Noncontiguous finished genome sequence and description of Streptococcus timonensis sp. nov. isolated from the human stomach.

Strain Marseille-P2915T, a Gram-positive, facultative anaerobic and nonmotile coccus, was isolated from the gastric lavage of a patient with severe anaemia. The 16S rRNA and rpoB gene comparison exhibited a sequence identity of 98.7 and 92.6% with Streptococcus infantis strain JCM 10157T, respectively, collocating it within the 'Streptococcus mitis' group. On the basis of phenotypic and genomic analysis, we propose the validation of the type strain Streptococcus timonensis sp. nov. Marseille-P2915T (= DSM 103349 = CSUR P2915).

Introduction

The genus Streptococcus comprises 116 officially recognized species (http://www.bacterio.net/) partially clustered into six species group (pyogenic, anginosus, mitis, salivarius, bovis and mutans) on the basis of systematic 16S rRNA sequence study [1]. The exact taxonomic classification of each species within this genus remains challenging, especially in the mitis group, as a result of a high genetic and phenotypic similarity shared within different species [2], in particular with different species sharing a 16S rRNA sequence identity greater than 98.7% [3]. Because species belonging to this group gather together highly virulent species involved in pathologies as meningitis, endocarditis, pneumonia and low-pathogenic commensal species, the rapid identification of clinical isolates is mandatory. To reach this goal, several different molecular targets have been tested, including sodA [4], rnpB [5], tuf [6] and groEL [7]. The use of rpoB was initially proposed in our laboratory [8]. Since then, it has been validated by its use in the classification of different new streptococcal species [9], [10], [11].

Nowadays, the widespread use of matrix-assisted desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) in the clinical and research setting, coupled with the rapid development of next-generation sequencing technology, gives us a new and more complete insight into the taxonomy of the Streptococcus genus. In this context, a polyphasic approach to describe new bacterial species, termed taxonogenomics, was proposed in our laboratory in 2004, combining common phenotypic tests such as API strips, whole genomic sequencing and MALDI-TOF MS spectra [12].

Here we present a phenotypic and genetic comparison of 16S rRNA genes, rpoB genes as well as whole-genomic-level analysis which led us to propose the validation of Streptococcus timonensis strain Marseille-P2915T (= DSM 103349 = CSUR P2915) as a new member within the genus Streptococcus.

Materials and Methods

Sample collection

In the context of a study aimed at the description of different gut microflora levels by culturomics [13], a gastric lavage sample from a 60-year-old patient was collected during a gastroscopy performed for medical reasons (severe anaemia). The patient had been receiving long-term proton pump inhibitor therapy. Informed and signed consent, approved by the Institut Fédératif de Recherche IFR48 (Faculty of Medicine, Marseille, France), under agreement 09-022, was obtained from the patient. After collection, the sample was immediately placed in an antioxidant transport medium [14] and plated within 2 hours.

Isolation and identification of strain

Strain Marseille-P2915T's first growth was obtained in April 2016 on 5% sheep's blood–enriched Columbia agar medium (bioMérieux, Marcy l'Etoile, France) under aerobic conditions at 37°C. Once isolated on pure culture, proteomic analysis was carried out with MALDI-TOF MS as previously described [15], [16] using a Microflex spectrometer (Bruker Daltonics, Bremen, Germany). Strain Marseille-P2915T spectra was thus obtained, imported into the MALDI BioTyper software (version 3.0, Bruker) and processed by standard pattern matching (with default parameter settings) against the main spectra of 7567 bacteria. The comparison with the BioTyper database spectra enabled the rapid matching of the analysed species with those present in the database. The resulting score, if >2, enabled the identification at the species level, while a score of <1.7 did not enable any identification.

To obtain the 16S rRNA sequence of strain Marseille-P2915T, PCR analysis was performed using a GeneAmp PCR System 2720 thermal cycler (Applied Biosystems, Bedford, MA, USA) as previously described [17]. The sequencing step was performed on an ABI Prism 3130xl Genetic Analyser capillary sequencer (Applied Biosystems). Chromas Pro 1.34 software (Technelysium, Tewantin, Australia) was used to fix the sequences, and a BLASTn (Basic Local Alignment Search Tool) search [18] was performed against the National Center for Biotechnology Information (NCBI) database (default parameters, uncultured/environmental sample sequences excluded).

The rpoB gene sequence of strain Marseille-P2915T was obtained using the 1730_F and 3700_R primers as previously described [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19]. Then a BLAST search was performed against the NCBI database using default parameters.

Phylogenetic analysis

On the basis of the BLAST research results, 16S rRNA reference sequences (http://www.bacterio.net/streptococcus.html) of the closest species with standing in nomenclature were downloaded from NCBI. Because there is no officially recognized reference for rpoB, gene sequences were downloaded directly from the NCBI database after the BLASTn search. Sequences were then aligned using Muscle v3.8.31 with default parameters, and phylogenetic inferences were obtained using the maximum-likelihood method with 1000 bootstrap replicates within MEGA6 software.

