bacillus andreraoultii strain sit1 (= csur p1162  =  dsm 29078 ) is the type strain of b.  andreraoultii sp .
this bacterium was isolated from the stool of a 2-year - old nigerian boy with kwashiorkor , a severe form of acute malnutrition , and is part of an effort called culturomics to cultivate all bacterial species from the human gut , .
this is a gram - positive , aerobic or facultatively anaerobic , motile , spore - forming , indole - negative and rod - shaped bacillus .
the ruling taxonomic classification of prokaryotes is based on a combination of phenotypic and genotypic criteria , . however , the three essential criteria that are used ( 16s rrna gene - based phylogeny , g+c content and dna - dna hybridization ( ddh ) ) ,   exhibit several drawbacks .
the number of sequenced bacterial genomes has rapidly increased due to the decrease in cost of sequencing ( to date , almost 40  000 bacterial genomes have been sequenced ) .
therefore , the genomic data , the matrix - assisted laser desorption / ionization time - of - flight mass spectrometry ( maldi - tof ) spectrum   and phenotypic criteria have been proposed for the description of new bacterial species , .
the genus is composed of 331 species with validly published names ( bacillus , 
 http://www.doi.namesforlife.com/ ) .
species of the genus bacillus are ubiquitous bacteria isolated from various environments including soil , gastrointestinal tracts of various insects and animals , vegetation , fresh- and seawater and food . in human beings
, the bacillus species may exist in opportunistic form in immunocompromised patients   or in pathogenic form , such as b.  cereus ( food poisoning ) and b.  anthracis ( anthrax ) .
other species may also be found in various human infections , including pneumonia , endocarditis , and ocular , cutaneous , bone or central nervous system infections and bacteraemia .
the following is a summary classification and a set of features for bacillus andreraoultii sp . nov .
a stool sample was obtained from a 2-year - old nigerian boy with kwashiorkor , a severe form of acute malnutrition , who was admitted to the emergency room in the national hospital in niamey , the capital city of niger , in october 2013 .
the study was approved by the local ethics committee of the institut fdratif de recherche ifr48 , faculty of medicine , marseille , france , under agreement 09 - 022 .
then the stool was sent to marseille , where it was kept at  80c until laboratory culture isolation .
enriched columbia agar ( biomrieux , marcy ltoile , france ) in an anaerobic atmosphere at 37c after 10 days of stool specimen incubation in a culture bottle containing a blood - enriched columbia agar liquid medium ( biomrieux )
. growth of the strain was tested under anaerobic conditions using genbag anaer system ( biomrieux ) , and under aerobic conditions , with or without 5% co2 .
different growth temperatures ( 25 , 30 , 37 , 45 , 55c ) were also tested .
maldi - tof protein analysis was carried out as previously described   using a microflex spectrometer ( bruker daltonics , leipzig , germany ) . in brief , a pipette tip was used to pick one isolated bacterial colony from a culture agar plate and spread it as a thin film on an msp 96 maldi - tof target plate ( bruker ) .
each smear was overlaid with 2 l of matrix solution ( saturated solution of alpha - cyano-4-hydroxycinnamic acid in 50% acetonitrile and 2.5% trifluoracetic acid ) and allowed to dry for 5 minutes .
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 ) .
the 20 sit1 spectra were imported into maldi biotyper 3.0 software ( bruker ) and analysed by standard pattern matching ( with default parameter settings ) against the main spectra of 7379 bacteria .
the method of identification included the m / z from 3000 to 15  000 da .
for every spectrum , a maximum of 100 peaks were compared with spectra in the database .
the resulting score enabled the identification of species whether tested or not : 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 .
that was performed using geneamp pcr system 2720 thermal cyclers ( applied biosystems , bedford , ma , usa ) and abi prism 3130xl genetic analyser capillary sequencer ( applied biosystems ) respectively .
the 16s rrna nucleotides sequence was corrected using chromas pro 1.34 software ( technelysium , tewantin , australia ) , and the blastn searches were performed in the online pubmed national center for biotechnology information ( ncbi ) database ( http://blast.ncbi.nlm.nih.gov.gate1.inist.fr/blast.cgi ) .
