the genus necropsobacter ( christensen et  al . 2011 ) was first described in 2011 . at this time
members of the genus necropsobacter were previously associated with the sp group that comprised mainly strains isolated from rabbits , rodents and humans . because necropsobacter rosorum was the only described species in this genus with no genome available , we first sequenced its genome for genomic comparison . necropsobacter massiliensis strain ff6 (= collection
de souches de lunit des rickettsies ( csur ) p3511  =  deutsche sammlung von mikroorganismen ( dsm )  =  27814 ) was isolated from a patient with a cervical abscess hospitalized at hpital principal in dakar , senegal .
n.  massiliensis is gram negative , aeroanaerobic , indole negative , nonmotile , and coccobacillus .
this bacterium was cultivated as part of the implementation of matrix - assisted laser desorption / ionization time - of - flight mass spectrometry ( maldi - tof ) in hpital principal , dakar , aiming at improving the routine laboratory identification of bacterial strains in senegal .
the current taxonomic classification of prokaryotes relies on a combination of phenotypic and genotypic characteristics , , including 16s rrna sequence similarity , g+c content and dna - dna hybridization .
however , these tools suffer from various drawbacks , mainly as a result of their threshold values , which are not applicable to all species or genera , . with the development of cost - effective high - throughput sequencing techniques ,
recently we developed a strategy , taxonomogenomics , in which genomic and phenotypic characteristics , notably the maldi - tof spectrum , are systematically compared to the phylogenetically closest species with standing in nomenclature , , .
here we present a summary classification and a set of features for necropsobacter massiliensis sp .
since july 2012 , the hpital principal in dakar , senegal , has been equipped with a maldi - tof ( vitek ms ruo ; biomrieux , marcy letoile , france ) to improve the microbiology laboratory work flow by enabling rapid bacterial identification .
isolates that are poorly identified using maldi - tof are referred to the urmite laboratory in marseille , france , for further identification .
strain ff6 ( table  1 ) was isolated by cultivation on 5% sheep 's blood  enriched columbia agar ( biomrieux ) from the cervical abscess of a 4-year - old senegalese boy .
strain ff6 exhibited a 95.17% 16s rrna sequence identity with necropsobacter rosorum 
 , the phylogenetically closest bacterial species with a validly published name ( fig .  1 ) .
these values were lower than the 98.7% 16s rrna gene sequence threshold recommended by meier - kolthoff et  al .   to delineate a new species within phylum firmicutes without carrying out dna - dna hybridization .
different growth temperatures ( 25c , 30c , 37c , 45c and 56c ) were tested .
growth was obtained between 37c and 45c , with the optimal growth temperature being 37c .
growth of the strain was tested under anaerobic and microaerophilic conditions using genbag anaer and genbag microaer systems , respectively ( biomrieux ) , and under aerobic conditions with or without 5% co2 .
colonies were 1  mm in diameter , grey and nonhaemolytic on 5% sheep 's blood 
necropsobacter massiliensis is gram negative , coccobacillus , not motile , and unable to form spores ( fig .  2 ) .
under electron microscopy , cells had a mean length of 1.5  m ( range , 0.92.1  m ) and a mean diameter of 0.4  m ( range , 0.20.6  m ) ( fig .  3 ) .
strain ff6 was oxidase positive and catalase negative . using an api zym strip ( biomrieux ) , positive reactions were observed for alkaline phosphatase , esterase , leucine arylamidase , phosphatase acid , -glucosidase and naphthol - as - bi - phosphohydrolase .
negative reactions were noted for -galactosidase , -glucuronidase , -mannosidase , -fucosidase , n - acetyl--glucosaminidase , lipase , -chrymotrypsin and cystine arylamidase .
using api 50ch , positive reactions were observed for glycerol , ribose , d - xylose , d - mannose , d - glucose , inositol , n - acetyl glucosamine , d - fructose , d - maltose d - melibiose , d - trehalose , d - saccharose , d - raffinose , starch , potassium 5-ketogluconate , alkaline phosphatase , esterase , leucine arylamidase , phosphatase acid , -glucosidase and naphthol - as - bi - phosphohydrolase .
