metagenomics is currently regarded as the reference method to study the gut microbiota , but culturomics is a complementary approach allowing the cultivation of new species .
indeed , advances in culture   and identification by amplification and sequencing of the 16s rrna   combined with routine matrix - assisted laser desorption / ionization time - of - flight mass spectrometry ( maldi - tof ms ) analyses , have resulted in a dramatic increase of bacterial species identified by culture .
most studies showing the relationship between human diseases and the digestive microbiota have been made from stool sample analysis .
however , analysis of faeces does not appear to be the best method to examine the gastrointestinal microbiota : the bacterial composition and concentration are different between stools and the duodenum .
we thus compared the culturomes of the stomach , duodenum , ileum , and right and left colon from six patients undergoing upper endoscopy and colonoscopy for various indications . while doing so , we isolated the strain marseille - p2749 from the duodenum of a patient with iron deficiency .
the genus fusobacterium included 20 species inhabiting the oral cavity and digestive tract of animals or humans .
fusobacterium nucleatum is the type species of this genus and the most frequently isolated from humans , and fusobacterium periodonticum was first isolated in 1983 from the human oral cavity .
however , our strain did not fulfil the usual criteria to be assigned to a previously known species of this genus .
the classic definition proposes to define a new species by dna - dna hybridization ( ddh ) , a nonreproducible and expensive method that is possibly only in a few laboratories . among others , we proposed since 2012 to systematically add the maldi - tof spectrum and the genome sequence to define a new species .
the main characteristics of strain marseille - p2749 (= csur p2749  =  dsm 103085 ) are reported with a phylogenetic and phenotypic analysis , along with a description of the complete genome .
using the culturomics approach , as a part of the human microbiome study , strain marseille - p2749 was first cultivated from the duodenum liquid sample of a 60-year - old man living in marseille who underwent digestive endoscopy to diagnose iron - deficiency anaemia .
a signed informed consent was obtained from the patient at the gastroenterology service of the hpital nord ( marseille , france ) , and the ethics committee of the institut fdratif de recherche ifr48 validated the study under number 2016 - 010 .
after collection in sterile conditions during an upper endoscopic examination , the sample was immediately preincubated in a blood culture bottle ( bd bactec , plus anaerobic / f media ; becton dickinson , le pont de claix , france ) previously enriched with 5  ml of sheep s blood , 0.2  m filtered rumen and three antioxidants ( uric acid 0.1  g / l , glutathione 0.1 
g / l ; sigma - aldrich , saint - quentin fallavier , france ) .
the initial growth of strain marseille - p2749 was obtained in anaerobic conditions ( anaerogen compact ; oxoid , thermo scientific , dardilly , france ) after a 1-day preincubation and a 1-day incubation at 37c of this liquid medium seeded on a 5% sheep s blood 
enriched columbia agar ( columbia agar  + 5% sheep s blood ; biomrieux , marcy letoile , france ) .
colonies obtained were then identified with our systematic maldi - tof ms screening on a microflex spectrometer ( bruker daltonics , bremen , germany ) as previously described .
when a colony was unidentifiable , a 16s rrna gene amplification was performed by standard pcr using fd1-rp2 universal primers ( eurogentec , angers , france ) as previously described .
the complete 16s rrna gene was then sequenced using internal primers ( 536f , 536r , 800f , 800r , 1050f , 1050r ) as previously described .
sequences were finally corrected using the codon code aligner software ( http://www.codoncode.com ) and then blasted ( basic local alignment search tool ) against the genbank nucleotide collection ( nr / nt ) or against genbank 16s ribosomal rna sequences ( http://blast.ncbi.nlm.nih.gov/blast.cgi ) .
after the 16s rrna gene sequence comparison , the strain is considered as a new species if the percentage of similarity is 98.7% .
a custom python script was used to automatically retrieve all species from the same family of strain marseille - p2749 and download 16s sequences from national center for biotechnology information ( ncbi ) by parsing ncbi results and the ncbi taxonomy page .
it only keeps sequences from type strains found on the list of prokaryotic names with standing in nomenclature ( lpsn ) website ( http://www.bacterio.net/ ) .
five temperatures ( room temperature , 28 , 37 , 45 and 55c ) and three atmospheres ( anaerobic ( anaerogen compact ; oxoid , thermo scientific ) , microaerophilic ( campygen compact ; oxoid , thermo scientific ) and aerobic in a plastic pouch to maintain a humid atmosphere ) were realized .
