a stool sample was obtained from a healthy 2-year - old boy living in senegal .
the stool was sent to and frozen in marseille at  80c until laboratory culture isolation .
strain sit12 was isolated in may 2015 by cultivation on marine medium in anaerobic atmosphere after 21 days incubation .
growth of the strain was tested under anaerobic and microaerophilic conditions using genbag anaer and genbag microaer systems respectively ( biomrieux , marcy ltoile , france ) , and in the presence of air , with or without 5% co2 , but growth was achieved only under anaerobic conditions .
m.  massiliensis grew at mesophilic temperatures between 25 and 42c after 48 hours incubation on columbia agar with 5% sheep s blood , chocolate agar and mller - hinton agar .
growth occurred at ph 6 , 6.5 , 7 and 8.5 and exhibited tolerance for nacl until a concentration of 5 
gram staining and electron microscopy of m.  massiliensis were performed using a technaig cryo ( fei company , limeil - brevannes , france ) at an operating voltage of 200 kev .
the cells were of coccus morphology , 0.5  m in diameter and occurred in pairs and short chains ( fig .  2 ) .
the sporulation test was performed using a thermic shock ( 80c during 30 minutes ) , but no free spores were observed and no viable cells could be recovered from sporulating cultures .
api zym and api 50 ch ( biomrieux ) gallery systems were used to perform biochemical assays .
distinguishing results from biochemical tests between m.  massiliensis sit 12 and murdochiella asaccharolitica are listed in table  1 .
the antibiotic susceptibility was studied using antibiotics discs ( i2a , montpellier , france ) .
murdochiella massiliensis sit12 was resistant to fosfomycin , tobramycin , naxidic acid and colistin but was susceptible to gentamicin , ciprofloxacin , trimethoprim 
sulfamethoxazole , teicoplanin , rifampicin , ceftazidime , erythromycin , imipenem , tazocillin and aztreonam . using 16s rrna phylogeny analyses
, we demonstrated that strain sit12 exhibited a 97% 16s rrna sequence identity with murdochiella asaccharolytica ( eu483153 ) and levyella massiliensis ( hm587324 ) species ( fig . 
investigation of the most closely related described species revealed that the novel species strain was member of the phylum firmicutes .
the major fatty acids for strain sit12 are mainly composed of 16 or 18 carbons : 16:0 ( 34% ) , 18:1n9 ( 28% ) , 18:2n6 ( 19% ) and 18:0 ( 12% ) .
moreover , several fatty acids are described with unusual longer chains such as 20:4n6 , 20:5n3 and 22:6n3 ( < 1% ) ( table  2 ) .
the sit12 spectra were imported into maldi biotyper 3.0 software ( bruker daltonics , leipzig , germany ) and analysed by standard pattern matching ( with default parameter settings ) against 7765 spectra of bacteria .
a maximum of 100 peaks were compared with spectra in the database for every spectrum .
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 ms score was obtained for strain sit12 against the bruker database , suggesting that our isolate was not a member of a known species .
genomic dna of m.  massiliensis was sequenced on the miseq technology ( illumina , san diego , ca , usa ) with the mate - pair strategy .
automated cluster generation and sequencing run were performed in a single 39-hour run at a 2   151  bp read length .
total information of 2.9 gb was obtained from a 297k / mm cluster density , with a cluster passing quality control filters of 97% ( 5 808 000 passing filter paired reads ) . within this run ,
the 440 495 paired reads were trimmed , then assembled in two scaffolds using the spades assembler .
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 trnascanse tool   was used to find trna genes , whereas ribosomal rnas were found by using rnammer .
of the 1478 predicted genes , 1426 were protein - coding genes and 52 were rnas ( two genes were 5s rrna , two 16s rrna , one 23s rrna and 47 trna ) . a total of 1002 genes ( 70.27% ) were assigned as putative function ( by cogs or by nr blast ) .
the resistome was analysed with the argannot ( antibiotic resistance gene - annotation ) database .
the exhaustive bacteriocin database available in our laboratories ( bacteriocins from the urmite database ) was performed by collecting all currently available sequences from the databases and from the national center for biotechnology information .
protein sequences from this database allowed putative bacteriocins from human gut microbiota to be identified using blastp methodology .
analysis of presence of polyketide synthases  nonribosomal peptide synthetases ( pks / nrps ) was performed by discriminating the gene with large size using a database realized in our laboratory ; predicted proteins were compared against nonredundant genbank database using blastp and then examined using antismash .
murdochiella massiliensis did not contain bacteriocins or nrps , and the analysis of the resistome showed no resistance genes .
the results indicated the presence of an incomplete phage with 48.2% g+c content and a complete phage with 49.5% g+c content ( fig . 
the draft gene sequence of m.  massiliensis is smaller than those of anaerococcus hydrogenalis , peptoniphilus indolicus , parvimonas micra and helcococcus kunzii ( 1642 , 1889 , 2238 , 1704 and 2096 mb respectively ) , but larger than those of helcococcus sueciensis ( 1574 mb ) .
the g+c content of m.  massiliensis is larger than those of a.  hydrogenalis , p. indolicus , h. sueciensis , p. micra and h. kunzii ( 48.97 , 29.64 , 31.69 , 28.4 , 28.66 and 29.35% respectively ) .
the gene content of m.  massiliensis is smaller than those of a.  hydrogenalis , h. sueciensis , p. micra and h.  kunzii ( 1.426 , 2.069 , 2.269 , 1.445 , 1.678 and 1.882 respectively ) .
on the basis of taxonogenomic analyses , we formally propose the creation of murdochiella massiliensis sp . nov . that contains the strain sit12 .
the murdochiella massiliensis name come from massilia , the ancient roman name for marseille , france , where the type strain was isolated .
the strain was anaerobic , gram positive , non  endospore forming , nonmotile and coccus shaped .
the genome is 1 642 295  bp long , and g+c content is 48.9% .
the 16s rrna gene sequence and whole - genome shotgun sequence of m.  massiliensis strain sit12 are deposited in genbank under accession numbers ln866998 and fizw00000000.1 respectively .
the type strain sit12 (= csur p1987  =  dsm 29078 ) was isolated from the stool of a healthy 2-year - old senegalese boy .
 murdochiella massiliensis strain sit12 (= csur p1987  =  dsm 29078 ) is the type strain of m.  massiliensis sp . 
 nov . 
 this bacterium was isolated from the stool of a healthy 2-year - old senegalese boy . 
 m.  massiliensis is an anaerobic , gram - positive coccus . 
 the genome size of m.  massiliensis strain sit12 is 1 642 295  bp with 48.9% g+c content and assembled into two scaffolds . 
Organism Information
Genome description
Conclusion
Description of 