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Anaerotruncus massiliensis sp. nov., a succinate-producing bacterium isolated from human stool from an obese patient after bariatric surgery

A new bacterium, strain AT3T, was isolated by microbial culturomics from a faecal sample from a Frenchman after bariatric surgery. The isolate exhibited 96.6% 16S ribosomal RNA gene nucleotide sequence similarity with Anaerotruncus colihominis strain WAL 14565T = CCUG 45055T = CIP 107754T. Phenotypic and genomic characteristics showed that the new strain represents a novel species, for which the name Anaerotruncus massiliensis sp. nov. is proposed. The type strain is strain AT3T = CSUR P2007T = DSM 100567T.


Introduction
Obesity is a major public health problem [1,2]. It increases the risk of metabolic diseases such as type 2 diabetes and cardiovascular diseases such as high blood pressure [3]. The global obesity rate has been steadily increasing since 1980 [4]. The influence of the gut microbiota in human health and disease has been revealed in the recent years. Recently obesity has been associated with gut microbiota dysbiosis [5][6][7].
The various treatment regimens proposed to treat obesity are mainly dietary measures as well as medical and surgical treatments. Bariatric surgery has been documented as one of the most effective treatments for obesity and has been associated with increased microbial diversity [8,9]. We studied stool samples from obese patients before and after bariatric surgery using the microbial culturomics approach [10]. During this study, a novel Gram-negative anaerobic bacterium was isolated from a stool sample collected after bariatric surgery from an obese man.
Here we characterize a novel bacterial species, strain AT3, belonging to Anaerotruncus genus [11], together with the genome sequencing assembly, annotation and comparison. At the time we described this new species, the genus Anaerotruncus only contained one species, the type species Anaerotruncus colihominis strain WAL 14565 T = CCUG 45055 T = CIP 107754 T [11]. enriched with rumen fluid and sheep's blood, then seeded on 5% sheep's blood Columbia agar (bioMérieux, Marcy l'Etoile, France) as described elsewhere [12]. Strain purification and identification were performed as previously described [12,13].
The 16S ribosomal RNA (rRNA) gene sequencing of the strain AT3 was performed as previously reported [14] using the fD1-rP2 primers, a GeneAmp PCR System 2720 thermal cycler (Applied Biosystems; Thermo Fisher Scientific, Waltham, MA, USA) and an ABI Prism 3130-XL capillary sequencer (Applied Biosciences, Saint Aubin, France). For taxonomic assignment, CodonCode Aligner (CodonCode, Centerville, MA, USA) software was used to correct sequences, and BLASTn searches were performed by the National Center for Biotechnology Information (NCBI) web server (http://blast.ncbi.nlm.nih.gov. gate1.inist.fr/Blast.cgi) for neighbour search. Sequences were aligned using CLUSTAL W [15] and phylogenetic inferences were obtained using the maximum likelihood method [16] by MEGA 7 software [17].
The colonies' morphology and pigmentation were observed after cultivation of the strain on a 5% sheep's blood Columbia agar plate (bioMérieux) at 37°C in anaerobic condition for 48 hours. Phenotypic characteristic analysis of the strain was performed as previously described [12,18]. The growth of the strain for temperature ranged from 25 to 55°C when assessed on sheep's blood-enriched Columbia agar (bioMérieux) under different atmospheric conditions. The pH growth of strain AT3 was assessed at various pH range from 6 to 8.5 under anaerobic condition at 37°C. The saltiness of the strain using various concentrations (5, 10, 50, 75 and 100 g/L) of NaCl on Schaedler agar with 5% sheep's blood (bioMérieux) under an anaerobic atmosphere at 37°C was also tested.
Biochemical assays using API Gallery strips-API ZYM, API 20A, API 50 CH and rapid ID 32 A (bioMérieux)-were performed according the manufacturer's instruction.
Cellular fatty acid methyl ester and carbohydrate metabolism end product of strain AT3 were assessed as previously reported [12]. Antimicrobial susceptibility of the strain was also tested according the European Committee on Antimicrobial Susceptibility Testing (EUCAST) recommendation [19,20] using 12 antibiotics.
Genomic DNA of strain AT3 was sequenced on MiSeq Technology (Illumina, San Diego, CA, USA) with the mate-pair strategy and assembly deployed as previously described [18,21]. The genome annotation of strain AT3 was performed as previously described [12] and compared to the genomes of his relatives species. For the comparison, genomes of closest species were automatically retrieved from the 16S rRNA tree and thus, Anaerotruncus colihominis strain WAL 14565 T (= CCUG 45055 T = CIP 107754 T ) (ABGD00000000) [11], Hydrogenoanaerobacterium saccharovorans strain SW512 T (= AS 1.5070 T = JCM 14861 T ) (FOCG00000000) [22], Ruthenibacterium lactatiformans strain 585-1 T (= DSM 100348 T = VKM B-2901 T ) (JXXK00000000) [23], Acutalibacter muris strain KB18 T (= DSM 26090 T = KCTC 15540 T ) (CP021422) [24], Acetanaerobacterium elongatum strain Z7 T (= JCM 12359 T = AS 1.5012 T ) (FNID00000000) [25], Ruminococcus flavefaciens strain C94 T (= ATCC 19208 T ) (JAEF00000000) [26] and Ruminococcus champanellensis strain 18P13 T (= DSM 18848 T = JCM 17042 T ) (FP929052) [27] were selected. For each selected genome, complete genome sequence, proteome genome sequence and Orfeome genome sequence were retrieved from NCBI's FTP. All proteomes were analysed with proteinOrtho [28]. Then for a couple of genomes, a similarity score was computed. This score was the mean value of nucleotide similarity of average genomic identity of orthologous gene sequences between the two genomes studied [29]. An annotation of the entire proteome was performed to define the distribution of functional classes of predicted genes according to the clusters of orthologous groups of proteins (using the same method as for the genome annotation). The genome of strain AT3 was locally aligned 2 by 2 using the BLAT algorithm [30,31] against each the selected genomes previously cited. Digital DNA-DNA hybridization values were estimated from a generalized model [32]. Average amino acid identity was also calculated on the basis of the overall similarity between two genomic data sets of the online proteins (http:// enve-omics.ce.gatech.edu/aai/index) [33,34].

