paenibacillus antibioticophila strain gd11 (= dsm 28228  = csur p1358 ) is the type strain of paenibacillus antibioticophila sp . nov . it is a gram - positive , aerobic , indole - negative rod - shaped bacterium isolated as part of a culturomics study , . this bacterium was isolated from a 63-year - old woman with multidrug - resistant tuberculosis who was receiving a broad - spectrum antibiotic regimen at the time of stool collection , as recently reported . the current classification of prokaryotes is based on a combination of phenotypic and genotypic characteristics ,   which includes 16s rrna gene phylogeny , g+c content and dna  dna hybridization . although these tools are considered to be the reference standard , they have several pitfalls , . as a result of the declining cost of sequencing , there has been rapid growth in the number of bacterial genomes being sequenced , and we thus recently proposed to add genomic information to phenotypic criteria for the description of new bacterial species , , , , , , , , , , , , , , , , , , , , , , , , , , , , , . the genus paenibacillus initially included gram - variable , facultative anaerobic endospore - forming bacteria that were reclassified as a separate genus in 1993   after being grouped with the genus bacillus . to date , the genus consists of 170 described species and four subspecies that have been isolated from a variety of environments such as soil , water , rhizosphere , vegetable matter , forage and insect larvae , , . one paenibacillus species was cultured for the first time from human faeces as a part of a culturomics study . here we present a summary of the classification and set of features for paenibacillus antibioticophila sp . a stool sample was collected from a 63-year - old woman with a pulmonary form of multidrug - resistant tuberculosis . the study was approved by the ethics committee of the institut fdratif de recherche ifr48 , faculty of medicine , marseille , france , under agreement 09 - 002 . strain gd11 ( table  1 ) was isolated in march 2012 by cultivation on 5% sheep 's blood agar in aerobic conditions at 37c after a 21-day preincubation in a blood culture bottle with sterile cow rumen fluid and sheep 's blood . strain gd11 exhibited a 97.6% 16s rrna sequence identity with p.  puldeungensis ( genbank accession no . nr117451 ) , the phylogenetically closest bacterial species with standing in nomenclature ( fig .  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 at different temperatures ( 25 , 30 , 37 , 45 and 56c ) was tested ; no growth was observed at 45c or 56c . enriched columbia agar ( biomrieux , marcy letoile , france ) under anaerobic and microaerophilic conditions using the genbag anaer and genbag microaer systems , respectively ( biomrieux ) , and under aerobic conditions , with or without 5% co2 . cells grown on agar were soft and translucent after 24 hours and had a mean width of 0.49  m and mean length of 2.67  m ( fig . using an api zym strip ( biomrieux ) , positive reactions were observed for esterase ( c4 ) , esterase lipase ( c8 ) , naphthol - as - bi - phosphohydrolase , -galactosidase , -galactosidase and -glucosidase . using rapid id32a , positive reactions were observed for -glucosidase , -arabinosidase , -glucuronidase , n - acetyl--glucosaminidase , nitrate reduction , glutamic acid decarboxylase , fermentation of mannose and raffinose . using an api 50 ch strip ( biomrieux ) , positive reactions were recorded for esculin hydrolysis and fermentation of l - arabinose , d - ribose , d - xylose , methyl-d - xylopranoside , d - galactose , d - glucose , d - fructose , d - mannose , l - rhamnose , d - mannitol , n - acetylglucosamine , amygdalin , arbutin , salicin , d - cellobiose , d - maltose , d - lactose , d - melibiose , d - saccharose , d - trehalose , inulin , d - melezitose , d - raffinose , starch , glycogen and d - lyxose . using an api zym strip ( biomrieux ) , negative reactions were observed for acid phosphatase , alkaline phosphatase , leucine arylamidase , valine arylamidase , cystine arylamidase , lipase ( c14 ) , trypsin , -chymotrypsin , -glucosidase , -mannosidase , and -fucosidase . using rapid api 32a , negative reactions were observed for arginine dihydrolase , urease , production of indole , leucine arylamidase , histidine arylamidase , phenylalanine arylamidase , tyrosin arylamidase , alanine arylamidase -mannosidase , -glucosidase , -fucosidase . an api  50 ch strip ( biomrieux ) , negative reactions were recorded for fermentation of erythritol , d - arabinose , l - xylose , d - adonitol , l - sorbose , dulcitol , inositol , d - sorbitol , xylitol , d - turanose , d - tagatose , d - fucose , l - fucose , d - arabitol , l - arabitol , potassium gluconate , potassium 2-ketogluconate , potassium 5-ketogluconate and potassium-5-ketogluconate . clavulanic acid , ceftriaxone , imipenem , vancomycin , rifampicin , erythromycin , gentamicin , ciprofloxacin and trimethoprim  sulfamethoxazole , but resistant to metronidazole . by comparison with p. puldeungensis strain cau 9324 , its phylogenetically closest neighbor , p.  antibioticophila differed in alkaline phosphates , acid phosphatase , oxidase and -glucosidase ( table  2 ) . matrix - assisted laser desorption / ionization time - of - flight mass spectrometry ( maldi - tof ) protein analysis was