paenibacillus ihumii strain at5 (=  csur p1981  =  dsm 100664 ) is the type strain of p.  ihumii sp . nov .
this isolate is part of an exploratory study of the gut flora from obese patients before and after bariatric surgery using a microbial culturomics approach , the aim of which is to exhaustively explore the microbial ecosystem of gut flora by using different culture conditions .
this bacterium was isolated from a stool sample collected before bariatric surgery from a 33-year - old frenchwoman living in marseille with morbid obesity .
the conventional parameters used in the delineation of bacterial species include 16s rrna sequence identity and phylogeny , genomic ( g  +  c content ) diversity and dna - dna hybridization ( ddh ) , .
however , these methods present some shortfalls , mainly due to their cutoff values , which vary according to species or genera . the advent of new technology tools such as high - throughput sequencing has enabled us to access descriptions of many bacterial species in the public nucleotide sequence library .
recently we proposed including genomic data in a polyphasic approach to describe new bacterial taxa ( taxonogenomics ) .
this strategy considers phenotypic characteristics , genomic analysis and the matrix - assisted laser desorption / ionization time - of - flight mass spectrometry ( maldi - tof ) spectrum and comparison , .
these characteristics support the circumscription and description of the species p.  ihumii as a novel bacterium . here
a stool sample was collected from a 33-year - old obese frenchwoman ( body mass index 38.6  kg / m ; 100  kg , 1.61  m tall ) in november 2011 .
written consent was obtained from the patient at the nutrition , metabolic disease and endocrinology service at timone hospital , marseille , france .
the study and consent procedures were approved by the local ifr 48 ethics committee under consent 09 - 022 , 2010 .
the stool sample was stored at  80c after collection and studied using microbial culturomics as previously reported .
the sterile stool extract was preincubated in blood culture bottles enriched with rumen fluid and sheep 's blood as described elsewhere .
the culture was monitored for 30 days . on various days ( days 1 , 3 , 7 , 10 , 15 , 21 and 30 )
, a seeding of the preincubated product was done on sheep 's blood  enriched columbia agar ( biomrieux , marcy l'etoile , france ) and incubated for 24 hours in an aerobic atmosphere at 37c .
, one isolated bacterial colony was picked up with a pipette tip from a culture agar plate and spread as a thin smear on an mtp 384 maldi - tof target plate ( bruker daltonics , leipzig , germany ) .
each smear was overlaid with 2  l of matrix solution ( saturated solution of -cyano-4-hydroxycinnamic acid in 50% acetonitrile , 2.5% trifluoroacetic acid ) and allowed to dry for 5 minutes .
spectra were recorded in the positive linear mode for the mass range from 2000 to 20  000 da ( parameter settings : ion source 1 ( isi ) , 20  kv ; is2 , 18.5  kv ; lens , 7  kv ) .
the 16s rrna pcr , coupled with sequencing , was performed using geneamp pcr system 2720 thermal cyclers ( applied biosystems , foster city , ca , usa ) and abi prism 3130xl genetic analyser capillary sequencer ( applied biosystems ) respectively .
chromas pro 1.34 software ( technelysium , tewantin , australia ) was used to correct sequences , and blastn searches were performed in national center for biotechnology information ( ncbi ; http://blast.ncbi.nlm.nih.gov.gate1.inist.fr/blast.cgi ) .
sequences were then aligned using clustal w , and phylogenetic inferences were obtained using the neighbour - joining method with mega 6 ( molecular evolutionary genetics analysis version 6 ) software .
the numbers of nodes correspond to the percentages of bootstrap values obtained by repeating the analysis 1000 times in order to generate a consensus tree .
different growth temperatures ( 25 , 28 , 37 , 45 and 55c ) were tested on sheep 's blood  enriched columbia agar ( biomrieux ) .
growth of this strain was tested in an anaerobic atmosphere using the genbag anaer system ( biomrieux ) , in a microaerophilic atmosphere using genbag microaer system ( biomrieux ) and in an aerobic atmosphere with or without 5% co2 .
the saltiness of this species was tested using 5% nacl on schaedler agar with 5% sheep 's blood ( biomrieux ) in an aerobic atmosphere .
biochemical assays were performed using api gallery systems : api zym ( biomrieux ) , api 20ne ( biomrieux ) and api50 ch ( biomrieux ) .
detection of catalase ( biomrieux ) and oxidase ( becton dickinson , franklin lakes , nj , usa ) was also conducted .
