MEPHI S Ndongo
email: sokhnandongo@gmail.com

M Beye

G Dubourg

T T Nguyen

C Couderc

D P Fabrizio

P.-E Fournier

D Raoult

E Angelakis

TAXONOGENOMICS: GENOME OF A KNOWN ORGANISM Genome analysis and description of Xanthomonas massiliensis sp. nov., a new species isolated from human faeces

Keywords: Culturomics, genome, human gut microbiota, taxonogenomics, Xanthomonas massiliensis

Xanthomonas massiliensis strain SN6 T is a Gram-negative bacterium which is aerobic, motile and nonsporulating. This new species isolated from human faeces exhibited the characteristic traits of members of this genus, such as yellow pigmentation and viscosity. Here we present the main phenotypic characteristics and the taxonogenomics description of this strain. The genome is 3 690 720 bp long with DNA G + C content of about 70.52%.

Introduction

The first member of the genus Xanthomonas was described by Dowson [START_REF] Dowson | On the systematic position and generic names of the Gram negative bacterial plant pathogens[END_REF], and the genus [START_REF] Dye | Genus 11. Xanthomonas Dowson 1939[END_REF] contains plant-associated bacteria that establish neutral, commensal or pathogenic relationships with plants. Taxonomically, the members of this genus were revised several times because the taxonomy had been previously based on host specificity. Each bacterium isolated from a new host was considered as a new species. Dye and Lelliott [START_REF] Dye | Genus 11. Xanthomonas Dowson 1939[END_REF] reduced the number of species from about 120 to the following five: Xanthomonas campestris, Xanthomonas albilineans, Xanthomonas axonopodis, Xanthomonas fragariae and Xanthomonas ampelina. The others were grouped together as nomenspecies in the X. campestris group (pathovar). In 1995, Vauterin et al. [START_REF] Vauterin | Reclassification of Xanthomonas[END_REF] partially clarified the classification and described 20 species among the three former species, X. axonopodis, X. fragariae and X. albilineans, and 62 pathovars of X. campestris, on the basis of DNA ± DNA hybridization data and biochemical and physiological tests. However, members of the genus Xanthomonas can be differentiated from members of the phylogenetically closest genus Pseudoxanthomonas by the absence of reduction of nitrates to nitrites and the presence of C13:0 iso 3-OH fatty acid [START_REF] Yang | Application of fatty acid methyl esters for the taxonomic analysis of the genus Xanthomonas[END_REF].

Members of the Xanthomonas genus were known exclusively as plant-associated organisms and did not durably colonize other niches [START_REF] Darrasse | Genome sequence of Xanthomonas fuscans subsp. fuscans strain 4834-R reveals that flagellar motility is not a general feature of xanthomonads[END_REF]. However, during the study of the bacterial diversity of the human microbiota by culturomics [START_REF] Lagier | Culture of previously uncultured members of the human gut microbiota by culturomics[END_REF], a strain of Xanthomonas was isolated from the stool sample of an obese French patient. It is the first Xanthomonas isolate identified in humans to date. Here we report the characterization of strain SN6 as a novel species of the genus Xanthomonas, Xanthomonas massiliensis strain SN6 (= CSUR P2129 = DSM 100900), with a description of the complete genomic sequence and its annotation. culturomics in September 2015. The strain was isolated from a 41-year-old obese Frenchwoman hospitalized in September 2012 at the La Timone Hospital in Marseille, France. This study and the assent procedure were validated by the ethics committee of the IFR48 Federative Research Institute Marseille under number 09-022, and we obtained the signed consent of the patient.

After collecting the stool, a portion of a sample was stored at -80°C until use. In June 2015, the stool sample was cultivated as part of an exploration of the human microbiome centred on microaerophilic bacteria the. Part of the frozen aliquot of the specimen (approximately 1 g) was taken out and diluted in 900 μL of phosphate-buffered saline (Life Technologies, Carlsbad, CA, USA) following ten serial dilutions to obtain 1/10. Inoculum (50 μL) was seeded on Columbia agar supplemented with 5% sheep's blood (bioMérieux, Marcy l'Etoile, France) and incubated under microaerophilic conditions (7% O 2 , 5% H 2 , 10% CO 2 , 85% N 2 ) using the generator CampyGen (Thermo Scientific, Villebon-sur-Yvette, France) at 37°C for 48 hours.

Strain identification

After 48 hours of incubation in microaerophilic conditions, pure colonies were isolated on Columbia agar and identified by proteomic analysis using matrix-assisted desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) following the same protocol as previously described by Seng et al. [START_REF] Seng | Ongoing revolution in bacteriology: routine identification of bacteria by matrix-assisted laser desorption ionization time-of-flight mass spectrometry[END_REF] with a Microflex spectrometer (Bruker Daltonics, Leipzig, Germany). All obtained spectra of strain SN6 were imported into the MALDI BioTyper software (version 2.0; Bruker) and analysed by standard pattern matching (with default parameter settings) and compared to those of the BioTyper database and our own collection. Thus, a score of >2 allowed identification at the species level, and a score of <1.7 did not allow any identification.

If the identification of the spectrum from colonies selected and purified several times by subculturing on Columbia agar failed, then the 16S rRNA gene was amplified and complete genome sequencing was carried out as previously described [START_REF] Morel | Complementarity between targeted real-time specific PCR and conventional broad-range 16S rDNA PCR in the syndrome-driven diagnosis of infectious diseases[END_REF].

The nucleotide sequence obtained was corrected using Chromas Pro 1.34 software (Technelysium, Tewantin, Australia) and compared to the nucleotide database using the BLAST similarities web services in the online PubMed National Center for Biotechnology Information (NCBI) database (http://blast. ncbi.nlm.nih.gov.gate1.inist.fr/Blast.cgi). As suggested previously, similarity level thresholds of 98.65% and 95% allowed the definition of a new species or a new genus, respectively [START_REF] Stackebrandt | Taxonomic parameters revisited: tarnished gold standards[END_REF][START_REF] Kim | Towards a taxonomic coherence between average nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes[END_REF].

Growth conditions and morphologic characterization

Strain SN6 was isolated at first under microaerophilic conditions (CampyGen) at 37°C for 48 hours on Columbia medium supplemented with 5% sheep's blood (COS) agar (bioMérieux) and we also tested its growth under aerobic and anaerobic conditions generated by AnaeroGen (bioMérieux). The minimum and maximum growth temperature ranges (28°C, 37°C, 45°C, 55°C) were determined as well as maximum salinity levels (0-5, 50-75, 100 g/L NaCl). The ability of the strain to grow on media with different pH was also tested. The colonies appeared on day 3 after culture on Columbia agar, and their diameter was measured. Cell morphology, Gram staining and motility were observed in fresh colonies using a DM1000 photonic microscope (Leica Microsystems, Nanterre, France) with a 40 × objective lens. Sporulation was tested by thermal shock, which consists of exposing the bacterium to a temperature of 60°C for 20 minutes and then watching its growth after 48 hours. Negative staining was carried out with detection Formvar-coated grids placed on a drop of 40 μL of bacterial suspension (after an overnight fixation in glutaraldehyde 2.5%) and incubated at 37°C for 30 minutes, followed by a 10-second incubation in 1% ammonium molybdate. The grids were dried on blotting paper and finally observed using a Tecnai G20 transmission electron microscope (FEI Company, Limeil-Brévannes, France).

