Listeria tempestatis sp. nov. and Listeria rocourtiae
subsp. hofi subsp. nov.

Phillip Brown, Alexandra Moura, Guillaume Valès, Nathalie Tessaud-Rita, Jeffrey Niedermeyer, Cameron Parsons, Alexandre Leclercq, Angela Harris, Ryan Emanuel, Sophia Kathariou, et al.

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To cite this version:

Phillip Brown, Alexandra Moura, Guillaume Valès, Nathalie Tessaud-Rita, Jeffrey Niedermeyer, et al. Listeria tempestatis sp. nov. and Listeria rocourtiae subsp. hofi subsp. nov.. International Journal of Systematic and Evolutionary Microbiology, 2025, 75 (5), pp.006774. 10.1099/ijsem.0.006774. pasteur-05303919

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*Listeria tempestatis* sp. nov. and *Listeria rocourtiae* subsp. *hofi* subsp. nov.

Phillip Brown¹,†, Alexandra Moura²,³,†, Guillaume Valès², Nathalie Tessaud-Rita², Jefffrey Niedermeyer⁴, Cameron Parsons⁴, Alexandre Leclercq², Angela Harris⁵, Ryan E. Emanuel⁶, Sophia Kathariou¹,⁴,*† and Marc Lecuit²,³,⁷,*‡

**Abstract**

In September 2018, Hurricane Florence resulted in major flooding in North Carolina, USA. Efforts to isolate *Listeria monocytogenes* and other *Listeria* spp. from Hurricane Florence floodwaters repeatedly yielded non-haemolytic *Listeria*-like isolates that could not be readily assigned to known *Listeria* taxa. Whole-genome sequence analyses against the 28 currently known *Listeria* species confirmed that the isolates constitute two new taxa within the genus *Listeria*. Taxon I, with one isolate, showed the highest similarity to *Listeria goaensis*, with an average nucleotide identity BLAST of 85.3±4.4% and an in silico DNA-DNA hybridization (isDDH) of 32.4% (range: 30–35%), differing from the latter by its ability to reduce nitrate, ferment D-ribose and sucrose, and by its inability to produce catalase or ferment D-trehalose and D-lactose. Taxon II, represented by 11 isolates, showed the highest similarity to *Listeria rocourtiae*, with an average nucleotide identity BLAST of 92.64±3.8% and an isDDH of 49.9% (range: 47.3–52.5%), differing from the latter by its ability to ferment L-arabinose and its inability to ferment L-rhamnose, D-galactose, D-lactose and D-melibiose. The names *Listeria tempestatis* sp. nov. and *Listeria rocourtiae* subsp. *hofi* subsp. nov. are proposed for taxon I and II, respectively, with type strains CLIP 2022/01175T (F6L-1A=CIP 112444T = DSM 117029T) and CLIP 2022/01000T (F66L-1A=CIP 112443T = DSM 117030T), respectively. Both taxa lack known *Listeria* pathogenic islands, suggesting a lack of pathogenicity for humans.

INTRODUCTION

The genus *Listeria* belongs to the phylum *Bacillota*, consisting of Gram-positive, rod-shaped bacteria that are typically catalase positive, oxidase negative and have a low G+C content [1]. Currently, 28 *Listeria* species have been described, with 22 identified only since 2010, largely due to increased efforts to isolate *Listeria* from diverse sources, especially aquatic ecosystems, the increased availability of whole-genome sequencing and the use of whole-genome sequence data in taxonomic classification. The genus *Listeria* can be partitioned into two major clades [2, 3]: (i) *Listeria sensu stricto*, with ten species (*L. monocytogenes* [4], *L. innocua* [5], *L. welshimeri* [6], *L. seeligeri* [6], *L. ivanovii* [7], *L. marthii* [8], *L. cossartiae* [9], *L. farberi* [9], *L. immobilis* [9] and ‘*L. swaminathanii*’ [10]) and (ii) *Listeria sensu lato*, containing 18 species (*L. grayi* [11], *L. rocourtiae* [12], *L. weihenstephanensis* [13], *L. aquatica* [14], *L. cornellensis* [14], *L. fleishchmannii* [14], *L. floridensis* [14], *L. grandensis* [14], *L. riparia* [14], *L. booriae* [15], *L. newyorkensis* [15], *L. costaricensis* [16], *L. goaensis* [17], *L. thailandensis* [18], *L. valentina* [19], *L. rustica* [9], *L. portnoyi* [9] and *L. ilorinensis* [20]). All species are

**Author affiliations:** ¹Department of Plant and Microbial Biology, North Carolina State University, Raleigh, North Carolina, USA; ²Institut Pasteur, Université Paris Cité, *Listeria* National Reference Center and WHO Collaborating Center, Paris, France; ³Institut Pasteur, Université Paris Cité, Inserm U1117, Biology of Infection Unit, Paris, France; ⁴Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State University, Raleigh, North Carolina, USA; ⁵Department of Civil, Construction and Environmental Engineering, North Carolina State University, Raleigh, North Carolina, USA; ⁶Nicholas School of the Environment, Durham, North Carolina, USA; ⁷Necker-Enfants Malades University Hospital, Division of Infectious Diseases and Tropical Medicine, APHP, Institut Imagine, Paris, France.

