Introduction {#sec1}
============

As a part of the rebirth of culture, the culturomics approach has enabled the discovery of hundreds of new species isolated from human gut [@bib1], thus contributing to a dramatic increase in the repertoire of bacteria associated with humans. Taxono-genomics was recently introduced \[1\] to describe these new taxa, combining phenotypic characteristics such as mass spectrum by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) analysis, biochemical properties, and genomic characteristics including 16S rRNA gene phylogeny, DNA--DNA hybridization (DDH), and G+C content [@bib2], [@bib3]. Among strategies adopted to enhance the diversification of the specimen is to improve culturomic performances [@bib1], [@bib4]. In particular, inclusion of sputum samples allows description of the respiratory microbiota of healthy people, thus expanding the known repertoire of bacteria colonizing the respiratory tract.

We recently isolated *Enterococcus timonensis* strain Marseille-P2817^T^. This new species, belonging to the genus *Enterococcus*, was cultured from a sputum sample from a healthy man in Marseille as part of a culturomics study [@bib5]. The history of the enterococci began in 1899 with Thiercelin [@bib6], [@bib7] when they were classified in the genus *Streptococcus* (Lancefield group D) until 1984 [@bib6], [@bib7]. Based on genome analysis, *Streptococcus faecalis* and *Streptococcus faecium* have been transferred to a new genus [@bib5]. Since then, urinary tract infections, diverticulitis, bacterial endocarditis, bacteraemia, and meningitis are important clinical infections caused by *Enterococcus* spp. [@bib6], [@bib7]. However, enterococcus-associated lower respiratory tract infections are very rare [@bib8]. At the time of writing, according to the List of Prokaryotic Names with Standing in Nomenclature (LPSN; <http://www.bacterio.net>), the genus *Enterococcus* consists of 58 species and two subspecies.

Herein we present a summary classification and a set of characteristics for *Enterococcus timonensis* strain Marseille-P2817^T^ (= DSM 103162, = CSUR P2817). In addition, we propose the description of the complete genome sequence and annotation.

Materials and methods {#sec2}
=====================

Ethics and sample collection {#sec2.1}
----------------------------

In February 2016, a sputum sample was obtained from a healthy 37-year-old French adult man living in Marseille, France. Informed and signed consent was obtained from the patient and the study was approved by the Institut Fédératif de Recherche 48, Faculty of Medicine, Marseilles, France, under agreement number 2016-011.

Strain isolation and identification by MALDI-TOF MS and 16S rRNA gene sequencing {#sec2.2}
--------------------------------------------------------------------------------

Sputum samples were cultured using 18 different culture conditions of culturomics [@bib4]. Strain Marseille-P2817^T^ was isolated on 5% sheep-blood-enriched Columbia agar (bioMérieux, Marcy l**'**Étoile, France), 10 days after being cultured in a culture bottle containing a blood-enriched Columbia agar liquid medium (BACTEC™ Plus Aerobic/F Culture Vials (Becton, Dickinson and Company)) with 4 mL rumen fluid at 30°C. This bacterium was tested for identification with a Microflex spectrometer (Brüker Daltonics, Leipzig, Germany) as previously described [@bib9], [@bib10] and the 12 spectra generated were compared to the 7,567 different bacteria found in our database by standard pattern matching (for which default parameter settings were applied), using MALDI BioTyper database software (version 2.0, Brüker). A resulting score of \<1.7 didn\'t enable identification, and a 16S rRNA gene sequencing was performed as previously described [@bib11]. Codon Code Aligner software (<http://www.codoncode.com>) was used to assemble and correct sequences, and BLASTn searches were performed in the National Center for Biotechnology Information (NCBI; <http://blast.ncbi.nlm.nih.gov.gate1.inist.fr/Blast.cgi>). Concerning similarity levels, for thresholds of 98.65% and 95%, a new species or a new genus was suggested respectively as proposed by Meier-Kolthoff et al., 2013 [@bib12] and Kim et al., 2014 [@bib13].

Phylogenetic analyses {#sec2.3}
---------------------

We used a custom python script to automatically retrieve all species belonging to the family of the new species and then downloaded 16S sequences from the NCBI by parsing NCBI eUtils results and NCBI taxonomy page. Only sequences from type strains were kept. In case of multiple sequences for one type strain, the sequence with the best identity rate from the BLASTn alignment with our sequence was selected. The 16S sequences are then separated into two groups: one containing the sequences of strains from the same genus (group a) and one containing the others (group b). The 15 closest strains from group a and the closest one from group b are finally kept. If the script is unable to get 15 sequences from group a, it selects more sequences from group b to get at least nine strains from both groups.

Growth conditions {#sec2.4}
-----------------

To assess its range of growth temperatures, strain Marseille-P2817^T^ was cultured at different temperatures (25, 30, 37, 42 and 57°C) on 5% sheep-blood-enriched Columbia agar (bioMérieux) under aerobic, anaerobic and microaerophilic conditions using GENbag Anaer and GENbag miroaer systems (bioMerieux). Aerobic growth was achieved with and without 5% CO~2~. Also, a salinity test was performed at different concentrations (5, 10, 50, 75 and 100 g/L), and four different pHs (6, 6.5, 7 and 8.5) were tested.

