“Caecibacter massiliensis” gen nov , sp nov isolated from the human right colon Accepted Manuscript “Caecibacter massiliensis” gen nov , sp nov isolated from the human right colon D Ricaboni, M Mailhe[.]
Accepted Manuscript “Caecibacter massiliensis” gen nov., sp nov isolated from the human right colon D Ricaboni, M Mailhe, A Benezech, F Cadoret, P.-E Fournier, D Raoult PII: S2052-2975(16)30133-0 DOI: 10.1016/j.nmni.2016.11.021 Reference: NMNI 264 To appear in: New Microbes and New Infections Received Date: November 2016 Revised Date: 14 November 2016 Accepted Date: 18 November 2016 Please cite this article as: Ricaboni D, Mailhe M, Benezech A, Cadoret F, Fournier P-E, Raoult D, “Caecibacter massiliensis” gen nov., sp nov isolated from the human right colon, New Microbes and New Infections (2016), doi: 10.1016/j.nmni.2016.11.021 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 “Caecibacter massiliensis” gen nov., sp nov isolated from the human right colon Running title : «Caecibacter massiliensis» RI PT D Ricaboni1-2,M Mailhe1, A Benezech3, F Cadoret1, P.-E Fournier1, D Raoult1* Aix-Marseille Université, URMITE, UM63, CNRS7278, IRD198, Inserm 1095, Institut Hospitalo-Universitaire Méditerranée-Infection, Faculté de médecine, 27 Boulevard Jean SC Moulin, 13385, Marseille cedex 05, France Department of Biomedical and Clinical Sciences, 3rd Division of Clinical Infectious Service de Gastroenterologie, Hopital Nord, Assistance Publique-Hopitaux de Marseille, 13915 Marseille, France Corresponding author E-mail address: didier.raoult@gmail.com TE D * M AN U Disease, University of Milan, Luigi Sacco Hospital, Italy AC C EP Keywords: Culturomics, taxono-genomics, “Caecibacter massiliensis”, human gut ACCEPTED MANUSCRIPT Abstract: We report here the main characteristics of “Caecibacter massiliensis” strain Marseille-P-2974T (CSURP2974) that was isolated from a human right colon sample AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT In the context of an ongoing culturomics [1] and metagenomic study which targets the modifications of the human microbiome in the different gastrointestinal tracts, we isolated from the right colon lavage sample of a 27-year-old obese patient a bacterial strain that escaped identification by our systematic matrix-assisted laser desorption-ionization time-of-flight screening mass spectrometry on a Microflex spectrometer (Bruker Daltonics, Brenen, Germany) [2] The patient 11 gave a signed informed consent and the study was validated by the ethics committee of the Institut 12 Fédératif de Recherche IFR48 under number 2016-010 13 Strain Marseille-P2974T first growth was obtained on 5% sheep blood-Columbia agar medium 14 (bioMérieux, Marcy l’Etoile, France) under anaerobic atmosphere (anaeroGEN, Oxoid Ltd, 15 Dardilly, France) after a 7-day enrichment of the fresh right colon lavage sample in an anaerobic 16 hemoculture bottle (Becton Dickinson, Pont de Claix, France) added with 5mL of sterile sheep 17 blood (bioMérieux) and 5mL of 0,2 µm filtered (Thermo Fisher Scientific, Villebon-sur-Yvette, 18 France) rumen at 37°C 19 After 96 hours of anaerobic incubation on 5% sheep blood-enriched agar (bioMérieux) at 37°C, 20 colonies were circular, convex with entire edges and whitish Mean diameter was 0.2-0.8 mm 21 Neither hemolysis nor pigmentation were observed 22 Bacterial cells were Gram-negative, non-motile cocci with a mean diameter ranging from 0.8 to 23 1.2µm Strain Marseille-P2974T tested