bacillus andreraoultii strain sit1 was observed , after negative colouration , using a morgani 268d ( philips , amsterdam , the netherlands ) transmission electron microscope at an operating voltage of 60 kv .
the gram colouration was performed using color gram 2 kit ( biomrieux ) and observed by using the dm1000 photonic microscope ( leica microsystems , wetzlar , germany ) with a 100 oil - immersion objective lens .
the sporulation test was done doing a thermic shock ( 80c during 30 minutes ) . to evaluate the motility of bacillus andreraoultii , fresh colonies
were observed between blades and slats using a dm1000 photonic microscope ( leica ) with a 40 objective lens .
api zym , api 20 ne and api 50 ch ( biomrieux ) gallery systems were used to perform biochemical assays .
oxidase ( becton dickinson , franklin lakes , nj , usa ) and catalase assays ( biomrieux ) were done separately .
the antibiotic susceptibility was tested using sirscan discs antibiotics ( i2a , montpellier , france ) .
genomic dna extraction of bacillus andreraoultii strain sit1 was performed according to the method previously described .
the dna concentration was 401.63 ng/l as measured by a qubit fluorometer using the high - sensitivity kit ( life technologies , carlsbad , ca , usa ) .
genomic dna of b.  andreraoultii 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 gdna was quantified by a qubit assay with the high - sensitivity kit ( life technologies ) to 87.67 ng/l .
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 pattern of 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 4.851 kb .
the circularized dna was mechanically sheared into small fragments with an optimal size of 684 bp on the covaris device s2 in microtubes ( covaris , woburn , ma , usa ) .
the library profile was visualized on a high sensitivity bioanalyzer labchip ( agilent ) , and the final concentration library was measured at 64.49 nmol / l .
after denaturation 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 a single 39-hour sequencing run were performed at a 2    251 bp read length .
all 8.2 gb of information was obtained from a 947k / mm cluster density , with a cluster passing quality control filters with 99% ( 18  111  784 clusters ) . within this run ,
the 1  513  908 paired reads were filtered according to the read qualities .
the reads obtained from applications were trimmed , and an optimal assembly of 14 scaffolds and 58 contigs was obtained through the soapdenovo software , which generated a genome size of 4.09 mb .
open reading frames ( orfs ) were predicted using prodigal   with default parameters , but the predicted orfs were excluded if they spanned a sequencing gap region ( contain n ) .
the predicted bacterial protein sequences were searched against the clusters of orthologous groups ( cogs ) database using blastp ( e - value 1e-03 , coverage of 0.7 , and an identity percentage of 30% ) .
if no hit was found , it search against the nr database using blastp with an e - value of 1e-03 , coverage of 0.7 and an identity percentage of 30% .
if sequence lengths were smaller than 80 amino acids , we used an e - value of 1e-05 .
the trnascanse tool   was used to find trna genes , whereas ribosomal rnas were found by using rnammer .
for each selected genome , complete genome sequences , proteome genome sequences and orfeome genome sequences were retrieved from the ftp site of ncbi .
this score is the mean value of nucleotide similarity between all couple of orthologues in the two genomes studied ( average genomic identity of orthologous gene sequences , agios ) .
the resistome was analysed with the arg - annot ( antibiotic resistance gene - annotation ) database and blastp in genbank .
the exhaustive bacteriocin database available in our laboratories ( bacteriocins from the urmite database ) ( http://drissifatima.wix.com/bacteriocins ) was performed by collecting all currently available sequences from the databases and from ncbi .
protein sequences from this database allowed putative bacteriocins from human gut microbiota to be identified using blastp methodology .
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 .
no significant maldi - tof score was obtained for strain sit1 against the bruker database , suggesting that our isolate was not a member of a known species .
we added the spectrum from strain sit1 to our database ( figure  1 ) .
then a gel view was performed to show the spectral differences with other members of the genus bacillus ( figure  2 ) . using 16s rrna phylogeny analyses
, we demonstrated that strain sit1 exhibited a 96% 16s rrna sequence identity with bacillus thermoamylovorans ( genbank accession no .
hm030742 ) , the phylogenetically closest bacterial species with standing in nomenclature ( figure  3 ) .
