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Genome analysis of Listeria ivanovii strain G770 that caused a deadly aortic prosthesis infection

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
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 [1]. The second pathogenic species is L. ivanovii, which mostly responsible for abortions, stillbirths and neonatal septicaemias in sheep and cattle [1]. 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). The eighth case was not detailed [3].
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.

Patient and Methods
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 the patient had developed fatigue, fever, chills and concomitant back pain. 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. CP009577). 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. However, his condition deteriorated, and the patient died on 23 January.

Genome sequencing
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/μL. Genomic sequencing was performed using the paired-end strategy. 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) completed the tag adapters and introduced dual-index barcodes. 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 pairedend reads was obtained for this project, with a 871K/mm 2 cluster density with a cluster passing quality control filters of 80.5%. The reads were trimmed and filtered according to the read qualities.

Bioinformatic analysis
The reads obtained after sequencing were assembled using the A5 assembler [9]. Then a finishing step was performed with the Mauve aligner software and CLC bioserver [10]. After assembly and finishing, the size of the genome was 2.9 Mb. 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 [11] and Clusters of Orthologous Groups (COGs) database using BLASTP. tRNAs and rRNAs were detected using tRNAscan-SE v.1.21 [12] and RNAmmer 1.2 [13], 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 [14] using the neighbour-joining method and MEGA 6 software [15]. 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 [16].

Nucleotide sequence accession number
The genome sequence from L. ivanovii strain G770 was deposited in GenBank under accession number CVPH01 000001-CVPH01000017.

Results
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. londoniensis strains (Fig. 1). 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. The G770 strain exhibited GGDC values of 99.3% with other L. ivanovii subsp. ivanovii strains and 51.2 to 51.7% with L. ivanovii subsp. londoniensis strains (Supplementary Table S1).
As detailed in Table 2, strain G770 exhibited much less differences with strains from L. ivanovii subsp. ivanovii than with those from subsp. londoniensis. Of the G770-specific genes, 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 G770-specific 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. S1).
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). These genes were also present in all compared L. ivanovii genomes.
The genome of strain G770 harboured one CRISPR region ( Supplementary Fig. 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 [17].

Discussion
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, the LIPI-1 cluster is the major pathogenesis factor. All Listeria species lacking this gene cluster are not pathogenic [18]. In L. 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 Heat repeat-containing pbs lyase (C) Hypothetical protein (not in COGs) Hypothetical protein (not in COGs) Hypothetical protein (not in COGs) Multidrug ABC transporter ATP-binding protein (V) CRISPR-associated protein Cst1 (not in COGs) 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 [19]. Studies have shown that mutations in the prfA gene inhibit bacterial cell-to-cell spread and reduce bacterial virulence in L. monocytogenes [20]. Mutations in different parts of ActA, notably the N-terminal region, cause several unusual motility and actin polymerization phenotypes in L. monocytogenes [21]. 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. ivanovii (Supplementary Table S3). The differences in ActA domains are displayed in Supplementary Fig. S1. However, the ActA domains involved in virulence are conserved in all strains [21]. 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 [22]. 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 [23]. Again, we found no difference in SSI-1 content among studied strains. Finally, we detected CRISPRs in strain G770 ( Supplementary Fig. S2). The presence of CRISPR regions confers to Listeria species an adaptive immunity that protects them against invading bacteriophages and plasmids [24]. However, no relationship between the number of CRISPRs and virulence has been reported for these bacteria.
In addition, strain G770 exhibited 21 specific genes ( Table 3). 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 [22]. 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 restrictionmodification system is found in L. ivanovii. Restrictionmodification systems effectively allow discrimination of self and nonself in bacteria [25]. These systems protect against phages, plasmids and transposons [25]. 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 [25]. One study showed that bacteria that have acquired a competent restriction-modification system acquire new properties especially during colonization of new habitats [8]. Therefore, we assume that the type I restriction-modification system identified in strain G770 may have conferred to it an increased virulence.
Strain G770 also exhibited a vanZ gene. VanZ is an accessory protein that is part of the Tn1546 transposon [26]. The presence of this transposon confers resistance to glycopeptides in Enterococcus faecium [26]. In the absence of the other components of the Tn1546 transposon, VanZ confers low-level resistance to teicoplanin (not to vancomycin) [27]. However, no case of resistance to vancomycin has been reported for strains of Listeria spp. originating from humans, food or the environment, and strain G770 was not resistant to glycopeptides [28].
Overall, the genomic analysis of L. ivanovii subsp. 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.