Results
The spectrum generated from clean isolate spots was unable to match those in Bruker's and our own database, so the strain's 16S RNA gene was sequenced. BLASTn searches performed using the 16S rRNA gene sequences showed that strain AT3 exhibited 96.6% sequence similarity with A. colihominis strain WAL 14565 T = CCUG 45055 T = CIP 107754 T [11], classified in the Ruminococcaceae family created by Rainey [35]. A maximum likelihood phylogenetic tree based on 16S rRNA gene sequences showed relationships between strain AT3 and some related taxa (Fig. 1). The numbers of nodes correspond to the percentages of bootstrap values obtained by repeating the analysis to 1000 times to generate a consensus tree.
This new isolate was a Gram-negative-staining, nonmotile and rod-shaped bacterium. The strain was catalase positive but oxidase negative. The growth of the strain occurred between 28 to 45°C, but optimal growth was observed at 37°C after 72 hours' incubation in anaerobic atmosphere on Columbia agar with 5% sheep's blood (bioMérieux). No growth was observed in aerobic atmosphere. No growth of this bacterium was observed using 10 g/L of NaCl concentration on Schaedler agar (bioMérieux). The pH for growth ranged from 6 to 8.5, but the optimal pH was 7.5. The colonies were opalescent, with a mean diameter of 0.5 to 1.5 mm. Haemolysis was not observed on Columbia agar plate (bioMérieux). Cells had a diameter ranging from 0.3 to 2.9 μm by electron microscopy (Fig. 2).

