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Aus dem Institut für Tierernährung des Fachbereichs Veterinärmedizin der Freien Universität Berlin Isolation of lactic acid-related bacteria from the pig mucosal proximal gastrointestinal tract, including Olsenella umbonata sp. nov. and Veillonella magna sp. nov. Inaugural-Dissertation zur Erlangung des Grades eines Doktors der Veterinärmedizin an der Freien Universität Berlin vorgelegt von Mareike Kraatz Tierärztin aus Berlin Berlin 2011 Journal-Nr.: 3431 Gedruckt mit Genehmigung des Fachbereichs Veterinärmedizin der Freien Universität Berlin Dekan: Univ.-Prof. Dr. Leo Brunnberg Erster Gutachter: Univ.-Prof. a. D. Dr. Ortwin Simon Zweiter Gutachter: Univ.-Prof. Dr. Lothar H. Wieler Dritter Gutachter: Univ.-Prof. em. Dr. Dr. h. c. Gerhard Reuter Deskriptoren (nach CAB-Thesaurus): anaerobes; Bacteria; catalase; culture media; digestive tract; digestive tract mucosa; food chains; hydrogen peroxide; intestinal microorganisms; isolation; isolation techniques; jejunum; lactic acid; lactic acid bacteria; Lactobacillus; Lactobacillus plantarum subsp. plantarum; microbial ecology; microbial flora; mucins; mucosa; mucus; new species; Olsenella; Olsenella profusa; Olsenella uli; oxygen; pigs; propionic acid; propionic acid bacteria; species composition; stomach; symbiosis; taxonomy; Veillonella; Veillonella ratti Tag der Promotion: 21. Januar 2011 Diese Dissertation ist als Buch (ISBN 978-3-8325-2789-1) über den Buchhandel oder online beim Logos Verlag Berlin (http://www.logos-verlag.de) erhältlich. This thesis is available as a book (ISBN 978-3-8325-2789-1) via the book trade or online at Logos Verlag Berlin (http://www.logos-verlag.de). Allen gewidmet, von denen ich lernte —Günter Grass, Beim Häuten der Zwiebel (2006) Contents Chapter I Scope and intention 10 Outline 12 Co-authors 14 Chapter II Lactobacilli and other lactic acid-related bacteria in the mucosal proximal gastrointestinal tract of pigs: a review of ecology for two derivative approaches for isolation of novel species 19 Chapter III LHP: a new medium for the specific direct isolation of lactobacilli and screening for strains of novel species with haem-independent manganese catalase activity on hydrogen peroxide 51 Chapter IV PGM-plus and PGM: new mucin-based media in the specific isolation of lactic acid-related bacteria from the mucosal stomach and jejunum of a pig, including Olsenella umbonata sp. nov. and Veillonella magna sp. nov. 78 Chapter V Olsenella umbonata sp. nov., a microaerotolerant anaerobic lactic acid bacterium from the sheep rumen and pig jejunum, and emended descriptions of Olsenella, Olsenella uli and Olsenella profusa 98 Chapter VI Veillonella magna sp. nov., isolated from the jejunal mucosa of a healthy pig, and emended description of Veillonella ratti 124 Chapter VII Lactic acid-related bacterial community from the mucosal stomach and jejunum of a pig, including Olsenella umbonata sp. nov. and Veillonella magna sp. nov. 140 Chapter VIII General discussion 149 Chapter IX Summary / Zusammenfassung 189 References 191 Acknowledgements / Danksagung 192 Statement of originality / Selbständigkeitserklärung 193 v Abbreviations 16S 16Svedberg adj. adjective Ala alanine ANI Anaerobe Identification AP alkaline phosphatase approx. approximately ATCC American Type Culture Collection ATP adenosine triphosphate BHG brain-heart-glucose BLAST Basic Local Alignment Search Tool bp base pair °C degree Celsius C4/C8/C14 4/8/14 carbon atoms ca. circa Ca calcium CaCl2 calcium chloride CCUG Culture Collection University of Gothenburg CDC Centers for Disease Control CFU colony-forming units CIP Collection de l’Institut Pasteur CO2 carbon dioxide CoA coenzyme A CuSO4 copper sulphate d day (time) D- D-configurated DDBJ DNA Data Bank of Japan DIG digoxigenin DNA deoxyribonucleic acid DSM/DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen edn. edition EDTA ethylenediaminetetraacetic acid e.g. exempli gratia (for example) EMBL European Molecular Biology Laboratory et al. et alii, et aliae (and others) FAA fastidious anaerobe agar FCM-FISH flow cytometry-fluorescent in situ hybridization vi fem. feminine (gender) FEMS Federation of European Microbiological Societies FeSO4 iron sulphate Fig. figure g gram GC gas chromatography G+C-content guanine and cytosine content GI gastrointestinal JCM Japan Collection of Microorganisms Jr. junior h hour (time) HAP hyper-ammonia-producing HCl hydrochloric acid H2O water H2O2 hydrogen peroxide HPLC high-pressure liquid chromatography H2S hydrogen sulphide i.e. id est (that is) IJSEM International Journal of Systematic and Evolutionary Microbiology INRA Institut National de la Recherche Agronomique kDa kilodalton K2HPO4 potassium monohydrogen phosphate KH2PO4 potassium dihydrogen phosphate l litre L. Latin L- L-configurated LAB lactic acid-producing bacteria LAMVAB Lactobacillus Anaerobic MRS with Vancomycin and Bromocresol green LARB lactic acid-related bacteria LDH lactate dehydrogenase LHP Lactobacillus-hydrogen peroxide LMG Laboratorium voor Microbiologie en Microbiële Genetica µg microgram µl microlitre µm micrometer m. masculine (gender) M molar max maximal ME minimum-evolution vii MEGA Molecular Evolutionary Genetics Analysis meq milliequivalent mg milligram MgSO4 magnesium sulphate min minute (time) or minimal (OD) ml millilitre mm millimetre mM millimolar mmol millimol MnSO4 manganese sulphate MP maximum-parsimony MRS de Man-Rogosa-Sharpe MRS-Van MRS-Vancomycin n number (quantity) n. noun N2 dinitrogen (nitrogen gas) N- nitrogen NaCl sodium chloride NAD+/NADH nicotinamide adenine dinucleotide Na2HPO sodium monohydrogen phosphate NaH2PO4 sodium dihydrogen phosphate NCBI National Center for Biotechnology Information NCIMB National Collection of Industrial, food and Marine Bacteria ng nanogram NJ neighbour-joining N.L. Neo-Latin nm nanometre nmol nanomol no. number nov. nova (novel, new) O2 dioxygen (oxygen gas) O- oxygen-linked OD/ODmax optical density /maximal optical density p- para p./pp. page/pages PBS phosphate-buffered saline PCR polymerase chain reaction PGM porcine gastric mucin PGMM porcine gastric mucus-mucosa viii pH pondus Hydrogenii; negative decadic logarithm of the molar concentration of dissolved hydronium ions PHYLIP PHYLogeny Inference Package PJMM porcine jejunal mucus-mucosa pKa negative decadic logarithm of the acid dissociation constant Ka PY peptone-yeast extract PYG peptone-yeast extract-glucose rDNA ribosomal deoxyribonucleic acid RDP Ribosomal Database Project Rogosa’s SL Rogosa’s selective Lactobacillus rRNA ribosomal ribonucleic acid s second (time) SB Slanetz and Bartley SCFA short-chain fatty acids SD standard deviation SEM scanning electron microscopy sp./spp. species/species pluralis subsp. subspecies T type (type strain of species) TES Tris-EDTA-sucrose TMB tetramethylbenzidine Tris tris(hydroxymethyl)aminomethane UBM unsupportive basal medium UK United Kingdom UPGMA unweighted pair-group method with arithmetic mean URAS unreduced aerobically sterilised UV ultraviolet vol. volume v/v volume/volume wk week (time) w/v weight/volume ix Chapter I Scope and intention The present thesis is about lactobacilli, other lactic acid-producing and lactic acid-fermenting bacteria [together termed herein ‘lactic acid-related bacteria’ (LARB)] from the pig mucosal proximal gastrointestinal (GI) tract. It describes the theoretical background for the design and application of two approaches for isolation of novel species of these groups of bacteria from the mucosae of the stomachs and jejuna of healthy domestic pigs. These approaches were developed based on what is invariably present in the otherwise often empty (or jejune) pig proximal gut: oxygen and mucins. As one