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 .
thus , this bacterium was considered as a new species called bacillus andreraoultii strain sit1 belonging family bacillaceae ( table  1 ) .
b.  andreraoultii growth occurred at all temperatures tested ; however , optimal growth was observed between 37 and 45c after 24 hours of incubation .
gram staining showed rod - shaped , gram - positive bacilli ( figure  4 ) .
cells grown on agar are smooth and greyish after 24 hours of incubation , and they have an average width and length of 0.5 m and 3 m , respectively , and exhibited flagella ( figure  5 ) .
strain sit1 showed catalase activity but was negative for oxidase . using an api zym strip ,
positive reactions were observed for alkaline phosphatase , esterase ( c4 ) , esterase lipase ( c8 ) , acid phosphatase , naphthol - as - bi - phosphohydrolase , -galactosidase , -galactosidase , -glucosidase and -glucosidase .
negative reactions were observed for lipase ( c14 ) , leucine arylamidase , valine arylamidase , cystine arylamidase , trypsin , -chymotrypsin , -glucuronidase , n - acetyl- -glucosaminidase , -mannosidase and -fucosidase .
using an api 20 ne strip , the nitrate reductase  hydrolysis reaction ( -glucosidase , esculine ) , and -galactosidase and assimilation reaction ( potassium gluconate ) were also positive .
negative reactions were found for urease , indole , arginine dihydrolase and fermentation ( glucose ) . using an api 50 ch strip ,
positive reactions were recorded for l - arabinose , d - ribose , d - xylose , d - glucose , d - fructose , d - mannose , n - acetylglucosamine , arbutine , esculine , salicine , d - celiobiose , d - maltose , d - saccharose , d - trehalose and amidon .
negative reactions were recorded for glycerol , erythritol , d - arabinose , l - xylose , d - adonitol , methyl-d - xylopyranoside , d - galactose , l - sorbose , l - rhamnose , dulcitol , inositol , d - mannitol , d - sorbitol , methyl-d - mannopyranoside , methyl-d - glucopyranoside , amygdalin , d - lactose , d - melibiose , inulin , d - melezitose , d - raffinose , glycogen , xylitol , gentiobiose , d - turanose , d - lyxose , d - tagatose , d - fucose , l - fucose , d - arabitol , l - arabitol , potassium gluconate , potassium 2-ketogluconate and potassium 5-ketogluconate .
bacillus andreraoultii sit was resistant to trimethroprim - sulphamethoxazole , macrolide ( erythromycin ) , vancomycin and the third generation of cephalosporin ( ceftriaxone ) but was susceptible to fosfomycin , imipenem , penicillin , amoxicillin , gentamicin , ciprofloxacin , doxycycline and rifampicin .
five species with validly published names in the bacillus genus were selected to make a phenotypic comparison with b.  andreraoultii ( table  2 ) .
bacillus andreraoultii sit1 was selected for sequencing on the basis of its phenotypic differences , phylogenetic position and 16s rrna sequence similarity to other members of the bacillus genus .
it was part of a culturomics study aimed at isolating all bacterial species from human digestive flora in patients with kwashiorkor , an acute form of malnutrition .
accession number of b.  andreraoultii genome is ccfj00000000 and consists of 14 scaffolds and 58 contigs ( figure  6 ) .
table  3 shows the project information and its association with minimum information about a genome sequence ( migs ) version 2.0 compliance .
the genome is 4  092  130 bp long with 35.42% g+c content . on the 3843 predicted genes , 3718 were protein - coding genes and 116 were rnas genes .
the remaining genes were annotated as hypothetical proteins ( 712 genes , > 19.15% ) .