tolerance of this strain to salt ( 10 , 15 and 20% ) and ph ( ph 5 , 5.5 , 6 , 6.5 , 7 , 7.5 and 8) was also managed under an anaerobic atmosphere . for the sporulation test , a heat shock at 80c for 20 minutes was performed .
then gram staining , motility and sporulation were observed using a light microscope ( dm1000 ; leica microsystems , nanterre , france ) .
cells were fixed with 2.5% glutaraldehyde in 0.1  m cacodylate buffer for 1 hour at 4c .
a drop of cell suspension was deposited for 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 g cryo transmission electron microscope ( fei company , limeil - brevannes , france ) operated at 200 kev .
catalase and oxidase tests ( bd catalase reagent and bd oxidase reagent ; becton dickinson ) were performed . moreover , biochemical traits and enzyme activities were analyzed using api gallery systems ( api 20a , api 50ch and api zym test strips ; biomrieux ) according to the manufacturer s instructions .
cellular fatty acid methyl ester ( fame ) analysis was performed by gas chromatography / mass spectrometry ( gc / ms ) as previously described .
two samples were prepared with 40  mg of bacterial biomass per tube collected from eight culture plates .
fame were separated using an elite 5-ms column and monitored by mass spectrometry ( clarus 500-sq 8  s ; perkinelmer , courtaboeuf , france ) .
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 fames mass spectral database ( wiley , chichester , uk ) .
antibiotic susceptibility was tested using a disk diffusion method   on mueller - hinton e agar ( biomrieux ) .
seventeen antibiotics were tested ( i2a , montpellier , france ) : vancomycin 30  g , colistin 50  g , gentamycin 15  g , amoxicillin 25  g , amoxicillin / clavulanic acid 30  g , penicillin 10  u , clindamycin 15  g , metronidazole 4  g , trimethoprim / sulfamethoxazole 25  g , oxacillin 5  g , imipenem 10  g , tobramycin 10  g , fosfomycin 50  g , ceftriaxone 30  g , rifampicin 30  g , doxycycline 30  g and erythromycin 15  g
. genomic dna ( gdna ) of strain marseille - p2749 was sequenced using miseq technology ( illumina , san diego , ca , usa ) with mate pair strategy .
the gdna was barcoded and mixed with 11 other projects with the nextera mate pair sample prep kit ( illumina ) , then quantified by qubit assay with the high - sensitivity kit ( life technologies , carlsbad , ca , usa ) to 61.6  ng/l . using the nextera mate pair illumina guide ,
the mate pair library was prepared with 1.5  g of fragmented and tagged genomic dna sample , with a mate pair junction adapter .
the pattern of fragmentation was validated on an agilent 2100 bioanalyzer ( agilent technologies , santa clara , ca , usa ) with a dna 7500 labchip .
the dna fragments were ranged in size from 1.5 to 11  kb with an optimal size of 6.13  kb .
no size selection was performed , and 441.5  ng of tagmented fragments were circularized .
the circularized dna was mechanically sheared with optima on a bimodal curve at 896 and 1663  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 24.52 
after a denaturation step and dilution at 15  pm , the pool of libraries was loaded .
automated cluster generation and sequencing run were performed in a single 39-hour run with a 2    251  bp read length .
total information of 7.2 gb was obtained from a 765k / mm cluster density with a cluster passing quality control filters of 94.7% ( 14  162  000 passing filter paired reads ) . within this run ,
the 1  012  974 paired reads were trimmed then assembled into six scaffolds .
open reading frame ( 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 database ( cogs ) databases using blastp .
the trnas and rrnas were predicted using the trnascan - se   and rnammer   tools respectively .
signal peptides and numbers 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 mean level of nucleotide sequence similarity at the genome level between strain marseille - p2749 and other bacteria ( fusobacterium perfoetens atcc 29250 jhxw00000000 , fusobacterium nucleatum atcc 49256 cp003700 , fusobacterium varium atcc 8501 acie00000000 , fusobacterium mortiferum atcc 25557 acdb00000000 , fusobacterium russii atcc 25533 armk00000000 and ilyobacter polytropus dsm 2926 cp002281 ) was estimated using average genomic identity of orthologous gene sequences ( agios ) software
. digital ddh , which exhibits high correlation with ddh , was also performed to evaluate genomic similarity with the closest strains ( http://ggdc.dsmz.de/ ) .
after a 1-day preincubation period at 37c in a blood culture bottle with sheep s blood , filtered rumen and antioxidants , and a 1-day anaerobic incubation period of cultivation of this enriched liquid medium seeded on colombia agar with 5% sheep s blood , the strain marseille - p2749 was first isolated .
maldi - tof ms was unable to provide reliable identification for colonies from this strain because its spectrum was not part of the database ( fig . 
