we propose anaerosalibacter massiliensis strain nd1 (= csur p762  =  dsm 27308 ) as the type strain of a.  massiliensis sp .
nov . , a new species within the genus anaerosalibacter ( rezgui et  al . ,
strain nd1 was isolated from the stool sample of a 49-year - old brazilian man as part of a culturomics study aiming at cultivating individually all bacterial species of the human gut microbiota , .
anaerosalibacter massiliensis is a gram - positive , obligate anaerobic , moderately halophilic and motile rod - shaped bacillus .
the genus anaerosalibacter ( rezgui et  al . , 2012 ) was created in 2012 and contains , to date , only one species , a.  bizertensis ( rezgui et  al . ,
2012 ) , an obligate anaerobic , gram - positive and rod - shaped bacillus that was isolated from sludge in bizerte , tunisia .
this polyphasic strategy combines phenotypic characteristics that may be obtained by most clinical microbiology laboratories wordwide , the matrix - assisted laser desorption / ionization time - of - flight mass spectrometry ( maldi - tof ) spectrum , and the analysis and comparison of the complete genome sequence . to date , taxonogenomics has enabled us to validly publish 13 bacterial names , , , , , , , , , , , , , , , .
strain nd1 (= csur p762  =  dsm 27308 ) , including its phenotype and genome sequence . on the basis of these characteristics
, we found that strain nd1 is sufficiently different from a.  bizertensis to be classified as a new anaerosalibacter species , and we propose the creation of the species anaerosalibacter massiliensis sp .
a stool sample was collected from a healthy 49-year - old brazilian volunteer living in manaus , brazil .
the patient signed informed consent , and the agreement of the local ethics committee of the ifr48 ( marseille , france ) was obtained under agreement 09 - 022 .
the faecal sample was frozen at  80c after collection and shipped to marseille , france .
a.  massiliensis strain nd1 ( table  1 ) was isolated in november 2013 by cultivation on 5% sheep 's blood  enriched agar ( biomrieux , marcy ltoile , france ) in anaerobic conditions after 14 days of preincubation of the stool specimen in a blood bottle culture , with addition of 5  ml rumen sheep .
strain nd1 exhibited a 98.05% 16s rrna sequence identity with a.  bizertensis ( genbank accession no .
hq534365 ) , the phylogenetically closest bacterial species with a validly published name ( figure  1 ) .
this value was lower than the 98.7% 16s rrna gene sequence threshold recommended by stackebrandt and ebers   to delineate a new species without carrying out dna - dna hybridization .
growth occurred between 37 and 45c , but optimal growth was observed at 37c after 48 hours of incubation in anaerobic conditions .
the colonies were 1.3  mm in diameter and moderately opaque on 5% sheep 's blood  enriched agar ( biomrieux ) .
growth of the strain was tested under anaerobic and microaerophilic conditions using genbag anaer and genbag microaer systems respectively ( biomrieux ) and under aerobic conditions with and without 5% co2 .
gram staining showed rod - shaped , gram - positive bacilli able to form spores ( figure  2 ) .
cells grown on agar were moderately opaque and exhibited ranges in diameter and length of 0.51 and 25  m respectively in electron microscopy ( figure  3 ) .
we also observed an oval and terminal spore of a 0.7    0.8  m , causing a terminal swelling ( figure  3 ) .
strain nd1 exhibited neither catalase nor oxidase activities . using api rapid i d 32a ( biomrieux ) , positive reactions were observed for arginine dihydrolase , n - acetyl--glucosaminidase and pyroglutamic acid arylamidase .
negative reactions were observed for urease , indole , nitrate reduction , l - arabinose , ribose , mannose , d - lactose , d - fructose , d - maltose and sucrose activities . using an api 50ch strip ( biomrieux ) , positive reactions were obtained for mannitol , arbutine , lactose and glycogen .
anaerosalibacter massiliensis is susceptible to penicillin g , amoxicillin , gentamicin , amoxicillin / clavulanate , ciprofloxacine , metronidazole , ceftriaxone , imipenem , erythromycin , rifampicin and doxycycline but resistant to trimethoprim / sulfamethoxazole .
compared to a.  bizertensis and representative species from other members of the genus clostridium , a. massiliensis strain nd1 differed in a combination of nitrate reductase and -galactosidase activities as well as arginine use ( table  2 ) .
maldi - tof protein analysis was carried out as previously described   using a microflex spectrometer ( bruker daltonics , leipzig , germany ) .
twelve individual colonies were deposited on a mtp 384 maldi - tof target plate ( bruker ) .
the 12 nd1 spectra were imported into maldi biotyper 2.0 software ( bruker ) and analysed by standard pattern matching ( with default parameter settings ) against the main spectra of 4706 bacteria , including one spectrum from anaerosalibacter bizertensis , used as reference data in the biotyper database .
a score enabled the presumptive identification and discrimination of the tested species from those in the database ; a score of > 2 with a validated species enabled the identification at the species level and a score of < 1.7 did not enable identification . for strain nd1 ,
no significant score was obtained , suggesting that our isolate was not a member of any known species ( fig .  4 , fig .  5 ) .
we added the spectrum from strain nd1 to our database ( figure  4 ) .
finally , the gel view showed the spectral differences with other members of the family clostridiaceae ( figure  5 ) .
gc / ms analyses were carried out on a clarus 500 gas chromatograph equipped with a sq8s  ms detector ( perkin elmer , courtaboeuf , france ) .
two microlitres of fame extracts were volatized at 250c ( split 20  ml / min ) in a focus liner with wool and separated on an elite-5ms column ( 30  m , 0.25  mm i.d .
