the genus weeksella ( holmes et  al . , 1986 ) was first described in 1986 . to date , this genus includes one species , weeksella virosa , which has been isolated from human clinical specimens , .
the current classification of prokaryotes relies on a polyphasic strategy combining phenotypic and genotypic characteristics , .
these include 16s rrna sequence similarity , g  +  c content and dna - dna hybridization ( ddh ) .
however , these tools have significant drawbacks , notably that the recommended threshold values do not apply to all species or genera , .
thanks to the progress made in sequencing technologies and their lowering costs , almost 40  000 bacterial genome sequences are currently available , covering many phyla .
recently we proposed to integrate phenotypic characteristics , notably the maldi - tof spectrum , and genomic analysis and comparison in the taxonomic description of bacterial species , , .
strain ff8 (= csur p860  =  dsm 28259 ) was isolated from the urine of a 65-year - old man treated at the hpital principal de dakar , senegal .
that is a gram - negative bacterium , aerobic , indole negative , nonmotile and rod shaped .
this bacterium was cultivated as part of the maldi - tof implementation at hpital principal de dakar aiming to improve the routine laboratory identification of microorganisms . here
in july 2013 , a urine sample was collected from a 65-year - old senegalese man with acute cystitis . from this clinical sample strain ff8 ( table  1 ) was isolated by cultivation on 5% sheep 's blood 
when analysed by maldi - tof , no identification was obtained because the strain displayed low scores .
strain ff8 exhibited a 98.38% 16s rrna sequence similarity with weeksella virosa strain dsm 16922 ( genbank accession number nr_074495 ) , the phylogenetically closest bacterial species with standing in nomenclature .
these values were lower than the 98.7% 16s rrna gene sequence threshold recommended by meier - kolthoff et  al . , 2013 , to delineate a new species within phylum bacteroidetes 
 ,   without carrying out ddh .
a phylogenetic tree based on the 16s rrna sequence highlights the position of weeksella massiliensis strain ff8 among the family flavobacteriaceae ( fig .  1 ) .
different growth temperatures ( 25 , 30 , 37 , 45 and 56c ) were tested .
growth of the strain was tested also under anaerobic and microaerophilic conditions using genbag anaer and genbag microaer systems , respectively ( biomrieux ) , and under aerobic conditions , with or without 5% co2 .
the colonies were opaque , light yellow in color with a smooth surface , not haemolytic on 5% sheep 's blood  enriched columbia agar ( biomrieux ) and approximately 2  mm in diameter .
cells are gram - negative , non - spore - forming rods of regular shape with rounded ends ( fig .  2 ) and have mean diameter of 0.3  m ( range , 0.20.5  m ) and a mean length of 1.5  m ( range , 0.82.1  m ) ( fig .  3 ) .
this bacterium possesses catalase and oxidase . using an api zym strip ( biomrieux ) ,
lipase , leucine arylamidase , acid phosphatase and naphthol - as - bi - phosphohydrolase .
negative reactions were noted for -chymotrypsin , cystine arylamidase , valine arylamidase , trypsin , -glucosidase , -glucosidase , -galactosidase , -galactosidase , -glucuronidase , -mannosidase , -fucosidase and n - acetyl--glucosaminidase .
strain ff8 is susceptible to ceftriaxone , amoxicillin / clavulanic acid , penicillin , imipenem , gentamicin and doxycycline but resistant to nitrofurantoin , vancomycin , trimethoprim / sulfamethoxazole and metronidazole .
the minimum inhibitory concentrations ( mics ) for some antibiotics tested by weeksella massiliensis strain ff8 t sp . nov .
a comparison of phenotypic characteristics with w.  virosa , bergeyella zoohelcum 
  and moheibacter sediminis 
  is presented in table  3 .
maldi - tof protein analysis was performed with a microflex lt ( bruker daltonics , leipzig , germany ) , as previously reported , .
the scores previously established by bruker allowing validating ( or not ) the identification of species compared to the database of the instrument were applied .
briefly , a score of  2.000 with a species with a validly published name provided allows the identification at the species level ; a score of  1.700 and <  2.000 allows the identification at the genus level ; and a score of < 1.700 does not allow any identification .
two microliters of matrix solution ( saturated solution of -cyano-4-hydroxycinnamic acid ) in 50% acetonitrile and 2.5% trifluoroacetic acid were distributed on each smear and submitted at air drying for 5 minutes .
