culturomics was recently introduced in our laboratory ( urmite , marseille , france ) as an alternative method to expand the human gut repertoire , thanks to the multiplication of culture conditions with a rapid identification method by matrix - assisted laser desorption / ionization time - of - flight mass spectrometry ( maldi - tof ) .
consequently , new genera and species of bacteria are found thanks to this technique , , , , .
the characterization of new bacteria was previously based on a combination of phylogenetic and genotypic characteristics , including 16s rrna sequence similarity , gc percentage and dna - dna hybridization , , , .
unfortunately , significant limits such as their threshold values , which were not applicable to all species or genera , did not promote their utilization .
the advent of high - throughput sequencing techniques made many bacterial genome sequences available in public databases .
our laboratory recently developed a taxonogenomic approach based on a systematic comparison of phenotypic ( especially the maldi - tof spectrum ) characteristics and genomic characteristics with the phylogenetically closest bacteria found in the databases , , , , .
here we present an analysis of the characteristics that allowed us to describe gabonia massiliensis strain gm3 , a bacterium isolated from a stool specimen from a healthy gabonese male youth and classified into the porphyromonadaceae family , created in 2011 by krieg  .
we describe the classification , biochemical features and complete genomic sequencing and annotation of g.  massiliensis gen .
, sp . nov . strain gm3 (= csur p1909  =  dsm 100571 ) .
the stool sample was collected in lbamba ( gabon ) in january 2015 after approval from the national ethics committee of gabon ( registration 0023/2013/sg / cne ) and ifr48 ( marseille , france ) ( registration 09 - 022 ) and after receipt of the patient 's signed consent .
the specimen was taken from a healthy 16-year - old gabonese male youth ( body mass index , 19.03  kg / m ) .
he was a member of the nzebi tribe , which is one of the biggest tribes in gabon .
the stool sample was stored at  80c until it was sent to urmite ( marseille , france ) . in april 2015 ,
the stool sample was diluted in phosphate - buffered saline ( life technologies , carlsbad , ca , usa ) .
obtained inoculum was preincubated anaerobically at 37c in an anaerobic blood culture bottle ( biomrieux , marcy ltoile , france ) enriched with blood and rumen .
after the preincubation stage , 100  l was incubated for 24 to 48 hours on 5% sheep 's blood  enriched columbia agar ( biomrieux ) at 37c under anaerobic conditions using an anaerobic atmosphere generator system ( genbag anaer ; biomrieux ) .
enriched columbia agar solid medium ( biomrieux ) and identified by maldi - tof and 16s rrna sequencing .
maldi - tof identification consisted of picking one isolated bacterial colony with a pipette tip from a culture agar plate and spreading it as a thin film on an mtp 384 maldi - tof target plate ( bruker daltonics , leipzig , germany ) , , , , .
then 2  l of matrix solution ( saturated solution of -cyano-4-hydroxycinnamic acid in 50% acetonitrile and 2.5% trifluoroacetic acid ) was overlaid on each smear and allowed to dry for 5 minutes .
measurements and maldi - tof analysis were conducted as previously described , , , , .
identification depended on the score obtained ; a score of 2 with a validly published species enabled identification at the species level ; a score of 1.7 but < 2 enabled identification at the genus level ; and a score of < 1.7 did not enable any identification , , , , .
if the colonies corresponded to a new bacterium , confirmation of the identification had to occur with a 16s rrna pcr coupled with sequencing .
that was performed using geneamp pcr system 2720 thermal cyclers ( applied biosystems , bedford , ma , usa ) and abi prism 3130xl genetic analyzer capillary sequencer ( applied biosystems ) , respectively .
if the confirmation was positive , the spectrum of the new bacterium was entered into the bruker database .
the 16s rrna nucleotide sequence was corrected using chromas pro 1.34 software ( technelysium , tewantin , australia ) , and the blastn searches were performed by the national center for biotechnology information tool ( http://blast.ncbi.nlm.nih.gov.gate1.inist.fr/blast.cgi ) .
for phylogeny , sequences were aligned using clustalw , and phylogenetic inferences were obtained using the neighbour - joining method within mega software .
numbers at the nodes are percentages of bootstrap values obtained by repeating the analysis 1000 times to generate a majority consensus tree .
the 16s rrna for g.  massiliensis strain gm3 was deposited in genbank under accession number ln849789 .
strain gm3 was cultivated on 5% sheep 's blood  enriched colombia agar ( biomrieux ) in anaerobic conditions at different temperatures ( 28 , 37 , 45 and 55c ) to assess its range of growth temperatures .