Phenotypic and biochemical characterization

Growth conditions

Growth of the strain was tested on sheep's blood–enriched Columbia agar (bioMérieux) under anaerobic conditions using anaeroGEN (Oxoid, Basingstoke, UK), microaerophilic (CampyGen, Oxoid) and aerobic conditions. Different growth temperatures (20, 28, 37, 45 and 55°C) were also tested. The acceptance limit of salinity by strain Marseille-P2915T was tested on Columbia agar using 10, 15 and 20% of NaCl concentrations. Moreover, seven pHs were tested: 5.0; 5.5; 6.0; 6.5; 7.0; 7.5 and 8.

Microscopy

The Gram coloration was performed using color Gram 2 kit (bioMérieux) and observed using the DM1000 photonic microscope (Leica Microsystems, Wetzlar, Germany) with a 100× oil-immersion objective lens. The ability to produce spores was studied by thermal shock (80°C during 20 minutes). The motility test was performed by observing fresh colonies between blades and slats using a DM1000 photonic microscope (Leica Microsystems) with a 100× oil-immersion objective lens. Transmission electron microscopic images were obtained using a Tecnai G20 (FEI Company, Limeil-Brevannes, France) at an operating voltage of 200 keV. Briefly, cells were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer for at least 1 hour at 4°C. A drop of cell suspension was deposited for approximately 5 minutes on glow-discharged formvar carbon film on 400 mesh nickel grids (FCF400-Ni, EMS, Hatfield, PA, USA). The grids were dried on blotting paper, and cells were negatively stained for 10 seconds with 1% ammonium molybdate solution in filtered water at room temperature.

Biochemical assays

A basic biochemical study was performed using the API Gallery systems: API ZYM, API strep and API 50CH (L medium) according to the manufacturer's instructions (bioMérieux). Oxidase (Becton Dickinson, Franklin Lakes, NJ, USA) and catalase assays (bioMérieux) were done separately.

Antibiotic susceptibility

The antibiogram profile of strain Marseille-P2915T was obtained with the disk diffusion method following European Committee on Antimicrobial Susceptibility Testing (EUCAST) 2016 recommendations. Tested antibiotics included: penicillin G 10 IU, amoxicillin 25 μg, ceftriaxone 30 μg, erythromycin 15 IU, rifampicin 30 μg, sulfamethoxazole 23.75 μg, trimethoprim 1.25 μg, imipenem 10 μg and vancomycin 30 μg.

Fatty acids analysis

Cellular fatty acid methyl ester (FAME) analysis was performed by gas chromatography mass spectrometry (GC/MS). Two samples were prepared with approximately 40 mg of bacterial biomass per tube collected from several 5% sheep's blood–enriched Columbia agar plates. FAMEs were prepared as described by Sasser [20]. GC/MS analyses were carried out as previously described [21]. FAMEs were separated using an Elite 5-MS column and monitored by mass spectrometry (Clarus 500-SQ 8 S, PerkinElmer, Waltham, MA, USA). A spectral database search was performed using MS Search 2.0 operated with the Standard Reference Database 1A (National Institute of Standards and Technology, Gaithersburg, MD, USA) and the FAME mass spectral database (Wiley, Chichester, UK).

Genomic DNA extraction and genome sequencing and assembly

After a pretreatment by a lysozyme incubation at 37°C, DNA was extracted on the EZ1 biorobot (Qiagen, Germantown, MD, USA) with a EZ1 DNA tissues kit. 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 ng/μL. gDNA of strain Marseille-P2915T was sequenced with the MiSeq Technology apparatus (Illumina, San Diego, CA, USA) with the mate pair strategy. The gDNA was barcoded in order to be mixed with 11 other projects with the Nextera Mate Pair sample prep kit (Illumina).

The mate pair library was prepared with 1.5 μg of 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, Santa Clara, CA, USA) with a DNA 7500 labchip. The DNA fragments ranged in size from 1.5 to 11 kb with an optimal size of 6.920 kb. No size selection was performed, and 678.5 ng of tagmented fragments were circularized. The circularized DNA was mechanically sheared to small fragments with optima on a bimodal curve at 675 and 1445 bp on the Covaris S2 device in T6 tubes (Covaris, Woburn, MA, USA). The library profile was visualized on a High Sensitivity Bioanalyzer LabChip (Agilent Technologies), and the final concentration library was measured at 42.2 nmol/L.

The libraries were normalized at 2 nM and 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 run were performed in a single 39-hour run at a 2 × 151 bp read length. Total information of 7.9 Gb was obtained from a 863K/mm2 cluster density with a cluster passing quality control filters of 94% (15 627 000 passing filter paired reads). Within this run, the index representation for Streptococcus timonensis strain Marseille-P 2915T was determined to be 11.94%. The 1 865 795 paired reads were trimmed, then assembled in four scaffolds.