carried out as previously described . briefly , using a pipette tip , one isolated bacterial colony from a culture on agar plate was transferred and spread as a thin film on a msp 96 maldi - tof target plate ( bruker daltonics , leipzig , germany ) . each smear was overlaid with 2 l of matrix solution ( saturated solution of alpha - cyano-4-hydroxycinnamic acid ) in 50% acetonitrile , 2.5% trifluoroacetic acid , and dried for 5 minutes . microflex spectrometer ( bruker ) was used for measurements , and spectra were then 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 gd11 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 , including 129 spectra from 70 paenibacillus species . the method of identification included the m / z from 3000 to 15  000 da . a maximum of 100 peaks were compared with spectra in the database for every spectrum . the resulting score enabled the identification of tested species ( 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 . no significant maldi - tof score was obtained for strain gd11 against the bruker database , suggesting that our isolate was not a member of a known species . finally , the spectral differences with other members of the genus paenibacillus were highlighted by gel view ( fig . as part of a culturomics study isolating all bacterial species from the human digestive flora from patients with multidrug - resistant tuberculosis and treated with broad - spectrum antibiotics , this organism was isolated and selected for sequencing on the basis of its phenotypic differences , phylogenetic position and 16s rrna sequence similarity to other members of the genus paenibacillus . the genbank bioproject number is prjeb1962 and consists of 131 large contigs in nine scaffolds . table  3 shows the project information and its association with minimum information about a genome sequence ( migs ) 2.0 compliance . paenibacillus antibioticophila strain gd11 (= dsm 28228  =  csur p1358 ) was cultured aerobically on columbia agar ( biomrieux ) . a total of 200 l of bacterial suspension was diluted in 1  ml tris - edta ( te ) buffer for lysis treatment . after a lysozyme incubation of 30 minutes at 37c , the lysis was performed with laurylsarcosylby 1% final and rnasea treatment at 50 g/l final concentration during 1 hour at 37c , followed by an overnight proteinase k incubation at 37c.the dna was purified three times by phenol the concentration was measured by the quant - it picogreen kit ( invitrogen ; life technologies , carlsbad , ca , usa ) on the genios_tecan fluorometer at 48  ng/l . genomic dna of paenibacillus antibioticophila strain gd11 was sequenced on 454_roche_titanium ( roche , basel , switzerland ) . this project was loaded twice on a  region for the 5  kb insert libraries on ptp picotiterplates . the library was constructed with 2.5  g of dna according to the 454_roche_titanium paired end protocol and manufacturer . it was mechanically fragmented on the covaris device ( kbioscience  lgc genomics , teddington , uk ) through minitube - red 5  kb . the dna fragmentation was visualized through the agilent 2100 bioanalyzer on a dna labchip 7500 , with an optimal size of 4.7  kb . circularization and nebulization were performed and generated a pattern with optimal at 490  bp . after pcr amplification through 17 cycles followed by double size selection , the library was then quantified on the quant - it ribogreen kit ( invitrogen ) on the genios_tecan fluorometer at 759  pg/l . the 5  kb paired end library was clonal amplified with 0.25 and 0.5 cpb in 4 empcr reactions per condition , with the gs titanium sv empcr kit ( lib - l ) v2 . the yield of the empcr was 5.21% and 5.69% , respectively , according to the quality expected by the range of 5 to 20% from the roche procedure . twice , 790  000 beads were loaded on the gs titanium picotiterplates ptp kit 70    75 and sequenced with the gs titanium sequencing kit xlr70 . the runs were performed overnight and then analysed through the gsrunbrowser and newbler assembler _ roche . the global 411  382 passed filter sequences generated 150.40 mb with a length average of 354  bp . these sequences were assembled on the gsassembler from roche with 90% identity and 40  bp as overlap . it led to nine scaffolds and 131 large contigs ( > 1500  bp ) , generating a genome size of 5.6 mb , which corresponds to a coverage of 19.96 genome equivalent . 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-03 for alignment length greater than 80 aa . if alignment lengths were smaller than 80 aa , we used an e value of 1e-05 . here , we compared the genome sequence of p.  antibioticophila strain gd11 with those of paenibacillus barengoltzii strain g22 ( genbank accession no . assd00000000.1 ) , which were identified using the proteinortho software ( version 1.4 )   using a 30% protein identity and 1e-05 e value . the average percentage of nucleotide sequence identity ( table  4 ) between corresponding orthologous sets was determined using the needleman - wunsch algorithm global alignment technique . artemis   was used for data management , and dna plotter   was used for visualization of genomic features . the genome of p. antibioticophila strain gd11 is 5  562  631  bp long with a 49.1% g+c content ( fig . 