ORIGINAL ARTICLE Description of Gracilibacillus phocaeensis sp. nov., a new halophilic bacterium isolated from Senegalian human stool I. I. Ngom1,2,I.Hasni1,2,4, B. Senghor1,2,C.I.Lo2,3,N.Armstrong1,2, C. Sokhna2,3,D.Raoult1,2, P.-E. Fournier2,3 and J.-C. Lagier1,2 1) Aix-Marseille Université, IRD, APHM, MEPHI, 2) IHU-Méditerranée Infection, 3) Aix-Marseille Université, IRD, APHM, SSA, VITROME, Marseille and 4) Amoeba R&D Department, Chassieu, France Abstract Using the taxonogenomics method, we describe Gracilibacillus phocaeensis strain Marseille-P3801, a new species previously isolated from a salty stool of a 20-year-old man from N’Diop, Senegal. It is a Gram-positive, aerobic and motile bacillus. The major fatty acids are C15:0- anteiso (59%), C16:0 (16%) and C17:0-anteiso (11%). Strain Marseille-P3801 exhibits a 98.45% sequence similarity with Gracilibacillus thailandensis strain TP2-8, the phylogenetically closest species. Its genome is 4.66 Mb with 39.6 mol% G + C content. © 2020 The Authors. Published by Elsevier Ltd. Keywords: Culturomics, genome, Gracilibacillus phocaeensis sp. nov., gut, taxonogenomics Original Submission: 19 August 2020; Revised Submission: 5 October 2020; Accepted: 19 October 2020 Article published online: 27 October 2020 The culturomics approach, which is based on the multiplica- Corresponding author: J.-C. Lagier, Institut Hospitalo- tion of culture conditions (variation of media, temperature and Universitaire Méditerranée-Infection, 19-21 Boulevard Jean Moulin, 13385, Marseille cedex 05, France. atmosphere) with a more rapid bacterial identification by matrix- E-mail: [email protected] assisted desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) [7] was used to explore the human gut halo- philic microbiota. Our culturomic approach included the use of high-salt-containing culture media, which allowed us to isolate a Introduction new moderately halophilic bacterial strain, Marseille-P3801T, which belongs to the genus Gracilibacillus [8]. The genus Gracili- bacillus currently compromises 13 species with valid standing in The genus Gracilibacillus has been described as moderately nomenclature [9]. Gracilibacillus species were isolated from halophilic, motile endospore-forming bacteria [1]. Moder- diverse salty environmental samples, including seawater, salty ately halophilic bacteria have been found in a variety of lakes [4,10,11], soil [12,13], food [1,14,15] and gut microbiota [6]. fermented foods; indeed, one of the most important food Various parameters, including phenotypic and genotypic char- preservation methods in history has been the use of salt [2]. acteristics, such as DNA-DNA hybridization, are used to define a Saltisthemainsourceofsodiuminourdiet.Somegut new species, but they have certain limitations [16]. In our study, we bacteria, such as Lactobacillus, are highly sensitive to salt [3]. sought to get around these limitations by using a taxonogenomic However, it has been demonstrated to favour the emergence approach that includes phenotypic characteristics, proteomic in- and growth of other, mainly halophilic, bacteria, including formation obtained by MALDI-TOF MS and analysis of the com- Gracilibacillus [4]. Halophilic and halotolerant bacteria are plete genome sequence. From this information, we were able to most commonly isolated from the human gut microbiota construct a complete description of a new halophilic species, [5,6]. G. phocaeensis, with type strain Marseille-P3801T (= CSUR P3801). New Microbe and New Infect 2020; 38: 100799 © 2020 The Authors. Published by Elsevier Ltd This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) https://doi.org/10.1016/j.nmni.2020.100799 2 New Microbes and New Infections, Volume 38 Number C, --- 2020 NMNI Materials and methods Blast.cgi) search was further performed against the GenBank nucleotide collection. If the 16S rRNA sequence similarity value was lower than 98.65% with the most closely related Bacterial strains and growth conditions species with standing in nomenclature, as proposed by Strain Marseille-P3801T was isolated from stool of a 20-year- Stackebrandt [21], the strain was proposed as belonging to a ’ old man from N Diop, Senegal. The study was approved by the new species [22]. ethics committee of the Institut Hospitalo-Universitaire Médi- terranée Infection (approval 2016-011), and the patient pro- Phenotypic and biochemical characteristics vided written informed consent. The morphology of strain Marseille-P3801T was revealed by The percentage of salt in the stool sample was determined negative staining observed with a Hitachi SU5000 scanning elec- fi using a salinity refractometer (Thermo Fisher Scienti c, tron microscope (Hitachi Group, Krefeld, Germany) and Gram Villebon-sur-Yvette, France) by diluting 1 g of NaCl in 10 mL of staining observed on a Leica DM2500 photonic microscope distilled water and centrifuging it for 10 minutes at 5000g. For a (Leica Microsystems, Nanterre, France) with a 100× oil- μ second run, 100 L of supernatant was deposited in the immersion objective. Sporulation, motility catalase and oxidase refractometer; the results were in a straight line, displayed on were tested, as previously reported [23,24]. To determine the screen as per-mille values, then reported in percentage of optimal growth conditions, strain Marseille-P3801 was cultivated NaCl. To culture the bacteria from stool samples, we used an in Müller-Hinton agar (Sigma-Aldrich) by varying the NaCl con- aerobic blood culture bottle (Becton Dickinson, Le Pont-de- centrations (from 5% to 20% (w/v)) as well as the pH (5, 5.5, 6, Claix, France) containing a halophilic medium prepared in a 6.5, 7, 7.5 and 8). It was also seeded on 5% sheep’s fi modi ed Columbia broth (Sigma-Aldrich, Saint-Quentin- blood–enriched Columbia agar (bioMérieux, Marcy l’Etoile, Fallavier, France), by adding (per litre): 1% (w/v) MgSO4, 0.1% France) and incubated under different temperatures (25, 28, 37, (w/v) MgCl2, 0.4% (w/v) KCl, 0.1% (w/v) CaCl2, 0.05% (w/v) 45 and 55°C). API 50 CH, ZYM and 20 NE test strips (bio- NaHCO3, 0.2% (w/v) of glucose, 0.5% (w/v) of yeast extract Mérieux) were used according to the manufacturer’s instructions – (Becton Dickinson) and 10 15% (w/v) NaCl according to the to study the carbohydrate metabolism, enzyme activity and required salinity, with pH adjusted to 7.5. This was incubated biochemical characteristics of strain Marseille-P3801. for 3 days in an aerobic atmosphere at 37°C [8]. All strains Cellular fatty acid methyl ester (FAME) analysis was performed fi were rst isolated in a halophilic culture medium with 15% (w/ by gas chromatography/mass spectrometry (GC/MS). Two sam- v) NaCl. ples were prepared with approximately 90 mg of bacterial The initial growth of colonies on agar was obtained after 24 biomass per tube, collected from several culture plates. FAMEs ’ hours incubation at 37°C under aerobic conditions. The oxy- were prepared as described by Sasser [25]. GC/MS analyses were gen requirement was evaluated by incubating strain Marseille- carried out as previously described [26]. Briefly, FAMEs were P3801T under aerobic, microaerophilic and anaerobic condi- separated using an Elite 5-MS column and monitored by MS tions using AnaeroGen (Atmosphere Generation Systems, (Clarus 500-SQ 8 S, PerkinElmer, Courtaboeuf, France). A fi Dardilly, France) at 37°C. The isolated colonies were identi ed spectral database search was performed using MS Search 2.0 using MALDI-TOF MS as previously described [17]. For the operated with the Standard Reference Database 1A (National fi unidenti ed colonies, the 16S ribosomal RNA (rRNA) gene was Institute of Standards and Technology (NIST), Gaithersburg, MD, sequenced, and the obtained sequence was matched against the USA) and the FAME MS database (Wiley, Chichester, UK). National Center for Biotechnology Information (NCBI) data- base using the BLAST algorithm [18]. Extraction, sequencing and assembly of genome Genomic DNA of Gracilibacillus phocaeensis was extracted in two 16S rRNA gene sequencing and phylogenetic analysis steps; first a mechanical treatment was performed by washing The 16S rRNA gene sequence of the strain was determined with glass beads and acid (G4649-500g; Sigma-Aldrich) using a for subsequent phylogenetic analysis. The genomic DNA of the FastPrep BIO 101 instrument (Qbiogene, Strasbourg, France) at fi strain was ampli ed by PCR using the primer pair fD1 and rP2 maximum speed (6.5 m/s) for 90 seconds. After 30 minutes’ (Eurogentec, Angers, France) [19] and sequenced with the incubation of the lysozyme at 37°C, the DNA was extracted by MiSeq Technology (Illumina, San Diego, CA, USA) as previ- the EZ1 biorobot (Qiagen, Germantown, MD, USA) with the ously described [20]. The 16S rRNA nucleotide sequences EZ1 DNA tissue kit. The elution volume was 50 μL. Genomic were assembled and corrected using CodonCode Aligner DNA was evaluated with a Qubit test (Life Technologies, software (https://www.codoncode.com/). A BLAST (Basic Carlsbad, CA, USA). The mate pair library was prepared with Local Alignment Search Tool; http://blast.ncbi.nlm.nih.gov/ 1.5 μg of genomic DNA using the Nextera mate pair Illumina © 2020 The Authors. Published by Elsevier Ltd, NMNI, 38, 100799 This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). NMNI Ngom et al. Gracilibacillus phocaeensis sp. nov. 3 guide. The DNA sample was simultaneously fragmented and SOAPdenovo2 [29] on trimmed (Trimmomatic) [30] or raw data. tagged with a mate pair junction adapter. The pattern of frag- To reduce assembly gap, GapCloser software was used. Scaffolds mentation was validated on an Agilent 2100 BioAnalyzer (Agilent less than 800 bp and scaffolds with a depth value of <25% of the Technologies, Santa Clara, CA, USA) with a DNA LabChip 7500 mean depth were discarded. The best assembly was selected using kit.