Genome annotation and comparison

Open reading frames (ORFs) were predicted using Prodigal (http://prodigal.ornl.gov/) with default parameters. However, the predicted ORFs were excluded if they spanned a sequencing gap region. The predicted bacterial protein sequences were searched against the GenBank [22] and Clusters of Orthologous Groups (COGs) databases using BLASTP. The tRNAs and rRNAs were predicted using the tRNAScan-SE [23] and RNAmmer [24] tools, respectively. Signal peptides and number of transmembrane helices were predicted using SignalP [25] and TMHMM [26], respectively. Mobile genetic elements were predicted using PHAST [27] and RAST [28]. ORFans were identified if their BLASTP E-value was lower than 1e-03 for an alignment length greater than 80 amino acids. If alignment lengths were smaller than 80 amino acids, an E value of 1e-05 was used. Such parameter thresholds have already been used in previous works to define ORFans. Artemis [29] and DNA Plotter [30] were used for data management and visualization of genomic features, respectively. The Mauve alignment tool (version 2.3.1) was used for multiple genomic sequence alignment [31].

The mean level of nucleotide sequence similarity at the genome level between strain Marseille-P2915T and other bacteria (Streptococcus oralis strain ATCC 35037 (ADMV00000000.1); Streptococcus infantis ATCC 700779 (AEVD00000000.1); Streptococcus pseudopneumoniae ATCC BAA960 (AICS00000000.1); Streptococcus sanguinis SK1 (CP000387.1); Streptococcus mitis ATCC 49456 (AEDX00000000.1); Streptococcus parasanguinis ATCC 15912 (CP002843.1); Streptococcus tigurinus strain AZ_3a (AORU00000000.1); Streptococcus pneumoniae strain R6 (AE007317.1); Streptococcus dentisani strain 7747 (CAUK00000000.1)) was estimated by average genomic identity of orthologous gene sequences (AGIOS) software [12]. Overall, this software combines two other software packages: Proteinortho [32] (to detect orthologous proteins between genomes compared two by two, then to retrieve the corresponding genes) and the Needleman-Wunsch global alignment algorithm (to determines the mean percentage of nucleotide sequence identity among orthologous ORFs). To evaluate genomic similarity within the strains, we also performed digital DNA-DNA hybridization (http://ggdc.dsmz.de/), which exhibits high correlation with DNA-DNA hybridization [33].

Results

After three failed identification attempts with MALDI-TOF MS, the reference spectrum of strain Marseille-P2915T (Fig. 1) was entered in our database and its 16S rRNA sequenced. The 16S rRNA comparison showed a sequence identity of 99.0% with Streptococcus dentisani strain 7747T and 98.8% with Streptococcus tigurinus strain AZ-3aT (Fig. 2). Because the 16S rRNA phylogenetic analysis did not distinguish within different species belonging to the Streptococcus mitis group, the rpoB gene was sequenced, resulting in a 92.6% sequence identity with the closest Streptococcus with an available rpoB sequence, Streptococcus infantis strain ATCC 700779T (Fig. 3). A gel view comparing the spectrum of strain Marseille-P2915T with other Streptococcaceae species is shown in Fig. 1(b). The Streptococcus timonensis strain Marseille-P2915T 16S rRNA accession number from European Molecular Biology Laboratory (EMBL)-European Bioinformatics Institute (EBI) is LT576411. The MALDI-TOF MS reference spectrum is available online and in the public domain (http://www.mediterranee-infection.com/article.php?laref=256&titre=urms-database).

Fig. 1

Matrix-assisted Laser desorption ionization–time of flight mass spectrometry analysis of Streptococcus timonensis strain Marseille-P2915T. (a) Reference mass spectrum from S. timonensis strain Marseille-P2915T. (b) Gel view comparing S. timonensis sp. nov. strain Marseille-P2915T spectra with other members of Streptococcus genus. Gel view displays raw spectra of loaded spectrum files arranged in pseudo-gel-like look. x-axis records m/z value. Left y-axis displays running spectrum number originating from subsequent spectra loading. Peak intensity is expressed by greyscale scheme code. Colour bar and right y-axis indicate relationship between colour in which peak is displayed and peak intensity in arbitrary units. Displayed species are indicated at left.

Fig. 2

Phylogenetic tree based on 16S rRNA showing position of Streptococcus timonensis strain Marseille-P2915T relative to other type strains within genus Streptococcus. Strains and their corresponding GenBank accession numbers for 16S rRNA genes are in parentheses. Tree was constructed by maximum likelihood method with Kimura two-parameter model and 1000 bootstrap replications using MEGA6 software and rooted by using Lactobacillus casei strain BL 23 (HM162415.1) as outgroup. Only bootstrap values of >95% are shown. Scale bar represents 0.02% nucleotide sequence divergence.

