bacterial vaginosis is a common yet poorly understood condition affecting women of childbearing age in both industrialized and developing countries .
bacterial vaginosis is characterized simultaneously by an abnormal loss of the normal doderlen flora accompanied by an unexplained overgrowth of anaerobic bacteria that were previously minor in the vagina , . in pregnant women ,
this vaginal dysbiosis is the consequence of certain complications such as miscarriage , preterm birth or chorioamnionitis .
bacterial vaginosis is mostly treated with antibiotics , mainly metronidazole and clindamycin , but treatment frequently fails ; the relapse rate is estimated at 50% at 6 months , . in order to describe the vaginal flora as fully as possible and to better understand the condition in order to provide better treatment ,
we studied the vaginal microbiota from healthy women and patients with bacterial vaginosis using the culturomics concept .
this is based on the multiplication of culture conditions ( atmosphere , media , and temperature ) and a rapid bacterial identification using matrix - assisted desorption ionization 
, it was only in 1992 that the name of this family was validated by reichenbach .
some species are found in soil and the marine environment , while others are pathogens found in fish and the human urogenital tract .
hence , in order to describe a new bacterium , our laboratory introduced taxonogenomics , a new approach that complements classic features with the proteomic information obtained by maldi - tof ms and the description of the annotated whole genome , . in the following section , we describe the vaginella massiliensis strain marseille p2517 (= dsm 102346  =  csur p2517 ) , a new genus isolated from a vaginal swab taken from a healthy 22-year - old french woman without bacterial vaginosis .
as previously described , a vaginal sample was taken from a healthy 22-year - old french woman without bacterial vaginosis at la timone hospital in marseille ( france ) in january 2016 using a sigma transwab ( medical wire , corsham , united kingdom ) .
the study was authorized by the local ifr48 ethics committee ( marseille , france ) under agreement number 09 - 022 .
the vaginal sample was first preincubated in a blood culture bottle ( bd diagnostics , le pont - de - claix , france ) supplemented with 4  ml rumen and filtered at 0.2  m using a pore filter ( thermo fisher scientific , villebon - sur - yvette , france ) and 3  ml sheep 's blood ( biomrieux , marcy letoile , france ) .
after 7 days of preincubation , 50 l of the supernatant was inoculated on chocolat polyvitex ( pvx ) agar ( bd diagnostics ) .
after 2 days of incubation at 37c in aerobic conditions , purified colonies were deposited in duplicate on a msp 96 maldi - tof ms target plate ( bruker daltonics , leipzig , germany ) , and , as previously described , 1.5 l of matrix solution was added to each spot .
identification was carried out using a microflex spectrometer ( bruker ) , which compares the protein spectra found to those in the bruker database ( constantly updated with spectra of new species discovered in our laboratory ) .
in contrast , if no spectra match the database , and for unidentified bacteria with a clear spectrum , 16s rrna gene sequencing is performed . as stackebrandt and
ebers suggested , if the 16s rrna sequence similarity value is lower than 95% or 98.7% , the strain is defined as a new genus or species , respectively .
a custom python script was used to automatically retrieve all species from the same order of the new genus and to download 16s sequences from national center for biotechnology information ( ncbi ) by parsing ncbi eutils results and the ncbi taxonomy page .
this only retains sequences from type strains . in the event of multiple sequences for one type strain
, it selects the sequence obtaining the best identity rate from the blastn alignment with our sequence .
the script then separates 16s sequences into two groups : one containing the sequences of strains from the same family ( group a ) and one containing the others ( group b ) .
finally , it only retains the 15 closest strains from group a and the closest one from group b. if it is impossible to retrieve 15 sequences from group a , the script selects more sequences from group b to achieve at least nine strains from both groups .
the ideal growth was tested by cultivating the strain marseille p2517 on colombia agar with 5% sheep 's blood incubated at different temperatures ( 25 , 28 , 37 , 45 and 56c ) and different atmospheres ( anaerobic , microaerophilic and aerobic ) .
the anaerobic and microaerophilic atmospheres were generated using , respectively , genbag anaer and genbag microaer systems ( biomrieux ) .
salinity and ph conditions were also tested at different concentrations of nacl ( 0 , 5 , 15 and 45% ) and different ph ( 5 , 6 , 6.5 , 7 and 8.5 ) .
sporulation , motility , gram stain , catalase and oxidase tests were performed using standard test procedures ( https://www.gov.uk/government/collections/standards-for-microbiology-investigations-smi#test-procedures ) . in order to observe cell morphology ,
samples were fixed with 2.5% glutaraldehyde in 0.1  m cacodylate buffer for at least an hour at 4c .
one drop of cell suspension was deposited for approximately 5 minutes on glow - discharged formvar carbon film on 400  mesh nickel grids ( fcf400-ni , ems ) .
