culturomics is a new approach introduced in our laboratory in 2012 that aims to study the diversity of the microorganisms colonizing the human gut .
recently culturomics was applied to explore the bacterial diversity of patients with kwashiorkor . during this study , we isolated the strain mt9 (=  csur p1511  =  dsm 102954 ) , which belongs to a new bacterial genus within the family clostridiaceae .
this family was created by ernst august pribram in 1933   and contains as its core the genus clostridium and 36 other genera .
they have been isolated from highly diverse habitats , including freshwater and marine sediments , salt lakes , various sites of the human body , manure piles , sewage sludge and different types of faeces .
so far , the criteria used to classify new species based on 16s rdna sequencing and dna - dna hybridization ( ddh ) , are limited due to the low cutoff between species and genus .
recently we proposed a new polyphasic approach associating phenotypic characterization , matrix - assisted desorption ionization 
time of flight mass spectrometry ( maldi - tof ms ) , and genotypic characteristics to better define and classify new species .
in addition , characterizing new species by sequencing the whole genome allows a better distinction between the closest related species , comparing the whole genome and all of its coded function ,   and genomic data to become available for new bacterial species . in this culturomics study
, we isolated the strain mt9 (= csur p1511  =  dsm 102954 ) , which is the first representative of a new bacterial genus within the family clostridiaceae . here
the stool specimens were collected from a 1-year - old senegalese patient with kwashiorkor after defecation in sterile plastic containers . the stool specimen was then formed into aliquots and stored at  80c until use .
the study and the assent procedure were approved by the national ethics committee of senegal and the local ethics committee of the ifr48 ( marseille , france ) under numbers 11 - 017 and 09 - 022 , respectively .
the stool sample was cultivated in anaerobic conditions and preincubated in a blood culture bottle ( becton dickinson , le pont - de - claix , france ) at 37c for 15 days after a thermic shock at 80c for 20 minutes .
then 1  ml of the enriched culture was spread on a 5% sheep 's blood 
enriched columbia agar ( biomrieux , marcy l'etoile , france ) and incubated at 37c for 72 hours .
the obtained colonies were purified and identified using maldi - tof ms as previously described , .
this proteomic analysis was conducted using a microflex spectrometer ( bruker daltonics , leipzig , germany ) with a mtp 96 maldi - tof target plate ( bruker ) .
the obtained spectra were imported into maldi biotyper 2.0 software ( bruker ) and analysed by standard pattern matching ( with default parameter settings ) against the 7567 references contained in our database ( bruker database incremented with our data ) .
when the identification score was 1.9 , the species level was determined ; a score between 1.9 and 1.7 allowed identification only at the genus level ; and a score < 1.7 gave no identification . in this last case ,
the 16s rrna gene was sequenced as previously described   in order to get a molecular identification .
stackebrandt and ebers   determined a 98.7 and 95% similarity level threshold to define a new species and a new genus respectively without performing ddh .
growth of the strain mt9 was tested under anaerobic and microaerophilic conditions generated using genbag anaer and genbag microaer systems ( biomrieux ) respectively , and in aerobic conditions with or without 5% co2 .
optimal salt concentration was determined by growing the strain at 0 , 0.5 , 1 and 1.5% nacl .
different ph conditions were tested : 6 , 6.5 , 7 , 7.5 , 8 and 8.5 . the biochemical characteristics of the strain mt9 were studied using api zym , api 20 ne and api 50 ch strips ( biomrieux ) according to the manufacturer 's instructions .
clavulanic acid , clindamycin , metronidazole , vancomycin , doxycycline , imipenem , rifampicin and trimethoprim / sulfamethoxazole using antibiotic discs ( i2a , montpellier , france ) .
the surface colonies of the strain mt9 was observed after 72-hour incubation under anaerobic conditions at 37c . in order to observe cell morphology ,
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 ; electron microscopy sciences ( 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 .
electron micrographs were acquired with a tecnai g20 cryo ( fei company , limeil - brevannes , france ) transmission electron microscope operated at 200 kev . to determine the gram stain
strain mt9 was observed under a dm1000 photonic microscope ( leica microsystems , nanterre , france ) after gram colouration as well as a fresh growing culture to assess its motility .
two samples were prepared with approximately 15  mg of bacterial biomass per tube collected from several culture plates .
briefly , fame were separated using an elite 5-ms column and monitored by ms ( 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 ( nist ) , gaithersburg , usa ) and the fames mass spectral database ( wiley , chichester , uk ) .
after a pretreatment by a lysozyme incubation at 37c for 2 hours , dna of strain mt9 was extracted on the ez1 biorobot ( qiagen , germantown , md , usa ) with ez1 dna tissues kit .
genomic dna ( gdna ) was quantified by a qubit assay with the high sensitivity kit ( life technologies , carlsbad , ca , usa ) to 31.7  ng/l .
gdna of strain mt9 was sequenced on the 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 7.670  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 804  bp on a 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 9.39 
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 in a 2    251  bp read length .