1(a ) ) . the phylogenetic analysis , based on 16s rrna , showed that strain marseille - p2749 exhibited a 95.9% sequence similarity   with fusobacterium periodonticum strain atcc 33693 ( genbank accession no .
x55405 ) , the phylogenetically closest species with standing in nomenclature . because the 16s rrna sequence divergence was > 1.3% from its phylogenetically closest species with standing in nomenclature ,
we propose the creation of a new species which putatively classifies it as a member of the fusobacterium genus within the fusobacteriaceae family in the fusobacteria phylum .
the phylogenetic tree shows the position of strain marseille - p2749 relative to other phylogenetically close neighbours , based on the 16s rrna gene sequence ( fig .  2 ) .
the 16s rrna sequence of strain marseille - p2749 was deposited in european molecular biology laboratory 
european bioinformatics institute ( embl - ebi ) under accession number lt576389 ( table  1 ) . to compare the spectrum of strain marseille - p2749 and the spectra of other related bacteria , a gel view was performed ( fig . 
the optimum growth of strain marseille - p2749 was observed after a 48 hours incubation at 37c on a 5% sheep s blood 
this strain was also able to grow in microaerophilic conditions , from 28 to 37c or from ph 5.5 to ph 8 .
colonies were circular , white and opalescent ; they measured 0.6 to 0.8  mm in diameter .
3 ) , with a length ranging from 1.7 to 2.5  m and a width ranging from 0.3 to 0.4  m ( fig .  4 ) .
classification and general features of strain marseille - p2749 are summarized in table  2 . using the api 50ch gallery system ,
positive reactions were observed for esculin ( ferric citrate ) , d - arabinose , d - raffinose , gentiobiose , d - tagatose and potassium 5-ketogluconate , whereas negative reactions were observed for fructose , glucose , lactose , maltose and mannose .
the api zym system showed positive reactions for alkaline phosphatase , acid phosphatase , esterase ( c4 ) , lipase esterase ( c8 ) , -chymotrypsin and naphtol - as - bi - phosphohydrolase , but negative reactions for -galactosidase , -glucuronidase and n - acetyl - glucosaminidase .
moreover , in the api 20a system , the strain showed indole production but not for gelatin .
two specific 3-hydroxy fatty acids were described : 14:0 3-oh ( 9% ) and 16:0 3-oh ( 5% ) .
an unusual branched unsaturated fatty acid was also detected : 15:1n5 anteiso ( 1% ) . cellular fatty acid composition of strain marseille - p2749 is compared to other fusobacterium 
  in table  4 .
strain marseille - p2749 was susceptible to colistin , amoxicillin , amoxicillin / clavulanic acid , penicillin , clindamycin , metronidazole , oxacillin , imipenem , tobramycin , ceftriaxone , rifampicin and doxycycline , whereas it was resistant to vancomycin , gentamicin , trimethoprim / sulfamethoxazole , fosfomycin and erythromycin . the genome length is 1  809  169  bp with 27.33  mol% g+c content ( table  5 and fig .  5 ) , and it is composed of six scaffolds ( seven contigs ) .
of the 1703 genes , 1646 were protein - coding genes ( 96.65% ) and 57 were rnas ( 48 were trna genes and nine were rrna genes ) .
a total of 1274 genes ( 77.39% ) were assigned as putative function ( by cogs or by nonredundant protein sequence blast ) , and 27 genes ( 1.64% ) were associated with orfans .
no gene was associated with resistance , and four genes were associated with polyketide synthase or nonribosomal peptide synthase .
the draft genome sequence of strain marseille - p2749 ( 1.81 mb ) was smaller than all the compared genomes : fusobacterium russii atcc 25533 ( 1.94 mb ) , fusobacterium perfoetens
atcc 29250 ( 2.10 mb ) , fusobacterium nucleatum atcc 49256 ( 2.12 mb ) , fusobacterium mortiferum atcc 25557 ( 2.67 mb ) , ilyobacter polytropus dsm 2926 ( 3.13 mb ) and fusobacterium varium atcc 8501 ( 3.30 mb ) .