*Correspondence: Sophia Kathariou, skathar@ncsu.edu; Marc Lecuit, marc.lecuit@pasteur.fr

**Keywords:** average nucleotide identity (ANI); Firmicutes; *in silico* DNA-DNA hybridization (isDDH); *Listeria*; new taxa; whole-genome sequencing.

**Abbreviations:** AAI, average amino acid identity; ANI, average nucleotide identity; BHI, brain heart infusion; BLAST, basic local alignment search tool; CAMP, Christie, Atkins, Munch-Petersen test; CIP, Collection of Institut Pasteur; CLIP, Collection *Listeria* Institut Pasteur; DDBJ, DNA Data Bank of Japan; DSMZ, German Collection of Microorganisms and Cell Cultures; EMBL, European Molecular Biology Laboratory; isDDH, *in silico* DNA-DNA hybridization; N50, minimum contig length covering 50% of the genome; PCR, polymerase chain reaction; PI-PLC, phosphatidylinositol-specific phospholipase C; UPGMA, unweighted pair group method with arithmetic mean.

The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences and draft genomes of the type strains are OR195332 and CAUYTK01 (*L. tempestatis* sp. nov. CLIP 2022/01775T) and OR195334 and CAUYTL01 (*L. rocourtiae* subsp. *hofi* subsp. nov. CLIP 2022/01000T), respectively.

†These authors contributed equally to this work

‡These authors share senior authorship.

Two supplementary figures and one supplementary table are available with the online version of this article.
006774 © 2025 The Authors

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non-pathogenic except for *L. monocytogenes*, which is considered the only human pathogen in the genus *Listeria*, and *L. ivanovii*, which predominantly infects ruminants and, rarely, humans [21]. *L. monocytogenes* represents an important concern for food safety and public health due to the severe symptoms and high case-fatality and hospitalization rates of listeriosis [2, 22].

In September 2018, Hurricane Florence caused massive flooding of many inland rivers and other locations in eastern North Carolina, USA, a largely rural region highly dense in facilities producing and processing food animals, especially swine and poultry [23, 24]. Samples from Hurricane Florence floodwaters were collected and analysed for *L. monocytogenes* and other *Listeria* species, as well as other bacterial pathogens [25, 26]. Several *Listeria*-like isolates that could not be readily assigned to published *Listeria* taxa were identified. Here, genotypic and phenotypic analyses were conducted to designate appropriate taxonomic classifications for 12 of these isolates.

# METHODS

## Bacterial strains and growth conditions

Unless stated otherwise, all isolates were grown in brain heart infusion (BHI) at 37°C for 24h, and strains used as controls were *L. booriae* CLIP 2016/00711, *L. ivanovii* subsp. *ivanovii* CLIP 12510T, *L. ivanovii* subsp. *londonensis* CLIP 12229T, *L. valentina* CLIP 2019/00642T (DSM 110544'), *L. thailandensis* CLIP 2015/00305T (DSM 107638') and/or *L. monocytogenes* CLIP 74910 (ATCC 19115), as described below. *L. rocourtiae* CIP 109804T and *L. goaensis* CLIP 2022/00569T (KCTC 33909T), the closest species to the new taxa, were included in all experiments.

Floodwater samples were analysed for *Listeria* spp. as previously described [27]. Briefly, for primary enrichments, 1.25 ml of the water samples were mixed with 11.25 ml of half Fraser broth supplemented with half Fraser selective supplement (Oxoid, Hampshire, UK) and incubated at 30°C for 24–48 h. Secondary enrichments consisted of 100 µl of the primary enrichment in 10 ml of full Fraser broth with full Fraser selective supplement (Oxoid) and were incubated at 37°C for 48 h. The primary and secondary enrichments (20 µl) were plated on modified Oxford medium (MOX; Becton, Dickinson and Co., Sparks, MD, USA) and incubated at 37°C for 48 h. Colonies typical of *Listeria* spp. (round, black in colour, smooth, flat or slightly convex in shape) were purified on blood agar plates (trypticase soy agar with 5% sheep blood; Remel, San Diego, CA, USA) and analysed via multiplex PCR to determine tentative serogroup designations as described [28, 29]. Twelve *Listeria*-like isolates that failed to yield an amplicon were sent to the World Health Organization Collaborating Centre *Listeria* (Institut Pasteur, Paris, France) for identification and further characterization.