a thermal shock at 100c for half an hour was carried out in order to test sporulation .
a fresh colony was observed between blades and slats using a leica dm 1000 photonic microscope ( leica microsystems , wetzlar , germany ) at 40 to assess the motility of the bacteria .
transmission electron microscopy using a tecnai g20 device ( fei company , limeil - brevannes , france ) at an operating voltage of 60  kv was performed to observe the p.  ihumii strain at5 after negative colouration .
gram staining was performed and observed using a leica dm 2500 photonic microscope with a 100 oil - immersion objective lens .
antibiotic susceptibility of the strain was tested using 18 antibiotics , including amoxicillin 25  g , amoxicillin 20  g / clavulanic acid 10  g , cefalexin 30  g , ceftriaxone 30  g , ciprofloxacin 5  g , doxycycline 30  iu , erythromycin 15  iu , nitrofurantoin 300  g , gentamicin 500  g , gentamicin 15  g , imipenem 10  g , metronidazole 4  g , oxacillin 5  g , penicillin g 10  iu , rifampicin 30  g , trimethoprim 1.25  g / sulfamethoxazole 23.75  g , tobramycin 10  g and vancomycin 30  g ( i2a , montpellier , france ) .
the scan 1200 was used to interpret the results ( interscience , saint - nom - la - bretche , france ) .
ihumii genomic dna ( gdna ) was sequenced on the miseq technology ( illumina , san diego , ca , usa ) using 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 using the high sensitivity kit ( thermo fisher scientific , waltham , ma ) to 145  ng/l .
the mate pair library was prepared with 1  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 ) using a dna 7500 lab chip .
dna fragments were ranged in size from 1.5 to 11  kb , with an optimal size of 3.987  kb .
no size selection was performed , and only 334  ng of tagmented fragments were circularized .
the circularized dna was mechanically sheared into small fragments with an optimal size of 1051  bp on the covaris device s2 in t6 tubes ( covaris , woburn , ma , usa ) .
the library profile was visualized on a high sensitivity bioanalyzer labchip ( agilent ) , and the final concentration library was measured at 2.90 
the libraries were normalized at 2  nm and pooled . following 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 a sequencing run were performed in a single 2    301  bp run . in total
, 7.3 gb of information was obtained from a 511k / mm cluster density , with a cluster passing quality control filters of 97.0% ( 12 
the 1  210  259 paired reads were trimmed and assembled to 12 scaffolds using the spades software .
open reading frames ( orfs ) were predicted using prodigal   with default parameters . nevertheless , the predicted orfs were excluded if they spanned a sequencing gap region ( containing n ) .
the predicted bacterial protein sequences were searched against the genbank database and the clusters of orthologous groups ( cogs ) database using blastp ( e value 1e-03 , coverage 0.7 and 30% identity ) . if no hit was found , it searched against the nr database using blastp with an e value of 1e-03 , coverage 0.7 and 30% identity .
if the sequence length was smaller than 80 amino acids , we used an e value of 1e-05 .
the trnas and rrnas were predicted using the trna scan - se and rnammer tools respectively , .
signalp and tmhmm were used to foresee the signal peptides and the number of transmembrane helices respectively , .
orfans were identified if 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 dna plotter were used for data management and visualization of genomic features respectively , .
genomes were automatically retrieved from the 16s rrna tree using xegen software ( phylopattern ) .
for each selected genome , complete genome sequence , proteome genome sequence and orfeome genome sequence were retrieved from the ftp site of national center for biotechnology information ( ncbi ) .
this score is the mean value of nucleotide similarity between all orthologous pairs in the two genomes studied ( average genomic identity of orthologous gene sequences , agios ) . for the genomic comparison , we used p.  ihumii strain at5 ( cyxk00000000 ) , p.  fonticola strain dsm21315 ( armt00000000 ) , p.  peoriae strain kctc 3763 ( cp011512 ) , p.  stellifer strain dsm 14472 ( cp009286 ) , p.  terrae strain hpl-003 ( cp003107 ) and p.  borealis strain dsm 13188 ( cp009285 ) .