Biochemical, sporulation and motility assays {#sec2.5}
--------------------------------------------

API Gallery systems---API ZYM, API 20 NE and API 50CH---were used to investigate biochemical analyses according to the manufacturer\'s instructions (bioMérieux). Catalase (bioMérieux) and oxidase (Becton Dickinson, Franklin Lakes, NJ, USA) tests were also performed separately. A thermal shock at 80°C for 30 min was done on bacterial colonies (diluted in phosphate-buffered saline) to check for sporulation ability in this bacterium. A DM1000 photonic microscope (Leica Microsystems, Nanterre, France) was used to assess the motility of the bacteria by observing the fresh colony between blades and slats with a 40 × objective lens.

Antibiotic susceptibility {#sec2.6}
-------------------------

The antibiotic susceptibility of strain Marseille-P2817^T^ was tested using the E-test strips method according to EUCAST 2015 recommendations (<http://www.eucast.org/>). Eighteen different antibiotics were used, including teicoplanin (TP) 0.016--256 μg/mL, daptomycin (DPC) 0.016--256 μg/mL, metronidazole (MZ) 0.016--256 μg/mL, rifampicin (RI) 0.002--32 μg/mL, imipenem (IP) 0.002--32 μg/mL, minocycline (MC) 0.016--256 μg/mL, benzylpenicillin (PG) 0.002--32 μg/mL, benzylpenicillin (PG) 0.016--256 μg/mL, vancomycin (VA) 0.016--256 μg/mL, cefotaxime (CT) 0.002--32 μg/mL, amikacin (AK) 0.016--256 μg/mL, erythromycin (EM) 0.016--256 μg/mL, ceftriaxone (TX) 0.016--256 μg/mL, amoxicillin(AC) 0.016--256 μg/mL, tobramycin (TM) 0.016--256 μg/mL, fosfomycin (FM) 0.064--1024 μg/mL, doxycycline (DC) 0.016--256 μg/mL and ertapenem (ETP) 0.002--32 μg/mL. Breakpoint tables for the interpretation of MICs and inhibition zone diameters (version 7.1, 2017) were used to interpret the results; these are available at [*http://www.eucast.org*](http://www.eucast.org){#intref0030}.

Microscopy {#sec2.7}
----------

The cells were first fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer for at least 1 h at 4°C. A drop of cell suspension was deposited for approximately 5 min on glow-discharged formvar carbon film on 400 mesh nickel grids (FCF400-Ni, EMS). The latter were then dried on blotting paper and cells were negatively stained for 10 s with 1% ammonium molybdate solution in filtered water at room temperature. We then acquire electron micrographs using a Tecnai G20 Cryo (FEI) transmission electron microscope operated at 200 keV.

Fatty acid methyl ester (FAME) analysis by gas chromatography/mass spectrometry (GC/MS) {#sec2.8}
---------------------------------------------------------------------------------------

Two samples were prepared with approximately 20 mg of bacterial biomass per tube harvested from several petri dish cultures. The method described by Sasser et al. (2006) was used to prepare FAMEs [@bib14]. We conducted GC/MS as previously described [@bib15]. Briefly, we separated FAMEs using an Elite 5-MS column monitored by mass spectrometry (Clarus 500 - SQ 8 S, Perkin Elmer, Courtaboeuf, France). We performed a spectral database search using MS Search 2.0 operated with the Standard Reference Database 1A (NIST, Gaithersburg, USA) and the FAMEs mass spectral database (Wiley, Chichester, UK).

DNA extraction and genome sequencing {#sec2.9}
------------------------------------

DNA of strain Marseille-P2817^T^ was extracted on the EZ1 biorobot (Qiagen) with EZ1 DNA tissues kit following a pretreatment by a lysozyme incubation at 37°C for 2 h. The elution volume was 50 μL. Genomic DNA (gDNA) was quantified by a Qubit assay with the high-sensitivity kit (Life technologies, Carlsbad, CA, USA) to 46.6 ng/μL. Then gDNA was sequenced by the MiSeq Technology (Illumina Inc, San Diego, CA, USA) with the mate pair strategy. The gDNA was barcoded in order to be mixed with 11 other projects using the Nextera Mate Pair sample prep kit (Illumina). We prepared the mate pair library with 1.5 μg gDNA using the Nextera mate pair Illumina guide. The gDNA sample was simultaneously fragmented and tagged with a mate pair junction adapter. The pattern of the fragmentation was validated on an Agilent 2100 BioAnalyzer (Agilent Technologies Inc, Santa Clara, CA, USA) with a DNA 7500 labchip. The size of the DNA fragments ranged from 1.5 kb to 11 kb with an optimal size of 7.710 kb. No size selection was performed, and 600 ng of tagmented fragments were circularized. The circularized DNA obtained was then mechanically sheared into small fragments with optima on a bimodal curve at 843 and 1565 bp on the Covaris device S2 in T6 tubes (Covaris, Woburn, MA, USA). We visualized the library profile on a High Sensitivity Bioanalyzer LabChip (Agilent Technologies Inc, Santa Clara, CA, USA). The final concentration library was measured at 59.91 nmol/L. The libraries were normalized at 2 nM and then pooled. After a denaturation step and dilution at 15 pM, the pool of libraries was loaded onto the reagent cartridge and then onto the instrument along with the flow cell. Automated cluster generation and sequencing runs were performed in a single 39-h run in a 2 × 151-bp. We obtained a total information of 8.3 Gb from a 910 K/mm^2^ cluster density with a cluster passing quality control filters of 92.8% (16 316 000 passing filter paired reads). We determined the index representation for strain Marseille-P2817^T^ within this run, which was of 8.06%. Finally, the 1 315 710 paired reads were trimmed and then assembled.