, 0.25  mm film thickness ) using a linear temperature gradient ( 70 to 290c at 6c / min ) , enabling the detection of c4 to c24 fatty acid methyl esters .
full scan monitoring was performed from 45 to 500  m / z . all data were collected and processed using turbomass 6.1 ( perkin elmer ) .
fatty acid methyl esters were identified by spectral database search using ms search 2.0 operated with the standard reference database 1a ( national institute of standards and technology [ nist ] , gaithersburg , md , usa ) and the fames mass spectral database ( wiley , chichester , uk ) .
a 37-component fame mix ( supelco ; sigma - aldrich , saint - quentin fallavier , france ) was used for retention time correlations with estimated nonpolar retention indexes from the nist database ; fame identifications were confirmed using this index .
small proportions of four other fatty acids were also detected ( table  3 ) .
the organism was selected for sequencing on the basis of its phylogenetic position and 16s rrna similarity to a.  bizertensis and other members of the family clostridiaceae .
it is part of a culturomics study of human digestive flora that aims to isolate all bacterial species within human faeces .
it was the first genome of anaerosalibacter species and the first genome of a.  massiliensis sp .
table  4 shows the project information and its association with minimum information about a genome sequence ( migs ) version 2.0 compliance .
, strain nd1 (= csur p762  =  dsm 27308 ) , was grown on 5% sheep 's blood 
then 200 l of this suspension was diluted in 1  ml te buffer for lysis treatment that included a 30-minute incubation with 2.5  g/l lysozyme at 37c , followed by an overnight incubation with 20  g/l proteinase k at 37c .
extracted dna was then purified using three successive phenol  chloroform extractions and ethanol precipitations at  20c overnight .
genomic dna of anaerosalibacter massiliensis was sequenced on the miseq sequencer ( illumina , san diego , ca , usa ) using two sequencing strategies : paired end and mate pair
. the paired end and the mate pair strategies were barcoded in order to be mixed respectively with 11 other genomic projects prepared with the nextera xt dna sample prep kit ( illumina ) and 11 other projects with the nextera mate pair sample prep kit ( illumina ) .
genomic dna was quantified at 35.3  ng/l using the qubit assay ( life technologies , carlsbad , ca , usa ) and diluted to 1  ng/l as input to prepare the paired - end library .
after tagmentation to fragment and tag the dna , limited cycle pcr amplification ( 12 cycles ) completed the tag adapters and introduced dual - index barcodes .
after purification using ampure xp beads ( beckman coulter , fullerton , ca , usa ) , the library was normalized using specific beads according to the nextera xt protocol ( illumina ) .
the library was loaded onto the reagent cartridge and then onto the instrument along with the flow cell .
automated cluster generation and paired - end sequencing with dual index reads were performed in a single 39-hour run at a 2    250  bp read length .
the sequencing generated 5.7 gb , of which 732  000 reads were from a.  massiliensis .
the mate pair library was prepared with 1  g of genomic dna using the nextera mate pair illumina guide .
the genomic dna sample was simultaneously fragmented and tagged with a mate pair junction adapter .
the profile of the fragmentation was validated on an agilent 2100 bioanalyzer ( agilent technologies , santa clara , ca , usa ) with a dna 7500 lab chip .
the dna fragments ranged in size from 1 to 11  kb , with an optimal size of 5  kb .
no size selection was performed , and 600  ng of tagmented fragments were circularized .
the circularized dna was mechanically sheared to small fragments with optimum at 692  bp on the covaris s2 device in microtubes ( covaris , woburn , ma , usa ) .
the library profile was visualized on a high sensitivity bioanalyzer labchip ( agilent technologies ) .
after a denaturation step and dilution at 10  pm , the pool of libraries was loaded onto the reagent cartridge and then onto the miseq instrument along with the flow cell . automated cluster generation and
the sequencing run were performed in a single 42-hour run at a 2    250  bp read length .
the mate pair sequencing generated 3.2 gb , 925  000 reads of which were from a.  massiliensis .
the reads obtained from both applications were trimmed , and the optimal assembly was obtained through the spades software with eight scaffolds , which generated a genome size of 3.28 mb . the gc% was calculated at 29% .
open reading frames ( orfs ) were predicted using prodigal   with default parameters , but the predicted orfs were excluded if they spanned a sequencing gap region .
the predicted bacterial protein sequences were searched against the genbank database   and the clusters of orthologous groups ( cogs ) databases 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-03 for alignment length greater than 80 amino acids .
if alignment lengths were smaller than 80 amino acids , we used an e value of 1e-05 .
