TAXONOGENOMICS: GENOME OF A NEW ORGANISM Rubeoparvulum massiliense gen. nov., sp. nov., a new bacterial genus isolated from the human gut of a Senegalese infant with severe acute malnutrition M. Tidjani Alou1, J. Rathored1, J.-C. Lagier1,2,3, S. Khelaifia1, C. Michelle1, C. Sokhna2, A. Diallo2, A. B. Diallo4, P.-E. Fournier1, D. Raoult1,5 and S. Edouard1 1) Aix-Marseille Université, URMITE, UM63, CNRS7278, IRD198, INSERM 1095, Faculté de médecine, 2) Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes IRD 198, CNRS 7278, Aix-Marseille Université, Marseille, France, 3) Campus Commun UCAD-IRD of Hann, Dakar, Senegal, 4) Laboratoire de microbiologie, département de biologie, Université Abdou Moumouni de Niamey, Niamey, Niger and 5) Special Infectious Agents Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah, Saudi Arabia Abstract Rubeoparvulum massiliense strain mt6T was isolated from the gut microbiota of a severely malnourished boy from Senegal and consisted of facultative anaerobic, spore-forming, nonmotile and Gram-negative rods. R. massiliense showed a 92% similarity with the 16S rRNA of Bacillus mannanilyticus. The genome of strain mt6T is 2 843 796 bp long with a 43.75% G+C content. It contains 2735 protein-coding genes and 76 RNA genes, among which are nine rRNA genes. © 2016 Published by Elsevier Ltd on behalf of European Society of Clinical Microbiology and Infectious Diseases. Keywords: Culturomics, genome, gut microbiota, Rubeoparvulum massiliense, taxonogenomics Original Submission: 18 October 2016; Accepted: 9 November 2016 Article published online: 17 November 2016 irritable bowel syndrome, obesity [3,4] and severe acute Corresponding author: S. Edouard, Aix-Marseille Université, malnutrition [5–7]. URMITE, UM63, CNRS7278, IRD198, INSERM 1095, Faculté de médecine, 27 Boulevard Jean Moulin, 13385 Marseille cedex 05, A new cultural approach, microbial culturomics, based on France the multiplication of culture conditions with a variation of E-mail: [email protected] temperature, media and atmosphere, was developed in our laboratory in order to explore as exhaustively as possible a microbial ecosystem [8,9]. Using this new approach, we isolated a new member of the Bacillaceae family. At this time, 52 vali- Introduction dated genera are part of the Bacillaceae family, which was created in 1895 by Fisher; Bacillus is its type genus, described by The human microbiome is defined as the sum of all microbes Cohn in 1872 [10]. Most species of this family are found in the colonizing the human body [1]. The gut microbiota is one of environment (soil, water and plants) and are opportunistic the largest microbial ecosystems of the human body, con- pathogens in humans, except Bacillus anthracis, which is well sisting of 1014 microbial cells with a microbiome 150 times known as being highly pathogenic. The Bacillaceae family in- larger than the human genome [2]. The gastrointestinal cludes Gram-positive, rod-shaped, mostly aerobic and faculta- microbiota colonization starts before birth with the maternal tive anaerobic genera [11]. By adding the description of the microbiota, and its early composition is influenced by the assembled and annotated genome of the species and the pro- mode of birth. Its composition matures rapidly for the first teomic description of the strain with the matrix-assisted year and reaches adult form by 3 years [2,3]. A disruption of desorption ionization–time of flight mass spectrometry its equilibrium has been proven to be implicated in a growing (MALDI-TOF MS) profile to the classical description principles number of pathologies such as inflammatory bowel disease, (phylogenetic relationships based on the 16S rRNA sequence, New Microbe and New Infect 2017; 15: 49–60 © 2016 Published by Elsevier Ltd on behalf of European Society of Clinical Microbiology and Infectious Diseases This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) http://dx.doi.org/10.1016/j.nmni.2016.11.003 50 New Microbes and New Infections, Volume 15 Number C, January 2017 NMNI phenotypic and genotypic characteristics), a new concept of temperatures (25, 30, 37, 45 and 56°C) were tested under description called taxonogenomics was developed in our lab- anaerobic and microaerophilic atmospheres using GENbag oratory [12]. anaer and GENbag microer systems respectively (bio- Here we describe the genus Rubeoparvulum, the type species Mérieux, Marcy l’Étoile, France). Aerobic growth was tested of which is Rubeoparvulum massiliense strain mt6 (= CSUR with and without 5% CO2. Growth was also tested at various P1473 = DSM 100479) from a stool sample collected in a 2- pHs (6, 6.5, 7, 7.5, 8 and 8.5) using a pH-adjusted Colombia month-old infant living in Senegal and presenting with kwashi- agar (bioMérieux). Salt tolerance was also tested with 0.5, 1, orkor, a type of severe acute malnutrition. 