Genome assembly, annotation and comparison {#sec2.10}
------------------------------------------

The assembly of the genome was carried out using a pipeline combining different softwares (Velvet [@bib16], Spades [@bib17] and Soap Denovo [@bib18]) on trimmed (MiSeq and Trimmomatic [@bib19] softwares) or untrimmed data (MiSeq software only). For each of the six assemblies performed, GapCloser [@bib20] was used to reduce gaps. We identified contamination with Phage Phix (BLASTn against Phage Phix174 DNA sequence) which was then eliminated. The scaffolds (\<800 bp) were then removed, and scaffolds with a depth value \< 25% of the mean depth were removed as they were identified as possible contaminants. The best assembly was selected by using different criteria (number of scaffolds, N50, number of N). For the strain Marseille-P2817^T^, Spades gave the best assembly, with depth coverage of 239x.

We predicted open reading frames (ORFs) using Prodigal [@bib20] with default parameters, but the predicted ORFs were excluded if they spanned a sequencing gap region (contains N). We searched for the predicted bacterial protein sequences against the clusters of orthologous groups (COGs) database using BLASTP (*E*-value 1e^−03^, coverage of 0.7 and identity percentage of 30%). If no hit was found, we searched against the NR database using BLASTP with *E*-value of 1e^−03^, coverage of 0.7 and identity percentage of 30%. If sequence lengths were \<80 amino acids, an *E* value of 1e^−05^ was used. The tRNA genes and rRNAs were found using tRNAScanSE tool [@bib21] and RNAmmer [@bib22], respectively, while we predicted lipoprotein signal peptides and the number of transmembrane helices using Phobius [@bib22]. ORFans were identified if all the performed BLASTP did not give positive results (*E*-value \<1e^−03^ for ORFs with sequence size \>80 aa or *E*-value \<1e^−05^ for ORFs with sequence length \<80 amino acids). The HMMscan of the HMMER3 suite [@bib23] were used to search PFAM conserved domains (PFAM-A and PFAM-B domains) on each protein. We searched PKS and NRPS against the ClusterMine360 [@bib24] and analysed the resistome by using ARG-ANNOT database [@bib25]. Toxin and antitoxin were found using a database composed of TADB database [@bib26] and homemade toxin and antitoxin of *Rickettsia* and *Wolbachia* (which were provided by Cristina Socolovschi). The mobilome was analysed using Phantome [@bib27], ICEs (ICEberg) [@bib28], ACLAME [@bib29], GYSPSYDB [@bib30] and CRISPR [@bib31]. For the latter we used spacer and repeat database to reconstruct CRISPR module. A homemade database [@bib32] was used to find bacteriocin. Virulence factors were analysed by using both VFDB [@bib33] and MvirDB [@bib34]. Species which had to be compared were automatically retrieved from the 16s RNA tree using Phylopattern [@bib35]. For each selected species, complete genome sequence, proteome and ORFeome sequences were retrieved from the FTP of NCBI. If no complete and available genome was found for one specific strain, a complete genome of the same species was used. If ORFeome and proteome were not predicted, Prodigal was used with default parameters to predict them. Protein Ortho was used to analyse all proteomes [@bib36]. Then for each couple of genomes, a similarity score was computed. This score is the mean value of nucleotide similarity between all couples of orthologous genes between the two genomes studied (AGIOS) [@bib3]. An annotation of the entire proteome was performed to define the distribution of functional classes of predicted genes according to the COG of proteins (using the same method as for the genome annotation). Two parameters were determined to evaluate the genomic similarity among the compared strains, dDDH that exhibits a high correlation with DDH [@bib12], [@bib37] and AGIOS [@bib3] which was designed to be independent from DDH. We used the GGDC web server to perform Genome-to-Genome Distance Calculator (GGDC) [@bib12]. Annotation and comparison processes were performed in the Multi-Agent software system DAGOBAH [@bib38] that included Figenix [@bib39] libraries which provided pipeline analysis.

Results {#sec3}
=======

Strain identification and phylogenetic analysis {#sec3.1}
-----------------------------------------------

Strain Marseille-P2817^T^ ([Table 1](#tbl1){ref-type="table"}) was first isolated in March 2016 by a 10-day preincubation in a blood culture bottle supplemented with 4 mL rumen fluid. This bacterium was isolated and cultivated on 5% sheep-blood-enriched Columbia agar in an aerobic atmosphere at 30°C.Table 1Classification and general features of *Enterococcus timonensis* strain Marseille-P2817^T^Table 1PropertiesTermCurrent classificationDomain: *Bacteria*Phylum: *Firmicutes*Class: *Bacilli*Order: *Lactobacillales*Family: *Enterococcaceae*Genus: *Enterococcus*Species: *Enterococcus timonensis*Type strain: Marseille-P2817^T^Gram stainPositiveCell shapeCoccusMotilityMotileSporulationNon-sporulatingTemperature rangeMesophileOptimum temperature37°COxygen requirementFacultative anaerobeHabitatHuman lungIsolationHuman sputum