Biochemical characterization

The biochemical properties of strain SN6 were characterized using API ZYM, API 20NE and API 50CH strips, according to the manufacturer's instructions (bioMérieux) for testing of carbon source utilization and enzyme activity. The presence of catalase and oxidase activities was tested by using a BBL Dry-Slide (Becton, Le Pont de Claix, France) according to the manufacturer's instructions. The analysis of cellular fatty acid methyl ester composition was performed by gas chromatography/mass spectrometry (GC/MS). Two samples of approximately 100 mg of bacterial biomass per tube collected from five fresh culture plates were used for the extraction of cellular fatty acid methyl esters with the protocol described by Sasser [START_REF] Sasser | Bacterial identification by gas chromatographic analysis of fatty acids methyl esters (GC-FAME)[END_REF]. GC/MS analyses were carried out as described by Dione et al. [START_REF] Dione | Genome sequence and description of Anaerosalibacter massiliensis sp[END_REF].

Antibiotic susceptibility

The sensitivity to classical antibiotics was tested to determine the antibiogram profile of strain SN6 using the disc diffusion method following the European Committee on Antimicrobial Susceptibility Testing 2016 recommendations (http://www. eucast.org). A suspension of 0.5 McFarland of the species was grown on Mueller-Hinton agar in a petri dish (bioMérieux), and the discs used were provided by i2a (Montpellier, France). The reading of inhibition diameters according to manual measurement by using a ruler was done after 48 hours of incubation at 37°C under aerobic conditions with the Sirscan system (i2a).

Genome sequencing and assembly

A EZ1 DNA tissue kit was used to extract the DNA of strain SN6 on the EZ1 biorobot (Qiagen, Courtaboeuf, France) after pretreatment by lysozyme incubation at 37°C as previously described [START_REF] Lagier | Non contiguous-finished genome sequence and description of Cellulomonas massiliensis sp[END_REF].

Genomic DNA (gDNA) was quantified by a Qubit assay with the high sensitivity kit (Life Technologies) and sequenced on the MiSeq Technology (Illumina, San Diego, CA, USA) with the mate-pair strategy as previously described [START_REF] Lagier | Non contiguous-finished genome sequence and description of Clostridium jeddahense sp[END_REF]. The Nextera Mate sample collection kit (Illumina) was used to mix DNA previously barcoded with 11 other projects. An assembly of six different software packages (Velvet [START_REF] Zerbino | Velvet: algorithms for de novo short read assembly using de Bruijn graphs[END_REF], Spades [START_REF] Bankevich | SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing[END_REF] and Soap Denovo [START_REF] Luo | SOAPdenovo2: an empirically improved memory-efficient short-read de novo assembler[END_REF]), on trimmed (MiSeq and Trimmomatic [START_REF] Bolger | Trimmomatic: a flexible trimmer for Illumina sequence data[END_REF] or untrimmed data (only MiSeq software) was created from a pipeline and allowed to perform genome assembly. GapCloser was used to reduce the gaps of each of the six assemblies performed [START_REF] Luo | SOAPdenovo2: an empirically improved memory-efficient short-read de novo assembler[END_REF]. The contamination with Phage Phix was identified by Blastn against Phage Phix174 DNA sequence and then eliminated. Finally, all scaffolds smaller than 800 bp or with a depth value lower than 25% of the mean depth were removed (identified as possible contaminants). On the basis of different criteria (number of scaffolds, N50, number of N), the best assembly was selected. For strain SN6, Spades gave the best assembly, with a depth coverage of 267.

Genome annotation and comparison

Prodigal allowed to predict open reading frames (ORFs) using default parameters [START_REF] Hyatt | Prodigal: prokaryotic gene recognition and translation initiation site identification[END_REF] and those that were spanning a sequencing gap region (contained N) were excluded. BLASTP with an E value of 1e-03, coverage of 0.7 and 30% identity was used to search the predicted bacterial protein sequences against the Clusters of Orthologous Groups (COGs) database.

If no hit was found, it searched against the NR database using BLASTP (E value of 1e-03, coverage of 0.7 and 30% identity) and an E value of 1e-05 was used if the sequence's length was shorter than 80 aa. The tRNAScanSE [START_REF] Lowe | tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence[END_REF] and RNAmmer [START_REF] Lagesen | RNAmmer: consistent and rapid annotation of ribosomal RNA genes[END_REF] tools were used to find transfer RNA genes and ribosomal RNA genes, respectively. The number of transmembrane helices and the lipoprotein signal peptides were predicted using Phobius [START_REF] Käll | A combined transmembrane topology and signal peptide prediction method[END_REF]. ORFans were identified if all the BLASTP performed did not yield positive results (E value smaller than 1e-05 for ORFs with sequence length inferior to 80 aa or E value smaller than 1e-03 for ORFs with sequence size larger than 80 aa). These different parameter thresholds had already been used in previous works to define ORFans.

The genomes of each species from the 16S RNA tree used in the comparison were automatically retrieved using Xegen software (PhyloPattern), and the NCBI FTP was used to recover the complete genome sequence, proteome sequence and Orfeome [START_REF] Gouret | PhyloPattern: regular expressions to identify complex patterns in phylogenetic trees[END_REF]. When the complete genome of one specific strain was not available, we used the complete genome of the same species. All proteomes were analysed with proteinOrtho [START_REF] Lechner | Proteinortho: detection of (co-)orthologs in large-scale analysis[END_REF]. For each couple of genomes, a similarity score defined by the mean value of nucleotide similarity between all couples of orthologous genes was computed by average genomic identity of orthologous gene sequences (AGIOS) software. The AGIOS values were calculated from the genome of Xanthomonas and Stenotrophomonas genera. The genome of Xanthomonas massiliensis strain SN6 (FCOY00000000) was compared to that of Xanthomonas vesicatoria ATCC_35937_LMG_911T (AEQV00000000), Xanthomonas gardneri DSM_19127 (AEQX00000000), Xanthomonas axonopodis LMG_538T (JPYE00000000), Xanthomonas sacchari LMG_471T(CP010409), Xanthomonas campestris ATCC_33913 (AE008922), Stenotrophomonas acidaminiphila AMX19 (CP012900) and Stenotrophomonas maltophilia IAM_12423(CP008838). Genome-to-Genome Distance Calculator (GGDC) analysis was also performed using the GGDC web server as previously reported by Meier-Kolthoff et al. [START_REF] Meier-Kolthoff | Genome sequence-based species delimitation with confidence intervals and improved distance functions[END_REF].