total information of 6.1 gb was obtained from a 653k / mm cluster density with a cluster passing quality control filters of 96.1% ( 12 
open reading frames ( orfs ) were predicted using prodigal   with default parameters , but the predicted orfs were excluded if they were spanning a sequencing gap region .
the predicted bacterial protein sequences were searched against the genbank database and the clusters of orthologous groups ( cogs ) database using blastp .
the trnascanse tool   was used to find trna genes , whereas ribosomal rnas were found by using rnammer   and blastn against the genbank database .
lipoprotein signal peptides and the number of transmembrane helices were predicted using signalp and tmhmm respectively .
orfans were identified if their blastp e value was lower than 1e for alignment length greater than 80 amino acids .
if alignment lengths were smaller than 80 amino acids , we used an e value of 1e .
we compared the genome of strain mt9 to those of other members of the order clostridiales .
their genomes were automatically retrieved from the 16s rrna tree using xegen software ( phylopattern ) .
for each selected genome , the complete genome sequence , proteome genome sequence and orfeome genome sequence were retrieved from the ftp of national center for biotechnology information .
this score is the mean value of nucleotide similarity between all couples 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 between anaeromassilibacillus genus and closely related genera
, we determined two parameters : digital dna - dna hybridization ( dddh ) , 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   that included figenix   libraries to provide pipeline analysis .
strain mt9 ( table  1 ) was isolated in january 2015 by anaerobic cultivation at 37c on 5% sheep 's blood 
maldi - tof ms results showed that the score obtained was under 1.7 for the strain mt9 , thus suggesting that our isolate was not a member of a known species listed in our database .
16s rrna gene sequencing and comparison demonstrate that the strain mt9 ( ln866991 ) exhibited 93.4% nucleotide sequence similarity with clostridium leptum ( nr114789 ) ( fig .  1 ) .
this value was lower than the 95% 16s rrna gene sequence threshold recommended by stackebrandt and ebers   to delineate a new genus without carrying out ddh .
we incremented our database with the spectrum from strain mt9 ( fig .  2 ) .
colonies of the strain mt9 were 1 to 2  mm in diameter on columbia agar , and opaque white in colour .
motile and spore - forming , 2.40  m in length and 0.9  m in diameter ( fig .  5 ) .
strain mt9 grows in a range from 25 to 37c , with an optimum at 37c .
nacl is not required for growth , but strain mt9 was only able to grow between 0 and 0.5% nacl .
using the api zym gallery , positive reactions were observed for alkaline phosphatase , esterase , esterase lipase , valine arylamidase , acid phosphatase , naphtol - as - bi - phosphohydrolase , -galactosidase , -galactosidase , -glucosidase , -glucosidase , n - acetyl--glucosaminidase and -mannosidase .
the api 50ch gallery showed positive reactions for d - ribose , d - adonitol , dulcitol , d - sorbitol , amygdalin , arbutin , esculin , d - cellobiose , d - maltose , inulin , d - raffinose , amidone , glycogen , xylitol , gentiobiose , d - tagatose and potassium 5-ketogluconate . using the api 20 ne , enzymatic activities were observed for gelatin hydrolysis and -galactosidase .
antibiotic susceptibility test demonstrated that strain mt9 is susceptible to vancomycin , doxycycline , ceftriaxone , imipenem , amoxicillin 
clavulanic acid , penicillin and rifampicin , and is resistant to trimethoprim / sulfamethoxazole , ciprofloxacin , oxacillin , cefoxitin , colistin and fosfomycin .
the phenotypic and biochemical features of the strain mt9 were compared to the features of closely related species ( table  2 ) .
the major fatty acids were hexadecanoic acid ( 32% ) , tetradecanoic acid ( 27% ) and 9-octadecenoic acid ( 26% ) .
minor amounts of unsaturated , branched and other saturated fatty acids were also detected ( table  3 ) .
the 3  511  289  bp long genome contains 52.94% of g+c content ( fig .  6 and table  4 ) and
is composed of 12 scaffolds ( composed of 15 contigs ) . of the 3095 predicted genes ,
3046 were protein - coding genes and 49 were rnas ( two genes are 5s rrna , one gene is 16s rrna , one gene is 23s rrna and 45 genes are trna genes ) .
a total of 1773 genes ( 58.21% ) were assigned as putative function ( by cogs or by nr blast ) .
the genomic characteristics of strain mt9 were compared to closely related species ( table  6 ) .
the draft genome sequence of strain mt9 ( 3.51  mb ) is smaller than those of oscillibacter valericigenes , clostridium cellulolyticum , flavonifractor plautii , acetivibrio cellulolyticus , clostridium clariflavum and clostridium papyrosolvens ( 4.47 , 4.07 , 3.81 , 6.15 , 4.90 and 4.40  mb respectively ) , but larger than those of ethanoligenens harbinense , faecalibacterium prausnitzii and clostridium leptum ( 3.01 , 3.32 and 2.82  mb respectively ) .
