the genus streptococcus contains 113 recognized species and 22 subspecies ( http://www.bacterio.net/streptococcus.html ) as of 5 february 2016 . these species are gram - positive cocci , chain - forming , facultative anaerobes and catalase negative .
previous studies have reported that a large number of streptococcus species colonize the oral cavities of humans and animals . among oral streptococci isolated from animals , we note : streptococcus ursoris isolated from the oral cavities of bears , streptococcus orisratti isolated from the surface of the lower molars of sprague - dawley rats , streptococcus oriloxodontae isolated from the oral cavities of elephants , and streptococcus mutans isolated from dental plaque .
recently , with next - generation sequencing technology able to sequence whole genomes in a short time , and mass spectrometric analysis of bacteria , we have had easy access to genetic and proteomic information .
therefore , we propose a polyphasic approach combining genomic properties in combination with matrix - assisted laser - desorption / ionization time - of - flight ( maldi - tof ) spectra and phenotypic characteristics to describe new bacterial species .
the strain ff10 (= csur p1489  =  dsm 100884 ) was isolated from the oral cavity of the nile monitor ( varanus niloticus ) in dakar , senegal . here , we present a summary classification and a set of features for streptococcus varani sp .
strain ff10 (= csur p1489  =  dsm 100884 ) , including the description of its complete genome and annotation .
in 2014 , a sample was collected from the oral cavity of a lizard reptile named 
nile monitor ( varanus niloticus ) in dakar , senegal , and stored at 80c . in october 2014 , the strain ff10 ( table  1 ) was isolated from this sample by cultivation on 5% sheep blood - enriched columbia agar ( biomrieux , marcy l'etoile , france ) at 37c with 5% co2 . to identify the strain , maldi - tof ms protein analysis was performed as previously described   using a microflex spectrometer ( bruker daltonics , leipzig , germany ) .
the 12 ff10 spectra 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 . from the resulting scores ,
the tested species may or may not be identified compared with the instrument 's database ; a score  2 with a validly published species enables identification at the species level ; a score  1.7 and  <  2 allows identification at the genus level ; and a score < 1.7 does not enable any identification . for strain ff10 , the scores obtained were lower than 1.306 , suggesting that our strain was not a member of any known species . the reference mass spectrum of s.  varani strain ff10 was added to our database ( fig .  1 ) . a gel view comparing the spectrum of strain ff10 with those of other streptococcaceae species
the bacterium was identified using 16s rrna pcr coupled with sequencing , as previously described .
strain ff10 exhibited 96% 16s rrna sequence similarity with streptococcus minor strain dsm 17118 ( genbank accession number : ay232832 ) , the phylogenetically closest bacterial species with standing in nomenclature - validated streptococcus species ( fig . 
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 .
different growth temperatures ( 25c , 30c , 37c , 45c 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 .
strain growth was observed under anaerobic and microaerophilic conditions but optimal growth was observed under aerobic conditions .
colonies were translucent and yellow with a regular surface , haemolytic on 5% sheep blood - enriched columbia agar ( biomrieux ) , and approximately 1  mm in diameter .
cells were gram - positive cocci , unable to form spores ( fig .  4 ) , and with mean diameter of 0.6  m ( range 0.40.8  m ) and mean length of 1.3  m ( range 0.71.9  m ) ( fig .  5 ) .
this bacterium , ff10 , exhibits neither catalase nor oxidase activities . using an api zym strip ( biomrieux )
, positive reactions were observed for alkaline phosphatase , esterase , esterase - lipase , lipase , leucine arylamidase , acid phosphatase , -glucosidase , -galactosidase , -mannosidase , and -fucosidase .
negative reactions were noted for cystine arylamidase , valine arylamidase , trypsin , -chymotrypsin , -glucosidase , -galactosidase , -glucuronidase , naphthol - as - bi - phosphohydrolase , and n - acetyl--glucosaminidase . using an api 20 ne strip ( biomrieux ) ,
positive reactions were observed only for the esculin hydrolysis test whereas negative reactions were observed for nitrate reduction , urease , indole production , arginine dihydrolase , glucose fermentation , arabinose , mannose , mannitol , n - acetyl - glucosamine , maltose , gluconate , caprate , adipate , malate , citrate , phenyl - acetate assimilation , and gelatine hydrolysis .