such parameter thresholds have already been used in previous works to define orfans . because no genome was available for a.  bizertensis 
 , the only anaerosalibacter species with standing in nomenclature
, we compared the genome from a.  massiliensis strain nd1 to those of other members of the family clostridiaceae , including clostridium ultunense strain esp ( cara00000000 ) , c.  acidurici strain 9a ( cp003326 ) , 
 c.  dakarense strain ff1 ( genbank accession no .
the last two species have as yet no standing in nomenclature but were proposed as new species ,   and are phylogenetically close to a.  massiliensis .
ortholog sets composed of one gene from each of these five genomes were identified using proteinortho 1.4 software   using threshold values of 30% protein identity and a 1e-05 e value .
the average percentages of nucleotide sequence identity between corresponding orthologous sets were determined using the needleman - wunsch algorithm global alignment technique .
artemis   was used for data management , and dnaplotter   was used for visualization of genomic features .
the genome of a.  massiliensis strain nd1 is 3  197  911  bp long with a 29.70% g+c content ( figure  6 , table  5 ) . of the 3333 predicted genes ,
six rrna genes ( two identical 16s rrnas , two identical 23s rrnas and two 5s rrnas ) and 56 predicted trna genes were identified in the genome . a total of 2376 genes ( 71.37% ) were assigned a putative function .
the distribution of genes into cogs functional categories is presented in table  6 and figure  7 .
we compared the genome of a.  massiliensis strain nd1 those of c.  acidurici strain 9a ( genbank accession no .
cp003326 ) , , c.  dakarense strain ff1 ( cbtz00000000 ) , c.  senegalense strain jc122 ( caev00000000 )   and c.  ultunense strain esp ( cara00000000 ) .
the draft genome of a.  massiliensis has a larger size than that of c.  acidurici ( 3.19 and 3.11 mb respectively ) but is smaller than those of c.  dakarense , c. senegalense and c.  ultunense ( 3.73 , 3.89 and 6.13 mb respectively ) .
the g+c content of a.  massiliensis is higher than those of c.  dakarense and c.  senegalense ( 29.70 , 27.98 and 26.8% respectively ) but lower than those of c.  acidurici and c. ultunense ( 29.9 and 40.9% respectively ) .
the gene content of a.  massiliensis is larger than those of c.  acidurici ( 3330 and 2957 genes respectively ) and smaller than those of c.  dakarense , c. senegalense and c.  ultunense ( 3916 , 3761 and 6744 genes respectively ) .
in addition , a.  massiliensis shared 3271 , 2839 , 3808 , 3704 and 5711 orthologous genes with c.  acidurici , c. dakarense , c. senegalense and c.  ultunense respectively .
the average nucleotide sequence identity ranged from 71.49% to 66.45% between a.  massiliensis and other members of the family clostridiaceae ( table  7 ) .
on the basis of phenotypic , phylogenetic and genomic analyses , we formally propose the creation of anaerosalibacter massiliensis sp .
this bacterial strain was isolated from the faecal flora of a 49-year - old brazilian man in good health .
n. , from massiliensis , massilia , the latin name for marseille , where a.  massiliensis was first isolated ) .
colonies were 1.3  mm in diameter and moderately opaque on 5% sheep 's blood  enriched agar .
cells are gram positive , rod shaped , motile and obligate anaerobic , with a mean diameter and length of 0.97 and 2.71  m respectively .
a.  massiliensis is negative for catalase , oxidase , urease , indole , nitrate reduction , l - arabinose , ribose , mannose , d - lactose , d - fructose , d - maltose and sucrose activities but positive for arginine dihydrolase , n - acetyl--glucosaminidase , pyroglutamic acid arylamidase , mannitol , arbutine , lactose and glycogen activities .
cells were susceptible to penicillin g , amoxicillin , amoxicillin / clavulanate , ceftriaxone , imipenem , gentamicin , ciprofloxacine , metronidazole , erythromycin , rifampicin and doxycycline but resistant to trimethoprim / sulfamethoxazole .
the 16s rrna and genome sequences are deposited in genbank under accession numbers hg315673 and ccez01000001- ccez010000082 respectively .
the type strain nd1 (= csur p762  =  dsm 27308 ) was isolated from the faecal flora of a 49-year - old healthy brazilian man .
 anaerosalibacter massiliensis sp . nov . strain nd1 
 t (= csur p762  =  dsm 27308 ) is the type strain of a.  massiliensis sp . nov . 
 , a new species within the genus anaerosalibacter . 
 this strain , the genome of which is described here , was isolated from the faecal flora of a 49-year - old healthy brazilian man . 
 anaerosalibacter massiliensis is a gram - positive , obligate anaerobic rod and member of the family clostridiaceae . with the complete genome sequence and annotation , 
 we describe here the features of this organism . 
 the 3  197  911  bp long genome ( one chromosome but no plasmid ) contains 3271 protein - coding and 62 rna genes , including six rrna genes . 
Introduction
Organism Information
Genome Sequencing Information
Genome properties
Conclusions
Description of 