Results

Strain identification and phylogenetic analyses

The first colonies of strain SN6 were isolated after direct inoculation of the stool sample on Columbia agar plates under microaerobic condition at 37°C for 48 hours. The bacterial spectrum obtained by MALDI-TOF MS did not match against the Bruker or our own database. Thus, it was incremented in our database (Fig. 1). The 16S rRNA sequenced showed that strain SN6 was phylogenetically clustered in the genus of Xanthomonas and presented a sequence identity of 98.08% with Xanthomonas campestris strain ATCC33913 (NR_074936), the phylogenetically closest species with standing in nomenclature [START_REF] Vauterin | Reclassification of Xanthomonas[END_REF] (Fig. 2), which putatively classifies strain SN6 as a member of a new species within the genus Xanthomonas in the phylum Proteobacteria. Thus, we propose the creation of the new species Xanthomonas massiliensis (Table 1). The 16S rRNA gene of Xanthomonas massiliensis strain SN6 is 1508 bp long and was deposited with the accession number AA00102 in the 16S IHU bank and LN881611 in GenBank. A comparison between the spectrum of the strain's protein level and that of the closely related species on the 16S rRNA tree and present in our database was performed in a gel view (Fig. 3).

Phenotypic characteristics

Xanthomonas massiliensis strain SN6 grows between 28°C and 42°C; optimal growth was observed under aerobic conditions on COS at 37°C and pH7 after 48 hours of incubation. A smaller growth rate was observed under microaerobic NMNI atmosphere after 48 hours of incubation, and no growth was observed under anaerobic conditions. Also, a smaller growth rate was observed at pH 7 and 8.5, and no growth was observed above 5% salinity. Colonies of the strain were yellowish, circular, viscous and smooth, nonhaemolytic and approximately 1 to 2 mm in diameter on Columbia agar under aerobic conditions after 48 hours. The colonies became khaki green after 4 days of incubation. The yellow pigments, which are mono-or dibromo arylpolyenes called xanthomonadins [START_REF] Andrewes | Structure of xanthomonadin I, a novel dibrominated arylpolyene pigment produced by the bacterium Xanthomonas juglandis[END_REF], are characteristic of this genus. Bacterial cells were Gram negative, rod shaped, motile and non-spore forming. Observed under electron microscopy, they occur singly or in chains and measure 0.6 μm in diameter and 1.8 to 2.0 μm in length (Fig. 4).

Biochemical analysis

The catalase activity test was positive, but the oxidase test was negative. Using API ZYM strip for the research of enzymatic activities of strain SN6, positive reactions were detected for alkaline phosphatase, esterase lipase (C8), leucine arylamidase, valine arylamidase, trypsin, acid phosphatase, naphthol-AS-BIphosphohydrolase, β-galactosidase, β-glucosidase, N-acetylβ-glucosaminidase and α-mannosidase. Esterase (C4), lipase (C14), cystine arylamidase, α-chymotrypsin, α-galactosidase, β-glucuronidase, α-glucosidase and α-fucosidase activities did not show any sign of activity. The study of carbohydrate and its derivatives metabolism using API 50CH showed no fermentation of substrates, except for esculin. API 20NE strip revealed that there was neither nitrate reduction nor indole production, and urease was also negative. The reduction of nitrate to nitrite also makes it possible to differentiate the genus Xanthomonas from the genus Pseudoxanthomonas. On the same strip, positive reactions were observed for gelatin hydrolysis, malate and N-acetylglucosamine, and it also allowed to confirm the assimilation of esculin and β-galactosidase. A panel of 15 antibiotics was tested, and strain SN6 was sensitive to vancomycin, ceftriaxone, ciprofloxacin, clindamycin, doxycycline, erythromycin, gentamicin, penicillin, rifampicin, colistin, fosfomycin and trimethoprim/sulfamethoxazole but resistant to oxacillin, teicoplanin and metronidazole. Table 2 compares the phenotypic characteristics of strain SN6 with those of closely related species.

According to the cellular fatty acid methyl ester analysis, the most abundant fatty acid by far was branched 13-methyl-tetradecanoic acid (58%). Many other branched structures were also described for this strain. Several specific 3-OH structures were detected. Minor amounts of unsaturated and saturated fatty acids were also identified. Regarding the differentiation between the species of the genera Pseudoxanthomonas and Xanthomonas, Xanthomonas massiliensis strain SN6 contains up to 4.5 ± 0.2% of 3-hydroxy-11-methyl-dodecanoic acid (C13:0 iso 3-OH) compared to the other species, which have none or only traces (Table 3). Stenotrophomonas rhizophila, Xanthomonas gardneri, Xanthomonas sacchari, Xanthomonas campestris and Xanthomonas axonopodis (3137, 3617, 4927, 4565, 3938, 4228, 4168, 4181 and 3904, respectively), but larger than the genome of Pseudoxanthomonas suwonensis (3132). This comparison of genomes between X. massiliensis and the other genetically closest species is shown in Table 6. In all genomes compared, the distribution of genes into COGs categories is identical (Fig. 6). Among Xanthomonas species with standing in nomenclature, AGIOS values ranged from 64.76% between Xanthomonas campestris pv. campestris and Stenotrophomonas acidaminiphila to 79.65% between Xanthomonas sacchari and Pseudoxanthomonas suwonensis. When comparing Xanthomonas massiliensis sp. nov. to other species, AGIOS values were in the same range, from 66.21% with Xanthomonas vesicatoria to 80.88% with Xanthomonas sacchari (Table 7). Among the species with standing in nomenclature, we found that by using the digital DNA-DNA hybridization (dDDH) with the GGDC software, values ranged from 20.8% between Xanthomonas vesicatoria and Pseudoxanthomonas suwonensis to 32.1% between Xanthomonas gardneri and Xanthomonas axonopodis. When comparing Xanthomonas massiliensis to other species, the dDDH value ranged FIG. 5. Graphical circular map of genome of Xanthomonas massiliensis strain SN6 T . From outside to center: Genes on forward strand coloured by COGs categories (only genes assigned to COGs), genes on reverse strand coloured by COGs categories (only gene assigned to COGs), RNA genes (tRNAs green, rRNAs red), GC content and GC skew. COGs, Clusters of Orthologous Groups database. FIG. 6. Distribution of functional classes of predicted genes according to clusters of orthologous groups of proteins.

NMNI

Conclusion

Phenotypic characteristics as well as phylogenetic and genomic analyses of strain SN6 suggest that it represents a novel species within the Xanthomonas genus, for which the name Xanthomonas massiliensis sp. nov. is proposed. This bacterial strain was isolated from the faecal flora of an obese Frenchwoman, and the description was based on a single isolate.