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 ) .
was locally aligned two by two by using the blat algorithm ,   against each of the selected genomes previously cited , and ddh values were estimated from a generalized model .
the ddh threshold is less than 70% for a species to be considered as new species .
annotation and comparison processes were performed using the multi - agent software system dagobah , including figenix   libraries that provide pipeline analysis .
the p.  ihumii strain at5 is a gram - negative motile rod which is catalase and oxidase negative ( fig .  1 ) .
the growth of the strain occurred between 28 to 55c , but optimal growth was observed at 37c after 24 hours of incubation in an aerobic atmosphere .
the colonies were approximately 1 to 2  mm in diameter and grey on 5% sheep 's blood  enriched columbia agar .
cells had a diameter ranging from 0.50 to 1.75  m , with a mean diameter of 1  m measured using electron microscopy ( fig .  2 ) .
no growth of this bacterium was observed using 5% nacl on schaedler agar with 5% sheep 's blood .
this bacterium is a facultative anaerobe bacillus but can also grow in a microaerophilic atmosphere .
of all the 18 antibiotics tested , the p.  ihumii strain at5 was susceptible to all of them except metronidazole and tobramycin . using an api zym strip , we observed that p.  ihumii possesses alkaline phosphatase , esterase ( c4 ) , esterase lipase ( c8 ) , naphthol - as - bi - phosphohydrolase , -galactosidase ( melibase ) , -galactosidase ( hydrolase ) , -glucosidase ( maltase ) and -glucosidase ( cellulose ) activities .
however , there are no activities of lipase ( c14 ) , leucin arylamidase , valine arylamidase , cystine arylamidase , trypsin , -chymotrypsin , phosphatase acid , -glucuronidase , n - acetyl--glucosaminidase , -mannosidase and -fucosidase . using an api 20 ne strip
, positive reactions were obtained for nitrate reduction , -galactosidase , arabinose , -glucosidase , mannose , mannitol , n - acetyl - glucosamine and maltose .
negative reactions were obtained for l - tryptophan , d - glucose , l - arginine , urea , gelatine , potassium gluconate , capric acid , adipic acid , malic acid , trisodium citrate and phenyl acetic acid . using an api 50 ch strip , we demonstrated that p.  ihumii is able to ferment l - arabinose , d - ribose , d - xylose , methyl-d - xylopranoside , d - galactose , d - glucose , d - fructose , d - mannose , d - mannitol , methyl-d - glucopyranoside , n - acetylglucosamine , amygdalin , arbutin , esculin ferric citrate , salicin , d - cellobiose , d - maltose , d - lactose , d - melibiose , d - saccharose , d - trehalose , d - raffinose , starch , glycogen , gentiobiose and d - lyxose . no fermentation were recorded for glycerol , erythritol , d - arabinose , l - xylose , d - adonitol , l - sorbose , l - rhamnose , dulcitol , inositol , d - sorbitol , methyl--d - mannopyranoside , inulin , d - melezitose , xylitol , d - turanose , d - tagatose , d - fucose , l - fucose , d - arabitol , l - arabitol , potassium gluconate , potassium 2-ketogluconate and potassium 5-ketogluconate . table  2 presents a comparison of the different characteristics with other representatives of the paenibacillus genus .
no match could be found from the spectrum generated from clean p.  ihumii strain at5 spots and those in the bruker database ( fig . 
the phylogenetic analysis , performed using 16s rrna sequences , showed that p.  ihumii sp .
strain at5 exhibited 98.2% identity with paenibacillus lentus 
 , classified in the paenibacillaceae family created by ash in 1993 .
however , this percentage remains lower than the 98.7% 16s rrna gene sequence threshold recommended by stackebrandt and ebers   to delineate a new species .
this enables us to say that the p.  ihumii strain at5 is a new species within the paenibacillaceae family ( table  1 ) . a neighbour - joining phylogenetic tree ( fig . 
4 ) based on 16s rrna gene sequences shows the relationships between p.  ihumii and some related taxa .
a gel view was performed in order to observe spectra differences between p.  ihumii and other close bacteria ( fig . 
6 ) is 5  924  686  bp long with 50.20% g  +  c content ( table  3 ) .
it consists of 12 scaffolds ( composed of 16 contigs ) . of the 5274 predicted genes , 5194 were protein - coding genes and 80 were rnas ( six 5s rrna , five 16s rrna , two 23s rrna , 67 trna ) .
a total of 3812 genes ( 73.39% ) were assigned as having a putative function ( by cogs or by nr blast ) , and 253 genes ( 4.87% ) were identified as orfans .
nevertheless , 15 genes associated to polyketide synthase or nonribosomal peptide synthetase   were discovered through genome analysis and implicated in the production of secondary metabolites .
