virgibacillus massiliensis strain vm-5 (= csur p971  =  dsm 28587 ) is the type strain of v.  massiliensis sp .
this bacterium is a gram - positive , strictly aerobic rod , motile by a polar flagellum , isolated from the stool specimen of a healthy amazonian boy as part of the culturomics study aiming at cultivating halophilic bacteria from the human feces using a high - salt - concentration medium .
the usual parameters used to delineate a bacterial species include 16s rrna sequence identity and phylogeny , , genomic g  +  c content diversity and dna - dna hybridization , . nevertheless , these methods have limitations , notably because these similarity values vary greatly between species and genera .
in addition , chemotaxonomic analyses such as fatty acid profile , cell wall diagnostic diamino acid and sporangium morphology are only performed by a few laboratories , are only partially reproducible and thus are of no practical value to identify clinical isolates .
therefore , we deliberately decided not to use these methods but rather include parameters that could be compared among laboratories , including widely used phenotypic criteria , matrix - assisted laser desorption / ionization time - of - flight mass spectrometry ( maldi - tof ) spectrum and genome sequence
. the introduction of high - throughput sequencing techniques has allowed researchers to make genomic data available for many bacterial species , , , , .
we recently proposed a new method ( taxonogenomics ) consisting in a polyphasic approach to describe new bacterial species .
this strategy combines phenotypic characteristics including maldi - tof spectrum and genomic analysis , , , , . here
we present a summary classification and a set of features for the virgibacillus massiliensis sp .
, strain vm-5 (= csur p971  =  dsm 28587 ) , including the description of its complete genome sequence and annotation .
virgibacillus massiliensis is the first representative from the virgibacillus genus to be isolated from the human intestinal microbiota .
the genus virgibacillus was first described by heyndrickx et  al . in 1998 and currently consists of mainly gram - positive , motile , spore - forming , rod - shaped bacteria that are moderately halophilic .
members of the genus virgibacillus are found in various environments including sediment of a saline lake , , , , traditional salt - fermented seafood , a permafrost core collected from the canadian high arctic , a marine solar saltern , , , , biofilm formation on mural paintings , seawater , , field soil , a dairy product sample , a saline mud sample , residual wash water produced during the processing of spanish - style green table olive sewage , salt crust   and fermented fish .
stool specimens were collected from a 10-year - old amazonian boy , formed into aliquots and stored at  80c until use . the child and his parents provided informed consent .
the study and the assent procedure were approved by the ethics committees of the institut fdratif de recherche 48 , faculty of medicine , marseille , france , under agreement 09 - 022 .
the salt concentration of the stool specimen was determined using a digital refractometer ( fisher scientific , illkirch , france ) and the ph with a ph meter ( table  1 ) .
strain vm-5 ( table  1 ) was isolated in december 2013 by aerobic culture on a homemade culture medium consisting of a columbia agar culture medium ( sigma - aldrich , saint - quentin fallavier , france ) modified by adding ( per liter ) the following : mgcl2 6h2o , 5  g ; mgso4 7h2o , 5  g ; kcl , 2  g ; cacl2 2h2o , 1  g ; nabr , 0.5  g ; nahco3 , 0.5  g , glucose , 2  g and 100 
strain vm-5 ( genbank accession number hg931931 ) exhibited a 16s rrna sequence identity of 97.3% with virgibacillus olivae strain e308 ( nr043572 ) , its phylogenetically closest bacterial species with standing in nomenclature ( fig .  1 ) .
colonies were obtained on our homemade culture medium after 24 hours of incubation in aerobic conditions at 37c .
the colonies of strain vm-5 were circular , greyish , shiny and smooth , with a diameter of 2 to 5  mm .
they were motile by polar flagella , were terminal spore forming and most commonly occurred as single cells or in pairs .
strain vm-5   was mesophilic and grew at temperatures ranging from 15 to 45c , at an optimum temperature of 37c .
