the genus listeria is composed of 17 species , including two that are recognized pathogens . of these
, l.  monocytogenes is considered as the only human pathogen causing listeriosis , a highly lethal opportunistic infection caused by ingestion of contaminated food .
the second pathogenic species is l.  ivanovii , which mostly responsible for abortions , stillbirths and neonatal septicaemias in sheep and cattle .
however , rare cases of human infection by this species have been published . to date , eight human l.  ivanovii infections have been reported , including four cases of bacteraemia and one case each of mesenteric adenitis , uterine discharge and stillbirth ( table  1 ) .
here we report a rare case of l.  ivanovii vascular infection in a 78-year - old man .
we applied real - time genomics to compare the genetic content of this strain to those of other l.  ivanovii strains available in public databases .
on 29 november 2014 a 78-year - old man was hospitalized in timone hospital , marseille , france , for suspected infectious endocarditis . the patient had had a mechanical bentall prosthesis inserted in 2010 as well as a mitral valve regurgitation repaired in 1998 .
he also had a dissection of the descending thoracic aorta . over the past 10 days
transesophageal echocardiography revealed moderate leakages of the mitral and mechanical aortic valves but no vegetation .
however , because endocarditis was suspected , empirical intravenous amoxicillin ( 12  g / d ) and gentamicin ( 3  mg
/ kg / d ) was initiated after blood was collected for culture . on 4 december , as a result of a persistent inflammatory syndrome ( c - reactive protein level 264 
mg / l ) , a new aortic paraprosthetic leakage was detected by transesophageal echocardiography .
a thoracic computed tomographic scan revealed increased size of the previously known descending aorta dissection .
blood cultures and systematic serology assays ( aspergillus sp . , bartonella sp . , brucella sp . , legionella pneumophila , mycoplasma pneumoniae , q fever ) were negative .
antibiotics were changed to intravenous imipenem ( 3  g / d ) and vancomycin ( 2  g / d ) . on 24
december liposomal amphotericin b ( 3  mg / kg / d ) was added to treat a persistent biological inflammatory syndrome . on 30 december the patient underwent a right posterolateral thoracotomy for resection of a mediastinal abscess .
culture of peroperative specimens were positive for a listeria isolate that was identified as l.  ivanovii using 16s rrna sequencing ( 99.9% identity with genbank accession no .
the antibiotic therapy was changed again to intravenous amoxicillin ( 12  g / d ) and gentamicin ( 3  mg / kg / d ) . however , on 8 january 2015 the patient developed respiratory distress and was transferred to the intensive care unit , where atrial fibrillation and pleural effusion were diagnosed . on 14 january
the patient underwent pleural drainage , right axillofemoral bypass , reimplantation of the subclavian artery on the common carotid artery , removal of the thoracic endoprosthesis and closure of the aortic stump .
listeria ivanovii strain g770 was deposited in the csur collection ( wdcm 875 ) under reference p1995 .
genomic dna of listeria ivanovii was extracted using an ez - one automate ( qiagen , hilden , germany ) .
genomic dna was quantified by a qubit assay with the high sensitivity kit ( life technologies , carlsbad , ca , usa ) to 10.8  ng/l .
genomic dna ( 1  ng ) was used to prepare the paired - end library with the nextera xt dna sample prep kit ( illumina , san diego , ca , usa ) . the tagmentation step fragmented and tagged the dna . then limited - cycle pcr amplification ( 12 cycles )
after purification on ampure xp beads ( beckman coulter , fullerton , ca , usa ) , the library was sequenced on the miseq sequencer ( illumina ) in a single 39-hour run in 2    250  bp .
a total of 455  380 paired - end reads was obtained for this project , with a 871k / mm cluster density with a cluster passing quality control filters of 80.5% .
the reads obtained after sequencing were assembled using the a5 assembler . then a finishing step was performed with the mauve aligner software and clc bioserver .
open reading frames ( orfs ) were predicted by prodigal software ( http://prodigal.ornl.gov/ ) with default parameters .
functional annotation was done by comparison of orf sequences to the genbank   and clusters of orthologous groups ( cogs ) database using blastp .
trnas and rrnas were detected using trnascan - se v.1.21   and rnammer 1.2 , respectively .
the absence of plasmids was verified both by searching the gene annotation for any plasmid - related gene and by mapping all contigs against previously published listeria plasmid sequences .
to identify the most closely related l.  ivanovii genomes , we performed a phylogenetic analysis ( fig . 