## Species identification, genome sequencing and analysis

Initial efforts for species identification employed the MALDI-TOF mass spectrometry Sirius system and the MBT library version 12.0.0.0 (Bruker Daltonics, Bremen, Germany) [30]. For DNA sequence-based identification, genomic DNA was extracted using the NucleoSpin Tissue purification kit (Macherey-Nagel, Düren, Germany) from 0.9 ml of cultures grown overnight in brain heart infusion (BHI, Difco, France) at 35°C. DNA libraries were prepared using the Nextera XT DNA Sample kit (Illumina, California, USA) and sequenced with Illumina NextSeq 500 using a 2x150 bp paired-end run. Raw reads were trimmed with *fqCleanER* v.21.10 (https://gitlab.pasteur.fr/GIPhy/fqCleanER) as previously described [31] and assembled with *SPAdes* v.3.14 [32] with the automatic *k-mer*, --only-assembler and --careful options. Contigs longer than 300 bp were annotated with *Prokka* v.1.14 [33], and screening for plasmids utilized MOB-suite v.2.0.1 [34]. In addition, the 16S rRNA gene was amplified with primers 27F (5'-AGAGTTTGATC MTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') [35] and amplicons were Sanger sequenced.

Phylogenetic analyses were performed based on the nucleotide sequences of the 16S rRNA genes and on the core genome alignment of all *Listeria* species, defined using Roary v.3.13 [36] and a BLASTP identity cut-off of 80% to identify orthologs [20]. Maximum likelihood phylogenetic trees were constructed using IQ-Tree v.2.2 [37], and the best fit substitution models (TPM3u+F+I+G4 for 16S rRNA and GTR+F+I+G4 for the core genome) were identified using ModelFinder [38] and visualized in *MEGA* v.7.0 [39]. The average nucleotide identity (ANIb) shared with other *Listeria* species was determined using the enveomics package [40], with the *BLASTN* settings defined as in *JSpecies* v1.2.1 and *Goris et al.* [41]. Dendrograms based on the unweighted pair group method with arithmetic mean (UPGMA) method were obtained from the distance matrices using *BioNumerics* v.7.6 (Applied Maths, Belgium). The average *in silico* DNA-DNA hybridization (*isDDH*) similarities were calculated using the GGDC 2.1 web server and formula 2 [sum of all identities found in high-scoring segment pairs (HSP) divided by overall HSP length] [42]. *In silico* PCR-serogrouping profiles were obtained from the draft genomes as described previously [28, 36].

## Phenotypic characterization

Gram staining was performed with the Color Gram 2 kit (bioMérieux, Marcy l'Etoile, France), according to the manufacturer's instructions. Catalase and oxidase activities were determined using the API ID Color Catalase kit (bioMérieux) and the Bactident Oxidase test strips (Merck Millipore, France), respectively, according to the manufacturer's instructions. Respiratory characteristics were determined in tubes containing meat liver agar (Bio-Rad, France) at 30°C after 24 h, following the manufacturer's instructions. The presence of a capsule was assessed using India ink staining. Growth characteristics were determined on BHI agar and broth at 4°C for 10 days and at 22, 37 and 42°C for 7 days. Growth was considered positive if there was an increase in cell number of at least
1.0 log (c.f.u./ml⁻¹). Isolates were grown on Rapid'L.mono' agar (Bio-Rad) and Agar *Listeria* according to Ottaviani and Agosti (ALOA; bioMérieux) plates at 30 and 37°C for 24 h.

Motility was tested by stab inoculation in U-shaped glass tubes containing tryptic soy semi-solid agar (Bio-Rad), followed by incubation at 22 and 37°C for 7 days in aerobic conditions. Haemolytic activity was assessed both by growth on sheep blood agar plates as described above and by stabbing the isolates into Columbia agar plates (bioMérieux) containing 5% defibrinated horse blood, as described in the *Bacteriological Analytical Manual* [43], followed by incubation for 24 h at 37°C. The Christie, Atkins, Munch-Petersen (CAMP) test was performed as previously described, by streaking isolates and controls horizontally on Columbia agar containing 5% defibrinated sheep blood (bioMérieux), together with *Rhodococcus equi* NCTC 1621, which was streaked vertically. *L. ivanovii* was used as a positive control, while *L. rocourtiae* and *L. goaensis* were used as negative controls. Plates were incubated at 37°C for 24 h and examined for enhanced haemolysis at the confluence of the horizontal and vertical streaks.

