the human gut includes approximately 10 microorganisms , known as the intestinal microbiota , and contains four main phyla : firmicutes , bacteroidetes , actinobacteria and proteobacteria 
 .
this microbial ecosystem contributes to the host 's health , ,   but may also be involved in human disease , , . because intestinal conditions are difficult to reproduce in the laboratory , particularly a strongly reduced environment with a quasi absence of oxygen , most of this microbial universe has not yet been cultivated .
however , strict anaerobic microbes should not be neglected because they are the mainstay of the healthy mature anaerobic gut microbiota indispensable for a healthy life .
several studies have already been conducted to identify the microorganisms included in this ecosystem by using both culture methods and culture - independent methods , such as sequencing of the gene encoding 16s ribosomal rna and metagenomics .
however , genomics identifies dna or rna sequences but not living microbes . recently in our laboratory the culturomics and taxonogenomics approaches were established to unravel gut microbiota diversity while overcoming biases associated with culture - independent techniques . through this innovative strategy associating several culture conditions , mass spectrometry and molecular biology , more
than 200 new species have been isolated from the human gut . using this approach , we isolated for the first time prevotella phocaeensis strain sn19 (= csur p2259  =  dsm 103364 ) from the gut of a woman infected by clostridium difficile .
here we describe its phenotypic features along with the genome annotation and comparison with the closest species .
a stool specimen was collected from an 81-year - old white woman with clostridium difficile infection ( toxin b , ribotype 027 negative ) at la timone hospital ( marseille , france ) in november 2015 .
the stool sample was collected in sterile plastic containers and stored at  80c once aliquots were made .
consent was obtained , and the study was approved by the institut fdratif de recherche 48 ( faculty of medicine , marseille , france ) under agreement number 09 - 022 .
the patient was treated with metronidazole ; the outcome was a full recovery and no relapse .
growth of this isolate was obtained after 5 days ' incubation in a 5% sheep 's blood and 5% rumen - enriched medium in anaerobic atmosphere at 37c .
enriched columbia agar ( cos ; biomrieux , marcy l'etoile , france ) . the growth environment of strain sn19 was determined by testing different temperatures ( 25 , 28 , 30 , 37 and 56c ) in aerobic and anaerobic ( anaerogen ; oxoid , thermo scientific , dardilly , france ) conditions .
time of flight mass spectrometry ( maldi - tof ms ) analysis of strains sn19 was performed on a microflex mass spectrometer ( bruker daltonics , leipzig , germany ) as previously described .
the acquired spectrum was then loaded into the maldi biotyper software ( bruker ) and analyzed as previously described   by using the standard pattern - matching algorithm , which compared the spectrum acquired with that present in the library ( bruker database and ours , constantly updated ) , including 7463 species .
score values of 1.7 but < 2 indicated identification beyond the genus level , and score values of 2.0 indicated identification at the species level .
, we used the ez1 dna tissue kit using biorobot ez1 advanced xl ( qiagen , courtaboeuf , france ) .
the amplification of the 16s rrna gene was carried out using pcr technology and universal primers fd1 and rp2   ( eurogentec , angers , france ) .
then we realized the purification , sequencing and assembly of the amplified products as previously described .
sequences of 16s rrna genes were confronted with those which are available in genbank by blastn ( basic local alignment search tool ) ( http://www.ncbi.nlm.nih.gov/genbank/ ) .
when the percentage of identity was < 98.7% , the studied strain was considered to be a new species .
a custom python script was used to automatically retrieve all species from the same family of the new species and download 16s sequences from national center for biotechnology information ( ncbi ) by parsing ncbi eutils results and the ncbi taxonomy page .
it only keeps sequences from type strains . in case of multiple sequences for one type strain
, it selects the sequence obtaining the best identity rate from the blastn alignment with our sequence .
the script then separates 16s sequences in two groups : one containing the sequences of strains from the same genus ( group a ) , and one containing the others ( group b ) .
it only keeps the 15 closest strains from group a and the closest one from group b. if it is impossible to get 15 sequences from group a , the script selects more sequences from group b to get at least nine strains from both groups . for biochemical characterization of strain sn19
zym , api rapid i d 20ne and api 50ch ( biomrieux ) according to the manufacturer 's instructions .
