'Gracilibacillus phocaeensis' sp. nov., 'Sediminibacillus massiliensis' sp. nov. and 'Virgibacillus ndiopensis' sp. nov., three halophilic species isolated from salty human stools by culturomics.

We report the isolation of three bacterial strains that could not be identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry screening. 'Gracilibacillus phocaeensis' sp. nov., 'Sediminibacillus massiliensis' sp. nov. and 'Virgibacillus ndiopensis' sp. nov. are halophilic species isolated from salty human stools by culturomics.

Culturomics is a new approach using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF MS) for bacteria identification and aiming to cultivate individually all bacterial species from the human gut and also from other human mucosa microbiota. Thus, this approach has allowed a considerable increase in the gut microbiota repertoire, with the description of more than 247 new species in the last few years [1]. Here we report the isolation of three bacterial strains that could not be identified by our MALDI-TOF MS screening on a Microflex spectrometer (Bruker Daltonics, Bremen, Germany) [2], [3]. These strains were isolated in 2017 from the salty stools (>1.7% NaCl) of healthy Senegalese individuals. The study was approved by the ethics committee of the Institut Hospitalo-Universitaire Méditerranée Infection under number 2016-011, and all patients provided signed informed consent.

The percentage of NaCl in the stool specimens was determined using a salinity refractometer (Thermo Scientific, Villebon-sur-Yvette, France) by diluting 1 g in 10 mL of distilled water and centrifuging it for 10 minutes at 5000g. Then 100 μL of supernatant was deposited in the refractometer; the result was in a straight line, displayed on the screen in per mille and then reported in percentage of NaCl.

To cultivate the bacteria from stool samples, we used an aerobic blood culture bottle (Becton Dickinson, Le Pont-de-Claix, France) containing a halophilic medium prepared by modifying a Columbia broth medium (Sigma-Aldrich, Saint-Quentin-Fallavier, France), as detailed in our previous study [4]. The amount of solute per liter was determined by the following formula: concentration (in %, w/v) = 100 × [(mass solute in g)/(volume solution in mL)].

All strains were first isolated in a halophilic culture medium with 15% (w/v) NaCl.

The initial agar-grown colonies were obtained after 24 hours of incubation at 37°C in aerobic conditions. The 16S rRNA genes were sequenced using the universal primer pair fD1–rP2 as previously described [5] using a 3130-XL sequencer (Applied Biosciences, Saint-Aubin, France). Because all the strains exhibited a 16S rRNA sequence homology of <98.7% with their phylogenetically closest species, we thus propose the creation of these three new species according to the nomenclature [6].

Strain Marseille-P3801T was isolated from stool samples (2% NaCl) of a 20-year-old man from N'Diop. Strain Marseille-P3801T can grow in media ranging from 2 to 20% (w/v) NaCl (optimum at 7.5 (w/v) NaCl). The growing colonies are yellow and circular with a mean diameter of 2 mm. Bacterial cells were motile by using peritrichous flagella under electron microscopy, and were Gram positive, rod shaped and polymorphic, and catalase and oxidase positive. Strain Marseille-P3801T exhibited a 98.45% sequence identity with Gracilibacillus thailandensis strain TP2-8 (GenBank accession no. NR_116568.1) (Fig. 1) [7], which allowed us to classify it as a member of the genus Gracilibacillus within the family Bacillaceae in the phylum Firmicutes. Strain Marseille-P3801T is the type strain of the new species ‘Gracilibacillus phocaeensis’ (pho.ca.een'sis, N.L. masc. adj., from phocaeensis, related to the Phocaeans, the founders of Marseille).

FIG. 1

Phylogenetic tree showing position of ‘Gracilibacillus phocaeensis’ Marseille-P3801T relative to other phylogenetically close neighbours. 16S rRNA gene sequences were aligned using ClustalW, and phylogenetic inferences were obtained using maximum-likelihood method within MEGA software. Numbers at nodes are percentages of bootstrap values obtained by repeating analysis 500 times to generate majority consensus tree. Only bootstrap scores of ≥75 were retained. Scale bar indicates 0.005 nucleotide sequence divergence.

Strain Marseille-P3518T was isolated from stool samples (2% NaCl) of a 15-year-old boy from Dielmo. Agar-grown colonies were beige, circular and shiny with a mean diameter of 2 mm. Bacterial cells were Gram positive, rod shaped and polymorphic, and had positive catalase and oxidase reaction. Strain Marseille-P3518T exhibited a 97.4% sequence identity with Sediminibacillus albus strain NHBX5 (GenBank accession no. NR_044031.1) [8], the phylogenetically closest species with standing in nomenclature (Fig. 2), which putatively classifies it as a member of the genus Sediminibacillus within the family Bacillaceae in the phylum Firmicutes. Strain Marseille-P3518T is the type strain of the new species Sediminibacillus massiliensis (ma.si.lien'sis, L. masc. adj., from massiliensis, related to the university hospital in Marseille, France, where the strain was isolated).

FIG. 2

Phylogenetic tree showing position of ‘Sediminibacillus massiliensis’ Marseille-P3518T relative to other phylogenetically close neighbours. Sequences alignment and phylogenetic inferences were realized as explained in Fig. 1. Scale bar represents 0.01 nucleotide sequence divergence.

Strain Marseille-P3835T was isolated from in stool samples (3.7% NaCl) of a 11-year-old boy from N'Diop. Strain Marseille-P3835T is Gram positive, and catalase and oxidase positive. The strain was able to grow in 0.5 to 15% (w/v) NaCl, with an optimum growth at 5% (w/v) NaCl. The agar colonies are pink and circular, with a mean diameter of 2 mm. Strain Marseille-P3835T exhibited a 16S rRNA sequence similarity of 98.6% with Virgibacillus zhanjiangensis strain JSM 079157 (GenBank accession no. NR_116658.1) (Fig. 3) [9]. On the basis of this result, we propose to classify ‘Virgibacillus ndiopensis’ as a new representative of the Virgibacillus genus belonging to the family Bacillaceae, of the phylum Firmicutes. Strain Marseille-P3835T is the type strain of ‘Virgibacillus ndiopensis’ (ndiop.en'sis, L. masc. adj., from ndiopensis, related to N'Diop, a Senegalese village from which stool samples were collected).

FIG. 3

Phylogenetic tree showing position of ‘Virgibacillus ndiopensis’ Marseille-P3835T relative to other phylogenetically close neighbours. Sequences alignment and phylogenetic inferences were realized as explained for Fig. 1. Scale bar represents 0.002 nucleotide sequence divergence.

MALDI-TOF MS spectrum

The MALDI-TOF MS spectrum of strains is available online (http://www.mediterranee-infection.com/article.php?laref=256&titre=urms-database).

Nucleotide sequence accession number

The 16S rRNA gene sequences were deposited in GenBank under accession numbers ‘Gracilibacillus phocaeensis’ Marseille-P3801T (LT934503), ‘Sediminibacillus massiliensis’ Marseille-P3518T (LT671588) and ‘Virgibacillus ndiopensis’ Marseille-P3835T (LT883149).

Deposit in a culture collection

The strains were deposited in the Collection de Souches de l'Unité des Rickettsies (CSUR, WDCM 875) under the following accession numbers: ‘Gracilibacillus phocaeensis’ Marseille-P3801T (P3801), ‘Sediminibacillus massiliensis’ Marseille-P3518T (3518) and ‘Virgibacillus ndiopensis’ Marseille-P3835T (P3835).

Conflict of interest

None declared.