Nitrate reduction was determined by inoculation in nitrate broth (bioMérieux) and incubation at 37°C for 5 days, with *L. rocourtiae* used as a positive control, while *L. goaensis* and *L. monocytogenes* were used as negative controls. Carbohydrate fermentation profiles were determined with API *Listeria* and the API50CH system (bioMérieux), following the manufacturer's recommendations, using *L. monocytogenes*, *L. rocourtiae* and *L. goaensis* as controls and following incubation at 37°C for 24 h. The production of acetoin from glucose fermentation (Voges-Proskauer test) was assessed using API20E strips (bioMérieux) and recorded after incubation at 37°C for 24 h. *L. monocytogenes*, *L. booriae*, *L. rocourtiae* and *L. goaensis* were used as controls.

## Antimicrobial susceptibility

Susceptibility to antimicrobials was determined with the disc diffusion method on Mueller–Hinton agar plates (Bio-Rad) at 30°C, following the recommendations from the European Committee on Antibiotic Susceptibility Testing (EUCAST, https://www.eucast.org/). The following antibiotic discs (Bio-Rad) were used: amoxicillin (25 µg), ampicillin (10 µg), cefotaxime (30 µg), chloramphenicol (30 µg), ciprofloxacin (5 µg), clindamycin (2 µg), erythromycin (15 µg), fosfomycin (50 µg), fusidic acid (10 µg), gentamicin (15 µg), imipenem (10 µg), kanamycin (30 µg), levofloxacin (5 µg), penicillin G (6 µg), moxifloxacin (5 µg), nalidixic acid (30 µg), rifampicin (30 µg), streptomycin (10 µg), sulphonamides (200 µg), tetracycline (30 µg), trimethoprim (5 µg) and vancomycin (30 µg). The diameters of inhibition zones were measured with the automatic reader Scan 4000 (Interscience, France).

## RESULTS AND DISCUSSION

### MALDI-TOF identification

Species identification efforts using the MALDI-TOF mass spectrometry Sirius system and the MBT library version 12.0.0.0 [30] were inconclusive for nine isolates (scores <2.00, indicative of *Listeria* spp.) and indicative of *L. newyorkensis* for three isolates (scores 2.02–2.05).

### Genome sequence and analyses

Sequence metrics of the 12 isolates (Table 1) met the quality standards for taxonomic determination [44]. Draft assemblies ranged between 2.8 and 3.4 Mb and had a G+C content between 38.6 and 40.6 mol% (Table 1), similar to other species of the genus *Listeria* (2.6 and 3.5 Mb, 35.9 and 43.6 mol% G+C). No plasmids were detected in any of the 12 isolates. Sequence-based serogrouping by analysis of the multiplex PCR target sequences revealed the *prs* gene (serogroup L, typical of non-*L. monocytogenes* species and certain uncommon *L. monocytogenes* serotypes) in both new taxa. The 16S rRNA gene sequence similarity of the two taxa (Suppl. Fig. 1, available in the online version of this article) was 100% with *L. rocourtiae* and 99.4% with *L. fleischmannii*, thus above the proposed cut-off of 98.7–99.0% [45], below which strains do not belong to the same species, and confirming the lack of adequate resolution of 16S rRNA gene sequence analysis for species delimitation.

Core genome analyses based on the 305 genes present in 95–100% of *Listeria* species representatives (Fig. 1) showed the highest sequence similarity of the two taxa with *L. rocourtiae* and *L. goaensis*. Isolates shared less than 95% ANIb and 70% *isDDH* (Table 1 and Suppl. Fig. 2), the proposed genomic cut-offs for species identification [44], with all known species. The mostly closely related species to the first taxon (11 isolates: CLIP 2022/01000T, CLIP 2022/01784, CLIP 2022/01786, CLIP 2022/01787, CLIP 2022/01789, CLIP 2022/01790, CLIP 2022/01795, CLIP 2022/01796, CLIP 2022/01797, CLIP 2022/01798 and CLIP 2022/01799) with a validly published name was *L. rocourtiae* (CLIP 2022/01000T: two-way ANIb of 92.10±4.78%, based on 14,248 genome fragments; *isDDH* of 49.9% [47.3–52.5%]) and the most closely related species to the second taxon (one isolate: CLIP 2022/01775T) was *L. goaensis* (CLIP 2022/01775T: two-way ANIb of 85.26±4.36% based on 12,669 genome fragments; *isDDH* of 32.4% [30.0–35.0%]). These results confirmed that the 12 isolates constitute two distinct novel taxa within the *Listeria* genus, for which the names *Listeria rocourtiae* subsp. *hofi* subsp. nov. (11 isolates) and *Listeria tempestatis* sp. nov. (1 isolate) are proposed.