1 ) by comparing the sequences of the hlya gene   using the neighbour - joining method and mega 6 software .
subsequently we compared the genomic content of strain g770 and those of the most closely related l.  ivanovii strains available in genbank .
the genome sequence similarity between studied strains was evaluated with ggdc software ( http://ggdc.dsmz.de ) .
the analysis of virulence factors was performed by searching the genes already identified as responsible for virulence in l.  ivanovii ( six genes of the lipi-1 cluster , internalin genes , genes of stress survival islet 1 ( ss1 ) ) and by screening every gene that was specific to strain g770 for a putative role in virulence .
the crisprfinder algorithm was used to identify putative crispr ( clustered regularly interspaced short palindromic repeat ) loci . the genome sequence from l. 
the draft genome sequence of l.  ivanovii strain g770 consisted of 17 scaffolds after assembly and finishing . no plasmid was detected .
the chromosome size , g+c content and number of genes were 2  965  602 bases , 37.10% and 2946 genes , respectively . among these genes , 2850 were protein - coding genes and 96 were rnas ( 21 rrnas , 74 trnas , one tmrna ) .
a total of 2433 genes were assigned to cogs ( 82 , 25% ) . of these ,
2393 genes were assigned a putative function ( 81.2% ) , and 553 ( 18.8% ) were annotated as hypothetical proteins .
phylogenetically , two lineages were identified among l.  ivanovii strains , including one that was made of l.  ivanovii subsp .
ivanovii strains , including strain g770 , and a second made of l.  ivanovii subsp .
the percentages of nucleotide sequence similarity between strain g770 and l.  ivanovii strains for the hlya gene ranged from 94.35% with the two strains of subspecies londoniensis to 99.87% with the other three strains of subspecies ivanovii . for 16s rrna , the percentages of nucleotide similarity were 99.87 and 100% between strain g770 and the strains of the subspecies londoniensis and ivanovii , respectively .
londoniensis strains ( supplementary table  s1 ) . as detailed in table  2 ,
21 were absent from all other compared genomes l.  ivanovii strains ( table  3 ) .
in addition , strain g770 had 12 , 11 and 13 missing genes with regard to subspecies londoniensis ( table  2 ) .
supplementary table  s2 lists the missing in strain g770 . among the 21 strain g770specific genes , five encoded proteins similar to type i restriction - modification genes of l.  monocytogenes and one was mostly similar to vanz , a component of the vancomycin - resistant operon in staphylococcus pseudintermedius ( table  3 ) .
strain g770 also had five specific genes encoding metabolic enzymes such as helicases , transferases and acetyl  coenzyme a synthase .
finally , the strain exhibited six genes encoding hypothetical proteins and one encoding a membrane protein ( table  3 ) .
as other l.  ivanovii strains , strain g770 had a complete lipi-1 virulence cluster . however , although the sequences from the five lipi-1 genes prfa , llo , mpl , plcc and plcb of all compared genomes were identical , strain g770 differed from other strains in the acta gene sequence ( supplementary table  s3 ) .
nevertheless , the annotated domains of the acta protein were similar to those of acta proteins from other strains of the subspecies ivanovii ( supplementary fig . 
strain g770 , like all other compared l.  ivanovii strains , exhibited 16 internalin or internalin - like genes including inla , inlb and inlc ( supplementary table  s4 ) .
in addition , we identified in strain g770 five genes related to the ssi-1 ( supplementary table  s5 ) .
s2 ) , whereas the other three subspecies ivanovii strains exhibited three crispr regions each and the subspecies londoniensis strains wslc30151 and wslc30167 had two and three crisprs , respectively .