the isolate required nacl for growth and grew at salinity ranging from 5 to 200 
the growth of strain vm-5 was tested under aerobic atmosphere , in the presence of 5% co2 and in anaerobic and microaerophilic atmospheres created using genbag anaer and genbag microaer ( biomrieux , marcy letoile , france ) , respectively .
the strain was strictly aerobic and grew in the presence of 5% co2 but did not grow in microaerophilic or anaerobic atmosphere .
the size ( 2 to 6  m in length and 0.5  m in diameter ) and ultrastructure of cells were determined by negative staining transmission electron microscopy ( fig . 
the commercially available api zym , api 20ne ( biomrieux ) , was used to characterize the biochemical properties of the strain according to the manufacturer 's instructions .
api 50 ch strips were inoculated with a bacterial suspension in api 50chb / e medium supplemented by 10% nacl ( w / v ) and incubated at 37c for 48 hours .
negative reactions were observed for alkaline phosphatase , galactosidase , n - acetyl--glucosaminidase and urease activities .
substrate oxidation and assimilation were examined using an api 50ch strip ( biomrieux ) at 37c .
negative reactions were obtained for d - lactose , l - arabinose , d - galactose and d - ribose .
positive reactions were obtained for d - glucose , d - fructose , d - mannose , d - mannitol , d - maltose and d - sucrose .
phenotypic characteristics were compared to those of the most closely related species ( table  2 ) .
virgibacillus massiliensis differed from other virgibacillus species based on its use of nitrate reductase ( + ) , n - acetyl - glucosamine (  ) , d - mannose ( + ) , d - sucrose ( + ) and d - maltose ( + ) .
antimicrobial susceptibility testing demonstrated that strain vm-5 was susceptible to penicillin , ampicillin , amoxicillin , ceftriaxone , imipenem , doxycycline , rifampicin , vancomycin , nitrofurantoin , erythromycin , ciprofloxacin and gentamicin but was resistant to trimethoprim / sulfamethoxazole and metronidazole .
briefly , a pipette tip was used to pick one isolated bacterial colony from a culture agar plate and spread it as a thin film on a maldi - tof target plate ( bruker daltonics , leipzig , germany ) .
after air drying , 2  l of matrix solution ( saturated solution of -cyanohydroxycinnaminic acid in 50% aqueous acetonitrile containing 2.5% trifluoroacetic acid ) was applied to each spot .
all spectra were recorded in the positive linear mode for the mass range of 2000 to 20  000 da ( parameter settings : ion source 1 ( isi ) , 20  kv ; is2 , 18.5  kv ; lens , 7  kv ) . a spectrum was obtained after 675 shots with variable laser power .
the 12 spectra of strain vm-5 were imported into the maldi biotyper software ( version 2.0 , bruker ) and analysed by standard pattern matching ( with default parameter settings ) against the main spectra of 7335 bacteria including the spectra from the closely related species virgibacillus proomii strain dsm 13055 , v.  proomii strain 10403186 , v.  pantothenticus strain dsm 26 , v.  halodenitrificans dsm 10037 , oceanobacillus massiliensis dsm 24644 and o.  jeddahmassiliense strain dsm 28586 .
for every spectrum , a maximum of 100 peaks were compared with spectra in database .
the resulting score enabled the identification ( or not ) of tested species : a score of  2 with a validly published species enabled identification at the species level .
no significant maldi - tof score was obtained ( < 0.9 ) for strain vm-5 against the bruker database , suggesting that our isolate was not a member of a known species .
finally , the gel view showed the spectral differences with other members of the genus virgibacillus ( fig .  4 , fig .  5 ) .
the v. massiliensis genome was sequenced as part of a culturomics study aiming at isolating all bacterial species colonizing the human gut   and because of its potential classification as a new species within the virgibacillus genus .
the genome from v.  massiliensis strain vm-5 is the fourth genome of a virgibacillus species and the first genome of v.  massiliensis sp .
this genome consists of seven contigs and was deposited in genbank under accession numbers ccdp010000001 to ccdp010000007ccdp010000001ccdp010000002ccdp010000003ccdp010000004ccdp010000005ccdp010000006ccdp010000007 .