the grids were dried on blotting paper , and cells were negatively stained for 10 seconds with 1% ammonium molybdate solution in filtered water at room temperature .
electron micrographs were acquired using a tecnai g cryo ( fei company , limeil - brevannes , france ) transmission electron microscope operated at 200  kev .
biochemical characteristics were studied using api zym , api 20ne and api 50ch strips ( biomrieux ) according to the manufacturer s instructions . a cellular fatty acid methyl ester ( fame )
two samples were prepared with approximately 90  mg of bacterial biomass per tube collected from several culture plates .
gc / ms analyses were carried out using a clarus 500 gas chromatograph equipped with a sq8s  ms detector ( perkin elmer , courtaboeuf , france ) . fame extracts ( 2  ml ) were volatilized at 250c ( split 20  ml / min ) in a focus liner with wool and separated on an elite-5ms column ( 30  m , 0.25  mm i.d .
, 0.25  mm film thickness ) using a linear temperature gradient ( 70290c at 6c / min ) , enabling the detection of c4 to c24 fatty acid methyl esters .
full scan monitoring was performed from 45 to 500  m / z . all data were collected and processed using turbomass 6.1 ( perkin elmer ) .
fames were identified through a spectral database search using ms search 2.0 operated with the standard reference database 1a ( national institute of standards and technology ( nist ) , gaithersburg , md , usa ) and the fames mass spectral database ( wiley , chichester , uk ) .
retention time correlations with estimated nonpolar retention indices from the nist database were obtained using a 37-component fame mix ( supelco ; sigma - aldrich , saint - quentin fallavier , france ) ; fame identifications were confirmed using this index .
the antibiotic susceptibility of strain marseille p2517 was tested using the disk diffusion method ( sirscan discs , perols , france ) .
strain marseille p2517 was grown in aerobic conditions at 37c using columbia agar enriched with 5% sheep 's blood ( biomrieux ) after 48 hours on four petri dishes .
bacteria were resuspended in 500  l of tris - edta ( te ) buffer ; 150  l of this suspension was diluted in 350  l 10 te buffer , 25  l proteinase k and 50  l sodium dodecyl sulfate for lysis treatment .
dna was purified using phenol / chloroform / isoamylalcohol successively for extraction , followed by ethanol precipitation at  20c for at least 2 hours each .
genomic dna ( gdna ) of strain marseille p2517 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 ) .
gdna was quantified using a qubit assay with the high sensitivity kit ( life technologies , carlsbad , ca , usa ) to 153  ng/l .
the mate - pair library was prepared with 1.5  g of gdna using the nextera mate pair illumina guide .
the gdna sample was simultaneously fragmented and tagged with a mate - pair junction adapter .
the fragmentation pattern 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 1.5 to 11  kb with an optimal size of 7.455  kb
. no size selection was performed , and 410.7  ng of tagmented fragments were circularized .
the circularized dna was mechanically sheared to small fragments with an optimal size of 1115  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 technologies ) , and the final concentration library was measured at 12.49 
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 run were performed in a single 39-hour run at a 2   251  bp read length .
a total of 9.2 gb of information was obtained from a 1042k / mm cluster density with a cluster passing quality control filters of 91.6% ( 18 
the 1  241  784 paired reads were trimmed , then assembled in nine scaffolds .
open reading frames ( orfs ) were predicted using prodigal software   with default parameters . predicted orfs spanning a sequencing gap region ( containing n ) were excluded .
we predicted the bacterial proteome sequences using blastp ( e value 1e-03 , coverage 0.7 and identity percentage 30 ) against the clusters of orthologous groups ( cogs ) database .
a search against the nr database   was performed if no hit was found using blastp with an e value of 1e-03 coverage of 0.7 and an identity percentage of 30 .
the hmmscan analysis tools were used for searching pfam conserved domains ( pfam - a and pfam - b domains ) on each protein .
we used rnammer   and the trnascanse tool   to find ribosomal rnas genes and trna genes respectively . for visualization and for data management of genomic features , we used artemis   and dna plotter   respectively . for the mean level of nucleotide sequence similarity analysis at the genome level , we used magi homemade software .
it calculated the average genomic identity of orthologous gene sequences ( agios ) among compared genomes .
the proteinortho   software was incorporated into the magi homemade software for detecting orthologous proteins in pairwise genomic comparisons .
the corresponding genes were then retrieved , and the mean percentage of nucleotide sequence identity among orthologous orfs was determined using the needleman - wunsch global alignment algorithm .
genomes from the genus weeksella , empedobacter and elizabethkingia were used for the calculation of agios values .