Description of Xanthomonas massiliensis sp. nov.

Xanthomonas massiliensis (mas.si.li.en'sis, L. fem. adj. massiliensis, 'of Massilia,' the Latin name of Marseille where strain SN6 T was first cultivated).

X. massiliensis is a rod-shaped (0.6 × 1.8-2.0 μm), aerobic and Gram-negative bacterium occurring singly or in chains. Growth was also observed under microaerophilic conditions. Cells are motile with a flagellum and nonsporulating. Fresh colonies were yellow, circular, smooth and viscous with a diameter of 1 to 2 mm on COS. Optimal growth of strain SN6 occurred at 37°C under aerobic atmosphere with a pH of 7 but did not grow at 5% of salinity or under anaerobic conditions. The strain was catalase positive. Tests for nitrate reduction, indole production and urease were negative. API 50CH showed that the only substrate used as a carbon source was esculin. Positive reactions were detected for alkaline phosphatase, esterase lipase (C8), leucine arylamidase, valine arylamidase, trypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, β-galactosidase, β-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase, gelatin hydrolysis, malate and N-acetylglucosamine. The strain was sensitive to ceftriaxone, ciprofloxacin, clindamycin, doxycycline, erythromycin, gentamicin, penicillin, rifampicin, colistin, vancomycin, fosfomycin and trimethoprim/ sulfamethoxazole but resistant to metronidazole, oxacillin and teicoplanin. Predominant fatty acids were 13-methyl-tetradecanoic acid followed by 9-methyl-decanoic acid.

The DNA G + C content is about 70.52%. The 16S rRNA gene and genome sequences were deposited in GenBank under accession number LN881611 and FCOY00000000, respectively. The type strain is Xanthomonas massiliensis strain SN6 T (= CSUR P2129 = DSM 100900) and was isolated from human faeces.

FIG. 1 .

 1 FIG. 1. Reference mass spectrum from Xanthomonas massiliensis strain SN6 T .

FIG. 3 .

 3 FIG. 3. Gel view comparing Xanthomonas massiliensis strain SN6 T to other close species. Gel view displays raw spectra of strain SN6 T of loaded spectrum files arranged in a pseudo-gel like look. X-axis records m/z value. Left y-axis displays running spectrum number originating from subsequent spectra loading. Peak intensity is expressed by greyscale scheme code. Colour bar and right y-axis indicate relation between colour of peak and its intensity, in arbitrary units. Displayed species are indicated at left.

100

  Bold indicates comparison between strain and itself.DDH, DNA-DNA hybridization; GGDC, Genome-to-Genome Distance Calculator; HSP, high-scoring segment pairs; PS, Pseudoxanthomonas suwonensis; SA, Stenotrophomonas acidaminiphila; SM, Stenotrophomonas maltophilia; SR, Stenotrophomonas rhizophila; XA, Xanthomonas axonopodis; XCC, Xanthomonas campestris pv. Campestris; XG, Xanthomonas gardneri; XM, Xanthomonas massiliensis; XS, Xanthomonas sacchari; XV, Xanthomonas vesicatoria. a Confidence intervals indicate inherent uncertainty in estimating DDH values from intergenomic distances based on models derived from empirical test data sets (which are always limited in size). NMNI Ndongo et al. Xanthomonas massiliensis

TABLE 1 .

 1 Classification and general features of Xanthomonas massiliensis strain SN6 T

	Property	Term
	Current classification	Domain: Bacteria
		Phylum: Proteobacteria
		Class: Gammaproteobacteria
		Order: Xanthomonadales
		Family: Xanthomonadaceae
		Genus: Xanthomonas
		Species: massiliensis Type strain: sn8 T
	Gram stain	Negative
	Cell shape	Rod
	Motility	Motile
	Sporulation	Nonsporulating
	Temperature range	Mesophilic
	Optimum temperature	37°C
	Oxygen requirement	Aerobic/microaerobic
	Salinity	0-5 g/L
	pH	7-8.5
	Optimum pH	7
	Energy source	Chemoorganotrophic
	Pathogenicity	Unknown
	Isolation	Human faeces
	Habitat	Host associated
	Biosafety level	2

TABLE 3 .

 3 Cellular fatty acid composition (%)

	Fatty acid	Name	Mean relative % a
	15:0 iso	13-Methyl-tetradecanoic acid	57.6 ± 0.4
	11:0 iso	9-Methyl-decanoic acid	10.2 ± 0.5
	17:1 iso	15-Methylhexadecenoic acid	4.9 ± 0.2
	13:0 3-OH iso	3-hydroxy-11-methyl-Dodecanoic acid 4.5 ± 0.2
	16:1n7	9-Hexadecenoic acid	3.7 ± 0.2
	17:0 iso	15-methyl-Hexadecanoic acid	3.5 ± 0.1
	12:0 3-OH	3-Hydroxydodecanoic acid	3.3 ± 0.1
	16:0 9,10-methylene 2-hexyl-Cyclopropaneoctanoic acid	2.3 ± 0.2
	15:1 iso	13-Methyltetradecenoic acid	2.2 ± 0.3
	16:0	Hexadecanoic acid	1.7 ± 0.1
	11:0 3-OH iso	3-hydroxy-9-Methyl-decanoic acid	1.4 ± 0.2
	15:0 anteiso	12-methyl-Tetradecanoic acid	1.3 ± 0.1
	16:1n9	7-Hexadecenoic acid	TR
	18:1 iso	16-Methylheptadecenoic acid	TR
	13:0 iso	11-methyl-Dodecanoic acid	TR
	18:1n9	9-Octadecenoic acid	TR
	10:0	Decanoic acid	TR
	17:0 anteiso	14-methyl-Hexadecanoic acid	TR
	14:0	Tetradecanoic acid	TR

a Mean peak area percentage; TR, trace amounts < 1 %.

TABLE 4 .

 4 Nucleotide content and gene count levels of

	genome
		Genome (total)
	Attribute	Value	% of total a
	Size (bp)	3 690 720	100
	G + C content (%)	2 602 093	70.52
	Coding region (bp)	3 265 075	88.46
	Total genes	3196	100
	RNA genes	59	1.84
	Protein-coding genes	3137	100
	Genes with function prediction	2533	80.74
	Genes assigned to COGs	2202	70.19
	Genes with peptide signals	779	24.83
	Genes with transmembrane helices	634	20.21
	Genes associated to virulence	687	21.89
	ORFan genes	104	3.31
	Genes associated with PKS or NRPS	19	0.60
	Genes associated to toxin/antitoxin	99	3.15

COGs, Clusters of Orthologous Groups database; NRPS, nonribosomal peptide synthase; PKS, polyketide synthase. a Total is based on either size of genome in base pairs or total number of protein-coding genes in annotated genome.