the genbag anaer and genbag microaer systems ( biomrieux ) were respectively used to assess the ability of the bacterium to grow anaerobically or in microaerophilic conditions at 37c , using the same medium culture . for halophilia and ph testing , four nacl concentrations
( 0 , 5 , 15 and 45% ) and three different phs ( 5 , 7 and 8.5 ) were tested .
api gallery systems were performed with api zym , api 20 a and api 50ch ( biomrieux ) for biochemical assays .
oxidase ( becton dickinson , le pont de claix , france ) and catalase assays ( biomrieux ) were done separately .
sporulation was tested by performing thermal shock on bacterial colonies ( diluted in phosphate - buffered saline ) at 80c for 10 minutes .
the motility of strain gm3 was tested by observing its fresh colony between blades and slats using a dm1000 photonic microscope ( leica microsystems , nanterre , france ) with a 40 objective lens .
the antibiotic susceptibility of g.  massiliensis was tested using sirscan discs of amoxicillin , doxycycline , rifampicin , nitrofurantoin , vancomycin , amoxicillin / clavulanic acid , clindamycin , gentamicin , imipenem , erythromycin , metronidazole , trimethoprim / sulfamethoxazole , amikacin , ciprofloxacin and tobramycin antibiotics ( i2a , montpellier , france ) .
g.  massiliensis strain gm3 was observed after negative coloration using a tecnai g20 ( fei , limeil - brevannes , france ) transmission electron microscope at an operating voltage of 60  kv .
this coloration was observed by using the dm1000 photonic microscope ( leica microsystems ) with a 100 oil - immersion objective lens . using the mate - pair strategy , the genomic dna ( gdna ) of g.  massiliensis gm3 was sequenced on the miseq sequencer ( illumina , san diego , ca , usa ) , , , , . 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  g of gdna using the nextera mate - pair illumina guide , and the gdna sample was simultaneously fragmented and tagged 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 ranged in size from 1 to 10  kb , with an optimal size of 4.08  kb .
no size selection was performed , and only 464  ng of tagmented fragments were circularized , , , , .
the circularized dna was mechanically sheared to small fragments with an optimal size of 569  bp in microtubes on the covaris s2 device ( covaris , woburn , ma , usa ) .
the library profile was visualized on a high sensitivity bioanalyzer labchip ( agilent technologies ) , and the final library concentration was measured at 24.4 
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 . an automated cluster generation and sequencing run were performed in a single 39-hour run on 2    251  bp .
a total of 10.1  gb of information was obtained from a 1189k / mm cluster density with a cluster passing quality control filters of 99.1% ( 22  579  000 clusters ) .
the reads obtained were trimmed and then assembled using the clc genomics wb4 software , , , , . using prodigal ( http://prodigal.ornl.gov/ ) with default parameters , we predicted open reading frames ( orfs ) .
however , if they spanned a sequencing gap region , the predicted orfs were excluded .
the predicted bacterial protein sequences were searched against the genbank   and clusters of orthologous groups ( cogs ) databases using blastp . both trnas and rrnas
signal peptides and the number of transmembrane helices were predicted using signalp   and tmhmm , respectively .
orfans were identified when 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 , we used an e value of 1e-05 .
artemis   and a dnaplotter   were used for data management and visualization of genomic features , respectively .
finally , we used the mauve alignment tool ( version 2.3.1 ) for multiple genomic sequence alignments . the mean level of nucleotide sequence similarity at the genome level between g.  massiliensis and other bacteria was estimated using the average genomic identity of orthologous gene sequences ( agios ) homemade software . overall
, this software is combined with other software : proteinortho   ( to detect orthologous proteins between genomes compared two by two , and then retrieves the corresponding genes ) and the needleman - wunsch global alignment algorithm ( to determine the mean percentage of nucleotide sequence identity among orthologous orfs ) .
annotation and comparison analyses were performed using the multiagent software system dagobah   that includes figenix .
g. massiliensis strain gm3 was compared with bacteroides salanitronis strain dsm 18170 , alistipes shahii strain wal 8301 ,  alistipes finegoldii strain dsm 17242 , barnesiella viscericola strain dsm 18177 , ornithobacterium rhinotracheale strain ort - umn 88 , bacteroides fragilis strain ych46 , bacteroides dorei strain hs1 and helicobacter pylori strain 26695 .
the spectrum resulting from the 12 clean g.  massiliensis spots did not identify bacteria because there was no spectrum match with those of bruker database . using 16s rrna sequence ,
strain gm3 , exhibited 91% identification with barnesiella viscericola ( ab267809.1 ) ( fig .  1 ) .
the similarity of 16s rrna in these two bacteria led to classification of this new bacterium into the porphyromonadaceae family , created in 2011 by krieg ( table  1 ) .
this 16s rrna nucleotide similarity to barnesiella viscericola was lower than the threshold of 95% recommended by stackebrandt and ebers   to delineate a new genus . consequently , g.  massiliensis gen .
the spectrum resulting from g.  massiliensis was thus added into the bruker database ( fig . 
