nocardioides massiliensis strain gd13 (= dsm 28216  =  csur p894 ) is the type strain of n.  massiliensis sp .
this bacterium was isolated from the human faeces of a 62-year - old man admitted to intensive care for a guillain - barr syndrome as part of an effort called culturomics to cultivate all bacterial species from the human gut , .
the current taxonomic 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 the reference standard , they have several drawbacks , . as a result of the declining cost of sequencing , the number of sequenced bacterial genomes has rapidly grown ; to date , almost 40  000
hence , we recently proposed to use genomic information together with phenotypic criteria for the description of new bacterial species , , , , , , , , , , , , , . here
the first two species were nocardioides albus and nocardioides luteus 
 , . these gram - positive , aerobic and cardioform actinomycetes exhibit a high g+c content and are able to develop mycelium , .
currently the genus nocardioides contains 72 species , including eight described in 2013 and species from the genera pimelobacter and arthrobacter that were reclassified in the genus nocardioides 
 , , , , , , , , , , , , ,   ( lpsn , http://www.bacterio.net/ ) . members of the genus nocardioides are mostly environmental bacteria found in soil or plants . to our knowledge , this is the first report of isolation of a nocardioides species from humans .
a stool sample was collected from a 62-year - old man admitted to the intensive care unit of hpital nord , marseille , france , in march 2012 .
the study was approved by the ethics committee of the institut fdratif de recherche ifr48 ( faculty of medicine , marseille , france ) , under agreement 09 - 002 . at the time of sampling ,
the patient had received a 10-day course of imipenem ( 3  g per day ) .
strain gd13 ( table  1 ) was isolated in march 2012 by cultivation on pvx agar ( biomrieux , marcy ltoile , france ) in an aerobic atmosphere at 28c after 14 days of incubation .
strain gd13 exhibited a 98.07% 16s rrna sequence identity with n.  mesophilus ( genbank accession no .
ef466117 ) , 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 was tested at different temperatures ( 25 , 30 , 37 , 45 , 56c ) .
growth of the strain was tested under anaerobic and microaerophilic conditions using genbag anaer and genbag microaer systems ( biomrieux ) respectively , and under aerobic conditions , with or without 5% co2 .
gram staining showed gram - positive , rod - shaped bacteria unable to form spores ( figure  2 ) .
cells grown on agar were soft and yellowish in colour after 24 to 48 hours and could be grouped in small clumps ( figure  2 ) .
they had a mean width of 0.46  m and a mean length of 1  m ( figure  3 ) .
strain gd13 exhibits catalase activity , but was negative for oxidase . using an api zym strip ( biomrieux ) , positive reactions were observed for esterase ( c4 ) , esterase lipase ( c8 ) , acid phosphatase , alkaline phosphatase , leucine arylamidase , naphthol - as - bi - phosphohydrolase , -glucosidase and -glucosidase ; negative reactions were observed for -galactosidase , -galactosidase , valine arylamidase , cystine arylamidase , -glucuronidase , lipase ( c14 ) , trypsin , -chymotrypsin , n - acetyl--glucosaminidase , -mannosidase and -fucosidase . using rapid id32a ( biomrieux ) ,
positive reactions were observed for arginine arylamidase , proline arylamidase and -fucosidase and negative reactions were observed for arginine dihydrolase and n - acetyl--glucosaminidase .
using rapid id32a ( biomrieux ) , positive reactions were observed for arginine arylamidase , proline arylamidase and -fucosidase ; negative reactions were observed for nitrate reduction , urease , production of indole , arginine dihydrolase , n - acetyl--glucosaminidase , serine arylamidase , histidin arylamidase , phenylalanine arylamidase and tyrosin arylamidase . using an api 50 ch strip ( biomrieux ) ,
positive reactions were recorded for esculin hydrolysis and fermentation of d - lactose ; negative reactions were obtained for fermentation of glycerol , erythritol , d - arabinose , l - arabinose , d - ribose , d - xylose , l - xylose , d - adonitol , methyl-d - xylopyranoside , d - galactose , d - glucose , d - fructose , d - mannose , l - sorbose , l - rhamnose , dulcitol , inositol , d - mannitol , methyl-d - xylopyranoside , methyl-d - glucopyranoside , n - acetylglucosamine , amygdalin , arbutin , esculin ferric citrate , salicin , d - cellobiose , d - maltose , d - mellibiose , d - trehalose , inulin , d - melezitose , d - raffinose , amidon , glycogen , gentiobiose , d - turanose , d - lyxose , d - tagatose , d - fucose , l - fucose , l - arabitol , d - sorbitol , d - saccharose , xylitol , d - arabitol , potassium-5-ketogluconate and potassium 2-ketogluconate .