Strain Marseille-P2817^T^ was not identified by MALDI-TOF MS because its spectrum did not match any of the spectra present in our database. The 16S rRNA nucleotide sequence (accession number LT576388) exhibited 95.99% sequence identity with *Enterococcus hirae* strain ATCC9790 (Genbank accession number NR_075022), the closest validated species. Thus, we can classify strain Marseille-P2817^T^ as a new species in the *Enterococcus* genus ([Fig. 1](#fig1){ref-type="fig"}). A representative reference spectrum was therefore added to our IHU Méditerranée Infection database (<http://www.mediterranee-infection.com/article.php?laref=256&titre=urms-database>) ([Fig. 2](#fig2){ref-type="fig"}), and a gel view shows the comparison between the spectrum of strain Marseille-P2817^T^ and that of the phylogenetically closest species ([Fig. 3](#fig3){ref-type="fig"}).Fig. 1Phylogenetic tree showing the position of *Enterococcus timonensis* sp. nov. strain Marseille-P2817^T^ (= CSUR P2817, = DSM 103162) with respect to other close species. Sequences were aligned using Muscle v3.8.31 with default parameters, and phylogenetic inferences were obtained using the neighbour-joining method with 500 bootstrap replicates, within MEGA6 software. Only bootstraps \>95% were kept. Nevertheless, the scale bar represents a 0.5% nucleotide sequence divergence.Fig. 1Fig. 2Reference mass spectrum from *Enterococcus timonensis* strain Marseille-P2817^T^. Spectra from 17 individual colonies were compared and the reference spectrum was generated.Fig. 2Fig. 3Gel view comparing *Enterococcus timonensis* strain Marseille-P2817^T^ to other species within the *Enterococcus* genus. The gel view displays the raw spectra of loaded spectrum files arranged in a pseudo-gel. The x-axis records the m/z value. The left y-axis displays the running spectrum number originating from subsequent spectra loading. The peak intensity is expressed by a grey scale scheme code. The colour bar and the right y-axis indicate the relation between the peak colour and the intensity in arbitrary units. Displayed species are indicated on the left.Fig. 3

Phenotypic description {#sec3.2}
----------------------

Growth was observed at 25, 30 and 37°C under aerobic, microaerophilic and anaerobic conditions on blood-enriched Columbia agar, with optimal growth being obtained aerobically at 37°C after 24 h of incubation. Strain Marseille-P2817^T^ grew in saline conditions of 5 g/L, and weak growth was observed also at 10 g/L. The strain tolerated pH values of 6, 6.5, 7 and 8.5. The cells were motile and non-sporulating, and they formed smooth, convex, grey colonies with a mean diameter of 1 mm on blood-enriched Columbia agar. Under electron microscopy, the bacteria had a mean diameter of 0.65 μm and a length of 1.1 μm ([Fig. 4](#fig4){ref-type="fig"}).Fig. 4Electron micrographs of *Enterococcus timonensis* strain Marseille-P2817^T^, using a Tecnai G^20^ Cryo (FEI) transmission electron microscope operated at 200 keV. The scale bar represents 200 nm.Fig. 4

The major fatty acids were 9-octadecenoic acid (34%) and hexadecanoic acid (33%). Several fatty acids composed of 18 carbon atoms were also listed: 18:2n6 (16%); 18:0 (11%); 18:1n6 (1%) and 18:1n7 (TR). No branched structures were found ([Table 2](#tbl2){ref-type="table"}).Table 2Cellular fatty acid composition (%)Table 2Fatty acidsNameMean relative %[a](#tbl2fna){ref-type="table-fn"}18:1n99-Octadecenoic acid34.3 ± 0.516:0Hexadecanoic acid32.5 ± 0.518:2n69,12-Octadecadienoic acid15.6 ± 0.618:0Octadecanoic acid11.1 ± 0.314:0Tetradecanoic acid3.1 ± 0.118:1n612-Octadecenoic acid1.3 ± 0.112:0Dodecanoic acidTR18:1n711-Octadecenoic acidTR10:0Decanoic acidTR16:1n79-Hexadecenoic acidTR15:0Pentadecanoic acidTR[^1][^2]