then we tested catalase ( biomrieux ) and oxidase ( bd bbl dryslide ; becton dickinson , franklin lakes , nj , usa ) activities . to determine sporulation ,
thermal shock was performed on a bacterial suspension of this strain at 80c for 20 minutes ; then this suspension was seeded columbia blood agar .
incubation was done for 72 hours under anaerobic conditions at different temperatures : 28 , 37 , 42 and 56c .
two samples were prepared with approximately 30  mg of bacterial biomass per tube collected from several culture plates .
briefly , fatty acid methyl esters were separated using an elite 5-ms column and monitored by mass spectrometry ( clarus 500-sq 8  s ; perkin elmer , courtaboeuf , france ) . 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 , usa ) and the fames mass spectral database ( wiley , chichester , uk ) .
mobility and gram staining of strain sn19 were determined using an optical microscope ( leica , wetzlar , germany ) with a 40 and 100 oil - immersion objective lens , respectively . in order to observe the cells ' morphology ,
they 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 ; electron microscopy sciences ( ems ) , hatfield , pa , usa ) .
the grids were dried on blotting paper , and the cells were negatively stained for 10 seconds with 1% ammonium molybdate solution in filtered water at room temperature .
electron micrographs were acquired with a tecnai g20 cryo ( fei company , limeil - brevannes , france ) transmission electron microscope operated at 200 kev .
the in  vitro antibiotic susceptibility was tested by disk diffusion method on mueller - hinton agar with 5% sheep 's blood for the following antibiotics : imipenem 10  g , penicillin g 10  g , ciprofloxacin 5  g , teicoplanin 30  g , gentamycin 500  g , rifampicin 30  g , ceftriaxone 30  g , clindamycin 15  g , colistin 50  g , doxycycline 30  g , erythromycin 15  g , fosfomycin 50  g , oxacillin 5  g , metronidazole 4  g and trimethoprim / sulfamethoxazole 25  g . after pretreatment by lysozyme incubation at 37c , dna was extracted on the ez1 biorobot ( qiagen ) with ez1 dna tissues kit .
the genomic dna ( gdna ) was quantified by a qubit assay with a high - sensitivity kit ( life technologies , carlsbad , ca , usausa ) at 152.3  ng/l .
gdna of strain sn19 was sequenced by miseq technology ( 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 at 7.472  kb . no size selection was performed , and 600  ng of tagmented fragments were circularized .
the circularized dna was mechanically sheared to small fragments with an optimal at 954  bp on the covaris device s2 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 6.08 
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 .
the total information of 2.9 gb was obtained from a 297k / mm cluster density with a cluster passing quality control filter of 97% ( 5  808  000 passing filter paired reads ) . within this run ,
open reading frames ( orfs ) were predicted using prodigal   with default parameters , but the predicted orfs were excluded if they spanned a sequencing gap region ( contain n ) .
the predicted bacterial protein sequences were searched against the clusters of orthologous groups ( cogs ) using blastp ( basic local alignment search tool ; e value 1e-03 , coverage 0.7 and identity percentage 30% ) .
if no hit was found , it was searched against the nr database using blastp with e value of 1e-03 coverage 0.7 and an identity percentage of 30% , and if the sequence length was smaller than 80 aa , we used an e value of 1e-05 .
the trnascanse tool   was used to find trna genes , whereas ribosomal rnas were found by using rnammer .
orfans were identified if all the blastp performed did not give positive results ( e value smaller than 1e-03 for orfs with a sequence size larger than 80 aa or an e value smaller than 1e-05 for orfs with sequence length smaller than 80 aa ) .
genomes were automatically retrieved from the 16s rna tree using xegen software ( phylopattern ) . for each selected genome ,
the complete genome sequence , proteome genome sequence and orfeome genome sequence were retrieved from the ncbi 's ftp site .
this score is the mean value of nucleotide similarity between all couple of orthologues between the two genomes studied ( average genomic identity of orthologous gene sequences , agios ) .
an annotation of the entire proteome was performed to define the distribution of functional classes of predicted genes according to the clusters of orthologous groups of proteins ( using the same method as for the genome annotation ) . to evaluate the genomic similarity among studied prevotella strains , we determined two parameters , digital dna - dna hybridization ( ddh ) , which exhibits a high correlation with ddh ,   and agios , which was designed to be independent from ddh . annotation and comparison processes were performed in the multi - agent software system dagobah , which includes figenix   libraries that provided pipeline analysis .