TABLE 5 .

 5 Number of genes associated with 25 general COGs

	functional categories
	Code	Value	% of total	Description
	J	199	6.3436403	Translation
	A	1	0.031877592	RNA processing and modification
	K	142	4.5266175	Transcription
	L	93	2.9646158	Replication, recombination and repair
	B	1	0.031877592	Chromatin structure and dynamics
	D	32	1.020083	Cell cycle control, mitosis and meiosis
	Y	0	0	Nuclear structure
	V	83	2.64584	Defense mechanisms
	T	104	3.3152692	Signal transduction mechanisms
	M	158	5.0366592	Cell wall/membrane biogenesis
	N	37	1.1794709	Cell motility
	Z	1	0.031877592	Cytoskeleton
	W	34	1.0838381	Extracellular structures
	U	72	2.2951865	Intracellular trafficking and secretion
	O	119	3.7934332	Posttranslational modification,
				protein turnover, chaperones
	X	37	1.1794709	Mobilome: prophages, transposons
	C	164	5.227925	Energy production and conversion
	G	130	4.144087	Carbohydrate transport and metabolism
	E	193	6.152375	Amino acid transport and metabolism
	F	65	2.0720434	Nucleotide transport and metabolism
	H	103	3.283392	Coenzyme transport and metabolism
	I	146	4.654128	Lipid transport and metabolism
	P	157	5.0047817	Inorganic ion transport and metabolism
	Q	80	2.5502074	Secondary metabolites biosynthesis,
				transport and catabolism
	R	227	7.236213	General function prediction only
	S	150	4.7816386	Function unknown
	-	935	29.805548	Not in COGs
	COGs, Clusters of Orthologous Groups database.

NMNI

TABLE 6 .

 6 Genome comparison of closely related species to Xanthomonas massiliensis strain SN6 T .

	Organism	INSDC	Size (Mbp)	G + C %	Protein-coding genes
	Xanthomonas_ massiliensis	FCOY00000000.1	3.69	70.52	3137
	Xanthomonas_vesicatoria_ATCC_35937_LMG_911_T	AEQV00000000.1	5.53	64.06	4927
	Xanthomonas_gardneri_type_strain__DSM_19127	AEQX00000000.1	5.30	63.53	4228
	Xanthomonas_axonopodis_LMG_538-T	JPYE00000000.1	5.02	64.88	3904
	Xanthomonas_sacchari_LMG_471_T	CP010409.1	4.92	69.04	4168
	Xanthomonas_campestris_pv. _campestris_str. _ATCC_33913_ATCC_33913	AE008922.1	5.07	65.06	4181
	Stenotrophomonas_acidaminiphila_AMX19	CP012900.1	4.13	68.48	3617
	Stenotrophomonas_maltophilia_IAM_12423	CP008838.1	4.93	66.22	4565
	INSDC, International Nucleotide Sequence Database Collaboration.

TABLE 7 .

 7 Number of orthologous proteins shared between genomes (upper right) and AGIOS values obtained (lower left) 21.3% with Stenotrophomonas maltophilia to 23.5% with Xanthomonas sacchari (Table8).

	P. suwonensis	3132	1898	1925	1895	1678	1858	1914	1858	1902	1901
	X. gardneri	66.11	4228	2945	2231	1859	2390	2962	2032	2880	2219
	X. campestris	66.12	72.68	4181	2234	1848	2425	2970	2018	2909	2234
	S. maltophilia	77.64	66.59	65.63	4565	1865	2137	2239	2174	2195	2466
	X. massiliensis	79.82	67.19	66.64	78.60	3137	1788	1835	1778	1834	1854
	X. sacchari	79.65	67.94	67.01	79.13	80.88	4168	2440	1973	2427	2127
	X. vesicatoria	65.61	69.79	68.62	65.75	66.21	66.85	4927	2024	2934	2223
	S. acidaminiphila 69.27	64.47	64.76	69.67	69.57	69.80	67.53	3617	1983	2125
	X. axonopodis	66.01	70.07	63.74	66.33	66.92	67.70	71.02	67.35	3904	2197
	S. rhizophila	66.12	65.42	61.96	68.84	66.81	67.04	66.54	67.77	66.98	3938

P. suwonensis X. gardneri X. campestris S. maltophilia X. massiliensis X. sacchari X. vesicatoria S. acidaminiphila X. axonopodis S. rhizophila Numbers of proteins per genome are indicated in bold. AGIOS, average genomic identity of orthologous gene sequences. Pseudoxanthomonas suwonensis 4M1, Xanthomonas gardneri DSM 19127, Xanthomonas campestris pv. campestris ATCC33913, Stenotrophomonas maltophilia IAM12423, Xanthomonas massiliensis SN6 T , Xanthomonas sacchari LMG471 T , Xanthomonas vesicatoria ATCC35937_LMG911 T , Stenotrophomonas acidaminiphila AMX19, Xanthomonas axonopodis LMG538 T , Stenotrophomonas rhizophila ep10. from

© 2018 Published by Elsevier Ltd, NMNI, 26, 63-72 This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

New Microbes and New Infections, Volume 26 Number C, November 2018 NMNI © 2018 Published by Elsevier Ltd, NMNI, 26, 63-72 This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Acknowledgements

The authors thank the Xegen Company (http://www.xegen.fr/) for automating the genomic annotation process, and M. Lardiere (MEPHI) for English-language editorial review. This study was supported by the Fondation Méditerranée Infection and the French government under the 'Investissements d'avenir' with the reference Mediterranée Infection 10-IAHU-3.

Genome properties

The genome of Xanthomonas massiliensis strain SN6 is 3 690 720 bp long with 70.52% GC content (Table 4, Fig. 5). It is composed of four scaffolds (composed of seven contigs). Of the 3196 predicted genes, 3137 were protein-coding genes and 59 were RNAs (two were 5S rRNA, two were 16S rRNA, two were 23S rRNA, 53 were tRNA genes). A total of 2533 genes (80.75%) were assigned as putative function (by COGs or by NR BLAST). A total of 104 genes were identified as ORFans (3.32%). The remaining genes were annotated as hypothetical proteins (350 genes, 11.16%). The distribution of genes into the different COGs functional categories is provided in Table 5.

Genome comparison

Conflict of Interest

None declared.