negative reactions were observed for d - mannitol , d - sorbitol , l - xylose , d - adonitol , methyl -d - xylopyranose , d - melezitose , inulin , -galactosidase , -glucuronidase , -mannosidase , -fucosidase , n - acetyl--glucosaminidase , lipase , -chrymotrypsin and cystine arylamidase .
necropsobacter massiliensis strain ff6 is susceptible to amoxicillin , amoxicillin / clavulanic acid , ceftriaxone , gentamicin , nitrofurantoin , trimethoprim / sulfamethoxazole , rifampicin and ciprofloxacin but resistant to erythromycin , doxycycline and vancomycin .
five species validly published names in the pasteurellaceae family were selected to make a phenotypic comparison with necropsobacter massiliensis ( table  2 ) .
maldi - tof protein analysis was carried out as previously described ,   using a microflex lt ( bruker daltonics , leipzig , germany ) .
twelve individual colonies were deposited on a mtp 384 maldi - tof target plate ( bruker ) .
a total of 2  l of matrix solution ( saturated solution of alpha - cyano-4-hydroxycinnamic acid ) in 50% acetonitrile and 2.5% trifluoroacetic acid were distributed on each smear and air dried for 5 minutes at room temperature .
the 12 individual spectra from strain ff6 were imported into maldi biotyper software 2.0 ( bruker ) and analysed by standard pattern matching ( with default parameter settings ) against the main spectra of 6252 bacterial spectra .
the scores previously established by bruker daltonics allowing ( or not ) validating the identification of species compared to the database of the instrument were applied .
briefly , a score 2.000 with a species with a validly published name provided allows the identification at the species level ; a score of 1.700 to < 2.000 allows the identification at the genus level ; and a score of < 1.700 does not allow any identification .
thus , scores ranging from 1.2 to 1.3 were obtained , suggesting that this isolate was not a member of any known species .
the reference mass spectrum from strain ff6 was incremented in our database ( fig .  4 ) .
finally , the gel view showed the spectral differences with other members of the family pasteurellaceae ( fig . 
the organism was selected for sequencing on the basis of its 16s rrna similarity , phylogenetic position and phenotypic differences with other members of the family pasteurellaceae , and is part of a study aiming at using maldi - tof for the routine identification of bacterial isolates in hpital principal in dakar .
it is the second genome of a necropsobacter species and the first genome of necropsobacter massiliensis sp .
table  3 shows the project information and its association with migs version 2.0 compliance ; associated migs records are also summarized in supplementary table  s1 .
necropsobacter massiliensis strain ff6 (= csur p3511  =  dsm 27814 ) was grown aerobically on 5% sheep 's blood 
bacteria grown on four petri dishes were resuspended in 5    100  l of te buffer ; 150  l of this suspension was diluted in 350  l te buffer 10    , 25  l proteinase k and 50  l sodium dodecyl sulfate for lysis treatment .
the genomic dna concentration was measured at 30.06  ng/l using the qubit assay with the high sensitivity kit ( life technologies , carlsbad , ca , usa ) .
genomic dna of necropsobacter massiliensis ff6 was sequenced on the miseq sequencer ( illumina , san diego , ca , usa ) with the paired - end and mate - pair strategies .
the paired - end and the mate - pair strategies were barcoded in order to be mixed respectively with ten other genomic projects prepared with the nextera xt dna sample prep kit ( illumina ) and 11 other projects with the nextera mate - pair sample prep kit ( illumina ) .
genomic dna was diluted to 1  ng/l to prepare the paired - end library .
tagmentation  step fragmented and tagged the dna with an optimal size distribution at 1.5  kb .
then limited - cycle pcr amplification ( 12 cycles ) completed the tag adapters and introduced dual - index barcodes .
after purification on ampure xp beads ( beckman coulter , fullerton , ca , usa ) , the libraries were then 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 were performed in single 39-hour runs at 2    250  bp .
total information of 3.89  gb was obtained from a 416  k / mm cluster density with a cluster passing quality control filters of 95.4% ( 7  899  000 clusters ) . within this run ,
the index representation for necropsobacter massiliensis was determined to be 7.02% and to present 529  002 reads filtered according to the read qualities .