the g+c content of strain marseille - p2749 ( 27.33% ) was higher than that of f.  perfoetens ( 25.97% ) but smaller than the other compared genomes : f.  nucleatum ( 27.34% ) , f.  russii ( 28.64% ) , f.  mortiferum ( 29.07% ) , f.  varium ( 29.18% ) and i.  polytropus ( 34.37% ) .
the gene content of strain marseille - p2749 ( 1646 ) was also smaller than all compared genomes : f.  russii ( 1764 ) , f.  perfoetens ( 1949 ) , f.  nucleatum ( 2250 ) , f.  mortiferum ( 2538 ) , i.  polytropus ( 2880 ) and f.  varium ( 3008 ) .
strain marseille - p2749 shared 993 orthologous genes with f.  russii , 912 with f.  varium , 880 with f.  mortiferum , 855 with f.  nucleatum , 823 with f.  perfoetens and 811 with i.  polytropus .
the distribution of genes into cogs categories was similar in all compared genomes ( fig . 
the agios value ranged from 57.46 to 76.55% when the closest species were compared . for strain marseille - p2749 ,
the agios value ranged from 57.70% with i.  polytropus to 76.55% with f.  russii ( table  7 ) .
the ddh value was of 26.10% ( 23.828.6% ) with f.  nucleatum , 26.00% ( 23.728.5% ) with f.  periodonticum , 21.30% ( 19.023.7% ) with f.  russii , 21.10% ( 18.823.5% ) with i.  polytropus , 20.90% ( 18.623.3% ) with f.  mortiferum , 20.60% ( 18.423.1% ) with f.  varium and 18.00% ( 15.920.4% ) with f.  perfoetens
( table  8) . the probability of error to say that this strain is a new species ( ddh > 70% ) compared to fusobacterium periodonticum atcc 33693 ,
the type strain of the closest phylogenetic neighbour based on 16s rrna similarity ( 95.9% ) , was of 0.02% ( ggdc2.1 formula 2 ; dsmz website ( http://ggdc.dsmz.de/distcalc2.php ) ) .
on the basis of the phylogenetic , phenotypic , biochemical and genomic evidence presented , the creation of a novel species of fusobacterium is proposed , with the name fusobacterium massiliense sp .
this bacterium was first isolated from the duodenum of a 60-year - old man as part of the culturomics study of the gut microbiota from stored samples taken from the gastrointestinal tract .
n. massiliense ,  of massilia, the latin name of marseille , france , where the type strain was first isolated ) .
cells are gram - negative bacilli and fusiform shaped , with a length ranging from 1.7 to 2.5  m and a width ranging from 0.3 to 0.4  m .
colonies are circular , white and opalescent , with a diameter of 0.6 to 0.8  mm on colombia agar enriched with 5% sheep s blood after 48 hours growth .
positive reactions were observed for esculin , d - arabinose , d - raffinose , gentiobiose , d - tagatose and potassium 5-ketogluconate .
the api zym system showed positive reactions for alkaline phosphatase , acid phosphatase , esterase ( c4 ) , lipase esterase ( c8 ) , -chymotrypsin and naphtol - as - bi - phosphohydrolase .
cells are susceptible to colistin , amoxicillin , amoxicillin / clavulanic acid , penicillin , clindamycin , metronidazole , oxacillin , imipenem , tobramycin , ceftriaxone , rifampicin and doxycycline .
the length of the genome is of 1  809  169  bp , and the g+c content is 27.33  mol% .
the accession number of the 16s rrna gene sequence deposited in embl - ebi is lt576389 , and the accession number of the genome sequence is fmja01000000 .
the type strain marseille - p2749 (= csur p2749  =  dsm 103085 ) was first isolated from the duodenum liquid sample of a 60-year - old man .
 the strain marseille - p2749 t (= csur p2749  =  dsm 103085 ) was isolated as part of culturomics study from a liquid duodenum sample from a french man . 
 bacterial cells were gram - negative bacilli , fusiform shaped and non  spore forming , and they grew in microaerophilic and anaerobic atmosphere . 
 its genome is 1  809  169  bp long and contains 1646 protein - coding genes . 
 the dna g+c content was 27.33  mol% . 
 this strain exhibited a 95.9% sequence similarity with fusobacterium periodonticum , the phylogenetically closest species with standing in nomenclature . 
 strain marseille - p2749 t is suggested to be a novel species belonging to the genus fusobacterium , for which the name fusobacterium massiliense sp . 
 nov . is proposed . 
Introduction
Materials and Methods
Results
Conclusion