### Phenotypic characterisation

*L. tempestatis* sp. nov. and all isolates of *L. rocourtiae* subsp. *hofi* subsp. nov. were Gram-positive bacilli without endospores or capsules, facultative anaerobic and oxidase negative. On Rapid'L.mono' agar, colonies were white, indicating no phosphatidylinositol-specific
<table><caption>**Table 1.** Genome metrics of isolates obtained in this study</caption><thead><tr><th>Isolate*</th><th>Alias</th><th>Geographic location</th><th>Geographic coordinates</th><th>Isolation date</th><th>No. of reads</th><th>Coverage (x)</th><th>No. of contigs</th><th>Total length (bp)</th><th>N50</th><th>GC%</th><th>Closest species</th><th>ANIB with closest species</th><th>iSDH with closest species</th><th>SRA accession no.</th><th>BIGSdb id</th></tr></thead><tbody><tr><td>L. tempestatis sp. nov.</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>CLIP</td><td>F6L-1A</td><td>Grifton, NC, USA</td><td>35.38 N, -79.45 W</td><td>2018-09-21</td><td>5.94E+06</td><td>318</td><td>38</td><td>2.83E+06</td><td>3.04E+05</td><td>38.6</td><td>L. goensis</td><td>85.3±4.36% (30.0-35.0%)</td><td>32.4% (47.3-52.5%)</td><td>ERR12145462</td><td>98880</td></tr><tr><td>L. rocourtiae subsp. hofi subsp. nov.</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>CLIP</td><td>F66L-1A</td><td>Fairmont, NC, USA</td><td>34.55 N, -79.19 W</td><td>2018-10-18</td><td>9.58E+06</td><td>449</td><td>124</td><td>3.28E+06</td><td>6.60E+04</td><td>40.5</td><td>L. rocourtiae</td><td>92.1±4.8% (49.9% (46.9-52.2%))</td><td>49.5% (46.7-52.0%)</td><td>ERR12145451</td><td>84502</td></tr><tr><td>CLIP</td><td>F61L-2A</td><td>Lumberton, NC, USA</td><td>34.62 N, -79.01 W</td><td>2018-10-18</td><td>5.92E+06</td><td>277</td><td>112</td><td>3.24E+06</td><td>6.66E+04</td><td>40.6</td><td>L. rocourtiae</td><td>92.0±4.92% (49.5%)</td><td>49.5% (46.7-52.2%)</td><td>ERR12145452</td><td>98889</td></tr><tr><td>CLIP</td><td>F67L-1A</td><td>Fairmont, NC, USA</td><td>34.53 N, -79.17 W</td><td>2018-10-18</td><td>6.18E+06</td><td>290</td><td>111</td><td>3.30E+06</td><td>6.72E+04</td><td>40.6</td><td>L. rocourtiae</td><td>92.1±4.83% (49.4%)</td><td>49.4% (46.7-52.0%)</td><td>ERR12145453</td><td>98891</td></tr><tr><td>CLIP</td><td>F67L-2A</td><td>Fairmont, NC, USA</td><td>34.53 N, -79.17 W</td><td>2018-10-18</td><td>5.62E+06</td><td>263</td><td>104</td><td>3.23E+06</td><td>7.07E+04</td><td>40.5</td><td>L. rocourtiae</td><td>92.2±4.53% (49.9%)</td><td>49.9% (47.3-52.6%)</td><td>ERR12145454</td><td>98892</td></tr><tr><td>CLIP</td><td>F70L-1A</td><td>Goldsboro, NC, USA</td><td>35.38 N, -77.82 W</td><td>2018-10-18</td><td>5.52E+06</td><td>259</td><td>104</td><td>3.34E+06</td><td>7.33E+04</td><td>40.6</td><td>L. rocourtiae</td><td>92.1±4.86% (49.4%)</td><td>49.4% (46.7-52.0%)</td><td>ERR12145455</td><td>98894</td></tr><tr><td>CLIP</td><td>F70L-2A</td><td>Goldsboro, NC, USA</td><td>35.38 N, -77.82 W</td><td>2018-10-18</td><td>6.08E+06</td><td>285</td><td>352</td><td>3.36E+06</td><td>7.47E+04</td><td>40.5</td><td>L. rocourtiae</td><td>92.2±4.47% (49.9%)</td><td>49.9% (47.3-52.6%)</td><td>ERR12145456</td><td>98895</td></tr><tr><td>CLIP</td><td>F79L-2A</td><td>Goldsboro, NC, USA</td><td>35.38 N, -77.87 W</td><td>2018-10-18</td><td>5.92E+06</td><td>277</td><td>115</td><td>3.30E+06</td><td>7.34E+04</td><td>40.6</td><td>L. rocourtiae</td><td>92.0±4.93% (49.4%)</td><td>49.4% (46.8-52.0%)</td><td>ERR12145457</td><td>98900</td></tr><tr><td>CLIP</td><td>F84L-1A</td><td>Goldsboro, NC, USA</td><td>35.38 N, -77.87 W</td><td>2018-10-18</td><td>6.51E+06</td><td>305</td><td>101</td><td>3.29E+06</td><td>7.41E+04</td><td>40.6</td><td>L. rocourtiae</td><td>92.0±4.86% (49.4%)</td><td>49.4% (46.8-52.0%)</td><td>ERR12145458</td><td>98901</td></tr><tr><td>CLIP</td><td>F86L-2A</td><td>Piney Green, NC, USA</td><td>35.11 N, -78.48 W</td><td>2018-10-18</td><td>6.25E+06</td><td>293</td><td>103</td><td>3.23E+06</td><td>7.83E+04</td><td>40.5</td><td>L. rocourtiae</td><td>92.3±4.46% (49.9%)</td><td>50.0% (47.3-52.6%)</td><td>ERR12145459</td><td>98902</td></tr><tr><td>CLIP</td><td>F89L-2A</td><td>Comfort, NC, USA</td><td>35.00 N, -77.48 W</td><td>2018-10-18</td><td>6.43E+06</td><td>301</td><td>112</td><td>3.30E+06</td><td>6.72E+04</td><td>40.6</td><td>L. rocourtiae</td><td>92.0±4.91% (49.4%)</td><td>49.4% (46.8-52.0%)</td><td>ERR12145460</td><td>98903</td></tr><tr><td>CLIP</td><td>F101L-2A</td><td>Northwest, NC, USA</td><td>34.32 N, -78.18 W</td><td>2018-10-19</td><td>4.45E+06</td><td>208</td><td>110</td><td>3.29E+06</td><td>6.62E+04</td><td>40.6</td><td>L. rocourtiae</td><td>92.0±4.90% (49.4%)</td><td>49.4% (46.8-52.0%)</td><td>ERR12145461</td><td>98904</td></tr></tbody></table>

