Accepted Manuscript Non-contiguous finished genome sequence and description of Streptococcus timonensis sp. nov. Isolated from the human stomach Davide Ricaboni, Morgane Mailhe, Jean-Christophe Lagier, Caroline Michelle, Nicholas Armstrong, Fadi Bittar, Veronique Vitton, Alban Benezech, Didier Raoult, Matthieu Million PII: S2052-2975(16)30125-1 DOI: 10.1016/j.nmni.2016.11.013 Reference: NMNI 256 To appear in: New Microbes and New Infections Received Date: 23 September 2016 Revised Date: 26 October 2016 Accepted Date: 4 November 2016 Please cite this article as: Ricaboni D, Mailhe M, Lagier J-C, Michelle C, Armstrong N, Bittar F, Vitton V, Benezech A, Raoult D, Million M, Non-contiguous finished genome sequence and description of Streptococcus timonensis sp. nov. Isolated from the human stomach, New Microbes and New Infections (2016), doi: 10.1016/j.nmni.2016.11.013. 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 Non-contiguous finished genome sequence and description of Streptococcus timonensis sp. nov. isolated from the human stomach Davide Ricaboni 1,2 , Morgane Mailhe 1, Jean-Christophe Lagier 1, Caroline Michelle 1, Nicholas Armstrong 1, Fadi Bittar 1, Veronique Vitton 3, Alban Benezech 3, Didier Raoult 1,4 and Matthieu Million 1,* 1Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes, CNRS (UMR 7278), IRD (198), INSERM (U1095), AMU (UM63), Institut Hospitalo-Universitaire Méditerranée- Infection, Faculté de médecine, Aix-Marseille Université 2Department of Biomedical and Clinical Sciences, 3rd Division of Clinical Infectious Disease, University of Milan, Luigi Sacco Hospital, Italy 3Service de Gastroentérologie, Hôpital Nord, Assistance Publique-Hôpitaux de Marseille, 13915 Marseille, France 4Special Infectious Agents Unit, King Fahd Medical RMANUSCRIPTesearch Center, King Abdul Aziz University, Jeddah, Saudi Arabia *Corresponding author: Matthieu MILLION, Aix Marseille Université, URMITE, UM63, CNRS 7278, IRD 198, INSERM 1095, Marseille, France E-mail address: [email protected] Running title: Streptococcus timonensis sp. nov. ACCEPTED ACCEPTED MANUSCRIPT 1 Non-contiguous finished genome sequence and description of Streptococcus timonensis sp. nov. 2 isolated from the human stomach 3 Abstract: 71 4 Main text: 3190 5 Running title: Streptococcus timonensis sp. nov. 6 Keywords : Streptococcus timonensis; human gut microbiota; stomach; culturomics; 7 taxonogenomics. MANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT 8 Abbreviations 9 AGIOS: Average Genomic Identity of Orthologous gene Sequences 10 CSUR: Collection de Souches de l'Unité des Rickettsies 11 DDH: DNA-DNA Hybridisation 12 DSM: Deutsche Sammlung von Mikroorganismen 13 FAME: Fatty Acid Methyl Ester 14 GC/MS: Gas Chromatography/Mass Spectrometry 15 GGDC: Genome-to-genome distance calculator 16 LPSN: List of Prokaryotic Names with a Standing in Nomenclature 17 NCBI: National Center for Biotechnology Information 18 URMITE: Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes MANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT 19 Abstract 20 Strain Marseille-P2915 T, a Gram-positive, facultative anaerobic and non-motile coccus, was 21 isolated from the gastric lavage of a severe anaemia-suffering patient. The 16S rRNA and rpoB 22 gene comparison exhibited a sequence identity of 98.7% and 92.6% with Streptococcus infantis 23 strain JCM 10157 T,respectively, collocating it within the “ Streptococcus mitis ” group. Based on 24 phenotypic and genomic analysis, we propose the validation of the type strain Streptococcus 25 timonensis sp. nov. Marseille-P2915T (=DSM 103349 =CSUR P2915). MANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT 26 I. INTRODUCTION 27 The genus Streptococcus comprises 116 officially recognized species [1] partially clustered into six 28 “species group” (pyogenic, anginosus, mitis, salivarius, bovis and mutans) on the basis of 29 systematic 16S rRNA sequence study [2]. The exact taxonomic classification of each species within 30 this genus remains challenging especially in the mitis group due to a high genetic and phenotypic 31 similarity shared within different species [3], in particular with different species sharing a 16S 32 rRNA sequence identity superior to 98.7% [4]. Since species belonging to this group gather togheter 33 highly virulent species involved in pathologies as meningitis, endocarditis, pneumonia, as well as 34 low-pathogenic commensal species, the rapid identification of clinical isolates is mandatory. To 35 reach this goal, several different molecular targets have been tested such as sod A [5], rnp B [6], tuf 36 [7] and groEL [8]. The use of rpoB was intially proposed in our laboratory [9],since then it has 37 been validated by the use in the classification of different new streptococcal species [10-12]. 