2 ) , and a gel view was performed in order to see the spectra differences between other close bacteria ( fig .  3 ) .
then the 16s rrna sequence of g.  massiliensis was deposited in genbank under accession number ln849789 .
, strain gm3 (= csur p1909  =  dsm 100571 ) , is a gram - negative bacillus ( fig .  4 ) .
strain gm3 is a non motile , non - spore - forming , oxidase - negative but catalase - positive bacterium .
growth was observed from 28 to 45c , with optimal growth at 37c , and colonies were obtained after 48 hours of culture .
g.  massiliensis grew in salinity conditions of 0 and at a ph of 7 .
it exhibited translucent colonies with a diameter of 1.5  mm on 5% sheep 's blood  enriched colombia agar .
strain gm3 is a preferentially anaerobic bacillus but was able to grow in microaerophilic atmosphere and unable to grow in aerobic conditions .
individual cells had a mean diameter of 0.5  m and a length of 1.25  m when observed with electron microscopy ( fig .  5 ) .
using api 20a , we observed positive reactions with d - glucose , d - lactose , d - maltose , d - cellobiose , d - mannose , d - raffinose , d - rhamnose and d - trehalose but not with d - mannitol , d - sucrose , salicin , d - xylose , l - arabinose , glycerol , d - melezitose and d - sorbitol .
negative reactions were recorded for indole formation , urease and gelatin using the same gallery .
using api 50ch , we concluded that g.  massiliensis is able to ferment inositol , methyl-d - mannopyranoside , methyl-d - glucopyranoside , n - acetylglucosamine , esculin ferric citrate , d - cellobiose , d - maltose , d - lactose , d - sucrose , inulin , starch , d - arabitol and potassium-5-ketogluconate . results from the api zym gallery showed enzymatic activities of alkaline phosphatase , esterase ( c4 ) , esterase lipase ( c8 ) , acid phosphatase , naphthol - as - bi - phosphohydrolase , - and -galactosidase and n - acetyl--glucosaminidase .
antibiotic susceptibility testing revealed that g.  massiliensis strain gm3 is sensitive to nitrofurantoin , doxycycline , rifampicin , amoxicillin / clavulanic acid , and imipenem .
however , it remained resistant against amoxicillin , vancomycin , clindamycin , gentamicin , erythromycin / metronidazole , tobramycin , trimethoprim / sulfamethoxazole , amikacin and ciprofloxacin .
the main phenotypic and biochemical characteristics of g.  massiliensis compared to the closest bacteria are summarized in table  2 .
it was composed of 19 scaffolds ( composed of 66 contigs ) ( fig . 
the 3,288 predicted genes , 3,227 were protein - coding genes and 61 were rna genes ( two genes were 5s rrna , two genes were 16s rrna , two genes were 23s rrna and 55 genes were trna genes ) .
a total of 1,807 genes ( 56.00% ) were assigned as putative function ( by cogs or by nr blast ) , and 113 genes ( 3.50% ) were identified as orfans ( table  3 ) .
the remaining genes were annotated as encoding hypothetical proteins ( 1,229 genes , 38.08% ) .
nucleotide content and gene count levels of the genome are summarized in table  3 , while the distribution of genes into cogs functional categories is presented in table  4 .
the draft genome of gabonia massiliensis strain gm3 was larger in size ( 4.26  mb ) than bacteroides salanitronis strain dsm 18170 , alistipes shahii strain wal 8301 , alistipes finegoldii strain dsm 17242 , barnesiella viscericola strain dsm 18177 and ornithobacterium rhinotracheale strain ort - umn 88 ( 4.24 , 3.76 , 3.73 , 3.08 and 2.4 mb , respectively ) but smaller than bacteroides fragilis strain ych46 and bacteroides dorei strain hs1 ( 5.28 and 5.24 mb ) .
