Phenotypic characterization and analysis of complete genomes of two distinct strains of the proposed species L. swaminathanii | Scientific Reports -…

Since 2010, there have been multiple new species added to the Listeria genus, many originally isolated from natural environments16. 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 border17. 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 L7-0020T) 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 2.78Mb chromosome and 55Kb plasmid (total genome length of 2.84Mb) with a G+C content of 38.7%; UTK C1-0024 consists of a 2.95Mb chromosome with a G+C content of 38.6% (Table 1), which is consistent with FSL L7-0020T. Of the validly published type strains, the two isolates showed highest similarity to L. marthii (94.094.1%) (Fig.1); however, they were most closely related to L. swaminathanii FSL L7-0020T, with 98.798.8% 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.

ANI Similarity Dendrogram. Average nucleotide Identity (ANI) dendrogram of the recently isolated L. swaminathanii strains (bold), along with all described Listeria spp. type strains and representative from each of the L. monocytogenes lineages (indicated in parentheses). Horizontal distance represents ANI similarity (%) and vertical dashed lines indicate ANI values of 96 (yellow), 95 (orange), and 94% (red).

Chromosomal alignment of FSL L7-0020T, UTK C1-0024, and UTK C1-0015. Alignment shows three horizontal panels, one per strain. The colored portions inside each panel represents sequence similarity, with height corresponding to average conservation at that location. Regions that are conserved among all genomes are purple. Regions that are conserved among only two of the genomes are red (FSL L7-0020T and UTK C1-0024), green (FSL L7-0020T and UTK C1-0015), or yellow (UTK C1-0024 and UTK C1-0015). Regions without coloring were not aligned and likely contain loci that are present in only a single genome.

Both genomes contained the following antibiotic resistance genes: fosX, lin, norB, and sul. Virulence-associated genes involved with adherence (dltA, fbpA, lap, lapB, pdeE), bile-resistance (bsh, mdrM), immune modulation (lntA), intracellular survival (lplA1, oppA, pdeE, prsA2, purQ, svpA), invasion (iap, lpeA, pdeE), peptidoglycan modification (oatA, pdgA), regulation of transcription and translation (agrAC, cheAY, codY, fur, lisKR, stp, virRS), surface protein anchoring (lgt, lspA, srtAB), and teichoic acid biosynthesis (gltB, gtcA) were identified in both genomes, along with internalins inlGHJK, inlC2, and inlD (Supplementary Table S1). Genes associated with Listeria pathogenicity island LIPI-3 (llsABDGPXY) were only found in UTK C1-0024 (Supplementary Fig. S1), as well as gltA (teichoic acid biosynthesis). The internalin genes inlA and inlB and genes associated with Listeria pathogenicity islands LIPI-1, LIPI-2, or LIPI-4 were not detected in either.

A 56Kb plasmid was identified in UTK C1-0015. The plasmid has an Illumina read depth of 2.2 the overall median depth, indicating a copy number of two. The plasmid found in UTK C1-0015 shows a high similarity (86.03% nucleotide identity) to pLMIV from L. monocytogenes strain FSL J1-020846,47. However, pLMIV is approximately 21Kb longer than the plasmid found in UTK C1-0015; this is due to the presence of a region encoding four complete internalins and one internalin-like protein in pLMIV, this region is absent in the plasmid in UTK C1-0015 (Fig.3). Both plasmids are also similar to the plasmid in L. monocytogenes FSL J1-0158. Both FSL J1-0208 and FSL J1-0158 were originally isolated from clinical caprine sources46. Most genes in the plasmid found in UTK C1-0015 seem to encode proteins predicted to be involved in plasmid maintenance and conjugation46, with only a few putative cargo genes, most which are of unknown function and one encoding a DNA-methyltransferase.

Comparison of plasmid found in UTK C1-0015 to plasmids from FSL J1-020 and FSL J1-158. 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).

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. monocytogenes48,49,50. 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 L7-0020T (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 strain53.

Nucleotide similarity of monocin regions. BLAST comparisons of monocin regions from L. monocytogenes 10403S, UTK C1-0015, UTK C1-0024, and the L. swaminathanii type strain FSL L7-0020T. Genes are represented by green arrows. The shaded regions represent nucleotide similarity (see scale at bottom right).

Listeria spp. grow at a wide range of temperatures from 0 to 45C7,8,14,16 and can survive at temperatures below freezing (7C)54. In the current study, we performed growth assessments at 4, 7, 22, 30, 37, and 41C. These temperatures were chosen to encompass the known growth temperature range, with 4 and 7C specifically included because some species are unable to grow well at low temperatures (<7C) 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 24h of incubation, UTK C1-0015 and UTK C1-0024 showed optimal growth at 30C (9.2 and 9.4 log10 CFU/mL), followed by at 37C (8.9 and 9.0 log10 CFU/mL). At 41C, 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 L7-0020T. At 4C, the concentration increases of UTK C1-0015 and UTK C1-0024 after 11 d (6.4 and 6.8 log10 CFU/mL, respectively) were higher than the increases seen in FSL L7-0020T (4.1 log10 CFU/mL)16.

Listeria spp. are Gram-positive rods7; this was confirmed for UTK C1-0015 and UTK C1-0024. Both isolates were observed to grow under aerobic and anaerobic conditions at 30C after 24h; this is another expected result, as Listeria spp. are facultative aerobes7. Both strains were oxidase negative (Supplementary Table S3), as expected7, 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 L7-0020T is catalase negative16, 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. 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 L7-0020T.

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 -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).

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 L7-0020T 16 and indicates an 80% (t-value of 0.62) ID to L. monocytogenes according to the APIweb database. The control strains, L. monocytogenes 10403S and L. innocua ATCC 33090, generated the expected codes of 6510 and 7510, respectively.

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 is consistent with L. monocytogenes 10403S, L. innocua ATCC 33090, and FSL L7-0020T 16 (Supplementary Table S3). UTK C1-0015 and UTK C1-0024 were negative for all other tests, including indole, urease, and H2S production 16. All API 20 E results were consistent with FSL L7-0020T 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 L7-0020T (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 L7-0020T, and most sensu stricto Listeria species16. Both strains tested negative for glycerol and starch (amidon); this differed from the type strain16, 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 formation56. 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, 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 L7-0020T 16 and the negative control L. innocua ATCC 33090. hemolysis is typically only observed in L. monocytogenes, L. ivanovii, and L. seeligeri7,45.

When observed microscopically, both UTK C1-0015 and UTK C1-0024 appeared motile at 25C and nonmotile at 37C (Supplementary Table S3). Motility at 25C was confirmed with MTM tubes; both strains were clearly motile after 5days of incubation as evidenced by an umbrella-shaped growth pattern, characteristic of motile Listeria spp. These results were consistent with FSL L7-0020T 16 and other sensu stricto species, with the exception of L. immobilis (non-motile at 25C10). In L. monocytogenes, motility genes like flagellin are expressed at lower temperatures like 25C, but become restricted at 37C57.

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Phenotypic characterization and analysis of complete genomes of two distinct strains of the proposed species L. swaminathanii | Scientific Reports -...