Fig. 3

Phylogenetic tree based on rpoB gene sequence showing position of Streptococcus timonensis strain Marseille-P2915T relative to other strains within genus Streptococcus. Strains and their corresponding GenBank accession numbers for rpoB genes are in parentheses. Tree was constructed using maximum likelihood method with Kimura two-parameter model and 1000 bootstrap replications using MEGA6 software and rooted by using Enterococcus hirae strain ATCC 29187 (HQ611249.1) as outgroup. Only bootstrap values of >95% are shown. Scale bar represents 0.02% nucleotide sequence divergence.

On sheep's blood–enriched Columbia agar, after 48 hours of aerobic incubation, cells formed 0.5 to 1 mm punctiform, greyish, α-hemolytic colonies with undulated edges. Growth was achieved at all tested pHs (5.0; 5.5; 6.0; 6.5 and 7.0; and 7.5 and 8), whereas no growth was registered in any salt-containing medium (>5 g/L of NaCl). Growth was obtained from 20 to 37°C under aerobic, anaerobic, and microaerophilic conditions, whereas no growth was obtained at 45 or 55°C. Cells were Gram-positive cocci (Fig. 4(a)) with a mean diameter of 0.6 μm (range, 0.4–0.8 μm) (Fig. 4(b)). Catalase and oxidase tested negative. The sporulation and motility tests were negative. Table 1 summarizes the classification and main features of strain Marseille-P2915T.

Fig. 4

Phenotypic features of Streptococcus timonensis strain Marseille-P2915T. (a) Gram staining of S. timonensis strain Marseille-P2915T. (b) Transmission electron microscopy of S. timonensis strain Marseille-P2915T using Tecnai G20 (FEI Company) at operating voltage of 200 keV. Scale bar = 200 nm.

Table 1

Classification and general features of strain Streptococcus timonensis Marseille-P2915T

Property	Term
Classification	Domain Bacteria
	Phylum Firmicutes
	Class Bacilli
	Family Streptococcaceae
	Genus Streptococcus
	Species Streptococcus timonensis
	Type strain Marseille-P2915T
Gram stain	Positive
Cell shape	Cocci
Motility	Nonmotile
Sporulation	Non–spore forming
Temperature range	Mesophile
Optimum temperature	37°C
Habitat	Human gut
Oxygen requirement	Facultative anaerobe
Biotic relationship	Free-living
Pathogenicity	Unknown
Geographical location	Marseille
Sample collection	April 2016
Latitude	43.296346° N
Longitude	5.369889° E

Using an API 20 strip gallery, positive reactions were recorded only for leucyl-aminopeptidase and acid production from starch. Negative reactions were recorded for: Voges-Proskauer reaction, hippuric acid hydrolysis, esculin hydrolysis, pyrrolidinyl arylamidase, α-galactosidase, β-glucuronidase, β-galactosidase, alkaline phosphatase and arginine hydrolase; fermentation reactions were negative for d-ribose, l-arabinose, d-mannitol, d-sorbitol, d-lactose, d-trehalose, inuline and d-raffinose. Using an API ZYM strip, positive reactions were observed for esterase (C4) and leucine arylamidase. Negative reactions were recorded for alkaline phosphatase, acid phosphatase, α-galactosidase, β-galactosidase, β-glucosidase, N-acetyl-β-glucosaminidase and naphthol-AS-BI-phosphohydrolase, lipase (C14), esterase lipase (C8), valine arylamidase, cystine arylamidase, trypsin, α-chymotrypsin, β-glucuronidase, α-glucosidase, α-fucosidase and α-mannosidase. Using an API 50 CH strip, positive reactions were observed for d-glucose, d-fructose, d-maltose and d-saccharose. Negative reactions were recorded for d-ribose, l-arabinose, d-xylose, d-mannose, inositol, glycerol, arbutin, d-tagatose, d-arabinose, d-fucose, N-acetylglucosamine, salicin, d-cellobiose, d-trehalose, amidone, erythritol, l-xylose, d-adonitol, methyl- βd-xylopyranoside, d-galactose, l-sorbose, l-rhamnose, dulcitol, d-sorbitol, methyl-αd-mannopyranoside, methyl-αd-glucopyranoside, esculin, d-lactose, d-melibiose, inulin, d-melezitose, d-mannitol, amygdalin, d-raffinose, glycogen, xylitol, gentiobiose, d-turanose, d-lyxose, l-fucose, d-arabitol, l-arabitol, potassium gluconate, potassium 2-ketogluconate and potassium 5-ketogluconate. Strain Marseille-P2915T resulted susceptible to penicillin, ceftriaxone, imipenem, vancomycin and rifampicin. Susceptibility to erythromycin was classified as intermediate. Strain Marseille-P2915T was tested resistant to trimethoprim-sulfamethoxazole. Biochemical characteristics that differentiate strain Marseille-P2915T from other related species within the family Streptococcaceae are summarized in Table 2. The major fatty acid is hexadecanoic acid (43%). Abundant fatty acids were either saturated or unsaturated. Moreover, several branched fatty acids were also described at lower abundances. A complete report of fatty acid analysis of strain Marseille-P2915T is reported in Table 3.