Jean-Luc Clément
email: clement.jluc@wanadoo.fr

Frederico Solla

Antoine Tran

Christian Morin

Walid Lakhal

Jérôme Sales De Gauzy

Julien Leroux

Jean-Marie Gennari

François-Henri Parent

Gaby Kreichati

J.-L Clément

J.-M Gennari

F.-H Parent

S Wolf

I Obeid

Five-year outcomes of the First Distal Uninstrumented Vertebra after posterior fusion for Adolescent Idiopathic Scoliosis Lenke 1 or 2

Keywords:

  L'archive ouverte pluridisciplinaire

Background: Tilt of the First Distal Uninstrumented Vertebra (FDUV) reflects changes in the main curve and compensatory lumbar curve after posterior fusion to treat thoracic Adolescent Idiopathic Scoliosis (AIS). Hypothesis: FDUV tilt 5 years or more post-fusion depends chiefly on reduction of the main curve and on other factors such as selection of the last instrumented vertebra. Material and method: A multicenter retrospective cohort of 182 patients with Lenke 1 or 2 AIS treated with posterior instrumentation and followed up for a mean of 8 years and a minimum of 5 years was studied. The patients were divided into two groups based on whether tilt of the upper endplate of the FDUV was ≤ 5 • or > 5 • at last follow-up. Variables associated with tilt were identified by multiple logistic regression. Results: Six variables were significantly associated with FDUVtilt: percentage of correction at last follow-up, correction loss, lumbar modifier B, number of instrumented vertebrae, inclusion within the instrumentation of the distal neutral vertebra, and inclusion within the instrumentation of the lowest vertebra intersected by the central sacral vertical line. Discussion and conclusion: The main variables associated with FDUVtilt ≤ 5 • were a final correction percentage ≥ 60% and absence of correction loss between the postoperative period and last follow-up. Given the stable reduction provided by contemporary instrumentations, we recommend selective thoracic fusion of Lenke 1 or 2 AIS with lumbar modifiers A, B, and C. The lowest instrumented vertebra should be either the neutral vertebra or the vertebra intersected by the central sacral vertical line if it is distal to the neutral vertebra. Level of evidence IV: Retrospective multicenter study.

Introduction

Advances in spinal instrumentation systems have significantly improved the surgical correction of Adolescent Idiopathic Scoliosis (AIS). The treatment objectives remain the same: to correct the deformity, to achieve good spinal balance in the coronal and sagittal planes, and to prevent worsening of the instrumented and non-instrumented curves.

Preserving lumbar mobility is also an objective in patients with Lenke 1 or 2 AIS. Correction of the main curve results in spontaneous correction of the compensatory non-instrumented lumbar curve, which can however progress subsequently [START_REF] Richards | Lumbar curve response in type II idiopathic scoliosis after posterior instrumentation of the thoracic curve[END_REF][START_REF] Lenke | Spontaneous lumbar curve coronal correction after selective anterior or posterior thoracic fusion in adolescent idiopathic scoliosis[END_REF][START_REF] Patel | Spontaneous lumbar curve correction in selective thoracic fusions of idiopathic scoliosis: a comparison of anterior and posterior approaches[END_REF]. Optimal selection of the Lowest Instrumented Vertebra (LIV) is crucial. Based on a study of 42 patients with thoracic AIS, Suk et al. recommended selecting the Neutral Vertebra (NV) when it was the same or one level distal to the End Vertebra (EV) of the main curve and NV-1 if more than two levels separated the EV from the NV [START_REF] Suk | Determination of distal fusion level with segmental pedicle screw fixation in single thoracic idiopathic scoliosis[END_REF]. Wang et al. suggested selecting EV + 1 as the LIV [START_REF] Wang | Distal adding-on phenomenon in Lenke 1A scoliosis: risk factor identification and treatment strategy comparison[END_REF] and Matsumoto et al., the Last Touched Vertebra (LTV) defined as the lowest vertebra intersected by the Central Sacral Vertical Line (CSVL) [START_REF] Matsumoto | Postoperative distal adding-on and related factors in Lenke 1A curve[END_REF]. Miyanji et al. distinguished two curve patterns based on the direction of the L4 tilt [START_REF] Miyanji | Is the lumbar modifier useful in surgical decision making? Defining two distinct Lenke 1A curve patterns[END_REF]. Sarlak et al. argued for selecting the EV as the LIV [START_REF] Sarlak | Radiographic features of the Lenke 1A curves to help to determine the optimum distal fusion level selection[END_REF], whereas Takahashi et al. [START_REF] Takahashi | Selective thoracic fusion in adolescent idiopathic scoliosis: factors influencing the selection of the optimal lowest instrumented vertebra[END_REF] recommended choosing the level immediately below the stable vertebra.

These studies describe the strategies used by their authors, with the outcomes assessed after only 2 years. Clearly, there is no consensus. Nevertheless, there is general agreement that LIV selection is crucial to ensure optimal curve outcomes. It should be borne in mind that 2 years is too short for definitive prognostic conclusions.

We selected the tilt of the first distal non-instrumented vertebra (FDUVtilt) as a marker for curve outcomes. The objective of this study was two-fold: to identify the variables associated with FDUVtilt 5 years after instrumentation and to determine criteria for selecting the LIV.

Materials and methods

Patients

The study was approved by the appropriate ethics committee (Commission de Protection des Personnes) and by the French Data Protection Authority (Commission Nationale de l'Informatique et des Libertés).

The 10 participating centres included a total of 182 patients (156 females and 26males) who underwent posterior instrumentation to treat Lenke 1 (n = 141) or Lenke 2 (n = 41) AIS, at a mean age of 14.8 years and were then followed-up for at least 5 years (mean, 8.3 years). The lumbar modifier was A in 111 patients, B in 31 patients, and C in 40 patients. At last follow-up, completed Scoliosis Research Society health-related quality of life questionnaires (SRS-30) were collected from 115 patients.

Operative techniques

Hybrid hook-screw instrumentation was used in most patients. The mean number of instrumented vertebrae was 10.6 and the mean number of anchors was 11.7, yielding an implant density of 55%.

The LIV was between T11 and L4. Selective thoracic fusion was performed in 169 patients. The instrumentation was extended to the lumbar spine in 15 patients with B or C lumbar modifiers.

Study variables

Anteroposterior and lateral radiographs were obtained preoperatively, postoperatively, and at last follow-up. The clinical and radiographic data were entered into the KEOPS-Spine database [START_REF] Maillot | Reproducibility and repeatability of a new computerized software for sagittal spinopelvic and scoliosis curvature radiologic measurements: Keops[END_REF]. The measurements were then performed automatically by the Keops-Analyzer software after a senior surgeon (JLC) identified each vertebra, as well as the NV of the main curve and the LTV. The position of the LIV relative to the NV was recorded as follows: NV-1 if the LIV was the vertebra immediately cephalad to the NV, NV-2 if the LIV was the second vertebra cephalad to the NV, NV + 1 and NV + 2 if the LIV was the first or second vertebra distal to the NV, and so on. The same notation was used to record the position of the LIV relative to the LTV.

The primary outcome measure was FDUVtilt at last follow-up. The patients were divided into two groups depending on whether FDUVtilt was ≤ 5 • (64 patients, 35%, FDUV-5 • group) or > 5 • (118 patients, 65%, FDUV + 5 • group) (Table 1). All clinical and radiographic variables were analysed in these two groups.