Strain Marseille-P2817^T^ was both catalase-negative and oxidase-negative. Using API^®^ ZYM, positive reactions were recorded for alkaline phosphatase, esterase (C4), esterase lipase (C8), lipase (C14), valine arylamidase, leucine arylamidase, cystine arylamidase, α-chymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, α-galactosidase, β-glucuronidase, α-glucosidase, α-fucosidase and α-mannosidase, and negative reactions were observed for trypsin, β-galactosidase, β-glucosidase and N-acetyl-β-glucosaminidase. Using API^®^ 20NE reactions recorded were positive for urease and β-galactosidase and negative for tryptophan and arginine dihydrolase, nitrate was reduced, glucose fermented, aesculin and gelatine were hydrolysed, assimilations were reported for mannose, mannitol and potassium gluconate, but not for glucose, arabinose, N-acetylglucosamine, maltose, capric acid, adipic acid, malate, trisodium citrate and phenylacetic acid. Using API API^®^ 50CH positive reactions were recorded for glycerol, D-galactose, D-glucose, D-fructose, D-mannose, D-mannitol, N-acetylglucosamine, aesculin ferric citrate, salicin, D-cellobiose, D-maltose, D-lactose, D-saccharose and D-trehalose, and negative reactions were observed for erythritol, D-arabinose, L-arabinose, D-ribose, D-xylose, L-xylose, D-adonitol, methyl-βD-xylopyranoside, L-sorbose, L-rhamnose, dulcitol, inositol, D-sorbitol, methyl-αD-mannopyranoside, methyl-αD-glucopyranoside, amygdalin, arbutin, D-melibiose, inulin, D-melezitose, D-raffinose, amidon, glycogen, xylitol, gentiobiose, D-turanose, D-lyxose, D-tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol, potassium gluconate, potassium 2-ketogluconate and potassium 5-ketogluconate ([Table 3](#tbl3){ref-type="table"}).Table 3*Enterococcus timonensis* strain Marseille-P2817^T^ reactions results with API^®^ ZYM, API^®^ 20NE and API^®^ 50CHTable 3Oxidase---API 50CHCatalase---Glycerol+**API ZYM**Erythritol---Alkaline phosphatase+D-Arabinose---Esterase (C4)+L-Arabinose---Esterase lipase (C8)+D-Ribose---Lipase (C14)+D-Xylose---Leucine arylamidase+L-Xylose---Valine arylamidase+D-Adonitol---Cystine arylamidase+Methyl-βD-xylopyranoside---Trypsin---D-Galactose+α-Chymotrypsin+D-Glucose+Acid phosphatase+D-Fructose+Naphthol-AS-BI-phosphohydrolase+D-Mannose+α-Galactosidase+L-Sorbose---β-Galactosidase---L-Rhamnose---β-Glucuronidase+Dulcitol---α-Glucosidase+Inositol---β-Glucosidase---D-Mannitol+N-Acetyl-β-glucosaminidase---D-sorbitol---α-Mannosidase+Methyl-αD-mannopyranoside---α-Fucosidase+Methyl-α\
D-glucopyranoside---N-Acetylglucosamine+**API 20NE**Amygdalin---Reduction of nitrates+Arbutin---Indole production (tryptophan)---Aesculin ferric citrate+Fermentation (Glucose)+Salicin+Arginine dihydrolase---D-Cellobiose+Urease---D-Maltose+Hydrolysis (β-glucosidase) (aesculin)+D-Lactose+Hydrolysis (protease) (gelatin)+D-Melibiose---β-Galactosidase+D-Saccharose+Assimilation (glucose)---D-Trehalose+Assimilation (arabinose)---Inulin---Assimilation (mannose)+D-Melezitose---Assimilation (mannitol)+D-Raffinose---Assimilation (N-acetylglucosamine)---Amidon---Assimilation (maltose)---Glycogen---Assimilation (potassium gluconate)+Xylitol---Assimilation (capric acid)---Gentiobiose---Assimilation (adipic acid)---D-Turanose---Assimilation (malate)---D-Lyxose---Assimilation (trisodium citrate)---D-Tagatose---Assimilation (phenylacetic acid)---D-Fucose---L-Fucose---D-Arabitol---L-Arabitol---Potassium gluconate---Potassium 2-ketogluconate---Potassium 5-ketogluconate---

Susceptibility to antimicrobial agents was interpreted according to the EUCAST recommendations leading to the following MIC results. Cells were susceptible to teicoplanin 0.75 μg/mL, vancomycin 1 μg/mL and amoxicillin 0.038 μg/mL, and intermediate for benzylpenicillin (0.002--32 μg/mL) 0.75 μg/mL, benzylpenicillin (0.016--256 μg/mL) 1 μg/mL and imipenem 0.75 μg/mL. Cells were resistant to metronidazole 256 μg/mL, cefotaxime (0.002--32 μg/mL) 6.0 μg/mL, ceftriaxone (0.016--256 μg/mL) 16 μg/mL and ertapenem 2 μg/mL. MICs toward agents for which breakpoints do not exist are distributed as follows: daptomycin 2.0 μg/mL, rifampicin 1.0 μg/mL, minocycline 0.094 μg/mL, amikacin 48.0 μg/mL, erythromycin 0.75 μg/mL, tobramycin 3.0 μg/mL, fosfomycin 12.0 μg/mL, and doxycycline 0.38 μg/mL.