the genome of strain sn19 was compared with the one of closest species such as  prevotella oralis ( aepe00000000 ) , prevotella micans ( bakh00000000 ) , prevotella stercorea ( afzz00000000 ) , prevotella timonensis ( cbqq000000000 ) , prevotella shahii ( baiz00000000 ) , prevotella loescheii ( arjo00000000 ) , prevotella saccharolytica ( bakn00000000 ) , prevotella oulorum ( adgi00000000 ) and prevotella marshii ( aeei00000000 ) .
strain sn19 was first isolated in november 2015 from the stool that was used to diagnose a c.  difficile infection , stored at  80c and cultured on columbia agar enriched with 5% sheep 's blood at 37c during 5 days under anaerobic conditions .
1 ) did not match either bruker 's database or our database , and was consequently added ( http://www.mediterranee-infection.com/article.php?laref=256&titre=urms-database ) .
then the 16s rrna gene sequencing demonstrated that the type strain prevotella oralis atcc 33269 ( genbank accession no .
nr_042841.1 ) is the phylogenetically closest to strain sn19 and showed a 97.70% sequence identity with it ( fig .  2 ) .
this value was lower than the 98.7% 16s rrna gene sequence threshold advised by stackebrandt and ebers   to describe a new species without carrying out dna - dna hybridization .
therefore , we propose strain sn19 as the type strain of a new species within the prevotella genus , for which we proposed the name prevotella phocaeensis sp . nov .
a gel view allowed us to visualize spectral differences with other prevotella species ( fig . 
prevotella phocaeensis strain sn19 grew at 37c on colombia agar ( with 5% sheep 's blood ) after 5 days in blood culture bottles enriched with rumen fluid ( 5% ) and blood ( 5% ) in anaerobic conditions .
no growth occurred under aerobic conditions at 37c and aerobic and anaerobic conditions at 28 , 42 and 56c .
bacterial cells tolerate a ph of 6 to 8.5 and an amount of nacl of 0 to 5g / l .
colonies were haemolytic , circular , small , white and irregular with a diameter of about 1 to 1.5  mm on blood - enriched colombia agar .
bacterial cells observed under electron microscopy were sticks , ranging in length from 1.7 to 2  m ( fig . 
using api zym , positive reactions were observed for alkaline phosphatase , acid phosphatase , naphthol - as - bi - phosphohydrolase , -galactosidase , -glucuronidase , -glucosidase and n - acetyl--glucosaminidase .
reactions for esterase ( c4 ) , esterase lipase ( c8 ) , lipase ( c14 ) , leucine arylamidase , valine arylamidase , trypsin , -chymotrypsin , -galactosidase , -glucosidase , -fucosidase and -mannosidase were negative .
an api 50ch strip showed positive reactions for l - arabinose , d - ribose , l - xylose , d - adonitol , esculin ferric citrate , salicin , d - sucrose , d - raffinose , d - lyxose , d - tagatose , potassium 2-ketogluconate and potassium 5-ketogluconate .
negative reactions were recorded for d - xylose , d - galactose , d - fructose , l - sorbose , amygdalin , d - melibiose , d - trehalose , inulin , d - melezitose , starch , glycogen , xylitol , gentiobiose , d - fucose , l - fucose , glycerol , erythritol , d - arabinose , methyl--d - xylopyranoside , d - glucose , d - mannose , l - rhamnose , dulcitol , inositol , d - mannitol , d - sorbitol , methyl-d - mannopyranoside , methyl-d - glucopyranoside , n - acetyl - glucosamine , arbutin , d - cellobiose , d - maltose , d - lactose , d - turanose , d - arabitol , l - arabitol and potassium gluconate .
api20ne showed for strain sn19 a positive reaction for esculin ferric citrate and -galactosidase but negative reactions for potassium nitrate , l - tryptophan , d - glucose ( fermentation and assimilation ) , l - arginine , urea , gelatin , l - arabinose , d - mannose , d - mannitol , n - acetylglucosamine , d - maltose , potassium gluconate , capric acid , adipic acid , malic acid , trisodium citrate and phenylacetic acid .
