Abstract
Varibaculum timonense sp. nov. strain Marseille-P3369T (= CSURP3369) is a new species from the order Actinomycetales that has been isolated from a fresh stool sample of a healthy French woman.
Keywords: Actinomycetales, culturomics, gut microbiota, human microbiota, Varibaculum timonense sp. nov.
Introduction
Currently, the implications of bacterial diversity for normal physiological functions and for disease must be understood [1]. To explore the diversity of human intestinal bacteria, the culturomics approach, based on diversified culture conditions, was designed to isolate species never cultivated before and also to complete the metagenomics of 16S rRNAs [2], [3], [4]. A new taxonomic method called taxonogenomics has been developed for a description associating the analysis of complete sequences of the genome and the phenotypic characteristics of novel bacterial species [5]. By integrating this new approach, we give here a brief description of a new species within the genus Varibaculum, isolated from a fresh stool sample of a healthy French woman.
Isolation and growth conditions
In September 2016, we isolated, from a fresh stool sample of a 26-year-old healthy French woman, a bacterial strain (Marseille-P3369T). This strain was not identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The analysis was performed on a Microflex LT spectrometer (Bruker Daltonics, Bremen, Germany), as previously described [6]. Spectra obtained (Fig. 1) were imported and analysed using the Biotyper 3.0 software against the Bruker database, which was constantly updated with the MEPHI database [1]. Growth of colonies of the strain Marseille-P3369T was observed after 48 hours of incubation at 37°C on 5% sheep's blood agar (bioMérieux, Marcy l’Etoile, France) under strict anaerobic conditions generated by anaeroGEN (Oxoid, Dardilly, France) [7].
Fig. 1.
MALDI-TOF MS reference spectrum of Varibaculum timonense sp. nov. The reference spectrum was generated by comparison of spectra from 12 individual colonies.
Phenotypic characteristics
The bacterial strain Marseille-P3369T is not motile and not spore-forming. Its colonies are circular and white with a mean diameter of 0.5 mm. Cells were Gram-positive, small rod-shaped, and slightly curved, ranging in diameter from 0.35 to 0.4 μm (Fig. 2). Catalase and oxidase activities were not observed for strain Marseille-P3369T. The optimal growth of this strain was observed at 37°C and pH 7.5 under strict anaerobic conditions. Results issued from API ZYM and API 20A tests are shown in Table 1. The main biochemical characteristics of the closest Varibaculum species with standing in nomenclature are compared in Table 2. The major fatty acid found for this strain was hexadecanoic acid (52%) followed by 9-octadecenoic acid (22%), 9,12-octadecadienoic acid (12%) and octadecanoic acid (9%). Minor amounts of unsaturated, branched and other saturated fatty acids were also detected (Table 3).
Fig. 2.
Scanning electron microscopy (SEM) of stained Varibaculum timonense sp. nov. A colony was collected from agar and immersed into a 2.5% glutaraldehyde fixative solution. Then a drop of the suspension was directly deposited on a poly-l-lysine-coated microscope slide for 5 minutes and treated with 1% phosphotungstic acid aqueous solution (pH 2.0) for 2 minutes to increase SEM image contrast. The slide was gently washed in water; air-dried and examined in a tabletop SEM (Hitachi TM4000). The scale is shown on the figure.
Table 1.
Phenotypic characterization of Varibaculum timonense sp. nov., based on analytical profile index (API) tests
| Biochemical characteristics | Results |
|---|---|
| Alkaline phosphatase | + |
| Esterase (C-4) | + |
| Esterase lipase (C-8) | + |
| Lipase (C-14) | + |
| Leucine arylamidase | + |
| Valine arylamidase | − |
| Cystine arylamidase | − |
| Trypsine | + |
| α-chymotrypsine | − |
| Acid phosphatase | + |
| Naphthalo-AS-BI-phosphohydrolase | + |
| α-galactosidase | − |
| β-galactosidase | − |
| β-glucuronidase | − |
| α-glucosidase | + |
| β-glucosidase | − |
| N-acetyl-β-glucosaminidase | − |
| α-mannosidase | + |
| α-fucosidase | − |
| Indole production | − |
| Urease | + |
| Glucose | − |
| Mannitol | + |
| Lactose | + |
| Sucrose | + |
| Maltose | + |
| Salicin | + |
| Xylose | − |
| Arabinose | − |
| Gelatin | − |
| Esculin | + |
| Glycerol | − |
| Cellobiose | + |
| Mannose | − |
| Melezitose | + |
| Raffinose | − |
| Sorbitol | − |
| Rhamnose | − |
| Trehalose | − |
Table 2.