( (A A A A A A A A A A A A A A A A A A
The major cellular fatty of the new strain were the anteiso-C 15:0 (29%), C 16:0 (20.6%) and C 14:0 (19.5%). The different characteristics of strain AT3 T compared to its closet species are listed in Table 1. The composition of cellular fatty acids for this isolate is shown in Table 2. The end products of carbohydrate metabolism of the strain AT3 after 72 hours' incubation in Wilkins Chalgren anaerobe broth (MilliporeSigma, St Louis, MO, USA) were acetic acid (12.3 ± 0.5 mM), butanoic acid (7.6 ± 2.0 mM), isobutanoic acid (1.4 ± 0.1 mM), isopentanoic acid (1.0 ± 0.2 mM) and isohexanoic acids (1.0 ± 0.1 mM).
The draft genome of strain AT3 is 3 145 950 bp long with 63.6% G+C content (Fig. 3). It is composed of seven scaffolds (eight contigs). Within the 2927 predicted genes, 2868 were protein-coding genes and 59 were RNAs genes (four 5S rRNA genes, two 16S rRNA genes, two 23S rRNA genes and 51 tRNA genes). A total of 2106 genes (74.4%) were assigned to a putative function by COGs or by nonredundant blast. There were 398 genes (13.9%) associated with peptide signal proteins. ARG-ANNOT (Antibiotic Resistance Gene-Annotation) analysis found two genes (0.07%) associated with antimicrobial agent resistance [36] and four genes (0.14%) associated with polyketide synthase or nonribosomal peptide synthetases [37]. Using the online RAST Server version 2.0 tool, 20 genes were found to be associated with antibiotic resistance (four genes for fluoroquinolone resistance, four for tetracycline resistance and 12 for multidrug resistance efflux pumps), nine genes associated with invasion and intracellular resistance, five to copper homeostasis, nine to cobalt-zinc-cadmium resistance, two to mercuric reductase, one to mercury resistance operon, one to copper homeostasis:copper tolerance and one to cadmium resistance. No gene associated with virulence, toxins or bacteriocins has been found [38]. There were 193 genes (6.7%) identified as ORFans. The remaining 481 (16.8%) were   annotated as hypothetical proteins. The distribution of genes into COGs functional categories of strain AT3 with its nearest neighbours' species is presented in Table 3 and Fig. 4. The numbers of orthologous proteins shared between genomes is shown in Table 4. The genome of the strain was deposited in EMBL-EBI (European Molecular Biology Laboratory-European Bioinformatics Institute) under accession number OEJM0000000. The digital DNA-DNA hybridization values of strain AT3 compared to its closely related species ranged from 21.5% with A. muris to 33.7% with R. champanellensis and are shown in Table 5. The average amino acid identity between the strain AT3 and closely related species was evaluated (Table 6).   J  151  141  144  149  153  148  151  134  Translation  A  0  0  0  0  0  0  0  0  RNA processing and modification  K  201  222  191  282  256  195  181  138  Transcription  L  106  161  116  201  294  147  124  119  Replication, recombination and repair  B  0  0  0  1  0  0  0  0  Chromatin structure and dynamics  D  24  29  25  28  36  22  31  29 Cell cycle control, mitosis and meiosis Y 0 These values ranged from 47% with R. flavefaciens to 68.5% with A. colihominis.

Discussion
When we compared the strain AT3 with the closest relative species differed by the catalase production, we found that his genome G+C content (63.7 vs. 41.9 to 54.6 for A. colihominis, H. saccharovorans, R. lactatiformans, A. muris, A. elongatum, R. flavefaciens and R. champanellensis, its closest relative species). The strain AT3 strain is phylogenetically close to A. colihominis but differs from it in Gram staining; A. colihominis is Gram staining positive while the new strain is Gram staining negative.
The anteiso-C 15:0 (29%) is one of the major cellular fatty acid for the new strain, but it is absent from A. colihominis.
On the basis of the phenotypic, chemotaxonomic, genomic and phylogenetic characteristics, a novel bacterium isolated from the stool sample of a morbidly obese Frenchman after bariatric surgery, termed Anaerotruncus massiliensis sp. nov., is proposed. The type strain is strain AT3 T = CSURP P2007 T = DSM 100567 T . A preliminary report of main characteristics was previously published as a new species announcement [39], but the present study reports the full characterization of this putative new species.