the genome of strain marseille p2517 ( european molecular biology laboratory ( embl)/european bioinformatics institute ( ebi ) accession number flmr00000000 ) was compared to those of weeksella virosa dsm 16922 ( nc_015144.1 ) , empedobacter brevis atcc 43319 ( nz_arnt00000000.1 ) , empedobacter falsenii strain 282 ( nz_jsyq00000000.1 ) , elizabethkingia anophelis strain b2d ( jncg00000000.1 ) , and elizabethkingia meningoseptica atcc 13253 ( bard00000000.1 ) ; all these genomes were reannotated with prodigal .
the multiagent software system dagobah   was used to perform the annotation and comparison process , including figenix   libraries , which provide pipeline analysis .
genome - to - genome distance calculator ( ggdc ) analysis was performed using the ggdc web server , as previously reported  .
strain marseille p2517 was first cultivated in january 2016 after 7 days of preincubation in a blood culture bottle supplemented with sheep s blood and rumen under aerobic conditions and then inoculated on pvx agar incubated for 2 days at 37c in aerobic conditions .
scores of 1.35 and 1.5 were obtained with the maldi - tof ms identification , suggesting that this isolate was not in the database and consequently was not a known species . the 16s rrna sequence ( accession no .
lt223570 ) of our strain showed 93.03% nucleotide sequence similarity with weeksella virosa , the phylogenetically closest species with a validly published name ( fig .  1 ) .
because this 16s rrna nucleotide sequence similarity was lower than 95% , the threshold recommended by stackebrandt and ebers   for delineating a new genus , strain marseille p2517 was classified as a new genus , vaginella , with vaginella massiliensis as the type species ( table  1 ) .
2 ) was then incremented in our database and compared to other known species of the family flavobacteriaceae .
3 . cultivated on blood agar for 2 days at 37c under aerobic conditions , colonies of strain marseille p2517
are yellow , opaque , circular and smooth with a diameter of 1.7 to 2  mm .
the strain grows only under aerobic conditions at 25 , 28 and 37c , but optimal growth was observed at 37c after 48 hours of incubation .
its growth also requires a ph ranging from 6.5 to 8.5 and a nacl concentration lower than 5 
nonmotile and non  spore forming , strain marseille p2517 exhibits positive oxidase activity ; however , the catalase activity was negative . under the microscope , bacterial cells are gram negative and rod shaped ( fig .  4 ) , and individual cells have a diameter ranging 0.54 to 0.68  m and a length ranging 1.2 to 1.5  m ( fig .  5 , table  1 ) . using an api 20ne strip , we observed that nitrate and nitrite were not reduced , and urease and indole activities were absent .
there was also no assimilation from d - glucose , l - arabinose , d - mannose , d - mannitol , d - maltose and n - acetyl - glucosamine . api 50ch showed that strain marseille p2517 metabolized only d - glucose ; and acid was not produced from other carbohydrates : arabinose , d - ribose , d - xylose , d - adonitol , d - galactose , d - fructose , d - mannose , l - rhamnose , d - mannitol , d - sorbitol , inositol , methyl-d - mannopyranoside , methyl- d - glucopyranoside , amygdalin , arbutin , esculin ferric citrate , salicin , d - cellobiose , d - maltose , d - lactose , d - melibiose , d - trehalose , inulin , d - melezitose , d - raffinose , starch , glycogen , xylitol , gentiobiose , d - turanose , d - lyxose , d - tagatose , fucose , arabitol , potassium gluconate , potassium 2-ketogluconate and potassium 5-ketogluconate .
api zym revealed positive reactions for alkaline phosphatase , esterase , esterase lipase , lipase , leucine arylamidase , cystine arylamidase , valine arylamidase , trypsin , -chymotrypsin , acid phosphatase and naphtol - as - bi - phosphohydrolase .
reactions for other enzymes such as galactosidase (  and  ) and oxidases were negative .
fame analysis demonstrated that the most abundant compound was 13-methyl - tetradecanoic acid ( 60% ) .
this strain shows many saturated and unsaturated branched fatty acids ( iso and anteiso ) .
several hydroxy fatty acids were also present ( table  2 ) . resistant to gentamicin ( cn 500  g ) and metronidazole ( met 4 ) , cells were sensitive to ceftriaxone ( cro 30  g ) , ciprofloxacin ( cip 5  g ) , clindamycin ( da 15  g ) , colistin ( ct 50  g ) , doxycycline ( do 30  g ) , erythromycin ( e 15  g ) , fosfomycin ( pos 50  g ) , oxacillin ( ox 5  g ) , penicillin ( p 10  g ) , rifampicin ( ra 30  g ) , teicoplasmin ( tec 30  g ) , trimethoprim / sulfamethoxazole ( sxt 25  g ) , vancomycin ( va 30  g ) and imipenem ( mp 10 ) .