cells were susceptible to penicillin g , amoxicillin , amoxicillin / clavulanic acid , ceftriaxone , imipenem , vancomycin , rifampicin , erythromycin , gentamicin , ciprofloxacin and trimethoprim / sulfamethoxazole and resistant to metronidazole . compared to n.  mesophilus , its phylogenetically closest neighbor , n.  massiliensis differed in -glucosidase , -glucosidase , esterase  lipase production and utilization of d - glucose and d - mannose ( table  2 ) .
in addition , nocardioides species clearly differed from aeromicrobium massiliense , a representative species from another genus within the family nocardioidaceae .
matrix - assisted laser desorption / ionization time - of - flight mass spectrometry ( maldi - tof ) protein analysis was carried out as previously described . 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 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% 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 ) . a spectrum was obtained after 240 shots with variable laser power .
the 20 gd13 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 48 spectra from two nocardioides species .
it corresponded to the bruker database combined with our own database constantly incremented with new species .
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 or not of tested species : 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 gd13 against the bruker database and against our own database , suggesting that our isolate was not a member of a known species .
we added the spectrum from strain gd13 to our database ( figure  4 ) .
finally , the gel view showed the spectral differences with other members of the genus nocardioides and aeromicrobium massiliense , a member of the genus aeromicrobium that is closely related to the genus nocardioides ( figure  5 ) .
nocardioides massiliensis gd13 was selected for sequencing on the basis of its phenotypic differences , phylogenetic position and 16s rrna sequence similarity to other members of the genus nocardioides .
it was part of a culturomics study aimed at isolating all bacterial species from the human digestive flora from patients treated with broad - spectrum antibiotics .
table  3 shows the project information and its association with minimum information about a genome sequence ( migs ) 2.0 compliance .
nocardioides massiliensis strain gd13 (= dsm 28216  =  csur p894 ) was cultured aerobically on 5% sheep 's blood 
bacteria grown on four petri dishes were resuspended in sterile water and centrifuged at 4c at 2000    g for 20 minutes .
cell pellets were resuspended in 1  ml tris / edta / nacl ( 10  mm tris / hcl ( ph 7.0 ) , 10  mm edta ( ph 8.0 ) , 300  mm nacl ) and recentrifuged under the same conditions .
the pellets were then resuspended in 200 l tris - edta ( te ) buffer and proteinase k and kept overnight at 37c for cell lysis .
dna was purified with phenol / chloroform / isoamylalcohol ( 25:24:1 ) , followed by an overnight precipitation with ethanol at  20c .
dna concentration was 401.63  ng/l as measured by qubit fluorometer using the high - sensitivity kit ( life technologies , carlsbad , ca , usa ) .
genomic dna ( gdna ) of nocardioides massiliensis was sequenced on a miseq sequencer ( illumina , san diego , ca , usa ) with two applications : paired end and mate pair .
the paired end and the mate pair strategies were barcoded in order to be mixed respectively with 14 other genomic projects constructed according the nextera xt library kit ( illumina ) and 11 other projects with the nextera mate pair kit ( illumina ) .
the gdna was diluted such that 1  ng of each strain 's gdna was used to construct the paired - end library .
then limited - cycle pcr amplification completed the tag adapters and introduced dual - index barcodes .
after purification on ampure beads ( life technologies ) , the libraries were normalized on specific beads according to the nextera xt protocol ( illumina ) .
