assembled into complete closed genomes (contiguous sequences that comprise the entire genome). The genome of UTK C1-0015 consists of a 2.78 Mb chromosome and 55 Kb plasmid (total genome length of 2.84 Mb) with a G+C content of 38.7%; UTK C1-0024 consists of a 2.95 Mb chromosome with a G+C content of 38.6% (Table 1), which is consistent with FSL L7-0020 T . Of the validly published type strains, the two isolates showed highest similarity to L. marthii (94.0-94.1%) (Fig. 1); however, they were most closely related to "L. swaminathanii" FSL L7-0020 T , with 98.7-98.8% ANI, indicating that they belong to the same species. Examination of the chromosomal alignment of the two isolates and the type strain shows that, overall, there is a high level of conservation across the entire chromosome, with no large rearrangements or deletions (Fig. 2). However, there are some loci throughout that are present or absent in only one of the isolates.
PHASTER and PhageBoost were used to predict prophage sequences in the genomes. The genome UTK C1-0024 was predicted to house a prophage integrated near a tRNA-Lys gene. Blastn results show the prophage from UTK C1-0024 has an 88.58% identity to Listeria phage A500 with 60% coverage. Prophages and other mobile genetic elements can contribute to genome diversity and have been used to distinguish epidemic clones of L. monocytogenes [48][49][50] . Strain UTK C1-0015 was predicted to house a partial monocin locus of eight open reading frames 51,52 ; structural genes such as those that code for the tail tape measure protein or tail fibers were absent from the locus. The monocin locus from strain UTK C1-0015 shares a 99.405% identity to the monocin locus from FSL L7-0020 T (GCF_014229645.1). The UTK C1-0024 genome was predicted to house the full monocin locus of 18 open reading frames, similar to the monocin in L. monocytogenes strain 10403S (Fig. 4). Blastp queries using the monocin locus from UTK C1-0015 and UTK C1-0024 return hits to L. marthii, L. cossartiae, L. innocua, L. farberi, and L. monocytogenes strains with 100% coverage and > 89.90% identity, suggesting this is fairly dispersed across the sensu stricto clade of Listeria. Monocins are bacteriocins produced by the host that may be significant in establishing dominant strains in ecological niches, as they target closely related species, but remain inactive against the producing strain 53 .
Listeria spp. grow at a wide range of temperatures from 0 to 45 °C 7,8,14,16 and can survive at temperatures below freezing (− 7 °C) 54 . In the current study, we performed growth assessments at 4, 7, 22, 30, 37, and 41 °C. These temperatures were chosen to encompass the known growth temperature range, with 4 and 7 °C specifically included because some species are unable to grow well at low temperatures (< 7 °C) 4 . Strain UTK C1-0015 exhibited growth at all temperatures tested and strain UTK C1-0024 exhibited growth at all termperatures except 41 °C (Supplementary Table S2). After 24 h of incubation, UTK C1-0015 and UTK C1-0024 showed optimal growth at 30 °C (9.2 and 9.4 log 10 CFU/mL), followed by at 37 °C (8.9 and 9.0 log 10 CFU/mL). At 41 °C, UTK C1-0024 was enumerated daily for up to five days and no growth was observed, which is dissimilar to both UTK C1-0015 and FSL L7-0020 T . At 4 °C, the concentration increases of UTK C1-0015 and UTK C1-0024 after 11 d (6.4 and 6.8 log 10 CFU/mL, respectively) were higher than the increases seen in FSL L7-0020 T (4.1 log10 CFU/mL) 16 .
Listeria spp. are Gram-positive rods 7 ; this was confirmed for UTK C1-0015 and UTK C1-0024. Both isolates were observed to grow under aerobic and anaerobic conditions at 30 °C after 24 h; this is another expected result, as Listeria spp. are facultative aerobes 7 . Both strains were oxidase negative (Supplementary Table S3), as expected 7 , indicating a lack of cytochrome c oxidase. Additionally, both were catalase positive, indicating they produce the catalase enzyme that converts hydrogen peroxide into oxygen gas and water; however, FSL L7-0020 T is catalase negative 16 , a phenotype that has only been described in one other Listeria spp. (L. costaricensis) 55 . When kat gene from the reference, two isolates, and the type strain are aligned, there are nucleotide differences at 158 positions. 16 of the nucleotide differences differ between the type strain and one or both of the isolates. Table 1. Genome statistics. Genome statistics for the two "L. swaminathanii" strain hybrid assemblies described here and for the recently described type strain 16 . www.nature.com/scientificreports/ Four of those result in amino acid differences, with two between the type strain and both isolates. At amino acid position 72, the type strain has glutamic acid (polar, acidic) and the two isolates have lysine (polar, basic), a radical substitution. At amino acid position 92, the type strain has histidine and the other two arginine (both polar, basic), a conservative substitution. These amino acid differences may have an effect on the structure and function of the resulting protein, leading to the catalase-negative phenotype of FSL L7-0020 T . On MOX agar, UTK C1-0015 and UTK C1-0024 colonies were typical for Listeria spp.: gray to black colonies with sunken centers and black halos, indicating esculin hydrolysis. On Listeria CHROMagar, UTK C1-0015 and UTK C1-0024 were typical for Listeria spp.: blue colonies (indicating β-glucosiadase enzyme activity), but lacking opaque white halos typical for L. monocytogenes and L. ivanovii (indicating no phosphoatidylinositol-specific phospholipase C [PI-PLC] activity) (Supplementary Table S3).