this bacterium is resistant to ciprofloxacin and fosfomycin but susceptible to imipenem , penicillin , teicoplanin , gentamicin , rifampicin , ceftriaxone , clindamycin , colistin , doxycycline , erythromycin , oxacillin , metronidazole and trimethoprim / sulfamethoxazole .
phenotypic characteristics of strain sn19 were compared to those of the closely related prevotella species ( table  2 ) . by comparison with prevotella oralis , its phylogenetically closest neighbor ,
it differs in motility , l - arabinose , glucose , fructose and lactose . concerning the fatty acid methyl ester analysis by gc / ms , the major fatty acid was hexadecanoic acid ( 16:0 , 29% ) .
cellular fatty acid profile of strain sn19 was also compared with closely related prevotella species ( table  3 ) .
the genome is 2  922  117  bp long with 44.6% of g+c content ( fig .  5 ) .
it is composed of 12 scaffolds ( composed of 13 contigs ) . of the 2486 predicted genes , 2430 were protein - coding genes and 56 were rnas ( three genes are 5s rrna
, one gene is 16s rrna , three genes are 23s rrna and 49 genes are trna genes ) .
a total of 1403 genes ( 57.74% ) were distributed in putative function ( by cogs or by nr blast ) .
the remainder of the genes were annotated as hypothetical proteins ( 971 genes , 39.96% ) .
table  4 summarizes the properties and the statistics of the genome and table  5 the distribution of predicted genes of strain sn19 according to cogs categories .
the draft genome sequence of strain sn19 is smaller than that of prevotella saccharolytica , prevotella loescheii , prevotella timonensis , prevotella stercorea and prevotella shahii ( 2922 , 2976 , 3509 , 3156 , 3097 and 3490  mb respectively ) , but larger than that of prevotella oralis , prevotella oulorum , prevotella marshii and prevotella micans ( 2840 , 2806 , 2559 and 2427  mb respectively ) .
the g+c content of strain sn19 is smaller than that of p.  loescheii , p. oulorum , p. stercorea , p. marshii and p.  micans ( 44.60 , 46.62 , 46.78 , 49.00 , 47.46 and 45.49% respectively ) , but larger than that of p.  oralis , p. saccharolytica , p. timonensis and p.  shahii ( 44.54 , 43.30 , 42.38 , and 44.38% respectively ) .
the gene content of strain sn19 is smaller than that of p.  oralis , p. saccharolytica , p. loescheii , p. timonensis , p. oulorum , p. stercorea and p.  shahii ( 2430 , 2488 , 2762 , 2838 , 2770 , 2488 , 3017 and 3392 respectively ) , but larger than that of p.  marshii and p.  micans ( 2335 and 2329 respectively )
. furthermore , strain sn19 shares 1054 , 1241 , 1317 , 1209 , 1662 , 1404 , 1269 , 1254 and 1236 orthologous genes with p.  micans , p. stercorea , p. timonensis , p. shahii , p. oralis , p. loescheii , p. saccharolytica , p. oulorum and p.  marshii respectively ( table  6 ) .
agios values vary from 56.79% between p.  saccharolytica and p. stercorea , to 80.98 between p.  loescheii and p.  shahii among compared genomes except for strain sn19 . when this one was compared with other species within the prevotella genus , values ranged from 58.13 with p.  stercorea to 76.50 with p. oralis , thus confirming its new species status ( table  6 ) .
table  7 shows the pairwise comparison of strain sn19 with other prevotella species using the genome - to - genome distance calculator .
6 shows the distribution of functional classes of predicted genes on the chromosomes of strain sn19 and related species of prevotella genus according to the cogs of proteins .
it demonstrates that the distribution of genes into cogs categories is similar in all compared prevotella species .
strain sn19 was isolated from the stool sample of an 81-year - old white woman who was a patient at la timone hospital diagnosed with c.  difficile colitis with good outcome without faecal microbiota transplantation . on the basis of phenotypic , genomic and phylogenetic characteristics
, we have isolated a new species , strain sn19 , within the genus prevotella , whose closest species is prevotella oralis .