approach was rewarding, the thesis also contains the descriptions of two novel LARB species: Olsenella umbonata and Veillonella magna. I decided on this scope, because I had learned from my veterinary studies, and especially from the studies of animal nutrition, microbiology and pig diseases during my time of student research (Kraatz et al., 2004; Taras et al., 2005; Kraatz et al., 2006; Taras et al., 2006), that the intimate co-existence (symbiosis) of mucosa-associated lactic acid-producing bacteria (LAB) in the proximal GI tract is utterly relevant for both pig nutrition and health. Lactobacilli and other acid-tolerant LAB are dominant members of the indigenous microbiota in the pig proximal GI tract due to their ‘special foregut association’ (Walter, 2008) with the non-glandular, stratified squamous epithelium of the stomach pars oesophagea (Savage, 1977; Tannock, 1992; Tannock, 2005; Bomba et al., 2006; Krüger et al., 2008). The stomach and small intestine of pigs are lactic acid habitats, in that lactic acid is the main microbial fermentation product found therein (Simon, 2008). Being monogastric omnivores with a simple, quasi non-caecal GI system, pigs meet their energy and nutrient requirements predominantly by endogenous digestive processes in the stomach and proximal small intestine (Püschner & Simon, 1988; Simon, 2008). The glycocalyx of the jejunal brush-border presents the hotspot of terminal endogenous digestion and nutrient absorption (Moran, 1982; Püschner & Simon, 1988) as well as an interface of the mucosa-associated cell surface and mucus gel microhabitats (Lee, 1985; Laux et al., 2005; Linden et al., 2008), which are inhabited by the co-evolved, metabolically interactive and overall mutually beneficial indigenous microbiota (Tannock, 2005a; Ley et al., 2008; Little et al., 2008; Resta, 2009). 10 I Scope and intention, Outline,
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  • Sphingopyxis Italica, Sp. Nov., Isolated from Roman Catacombs 1 2

    Sphingopyxis Italica, Sp. Nov., Isolated from Roman Catacombs 1 2

    View metadata, citation and similar papers at core.ac.uk brought to you by CORE IJSEM Papers in Press. Published December 21, 2012 as doi:10.1099/ijs.0.046573-0 provided by Digital.CSIC 1 Sphingopyxis italica, sp. nov., isolated from Roman catacombs 2 3 Cynthia Alias-Villegasª, Valme Jurado*ª, Leonila Laiz, Cesareo Saiz-Jimenez 4 5 Instituto de Recursos Naturales y Agrobiologia, IRNAS-CSIC, 6 Apartado 1052, 41080 Sevilla, Spain 7 8 * Corresponding author: 9 Valme Jurado 10 Instituto de Recursos Naturales y Agrobiologia, IRNAS-CSIC 11 Apartado 1052, 41080 Sevilla, Spain 12 Tel. +34 95 462 4711, Fax +34 95 462 4002 13 E-mail: [email protected] 14 15 ª These authors contributed equally to this work. 16 17 Keywords: Sphingopyxis italica, Roman catacombs, rRNA, sequence 18 19 The sequence of the 16S rRNA gene from strain SC13E-S71T can be accessed 20 at Genbank, accession number HE648058. 21 22 A Gram-negative, aerobic, motile, rod-shaped bacterium, strain SC13E- 23 S71T, was isolated from tuff, the volcanic rock where was excavated the 24 Roman Catacombs of Saint Callixtus in Rome, Italy. Analysis of 16S 25 rRNA gene sequences revealed that strain SC13E-S71T belongs to the 26 genus Sphingopyxis, and that it shows the greatest sequence similarity 27 with Sphingopyxis chilensis DSMZ 14889T (98.72%), Sphingopyxis 28 taejonensis DSMZ 15583T (98.65%), Sphingopyxis ginsengisoli LMG 29 23390T (98.16%), Sphingopyxis panaciterrae KCTC12580T (98.09%), 30 Sphingopyxis alaskensis DSM 13593T (98.09%), Sphingopyxis 31 witflariensis DSM 14551T (98.09%), Sphingopyxis bauzanensis DSM 32 22271T (98.02%), Sphingopyxis granuli KCTC12209T (97.73%), 33 Sphingopyxis macrogoltabida KACC 10927T (97.49%), Sphingopyxis 34 ummariensis DSM 24316T (97.37%) and Sphingopyxis panaciterrulae T 35 KCTC 22112 (97.09%).