NCBI RefSeq and genome accessions
API test kits were used to characterize metabolic function of UTK C1-0015 and UTK C1-0024. The Listeria API kit is designed for species-level identification Listeria spp. based on enzymatic tests and sugar fermentations. For this test, both strains generated a code of 6110 (Supplementary Table S3), consistent with FSL L7-0020 T 16 and indicates an 80% (t-value of 0.62) ID to L. monocytogenes according to the APIweb The API 20 E kit is designed for identification of Enterobacteriaceae and other non-fastidious Gram-negative rods; however, this kit contains tests that can be used for genus-level identification of Listeria spp. and has been used previously in the characterization of novel Listeria spp. 10,16 . For this test, UTK C1-0015 and UTK C1-0024 were positive for acetoin production (Voges Proskauer) and D-glucose and amygdalin fermentation, which Comparison of the plasmids found in UTK C1-0015, FSL J1-020, and FSL J1-158, using pLMIV from J1-208 as the reference. The innermost black ring represents pLMIV. The middle rings represent FSL J1-158 (teal) and UTK C1-0015 (purple), with BLAST identity indicated by shading (see legend). The outermost ring contains gene annotations from pLMIV that are colored by functional category: green (plasmid replication and conjugation), red (internalins or internalin-related), blue (transposases or integrases), gray (hypothetical proteins), and black (other).  (Supplementary  Table S3). UTK C1-0015 and UTK C1-0024 were negative for all other tests, including indole, urease, and H 2 S production 16 . All API 20 E results were consistent with FSL L7-0020 T 16 . Nitrogen reduction was evaluated using both the API 20E kits and nitrogen broth; both strains were negative. The API 50 CH kit is designed for the study of carbohydrate and carbohydrate-derivative metabolism and API 50 CHB/E medium is designed for use with Bacillus and related genera, Enterobacteriaceae, and Vibrionaceae. Results for this test were consistent between UTK C1-0015, UTK C1-0024 and FSL L7-0020 T (Supplementary  Table S3), with four differences. UTK C1-0015 yielded a negative result for D-lactose, a result that differs from UTK C1-0024, FSL L7-0020 T , and most sensu stricto Listeria species 16 . Both strains tested negative for glycerol and starch (amidon); this differed from the type strain 16 , which is positive for both. UTK C1-0024 was positive for d-trehalose fermentation, while UTK C1-0015 and the type strain were negative. Examination of the genomes shows that a locus containing three genes associated with trehalose fermentation (treR, treC, and treP) is present in UTK C1-0024, but absent in the two other genomes. In L. monocytogenes, trehalose has been shown to increase biofilm formation 56 . The API 50CH test is a qualitative test and interpretation of results can vary, which is one major limitation of qualitative tests.
The complete lysis of red blood cells, β hemolysis, is associated with pathogenicity in Listeria spp. 7 On SBA, UTK C1-0015 and UTK C1-0024 were non-hemolytic, which is consistent with the non-hemolytic FSL L7-0020 T 16 and the negative control L. innocua ATCC 33090. β hemolysis is typically only observed in L. monocytogenes, L. ivanovii, and L. seeligeri 7,45 .
When observed microscopically, both UTK C1-0015 and UTK C1-0024 appeared motile at 25 °C and nonmotile at 37 °C (Supplementary Table S3). Motility at 25 °C was confirmed with MTM tubes; both strains were clearly motile after 5 days of incubation as evidenced by an umbrella-shaped growth pattern, characteristic of motile Listeria spp. These results were consistent with FSL L7-0020 T 16 and other sensu stricto species, with the exception of L. immobilis (non-motile at 25 °C 10 ). In L. monocytogenes, motility genes like flagellin are expressed at lower temperatures like 25 °C, but become restricted at 37 °C 57 .

Conclusions
In this study, we described two strains isolated from soil samples collected in the GSMNP, which belong to the recently proposed novel species "L. swaminathanii". By the addition of two additional strains to this species (bringing the total number described to three), the diversity of this species can be further evaluated and the characteristics of these two strains can be compared to those of the type strain FSL L7-0020 T . Additionally, we were able to provide complete closed genomes for both strains, including the plasmid found in UTK C1-0015,  License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.