The biochemical and phenotypic features of strain Marseille-P2817^T^ were compared to the corresponding features of other close representatives of the *Enterococcus* genus ([Table 4](#tbl4){ref-type="table"}). We observed that all the species were facultatively anaerobic, Gram-positive, and positive for N-acetylglucosamine, D-glucose, D-fructose, D-maltose, D-lactose.Table 4Differential characteristics of *Enterococcus timonensis* strain Marseille-P2817^T^*, E. hirae* strain ATCC 9790 [@bib43]*, E. gallinarum* strain NBRC 100675 [@bib44], *E. saccharolyticus* strain ATCC 43076 [@bib45], [@bib46], *E. casseliflavus* strain NBRC 100478 [@bib44], *E. rotai* strain LMG 26678 [@bib47], *E. silesiacus* strain LMG 23085 [@bib48] and *E. asini* strain ATCC 700915 [@bib49]Table 4Properties*E. timonensisE. hiraeE. gallinarumE. saccharolyticusE. casseliflavusE. rotaiE. silesiacusE. asini*Cell diameter (μm)0.65--1.1nananananananaOxygen requirementFacultative anaerobeFacultative anaerobeFacultative anaerobeFacultative anaerobeFacultative anaerobeFacultative anaerobenaFacultativeGram stainPositivePositivePositivePositivePositivePositivePositivePositiveSalt requirement+++++++---MotilityMotileNon-motileNon-motilenaMotileNon-motileNon-motileNon-motileEndospore formation---na---nana---na---**Production of:**Alkaline phosphatase+---na+na---------Catalase------------------v---Oxidase---nananananananaNitrate reductase+nananana---nanaUrease---nana---na+nanaα-Glucosidase+nana---na+nanaβ-Galactosidase+++---+++---**Acid from:**N-Acetylglucosamine++++++++L-Arabinose------+---+++---D-Ribose---++---+++---D-Mannose+++na+++---D-Mannitol+---++++------Sucrose---+++++------D-Glucose++++++++D-Fructose++++++++D-Maltose+++++v++D-Lactose++++++++G+C content (mol%)38.4636.939.8036.9042.4036.736.4044.70HabitatHuman lungChicken and pig intestinesIntestines of domestic fowlsFresh broccoliPlant materialDrinking waterSurface watersCaecum of donkeys[^3]

Genome properties {#sec3.3}
-----------------

The genome of strain Marseille-P2817^T^ (genome accession no. FNVY00000000) was 2 123 933 bp long with 38.46 mol% of G+C content ([Fig. 5](#fig5){ref-type="fig"}). It is composed of four scaffolds (composed of four contigs). Along the 2048 predicted genes, 1983 were protein-coding genes and 65 were RNAs (three genes were 5S rRNA, three genes were 23S rRNA, three genes were 16S rRNA, and 56 genes were tRNA genes). Putative functions were attributed to a total of 1507 genes (76%) (by COGs or by NR blast); 112 genes (5.65%) were identified as ORFans, other genes were annotated as hypothetical proteins (261 genes ≥13.16%) ([Table 5](#tbl5){ref-type="table"}). The properties and statistics of the genome are summarized in [Table 5](#tbl5){ref-type="table"}, [Table 6](#tbl6){ref-type="table"}. The distribution of genes into COGs functional categories is presented in [Table 6](#tbl6){ref-type="table"}.Fig. 5Graphical circular map of the genome. From outside to the centre: contigs (red/grey), clusters of orthologous groups (COG) category of genes on the forward strand (three circles), genes on the forward strand (blue circle), genes on the reverse strand (red circle), COG category on the reverse strand (three circles), G+C content.Fig. 5Table 5Nucleotide content and gene count levels of genomeTable 5AttributeGenome (total)[a](#tbl5fna){ref-type="table-fn"}Number% of total[a](#tbl5fna){ref-type="table-fn"}Size (bp)2 123 933100G+C content (mol%)816 80338.46Coding region (bp)1 887 34788.86Total genes2048100RNA genes653.17Protein-coding genes1983100Genes with function prediction150776.00Genes assigned to COGs139870.50Genes with peptide signals1969.88Gene associated to bacteriocine281.41Genes associated to mobilome90545.64Genes associated to virulence43221.79Genes associated to toxine/antitoxine864.34Genes with Pfam-A domains186791ORFan genes1125.64Genes with transmembrane helices45222.79Genes associated with PKS or NRPS10.05Number of antibiotic resistance genes00[^4][^5]Table 6Number of genes associated with 25 general clusters of orthologous groups (COGs) functional categoriesTable 6CodeValue% of total[a](#tbl6fna){ref-type="table-fn"}Description\[J\]1859.33Translation\[A\]00RNA processing and modification\[K\]1075.40Transcription\[L\]894.49Replication, recombination and repair\[B\]00Chromatin structure and dynamics\[D\]221.11Cell cycle control, mitosis and meiosis\[Y\]00Nuclear structure\[V\]723.63Defence mechanisms\[T\]572.87Signal transduction mechanisms\[M\]733.68Cell wall/membrane biogenesis\[N\]90.45Cell motility\[Z\]00Cytoskeleton\[W\]20.10Extracellular structures\[U\]180.91Intracellular trafficking and secretion\[O\]512.571Post-translational modification, protein turnover, chaperones\[X\]452.27Mobilome: prophages, transposons\[C\]532.67Energy production and conversion\[G\]1768.88Carbohydrate transport and metabolism\[E\]924.64Amino acid transport and metabolism\[F\]552.77Nucleotide transport and metabolism\[H\]562.82Coenzyme transport and metabolism\[I\]432.17Lipid transport and metabolism\[P\]733.68Inorganic ion transport and metabolism\[Q\]201.01Secondary metabolites biosynthesis, transport and catabolism\[R\]1276.40General function prediction only\[S\]1035.19Function unknown---58529.50Not in COGs[^6]