Statistical analyses

SAS version 9.0 software (SAS Institute, Cary, NC, USA) was used for the statistical analyses. Means were compared by applying the Wilcoxon test or Student test and percentages by applying the chisquare test. Pearson's test was chosen to assess correlations among study variables. Based on the results of the univariate analysis, published data, and personal opinion, 13 variables were selected as potentially relevant and used to build a multiple logistic regression model. Values of P lower than 0.05 were taken to indicate significant differences.

Results

Study variables in the two groups

The percentage of correction of the main curve at last followup was 60% in the FDUV-5 • group and 51% in the FDUV + 5 • (P = 0.0016) (Table 2). Loss of correction between the postoperative evaluation and last follow-up was 2% in the FDUV-5 • group and 8% in the FDUV + 5 • group (P = 0.0035). The two groups were not significantly different regarding the preoperative angle of the main curve or distal lumbar compensatory curve, curve FDUV: First Distal Uninstrumented Vertebra.

Table 4

Distribution of patients according to the location of the last instrumented vertebra relative to the neutral vertebra and last touched vertebra.

Location of LIV Global VNID-5 Mean number of anchors was 13 in the FDUV-5 • group and 11 in the FDUV + 5 • group (P = 0.04). The number of instrumented vertebrae was 11 in the FDUV-5 • group and 10 in the FDUV + 5 • group (P = 0.006). Implant density, which was not significantly different between the two groups, correlated positively with the percentage of correction (P < 0.00001) and negatively with correction loss (P = 0.0001).

The proportion of patients with the A lumbar modifier was higher in the FDUV + 5 • group (73%) than in theFDUV-5 • (27%) group, and the opposite was true for the proportions of patients with the B or C modifier (Table 3).

Among patients whose LIV was NV-1 or NV-2, 79% were in the FDUV + 5 • group. On the other hand, among patients whose LIV was NV or NV +, the proportion with FDUV-5 • was higher than when the LIV was at other levels (P = 0.045).

When the LIV was above the LTV, the proportion of FDUV + 5 • patients was 84% (P = 0.03) (Table 4).

Results of the multivariate analysis

Four variables showed significant and independent associations with FDUVtilt (Table 5).

No significant differences were found between the two groups for any of the other study variables including presence or absence of a neutral disc determined by closure of the first non-instrumented disc on the preoperative lateral-bending radiographs, spinal alignment in the coronal and sagittal planes, and sagittal parameters.

The analysis of the 115 available SRS 30 quality-of-life questionnaires showed no significant difference between the two groups.

Discussion

Study rationale

The 5 • FDUVtilt cut-off chosen to separate satisfactory from less satisfactory outcomes was based on a review of publications on the LIV. Adding-on, which indicates an unfavourable outcome, is defined as distal migration of the NV combined with either more than 5 mm of FDUV translation relative to the CSVL or a greater than 5 • increase in FDUVtilt1 year after surgery [START_REF] Wang | Distal adding-on phenomenon in Lenke 1A scoliosis: risk factor identification and treatment strategy comparison[END_REF]. These changes indicate worsening of both the main curve and the lumbar compensatory curve.

In our study, 3 patients had distal NV migration between the preoperative evaluation and last follow-up. All 3 patients had an FDUVtilt value greater than 5 • and were therefore in the FDUV + 5 • group.

Measuring lateral vertebral translation in mm requires calibration of the radiographs, which is not always available.

Finally, we felt that a 5 • increase in FDUVtilt was not sufficient to characterize curve outcomes. In our study, 43 patients had a 5 • increase in FDUVtilt during follow-up. In all of them, FDUVtilt was greater than 5 • at the initial postoperative evaluation. Among the 62 patients whose FDUVtilt decreased, 26 had less than 5 • of FDUVtilt postoperatively and remained in the FDUV-5 • group at last follow-up and 14 switched from more to less than 5 • of FDUVtilt due to the decrease. The remaining 22 patients had more than 5 • of FDUVtilt initially followed by a decrease that was not sufficient to bring them into the FDUV-5 • group 5 years after surgery. We are aware that the 5 • cut-off is a demanding criterion and that many patients whose FDUVtilt is greater than 5 • have satisfactory outcomes, particularly if the FDUVtilt value is declining. Nevertheless, we believe that the optimal outcome is defined as a fused segment seated on a horizontal vertebra.

Study limitations

The retrospective design is the main limitation. Strengths of the study are the long follow-up; multicenter recruitment; automatic computer-assisted analysis of radiographic parameters (Keops Analyser software [START_REF] Maillot | Reproducibility and repeatability of a new computerized software for sagittal spinopelvic and scoliosis curvature radiologic measurements: Keops[END_REF]), which provides better repeatability and reproducibility than do conventional measurements; and statistical analysis performed by an independent operator.

Parameters associated with FDUVtilt

Both percentage of correction and correction loss at last follow-up were associated with FDUVtilt, confirming our working hypothesis (Fig. 1). A percentage of correction ≥ 60% at last followup, which was associated with ≤ 20 • of final angulation, seems to be the requirement for obtaining an FDUVtilt value below 5 • (Fig. 2).

Furthermore, between the postoperative evaluation and last follow-up, FDUVtilt decreased in the FDUV-5 • group but increased in the FDUV + 5 • (Table 1), in correlation with the correction loss. An improvement in FDUVtilt is ascribable to the potential of the noninstrumented lumbar compensatory curve to improve over time, as reported previously [START_REF] Lowe | Distal junctional kyphosis of adolescent idiopathic thoracic curves following anterior or posterior instrumented fusion: Incidence, risk factors and prevention[END_REF][START_REF] Ilgenfritz | Lenke 1C and 5C spinal deformities fused selectively: 5 year outcomes of the uninstrumented compensatory curves[END_REF][START_REF] Ishikawa | Postoperative behavior of thoracolumbar/lumbar curve and coronal balance after posterior thoracic fusion for Lenke 1C and 2C adolescent idiopathic scoliosis[END_REF].

One might expect that an FDUVtilt value below 5 • after instrumentation can only be expected when the curve is moderate and flexible. However, in our study, the two groups were not significantly different regarding initial curve angulation or reducibility on lateral-bending radiographs.

Among lumbar modifiers, C was more common in the FDUV-5 • group and A in the FDUV + 5 • group (Table 3). Thus, FDUVtilt at last follow-up was less marked when the compensatory curve was initially more severe. This finding demonstrates the potential for the compensatory curve to correct spontaneously in response to correction of the main thoracic curve [START_REF] Ilgenfritz | Lenke 1C and 5C spinal deformities fused selectively: 5 year outcomes of the uninstrumented compensatory curves[END_REF][START_REF] Ishikawa | Postoperative behavior of thoracolumbar/lumbar curve and coronal balance after posterior thoracic fusion for Lenke 1C and 2C adolescent idiopathic scoliosis[END_REF]. It should, nevertheless, be interpreted with caution, as some patients with the B or C modifier underwent non-selective instrumentation.