Table 2

Differential characteristics of Streptococcus timonensis strain Marseille-P2915T compared to closely related strains

Trait	1	2	3	4	5	6	7	8
Amygdalin	−	−	−	−	−	−	+	−
Arbutin	−	−	−	−	−	−	+	−
Inulin	−	−	−	(+)	−	−	−	−
Lactose	−	+	+	+	(+)	+	−	+
Maltose	+	+	+	+	+	(+)	+	+
Raffinose	−	(+)	+	−	−	−	(+)	+
Ribose	−	−	+	−	−	−	−	−
Starch	(+)	−	−	−	−	−	(+)	+
Trehalose	−	−	−	+	(+)	−	+	v
α-Galactosidase	−	(+)	−	−	−	−	−	−
Alkaline phosphatase	−	(+)	−	+	−	−	−	−
VP-reaction	−	−	−	−	−	−	+	−
β-Glucosidase	−	−	−	−	(+)	−	+	−
Leucine-aminopeptidase	+	NA	NA	NA	NA	NA	NA	+
Cellobiose	−	−	−	−	−	−	+	−
Fructose	+	(+)	+	+	+	+	−	+
Galactose	−	+	+	+	+	+	−	+
Gentiobiose	−	−	−	−	−	−	+	−
Glucose	+	−	+	+	+	+	−	+
Hippuric acid	−	−	−	−	−	+	−	−
Mannose	−	(+)	(+)	+	+	+	−	+
Methyl β-d-xylopyranoside	−	−	+	+	+	+	−	−
Sucrose	+	(+)	+	+	+	+	−	+

Strains: 1, Streptococcus timonensis strain Marseille-P2915T; 2, S. dentisani strain 7747T; 3, S. mitis ATCC 49465T; 4, S. oralis ATCC35037T; 5, S. sanguinis ATCC 10556T; 6, S. infantis JCM 10157T; 7, S. salivarius ATCC 7073T; 8, S. tigurinus Az_3aT; data for strains 2 to 7 are from [34], while data for strain 8 are derived from [35].

+, positive reaction; −, negative reaction; (+), weakly positive reaction; NA, not available; v, variable.

Table 3

Fatty acid profiles of Streptococcus timonensis strain Marseille-P2915T

Fatty acid	Name	Mean relative %
16:0	Hexadecanoic acid	43.3 ± 1.5
18:1n9	9-Octadecenoic acid	19.5 ± 0.4
18:0	Octadecanoic acid	14.1 ± 0.1
14:0	Tetradecanoic acid	8.5 ± 0.1
18:2n6	9,12-Octadecadienoic acid	6.0 ± 0.3
18:1n7	11-Octadecenoic acid	2.7 ± 0.4
18:1n5	13-Octadecenoic acid	1.5 ± 0.3
17:0	Heptadecanoic acid	1.0 ± 0.2
12:0	Dodecanoic acid	1.0 ± 0.1
15:0	Pentadecanoic acid	TR
16:1n7	9-Hexadecenoic acid	TR
13:0	Tridecanoic acid	TR
17:1n7	10-Heptadecenoic acid	TR
13:0 iso	11-Methyl-dodecanoic acid	TR
16:1n5	11-Hexadecenoic acid	TR
16:1n9	7-Hexadecenoic acid	TR
17:0 anteiso	14-Methyl-hexadecanoic acid	TR
15:0 iso	13-Methyl-tetradecanoic acid	TR
17:0 iso	15-Methyl-Hexadecanoic acid	TR

TR, trace amounts <1%.

Genome properties

The draft genome of the strain Marseille-P2915T is 1 925 331 bp long (Fig. 5) with a 38.56% G+C content. It is composed of four scaffolds (comprising four contigs). Among the 2056 predicted genes, 1974 were protein-coding genes and 82 were RNAs (six genes are 5S rRNA, six genes are 16S rRNA, six genes are 23S rRNA and 64 genes are tRNA genes). A total of 1436 genes (72.75%) were assigned a putative function (by COGs or by NR BLAST). Twenty-two genes were identified as ORFans (1.11%). The remaining genes were annotated as hypothetical proteins (426 genes ≥21.58%). Genomic statistics are reported in Table 4, while Table 5 lists the distribution of genes into COGs functional categories (Fig. 5). The genome sequence has been deposited in EMBL-EBI under accession number FMIX01000000.

Fig. 5

Graphical circular map of genome of strain Streptococcus timonensis Marseille-P2915T. From outside to centre: contigs (red/grey), COGs category of genes on forward strand (three circles), genes on forward strand (blue circle), genes on reverse strand (red circle), COGs category on reverse strand (three circles), GC content.