catalase and oxidase negative Different temperatures (20°- 24 28°C-37°C-45°-55°C) and atmospheres (anaerobic, microaerophilic (CampyGEN,Oxoid Ltd) and 25 aerobic conditions) were tested on 5%-sheep blood-enriched Columbia agar (bioMérieux) Growth 26 was achieved only under anaerobic atmosphere at 37°C Sporulation test (20 at 80°C) was 27 negative 28 The 16S rRNA gene was sequenced using fD1-rP2 primers as previously described [3], using a 29 3130-XL sequencer (Applied Biosciences, Saint Aubin, France) Strain Marseille-P2974T exhibited 30 a 94.24 % sequence identity with Megasphaera cerevisiae strain JCM 6130T (Genbank accession 31 number: LC037207), the phylogenetically closest species with standing nomenclature (Fig 1), AC C EP TE D M AN U SC RI PT 10 ACCEPTED MANUSCRIPT which putatively classifies it as a member of a new genus within the family Veillonellaceae in the 33 phylum Firmicutes 34 The family Veillonellaceae is constitued by genera with validated and published names 35 (Allisonella, Anaeroglobus, Dialister, Megasphaera, Veillonella and Negativicoccus) Members of 36 this family are characterized by being Gram-negative cocci or cocco-bacilli with a microaerophilic 37 or anaerobic metabolism [4] The closest genus to strain Marseille-P2974T, Megasphaera, was 38 created in 1971 and all its six recognized species possess a strictly anaerobic metabolism [5] 39 Members of this genus are commonly found in rumen, human and pig intestine (Megasphaera 40 elsdenii), brewery associated environments (Megasphaera cerevisiae, Megasphaera paucivorans, 41 Megasphaera sueciensis) while Megasphaera micronuciformis was isolated from a human liver 42 abcess [6] 43 On the basis of the 16S rRNA sequence divergence of strain Marseille-P2974T with the 44 phylogenetically closest species with standing in nomenclature [7] we propose here the creation of 45 the new genus “Caecibacter” (Cae.ci.bac.ter L neut n caecum the caecum of the large intestine; 46 N.L masc n bacter a rod; N.L masc n Caecibacter, a rod from the large intesine) for which the 47 strain MarseilleP-2974T (=CSURP2974) is the type strain Strain Marseille-P2974T is the type strain 48 of “Caecibacter massiliensis” gen nov., sp nov., (mas.si.li.en’sis, L masc adj., massiliensis from 49 Massilia, the Roman name of Marseille) SC M AN U TE D EP AC C 50 RI PT 32 51 The MALDITOF spectrum of “Caecibacter massiliensis” strain Marseille-P2974 T is available at: 52 (http://www.mediterranee-infection.com/article.php?laref=256&titre=urms-database) 53 Nucleotide sequence accession number The 16S r RNA gene sequence was deposited in Genbank 54 under accession number LT576402 55 Deposit in a culture collection Strain Marseille-P2974T was deposited in the Collection de 56 Souches de l’Unité des Rickettsies (CSUR, WDCM 875) under number P2974 The deposit in 57 DSMZ collection is in progress ACCEPTED MANUSCRIPT Conflict of interest: 59 None to declare 60 Funding sources: 61 This work was funded by Mediterrannée-Infection Foundation AC C EP TE D M AN U SC RI PT 58 ACCEPTED MANUSCRIPT References 63 [1] Lagier JC, Hugon P, Khelaifia S, Fournier PE, La Scola B, Raoult D The rebirth of culture in 64 microbiology through the example of culturomics to study human gut microbiota Clin Microbiol 65 Rev 2015;28:237–64 66 [2] Seng P, Abat C, Rolain JM, Colson P, Lagier JC, Gouriet F, Fournier PE, Drancourt M, La Scola 67 B and Raoult D Identification of rare pathogenic bacteria in a clinical microbiology