using api 50 ch strip ( biomrieux ) , negative reactions were observed for the 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 , d - sorbitol , methyl-d - xylopyranoside , methyl-d - glucopyranoside , n - acetylglucosamine , amygdalin , arbutin , salicin , d - cellobiose , d - maltose , d - lactose , d - mellibiose , d - saccharose , d - trehalose , inulin , d - melezitose , d - raffinose , amidon , glycogen , xylitol , gentiobiose , d - turanose , d - lyxose , d - tagatose , d - fucose , l - fucose , d - arabitol , l - arabitol , potassium gluconate , potassium 2-ketogluconate and potassium-5-ketogluconate .
streptococcus varani strain ff10 was susceptible to penicillin , amoxicillin , amoxicillin / clavulanic acid , ticarcillin , ceftriaxone , cefalotin , imipenem , gentamicin , kanamycin , trimethoprim / sulfamethoxazole , erythromycin , doxycycline , metronidazole , vancomycin and rifampicin , but resistant to ciprofloxacin , nitrofurantoin and colistin .
the differential phenotypic characteristics with other streptococcus species ( s.  minor 
 , streptococcus plurextorum 
 , s.  ursoris 
  and s.  orisratti 
 ) are summarized in table  2 .
genomic dna ( gdna ) of s.  varani ff10 was sequenced on the miseq technology ( illumina inc . , san diego , ca , usa ) using the mate - pair strategy .
the gdna was bar - coded in order to be mixed with 11 other projects with the nextera mate pair sample prep kit ( illumina ) .
the gdna was quantified by a qubit assay with the high sensitivity kit ( thermo fisher scientific , waltham , ma , usa ) to 62.4  mg / l .
the mate - pair library was prepared with 1.5  g of genomic dna 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 inc . ,
the dna fragments ranged from 1.5  kb up to 11  kb with optimal size of 5.8  kb . no size selection was performed and 591  ng of tagmented fragments were circularized .
the circularized dna was mechanically sheared to small fragments , optimally at 697  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 inc . ) and the final concentration library was measured at 75.52  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 39-h run in a 2    251-bp .
total information of 5.9 gb was obtained from a 624  k / mm cluster density with a cluster passing quality control filters of 96.33% ( 12  040  000 clusters ) . within this run ,
open reading frames ( orfs ) were predicted using prodigal ( http://prodigal.ornl.gov/ ) with default parameters . however , the predicted orfs were excluded if they spanned a sequencing gap region .
the predicted bacterial protein sequences were searched against genbank   and clusters of orthologous groups ( cog ) databases using blastp .
the trnas and rrnas were predicted using the trnascan - se   and rnammer   tools , respectively .
signal peptides and numbers 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 > 80 amino acids . if alignment lengths were < 80 amino acids , we used an e - value of 1e-05 .
artemis 
  and dna plotter 
  were used for data management and visualization of genomic features , respectively .
the mauve alignment tool ( version 2.3.1 ) was used for multiple genomic sequence alignment .
the mean level of nucleotide sequence similarity at the genome level between s.  varani and other bacteria ( s.  orisratti ( arcg01000001.1 ) , s.  tigurinus ( aoru01000001.1 ) , streptococcus parasanguinis ( cp002843.1 ) , streptococcus agalactiae ( aeqq01000001.1 ) , s.  plurextorum ( auio01000001.1 ) , s.  minor ( aqyb01000001.1 ) , streptococcus oralis ( admv01000001.1 ) , streptococcus pyogenes ( ae004092.2 ) , streptococcus vestibularis ( aevi01000001.1 ) , streptococcus peroris ( aevf01000001.1 ) and lactococcus lactis ( nc_002662.1 ) ) was estimated using the average genomic identity of gene sequences ( agios ) home - made software .
overall , this software combines the functionality of other software programs : proteinortho 
  ( detects orthologous proteins between genomes compared two by two , then retrieves the corresponding genes ) and the needleman 
wunsch global alignment algorithm ( determines the mean percentage of nucleotide sequence identity among orthologous orfs ) .
the genome is 2  460  376  bp long with 40.4% gc content ( table  3 ) .