, strain vm-5 ( csur p971  =  dsm 28587 ) , was grown on a homemade culture medium at 37c in aerobic atmosphere .
bacteria grown on ten petri dishes were collected and resuspended in 4    100  l of tris - edta ( te ) buffer . 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 3 successive phenol  chloroform extractions and ethanol precipitations at  20c overnight .
the yield and concentration was measured by the quant - it picogreen kit ( invitrogen , waltham , ma , usa ) on a genios - tecan fluorometer at 40.5  ng/l .
genomic dna ( gdna ) of v.  massiliensis vm-5 was sequenced on the miseq sequencer ( illumina , san diego , ca , usa ) using the mate pair strategy .
the gdna was bar coded in order for it 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 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 .
no size selection was performed , and only 14  ng of tagmented fragments were circularized .
the circularized dna was mechanically sheared to small fragments with an optimal at 696  bp on the covaris device s2 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 instrument along with the flow cell . automated cluster generation and sequencing run were performed in a single 42-hour run in a 2    251  bp .
total information of 4.7 gb was obtained from a 488k / mm cluster density , with a cluster passing quality control filters of 97.2% ( 9  590  000 clusters ) . within this run ,
contigs obtained were combined together by sspace   and opera software   helped by gapfiller   to reduce the set . some manual refinements using clc genomics v7 software ( clc bio , aarhus , denmark ) and homemade tools in python improved the genome . finally , the draft genome of v.  massiliensis strain vm-5 consists of seven contigs .
noncoding genes and miscellaneous features were predicted using rnammer , aragorn , rfam , pfam   and infernal .
coding dna sequences ( cdss ) were predicted using prodigal , and functional annotation was achieved using blast+   and hmmer3   against the uniprotkb database .
the genome of v.  massiliensis strain vm-5 contains 4  353  177  bp with a g  +  c content of 36.87% ( fig . 
one hundred twenty - five rnas were detected , including five rrnas ( one 16s rrna , one 23s rrna , three 5s rrna ) , 42 trnas and 78 miscellaneous rnas .
overall , 4394 genes were identified , representing a coding capacity of 3  754  518  bp ( coding percentage , 86.25% ) . among these genes ,
322 ( 7.33% ) were identified as putative proteins and 1107 ( 25.19% ) were annotated as hypothetical proteins .
moreover , 4291 genes matched a least one sequence in the clusters of orthologous groups ( cogs ) database ,   with blastp default parameters .
we compared the genome of v.  massiliensis strain vm-5 to that of v.  halodenitrificans strain 1806 ( alef01000001 ) , which is currently the closest available sequenced genome based on 16s rrna comparison ( table  6 ) .
the draft genome sequence of v.  halodenitrificans strain 1806 had a smaller size compared to v.  massiliensis strain vm-5 ( 3.9 mb vs. 4.3 mb , respectively ) , a smaller total number of genes ( 3886 and 4394 genes , respectively ) , and a lower ratio of genes per mb ( 996.4 genes / mb vs. 1010 , respectively ) , but a higher g  +  c content ( 37.41% and 36.87 , respectively ) .
at the time of analysis , only four whole genome sequences of virgibacillus were available at the national center for biotechnology information .
therefore , whole genome comparison was done between v.  massiliensis , v. alimentarius ( genbank accession number nz_jfbd00000000 ) , v. halodenitrificans ( nz_alef01000000 ) and v.  senegalensis ( nz_ccxu01000000 ) ( table  7 ) . among virgibacillus genomes , that of v.  massiliensis ( 4.35 mb ) is the largest , followed by v.  halodenitrificans ( 3.92 mb ) , v.  senegalensis ( 3.92 ) and v.  alimentarius ( 3.05 mb ) . to estimate the mean level of nucleotide sequence similarity at the genome level between v.  massiliensis and the other four virgibacillus genomes , we calculated the average genomic identity of orthologous gene sequences ( agios ) values using an in - lab pipeline named marseille average genomic identity ( magi ) .