Table 4

Nucleotide content and gene count levels of the genome of Streptococcus timonensis strain Marseille-P2915T

Attribute	Genome (total)
	Value	% of total
Size (bp)	1 925 331	100
G+C content (%)	742 420	38.56
Coding region (bp)	1 731 074	89.9
Total genes	2056	100
RNA genes	82	3.98
Protein-coding genes	1974	96.01
Genes with function prediction	1436	72.75
Genes assigned to COGs	1221	61.85
Genes with peptide signals	182	9.21
Genes with transmembrane helices	445	22.54
Genes associated to PKS or NRPS	5	0.25
Genes associated to ORF	22	1.11
Genes associated to mobilome	887	44.93
Genes associated to toxin/antitoxin	72	3.64
Genes associated to resistance genes	0	0
Genes associated to virulence	422	21.37
Genes associated to bacteriocin	22	1.11
Genes with paralogues (E value: 1e-10)	289	14.64
Genes with paralogues (E value: 1e-25)	170	8.61
Genes associated to hypothetical proteins	426	21.58
Genes larger than 5000 nucleotides	6	0

COGs, Clusters of Orthologous Groups database; NRPS, nonribosomal peptide synthase; ORF, open reading frame; PKS, polyketide synthase.

Table 5

Number of genes associated with 26 general COGs functional categories

Code	Value	% of totala	Description
J	189	9.57	Translation
A	0	0	RNA processing and modification
K	93	4.71	Transcription
L	76	3.85	Replication, recombination and repair
B	0	0	Chromatin structure and dynamics
D	26	1.31	Cell cycle control, mitosis and meiosis
Y	0	0	Nuclear structure
V	42	2.12	Defense mechanisms
T	54	2.73	Signal transduction mechanisms
M	68	3.44	Cell wall/membrane biogenesis
N	9	0.45	Cell motility
Z	0	0	Cytoskeleton
W	2	0.10	Extracellular structures
U	17	0.86	Intracellular trafficking and secretion
O	55	2.78	Posttranslational modification, protein turnover, chaperones
C	34	1.72	Energy production and conversion
X	15	0.75	Mobilome: prophages, transposons
G	99	5.01	Carbohydrate transport and metabolism
E	122	6.18	Amino acid transport and metabolism
F	72	3.64	Nucleotide transport and metabolism
H	57	2.88	Coenzyme transport and metabolism
I	50	2.53	Lipid transport and metabolism
P	64	3.24	Inorganic ion transport and metabolism
Q	18	0.91	Secondary metabolites biosynthesis, transport and catabolism
R	105	5.31	General function prediction only
S	90	4.55	Function unknown
—	753	38.14	Not in COGs

COGS, Clusters of Orthologous Groups database.

Total is based on the total number of protein-coding genes in the annotated genome.

Comparison with other genomes

The draft genome sequence of strain Marseille-P2915T (1925 Mb) is smaller than that of Streptococcus pseudopneumoniae (2086 Mb), Streptococcus tigurinus (2185 Mb), Streptococcus pneumoniae (2161 Mb), Streptococcus parasanguinis (2154 Mb) and Streptococcus sanguinis (2303 Mb), but larger than that of Streptococcus dentisani (1884 Mb), Streptococcus infantis (1857 Mb), Streptococcus mitis (1916 Mb) and Streptococcus oralis (1914 Mb). The G+C content of S. timonensis (38.5%) is smaller than that of S. dentisani (41.1%), S. pseudopneumoniae (39.8%), S. infantis (38.9%), S. mitis (41.1%), S. tigurinus (40.3%), S. pneumoniae (39.7%), S. parasanguinis (41.7%), S. sanguinis (43.2%) and S. oralis (41.4%).

The gene content of strain Marseille-P2915T (1.974 genes) is smaller than that of S. pseudopneumoniae (2.113), S. tigurinus (2.114), S. pneumoniae (2.125), S. parasanguinis (2.022) and S. sanguinis (2.268), but larger than that of S. dentisani (1.797), S. infantis (1.876), S. mitis (1.793) and S. oralis (1.847). Within species with standing in nomenclature, AGIOS values ranged from 82.23% between S. mitis and S. oralis to 58.20% between S. sanguinis and S. infantis (Table 6). Genome-to-Genome Distance Calculator (GGDC) values [36] ranged from 59.1 ± 2.8 between S. pneumoniae and S. pseudopneumoniae to 23.1 ± 2.3 between S. sanguinis and S. parasanguinis (Table 7). Compared to the closest phylogenetic neighbour according to rpoB tree (Streptococcus infantis ATCC 700779T, Fig. 3), the probability that both are same species is lower than 5% (1.35%, GGDC 2.1 formula 2, http://ggdc.dsmz.de/).