p.  borealis , p. fonticola , p. peoriae , p. stellifer and p.  terrae are species closely related to p.  ihumii with available genomes ( table  1 ) and were consequently chosen for this comparative analysis .
the g  +  c content of p.  ihumii is smaller than that of p.  borealis and p. stellifer ( 50.20 , 51.39 and 53.54% respectively ) but larger than that of p.  fonticola , p. peoriae and p.  terrae ( 47.68 , 46.44 and 46.77% respectively ) .
the gene content of p.  ihumii is smaller than that of p.  fonticola , p. borealis and p.  terrae ( 5194 , 5645 , 6213 and 5525 respectively ) but larger than that of p.  peoriae and p.  stellifer ( 5122 and 4464 respectively ) .
7 shows that the distribution of genes into cogs categories was similar across all compared genomes .
in addition , p.  ihumii shared 2595 , 2141 , 1998 , 2217 and 2414 orthologous genes with p.  fonticola , p. peoriae , p. stellifer , p. terrae and p.  borealis respectively ( table  5 ) .
the agios values ranged from 57.59 to 87.31% among compared paenibacillus species with the exception of p.  ihumii .
when p.  ihumii was compared to other paenibacillus species , the agios value ranged from 57.88% for p.  fonticola to 67.99% for p.  peoriae ( table  5 ) .
ddh was 28%    2.43 for p.  fonticola , 17.5%    2.23 for p.  peoriae , 18.2%    2.26 for p.  stellifer , 17.6%    2.23 for p.  terrae and 17.6%    2.24 for p.  borealis ( table  6 ) .
these data confirm p.  ihumii as a unique species . on the basis of phenotypic , genomic and phylogenetic analyses
this bacterium was isolated from a stool sample of a 33-year - old morbidly obese frenchwoman living in marseille .
n. ihumii , based on the acronym ihumi , the institut hospitalo - universitaire mditerrane - infection in marseille , france , where the type strain was isolated ) .
cells are gram - negative , spore - forming , motile , rod - shaped bacilli with a size of 0.51.75  m .
colonies are grey , with a diameter of 12  mm on 5% sheep 's blood
it has an optimum growth temperature of 37c and is a facultative anaerobe , able to grow in a microaerophilic atmosphere . using api gallery systems ,
positive reactions were observed for alkaline phosphatase , esterase ( c4 ) , esterase lipase ( c8 ) , naphthol - as - bi - phosphohydrolase , -galactosidase , -galactosidase , -glucosidase , -glucosidase , nitrate reduction , -galactosidase , mannose , mannitol , n - acetyl - glucosamine , arabinose , maltose , l - arabinose , d - ribose , d - xylose , methyl-d - xylopranoside , d - galactose , d - glucose , d - fructose , d - mannose , d - mannitol , methyl-d - glucopyranoside , n - acetylglucosamine , amygdalin , arbutin , esculin ferric citrate , salicin , d - cellobiose , d - maltose , d - lactose , d - melibiose , d - saccharose , d - trehalose , d - raffinose , starch , glycogen , gentiobiose and d - lyxose .
cells are susceptible to amoxicillin , amoxicillin / clavulanic acid , cefalexin , ceftriaxone , ciprofloxacin , doxycycline , erythromycin , nitrofurantoin , gentamicin , imipenem , oxacillin , penicillin g , rifampicin , trimethoprim / sulfamethoxazole and vancomycin , and are resistant to metronidazole and tobramycin .
the length of the genome is 5  924  686  bp with 50% g  +  c content .
the 16s rrna gene sequence and whole - genome shotgun sequence of the p.  ihumii strain at5 were deposited in embl - ebi under accession numbers ln881615 and cyxk00000000 respectively .
the at5 type strain (=  csur p1981  =  dsm 100664 ) was isolated from a stool sample from an obese frenchwoman .
 paenibacillus ihumii sp . nov . 
 strain at5 
 (=  csur 1981  =  dsm 100664 ) is the type strain of p.  ihumii . 
 this bacterium was isolated from a stool sample from a morbidly obese french patient using the culturomics approach . 
 the genome of this gram - negative , facultative anaerobic , motile and spore - forming bacillus is 5  924  686  bp long . 
 genomic analysis identified 253 ( 5% ) of 3812 genes as orfans and at least 2599 ( 50.03% ) of 5194 orthologous proteins not shared with the closest phylogenetic species . 
Introduction
Materials and Methods
Results
Taxonomic and Nomenclatural Proposals