then the spectra from the 12 different colonies were imported into the maldi biotyper software ( version 2.0 , bruker ) and analysed by standard pattern matching ( with default parameter settings ) against the main spectra of 6252 bacteria .
scores ranging from 1.32 to 1.56 were obtained for strain ff8 , suggesting that it was not a member of any known species .
the reference mass spectrum from strain ff8 was incremented in our database ( fig .  4 ) .
the organism was selected for sequencing on the basis of its phylogenetic position , 16s rrna similarity and phenotypic differences with other members of the family flavobacteriaceae .
it was the second genome within weeksella genus and the first genome of w.  massiliensis sp .
table  4 shows the project information and its association with minimum information about a genome sequence ( migs , version 2.0 ) compliance ; associated migs records are also summarized in supplemental table  s1 .
weeksella massiliensis strain ff8 (= csur p860  =  dsm 28259 ) was grown aerobically on 5% sheep 's blood 
bacteria grown on four petri dishes were resuspended in 5    100  l of tris - edta buffer ; 150  l of this suspension was diluted in 350  l tris - edta buffer 10 , 25  l proteinase k and 50  l sodium dodecyl sulfate for lysis treatment .
extracted dna was then purified using 3 successive phenol  chloroform extractions ( thermo fisher scientific , waltham , ma , usa ) and ethanol precipitations at  20c overnight .
the genomic dna concentration was measured at 53.7  ng/l using the qubit assay with the high sensitivity kit ( life technologies , carlsbad , ca , usa ) .
genomic dna of weeksella massiliensis ff8 was sequenced on a miseq sequencer ( illumina , san diego , ca , usa ) using the mate pair strategy .
the genomic dna ( gdna ) was bar coded in order to be mixed with 11 other projects with the nextera mate - pair sample prep kit ( illumina ) .
the mate pair library was prepared with 1  g of genomic dna using the nextera mate - pair illumina guide .
the fragmentation pattern was validated on an agilent 2100 bioanalyzer ( agilent technologies , santa clara , ca , usa ) with a dna 7500 lab chip .
the dna fragment sizes ranged from 1 to 10  kb with an optimal size at 3.1  kb .
no size selection was performed , and only 143  ng of tagmented fragments were circularized .
the circularized dna was mechanically sheared to small fragments with an optimal at 645  bp on a covaris device s2 in microtubes ( covaris , woburn , ma , usa ) .
the library profile was visualized on a high sensitivity bioanalyzer labchip ( agilent technologies ) , and the final concentration library was measured at 6.03  nmol / l .
after 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 sequencing were performed in a single 27-hour run in a 2    151  bp .
total information of 6 gb was obtained from a 1158k / mm cluster density with a cluster passing quality control filters of 88.9% ( 21  834  000 clusters ) . within this run ,
the 1  613  495 paired reads were filtered according to the read qualities .
these reads were trimmed then assembled thought clcgenomicswb4 software . finally , the draft genome of weeksella massiliensis consists of 17 scaffolds made of 54 contigs and generated a genome size of 2.56 mb with g  +  c content of 35.9% .
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 for alignment length greater than 80 aa . if alignment lengths were smaller than 80 aa , we used an e value of 1e. such parameter thresholds have already been used in previous works to define orfans .
artemis   was used for data management , and dna plotter   was used for visualization of genomic features .
the mauve alignment tool ( version 2.3.1 ) was used for multiple genomic sequence alignment . to estimate the mean level of nucleotide sequence similarity at the genome level
, we used calculated the average genomic identity of orthologous gene sequences ( agios ) parameter using an in - lab pipeline named marseille average genomic identity ( magi ) .
briefly , this software combines the proteinortho software   for detecting orthologous proteins in pairwise comparisons of genomes , then retrieves the corresponding genes and determines the mean percentage of nucleotide sequence identity among orthologous orfs using the needleman - wunsch global alignment algorithm .
the script was created to calculate the average genomic identity of orthologous gene sequences ( agios ) between genomes by magi . the magi script created to calculate agios values written in perl and bioperl modules .
genome - to - genome distance ( ggdc ) analysis was also performed using the ggdc web server ( http://ggdc.dsmz.de/ ) as previously reported , .
the genome of w. massiliensis strain ff8 is 2  562  781  bp long with a 35.9% g  +  c content ( fig . 
three rrna genes ( one 16s rrna , one 23s rrna and one 5s rrna ) and 53 predicted trna genes were identified in the genome . a total of 1428 genes ( 58.38% ) were assigned a putative function .