Differential characteristics of Varibaculum timonensis sp. nov., Varibaculum cambriensis and Varibaculum anthropi
| Properties | V. timonensis sp. nov | V. cambriensis | V. anthropi |
|---|---|---|---|
| Cell diameter (μm) | 0.4-0.5 | NA | NA |
| Oxygen requirement | − | − | − |
| Gram stain | + | + | + |
| Salt requirement | − | − | − |
| Motility | − | − | − |
| Endospore formation | − | − | − |
| Alkaline phosphatase | + | − | − |
| Catalase | − | − | − |
| Indole | + | − | − |
| Nitrate reductase | − | + | − |
| Urease | + | − | + |
| β-galactosidase | − | − | − |
| N-acetyl-glucosamine | − | − | − |
| Arabinose | − | − | − |
| Lipase (C8) | + | + | NA |
| Trypsine | + | − | NA |
| Mannose | − | − | − |
| Mannitol | + | − | − |
| d-Glucose | − | + | + |
| d-Maltose | + | + | + |
| Source | Stool samples | Human samples | Human samples |
+, positive result; −, negative result; NA, data not available.
Table 3.
Fatty acid profiles (%) of Varibaculum timonensis strain Marseille-P3369
| Fatty acids | Name | Mean relative % a |
|---|---|---|
| 16:0 | hexadecanoic acid | 52.2 ± 1.0 |
| 18:1n9 | 9-octadecenoic acid | 21.5 ± 0.2 |
| 18:2n6 | 9,12-octadecadienoic acid | 11.5 ± 0.5 |
| 18:0 | octadecanoic acid | 8.6 ± 0.3 |
| 14:0 | tetradecanoic acid | 1.6 ± 0.1 |
| 18:1n5 | 13-octadecenoic acid | 1.5 ± 0.1 |
| 18:1n7 | 11-octadecenoic acid | TR |
| 15:0 | pentadecanoic acid | TR |
| 17:0 anteiso | 14-methyl-hexadecanoic acid | TR |
| 17:0 | heptadecanoic acid | TR |
| 16:1n7 | 9-hexadecenoic acid | TR |
| 15:0 iso | 13-methyl-tetradecanoic acid | TR |
| 17:0 iso | 15-methyl-hexadecanoic acid | TR |
TR = trace amounts <1%.
Mean peak area percentage.
Strain identification
To classify this bacterium, the 16S rRNA gene was amplified using the primer pair fD1 and rP2 (Eurogentec, Angers, France) and sequenced using the Big Dye® Terminator v1.1 Cycle Sequencing Kit and 3500xLGenetic Analyzer capillary sequencer (Thermofisher, Saint-Aubin, France), as previously reported [8]. The 16S rRNA nucleotide sequences were assembled and corrected using CodonCode Aligner software (http://www.codoncode.com). Strain Marseille-P3369T exhibited 98.32% similarity with Varibaculum cambriense strain CCUG 44998 (GenBank Accession no. NR_114873.1), its phylogenetically closest species with standing in nomenclature (Fig. 3). We consequently proposed classifying strain Marseille-P3369T as a new species within the genus Varibaculum belonging to the phylum Actinobacteria.
Fig. 3.