the g+c content ( 37.9% ) of gabonia massiliensis strain gm3 is larger than that of ornithobacterium rhinotracheale strain ort - umn 88 ( 37.2% ) but smaller than those of alistipes shahii strain wal 8301 , alistipes finegoldii strain dsm 17242 , barnesiella viscericola strain dsm 18177 , bacteroides fragilis strain ych46 and bacteroides dorei strain hs1 ( 57.2 , 56.6 , 51.7 , 46.6 , 43.3 and 41.6 , respectively ) . the gene content ( 3228 ) of gabonia
massiliensis strain gm3 is larger compared to those of ornithobacterium rhinotracheale strain ort - umn 88 , barnesiella viscericola strain dsm 18177 , alistipes shahii strain wal 8301 and alistipes finegoldii strain dsm 17242 ( 2,289 , 2,557 , 3,090 and 3,226 ) but smaller than that of bacteroides fragilis strain ych46 , bacteroides dorei strain hs1 and bacteroides salanitronis strain dsm 18170 ( 4,670 , 4,024 and 3,543 ) .
similarly , the number of protein - coding genes ( 3,227 ) of gabonia massiliensis strain gm3 were higher than those of ornithobacterium rhinotracheale strain ort - umn 88 , barnesiella viscericola strain dsm 18177 , alistipes shahii strain wal 8301 and alistipes finegoldii
strain dsm 17242 ( 2,210 , 2,464 , 2,996 and 3,108 ) but lower than those of bacteroides fragilis strain ych46 , bacteroides dorei strain hs1 and bacteroides salanitronis strain dsm 18170 ( 4,578 , 4,024 and 3,543 ) .
however , the distribution of genes into cogs categories was similar in all genomes compared ( fig . 
in addition , gabonia massiliensis strain gm3 shared 3,231 , 3,132 , 4,269 , 4,373 , 3,686 , 2,499 , 1,579 and 2,312 orthologous genes with alistipes finegoldii strain dsm 17242 , alistipes shahii strain wal 8301 , bacteroides dorei strain hs1 , bacteroides fragilis strain ych46 , bacteroides salanitronis strain dsm 18170 , barnesiella viscericola strain dsm 18177 , helicobacter pylori strain 26695 and ornithobacterium rhinotracheale strain ort - umn 88 ( table  5 ) .
although gabonia massiliensis is a draft genome , making the comparison complex , we propose a summary of the comparison of genome content of gabonia massiliensis and other bacteria in table  6 . among species with standing in nomenclature , agios values ranged from 83.25 between alistipes shahii strain wal 8301 and alistipes finegoldii strain dsm 17242 to 56.41 between gabonia massiliensis strain gm3 and alistipes finegoldii strain dsm 17242 ( table  5 ) .
to evaluate genomic similarity among the studied strains , we determined two parameters , agios ( table  5 ) , which was designed to be independent from dna - dna hybridization ( ddh ) , and digital ddh , which exhibits a high correlation with ddh ,   ( table  7 ) .
on the basis of phenotypic , phylogenetic and genomic analyses , we formally propose the creation of gabonia massiliensis gen .
this bacterium was isolated from a stool sample from a healthy gabonese male youth in marseille , france .
n. gabonia , the latin name of gabon , the sub - saharan african country where the stool specimen was collected ) .
this is a gram - negative , non - spore - forming and non motile bacillus .
it showed positive activity for alkaline phosphatase , esterase ( c4 ) , esterase lipase ( c8 ) , acid phosphatase , naphthol - as - bi - phosphohydrolase , - and -galactosidase and n - acetyl--glucosaminidase .
massiliensis , of massilia , the latin name of marseille , where g.  massiliensis was isolated ) .
individual cells exhibited a diameter of 0.5  m and a length of 1.25  m .
it is a gram - negative , non - spore - forming and nonmotile bacillus .
g.  massiliensis showed positive reactions for alkaline phosphatase , esterase ( c4 ) , esterase lipase ( c8 ) , acid phosphatase , naphthol - as - bi - phosphohydrolase , - and -galactosidase and n - acetyl--glucosaminidase .
it was sensitive for nitrofurantoin , doxycycline , rifampicin , amoxicillin / clavulanic acid and imipenem .
g.  massiliensis type strain gm3 (= csur p1909  =  dsm 100571 ) was isolated from a stool sample of a healthy gabonese male youth .
its 16s rrna sequence was deposited in genbank under accession number ln849789 , and the whole genome shotgun sequence has been deposited in genbank under accession number cypv00000000 .
 culturomics coupled with taxonogenomics is currently used to isolate and characterize new bacteria . here 
 we describe the features and complete genome sequence of gabonia massiliensis strain gm3 , an anaerobic gram negative , non - spore - forming and catalase - positive bacillus isolated from a stool specimen of a healthy gabonese male youth . belonging to a new genus called gabonia , it exhibits a genome of 4  261  752  bp including 37.9% gc 
 content and 3,288 predicted genes . 
Introduction
Material and Methods
Results
Conclusion
Taxonomic and nomenclatural proposals