Bacillus Niameyensis Sp. Nov., a New Bacterial Species Isolated from Human Gut
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Accepted Manuscript Bacillus niameyensis sp. nov., a new bacterial species isolated from human gut Maryam Tidjani Alou, Jaishriram Rathored, Sory Ibrahima Traore, Saber Khelaifia, Caroline Michele, Souleymane Brah, Bouli Ali Diallo, Didier Raoult, Jean-Christophe Lagier PII: S2052-2975(15)00076-1 DOI: 10.1016/j.nmni.2015.09.011 Reference: NMNI 75 To appear in: New Microbes and New Infections Received Date: 8 July 2015 Revised Date: 13 September 2015 Accepted Date: 14 September 2015 Please cite this article as: Alou MT, Rathored J, Traore SI, Khelaifia S, Michele C, Brah S, Diallo BA, Raoult D, Lagier J-C, Bacillus niameyensis sp. nov., a new bacterial species isolated from human gut, New Microbes and New Infections (2015), doi: 10.1016/j.nmni.2015.09.011. 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. 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ACCEPTED MANUSCRIPT Bacillus niameyensis sp. nov., a new bacterial species isolated from human gut Maryam Tidjani Alou 1,5 , Jaishriram Rathored 1,5 , Sory Ibrahima Traore 1,5 , Saber Khelaifia 1, Caroline Michele 1, Souleymane Brah 2, Bouli Ali Diallo 3, Didier Raoult 1,4 , Jean-Christophe Lagier 1¶ 1Aix-Marseille Université, URMITE, UM63, CNRS7278, IRD198, Inserm 1095, Faculté de médecine, 27 Boulevard jean Moulin, 13385 Marseille cedex 05, France. 2 Hopital National de Niamey, Niamey, Niger 3Laboratoire de microbiologie, département de biologie, Université Abdou Moumouni de Niamey 4Special Infectious Agents Unit, King Fahd MedicalMANUSCRIPT Research Center, King Abdulaziz University, Jeddah, Saudi Arabia 5 equally contribution Corresponding author: Jean-Christophe Lagier Aix-Marseille Université,ACCEPTED URMITE, UMR CNRS 7278, IRD 198, INSERM U1095, Faculté de Médecine, 27 Bd Jean Moulin, 13385 Marseille cedex 5 Email: [email protected] Phone: (33) 491 32 43 75 - Fax: (33) 491 38 77 72 ACCEPTED MANUSCRIPT 1 Title: Bacillus niameyensis sp. nov., a new bacterial species isolated from human gut 2 Abstract 3 Bacillus niameyensis sp. nov. strain SIT3 T (= CSUR P1266 =DSM 29725) is the type strain of 4 B. niameyensis sp. nov . This Gram-positive strain was isolated from the digestive flora of a 5 child suffering from kwashiorkor and is a facultative anaerobic rod, member of the 6 Bacillaceae family. This organism is hereby described alongside its complete genome 7 sequence and annotation. The 4,286,116 bp long genome (one chromosome but no plasmid) 8 contains 4,130 protein-coding and 66 RNA genes including 5 rRNA genes. 9 Keywords: Bacillus niameyensis , genome, culturomics, taxono-genomics 10 Abbreviations 11 CSUR: Collection de souches de l’Unité des Rickettsies 12 DSM: Deutsche Sammlung von MikroorganismenMANUSCRIPT 13 MALDI-TOF MS: Matrix-assisted laser-desorption/ionization time-of-flight mass 14 spectrometry 15 TE buffer: Tris-EDTA buffer 16 SDS: sodium dodecyl sulfate 17 URMITE: Unité des Maladies Infectieuses et Tropicales Emergentes 18 19 20 ACCEPTED 21 22 23 1 ACCEPTED MANUSCRIPT 24 1. Introduction 25 Bacillus niameyensis strain SIT3 T (=CSUR P1266=DSM 29725) is the type strain of B. 26 niameyensis sp. nov. This bacterium is a Gram-positive bacillus, spore-forming, facultative 27 anaerobic and motile. It was isolated from the stool of a child living in Niamey, Niger, 28 suffering from kwashiorkor. This isolation was part of a “culturomics” study of the gut 29 microbiota of children affected with severe acute malnutrition aiming to characterize their 30 microbiota. Culturomics studies aim to explore as exhaustively as possible a microbial 31 ecosystem using multiple culture conditions [1; 2]. 32 Phylogenetic relationships based on the 16s ribosomal RNA gene are currently used for 33 bacterial classification alongside phenotypic and genotypic characteristics [3-5]. However, 34 with the development of genomic sequencing and its decreasing cost, a new concept of 35 bacterial description has been used in our laboratory [6-12] combining a proteomics 36 description with the MALDI-TOF profile [13] associated with a biochemical and genomic 37 description of the new bacterial species. MANUSCRIPT 38 The genus Bacillus was established in 1872 by Cohn and encompasses over 200 described 39 species and sub-species belonging to the firmicutes phylum. Bacillus species are strictly 40 aerobic and facultative anaerobic rod-shaped bacteria that form heat-resisting endospores [13- 41 16]. They are often found in the environment (water, soil, air), food, plants and human clinical 42 samples [17]. Some Bacillus species, such as Bacillus thuringiensis, are known to be 43 pathogenic for insects and are used as biological control agents for crops [18]. 