here we compared the genome sequence of w.  massiliensis strain ff8 with those of weeksella virosa strain dsm 16922 ( genbank accession number cp002455 ) , chryseobacterium gleum strain atcc 35910 ( ackq00000000 ) , elizabethkingia meningoseptica atcc
the draft genome of w.  massiliensis has a larger size than that of w.  virosa ( 2.56 mb and 2.27 mb , respectively ) but smaller than those of c.  gleum , e. meningoseptica and e.  brevis ( 5.57 mb , 3.96 mb and 3.79 mb , respectively ) .
weeksella massiliensis has a higher g  +  c content than e.  brevis ( 35.89% and 32.7% , respectively ) but lower than w.  virosa , c. gleum and e.  meningoseptica ( 35.9% , 36.8% and 36.4% , respectively ) . because it has been suggested in the literature that the g  +  c content deviation is at most 1% within species ,
the gene content of w.  massiliensis is larger than that of w.  virosa ( 2446 and 2171 , respectively ) but smaller than those of c.  gleum , e. meningoseptica and e.  brevis ( 5369 , 3423 and 3655 , respectively ) .
however , the distribution of genes into cogs categories was similar in all compared genomes .
in addition , w.  massiliensis shared 2390 , 2049 , 5289 , 3369 and 3589 orthologous genes with w.  virosa , c. gleum , e. meningoseptica and e. brevis , respectively .
because only one genome was available for the genus weeksella , we used the genomes from closely related species for the calculation of agios values .
the agios values ranged from 66.45% to 73.15% between w.  virosa and other members of the family flavobacteriaceae ( table  7 ) .
w.  massiliensis exhibited similar values when compared to representatives of other genera , from 66.65% to 72.25% , but a higher value when compared to w.  virosa ( 87.85% ) .
in addition , digital ddh similarities between the genomes were calculated by the ggdc web server version 2.0 , as recommended ( table  8) , , , .
on the basis of phenotypic , phylogenetic and genomic analyses , we formally propose the creation of weeksella massiliensis sp . nov . that contains strain ff8 as type strain .
the strain was isolated from the urine of a 65-year - old senegalese man who sought care at the hpital principal de dakar , senegal .
n. massiliensis , of massilia , the latin name for marseille , where strain ff8 was cultivated ) .
colonies were 2  mm in diameter and are opaque and light yellow with a smooth surface on 5% sheep 's blood - enriched columbia agar .
cells are gram negative and not motile , with a mean diameter of 0.3  m ( range , 0.20.5  m ) and a mean length of 1.5  m ( range , 0.82.1  m ) .
lipase , leucine arylamidase , phosphatase acid and naphthol - as - bi - phosphohydrolase activities .
negative reactions were noted for -chymotrypsin , cystine arylamidase , valine arylamidase , trypsin , -glucosidase , -glucosidase , -galactosidase , -galactosidase , -glucuronidase , -mannosidase , -fucosidase and n - acetyl--glucosaminidase activities .
weeksella massiliensis strain ff8 is susceptible to ceftriaxone , amoxicillin / clavulanic acid , penicillin , imipenem , gentamicin and doxycycline but resistant to nitrofurantoin , vancomycin , trimethoprim  sulfamethoxazole and metronidazole .
the 16s rrna and genome sequences are deposited in genbank under accession numbers hg931340 and ccmh00000000 , respectively .
the type strain ff8 (= csur p860  =  dsm 28259 ) was isolated from the urine of a 65-year - old man with acute cystitis at hpital principal de dakar , senegal .
 strain ff8 t (= csur p860  =  dsm 28259 ) was isolated in dakar , senegal , from the urine of a 65-year - old man with acute cystitis . 
 this strain shows a similarity of sequence of 16s rrna of 98.38% with weeksella virosa , and its genbank accession numbers are hg931340 and ccmh00000000 . 
 matrix - assisted laser desorption / ionization time - of - flight mass spectrometry analysis had a poor score , ranging from 1.32 to 1.56 , that did not allow identification of the bacterium . using a polyphasic study made of phenotypic and genomic analyses , 
 strain ff8 t was a gram - negative , aerobic rod and a member of the family flavobacteriaceae . 
 the sequenced genome is 2  562  781  bp with one chromosome but no plasmid . 
 it exhibits a g  +  c content of 35.9% and contains 2390 protein - coding and 56 rna genes , including a complete rrna operon . on the basis of these data , 
 we propose the creation of weeksella massiliensis sp . nov . 
Introduction
Organism Information
Genome Sequencing Information
Genome Properties
Conclusions
Description of 