the pooled single strand 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 was performed in a single 39-hour run at a 2    250  bp read length . within this pooled run
total information of 3.27 gb was obtained from a 374k / mm density with 94.2% ( 6  719  000 clusters ) of the clusters passing quality control ( qc ) filters . from the genome sequencing process ,
the 248  710 produced illumina paired end reads for nocardioides massiliensis were filtered according to the read qualities .
the mate pair library was constructed from 1  g of gdna using the nextera mate pair illumina guide .
the profile of the fragmentation was validated on an agilent 2100 bioanalyzer ( agilent technologies , santa clara , ca , usa ) with a dna7500 lab chip .
the dna fragments ranged in size from 1 to 8.4  kb with a mean size of 3.7  kb .
the larger circularized dna molecules were physically sheared to smaller sized fragments with a mean size of 839  bp on the covaris device s2 in microtubes ( woburn , ma , usa ) .
the library 's profile and the quantitation were visualized on a high sensitivity bioanalyzer labchip .
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 was performed in a single 39-hour run at a 2    250  bp read length .
total mate paired run information of 3.2 gb was obtained from a 690k / mm density with 95.4% ( 13  264  000 clusters ) of the clusters passing qc filters . within this pooled run , the index representation for nocardioides massiliensis was determined to be 8.12% . from this genome sequencing process ,
the 1  027  586 produced illumina paired end reads of n.  massiliensis were filtered according to the read qualities .
the reads obtained from both applications were trimmed and the optimal assembly was obtained through the clc genomics wb4 software with 839 contigs which generated a genome size of 4.00 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 .
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 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 . for the genomic comparison
, we compared the genome sequence of n.  massiliensis strain gd13 ( genbank accession no .
aufn00000000 ) , nocardioides halotolerans strain dsm 19273 ( augt00000000 ) , nocardioides insulae strain dsm 17944 ( augu00000000 ) and aeromicrobium massiliense strain jc14 ( cahg00000000 ) .
to estimate the nucleotide sequence similarity at the genome level between n.  massiliensis and other members of the family nocardioidaceae , we determined the average genomic identity of orthologous gene sequences ( agios ) parameter using the magi ( marseille average genomic identity ) software as follows : orthologous proteins were detected using the proteinortho software ( version 1.4 )   using the following parameters : e value 1e-5 , 30% percentage of identity , 50% coverage and algebraic connectivity of 50% ; after fetching the corresponding nucleotide sequences of orthologous proteins for each pair of genomes , we determined the mean percentage of nucleotide sequence identity using the needleman - wunsch global alignment algorithm ; artemis   was used for data management , and dnaplotter   was used for visualization of genomic features .
the genome of n.  massiliensis strain gd13 is 4  006  620  bp long with a 71.0% g+c content ( table  4 , figure  6 ) .
of the 4179 predicted genes , 4132 were protein - coding genes and 47 were rnas .
three rrna genes ( one 16s rrna , one 23s rrna and one 5s rrna ) and 44 predicted trna genes were identified in the genome .
a total of 2783 genes ( 66.59% ) were assigned a putative function , and 382 genes ( 9.24% ) were identified as orfans .
the draft genome of n.  massiliensis has a larger size than that of a.  massiliense ( 4.00 and 3.32 mb respectively ) but smaller than n.  alkalitolerans , n. halotolerans and n.  insulae ( 4.85 , 4.86 and 4.06 mb respectively ) .
the g+c content of n.  massiliensis is higher than those of n.  insulae ( 71.0 and 70.8% respectively ) but less than that of a.  massiliense , n. alkalitolerans and n.  halotolerans ( 72.6 , 73.1 and 72% respectively ) .
the gene content of n.  massiliensis is larger than those of a.  massiliense and n.  insulae ( 4179 , 3328 and 3702 respectively ) and smaller than those of n.  alkalitolerans and n.  halotolerans ( 4634 and 4633 respectively ) .
however , the distribution of genes into cogs categories was similar in all compared genomes ( figure  7 ) .
