Strain identification and phylogenetic analysis
MALDI-TOF mass spectrometry could not correctly identify the strain Marseille-P3740. Thus, the obtained reference spectrum was added to the local database. Similarity analysis based on 16S rRNA of strain Marseille-P3740 against GenBank showed the highest nucleotide sequence similarities of 96.31 % sequence identity with Collinsella intestinalis strain JCM 10643 (Genbank accession number: NR_113165.1), which was the phylogenetically closest species. This value obtained is below the threshold of 98.65% recommended to delimit the species barrier in bacteria 24,26. Therefore, the strain Marseille-P3740 was considered as a potentially new species belonging to the genus Collinsella within the Coriobacteriaceae family in the phylum Actinobacteria (Figure 1).
Phenotypicproperties of the strain
After the isolation step, the strain Marseille-P3740 was cultured in order to obtain pure and isolated colonies on 5% sheep blood enriched Columbia agar. The colonies were small and transparent. Bacterial cells were Gram-positive. No spore forming after thermal shock (10 minutes at 80°C) for strain Marseille-P3740. In addition, the shape of the bacterium was demonstrated by a photomicrograph obtained with the Hitachi TM4000 instrument (Figure 2). Bacterial cells appeared to be rod-shaped with a mean length of 1 μm and a mean diameter of 0.5 μm. This bacterium is fastidious but grows under anaerobic conditions at pH 7 on 5% sheep blood enriched Columbia agar after 48 hours. No growth in aerobic or microaerophilic atmosphere was observed. However, it is also possible to obtain bacterial colonies at temperatures ranging from 28 to 45°C, knowing that optimal growth occurs at 37° C in an anaerobic atmosphere. The use of API ZYM and 50 CH (bioMérieux) galleries revealed that strain Marseille-P3740T presented enzymatic activities, such as acid phosphatase, alkaline phosphatase and naphthol-AS-BI-phosphohydrolase, while D-mannose, D-raffinose and D-trehalose were positive. However, negative reactions were observed with lipase, trypsin, achymotrypsin, α-mannosidase, α-fucosidase, α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase and β-glucosidase, erythritol, arabinose, D-ribose, D-adonitol, xylose, methyl-β-D-xylopranoside, D-galactose, glucose, arbutin, fructose, L-sorbose, L-rhamnose, D-lyxose, dulcitol, inositol, D-mannitol, esculin ferric citrate, methyl-αD-glucopranoside, N-acetyl-β-glucosaminidase, glycogen, methyl-αD-xylopranoside, amygdalin, salicin, D-cellobiose, D-maltose, glycerol, D-lactose, D-melibiose, inulin, D-melezitose, amidon, D-turanose, D-tagatose, gentiobiose, L-fucose, L-arabitol, potassium 2-ketogluconate and potassium gluconate. Strain Marseille-P3740T showed catalase-negative and oxidase-negative activities. The Table 1 comparing the main phenotypic criteria shows that the strain Marseille-P3740 differs from other Collinsella by not consuming glucose. Fatty acid methyl esther analysis was performed as previously described .The major fatty acid were hexadecanoic acid (41 %) and 9-octadecenoic acid (23.6 %). Very few other structures were described. Minor amounts of other unsaturated and saturated fatty acids were also detected, including unsaturated structures (Table 2).
Genome sequencing and comparison
The genome of Collinsella provencensis strain Marseille-P3740 is 1,737,922 bp long with a 58.2 mol% G+C content.
The genome size, the number of proteins and the number of genes of strain Marseille-P3740 were lower than those of the other genomes studied here (Table 3). On the other hand, it has a genome with a GC percentage higher than that of C. bouchesdurhonensis, but lower than that of C. intestinalis, C. vaginalis, C. stercoris and C. tanakaei (62.4, 64.4, 62.7 and 60.2 mol% respectively). Calculation of the degree of genomic similarity of strain Marseille-P3740 with closely related species showed that values ranged from 72.57% between C. bouchesdurhonensis and C. vaginalis to 82.09% between C. intestinalis and C. stercoris. On the other hand, C. provencensis compared to the other Collinsella species, showed values ranging from 78.01% with C. stercoris to 72.90% with C. bouchesdurhonensis. Therefore, we found that the OrthoANI values among closely related species (Figure 3) were below the value at the 95% threshold recommended for delineating species barrier in prokaryotes. However, the analysis of the DDH values calculated between the genomes of the Collinsella species studied here, showed that no value is close to the threshold value (70%) making it possible to delimit a new bacterial species. Indeed, 27.3% was the highest DDH value obtained between C. stercoris and C. phocaeensis, while the lowest DDH value obtained (21.6%) was shared between C. vaginalis and C. aerofaciens (Table 4).