38 Nowadays, the widespread use of matrix-assisted laser-desorption/ionization time-of-flight 39 (MALDI-TOF) in the clinical and research setting coMANUSCRIPTupled with the rapid developement of next 40 generation sequencing technology gives us a new and more complete insight into the taxonomy of 41 the Streptococcus genus. In this context, a polyphasic approach to describe new bacterial species 42 named “Taxono-genomic” was proposed in our laboratory in 2004 combining common phenotypic 43 tests such as API strips, whole genomic sequencing and MALDI-TOF spectra [13]. 44 In this manuscript, we present a phenotypic and genetic comparison of 16S rRNA genes, rpoB 45 genes as well as whole-genomic-level analysis which led us to propose the validation of 46 Streptococcus timonensis strain Marseille-P2915 T (=DSM 103349 = CSUR P2915) as a new 47 member within the ACCEPTEDgenus Streptococcus . 48 49 II. MATERIAL AND METHODS 50 1. Sample collection 51 In the context of a study aimed at the description of different gut microflora levels by culturomics 52 [14], a gastric lavage sample fromA aCCEPTED 60-year-old paMANUSCRIPTtient was collected during a gastroscopy 53 performed for medical reasons (severe anemia). It is worth to mention that the patient was under 54 long term proton pump inhibitor therapy. Informed and signed consent, approved by the Institut 55 Fédératif de Recherche IFR48 (Faculty of Medicine, Marseille, France), under agreement Number 56 09-022, was obtained from the patient. After collection, the sample was immediately placed in an 57 antioxidant transport medium [15] and plated within 2 hours. 58 2. Isolation and identification of the strain 59 Strain Marseille-P2915 T’s first growth was obtained in April 2016 on 5% sheep blood-enriched 60 Columbia agar medium (BioMérieux, Marcy l’Etoile, France) under aerobic conditions at 37°C. 61 Once isolated on pure culture, proteomic analysis was carried out with MALDI-TOF MS as 62 previously described [16-17] using a Microflex spectrometer (Bruker Daltonics, Leipzig, Germany). 63 Strain Marseille-P2915 T spectra was thus obtained, imported into the MALDI BioTyper software 64 (version 3.0, Bruker) and processed by standard pattern matching (with default parameter settings) 65 against the main spectra of 7,567 bacteria. The comparisonMANUSCRIPT with the BioTyper database spectra 66 enabled the rapid matching of the analyzed species with those present in the database. The resulting 67 score, if >2, enabled the identification at the species level while a score < 1.7 did not enable any 68 identification. 69 To obtain the 16S rRNA sequence of strain Marseille-P2915 T, a PCR was performed using a 70 GeneAmp PCR System 2720 thermal cycler (Applied Bio Systems, Bedford, MA, USA) as 71 previously described [18]. The sequencing step was performed on an ABI Prism 3130xl Genetic 72 Analyser capillary sequencer (Applied Bio Systems). Chromas Pro 1.34 software (Technelysium 73 Pty. Ltd., Tewantin,ACCEPTED Australia) was used to fix the sequences and a BLASTn research [19] was 74 performed against the NCBI database (default parameters, uncultured/environmental sample 75 sequences excluded). 76 The rpoB gene sequence of strain Marseille-P2915 T was obtained using the 1730_F and 3700_R 77 primers as previously described [9-20]. Then a BLAST research was performed against the NCBI 78 database using default parameters.A CCEPTED MANUSCRIPT 79 3. Phylogenetic analysis 80 Based on the BLAST research results, 16S rRNA reference sequences 81 (http://www.bacterio.net/streptococcus.html) of the closest species with standing in nomenclature 82 were downloaded from NCBI. As there is no officially recognized reference for rpoB , gene 83 sequences were downloaded directly from the NCBI database after the BLASTn search. Sequences 84 were then aligned using Muscle v3.8.31 with default parameters and phylogenetic inferences were 85 obtained using the maximum-likelihood method with 1000 bootstrap replicates within the MEGA6 86 software. 87 4. Phenotypic and biochemical characterization 88 Growth conditions 89 Growth of the strain was tested on sheep blood-enriched Columbia agar (BioMérieux) under 90 anaerobic conditions using anaeroGEN (Oxoid Ltd), microaerophilic (CampyGen, Oxoid) and 91 aerobic conditions. Different growth temperatures (20°C,MANUSCRIPT 28°C, 37°C,