Accepted Manuscript Virgibacillus senegalensis sp. nov. A new moderately halophilic bacterium isolated from the human gut El hadji Seck, Jaishriram Rathored, Saber Khelaifia, Olivier Croce, Catherine Robert, Carine Couderc, Fabrizio Di Pinto, Cheikh Sokhna, Didier Raoult, Dr Jean-Christophe Lagier PII: S2052-2975(15)00079-7 DOI: 10.1016/j.nmni.2015.09.014 Reference: NMNI 78 To appear in: New Microbes and New Infections Received Date: 12 August 2015 Revised Date: 23 September 2015 Accepted Date: 24 September 2015 Please cite this article as: Seck Eh, Rathored J, Khelaifia S, Croce O, Robert C, Couderc C, Di Pinto F, Sokhna C, Raoult D, Lagier J-C, Virgibacillus senegalensis sp. nov. A new moderately halophilic bacterium isolated from the human gut, New Microbes and New Infections (2015), doi: 10.1016/ j.nmni.2015.09.014. This is a PDF file of an unedited manuscript that has been accepted for publication. 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ACCEPTED MANUSCRIPT Virgibacillus senegalensis sp. nov . a new moderately halophilic bacterium isolated from the human gut El hadji Seck 1, Jaishriram Rathored 1, Saber Khelaifia 1, Olivier Croce 1, Catherine Robert 1, Carine Couderc 1, Fabrizio Di Pinto 1, Cheikh Sokhna 2, Didier Raoult 1,3 and Jean-Christophe Lagier 1* 1 Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes, UM 63, CNRS 7278, IRD 198, Inserm 1095, Institut Hospitalo-Universitaire Méditerranée-Infection, Faculté de médecine, Aix-Marseille Université 2 Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes IRD 198, CNRS 7278, Aix-Marseille Université, Marseille, France; Campus Commun UCAD-IRD of Hann, Dakar, Senegal. 3Special Infectious Agents Unit, King Fahd MedicalMANUSCRIPT Research Center, King Abdulaziz University, Jeddah, Saudi Arabia *Corresponding author: Dr Jean-Christophe LAGIER URMITE, UMR CNRS 7278, L’Institut de Recherche pour le Développement 198, INSERM U1095, Faculté de Médecine, Aix-Marseille Université, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 5, France. Tel: 00 33 4 91 32 49 50, Fax: 00 33 4 91 38 77 72. E-mail: [email protected] ACCEPTED MANUSCRIPT 1 Abstract 2 Virgibacillus senegalensis SK-1T (CSUR P1101 = DSM 28585), is the type strain of V. 3 senegalensis sp. nov. It is an aerobic, Gram positive, moderately halophilic, motile bipolar 4 flagellum, isolated from a healthy Senegalese male. Here, we describe the genomic and 5 phenotypic characteristics of this isolate. The 3,755,098 bp long genome (1 chromosome, no 6 plasmid) exhibits a G+C content of 42.9 % and contains 3,738 protein-coding and 95 RNA 7 genes. 8 9 Keywords: Keywords: Virgibacillus senegalensis ; genome; moderately halophilic bacteria; 10 human gut; culturomics; taxonogenomics. 11 Abbreviations 12 CSUR: Collection de Souches de l’Unité des Rickettsies 13 DSM: Deutsche Sammlung von MikroorganismenMANUSCRIPT 14 MALDI-TOF MS: Matrix-assisted laser-desorption/ionization time-of-flight mass 15 spectrometry 16 TE buffer: Tris-EDTA buffer 17 SDS: sodium dodecyl sulfate 18 URMITE: Unité des Maladies Infectieuses et Tropicales Emergentes ACCEPTED ACCEPTED MANUSCRIPT 19 Introduction 20 The concept of "microbial culturomics" is based on the variation of physicochemical 21 parameters of the culture conditions, so as to express the maximum of microbial diversity. It 22 is based on rapid methods for identification as MALDI-TOF and 16SrRNA amplification and 23 sequencing for unidentified colonies. This concept enriches considerably the gut microbiota 24 repertoire including new species not previously isolated from humans [1, 2]. 25 This isolation is part of the "culturomics study", using high salt containing culture conditions 26 to grow halophilic bacteria from human stool [1]. 27 The typical parameters used to define bacterial species comprise 16S rRNA 28 sequencing and phylogeny, G + C content genomic diversity and DNA hybridization (DDH). 29 However, some limitations have been noted [3-6]. Using the availability of data in genomics 30 through the development of new tools for sequencing DNA, we introduced a new taxonomic 31 method for the description of new bacterial speciesMANUSCRIPT. This concept that we named 32 taxonogenomics includes their genomic features [7] and proteomic information obtained by 33 MALDI-TOF-MS analysis [8-17]. 34 The genus Virgibacillus was first proposed by Heyndrickx in 1998 with the transfer of 35 Bacillus pantothenticus to Virgibacillus pantothenticus . [18]. To date, there are more than 25 36 recognized species [19]. These bacteria are positive, Gram variable rods which are ellipsoidal 37 to oval endospores and contained the DNA G + C content ranging from 36 to 43%. [20]. 