Table 6

Number of orthologous proteins shared between Streptococcus genomes (upper right), average percentage similarity of nucleotides 502 corresponding to orthologous proteins shared between genomes (lower left) and number of proteins per genome (bold)

	S. oralis	S. infantis	S. pseudopneumoniae	S. sanguinis	S. mitis	S. parasanguinis	S. tigurinus	S. pneumoniae	S. timonensis	S. dentisani
S. oralis	1847	1280	1290	1246	1449	1241	1377	1290	1285	1401
S. infantis	67.65	1876	1214	1145	1272	1199	1260	1203	1297	1278
S. pseudopneumoniae	67.39	67.27	2113	1163	1299	1200	1277	1325	1220	1292
S. sanguinis	6078	58.20	61.42	2268	1245	1235	1203	1176	1158	1238
S. mitis	82.23	65.24	65.83	62.81	1793	1254	1355	1307	1285	1392
S. parasanguinis	62.65	62.01	61.30	62.62	62.75	2022	1231	1213	1212	1248
S. tigurinus	75.57	68.32	67.28	59.43	69.27	62.57	2114	1268	1263	1369
S. pneumoniae	71.68	66.61	68.25	62.63	72.49	63.40	70.54	2125	1210	1285
S. timonensis	65.09	64.76	65.47	73.09	67.07	63.17	62.27	67.09	1974	1274
S. dentisani	69.44	61.94	67.78	73.88	71.69	63.24	66.11	69.08	81.41	1797

Table 7

Pairwise comparison of Streptococcus timonensis Marseille-P2915T with nine other Streptococcus species using GGDC, formula 2 (DDH estimates based on identities/HSP length)

	S. timonensis	S. oralis	S. infantis	S. pseudopneumoniae	S. sanguinis	S. mitis	S. parasanguinis	S. tigurinus	S. pneumoniae	S. dentisani
S. timonensis	100% ± 00	25.3% ± 2.8	37.4% ± 2.5	26.2% ± 2.6	25.2% ± 2.0	26.0% ± 2.4	26.0% ± 2.4	25.2% ± 2.4	25.8% ± 2.4	25.2% ± 2.4
S. oralis		100% ± 00	25.8% ± 2.4	31.8% ± 2.4	23.7% ± 1.9	31.9% ± 2.4	24.8% ± 2.3	49.8% ± 2.6	31.6% ± 2.4	44.8% ± 2.5
S. infantis			100% ± 00	26.4% ± 2.4	24.5% ± 2.4	26.2% ± 2.4	26.5% ± 2.4	25.7% ± 2.4	25.7% ± 2.4	25.5% ± 2.4
S. pseudopneumoniae				100% ± 00	24.2% ± 2.4	48.5% ± 2.6	25.1% ± 2.4	31.6% ± 2.4	59.1% ± 2.8	31.5% ± 2.4
S. sanguinis					100% ± 00	23.5% ± 2.4	23.1% ± 2.3	23.1% ± 2.3	23.3% ± 2.3	23.4% ± 2.4
S. mitis						100% ± 00	24.2% ± 2.4	31.7% ± 2.6	46.7% ± 2.5	31.7% ± 2.5
S. parasanguinis							100% ± 00	24.5% ± 2.4	25.6% ± 2.4	23.6% ± 2.4
S. tigurinus								100% ± 00	31.8% ± 2.4	43.1% ± 2.5
S. pneumoniae									100% ± 00	31.3% ± 2.4
S. dentisani										100% ± 00

DDH, DNA-DNA hybridization; GGDC, Genome-to-Genome Distance Calculator; HSP, high-scoring segment pairs.

Distribution of functional classes of predicted genes according to the COGs categories was realized for the closest species of strain Marseille-P2915T (Fig. 6) and showed a similar profile.

Fig. 6

Distribution of functional classes of predicted genes according to clusters of orthologous groups of proteins from Streptococcus timonensis strain Marseille-P2915T.

Discussion

A strong argumentation to support the recognition of strain Marseille-P2915T as a new species within the Streptococcus genus is brought by the low value of GGDC (formula 2) obtained with other close species (highest value 37.4% with Streptococcus infantis, probability that both are the same species <5%), particularly when compared to the high value obtained within different streptococcal species with standing in nomenclature (59.1% between Streptococcus pneumoniae and Streptococcus pseudopneumoniae and 48.5% between Streptococcus mitis and Streptococcus pseudopneumoniae) (Table 7). Other evidence comes from the phenotypic analysis obtained by the API strips, which showed a unique enzymatic profile (Table 2) compared to the eight most closely related species. In the family Streptococcaceae and in particular within the ‘Streptococcus mitis’ group, it is a common feature to share a high 16S rRNA similarity [3] with other streptococcal species (Table 8). Another important point that differentiates strain Marseille-P2915T from Streptococcus dentisani and Streptococcus tigurinus (the16S rRNA closest species with standing in nomenclature) is the difference in G+C content (2.6 and 1.8%, respectively). To our knowledge, there is no officially recognized threshold for rpoB gene sequence similarity to delineate a new species, but the 92.6% value obtained with Streptococcus infantis is well below the 96.7% of the rpoB's gene similarity between Streptococcus oralis (strain KCCM 41567) and Streptococcus cristatus (strain CIP 56.62) or the 96.4% gene similarity between Streptococcus salivarius (strain 735-09) and Streptococcus thermophilus (strain CIP 105446) (Table 9).

