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 .
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 , in particular with different species sharing a 16s rrna sequence identity greater than 98.7% . 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 , rnpb , tuf 
  and groel 
 .
, it has been validated by its use in the classification of different new streptococcal species , , . 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 .
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 - p2915 (= dsm 103349  =  csur p2915 ) as a new member within the genus streptococcus .
in the context of a study aimed at the description of different gut microflora levels by culturomics , a gastric lavage sample from a 60-year - old patient was collected during a gastroscopy performed for medical reasons ( severe anaemia ) .
informed and signed consent , approved by the institut fdratif 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   and plated within 2 hours .
strain marseille - p2915 's first growth was obtained in april 2016 on 5% sheep 's blood 
enriched columbia agar medium ( biomrieux , marcy l'etoile , france ) under aerobic conditions at 37c .
once isolated on pure culture , proteomic analysis was carried out with maldi - tof ms as previously described ,   using a microflex spectrometer ( bruker daltonics , bremen , germany ) .
strain marseille - p2915 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 - p2915
, pcr analysis was performed using a geneamp pcr system 2720 thermal cycler ( applied biosystems , bedford , ma , usa ) as previously described .
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   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 - p2915 was obtained using the 1730_f and 3700_r primers as previously described , , , , , , , , , , , . then a blast search was performed against the ncbi database using default parameters . 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 .
enriched columbia agar ( biomrieux ) under anaerobic conditions using anaerogen ( oxoid , basingstoke , uk ) , microaerophilic ( campygen , oxoid ) and aerobic conditions .
different growth temperatures ( 20 , 28 , 37 , 45 and 55c ) were also tested .
the acceptance limit of salinity by strain marseille - p2915 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 .
the gram coloration was performed using color gram 2 kit ( biomrieux ) and observed using the dm1000 photonic microscope ( leica microsystems , wetzlar , germany ) with a 100 oil - immersion objective lens . the ability to produce spores
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 4c .
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 .
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 ( biomrieux ) .
oxidase ( becton dickinson , franklin lakes , nj , usa ) and catalase assays ( biomrieux ) were done separately .
the antibiogram profile of strain marseille - p2915 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  g , ceftriaxone 30  g , erythromycin 15  iu , rifampicin 30  g , sulfamethoxazole 23.75  g , trimethoprim 1.25  g , imipenem 10  g and vancomycin 30  g . 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 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 ) . after a pretreatment by a lysozyme incubation at 37c , dna was extracted on the ez1 biorobot ( qiagen , germantown , md , usa ) with a ez1 dna tissues kit .
genomic dna ( gdna ) was quantified by a qubit assay with the high - sensitivity kit ( life technologies , carlsbad , ca , usa ) to 46  ng/l .
gdna of strain marseille - p2915 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  g of gdna using the nextera mate pair illumina guide .
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 
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 / mm 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 2915 was determined to be 11.94% .
the 1  865  795 paired reads were trimmed , then assembled in four scaffolds .
open reading frames ( orfs ) were predicted using prodigal ( http://prodigal.ornl.gov/ ) with default parameters .
the predicted bacterial protein sequences were searched against the genbank   and clusters of orthologous groups ( cogs ) databases using blastp .
the trnas and rrnas were predicted using the trnascan - se   and rnammer   tools , respectively .
signal peptides and number of transmembrane helices were predicted using signalp   and tmhmm , respectively .
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 .
artemis   and dna plotter   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 .
the mean level of nucleotide sequence similarity at the genome level between strain marseille - p2915 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 .
overall , this software combines two other software packages : proteinortho   ( 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 .
after three failed identification attempts with maldi - tof ms , the reference spectrum of strain marseille - p2915 ( fig . 
dentisani strain 7747 and 98.8% with streptococcus tigurinus strain az-3a ( 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 700779 ( fig .  3 ) .
a gel view comparing the spectrum of strain marseille - p2915 with other streptococcaceae species is shown in fig . 
the streptococcus timonensis strain marseille - p2915 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 ) . on sheep 's blood 
enriched columbia agar , after 48 hours of aerobic incubation , cells formed 0.5 to 1  mm punctiform , greyish , -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 37c under aerobic , anaerobic , and microaerophilic conditions , whereas no growth was obtained at 45 or 55c .
4(a ) ) with a mean diameter of 0.6  m ( range , 0.40.8  m ) ( fig . 
table  1 summarizes the classification and main features of strain marseille - p2915 . using an api 20 strip gallery ,
negative reactions were recorded for : voges - proskauer reaction , hippuric acid hydrolysis , esculin hydrolysis , pyrrolidinyl arylamidase , -galactosidase , -glucuronidase , -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 , -galactosidase , -galactosidase , -glucosidase , n - acetyl--glucosaminidase and naphthol - as - bi - phosphohydrolase , lipase ( c14 ) , esterase lipase ( c8 ) , valine arylamidase , cystine arylamidase , trypsin , -chymotrypsin , -glucuronidase , -glucosidase , -fucosidase and -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- d - xylopyranoside , d - galactose , l - sorbose , l - rhamnose , dulcitol , d - sorbitol , methyl-d - mannopyranoside , methyl-d - 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 - p2915 resulted susceptible to penicillin , ceftriaxone , imipenem , vancomycin and rifampicin .
biochemical characteristics that differentiate strain marseille - p2915 from other related species within the family streptococcaceae are summarized in table  2 .
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 - p2915 is reported in table  3 .
the draft genome of the strain marseille - p2915 is 1  925  331  bp long ( fig . 
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 ) .
genomic statistics are reported in table  4 , while table  5 lists the distribution of genes into cogs functional categories ( fig . 
the draft genome sequence of strain marseille - p2915 ( 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 - p2915 ( 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   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 700779 t , fig . 
3 ) , the probability that both are same species is lower than 5% ( 1.35% , ggdc 2.1 formula 2 , http://ggdc.dsmz.de/ ) . distribution of functional classes of predicted genes according to the cogs categories was realized for the closest species of strain marseille - p2915 ( fig . 
a strong argumentation to support the recognition of strain marseille - p2915 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   with other streptococcal species ( table  8) .
another important point that differentiates strain marseille - p2915 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 ) .
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 - p2915 is the type strain , and it was isolated from human stomach .
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 37c and at an optimum temperature of 37c .
after 48 hours of aerobic incubation on 5% sheep 's blood  enriched columbia agar , colonies are pinpoint , greyish and -haemolytic , with undulated edges and with a diameter of 0.5 to 1  mm .
streptococcus timonensis strain marseille - p2915 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 - p2915 was found to be susceptible to penicillin , ceftriaxone , imipenem , vancomycin and rifampicin .
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 - p2915 (= csur p2915  =  dsm 103349 ) , and it was isolated from a human stomach sample in marseille , france .
 strain marseille - p2915 t , 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 10157 t , 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 - p2915 t (= dsm 103349  =  csur p2915 ) . 
Introduction
Materials and Methods
Results
Discussion
Conclusion