38 These species were isolated from sediments of a salt lake [20-23], fermented seafood in the 39 traditional salt [24],ACCEPTED a permafrost core collected from the Canadian High Arctic [25], a Navy 40 solar salt marsh [26, 27], soil [28], seawater [29], field soil, a dairy product [30], residual 41 wash water produced during processing wastewater, Spanish style green table olives [31], 42 saline sample of mud, salt-crust [32], and Thaï fermented fish [33]. Here, we present a brief 43 classification and a set of features for strain SK-1T (CSUR P1101 = DSM 28585), with the ACCEPTED MANUSCRIPT 44 description of the complete genome sequence and annotation. We named Virgibacillus 45 senegalensis this new isolate. 46 Materials and methods 47 Sample and culture condition 48 The stool sample was collected from a healthy male Senegalese volunteer patient 49 living in N’diop (a rural village in the Guinean-Sudanian zone in Senegal). After the patient 50 gave signed informed consent, the sample was collected in a sterile pot and transported in our 51 laboratory. The study and the assent procedure were approved by the National Ethics 52 Committee of Senegal and by the Ethics Committees of the Institut Fédératif de Recherche 53 48, Faculty of Medicine, Marseille, France, (agreement number 09-022). Salt concentration of 54 the stool specimen was determined by a digital refractometer (Fisher scientific, Illkirch, 55 France) and the pH using a pH-meter (Cyberscan510PH, Eutech instruments, Singapore) 56 Strain SK-1T was isolated in February 2014 by aerobic culture on a home-made 57 culture medium consisting of a Columbia agar culturMANUSCRIPTe medium (Sigma-Aldrich, Saint-Quentin 58 Fallavier, France) modified by adding (per liter): MgCl2 6H2O, 5 g; MgSO4 7H2O, 5 g; KCl, 59 2 g; CaCl2 2H2O, 1g; NaBr, 0,5 g; NaHCO3, 0,5 g, glucose, 2 g and 100g/L of NaCl. The pH 60 was adjusted to 7.5 with 10M NaOH before autoclaving. 61 62 MALDI-TOF identification 63 An isolated colony was deposited in duplicate on a MALDI-TOF target to be 64 analyzed. A matrixACCEPTED of 1,5 µL (Saturated solution of α-cyano-4-hydroxycinnamic acid diluted 65 in 500 µl acetonitrile, 250 µl of acid Tri-Fluoro-Acetic to 10%, and 250 µl of HPLC water) was 66 used on each spot. This solution enables ionization and desorption of the homogeneous 67 biological sample with which it crystallizes. The analysis was performed by a Microflex 68 (Bruker Daltonik) and protein spectra were compared with those of the hospital database. A ACCEPTED MANUSCRIPT 69 score was assigned indicating the reliability of the identification of the bacteria: above 1.9 is 70 considered a proper identification. Conversely if the bacterium is not referenced in the 71 database, sequencing the 16S rRNA is used in order to achieve a correct identification [34]. 72 73 Identification by sequencing of 16S rRNA 74 Colonies non identified by the MALDI-TOF after three tests were suspended in 200 µl 75 of distilled water for DNA extraction by EZ1 DNA Tissue Kit (QIAGEN,Venlo, Pays-Bas). 76 The amplification of the 16S rRNA was done by standard PCR in a thermocycler using the 77 universal primer pair FD1 and rp2 according to the following amplification program: 78 activation of the polymerase (95°C for 5 min) followed by 40 cycles (95°C 30sec, 52°C 79 45sec, 72°C 2min) followed by 5 min at 72°C. The DNA amplified by this reaction was 80 revealed by electrophoresis on 1.5% agarose gel. Once validated, the PCR product was 81 purified and sequenced using the Big Dye Terminator Sequencing Kit using the following 82 internal primers: 536F, 536R, 800F, 800R, 1050F,MANUSCRIPT 1050R, as previously described [2]. 83 84 Phylogenetic analysis 85 Phylogenetic analysis based on 16S rRNA of our isolates was performed to identify its 86 phylogenetic affiliations with other near isolates, including other members of the genus 87 Virgibacillus . The MEGA 6 (Molecular Evolutionary Genetics Analysis) software allowed us 88 to construct a phylogenetic tree. Sequence alignment of the different species was performed 89 using CLUSTALACCEPTED W and the evolutionary distance was calculated with the Kimura two- 90 parameter model [35]. 91 92 Biochemistry, atmospheric and antimicrobial susceptibility tests ACCEPTED MANUSCRIPT 93 Biochemical tests were performed using the commercially available Api ZYM 94 (BioMerieux, Marcy-l’Etoile, France), API 50CH (BioMerieux, Marcy-l’Etoile, France), 20 95 NE (BioMerieux, Marcy-l’Etoile, France) strips. The incubation time was 48 hours for the last 96 two and 4h for Api ZYM. Growth of the strain SK-1T was tested in aerobic atmosphere, in 97 the presence of 5% CO2, and also in anaerobic and microaerophilic atmospheres, created 98 using AnaeroGenTM (Atmosphere Generation Systems, Dardily, France). Antibiotic 99 susceptibility was determined by Mueller-Hinton agar in a Petri dish (BioMerieux, Marcy- 100 l’Etoile, France).