the draft genome sequence of bacillus andreraoultii was smaller than those of bacillus halodurans c-125 , bacillus pseudofirmus of4 and lysinibacillus sphaericus c3 - 41 ( 4.09 , 4.20 , 4.25 and 4.82 mb respectively ) but larger than those of solibacillus silvestris stlb046 , bacillus pumilus safr-032 and bacillus coagulans 2 - 6 wk1 ( 3.98 , 3.70 and 3.07 mb , respectively ) .
the g+c content of bacillus andreraoultii was smaller than those of bacillus pumilus safr-032 , bacillus coagulans 2 - 6 , bacillus halodurans c-125 , bacillus pseudofirmus of4 and lysinibacillus sphaericus c3 - 41 ( 35.42 , 41.29 , 47.29 , 43.69 , 39.86 and 37.13% respectively ) .
the gene content of bacillus andreraoultii was smaller than those of bacillus halodurans c-125 , bacillus pseudofirmus of4 and lysinibacillus sphaericus c3 - 41 ( 3718 , 4052 , 4335 and 4771 , respectively ) but larger than those of bacillus pumilus safr-032 and bacillus coagulans 2 - 6 ( 3681 and 2971 respectively ) ( table  6 ) .
the distribution of genes into cogs categories in the genomes from all seven compared bacillus species , anoxybacillus flavithermus , lysinibacillus sphaericus and solibacillus silvestris ( figure  7 ) .
bacillus andreraoultii contained a bacteriocin ( colicin ) consisting of 175 amino acids harboured with the bacteriocins of paenibacillus and lactobacillus ( figure  8) and sharing 62% of the homology of bacillus vireti .
the results did not indicate the presence of nonribosomal peptide synthetases and polyketide synthases and phage .
we performed the analysis of the resistome of bacillus andreraoultii sit1 antibiotic classes of the macrolide 
streptogramin b ( mlsb ) antibiotics , such as atp - binding transporters ( abc ) lsab and msrd , major facilitator transporters mefa , transferases ( vata ) and two - component system vancomycin resistance vans / vanr , nora ( table  7 ) .
on the basis of phenotypic , phylogenetic and genomic analyses ( taxonogenomics ) , we formally propose the creation of bacillus andreraoultii sp . nov . that contains the strain sit1 .
this bacterium was isolated from the stool of a 2-year - old nigerian boy with kwashiorkor , a severe form of acute malnutrition .
the bacillus andreraoultii name come from andr raoult , who was a military doctor who worked with malnutrition and kwashiorkor in senegal and who described its specific features .
strain sit1 is aerobic , gram positive , endospore forming , motile and rod shaped .
after 24 hours of growth on 5% sheep 's blood  enriched columbia agar at 37c , bacterial colonies were smooth and greyish with a diameter of 0.1 to 0.3 mm . the cells had a mean width and length of 0.5 m and 3 m , respectively , and exhibited flagella . they were catalase positive and oxidase negative .
bacillus andreraoultii sit1 was resistant to trimethroprim - sulphamethoxazole , macrolide ( erythromycin ) , vancomycin and the third generation of cephalosporin ( ceftriaxone ) .
the genome is 4  092  130 bp long , and the g+c content is 35.42% .
the 16s rrna gene sequence and whole - genome shotgun sequence of b.  andreraoultii strain sit1 are deposited in genbank under accession nos . lk021120 and ccfj00000000 , respectively .
the type strain sit1 (= csur p1162  =  dsm 29078 ) was isolated from the stool of a 2-year - old nigerien boy with kwashiorkor , a severe form of acute malnutrition .
 bacillus andreraoultii strain sit1 t (= csur p1162  =  dsm 29078 ) is the type strain of b.  andreraoultii sp . nov . 
 this bacterium was isolated from the stool of a 2-year - old nigerian boy with a severe form of kwashiorkor . 
 bacillus andreraoultii is an aerobic , gram - positive rod . 
 we describe here the features of this bacterium , together with the complete genome sequencing and annotation . 
 the 4  092  130 bp long genome contains 3718 protein - coding and 116 rna genes . 
Introduction
Materials and methods
Results
Conclusion
Description of 
Conflict of Interest