Genome comparison {#sec3.4}
-----------------

The draft genome sequence of strain Marseille-P2817^T^ was compared to that of the closest species in the *Enterococcus* genus: *Enterococcus casseliflavus* strain NBRC 100478 (BCPT00000000), *Enterococcus gallinarum* strain FDAARGOS_163 (CP014067), *Enterococcus asini* strain ATCC_700915 (ASVU00000000), *Enterococcus dispar* strain ATCC_51266 (AHYR00000000), *Enterococcus saccharolyticus* subsp*. saccharolyticus* strain ATCC_43076 (ASWN00000000), *Enterococcus rotai* strain LMG_26678 (CP013655), *Enterococcus hirae* strain ATCC 9790 (CP003504), and *Enterococcus silesiacus* strain LMG_23085 (CP013614). It is smaller than the genomes of *E. silesiacus, E. rotai, E. hirae, E. asini, E. saccharolyticus* subsp. *saccharolyticus, E. dispar, E. gallinarum* and *E. casseliflavus* (2124, 3928, 3746, 2883, 2573, 2604, 2813, 3821 and 3498 MB respectively).

The G+C content of strain Marseille-P2817^T^ is lower than that of *E. asini, E. gallinarum* and *E. casseliflavus* (38.46, 44.72, 42.32 and 42.35% respectively) but larger than that of *E. silesiacus, E. rotai, E. hirae, E. saccharolyticus* subsp. *saccharolyticus* and *E. dispar* (36.41, 36.13, 36.75, 36.89 and 37.17% respectively). The protein-coding gene content of strain Marseille-P2817^T^ is smaller than that of *E. silesiacus, E. rotai, E. hirae, E. asini, E. saccharolyticus* subsp*. saccharolyticus, E. dispar, E. gallinarum* and *E. casseliflavus* (1983, 3559, 3253, 2669, 2430, 2582, 2637, 3333 and 3353 respectively).

To evaluate the genomic similarity among studied strains of the Enterococcaceae, we determined two parameters: AGIOS (average of genomic identity of orthologous gene sequences) [@bib4], which was designed to be independent from DDH, and dDDH, which exhibited a high correlation with DDH ([Table 7](#tbl7){ref-type="table"}, [Table 8](#tbl8){ref-type="table"}) [@bib25], [@bib26]. Strain Marseille-P2817^T^ shared 1049, 1176, 1215, 1064, 1117, 1159, 1065 and 1136 orthologous genes with *E. saccharolyticus, E. gallinarum, E. casseliflavus, E. rotai, E. silesiacus, E. asini, E. hirae* and *E. dispar* respectively ([Table 7](#tbl7){ref-type="table"}). AGIOS values ranged from 56.89 between *E. hirae* and *E. gallinarum* to 84.10% between *E. rotai* and *E. silesiacus* among compared species with standing in nomenclature (except strain Marseille-P2817^T^). AGIOS values of strain Marseille-P2817^T^ ranged from 58.18% with *E. rotai* to 66.31% with *E. casseliflavus* when compared to other species ([Table 7](#tbl7){ref-type="table"}). Concerning dDDH values of compared species (except strain Marseille-P2817^T^), they ranged from 20.50% (18.3--22.9) when estimated between *E. casseliflavus* and *E. saccharolyticus* to 28.50% (26.1--31%) when estimated between *E. dispar* and *E. hirae*. dDDH values of strain Marseille-P2817^T^ ranged from 20.00% (17.8--22.4%) with *E. saccharolyticus* to 25.80% (23.5--28.3%) with *E. dispar* when compared to other species ([Table 8](#tbl8){ref-type="table"}). Those values are under the 70% threshold, thus confirming the new species status [@bib40], [@bib41], [@bib42]. Furthermore, the distribution of genes into COGs categories was similar in all compared genomes ([Fig. 6](#fig6){ref-type="fig"}).Table 7Numbers of orthologous proteins shared between genomes (upper right), average percentage similarity of nucleotides corresponding to orthologous proteins shared between genomes (lower left), and numbers of proteins per genome (bold)Table 7*E. saccharolyticusE. gallinarumE. timonensisE. casseliflavusE. rotaiE. silesiacusE. asiniE. disparE. hiraeE. saccharolyticus***2582**14791049152813711423129513361235*E. gallinarum*60.09**3333**1176198015571636145615191436*E. timonensis.*60.1858.36**1983**121510641117115911361065*E. casseliflavus*61.6372.8366.31**3353**157116521,51415761464*E. rotai*61.2958.6158.1858.58**3253**1748130813581307*E. silesiacus*61.1958.4358.6959.0884.10**3559**139314461350*E. asini*59.0361.8860.0662.0556.9857.49**2430**14231252*E. dispar*58.2758.6959.2259.4760.2460.1461.59**2637**1352*E. hirae*6156.8958.5759.5162.4462.2157.3162.49**2669**Table 8Pairwise comparison of *Enterococcus timonensis* strain Marseille-P2817^T^ with other species using the Genome-to-Genome Distance Calculator (GGDC), formula 2 (DNA--DNA hybridization (DDH) estimates based on identities/high-scoring segment pairs (HSP) length)Table 8*E. timonensis.E. gallinarumE. saccharolyticusE. casseliflavusE. rotaiE. silesiacusE. asiniE. disparE. hiraeE. timonensis.*100% ± 0021.90% \[19.6--24.3%\]20.00% \[17.8--22.4%\]24.20% \[21.9--26.7%\]23.40% \[21.1--25.9%\]23.40% \[21.1--25.8%\]25.50% \[23.1--28%\]25.80% \[23.5--28.3%\]25.00% \[22.7--27.5%\]*E. gallinarum*100% ± 0021.20% \[19--23.7%\]21.30% \[19.1--23.8%\]21.10% \[18.9--23.6%22.70% \[20.4--25.1%\]24.20% \[21.9--26.6%\]25.30% \[23--27.8%\]25.00% \[22.6--27.4%\]*E. saccharolyticus*100% ± 0020.50% \[18.3--22.9%\]20.70% \[18.4--23.1%\]21.00% \[18.8--23.5%\]25.00% \[22.7--27.5%\]22.00% \[19.8--24.5%\]22.40% \[20.1--24.8%\]*E. casseliflavus*100% ± 0022.80% \[20.5-- 5.2%\]23.80% \[21.5--26.3%\]23.50% \[21.2--26%\]25.70% \[23.4--28.2%\]23.50% \[21.2--25.9%\]*E. rotai*100% ± 0026.90% \[24.6--29.4%\]25.20% \[22.9--27.7%\]22.40% \[20.1--24.8%\]23.40% \[21.1--25.9%\]*E. silesiacus*100% ± 0023.60% \[21.3--26%\]22.40% \[20.1--24.9%\]24.20% \[21.9--26.7%\]*E. asini*100% ± 0025.20% \[22.9--27.7%\]28.30% \[25.9--30.8%\]*E. dispar*100% \[00--00%\]28.50% \[26.1--31%\]*E. hirae*100% \[00--00%\]Fig. 6Distribution of functional classes of predicted genes in genomes from *Enterococcus timonensis, E. hirae, E. gallinarum, E. saccharolyticus, E. casseliflavus, E. rotai, E. silesiacus* and *E. asini* chromosomes according to clusters of orthologous groups of proteins.Fig. 6