The quality of the correction is well known to correlate with implant density [START_REF] Clements | Correlation of scoliosis curve correction with the number and type of fixation anchors[END_REF]. The number of anchors was fairly low in our study, because all patients had surgery before 2010. Implant density was not significantly associated with FDUVtilt. Nevertheless, FDUVtilt was associated with both the number of anchors and instrumentation length. This last is probably the relevant factor, as its increase necessarily leads to an increase in the number of anchors. However, in our study, implant density correlated negatively with correction loss and positively with the percentage of correction of the main curve. These correlations confirm the major role for the number of anchors in the quality of the reduction [START_REF] Clements | Correlation of scoliosis curve correction with the number and type of fixation anchors[END_REF].

Quality of life was not significantly different between the two groups. FDUVtilt is not perceived by the patient. It was not associated with any of the five SRS 30 domains.

Selecting the LIV

Our findings (Table 4) constitute a strong argument against instrumentations that are shorter than the NV. Long instrumentations are more satisfactory but compromise the mobility of the lumbar spine. Results were best when the LIV was at NV + 2.

The LTV is also important to consider. Among patients whose instrumentation was shorter than the LTV, 84% were in the FDUV + 5 • group.

Selection of the LIV involves striking the best compromise between preserving lumbar spine mobility and achieving a horizontal FDUV. Several criteria have been suggested based on studies with 2 years of follow-up [START_REF] Suk | Determination of distal fusion level with segmental pedicle screw fixation in single thoracic idiopathic scoliosis[END_REF][START_REF] Wang | Distal adding-on phenomenon in Lenke 1A scoliosis: risk factor identification and treatment strategy comparison[END_REF][START_REF] Matsumoto | Postoperative distal adding-on and related factors in Lenke 1A curve[END_REF][START_REF] Miyanji | Is the lumbar modifier useful in surgical decision making? Defining two distinct Lenke 1A curve patterns[END_REF][START_REF] Sarlak | Radiographic features of the Lenke 1A curves to help to determine the optimum distal fusion level selection[END_REF][START_REF] Takahashi | Selective thoracic fusion in adolescent idiopathic scoliosis: factors influencing the selection of the optimal lowest instrumented vertebra[END_REF]. With a mean follow-up of 8 years, our study argues against very short instrumentations. Stopping the instrumentation at NV-1 orNV-2 is associated with an increased risk of FDUVtilt > 5 • . We suggest stopping the instrumentation at the NV as a good compromise between instrumentation length and lumbar spine mobility. However, if the LTV is distal to the NV, we recommend extending the instrumentation down to the LTV. When the thoracic curve is long with an NV at L3 or L4, to preserve lumbar spine mobility, the instrumentation can be stopped at NV-1 provided the LTV is cephalad to the NV.

Conclusions

A final percentage of correction greater than 60% and absence of correction loss between the postoperative evaluation and last follow-up were the main factors associated with an FDUVtilt lower than 5 • in our study of 182 patients managed by posterior instrumentation for Lenke 1 or 2 AIS and followed-up for a mean of 8 years.

Fig. 1 .

 1 Fig. 1. Patient in the FDUV + 5 • group, Lenke 2A Adolescent Idiopathic Scoliosis, 14 years of age. Loss of correction. The last instrumented vertebra is at NV-2. (a) Preoperative radiographs: NV at L4; (b) Radiograph 1 month after T2-L2 instrumentation with the last instrumented vertebra at NV-2; (c) Radiographs 5 years after surgery.

Fig. 2 .

 2 Fig. 2. Patient in the FDUV-5 • group, Lenke 2A Adolescent Idiopathic Scoliosis, 14 years of age. Stable correction. Last instrumented vertebra is at NV + 1. (a) Preoperative radiographs: NV at T12; (b) Radiograph 1 month after T2-L1 instrumentation with the last instrumented vertebra at NV + 1; (c) Radiographs 5 years after surgery.

Table 1

 1 Distribution of the patients between the two groups defined based on tilt of the first distal uninstrumented vertebra.

	182 AIS Lenke 1 or 2	FDUV-5 •	FDUV + 5 •	P value
	Patients	64 (35%)	118 (65%)
	FDUVtilt postop. ( • )	3.5 •	7.6 •	<0.0001
	FDUVtilt at last FU ( • )	2.7 •	11.5 •	<0.0001
	Changefrom postop/to last FU	-0.8 •	+ 3.9 •	<0.0001

AIS: Adolescent Idiopathic Scoliosis; FDUV: First Distal Uninstrumented Vertebra; FDUVtilt: tilt of the First Distal Uninstrumented Vertebra, in degrees ( • ); postop.: postoperatively; FU: Follow-Up.

Table 2

 2 Preoperative angulation, reducibility, and percentage of correction in the two groups defined based on tilt of the first distal uninstrumented vertebra.

	182 AIS Lenke 1 or 2	All	FDUV-5 •	FDUV + 5 •	P value univariate	P value multivariate
	Preop. main curve angulation ( • )	54 •	53 •	54 •	NS	NS
	Initial correction (%)	60%	62%	59%	NS	NS
	Final correction (%)	54%	60%	51%	0.0016	0.026
	Loss of correction (%)	6%	2%	8%	0.0035	0.03
	Reducibilityof main curve (bending)	48%	47%	49%	NS	NS
	Preop. lumbar curve angulation ( • )	30 •	31 •	30 •	NS	NS

AIS: Adolescent Idiopathic Scoliosis; FDUV: First Distal Uninstrumented Vertebra; Preop.: preoperatively.

Table 3

 3 Distribution of lumbar modifiers in the twogroups defined based on tilt of the First Distal Uninstrumented Vertebra.

	Lumbar modifier	All (n = 182)	FDUV-5 • (n = 64)	FDUV + 5 • (n = 118)	P value chi 2
	A	111	30 (27%)	81 (73%)
	B	31	15 (48%)	16 (52%)	0.016
	C	40	19 (47%)	21 (53%)

Table 5

 5 Last Touched Vertebra defined as the last vertebra intersected by the central sacral vertical line. Results of the multivariate analysis of factors associated with tilt of the first distal instrumented vertebra at a mean of 8 years after surgery.

	•	VNID + 5 •	P value chi 2

We recommend selective thoracic fusion for Lenke 1 or 2 AIS, regardless of whether the lumbar modifier is A, B, or C. We believe the LIV should be the distal NV of the main thoracic curve or the LTV if this last is distal to the NV.

Disclosure of interest

The authors declare that they have no competing interest.