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 labchip .
the dna fragments ranged in size from 1  kb up to 6.6  kb , with an optimal size at 4.5  kb .
no size selection was performed , and 368  ng of tagmented fragments were circularized .
the circularized dna was mechanically sheared to small fragments on the covaris s2 device in microtubes ( covaris , woburn , ma , usa).the library profile was visualized on a high sensitivity bioanalyzer labchip ( agilent ) with an optimal peak at 672  bp .
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 39-hour run at 2    250  bp .
open reading frame ( orf ) prediction was carried out using prodigal   with default parameters .
functional assessment of protein sequences was performed by comparing them with sequences in the genbank   and the clusters of orthologous groups ( cogs ) databases using blastp .
trnas , rrnas , signal peptides and transmembrane helices were identified using trnascan - se 1.21 , rnammer , signalp   and tmhmm , respectively .
artemis   was used for data management , whereas dna plotter   was used for visualization of genomic features . in - house
orfans were sequences which have no homology in a given database , i.e. in nonredundant ( nr ) or identified if their blastp e value was lower than 1e-03 for alignment lengths greater than 80 amino acids .
phast was used to identify , annotate and graphically display prophage sequences within bacterial genomes or plasmids . to estimate the nucleotide sequence similarity at the genome level between necropsobacter massiliensis and another ten members of the pasteurellaceae family
, we determined the average genomic identity of orthologous gene sequences ( agios ) parameter as follows : orthologous proteins were detected using the proteinortho software   ( with the following parameters : e - value 1e-5 , 30% percentage identity , 50% coverage and 50% algebraic connectivity ) and genomes compared two by two . 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 .
genomes from the genus necropsobacter and closely related genera were used for the calculation of agios values .
the script created to calculate agios values was named magi ( marseille average genomic identity ) and is written in perl and bioperl modules .
ggdc analysis was also performed using the ggdc web server ( http://ggdc.dsmz.de ) as previously reported , . here , we compared the genome sequences of necropsobacter massiliensis strain ff6 ( genbank accession number cdon00000000 ) with those of n. rosorum strain p709 t ( ccmq00000000 ) , pasteurella multocida subsp .
multocida strain pm70 ( ae004439 ) , haemophilus influenzae strain rd kw20 ( l42023 ) , haemophilus ducreyi strain 35000hp ( ae017143 ) , histophilus somnus strain 129pt ( cp000436 ) , haemophilus parasuis strain sh0165 ( cp001321 ) , haemophilus parainfluenzae strain t3t1 ( fq312002 ) and aggregatibacter aphrophilus strain nj8700 ( cp001607 ) . the genome of necropsobacter massiliensis strain ff6 is 2  493  927  bp long with a 46.2% g+c content ( fig
2309 were protein coding genes and 54 were rna genes including 1 complete rrna operon .
the draft genome of necropsobacter massiliensis ( 2.49 mb ) has a lower size than that of n.  rosorum ( 2.52 mb ) but a larger size than those of p.  multocida ( 2.25 mb ) , h.  influenzae ( 1.83 mb ) , h.  ducreyi ( 1.69 mb ) , h.  somnus ( 2.00 mb ) , h.  parasuis ( 2.26 mb ) , h.  aphrophilus ( 2.31 mb ) and h.  parainfluenzae ( 2.08 mb ) .
the g+c content of necropsobacter massiliensis ( 46.2% ) was lower than that of n.  rosorum ( 48.9% ) but higher than those of p.  multocida ( 40.40% ) , h.  influenzae ( 38.15% ) , h.  ducreyi ( 38.22% ) , h.  somnus ( 37.20% ) , h.  parasuis ( 39.99% ) , h.  aphrophilus ( 42.23% ) and h.  parainfluenzae ( 39.57% ) . because it has been suggested in the literature that the g+c content deviation is at most 1% within species , these data are an additional argument for the creation of a new taxon .