laboratory: 68 impact of matrix-assisted laser desorption ionization-time of flight mass spectrometry J Clin 69 Microbiol 2013;51:2182-94 70 [3] Drancourt M, Bollet C, Carlioz A,Martelin R, Gayral J-P, Raoult D 16S ribosomal DNA 71 sequence analysis of a large collection of environmental and clinical unidentifiable bacterial 72 isolates J Clin Microbiol 2000; 38:3623-3630 73 [4] Rogosa M Transfer of Peptostreptococcus elsdenii Gutierrez et al to a New Genus, 74 Megasphaera [M elsdenii (Gutierrez et al.) comb nov.] Inter J Syst Evol Microbiol 1971;21:187– 75 76 [5] Marchandin H, Teyssier C, Campos J, Jean-Pierre H, Roger F, Gay B, et al Negativicoccus 77 succinicivorans gen nov., sp nov., isolated from human clinical samples, emended description of 78 the family Veillonellaceae and description of Negativicutes classis nov., Selenomonadales ord nov 79 and Acidaminococcaceae fam nov in the bacterial phylum Firmicutes Int J Syst Evol Microbiol 80 2010;60:1271–9 81 [6] Lanjekar VB, Marathe NP, Ramana VV, Shouche YS, Ranade DR Megasphaera indica sp nov., 82 an obligate anaerobic bacteria isolated from human faeces Int J Syst Evol Microbiol 83 2014;64:2250–6 84 [7] Kim M, Oh H-S, Park S-C, Chun J Towards a taxonomic coherence between average nucleotide 85 identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes Int J Syst 86 Evol Microbiol 2014;64:346–51 87 AC C EP TE D M AN U SC RI PT 62 ACCEPTED MANUSCRIPT Figure 1: Phylogenetic tree showing the position of “Caecibacter massiliensis” strain Marseille- 89 P2974T relative to other phylogenetically-close neighbors Sequences were aligned using 90 CLUSTALW, and phylogenetic inferences obtained using the maximum-likelihood method within 91 the MEGA software Numbers at the nodes are percentages of bootstrap values obtained by 92 repeating the analysis 1.000 times to generate a majority consensus tree Only the bootstraps score 93 at least 90 were retained AC C EP TE D M AN U SC RI PT 88 ACCEPTED MANUSCRIPT Megasphaera paucivorans (NR 043657) 100 93 Megasphaera sueciensis ( NR 043656) Megasphaera cerevisiae (LC037207) Anaeroglobus geminatus (NR 028812) RI PT Megasphaera massiliensis (NR 133027) Megasphaera indica (NR 134080) 100 100 94 Megasphaera elsdenii (NR 102980) Caecibacter massiliensis (LT576402) Veillonella ratti (NR 113377) Veillonella criceti (NR 113376) SC 100 98 Veillonella magna ( NR 044400) Veillonella atypica (NR 119170) 100 M AN U Veillonella parvula (NR 074980) 100 Veillonella tobetsuensis (NR 113570) Veillonella denticariosi (NR 044153) Veillonella dispar ( NR 115355) Sporomusa malonica (NR 117652) Selenomonas bovis (NR 044111) 100 TE D 100 Mitsuokella multacida ( LC036321) Dialister micraerophilus (NR 113356) AC C 0.02 EP 100 Dialister succinatiphilus (NR 041666) 99 Dialister invisus (NR 113355) Dialister pneumosintes (LC037225) Allisonella histaminiformans (NR 028862) ... MANUSCRIPT “Caecibacter massiliensis” gen nov. , sp nov isolated from the human right colon Running title : «Caecibacter massiliensis» RI PT D Ricaboni1-2,M Mailhe 1, A Benezech 3, F Cadoret 1, P.-E... succinicivorans gen nov. , sp nov. , isolated from human clinical samples, emended description of 78 the family Veillonellaceae and description of Negativicutes classis nov. , Selenomonadales ord nov 79... which the 47 strain MarseilleP-2974T (=CSURP2974) is the type strain Strain Marseille-P2974T is the type strain 48 of “Caecibacter massiliensis” gen nov. , sp nov. , (mas.si.li.en’sis, L masc adj.,