Accepted Manuscript Christensenella massiliensis, a new bacterial species isolated from the human gut Sokhna Ndongo, Saber Khelaifia, Pierre-Edouard Fournier, Didier Raoult PII: S2052-2975(16)30028-2 DOI: 10.1016/j.nmni.2016.04.014 Reference: NMNI 166 To appear in: New Microbes and New Infections Received Date: 26 April 2016 Revised Date: 29 April 2016 Accepted Date: 29 April 2016 Please cite this article as: Ndongo S, Khelaifia S, Fournier P-E, Raoult D, Christensenella massiliensis, a new bacterial species isolated from the human gut, New Microbes and New Infections (2016), doi: 10.1016/j.nmni.2016.04.014. 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 1 Christensenella massiliensis , a new bacterial species isolated from the human gut 2 Sokhna Ndongo 1, Saber Khelaifia 1, Pierre-Edouard Fournier 1 and Didier Raoult 1, 2 * 3 Running title: Christensenella massiliensis 4 1 Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes, CNRS (UMR 5 7278), IRD (198), INSERM (U1095), AMU (UM63) 6 2 Special Infectious Agents Unit, King Fahd Medical Research Center, King Abdulaziz 7 University, Jeddah, Saudi Arabia 8 9 *Corresponding author: Didier Raoult 10 URMITE, CNRS (UMR 7278), IRD (198), INSERM (U1095), AMU (UM63), Faculté de 11 Médecine, Aix-Marseille Université, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 5, 12 France. Tel: 00 33 4 91 32 49 50, Fax: 00 33 4 91 MANUSCRIPT38 77 72. E-mail: [email protected]. 13 Keywords: Culturomics, taxonomy, Christensenella massiliensis ACCEPTED 1 ACCEPTED MANUSCRIPT 1 Abstract 2 We described here the main characteristics of Christensenella massiliensis , strain Marseille- 3 P2438 T (CSUR P2438) isolated from the stool specimen of a 66-year-old patient. MANUSCRIPT ACCEPTED 1 ACCEPTED MANUSCRIPT 4 In this report, we present an early description of a new bacterium isolated as part of 5 culturomics study of the human gut microbiota [1] from stool specimen of a 66-year-old 6 patient hospitalized in November 2015 at the Timone Hospital in Marseilles, France. 7 Approval of the study by the local ethics committee of IFR48 (Marseille, France) was 8 obtained beforehand under 09-022 agreement. After obtaining the consent signed by the 9 patient, the stool sample was directly pre-incubated for 7 days in an anaerobic blood bottle 10 culture supplemented with 5 mL of rumen fluid. Then, isolated colonies of the strain were 11 obtained by subculturing on 5% sheep blood agar (bioMérieux, Marcy l’Etoile, France) in 12 anaerobic conditions generated by AnaeroGen TM (bioMérieux) after 4-day incubation. Our 13 systematic matrix-assisted laser desorption-ionization time-of-flight MALDI-TOF screening 14 on a MicroFlex spectrometer (Bruker Daltonics, Germany) [2] could not be able to identify 15 this strain. The strain Marseille-P2438 feature Gram negative bacilli, strict anaerobic, no 16 motile, and non-spore forming (0.3–0.5 × 1.2–1.5 µm). It presents no catalase and oxidase 17 activity. The colonies are yellow-white, circular andMANUSCRIPT about 0.1-0.2 mm in diameter after 4-day 18 incubation on 5% sheep blood agar. The optimal growth occurs at 37°C, pH 7 and 0.5% NaCl. 19 Using a 3130-XL sequencer (Applied Biosciences, Saint Aubin, France), the 16S rRNA gene 20 was sequenced using the universal primers fD1 and rp2 (Eurogentec, Angers, France) as 21 previously described [3]. Strain Marseille-P2438 (GenBank LT161898) exhibited a 97.5% 22 sequence identity with Christensenella minuta strain YIT 12065 T (GenBank accession number 23 NR112900), the phylogenetically closest species with standing in nomenclature (Figure 1), 24 which putativelyACCEPTED classifies it as a member within the genus Christensenella in the Firmicutes 25 phylum [4]. 