Table 8

16S rRNA similarity within: 1, Streptococcus timonensis strain Marseille-P2915T; 2, Streptococcus dentisani strain 7747T; 3, Streptococcus pneumoniae strain ATCC 33400T; 4, Streptococcus infantis strain JCM 10157T;5, Streptococcus tigurinus strain AZ 3aT; 6, Streptococcus mitis strain NCTC 3165T; 7, Streptococcus oralis 35037T; 8, Streptococcus parasanguinis strain ATCC 15912T; 9, Streptococcus sanguinis train SK1T; 10, Streptococcus salivarius strain ATCC 7073T

	1	2	3	4	5	6	7	8	9
1	1
2	99.0	1
3	98.8	98.9	1
4	98.7	98.9	98.5	1
5	99.0	98.7	98.4	98.6	1
6	97.0	96.9	96.3	96.6	97.0	1
7	98.3	98.4	98.9	98.6	98.3	97.0	1
8	96.8	97.0	97.0	97.0	97.3	96.5	97.1
9	98.1	97.9	97.8	97.8	98.5	97.4	97.7	97.2
10	95.4	95.2	95.3	95.6	95.3	95.5	95.3	95.3	95.7

Table 9

rpoB's gene similarity within: 1, Streptococcus infantis strain ChDC B 194; 2, Streptococcus peroris strain ChDC B648; 3, Streptococcus mitis strain ChDC B183; 4, Streptococcus pneumoniae strain NCTC 7465; 5, Streptococcus cristatus train CIP 56.62; 6, Streptococcus oligofermentans strain ChDC B685; 7, Streptococcus oralis strain KCCM 41567; 8, Streptococcus pseudopneumoniae strain CIP 1086; 9, Streptococcus oligofermentans strain ChCD B689; 10, Streptococcus infantarius strain GMRS55; 11, Streptococcus thermophilus strain CIP 105446; 12, Streptococcus salivarius strain 735-09; 13, Streptococcus timonensis strain Marseille-P2915T

	1	2	3	4	5	6	7	8	9	10	11	12
1	1
2	94.1	1
3	94.0	93.7	1
4	92.5	91.9	95.8	1
5	94.0	93	95.5	94.3	1
6	94.0	92.7	94.0	94.0	94.9	1
7	93.5	93.7	94.1	93.7	96.7	94.9	1
8	92.8	92.2	96.4	95.3	95.0	93.8	94.9	1
9	92.5	92.2	94.1	93.5	94.3	95.6	95.2	94.0	1
10	90.0	89.1	89.1	87.9	88.6	88.0	88.6	88.6	88.5	1
11	88.6	89.2	88.6	88.2	88.6	88.2	88.9	87.9	87.6	90.9	1
12	30.1	90.3	90.1	88.8	89.2	88.5	89.4	89.2	88.3	91.2	96.4	1
13	93.7	92.5	92.5	91.2	92.1	91.5	91.6	90.3	90.1	88.0	87.9	88.6

Conclusion

On the basis of the phenotypic, phylogenetic and genomic analyses, we propose the validation of Streptococcus timonensis sp. nov. within the family Streptococcaceae. Strain Marseille-P2915T is the type strain, and it was isolated from human stomach.

Description of Streptococcus timonensis strain Marseille-P2915T sp. nov.

Streptococcus timonensis (ti.mon.e′n.sis, L. gen. masc. ‘originating from La Timone,’ the hospital where the sample was collected). S. timonensis is a nonmotile, non-spore-forming, facultative anaerobe and Gram-positive coccus. Growth is achieved under aerobic, anaerobic and microaerophilic atmospheres in a temperature range of 20 to 37°C and at an optimum temperature of 37°C. After 48 hours of aerobic incubation on 5% sheep's blood–enriched Columbia agar, colonies are pinpoint, greyish and α-haemolytic, with undulated edges and with a diameter of 0.5 to 1 mm. Cells are roughly round with a 0.6 μm diameter. Catalase and oxidase are negative. Streptococcus timonensis strain Marseille-P2915T exhibits positive reactions for leucyl-aminopeptidase, esterase C4 and leucine arylamidase. It is able to ferment starch, d-glucose, d-fructose, d-maltose and d-saccharose. Strain Marseille-P2915T was found to be susceptible to penicillin, ceftriaxone, imipenem, vancomycin and rifampicin. The major fatty acid is hexadecanoic acid (43%). The G+C content of the genome is of 38.56%. The 16S rRNA sequence and whole-genome shotgun sequence are deposited in EMBL-EBI under accession numbers LT576411 and FMIX01000000, respectively. The type strain is Marseille-P2915T (= CSUR P2915 = DSM 103349), and it was isolated from a human stomach sample in Marseille, France.