briefly , this pipeline combines proteinortho 4 software ( with the following parameters : e - value 1e , 30% of identity , 50% coverage and algebraic connectivity of 50% ) for detecting orthologous proteins between genomes compared pairwise , then retrieves the corresponding gene nucleotide sequences and determines the mean percentage of nucleotide sequence identity among orthologous open reading frames using the needleman - wunsch global alignment algorithm .
similarity values at the genome level were also calculated using genome - to - genome distance ( ggdc ) calculator software .
the agios and ggdc values obtained using the magi pipeline and ggdc software , respectively , among the different studied genomes are summarized in table  8 .
the agios values among virgibacillus genomes ranged from 65.54 between v.  alimentarius and v.  senegalensis to 69.66% between v.  alimentarius and v. halodenitrificans .
the agios values obtained between v.  massiliensis and other compared species were within this range ( 65.78% with v.  senegalensis to 66.88% with v.  halodenitrificans ) .
the ggdc values among virgibacillus genomes ranged from 19.1% between v.  alimentarius and v.  halodenitrificans to 27.7% between v.  senegalensis and v. halodenitrificans .
the ggdc values obtained between v.  massiliensis and other compared species were also within a similar range ( 17.8% with v.  alimentarius to 26.8% with v.  senegalensis ) .
on the basis of phenotypic , phylogenetic and genomic analyses , we formally propose the creation of virgibacillus massiliensis sp .
this description was based on a single isolate , similarly to the descriptions of v.  halotolerans ( seiler and wenning , 2013 ) and v.  oceani ( yin et  al . , 2015 ) .
massiliensis , from massilia , the roman name for marseille , france , where the type strain was isolated ) .
strain vm-5 required nacl for growth and grew at salinity ranging from 5 to 200  g / l of nacl ( optimum , 100 
strain vm-5 grew in presence of 5% co2 but not in a microaerophilic or anaerobic atmosphere .
colonies were circular , greyish , shiny and smooth , with a diameter of 2 to 5  mm .
cells were motile by polar flagella , spore forming ( 2 to 6  m in length and 0.5  m in diameter ) and generally occurred as single cells or in pairs .
strain vm-5 was positive for nitrate reduction but negative for phosphatase alkaline activity , -galactosidase , n - acetyl--glucosaminidase and urease .
strain vm-5 was negative for ribose , l - arabinose and d - lactose assimilation and positive for d - glucose , d - fructose , d - mannose , d - mannitol , d - maltose and d - sucrose .
strain vm-5 was susceptible to penicillin , ampicillin , amoxicillin , ceftriaxone , imipenem doxycycline , rifampicin , vancomycin , nitrofurantoin , erythromycin , ciprofloxacin and gentamicin but was resistant to trimethoprim / sulfamethoxazole and metronidazole .
the 16s rrna and genome sequences are deposited in genbank under accession numbers hg931931 and ccdp010000001 to ccdp010000007ccdp010000001ccdp010000002ccdp010000003ccdp010000004ccdp010000005ccdp010000006ccdp010000007 , respectively .
the type strain vm-5 (= csur p971  =  dsm 28587 ) was isolated from the stool specimen of a healthy amazonian boy .
 strain vm-5 t was isolated from the stool specimen of a 10-year - old amazonian boy . 
 this bacterium is a gram - positive , strictly aerobic rod , motile by a polar flagellum . 
 here we describe its phenotypic characteristics and complete genome sequence . 
 the 4  353  177  bp long genome exhibits a g  +  c content of 36.87% and contains 4394 protein - coding and 125 predicted rna genes . 
 phylogenetically and genetically , strain vm - c is a member of the genus virgibacillus but is distinct enough to be classified as a new species . 
 we propose the creation of v.  massiliensis sp . 
 nov . 
 , whose type strain is strain vm-5 t ( csur p971  =  dsm 28587 ) . 
Introduction
Organism Information
Genome Sequencing Information
Insights into the genome sequence
Genome Comparison
Conclusion