<footnote>*CLIP, Collection Listeria Institut Pasteur.</footnote>
<figure><img src="image_6.png" /><figcaption>**Fig. 1.** Maximum likelihood phylogenetic analysis based on the core gene alignment of 35 *Listeria* reference strains and the 12 isolates of this study. Distance estimation was obtained by the GTR+F+I+G4 model [38], based on an alignment of 80,539 positions. Branch lengths represent the number of nucleotide substitutions per site, and bootstrap percentages of 1,000 replicates are shown. GenBank accession numbers are provided in brackets. *Listeria tempestatis* sp. nov. and *Listeria rocourtiae* subsp. *hofi* subsp. nov. are highlighted in bold.</figcaption></figure>

phospholipase C (PI-PLC) activity, surrounded by a yellow halo (D-xylose fermentation). On ALOA, colonies were blue (β-glucosidase activity) without halos (no PI-PLC activity).

Neither *L. tempestatis* sp. nov. nor any of the isolates of *L. rocourtiae* subsp. *hofi* subsp. nov. were motile at 22 or 37°C, consistent with the absence of motility genes in their genomes. *L. tempestatis* sp. nov. and all isolates of *L. rocourtiae* subsp. *hofi* subsp. nov. were also negative for haemolysis and the CAMP tests, consistent with the absence of known *Listeria* pathogenicity islands within their draft genomes, as observed in all other *Listeria sensu lato* species.
Similarly to most *Listeria* species except *L. costaricensis* and *L. ilorinensis*, all isolates of *L. rocourtiae* subsp. *hofi* subsp. nov. were catalase positive. In contrast, and differing in this regard from most *Listeria* species, including its closest relative *L. goaensis*, *L. tempestatis* sp. nov. was catalase negative, consistent with the absence of catalase genes in its genome.

*L. tempestatis* sp. nov. was able to grow at all tested temperatures except at 4°C, similarly to its closest species, *L. goaensis*. On the other hand, all isolates of *L. rocourtiae* subsp. *hofi* subsp. nov. were able to grow at all tested temperatures except at 42°C, similarly to *L. rocourtiae* subsp. *rocourtiae* subsp. nov.

Biochemical characterization of *L. rocourtiae* subsp. *hofi* subsp. nov. and *L. tempestatis* sp. nov. is summarized in Suppl. Table 1. *L. tempestatis* sp. nov. was able to reduce nitrate, contrary to its closest species, *L. goaensis*. All isolates of *L. rocourtiae* subsp. *hofi* subsp. nov. were able to reduce nitrate, similarly to *L. rocourtiae* subsp. *rocourtiae* subsp. nov.