these results and the divergence of the 16s rrna gene sequence with the closest species confirms that sn19 strain is a new species . also , genomic analysis identified 12 orfans ( table  4 ) and at least 768 ( 31.6% ) of 2430 orthologous proteins genes not shared with the closest phylogenetic neighbor , prevotella oralis ( table  6 ) , supporting the description of a new species .
strain sn19 differs from the phylogenetically closest species by its mobility , its major fatty acids and its inability to use certain sugars including mannose , glucose , fructose , maltose and lactose ( table  2 ) , suggesting that they are different species .
the composition of the intestinal microbiota shows an abundance of bacteria belonging to firmicutes and bacteroidetes phyla . in bacteroidetes
prevotella are more prevalent in populations with plant - based diet , suggesting that it contributes to their catabolism in the human gut .
possibly as a result of its link to a vegetarian diet , prevotella is found less often in the western population ; prevotella species in this population are mostly found in the oral cavity , . moreover , arumugam et  al .  
reported the existence of enterotypes in the human microbiome including prevotella as a main contributor of one of these enterotype regardless of age , sex or origin .
in addition , the abundance of prevotella in a body habitat is correlated by an improvement in glucose metabolism .
however , the presence of prevotella in the human gut was also linked to inflammation .
further studies are needed in order to decipher the role of prevotella phocaeensis in health and diseases and particularly plant catabolism , glucose regulation and its interaction with clostridium difficile in the human gut .
maybe the presence of p. phocaeensis , a probable member of the human mature anaerobic gut microbiota , explains the good outcome of the patient with clostridium difficile infection .
therefore , considering phenotypic differences with the closest species , the proteomic profile obtained with maldi - tof ms of strain sn19 , its phylogenetic analysis of the 16s rrna gene sequence , its genomic description and annotation , agios and digital ddh values , we propose the creation of strain sn19 as a new species named prevotella phocaeensis sp .
finally , we showed here that culturomics and taxonogenomics dramatically extend the discovery of human mature anaerobic gut microbiota previously associated with gut maturation and health .
phocaeensis ,  of phocaea, the latin name of marseille , adapted from the first greek name of marseille , phokaia , where the strain was isolated ) .
strict anaerobic , gram - negative , oxidase and catalase negative , not endospore forming , and with nonmotile rods , the colonies are haemolytic , circular , small and white , and have a diameter of about 1 to 1.5  mm on columbia agar  +  5% sheep 's blood .
strain sn19 grows at 37c after 72 hours of incubation and tolerates a ph of 6 to 8.5 and an amount of nacl of 0 to 5 
bacterial cells observed under electron microscopy were sticks ranging in length from 1.7 to 2  m , with a diameter lower to 0.5  m .
using the zym strip and api 50ch , positive reactions were observed for alkaline phosphatase , acid phosphatase , naphthol - as - bi - phosphohydrolase , -galactosidase , -glucuronidase , -glucosidase , n - acetyl--glucosaminidase and l - arabinose , d - ribose , l - xylose , d - adonitol , esculin ferric citrate , salicin , d - sucrose , d - raffinose , d - lyxose , d - tagatose , potassium 2-ketogluconate and potassium 5-ketogluconate .
p.  phocaeensis strain sn19 was susceptible to imipenem , penicillin , teicoplanin , gentamicin , rifampicin , ceftriaxone , clindamycin , colistin , doxycycline , erythromycin , oxacillin , metronidazole and trimethoprim / sulfamethoxazole .
the accession numbers of the sequences of 16s rrna and genome which were deposited in embl - ebi are ln998069 and fizg00000000 , respectively .
the type strain sn19 (= csur p2259  =  dsm 103364 ) was isolated from the stool sample of an 81-year - old white woman .
 prevotella phocaeensis sp . nov . strain sn19 
 t (= dsm 103364 ) is a new species isolated from the gut microbiota of patient with colitis due to clostridium difficile . 
 strain sn19 t is gram - negative rod - shaped bacteria , strictly anaerobic , nonmotile and non  endospore forming . 
 the predominance fatty acid is hexadecanoic acid . 
 its 16s rrna showed a 97.70% sequence identity with its phylogenetically closest species , prevotella oralis . 
 the genome is 2  922  117  bp long and contains 2486 predicted genes including 56 rna genes . 
Introduction
Material and Methods
Results
Discussion and Conclusion