2471 were protein - coding genes , and 45 were rnas ( one gene is 5s rrna , one gene is 16s rrna , one gene is 23s rrna , 42 genes are trna genes ) .
the remaining genes were annotated as hypothetical proteins ( 293 genes    11.64% ) .
the draft genome of s.  varani ( 2.46 mb ) is larger than those of s.  agalactiae ( 2.11 mb ) , s.  minor ( 1.93 mb ) , s.  oralis ( 1.90 mb ) , s.  orisratti ( 2.41 mb ) , s.  parasanguinis ( 2.15 mb ) , s.  peroris ( 1.63 mb ) , s.  plurextorum ( 2.10 mb ) , s.  pyogenes ( 1.85 mb ) , s.  tigurinus ( 2.18 mb ) , s.  vestibularis ( 1.84 mb ) .
the g+c content of s.  varani ( 40.40% ) is lower than those of s.  minor ( 41.10% ) , s.  oralis ( 44.40% ) , s.  parasanguinis ( 41.70% ) , s.  plurextorum ( 41.10 ) but higher than those of s.  agalactiae ( 35.30% ) , s.  orisratti ( 38.50% ) , s.  peroris ( 39.10% ) , s.  pyogenes ( 38.50% ) , s.  tigurinus ( 40.30% ) and s.  vestibularis ( 39.60% ) .
similarly , the protein - coding genes of s.  varani ( 2471 ) are larger than those of s.  agalactiae , s.  minor , s.  oralis , s.  orisratti , s.  parasanguinis , s.  peroris , s.  plurextorum , s.  pyogenes , s.  tigurinus and s.  vestibularis ( 2139 , 1903 , 1795 , 2319 , 1982 , 1608 , 2081 , 1776 , 2146 and 1812 , respectively ) .
the gene content of s.  varani ( 2516 ) is larger than that of s.  agalactiae , s.  minor , s.  oralis , s.  orisratti , s.  parasanguinis , s.  peroris , s.  plurextorum , s.  pyogenes , s.  tigurinus and s.  vestibularis ( 2164 , 1948 , 1830 , 2348 , 2055 , 1641 , 2116 , 1854 , 2197 and 1838 , respectively ) .
however , the cog category gene distribution is similar in all genomes compared ( fig . 
7 ) . among species with standing in nomenclature , agios values ranged from 81.54 between s.  peroris and s.  oralis to 65.60 between l.  lactis and s.  varani ( table  5 ) . to evaluate the genomic similarity among the strains
, we determined two parameters , dddh , which exhibits high correlation with ddh , and agios , which was designed to be independent of ddh ( table  6 ) .
on the basis of phenotypic , phylogenetic and genomic analyses , we formally propose the creation of streptococcus varani sp . nov . that contains the strain ff10 .
this bacterial strain has been isolated from the oral cavities of a lizard ( varanus niloticus ) in dakar , senegal .
n. varani , from varanus , the latin name from the lizard which the type strain was isolated ) is a non - motile , non - spore forming , facultative anaerobe , and gram - positive coccus .
the colonies are translucent and yellow with a regular surface , haemolytic on 5% sheep blood - enriched columbia agar , and approximately 1  mm in diameter .
cells have mean diameter of 0.6  m and mean length of 1.3  m .
streptococcus varani strain ff10 exhibits positive reactions for alkaline phosphatase , esterase , esterase - lipase , lipase , leucine arylamidase , acid phosphatase , -glucosidase , -galactosidase , -mannosidase , -fucosidase , and for the esculin hydrolysis test .
streptococcus varani strain ff10 is sensitive to penicillin , amoxicillin , amoxicillin / clavulanic acid , ticarcillin , ceftriaxone , cefalotin , imipenem , gentamicin , kanamycin , trimethoprim / sulfamethoxazole , erythromycin , doxycycline , metronidazole , vancomycin and rifampicin , but resistant to ciprofloxacin , nitrofurantoin and colistin .
the 16s rrna gene sequence and whole - genome shotgun sequence of strain ff10 are deposited in genbank under accession numbers ln810501 and gca_001375655 , respectively .
the type strain ff10 (= csur p1489  =  dsm 100884 ) was isolated from the oral cavity of a lizard ( varanus niloticus ) in dakar , senegal .
pef and ff conceived the study , participated in its design and coordination and helped to draft the manuscript .
 strain ff10 t (= csur p1489  =  dsm 100884 ) was isolated from the oral cavity of a lizard ( varanus niloticus ) in dakar , senegal . 
 here we used a polyphasic study including phenotypic and genomic analyses to describe the strain ff10 t . 
 results support strain ff10 t being a gram - positive coccus , facultative anaerobic bacterium , catalase - negative , non - motile and non - spore forming . 
 the sequenced genome counts 2.46 mb with one chromosome but no plasmid . 
 it exhibits a g+c content of 40.4% and contains 2471 protein - coding and 45 rna genes . on the basis of these data 
 , we propose the creation of streptococcus varani sp . nov . 
Introduction
Classification and Features of the Strain
Genome Description
Genome Annotation and Genome Analysis
Conclusion
Description of 
Authors' Contributions
Transparency Declaration