6 ) . of the 5155 predicted genes , 5084 were protein - coding genes and three were rnas . three rrna genes ( one 16s rrna , one 23s rrna and one 5s rrna ) and 68 predicted trna genes were identified in the genome . a total of 3814 genes ( 73.98% ) were assigned a putative function . the properties and the statistics of the genome are summarized in table  5 , table  6 . the genome of p.  antibioticophila strain gd11 was compared to each of paenibacillus barengoltzii strain g22 ( genbank accession no . the draft genome of p.  antibioticophila has a larger size than that of p.  barengoltzii , p. sanguinis and p.  zanthoxyli ( 5.56 , 4.75 , 4.8 and 5.05 mb , respectively ) . the g+c content of p.  antibioticophila is higher than those of p.  massiliensis , p.  panacisoli , p.  polymyxa , p.  senegalensis and p.  terrae ( 49.1 , 48.5 , 48.3 , 44.9 , 48.2 and 46.8% , respectively ) but less than that of p.  barengoltzii , p. sanguinis and p.  zanthoxyli ( 51.9 , 49.3 and 50.9% , respectively ) . the gene content of p.  antibioticophila is larger than those of p.  barengoltzii , p.  sanguinis , p.  senegalensis and p.  zanthoxyli ( 5155 , 4394 , 4209 , 4422 and 4878 , respectively ) . however , the distribution of genes into cogs categories was similar in all nine compared genomes ( fig . in addition , p.  antibioticophila shared 5084 , 4307 , 5055 , 5059 , 5068 , 4093 , 4278 , 5525 and 4676 orthologous genes with p.  barengoltzii , p. massiliensis , p. panacisoli , p. polymyxa , p. sanguinis , p. senegalensis , p. terrae and p. zanthoxyli , respectively ( table  6 ) . finally , no sequences coding for nonribosomal peptide synthetases or polyketide synthases were found within the p.  antibioticophila genome . the analysis of virome revealed the presence of one intact phage of 20.8  kb , and other three incomplete phages , of , respectively , 17 , 15.4 and 14.3  kb with 43.96 , 50.60 and 49.84% g+c content ( table  7 ) . on the basis of phenotypic , phylogenetic and genomic analyses , we formally propose the creation of paenibacillus antibioticophila sp . the strain has been isolated from the stool sample of a 63-year - old woman with multidrug - resistant tuberculosis in marseille , france . several other previously undescribed bacterial species were also cultivated from different faecal samples through diversification of culture conditions , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , thus suggesting that the human faecal flora of humans remains partially unknown . paenibacillus antibioticophila strain gd11 (= dsm 28228  =  csur p1358 ) is the type strain of the genus paenibacillus . it was isolated from the stool samples of a 63-year - old woman with a pulmonary form of multidrug - resistant tuberculosis hospitalized in an infectious diseases ward in marseille , france . the main scope of the culturomics study is to cultivate all the species within the human faeces . p.  antibioticophila is a gram - positive bacilli that does exhibits neither catalase nor oxidase activity . colonies were 0.5  mm in diameter , and cells have a mean width of 0.49  m and a mean length of 2.67  m . esterase ( c4 ) , esterase lipase ( c8 ) , naphthol - as - bi - phosphohydrolase , -galactosidase , -galactosidase and -glucosidase , -arabinosidase , -glucuronidase , n - acetyl--glucosaminidase , nitrate reduction , glutamic acid decarboxylase , fermentation l - arabinose , d - ribose , d - xylose , methyl-d - xylopranoside , d - galactose , d - glucose , d - fructose , d - mannose , l - rhamnose , d - mannitol , n - acetylglucosamine , amygdalin , arbutin , salicin , d - cellobiose , d - maltose , d - lactose , d - melibiose , d - saccharose , d - trehalose , inulin , d - melezitose , d - raffinose , starch , glycogen and d - lyxose were positive . acid phosphatase , alkaline phosphatase , leucine arylamidase , valine arylamidase , cystine arylamidase , lipase ( c14 ) , trypsin , -chymotrypsin , -glucosidase , -mannosidase , -fucosidase , arginine dihydrolase , urease , production of indole , leucine arylamidase , histidine arylamidase , phenylalanine arylamidase , tyrosin arylamidase , alanine arylamidase -mannosidase , fermentation erythritol , d - arabinose , l - xylose , d - adonitol , l - sorbose , dulcitol , inositol , d - sorbitol , xylitol , d - turanose , d - tagatose , d - fucose , l - fucose , d - arabitol , l - arabitol , potassium gluconate , potassium 2-ketogluconate , potassium 5-ketogluconate and potassium-5-ketogluconate were negative . clavulanic acid , ceftriaxone , imipenem , vancomycin , rifampicin , erythromycin , gentamicin , ciprofloxacin and trimethoprim  sulfamethoxazole and resistant to metronidazole . the 16s rrna gene sequence and whole - genome shotgun sequence of p.  antibioticophila strain gd11 is deposited in genbank under accession number kc158472 .
paenibacillus antibioticophila strain gd11 t sp . nov . is the type strain of a new species within the genus paenibacillus . 
 this strain , whose genome is described here , was isolated from human faeces of a 63-year - old woman with multidrug - resistant tuberculosis who was receiving numerous antibiotics at the time of stool collection . 
 p.  antibioticophila is a gram - positive aerobic bacterium . 
 we describe here the features of this bacterium , together with the complete genome sequence and annotation . 
 the 5  562  631  bp long genome contains 5084 protein - coding and 71 rna genes .