Carbohydrate fermentation profiles play an important role in *Listeria* characterization, as these profiles help differentiate between species based on metabolic capabilities. *L. tempestatis* sp. nov. was able to ferment amygdalin, arbutin, D-cellobiose, D-xylose, aesculin ferric citrate, D-fructose, gentiobiose, D-glucose, D-maltose, D-mannitol, methyl-α-D-glucopyranoside, D-mannose, N-acetylglucosamine, L-rhamnose, D-ribose, D-saccharose, salicin and xylitol. D-Arabitol fermentation was variable depending on substrate concentration [negative in API50CH (1.4 mg) but positive in API *Listeria* (0.4 mg); Suppl. Table 1].

All isolates of *L. rocourtiae* subsp. *hofi* subsp. nov. were able to ferment the following substrates: amygdalin, arbutin, D-cellobiose, D-xylose, aesculin ferric citrate, D-fructose, gentiobiose, D-glucose, L-arabinose, D-maltose (weak), methyl-α-D-glucopyranoside, D-mannose, N-acetylglucosamine, D-ribose, salicin and D-trehalose. Similarly to certain *Listeria sensu lato* species, including *L. booriae*, *L. rocourtiae* and *L. goaensis*, the Voges-Proskauer test was negative for *L. tempestatis* sp. nov. and for all isolates of *L. rocourtiae* subsp. *hofi* sp. nov.

UPGMA analysis of all species phenotypic traits also showed that *L. tempestatis* sp. nov. is closest to *L. goaensis*, differing from the latter only by its ability to ferment D-ribose and sucrose and its inability to ferment D-trehalose or D-lactose, as well as being positive for nitrate reductase and negative for catalase (Table S1). All isolates of *L. rocourtiae* subsp. *hofi* subsp. nov. differ from *L. rocourtiae* subsp. *rocourtiae* subsp. nov. by their ability to ferment L-arabinose and inability to ferment L-rhamnose, D-galactose, D-lactose and D-melibiose. These specific differences in carbohydrate metabolism are important species-differentiating factors and should be considered for precise identification in the absence of whole-genome sequencing.

*L. tempestatis* sp. nov. was sensitive to all tested antibiotics, except amoxicillin, ampicillin, cefotaxime and penicillin, which resulted in no detectable inhibition halo. All isolates of *L. rocourtiae* subsp. *hofi* subsp. nov. were sensitive to all tested antibiotics, except to clindamycin and fosfomycin, for which no inhibition halo was detected. Genes conferring resistance towards antibiotics or tolerance towards disinfectants were not detected. The observed resistance traits were shared with their closest respective species, suggestive of intrinsic resistance mechanisms as reported for these antibiotics in *L. monocytogenes* [46].

In conclusion, this study reports the discovery of two novel *Listeria* taxa, *L. tempestatis* sp. nov. and *L. rocourtiae* subsp. *hofi* subsp. nov., isolated from hurricane floodwaters, expanding our understanding of the *Listeria* genus. Continued investigations into *Listeria* diversity are crucial to identify characterise new taxa, especially from previously understudied sources such as the aquatic ecosystem. Such investigations can offer insights into *Listeria* evolution, ecology and adaptations and into potential impacts of this ubiquitous genus of bacteria on public health and food and water safety, particularly in the context of current global environmental changes.

## DESCRIPTION OF LISTERIA TEMPESTATIS SP. NOV.

*Listeria tempestatis* sp. nov. (tem.pes.ta'tis. L. gen. n. tempestatis, meaning 'of a storm'; named after the hurricane floodwaters from which the strain was originally isolated).

Cells are straight, Gram-stain-positive, non-spore-forming and non-encapsulated short rods. Facultative anaerobic, catalase negative and oxidase negative. Colonies are opaque with a flat shape and entire margins on BHI. On ALOA, colonies exhibit blue colour (due to β-glucosidase activity) without a surrounding halo (lack of PI-PLC activity), typical of *Listeria* non-haemolytic species. Growth occurs at 22-42°C, with optimal growth between 22 and 37°C. Non-motile at both 22 and 37°C. Negative for haemolysis and nitrite reduction but positive for nitrate reduction. Voges-Proskauer test negative. After 24 h, acid is produced from amygdalin, arbutin, D-cellobiose, D-xylose, aesculin ferric citrate, D-fructose, gentiobiose, D-glucose, D-maltose, methyl-α-D-glucopyranoside, D-mannose, N-acetylglucosamine, L-rhamnose, D-ribose, D-saccharose and salicin. Phenotypically, *L. tempestatis* sp. nov. can be differentiated from its current closest species, *L. goaensis*, by its ability to reduce nitrate and ferment D-ribose and sucrose, and its inability to produce catalase or ferment D-trehalose.

