Whole-Genome Sequencing and Bioinformatics as Pertinent Tools to Support Helicobacteracae Taxonomy, Based on Three Strains Suspected to Belong to Novel Helicobacter Species Elvire Berthenet, Lucie Bénéjat, Armelle Ménard, Christine Varon, Sabrina Lacomme, Etienne Gontier, Josette Raymond, Ouahiba Boussaba, Olivier Toulza, Astrid Ducournau, et al. To cite this version: Elvire Berthenet, Lucie Bénéjat, Armelle Ménard, Christine Varon, Sabrina Lacomme, et al.. Whole- Genome Sequencing and Bioinformatics as Pertinent Tools to Support Helicobacteracae Taxonomy, Based on Three Strains Suspected to Belong to Novel Helicobacter Species. Frontiers in Microbiology, Frontiers Media, 2019, 10, pp.2820. 10.3389/fmicb.2019.02820. inserm-03004847 HAL Id: inserm-03004847 https://www.hal.inserm.fr/inserm-03004847 Submitted on 13 Nov 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. fmicb-10-02820 December 5, 2019 Time: 16:13 # 1 ORIGINAL RESEARCH published: 06 December 2019 doi: 10.3389/fmicb.2019.02820 Whole-Genome Sequencing and Bioinformatics as Pertinent Tools to Support Helicobacteracae Taxonomy, Based on Three Strains Suspected to Belong to Novel Helicobacter Species Elvire Berthenet1,2†, Lucie Bénéjat1†, Armelle Ménard2, Christine Varon2, Sabrina Lacomme3, Etienne Gontier3, Josette Raymond4, Ouahiba Boussaba1, Edited by: Olivier Toulza5, Astrid Ducournau1, Alice Buissonnière1, Alban Giese2, Iain Sutcliffe, Francis Megraud1,2, Emilie Bessède1,2, Quentin Jehanne1,2 and Philippe Lehours1,2* Northumbria University, United Kingdom 1 French National Reference Center for Campylobacters and Helicobacters, Bordeaux, France, 2 Univ. Bordeaux, INSERM, 3 Reviewed by: Bordeaux Research in Translational Oncology, BaRITOn, U1053, Bordeaux, France, CNRS, INSERM, Bordeaux Imaging 4 Tomoo Sawabe, Center UMS 3420 – US4, Pôle d’Imagerie Électronique, Bordeaux, France, Bacteriology, Cochin Hospital, Institut Pasteur, 5 Hokkaido University, Japan University of Paris-Descartes, Paris, France, Clinique Vétérinaire AQUIVET, Eysines, France Bin-Bin Xie, Shandong University, China The present study describes three putative novel species received at the French *Correspondence: National Reference Center for Campylobacters & Helicobacters (CNRCH). The Philippe Lehours [email protected] CNRCH 2005/566H strain was isolated in 2005 from the feces of a patient †These authors have contributed with a hepatocellular carcinoma and gastroenteritis. Strain 48519 was isolated in equally to this work 2017 from the blood of a male patient suffering from a bacteremia. Strain Cn23e was isolated from a gastric biopsy from a dog suffering from chronic gastritis. Specialty section: This article was submitted to Biochemical and growth characteristics and electron microscopy for these three strains Evolutionary and Genomic were studied. Their genomes were also sequenced. gyrA based phylogeny built Microbiology, a section of the journal with 72 nucleotide sequences placed CNRCH 2005/566H among the unsheathed Frontiers in Microbiology enterohepatic helicobacters, close to Helicobacter valdiviensis; strain 48519 among the Received: 02 August 2019 sheathed enterohepatic helicobacters, close to Helicobacter cinaedi; and strain Cn23e Accepted: 20 November 2019 among gastric helicobacters, close to Helicobacter felis. 16S rRNA gene phylogeny Published: 06 December 2019 showed similar results, but with weak discriminant strength. Average nucleotide identity Citation: Berthenet E, Bénéjat L, Ménard A, and in silico DNA–DNA hybridization analyses revealed that CNRCH 2005/566H and Varon C, Lacomme S, Gontier E, 48519 strains belong to new putative species, but confirmed that Cn23e corresponds Raymond J, Boussaba O, Toulza O, Ducournau A, Buissonnière A, to H. felis. Cn23e was able to infect C57BL6 mice and to induce gastric inflammation. Giese A, Megraud F, Bessède E, The genomics data, together with their different morphological and biochemical Jehanne Q and Lehours P (2019) characteristics, revealed that these two strains represent novel Helicobacter species. Whole-Genome Sequencing and Bioinformatics as Pertinent Tools We propose the following names: ‘Helicobacter burdigaliensis,’ with the type strain to Support Helicobacteracae CNRCH 2005/566H ( =CECT 8850 =CIP 111660), and ‘Helicobacter labetoulli,’ with Taxonomy, Based on Three Strains Suspected to Belong to Novel the type strain 48519 ( =CCUG 73475 =CIP 1111659). This study highlights that the Helicobacter Species. diversity of the Helicobacteraceae family remains to be fully explored. Front. Microbiol. 