Accepted Manuscript Description of Clostridium phoceensis sp. nov., a new species within the genus Clostridium Michel Hosny, Samia Benamar, Guillaume Durand, Nicholas Armstrong, Caroline Michelle, Frédéric Cadoret, Bernard La Scola, Nadim Cassir PII: S2052-2975(16)30098-1 DOI: 10.1016/j.nmni.2016.09.008 Reference: NMNI 229 To appear in: New Microbes and New Infections Received Date: 17 June 2016 Revised Date: 9 September 2016 Accepted Date: 16 September 2016 Please cite this article as: Hosny M, Benamar S, Durand G, Armstrong N, Michelle C, Cadoret F, La Scola B, Cassir N, Description of Clostridium phoceensis sp. nov., a new species within the genus Clostridium, New Microbes and New Infections (2016), doi: 10.1016/j.nmni.2016.09.008. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. ACCEPTED MANUSCRIPT Description of Clostridium phoceensis sp. nov., a new species within the genus Clostridium Michel Hosny 1, Samia Benamar 1†, Guillaume Durand 1, Nicholas Armstrong 1, Caroline Michelle 1, Frédéric Cadoret 1, Bernard La Scola 1, Nadim Cassir 1*, 1 Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes (URMITE), UM63 CNRS 7278 IRD 198 INSERM U1095, IHU Méditerranée Infection, Pôle des Maladies Infectieuses, Assistance Publique-Hôpitaux de Marseille, Faculté de Médecine, 27 Bd Jean 9 Moulin, 13005 Marseille, France. † These authors contributed equally to this work. MANUSCRIPT *Corresponding authors: Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes, URMITE, UM63, CNRS 7278, IRD 198, INSERM 1095, Faculté de médecine, Aix-Marseille Université 27 Boulevard Jean Moulin, 13385 Marseille cedex 05, France Tel: +33 491 385 517, Fax: +33 491 387 772 E-mail: Nadim Cassir: [email protected] ACCEPTED Running head : Taxono-genomic and description of Clostridium phoceensis strain GD3 T ACCEPTED MANUSCRIPT 1 TAXONOGENOMICS: GENOME OF A NEW ORGANISM 2 3 Description of Clostridium phoceensis sp. nov., a new species within the genus Clostridium 4 5 6 Running head : Taxono-genomic and description of Clostridium phoceensis strain GD3T MANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT 7 Abstract 8 Clostridium phoceencis sp. nov, strain GD3 T (CSUR P1929, DSM 100334) is the type strain of C. 9 phoceensis sp. nov. , a new species within the genus Clostridium . This strain was isolated from the 10 gut microbiota of a 28-year-old healthy French man. Clostridium phoceensis is a Gram-negative 11 spore-forming, non-motile, strictly anaerobic bacterium. Here, we describe its complete genome 12 sequence and annotation, together with its phenotypic characteristics. 13 MANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT 14 Introduction 15 Human adult gut microbiota is estimated to consist of up to 100 trillion microorganisms, 16 comprising at least 500 different species, mostly anaerobic bacteria [1]. Although the advent of 17 modern molecular microbiological methods has expanded the degree of bacterial detection from 18 stool samples, it does not allow for the phenotypic description of new living species [2]. 19 Consequently, there has been renewed interest in culture methods [3]. 20 A new taxonomic approach known as ‘taxonogenomics’ has recently been proposed to 21 describe new isolated bacterial species [4]. This polyphasic strategy combines phenotypic 22 characteristics, the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry 23 (MALDI-TOF) spectrum, and the analysis and comparison of the complete genome sequence. 24 Since the creation of the genus Clostridium in 1880, more than 200 species have been 25 described [5]. While species belonging to the family Clostridiaceae are mainly associated with the 26 commensal digestive flora of mammals and can be commonly found in the environment, some are 27 major human pathogens, including toxigenic ClostridiumMANUSCRIPT botulinum, Peptoclostridium difficile, 28 Clostridium tetani and Clostridium perfringens [6]. 29 We propose Clostridium phoceencis sp. nov, strain GD3 T (CSUR P1929, DSM 100334) as 30 the type strain of C. phoceensis sp. nov. , a new species within the genus Clostridium . This strain 31 was isolated from the gut microbiota of a 28-year-old healthy French man as part of a ‘culturomics’ 32 study aiming at individually cultivating all bacterial species from a stool sample. Here, we describe 33 the characteristics of C. phoceensis sp. nov. strain GD3 T, including its phenotype and genome 34 sequence. 35 ACCEPTED 36 Material and Methods 37 Ethic and collection of the sample 38 The stool sample was taken from a 28-year-old healthy French man. The sample was collected as 39 part of a research study on the human gut microbiota. The study was approved by the Institut 40 Fédératif de Recherche 48 (agreementACCEPTED number 09–022, MANUSCRIPT Marseille, France) and the patient’s consent 41 was obtained. The sample was stored at -80°C in La Timone Hospital (Marseille, France). 