all the phenotypic characteristics of strain marseille p2517 were compared to those of the closely related flavobacteriaceae species , , ,   ( table  3 ) .
the draft genome of strain marseille p2517 is 2  434  475  bp long with 38.16  mol% g+c content ( table  4 , fig . 
it is composed of nine scaffolds ( composed of ten contigs ) . on the 2395 predicted genes ,
2324 were protein - coding genes and 71 were rnas ( five 5s rrna , four 16s rrna , two 23s rrna and 60 trna genes ) .
a total of 1618 genes ( 69.62% ) were assigned a putative function ( by cogs or by nr blast ) , and 123 genes were identified as orfans ( 5.29% ) .
comparison of the genome of our strain marseille p2517 with those of the closest species revealed that the draft genome sequence of strain marseille p2517 ( 2.34 mb ) was smaller than those of empedobacter falsenii ( 3.71 mb ) , empedobacter brevis ( 3.79 mb ) , elizabethkingia meningoseptica ( 3.84 mb ) and elizabethkingia anophelis ( 4.02 mb ) but larger than those of weeksella virosa ( 2.27 mb ) .
the g+c content of strain marseille p2517 was larger than those of all the compared genomes : e.  meningoseptica ( 36.4% ) , w.  virosa ( 35.9% ) , e.  anophelis ( 35.6% ) , e.  brevis ( 32.8% ) and e.  falsenii ( 32.1% ) .
the gene content of strain marseille p2517 ( 2395 ) was smaller than those of e.  falsenii , e. brevis , e. anopheles and e.  meningoseptica ( 3610 , 3633 , 4108 and 3500 respectively ) but larger than those of w.  virosa ( 2192 ) .
nevertheless , the distribution of genes into cogs categories was similar among all compared genomes ( fig . 
otherwise , the agios analysis showed that strain marseille p2517 shares between 1214 and 690 orthologous genes with its closely related species : 1214 , 1200 , 1142 , 702 and 690 with e.  brevis , e. falsenii , w. virosa , e. anopheles and e.  meningoseptica respectively ( table  6 ) .
analysis of the average percentage of nucleotide sequence identity between strain marseille p2517 and other species ranged from 67.26% with e.  meningoseptica and 75.10% with w.  virosa ( table  6 ) .
similar results were also observed for analysis of digital dna - dna hybridization ( table  7 ) .
phenotypic , phylogenetic analyses and genomic results enable us to propose that strain marseille p2517 may be the representative of a novel genus , vaginella , with vaginella massiliensis as the type strain .
the organism is an obligate aerobic , gram - negative and rod - shaped bacilli .
it has negative catalase activity , and nitrate not reduced , with no urease production and positive oxidase activity .
massiliensis , from massilia , the latin name of marseille , france , where the organism was first grown , identified , and characterized ) .
the organism is obligate aerobic , nonmotile , nonsporulating and mesophilic , with optimal growth at 37c .
vaginella cells are gram negative and rod shaped , with a mean diameter of 0.61  m and a length of 1.35  m ; it is oxidase positive and catalase negative ; the major fatty acid is 13-methyl - tetradecanoic acid ( 60% ) . on columbia
agar , colonies are yellow , opaque , circular , smooth and approximately 1.85  mm in diameter . nitrate reduction , urease and indole formation are negative
cells are susceptible to ceftriaxone , ciprofloxacin , clindamycin , colistin , doxycycline , erythromycin , fosfomycin , oxacillin , penicillin , rifampicin , teicoplasmin , trimethoprim / sulfamethoxazole , vancomycin and imipenem but are resistant to gentamicin and metronidazole .
the genome of vaginella massiliensis is 2  434  475  bp long and exhibits 38.16% g+c content .
its 16s rrna gene sequence and that of draft genome are both deposited in embl / ebi under accession numbers lt223570 and flmr00000000 , respectively .
the type strain marseille p2517 (= dsm 102346  =  csur p2517 ) was isolated from the vaginal swab of a healthy frenchwoman .
 an obligate aerobic , gram - negative , nonmotile and nonsporulating rod designated marseille p2517 was isolated from the vaginal flora . 
 we describe its features , annotate the genome and compare it to the closest species . 
 the 16s rrna analysis shows 93.03% sequence similarity with weeksella virosa , the phylogenetically closest species . 
 its genome is 2  434  475  bp long and presents 38.16% g+c . on the basis of these data , 
 it can be considered as a new genus in the flavobacteriaceae family , for which we proposed the name vaginella massiliensis gen . 
 nov . , sp . nov . 
 the type strain is marseille p2517 t . 
Introduction
Materials and Methods
Results
Conclusion