44 2. Materials andACCEPTED methods 45 2.1. Organism information; Classification and features 46 A stool sample was collected from a 2-year-old boy living in Niamey, Niger who suffered 47 from kwashiorkor, which is a form of severe acute malnutrition. Consent was obtained from 48 the child’s parents at the National Hospital of Niamey and the study was approved by the 2 ACCEPTED MANUSCRIPT 49 Institut Fédératif de Recherche 48, Faculty of Medicine, Marseille, France, under agreement 50 number 09-022. The patient did not receive antibiotics at the time of sample collection and the 51 fecal sample was stored at -80°C. 52 2.3. Strain identification 53 The stool sample was cultured using the “culturomics” concept [2]. MALDI-TOF MS was 54 used for colony identification as described below. In case of a failed identification using this 55 technic, the 16s ribosomal RNA was sequenced. Stackebrandt and Ebers suggested that a 56 similarity level lower than 98.7% defined a new species (Figure1) without performing DNA- 57 DNA hybridization [19]. 58 This similarity value is below the 16S rRNA threshold of 98.7% set by Stackebrandt and 59 Ebers to delineate a new species without carrying out DNA-DNA hybridization [19]. 60 2.2 Phenotypic characteristics 61 Phenotypic characteristics like Gram staining (Figure 2), sporulation, motility, catalase, 62 oxidase were highlighted as previously describedMANUSCRIPT [20]. Biochemical features of our strain 63 were investigated using API 20NE, ZYM and 50 CH strips according to the manufacturer’s 64 instructions (BioMerieux, Marcy l’Etoile, France). Various growth temperatures (25, 30, 37, 65 45 and 56°C) and atmospheres were tested. Growth under anaerobic and microaerophilic 66 conditions occurrence was tested using GENbag Anaer and GENbag miroaer systems, 67 respectively (BioMerieux). Aerobic growth was achieved with or without 5% CO 2. 68 In order to perform the electronic microscopy to observe our strain (Figure 3), detection 69 formvar coated gridsACCEPTED were deposited on a 40µL bacterial suspension drop and incubated 70 during 30 min at 37°C. The grids were incubated for 10 seconds on ammonium molybdate 71 1%, dried on blotting paper and then observed using a Morgani 268D at an operating voltage 72 of 60kV. 3 ACCEPTED MANUSCRIPT 73 Matrix-assisted laser-desorption/ionization time-of-flight (MALDI-TOF) MS protein analysis 74 was carried out as previously described [13; 21] using a Microflex spectrometer (Brüker 75 Daltonics, Leipzig, Germany). Twelve individual colonies were deposited on a MTP 96 76 MALDI-TOF target plate (Brüker). The twelve spectra were imported into the MALDI 77 BioTyper software (version 2.0, Bruker) and analyzed by standard pattern matching (with 78 default parameter settings) against the main spectra of 6.252 bacteria, including 199 spectra 79 from 104 validly named Bacillus species used as reference data in the BioTyper database. A 80 score enabled the presumptive identification and discrimination of the tested species from 81 those in a database: a score > 2 with a validated species enabled the identification at the 82 species level; and a score < 1.7 did not enable any identification. No significant score was 83 obtained for strain mt2 thus suggesting that our isolate was not a member of any known 84 species (Fig. 4 and 5). The reference spectrum for strain SIT3 T (Fig. 4) was incremented in 85 our database and then compared to other known species of the Bacillus genus. The differences 86 exhibited are showed in the obtained gel view (Fig.MANUSCRIPT 5). 87 2.4 Growth conditions and genomic DNA preparation 88 B. niameyensis strain SIT3 T (= CSUR P1266 =DSM29725) was grown on 5 % sheep blood- 89 enriched Columbia agar (BioMerieux) at 37°C in aerobic atmosphere. Bacteria grown on 90 three Petri dishes were harvested and resuspended in 4x100µL of TE buffer. Then, 200 µL of 91 this suspension was diluted in 1ml TE buffer for lysis treatment that included a 30- minute 92 incubation with 2.5 µg/µL lysozyme at 37°C, followed by an overnight incubation with 20 93 µg/µL proteinaseACCEPTED K at 37°C. Extracted DNA was then purified using 3 successive phenol- 94 chloroform extractions and ethanol precipitations at -20°C overnight. After centrifugation, the 95 DNA was resuspended in 160 µL TE buffer. 96 2.5. Genome sequencing and assembly 4 ACCEPTED MANUSCRIPT 97 Genomic DNA of Bacillus niameyensis was sequenced on the MiSeq Technology (Illumina 98 Inc, San Diego, CA, USA) with the mate pair strategy. The gDNA was barcoded in order to be 99 mixed with 11 others projects with the Nextera Mate Pair sample prep kit (Illumina). gDNA 100 was quantified by a Qubit assay with the high sensitivity kit (Life technologies, Carlsbad, CA, 101 USA) to 66.2 ng/µl .The mate pair library was prepared with 1µg of genomic DNA using the 102 Nextera mate pair Illumina guide.