the protein - coding genes of necropsobacter massiliensis is larger than those of p.  multocida , h. influenzae , h. ducreyi , h. somnus , h. parasuis , h. aphrophilus and h.  parainfluenzae ( 2012 , 1603 , 1717 , 1791 , 2021 , 2218 and 1975 , respectively ) but smaller than that of n.  rosorum ( 2311 ) .
in addition , necropsobacter massiliensis shared 2012 , 1603 , 1717 , 1791 , 2021 , 1975 , 2301 and 2218 orthologous genes with p.  multocida , h. influenzae , h. ducreyi , h. somnus , h. parasuis , h. aphrophilus and h.  parainfluenzae , respectively . among species with standing in nomenclature ,
agios values ranged from 66.32 between n.  rosorum and h.  ducreyi to 98.71% between p.  multocida and h.  parainfluenzae ( table  6 ) .
when compared to other species , necropsobacter massiliensis exhibited agios values ranging from 67.15 with h.  ducreyi to 84.44 with n.  rosorum .
we obtained similar results using the ggdc software , as dddh values ranged from 0.201 to 0.281 between studied species and were 0.275 between n.  rosorum .
on the basis of phenotypic , phylogenetic and genomic analyses , we formally propose the creation of necropsobacter massiliensis sp . nov . that contains strain ff6 .
the strain was isolated from a cervical abscess of a 4-year - old senegalese boy .
n. massiliensis , of massilia , the latin name of marseille , where this strain was characterized ) . on 5% sheep 's blood
 enriched columbia agar ( biomrieux ) , colonies were 1  mm in diameter and grey .
cells are gram negative and not motile , with a mean diameter of 0.4  m ( range , 0.20.6  m ) and a mean length of 1.5  m ( range , 0.92.1  m ) .
positive reactions were observed for glycerol , ribose , d - xylose , d - mannose , d - glucose , inositol , n - acetyl glucosamine , d - fructose , d - maltose d - melibiose , d - trehalose , d - saccharose , d - raffinose , starch , potassium 5-ketogluconate , alkaline phosphatase , esterase , leucine arylamidase , phosphatase acid , -glucosidase , naphthol - as - bi - phosphohydrolase and -glucosidase .
negative reactions were observed for d - mannitol , d - sorbitol , l - xylose , d - adonitol , methyl -d - xylopyranoside , d - melezitose , inulin , -galactosidase , -glucuronidase , -mannosidase , -fucosidase , n - acetyl--glucosaminidase , lipase , -chrymotrypsin , cystine arylamidase , -galactosidase , -glucuronidase , -mannosidase , -fucosidase , n - acetyl--glucosaminidase , lipase , -chrymotrypsin and cystine arylamidase .
necropsobacter massiliensis strain ff6 is susceptible to amoxicillin , amoxicillin / clavulanic acid , ceftriaxone , gentamicin , nitrofurantoin , rifampicin , trimethoprim / sulfamethoxazole and ciprofloxacin but resistant to erythromycin , doxycycline and vancomycin .
the 16s rrna and genome sequences of n.  massiliensis strain ff6 (= csur p3511  =  dsm 27814 ) are deposited in genbank under accession numbers hg428679 and cdon00000000 , respectively .
the type strain , ff6 , was isolated from a cervical abscess of a 4-year - old senegalese boy hospitalized in hpital principal in dakar , senegal .
 strain ff6 t was isolated from the cervical abscess of a 4-year - old senegalese boy , in dakar , senegal . 
 maldi - tof ms did not provide any identification . 
 this strain exhibited a 95.17% 16s rrna sequence identity with necropsobacter rosorum . using a polyphasic study including phenotypic and genomic analyses , strain ff6 t was an aero - anaerobic gram - negative cocobacillus , oxidase positive , and exhibited a genome of 2,493,927 bp ( 1 chromosome but no plasmid ) with a g+c content of 46.2% that coded 2,309 protein - coding and 53 rna genes . on the basis of these data , we propose the creation of necropsobacter massiliensis sp . 
 nov . 
Introduction
Organism Information
Genome Sequencing Information
Insights From Genome Sequence
Conclusions