in addition , n.  massiliensis shared 1447 , 1745 , 1693 and 1598 orthologous genes with a.  massiliense , n. alkalitolerans , n. halotolerans and n.  insulae . among nocardioides species , agios values ranged from 75.67% between n.  alkalitolerans and n.  insulae to 77.56% between n.  alkalitolerans and n.  halotolerans . when considering n.  massiliensis , agios values were 75.06 , 76.67 and 76.82 with n.  insulae , n. alkalitolerans and n.  halotolerans respectively , in the same range as among other nocardioides species ( table  6 ) .
on the basis of phenotypic , phylogenetic and genomic analyses , we formally propose the creation of nocardioides massiliensis sp .
the strain was isolated from a stool sample from a 62-year - old man admitted to intensive care in marseille , france , for guillain - barr syndrome and who had received 10 days of broad - spectrum antibiotics .
adj . , from massiliensis of massilia , the ancient roman name for marseille , france , where the type strain was isolated ) .
cells are gram - positive , nonsporulating , nonmotile and rod - shaped bacilli with a mean diameter and length of 0.46 and 1.0  m respectively .
colonies are 0.5  mm in diameter and are smooth and pale yellow on 5% sheep 's blood  enriched columbia agar ( biomrieux ) .
positive reactions are observed for esterase ( c4 ) , esterase lipase ( c8 ) , acid phosphatase , alkaline phosphatase , leucine arylamidase , naphthol - as - bi - phosphohydrolase , -glucosidase , -glucosidase , arginine arylamidase , proline arylamidase , esculin hydrolysis and fermentation of d - lactose .
negative reactions are observed for -galactosidase , -galactosidase , valine arylamidase , cystine arylamidase , -glucuronidase , lipase ( c14 ) , trypsin , -chymotrypsin , n - acetyl--glucosaminidase , -mannosidase , -fucosidase , arginine dihydrolase , n acetyl--glucosaminidase , nitrate reductase , urease , indole production , arginine dihydrolase , n acetyl--glucosaminidase , serine arylamidase , histidin arylamidase , phenylalanine arylamidase , tyrosin arylamidase , fermentation of glycerol , erythritol , d - arabinose , l - arabinose , d - ribose , d - xylose , l - xylose , d - adonitol , methyl-d - xylopyranoside , d - galactose , d - glucose , d - fructose , d - mannose , l - sorbose , l - rhamnose , dulcitol , inositol , d - mannitol , methyl-d - xylopyranoside , methyl-d - glucopyranoside , n - acetylglucosamine , amygdalin , arbutin , esculin ferric citrate , salicin , d - cellobiose , d - maltose , d - mellibiose , d - trehalose , inulin , d - melezitose , d - raffinose , amidon , glycogen , gentiobiose , d - turanose , d - lyxose , d - tagatose , d - fucose , l - fucose , l - arabitol , d - sorbitol , d - saccharose , xylitol , d - arabitol , potassium-5-ketogluconate and potassium 2-ketogluconate .
cells are susceptible to penicillin g , amoxicillin , amoxicillin - clavulanic acid , ceftriaxone , imipenem , vancomycin , rifampicin , erythromycin , gentamicin , ciprofloxacin and trimethoprim / sulfamethoxazole and resistant to metronidazole .
the 16s rrna and whole - genome sequence of n.  massiliensis strain gd13 are deposited in embl - ebi under the accession numbers hf952922 and ccxj00000000 respectively .
the type strain gd13 (= csur p894  =  dsm 28216 ) was isolated from the faecal flora of a 62-year - old man admitted to intensive care in marseille , france , for guillain - barr syndrome who had received 10 days of broad - spectrum antibiotics .
 nocardioides massiliensis sp . 
 nov strain gd13 t is the type strain of n.  massiliensis sp . 
 nov . , a new species within the genus nocardioides . 
 this strain was isolated from the faeces of a 62-year - old man admitted to intensive care for guillain - barr syndrome . 
 nocardioides massiliensis is a strictly aerobic gram - positive rod . 
 herein we describe the features of this bacterium , together with the complete genome sequence and annotation . 
 the 4  006  620  bp long genome contains 4132 protein - coding and 47 rna genes . 
Introduction
Organism Information and Classification
Genome Sequencing Information
Conclusion
Description of 