26 C. minuta strain YIT 12065T T is strictly anaerobic, Gram-negative, non-motile, non-spore- 27 forming and short straight rods isolated from human feces. Catalase and oxidase activities for 2 ACCEPTED MANUSCRIPT 28 C. minuta were negative. At to date, it is the first species of Christensenella genus published 29 with validated name [4]. 30 Strain Marseille-P2438 (DSM 102344) shows a 16S rRNA gene sequence divergence >1.3% 31 with its phylogenetically closest species with a validly published name standing in 32 nomenclature [5]. Based on these results, strain Marseille-P2438 T is proposed as a novel 33 species within the genus Christensenella , namely Christensenella massiliensis sp. nov. 34 35 MALDI-TOF-MS spectrum accession number . The MALDI-TOF-MS spectrum of 36 Christensenella massiliensi is available at: 37 http://www.mediterraneeinfection.com/article.php?laref=256&titre=urms-database 38 39 Nucleotide sequence accession number. The 16S rRNA gene sequence was deposited in 40 GenBank under accession number LT161898. MANUSCRIPT 41 42 Deposit in a culture collection. Strain Marseille-P2438 T was deposited in the Collection de 43 Souches de l’Unité des Rickettsies (CSUR) under number P2438. 44 45 46 ACCEPTED 3 ACCEPTED MANUSCRIPT 47 ACKNOWLEDGMENTS 48 This research is funded by the Mediterranee-Infection Foundation. MANUSCRIPT ACCEPTED 4 ACCEPTED MANUSCRIPT 49 References 50 [1] Lagier JC, Hugon P, Khelaifia S, Fournier PE, La Scola B, Raoult D. The rebirth of 51 culture in microbiology through the example of culturomics to study human gut microbiota. 52 Clin Microbiol Rev 2015;28:237-64. 53 [2] Seng P, Abat C, Rolain JM, Colson P, Lagier JC, Gouriet F, et al. Identification of rare 54 pathogenic bacteria in a clinical microbiology laboratory: impact of matrix-assisted laser 55 desorption ionization-time of flight mass spectrometry. J Clin Microbiol 2013;51:2182-94. 56 [3] Drancourt M, Bollet C, Carlioz A, Martelin R, Gayral JP, Raoult D. 16S ribosomal DNA 57 sequence analysis of a large collection of environmental and clinical unidentifiable bacterial 58 isolates. J Clin Microbiol 2000;38:3623-30. 59 [4] Morotomi M, Nagai F, Watanabe Y. Description of Christensenella minuta gen. nov., sp. 60 nov., isolated from human faeces, which forms a distinct branch in the order Clostridiales, and 61 proposal of Christensenellaceae fam. nov. Int J SystMANUSCRIPT Evol Microbiol 2012;62:144–9. 62 [5] Kim M, Oh HS, Park SC & Chun J. Towards a taxonomic coherence between average 63 nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of 64 prokaryotes. Int J Syst Evol Microbiol 2014;64:346-51. ACCEPTED 5 ACCEPTED MANUSCRIPT 65 Figure legend 66 Figure 1: Phylogenetic tree showing the position of Christensenella massiliensis strain 67 Marseille-P2438 T relative to other phylogenetically-close members of the family 68 Christensenellaceae . GenBank accession numbers are indicated in parentheses. Sequences 69 were aligned using CLUSTALW, and phylogenetic inferences obtained using the maximum- 70 likelihood method within the MEGA software. Numbers at the nodes are percentages of 71 bootstrap values obtained by repeating the analysis 500 times to generate a majority 72 consensus tree. Only values greater than 95% were displayed. The scale bar indicates a 2% 73 nucleotide sequence divergence. MANUSCRIPT ACCEPTED 6 ACCEPTED MANUSCRIPT 74 Christensenella minuta (NR_112900) 75 Christensenella massiliensis (LT_161898) 76 Catabacter hongkongensis (NR_104811) Acetomicrobium faecale (NR_104811) Tindallia texcoconensis (NR_043664) Alkaliphilus metalliredigens (NR_074633) MANUSCRIPT ACCEPTED 7 .