to date , only eight cases of human infections caused by l.  ivanovii have been reported in the literature . of these ,
two were diagnosed in patients over 60 years , and most occurred in immunocompromised patients ( two pregnant women , and one case each of aids , drug abuse , hepatic lymphoma and immunosuppression ; table  1 ) . in the remaining two cases of human l.  ivanovii infection , no underlying condition was described ( table  1 ) . to our knowledge
, we herein present the first case of l.  ivanovii vascular infection . in an effort to determine whether this unusual presentation was due to an increased virulence of strain g770 , we compared the genome of strain g770 to those of other l.  ivanovii strains . in listeria species ,
ivanovii , the lipi-1 cluster is made of six genes , including a pore - forming toxin ( listeriolysin o ) and two phospholipases ( plcc and plcb ) which cooperate to lyse the membrane of the phagocytic vacuole ; an actin - polymerization surface protein ( acta ) that is responsible for intracellular bacterial motility and spread ; a metalloprotease ( mpl ) that is involved in the maturation of proplcb ; and a transcriptional activator ( prfa ) that controls the expression of lipi-1 genes .
studies have shown that mutations in the prfa gene inhibit bacterial cell - to - cell spread and reduce bacterial virulence in l.  monocytogenes 
 .
mutations in different parts of acta , notably the n - terminal region , cause several unusual motility and actin polymerization phenotypes in l.  monocytogenes 
 .
strain g770 possessed all six genes of the lipi-1 cluster but varied in hly and acta when compared to other strains from l.  ivanovii subsp .
therefore , the observed differences in cluster lipi-1 or acta do not support an increased virulence of strain g770 . among other known virulence factors of listeria species , internalins such inla , inlb and inlc are essential for host invasion and cell - to - cell spread .
however , strain g770 did not exhibit differences in internalin content ( supplementary table  s4 ) compared to other l.  ivanovii strains .
in addition , strain g770 also possessed the stress survival islet 1 ( supplementary table  s5 ) , known in l.  monocytogenes to be linked to tolerance towards acidic , salt , bile and gastric stress .
again , we found no difference in ssi-1 content among studied strains . finally , we detected crisprs in strain g770 ( supplementary fig . 
the presence of crispr regions confers to listeria species an adaptive immunity that protects them against invading bacteriophages and plasmids .
however , no relationship between the number of crisprs and virulence has been reported for these bacteria .
the best matches for 12 of these genes were found in both listeria seeligeri and l.  monocytogenes .
only one specific gene ( scaffold1.1_178 ) was not found in the genus listeria , thus confirming the highly conserved nature of genomes in this genus . of these additional genes ,
six may have roles in virulence or resistance of strain g770 , including five genes similar to a type i restriction - modification system of listeria monocytogenes .
this is the first time that a type i restriction - modification system is found in l.  ivanovii .
in addition , type i restriction - modification systems play roles in various cellular processes including host defence and virulence , and they even control the speed of evolution of the organism .
one study showed that bacteria that have acquired a competent restriction - modification system acquire new properties especially during colonization of new habitats .
therefore , we assume that the type i restriction - modification system identified in strain g770 may have conferred to it an increased virulence .
the presence of this transposon confers resistance to glycopeptides in enterococcus faecium 
 . in the absence of the other components of the tn1546 transposon
however , no case of resistance to vancomycin has been reported for strains of listeria spp . originating from humans , food or
ivanovii strain g770 , which caused a deadly aortic infection , enabled identification of specific characteristics among other l.  ivanovii strains , including a type i restriction - modification system that may have conferred to it an increased virulence .
 we sequenced the genome of listeria ivanovii strain g770 , which caused a deadly infection of the thoracic aortic prosthesis of a 78-year - old man . 
 the 2.9 mb genome exhibited 21 specific genes among l.  ivanovii strains , including five genes encoding a type i restriction modification system and one glycopeptide resistance gene . 
Introduction
Patient and Methods
Results
Discussion