5, 7.5 and 10% (w/v) NaCl. Morphologic, biochemical and antibiotic susceptibility Materials and Methods tests Phenotypic characteristics (Gram staining, sporulation, Ethics and sample collection motility) were determined as previously described [8].The The strain mt6 was isolated from a stool taken from a severely catalase (bioMérieux) and oxidase (Becton Dickinson, Le Pont malnourished 2-month-old boy with a height-for-age score de Claix, France) activities were also tested. Cell morphology of −5.87 who had nutritional edoema. Collection was per- was observed after negative staining of bacteria using a Tecnai formed in Senegal in April 2014. This sampling was undertaken G20 transmission electron microscope (FEI Company, Limeil- T as part of an exploratory study of the human gut microbiota in Brevannes, France). The biochemical features of strain mt6 African children with malnutrition. The study was approved by were investigated with API 50CH, API ZYM and API 20A ’ the local IFR 48 ethics committee under agreement 09-022. strips (bioMérieux) according to the manufacturer sin- The boy’s parents provided informed oral consent. The sample structions. Cellular fatty acid methyl ester (FAME) analysis was stored at −80°C after collection. was performed by gas chromatography mass spectrometry (GC/MS). Strain mt6 was grown on 5% sheep’s blood– Strain identification by MALDI-TOF MS and 16S rRNA enriched Colombia agar (bioMérieux) for the fatty acid anal- sequencing ysis, which was carried out by GC/MS. Approximately 67 mg In order to explore as exhaustively as possible the bacterial of bacterial biomass was each collected from several culture diversity of the faecal sample, the culturomics concept was used plates. Cellular FAMEs were prepared as described by Sasser to culture this sample using 18 culture conditions [8]. The (http://www.midi-inc.com/pdf/MIS_Technote_101.pdf). purified colonies obtained were identified using MALDI-TOF Briefly, GC/MS analyses were realized by a Clarus 500 gas MS as described previously [13,14]. Colonies were deposited chromatographequippedwithaSQ8SMSdetector(Perkin on a MTP 96 MALDI-TOF MS target plate (Bruker Daltonics, Elmer, Courtaboeuf, France). A total of 2 μLofFAMEex- Leipzig, Germany), which was analysed with a Microflex spec- tracts were volatized at 250°C (split 20 mL/min) in a Focus trometer (Bruker Daltonics). The spectra obtained were liner with wool and separated on an Elite-5MS column (30 m, matched against the references of the 7567 bacteria contained 0.25 mm i.d., 0.25 mm film thickness) using a linear temper- in the database by standard pattern matching (with default ature gradient (70 to 290°C at 6°C/min), allowing the parameter settings) with MALDI BioTyper database software detection of C4 to C24 fatty acid methyl esters. Helium 2.0 (Bruker Daltonics). An identification score over 1.9 with a flowing at 1.2 mL/min was used as carrier gas. The MS inlet validated species allowed the identification at the species level, line was set at 250°C and EI source at 200°C. Full scan and a score under 1.7 did not enable any identification. The 16S monitoring was performed from 45 to 500 m/z.Alldatawere rRNA gene was amplified and sequenced as previously collected and processed using Turbomass 6.1 (Perkin Elmer). described [15]. The obtained 16S rRNA sequence was FAMEs were identified by a spectral database search using MS compared to those in GenBank (http://blast.ncbi.nlm.nih.gov. Search 2.0 operated with the Standard Reference Database gate1.inist.fr/Blast.cgi) to determine the percentage of 1A (National Institute of Standards and Technology (NIST), sequence similarity with the closest bacteria. A new species or Gaithersburg,MD,USA)andtheFAMEsmassspectraldata- genus was defined by a similarity level of the 16S rRNA base (Wiley, Chichester, UK). Retention time correlations sequence under 98.65% or 95% respectively [16]. with estimated nonpolar retention indexes from the NIST database were obtained using a 37-component FAME mix Growth conditions (Supelco; Sigma-Aldrich, Saint-Quentin Fallavier, France); The ideal growth conditions of strain mt6T were determined FAME identifications were confirmed using this index. Anti- by testing different culture conditions. Five growth biotic susceptibility testing was performed using a disk © 2016 Published by Elsevier Ltd on behalf of European Society of Clinical Microbiology and Infectious Diseases, NMNI, 15,49–60 This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). NMNI Tidjani Alou et al. Rubeoparvulum massiliense gen. nov., sp. nov. 51 diffusion method according to European Committee on Genome annotation and comparison Antimicrobial Susceptibility Testing (EUCAST) 2015 recom- Open reading frames (ORFs) were predicted using Prodigal mendations [17]. Inhibition diameters were measured using [18] with default parameters, but the predicted ORFs were the Scan1200 scanner (Interscience, Saint-Nom-La Bretêche, excluded if they spanned a sequencing gap region. The pre- France). dicted bacterial protein sequences were searched against the GenBank [19] and the Clusters of Orthologous Groups Genomic DNA (gDNA) preparation (COGs) databases using BLASTP (E value 1e-03, coverage 0.7 For gDNA preparation, R.