42 Strain isolation and identification (MALDI-TOF MS and 16S rRNA sequencing) 43 The fecal sample was treated using the concept of culturomics [7]. The colonies obtained were 44 identified using MALDI-TOF MS [8,9] (Brüker Daltonics, Leipzig, Germany) and analyzed using a 45 Microflex spectrometer (Brüker Daltonics), leading to the protein spectrum being obtained. A score 46 under 1.7 did not enable any identification. Subsequently, 16S rRNA was sequenced and the 47 sequence was matched using BLAST against NCBI [10]. DNA was extracted using the EZ1 Tissue 48 Extraction Kit (Qiagen, Hilden, Germany) and sequences were aligned using ChromasPro version 49 1.6 (Technelysium Pty Ltd, South Brisbane, Queensland, Australia). 50 Growth conditions 51 The growth condition of the strain was determined by testing different temperatures and 52 atmospheres. Five growth temperatures (ambient, 28, 37, 45 and 56°C) were tested under anaerobic 53 (GENbag Anaer) microaerophilic atmospheres (GENbagMANUSCRIPT microer) (BioMerieux, Marcy l’Etoile, 54 France) and aerobic condition on 5% sheep blood aga r (BioMerieux, France). Colonies were 55 obtained after thermal shock for 20 minutes at 80°C, in an anaerobic blood culture bottle (Bactec TM 56 Lytic/10 Anaerobic/F) supplemented with 5% sheep blood at 37°C. 57 Phenotypic, biochemical and antibiotic susceptibility tests 58 Phenotypic characteristics: Gram staining, motility, catalase and oxidase were determined as the 59 methods described by Lagier et al. [3]. Sporulation was tested using a thermal shock on bacterial 60 colonies (diluted in PBS) for 10 minutes at 80°C. The biochemical characteristics were tested using 61 API 50CH, API ZYMACCEPTED and API 20A strips (BioMérieux, France). Antibiotic susceptibility referred 62 to EUCAST 2015 recommendations. 63 Fatty acid methyl ester (FAME) analysis by GC/MS 64 Fatty acid methyl ester analysis was performed by GC/MS. Two samples were prepared with 2 mg 65 of bacterial biomass from several culture plates. Two samples were prepared with approximately 2 66 mg of bacterial biomass per tube harvestedACCEPTED from MANUSCRIPTseveral culture plates. Fatty acid methyl esters 67 were prepared as described by Sasser et al. [11]. GC/MS analyses were carried out as described by 68 Dione et al. [12]. Briefly, fatty acid methyl esters were separated using an Elite 5-MS column and 69 monitored by mass spectrometry (Clarus 500-SQ8S, Perkin Elmer, Courtaboeuf, France). A spectral 70 database search was performed using MS Search 2.0 operated using the Standard Reference 71 Database 1A (NIST, Gaithersburg, USA) and the FAMEs mass spectral database (Wiley, Chichester, 72 UK). 73 Microscopy 74 Individual cells of C. phoceensis strain GD3 T were captured using a Tecnai G20 electron 75 microscopy (FEI company, Limeil-Brevannes, France). A Color Gram 2 Kit (Biomerieux) was used 76 to perform the Gram coloration observed with a 100X oil-immersion objective lens using the 77 DM1000 photonic microscope (Leica Microsystems) [13,14]. 78 Genome project history 79 The organism was selected for sequencing because it MANUSCRIPThad been isolated from a healthy person for 80 the first time and on the basis of its 16S rRNA similarity, phylogenetic position, and phenotypic 81 differences with other members of the genus Clostridia. The Genbank accession number is 82 CVUG01000000 and consists of 10 scaffolds with a total of 16 contigs [15]. 83 Genome sequencing and assembly 84 The genomic DNA of Clostridium phoceensis GD3 T was sequenced on the MiSeq sequencer 85 (Illumina, San Diego, CA, USA) using the mate-pair strategy. The gDNA was barcoded in order to 86 be mixed with 11 other projects using the Nextera mate-pair sample prep kit (Illumina). The mate- 87 pair library was preparedACCEPTED with 1.5 µg of genomic DNA using the Nextera Mate-Pair Illumina guide. 88 The gDNA sample was simultaneously fragmented and tagged using a mate-pair junction adapter. 89 The pattern of the fragmentation was validated on an Agilent 2100 BioAnalyzer (Agilent 90 Technologies, Santa Clara, CA, USA) with a DNA 7500 labchip. The DNA fragments ranged in 91 size from 1 kb up to 10 kb with an optimal size at 4.490 kb. No size selection was performed and 92 only 600 ng of tagmented fragmentsACCEPTED were circularize MANUSCRIPTd. The circularized DNA was mechanically 93 sheared to small fragments with an optimal size of 938 bp on the Covaris S2 device in microtubes 94 (Covaris, Woburn, MA, USA). The library profile was visualized on a High Sensitivity Bioanalyzer 95 LabChip (Agilent Technologies) and the final library concentration was measured at 4.457 nmol/L. 96 The libraries were normalized at 2nM and pooled. After a denaturation step and dilution at 15 pM, 97 the pool of libraries was loaded onto the reagent cartridge and then onto the instrument along with 98 the flow cell.