Conclusion {#sec4}
==========

In conclusion, based on the phenotypic, phylogenetic and genomic analyses, we suggest the creation of a new species, *Enterococcus timonensis* sp. nov., that contains the strain Marseille-P2817^T^. This bacterial strain has been isolated from the sputum of a healthy adult man from Marseilles, France.

Taxonomic and nomenclatural proposals {#sec5}
=====================================

Description of *Enterococcus timonensis* sp. nov {#sec5.1}
------------------------------------------------

*Enterococcus timonensis* (ti.mo.nen\'sis, N.L. masc. adj., *timonensis* from the Latin name of the Hôpital de la Timone, hospital in Marseille, where strain Marseille-P2817^T^ was isolated) exhibited smooth, convex, grey colonies with a diameter of 1 mm on 5% sheep-blood-enriched Columbia agar. Cells showed a mean diameter of 0.65 μm and a length of 1.1 μm. This bacterium is a Gram-positive, non-spore-forming and motile coccus. Optimal growth was obtained aerobically at 37°C after 24 h of incubation. It is both oxidase- and catalase-negative. The reactions were positive for alkaline phosphatase, esterase (C4), esterase lipase (C8), lipase (C14), leucine arylamidase, valine arylamidase, cystine arylamidase, α-chymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, α-galactosidase, β-glucuronidase, α-glucosidase, α-mannosidase, α-fucosidase, β-galactosidase, nitrate reduction, aesculin hydrolysis, gelatine hydrolysis, assimilation of mannose, mannitol and potassium gluconate and fermentation of glycerol, D-galactose, D-glucose, D-fructose, D-mannose, D-mannitol, N-acetylglucosamine, aesculin ferric citrate, salicin, D-cellobiose, D-maltose, D-lactose, D-saccharose and D-trehalose. The major fatty acids were 9-octadecenoic acid (34%) and hexadecanoic acid (33%).

This strain exhibited a G+C genome content of 38.46 mol%. The 16S rRNA gene sequence and whole-genome shotgun sequence have been deposited in EMBL-EBI under accession numbers LT576388 and FNVY00000000, respectively. The type strain Marseille-P2817^T^ (= CSUR P2817 = DSM 103162) was isolated from the sputum of a French adult man living in Marseille.

Transparency declaration {#sec6}
========================

The authors declare no conflict of interest. This work has benefited from French State support, managed by the 'Agence Nationale pour la Recherche' including the 'Programme d'Investissement d\'avenir' under the reference Méditerranée Infection 10-IAHU-03. This work was also supported by the Région Provence-Alpes-Côte d\'Azur and Fonds Européen de Développement Regional - Plateformes de Recherche et d\'Innovation Mutualisées Méditerranée Infection (FEDER PRIMI).

The authors thank the Xegen Company ([www.xegen.fr](http://www.xegen.fr){#intref0040}) for automating the genomic annotation process. This study was funded by the Fondation Méditerranée Infection.

We thank Magdalen Lardiere for reviewing the English language.

[^1]: TR, trace amounts \<1%.

[^2]: Mean peak area percentage.

[^3]: na, data not available; v, variable.

[^4]: COG, clusters of orthologous groups; PKS, polyketide synthase; NRPS, non-ribosomal peptide-synthetase.

[^5]: Total is based on either the size of the genome (in base pairs) or the total number of protein coding genes in the annotated genome.

[^6]: The total is based on the total number of protein-coding genes in the annotated genome.