the g+c content of strain mt9 ( 52.94% ) is smaller than those of o.  valericigenes , e. harbinense , f. prausnitzii and f.  plautii ( 53.19 , 55.56 , 56.82 and 61.07% respectively ) , but larger than those of c.  cellulolyticum , c. leptum , a. cellulolyticus , c. clariflavum and c.  papyrosolvens ( 37.40 , 50.25 , 35.55 , 35.72 and 37.13% respectively ) .
the gene content of strain mt9 ( 3046 ) is smaller than those of o.  valericigenes , c. cellulolyticum , f. plautii , a. cellulolyticus , c. clariflavum and c.  papyrosolvens ( 4723 , 3390 , 4278 , 5082 , 3892 and 3867 respectively ) , but larger than those of e.  harbinense , f. prausnitzii and c.  leptum ( 2701 , 2756 and 2482 respectively ) .
strain mt9 shared respectively 866 , 787 , 841 , 789 , 766 , 818 , 681 , 780 and 772 orthologous genes with f.  plautii , a. cellulolyticus , c. leptum , c. papyrosolvens , e. harbinense , o. valericigenes , f. prausnitzii , c. cellulolyticum and c. clariflavum .
the comparison of the strain mt9 to other species resulted in agios values ranging from 57.07% with a.  cellulolyticus to 67.67% with c.  leptum ( table  7 ) .
dddh was estimated between strain mt9 's genome and the other compared genomes , and results ranged from 19.6% with f.  plautii to 30.4% with c.  clariflavum ( table  8) .
these values are in the range of the dddh values of compared species except our strain ( 18.832.2% ) and under the 70% threshold used to delineate a new species , thus confirming the status of the strain mt9 as a previously unknown member of the clostridiaceae family .
the distribution of genes into cogs categories was similar , but not identical , in all compared genomes ( fig . 
for instance , o.  valericigenes presented more genes dedicated to the dna replication , recombination and repair .
the new polyphasic approach based on phenotypic , phylogenetic and genomic analyses ( taxonogenomics ) represented here support the affiliation of the strain mt9 to a new bacterial genus .
, sp . nov . , is the first representative of this new genus for which the strain mt9 is the type strain .
this strain was isolated from the stool specimen of a 1-year - old patient with kwashiorkor living in senegal .
n. , anaero , from anar , the greek term for absence of oxygen , massili for massilia , the latin name of marseille , and bacillus ,  little rod ) .
cells are gram - negative , motile , strictly anaerobic , spore - forming , rod - shaped bacilli .
adj . , senegalensis ,  originating from senegal, the country from which the stool sample was collected ) .
cells are gram - negative , motile , spore - forming , rod - shaped bacilli with a length of 2.4  m and a diameter of 0.9  m . colonies were 1 to 2  mm in diameter on columbia agar , and opaque white in colour .
a.  senegalensis strain mt9 is strictly anaerobic , grows at 37c ( optimum ) and ph 7 ( optimum ) and nacl is not required .
positive reactions were observed for alkaline phosphatase , esterase , esterase lipase , valine arylamidase , acid phosphatase , naphtol - as - bi - phosphohydrolase , galactosidase , -galactosidase , -glucosidase , -glucosidase , n - acetyl--glucosaminidase , -mannosidase , d - ribose , d - adonitol , dulcitol , d - sorbitol , amygdalin , arbutin , esculin , d - cellobiose , d - maltose , inulin , d - raffinose , amidone , glycogen , xylitol , gentiobiose , d - tagatose and potassium 5-ketogluconate .
antibiotic susceptibility test showed that a.  senegalensis strain mt9 is susceptible to vancomycin , doxycycline , ceftriaxone , imipenem , amoxicillin 
clavulanic acid , penicillin , and rifampicin , and resistant to trimethoprim / sulfamethoxazole , ciprofloxacin , oxacillin , cefoxitin , colistin and fosfomycin .
the 3  511  289  bp long genome exhibits a g+c content of 52.94% .
the 16s rrna and genome sequences are deposited in embl - ebi under accession numbers ln866991 and cwip00000000 , respectively .
the type strain mt9 (= csur p1511  =  dsm 102954 ) was isolated from the gut microbiota of a 1-year - old senegalese patient with kwashiorkor .
 using the culturomics strategy , a wide range of anaerobic bacteria was discovered including anaeromassilibacillus senegalensis strain mt9 t (=  csur p1511  =  dsm 102954 ) , isolated from the gut microbiota of a 1-year - old senegalese patient with kwashiorkor . 
 this gram - negative strain is a strictly anaerobic , spore - forming rod motile by a polar flagellum . 
 the 3  511  289  bp long genome of this strain contains 3046 protein - coding and 49 rna genes , including 45 trna and four rrna genes , and exhibits a g+c content of 52.94% . here 
 we describe the features of this organism , together with the complete genome sequence and annotation . 
Introduction
Materials and Methods
Results
Conclusion