10:2820. doi: 10.3389/fmicb.2019.02820 Keywords: whole-genome sequencing, novel species, Helicobacter genus, taxonomy, gyrA Frontiers in Microbiology| www.frontiersin.org 1 December 2019| Volume 10| Article 2820 fmicb-10-02820 December 5, 2019 Time: 16:13 # 2 Berthenet et al. Putative New Helicobacter Species INTRODUCTION each of the three strains were conserved at −80◦C in an in-house brucella broth with 25% glycerol. To date, the Helicobacter genus is comprised of 41 validated The strain CNRCH 2005/566H has been assigned to the species1. The type species for the Helicobacter genus is Collection de l’Institut Pasteur (CIP) and the Spanish Type Helicobacter pylori, well-known for its link with gastritis, peptic Culture Collection (CECT) under the identification numbers ulcer disease and gastric cancer in humans (Malfertheiner et al., 11160 and 8850, respectively. The strain 48519 has been 2017). This species was the first to be described for this assigned to the CIP and Culture Collection of Gothenburg genus, by Marshall and Warren(1984). It was originally named University (CCUG) under the identification numbers 111659 and Campylobacter pyloridis. Species belonging to the Helicobacter 73475, respectively. genus are curved, spiral or fusiform rods with size ranging from 1.5 to 10 mm long and 0.2 to 1.2 mm wide. When cultured for too Biochemical Characterization long, they often become coccoidal. They are non-spore-forming Enzymatic activities were assessed by using the API R Campy and Gram negative and show flagellar activity (On et al., 2015). strip (bioMérieux, Marcy-l’Étoile, France). The presence The Helicobacter genus encompasses a large variety of of catalase and oxidase was investigated. A disk diffusion species (De Witte et al., 2016; Smet et al., 2018). Two method was used to assess the susceptibility to nalidixic species categories are based on the niche occupied: gastric and acid (30 mg) and cephalothin (30 mg) Antimicrobial enterohepatic. All known gastric helicobacters possess sheathed susceptibility testing was performed according to the flagella, but enterohepatic species can be divided into sheathed European Committee for Antimicrobial Susceptibility Testing and unsheathed subtypes (Solnick and Vandamme, 2001). Some (EUCAST) recommendations on in-house Mueller-Hinton agar species are host-specific while others can colonize different hosts. supplemented with 5% defibrinated sheep blood (Thermo-Fisher Helicobacter species are able to colonize alternative sites of Scientific) (MH-F) and 20 mg/L of b-nicotinamide adenine the digestive tract in various hosts (mammals, birds, reptiles) dinucleotide (Sigma Aldrich, Merck, Darmstadt, Germany), (Dewhirst et al., 1994; Collado et al., 2014). under the same atmosphere and temperature conditions, as 16S rRNA sequencing, a powerful tool for taxonomy, was previously described (Sifré et al., 2015). MICs were determined shown to be unreliable for the identification of Helicobacter for each isolate with E-test R strips (bioMérieux). For levofloxacin, species as well as for other bacteria (Fox et al., 1992; Vandamme clarithromycin, rifampicin, amoxicillin, tetracycline, and et al., 1996; Jalava et al., 1997; Dewhirst et al., 2005), in part due metronidazole the cut-offs of the “Comité de l’antibiogramme to horizontal gene transfers. Therefore, gyrA based phylogeny de la Société Française de Microbiologie” (CA-SFM) (V.2.0. May is currently the preferred method, to take into account the 2019) were employed2. The reference strain H. pylori CCUG variability in the Helicobacter genus (Ménard et al., 2016). 17874 was used as a quality control strain. Strains were stored at The French National Reference Center for Campylobacters & −80◦C in brucella broth supplemented with 25% glycerol. Helicobacters (CNRCH) collects strains of Campylobacter and Helicobacter related species sent by private clinical laboratories Imaging and public hospitals. In addition to this routine activity, The morphology, cell size, and presence of flagella were the CNRCH sometimes receives strains that collaborators determined by transmission electron microscopy. Bacteria failed to identify. In recent years, three helicobacter isolates were scraped and introduced into a fixative solution of were suspected to belong to novel species. These strains 2.5% glutaraldehyde in 0.1M cacodylate buffer (pH 7.4) and were characterized in the laboratory and their genomes were incubated for 1 h at room temperature. After centrifugation sequenced.