The type strain CLIP 2022/01775 (F6L-1A) was isolated on 18 October 2018 from floodwaters produced by Hurricane Florence in eastern North Carolina, USA. The genomic DNA G+C content of the type strain is 38.64 mol%. The type strain is deposited at the Leibniz Institute DSMZ - German Collection of Microorganisms (DSM 117029T) and the Collection of Institut Pasteur (CIP 112444T). The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequence and the draft genome of the type strain are OR195332 and CAUYTK01, respectively.
## DESCRIPTION OF LISTERIA ROCOURTIAE SUBSP. ROCOURTIAE SUBSP. NOV.

*Listeria rocourtiae* subsp. *rocourtiae* nov. (ro.cour'ti.ae. N.L. fem. gen. n. *rocourtiae*, named after the French bacteriologist Jocelyne Rocourt).

General characteristics have been previously described [12]. Cells are Gram-stain-positive, non-spore-forming short rods. Facultative anaerobic, non-haemolytic, catalase positive, nitrate reduction positive and oxidase negative. Colonies are round with a low convex and entire margin on trypto-casein-soy agar when incubated at 30°C. Growth occurs at 4-37°C. After 24 h, acid is produced from L-galactose, D-glucose, glycerol, D-lactose, D-maltose, D-mannitol, D-melibiose, methyl α-D-glucopyranoside, L-rhamnose, D-ribose, D-xylose, N-acetylglucosamine, amygdalin, arbutin, D-cellobiose, D-fructose, D-mannose and salicin.

The type strain is strain CIP 109804T (=DSM 22097T =Allerberger 700284/02T).

## DESCRIPTION OF LISTERIA ROCOURTIAE SUBSP. HOFI SUBSP. NOV.

*Listeria rocourtiae* subsp. *hofi* subsp. nov. (ho'fi N.L. masc. n. *hofi*, named in honour of Dr Herbert Hof, for his research and other pioneering contributions on *Listeria monocytogenes* and listeriosis).

Cells are straight, Gram-stain-positive, non-spore-forming and non-encapsulated short rods. Facultative anaerobic, catalase positive and oxidase negative. Colonies are opaque with a flat shape and entire margins on BHI. On ALOA, colonies exhibit blue colour (due to β-glucosidase activity) without a surrounding halo (lack of PI-PLC activity), typical of non-haemolytic *Listeria* species. Growth occurs at 4-37°C, with optimal growth at 30°C. Non-motile at both 22 and 37°C. Negative for haemolysis and nitrite reduction but positive for nitrate reduction. Voges-Proskauer test negative.

After 24 h, acid is produced from amygdalin, arbutin, D-cellobiose, D-xylose, aesculin ferric citrate, D-fructose, gentiobiose, D-glucose, L-arabinose, D-maltose (weak), methyl-α-D-glucopyranoside, D-mannose, N-acetylglucosamine, D-ribose, salicin and D-trehalose. D-Arabitol fermentation is variable depending on substrate concentration, being negative in API50CH (1.4 mg) but positive in API *Listeria* (0.4 mg). The production of acid from glycerol is variable among the isolates. Phenotypically, all isolates of *L. rocourtiae* subsp. *hofi* subsp. nov. can be differentiated from *L. rocourtiae* subsp. *rocourtiae* subsp. nov. by their ability to ferment L-arabinose and inability to ferment L-rhamnose, D-galactose, D-lactose and D-melibiose.

The type strain CLIP 2022/01000 (F66L-1A) was isolated on 18 October 2018 from floodwaters produced by Hurricane Florence in eastern North Carolina, USA. The genomic DNA G+C content of the type strain is 40.48 mol%. The type strain is deposited at the Leibniz Institute DSMZ - German Collection of Microorganisms (DSM 117030T) and the Collection of Institut Pasteur (CIP 112443T). The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequence and draft genome of the type strain are OR195334 and CAUYTL01, respectively.

### Funding information

This work was supported by Institut Pasteur, Inserm and Santé Publique France. This work was partially supported by the International Life Sciences Institute (ILSI) North America Food Microbiology Committee. ILSI North America had no role in the design, analysis, interpretation or presentation of the data and results. Partial support was also provided by the College of Agriculture and Life Sciences and the Department of Food, Nutrition and Bioprocessing Sciences at North Carolina State University, Raleigh, NC USA and by award 2018-07464 from the USDA National Institute of Food and Agriculture. Any opinions, findings, conclusions or recommendations expressed are those of the authors and do not necessarily reflect the view of the USDA.

### Acknowledgements

The authors wish to thank the P2M platform (Institut Pasteur, Paris, France) for genome sequencing and the Leibniz Institute DSMZ-German Collection for Microorganisms and Cell Cultures GmbH (Braunschweig, Germany) and the Collection of Institut Pasteur (Paris, France) for depositing the type strains in their collections.

### Conflicts of interest

The authors declare no conflict of interests.

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