Phylogenetic tree highlighting the position of Varibaculum timonense sp. nov., relative to the most closely related type strains within the genus Varibaculum. GenBank Accession numbers of 16S rRNA are indicated in parentheses. Sequences were aligned using MUSCLE with default parameters, phylogenetic inference were obtained using the maximum likelihood method and MEGA 7 software. Numbers at the nodes are percentages of bootstrap values obtained by repeating the analysis 1000 times to generate a majority consensus tree. The scale bar indicates a 2% nucleotide sequence divergence.
Genome sequencing
Genomic DNA was extracted using the EZ1 biorobot with the EZ1 DNA tissue kit (Qiagen, Hilden, Germany) and then sequenced on a MiSeq sequencer (Illumina Inc., San Diego, CA, USA) with the Nextera Mate Pair sample prep kit and Nextera XT Paired End (Illumina), as previously described [9]. The assembly was performed using a pipeline containing several softwares (Velvet [10], Spades [11] and Soap Denovo [12]), and trimmed data (MiSeq and Trimmomatic [13] softwares) or untrimmed data (only MiSeq software). GapCloser was used to reduce assembly gaps. Scaffolds <800 bp and scaffolds with a depth value < 25% of the mean depth were removed. The best assembly was selected by using different criteria (number of scaffolds, N50, number of N). The genome of Strain Marseille-P3369T was 2.73 Mb with 33.2% G + C content. The degree of genomic similarity of the strain with closely related species was calculated using OrthoANI software [14]. OrthoANI values among closely related species (Fig. 4) ranged from 58.83% between Actinomyces neuii and Varibaculum timonense to 77.54% between Actinomyces odontolyticus and Actinomyces georgiae. When Varibaculum timonense was compared with these closely related species values ranged from 58.83% with Actinomyces neuii to 66% with Actinomyces georgiae.
Fig. 4.
Heatmap generated with OrthoANI values calculated using the OAT software between Varibaculum timonense sp. nov. and other closely related species with standing in nomenclature.
Conclusion
On the basis of unique phenotypic features, including MALDI-TOF spectrum, a 16S rRNA sequence divergence >1.3%, and an OrthoANI value < 95% with the phylogenetically closest species with standing in nomenclature, we formally proposed strain Marseille-P3369T as the type strain of Varibaculum timonense sp. nov., which is a new species in the genus Varibaculum.
Description of Varibaculum timonense strain Marseille-P3369T sp. nov.
Strain Marseille-P3369T is the type strain of Varibaculum timonense sp. nov. (ti.mo.nen'se, N.L. neut. adj. timonense, related to Timone, the name of the main university hospital in Marseille, France, from where the strain was isolated). Varibaculum timonense is a strict anaerobic, non-motile and non-sporulating Gram-stain-positive rod bacterium. Strain Marseille-P3369T grows under anaerobic conditions at temperatures ranging between 37°C and 45°C, with an optimal temperature of 37°C. It exhibits neither catalase nor oxidase activities. The genome of Strain Marseille-P3369T was 2.73 Mb with 33.2% G + C content. The potential pathogenicity of the type strain Marseille-P3369T (= CSURP3369) is unknown. It was isolated from the fresh stool sample of a 26-year-old French healthy woman.
Nucleotide sequence accession number
The 16S rRNA gene and genome sequences were deposited in GenBank under Accession numbers LT797538 and FWDK00000000, respectively.
Deposit in culture collections
Strain Marseille-P3369T was deposited in two different strain collections under the following number (= CSURP3369).
Conflicts of interest
None to declare.
Funding sources
This study was supported by the Institut Hospitalo-Universitaire (IHU) Méditerranée Infection, the National Research Agency under the programme Investissements d'avenir, reference ANR-10-IAHU-03, the Région Provence Alpes Côte d’Azur and European funding FEDER PRIMI.
Ethics and consent
The study was approved by the ethics committee of the Institut Federatif de Recherche 48 under reference 2016-010. The woman provided written consent.
Acknowledgements
The authors thank Catherine Robert for the sequencing of the genome, Aurelia Caputo for submitting the genomic sequence to GenBank, and Stéphane Alibar for the cultivation of the strain.
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