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Aus Dem Institut Für Tierhygiene Und Öffentliches Veterinärwesen Der Veterinärmedizinischen Fakultät Der Universität Leipzig

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Aus Dem Institut Für Tierhygiene Und Öffentliches Veterinärwesen Der Veterinärmedizinischen Fakultät Der Universität Leipzig Aus dem Institut für Tierhygiene und Öffentliches Veterinärwesen der Veterinärmedizinischen Fakultät der Universität Leipzig Phänotypische Charakterisierung der Spezies des Genus Burkholderia mittels biochemischer Feintypisierung und in vitro-Resistenztestung Inaugural-Dissertation zur Erlangung des Grades eines Doctor medicinae veterinariae (Dr. med. vet.) durch die Veterinärmedizinische Fakultät der Universität Leipzig eingereicht von Martina B. Oberdorfer aus Ulm Leipzig, 2007 Mit Genehmigung der Veterinärmedizinischen Fakultät der Universität Leipzig Dekan: Prof. Dr. Karsten Fehlhaber Betreuer: Prof. Dr. Uwe Truyen Gutachter: Prof. Dr. Uwe Truyen Institut für Tierhygiene und Öffentliches Veterinärwesen Veterinärmedizinische Fakultät der Universität Leipzig PD Dr. Heinrich Neubauer Direktor und Professor Institut für bakterielle Infektionen und Zoonosen Friedrich-Loeffler-Institut Jena Prof. Dr. Rolf Bauerfeind Institut für Hygiene und Infektionskrankheiten der Tiere Veterinärmedizinische Fakultät der Justus-Liebig-Universität Gießen Tag der Verteidigung: 18. Juli 2006 Meinem Mann in Liebe und Dankbarkeit Inhaltsverzeichnis 1 Einleitung 1 2 Literaturübersicht 3 2.1 Geschichte und Taxonomie des Genus Burkholderia 3 2.2 Pathogene Burkholderia -Spezies 4 2.2.1 Burkholderia mallei 4 2.2.2 Burkholderia pseudomallei 14 2.3 Fakultativ pathogene Burkholderia -Spezies 18 2.3.1 Burkholderia c epacia -Komplex 18 2.3.1.1 Burkholderia cepacia 19 2.3.1.2 Burkholderia multivorans 19 2.3.1.3 Burkholderia cenocepacia 19 2.3.1.4 Burkholderia stabilis 20 2.3.1.5 Burkholderia vietnamiensis 20 2.3.1.6 Burkholderia dolosa 20 2.3.1.7 Burkholderia ambifaria 20 2.3.1.8 Burkholderia anthina 20 2.3.1.9 Burkholderia pyrrocinia 20 2.3.2 Burkholderia fungorum 20 2.3.3 Burkholderia gladioli 21 2.3.4 Burkholderia thailandensis 22 2.4 Apathogene Burkholderia -Spezies (Mensch und Tier) 22 2.4.1 Burkholderia andropogonis 22 2.4.2 Burkholderia caledonica 22 2.4.3 Burkholderia caribensis 22 2.4.4 Burkholderia caryophylli 23 2.4.5 Burkholderia glathei 23 2.4.6 Burkholderia glumae 23 2.4.7 Burkholderia graminis 23 2.4.8 Burkholderia hospita 23 2.4.9 Burkholderia kururiensis 23 2.4.10 Burkholderia phenazinium 24 2.4.11 Burkholderia phenoliruptrix 24 2.4.12 Burkholderia phymatum 24 2.4.13 Burkholderia phytofirmans 24 2.4.14 Burkholderia plantarii 24 2.4.15 Burkholderia sacchari 25 2.4.16 Burkholderia sordicola 25 2.4.17 Burkholderia terricola 25 2.4.18 Burkholderia tropica 25 2.4.19 Burkholderia tuberum 25 2.4.20 Burkholderia ubonensis 26 2.4.21 Burkholderia unamae 26 2.4.22 Burkholderia xenovorans 26 2.5 Biochemische Phänotypisierung 27 2.5.1 Differenzierung und biochemische Charakteristika 28 2.5.2 Kommerzielle Testsysteme 31 2.6 Resistenztestung 34 2.6.1 In vitro-Resistenzbestimmung 34 2.6.1.1 Verfahren zur Resistenzbestimmung 35 2.6.2 Resistenzen bei pathogenen Burkholderia -Spezies in vitro und in vivo 35 2.6.2.1 Burkholderia mallei und Burkholderia pseudomallei 35 2.6.3 Resistenzen bei Burkholderia -Spezies 41 2.7 Diagnostische Nachweisverfahren 42 2.7.1 Burkholderia mallei 42 2.7.2 Burkholderia pseudomallei 43 2.7.3 Burkholderia-Spezies 45 3 Material und Methoden 48 3.1 Taxa Profile-Mikrotitrationsplatten der Firma Merlin, Bornheim-Hersel , 48 Deutschland, zur biochemischen Feintypisierung 3.1.1 Profile A-Platte: Aminosäuren 49 3.1.2 Profile C-Platte: Kohlenhydrate 49 3.1.3 Profile E-Platte: Enzyme 49 3.2 GENARS GN4-Platte der Firma Merlin, Bornheim-Hersel, Deutschland , 49 zur in vitro-Resistenztestung 3.3 Bakterienstämme 50 3.4 Suspensionsmedium 52 3.5 Photometer 55 3.6 Computersoftware 56 3.7 Plattenbelegung 56 3.8 Vorversuch 57 3.9 Auswertung 58 3.9.1 Taxa Profile-Mikrotitrationsplatten 58 3.9.2 GENARS GN4-Platte 59 4 Ergebnisse 61 4.1 Biochemische Feintypisierung der Spezies des Genus Burkholderia 61 4.1.1 Unterteilung in Biovare 61 4.1.2 Nicht-auswertbare Substrate 62 4.1.2.1 Profile A-Mikrotitrationsplatte 63 4.1.2.2 Profile C-Mikrotitrationsplatte 63 4.1.2.3 Profile E-Mikrotitrationsplatte 64 4.1.3 Auswertbare Substrate 64 4.1.3.1 Profile A-Mikrotitrationsplatte 65 4.1.3.2 Profile C-Mikrotitrationsplatte 65 4.1.3.3 Profile E-Mikrotitrationsplatte 65 4.1.4 Minimalergebnis 66 4.2 In vitro-Resistenztestung der Spezies des Genus Burkholderia 81 5 Diskussion 85 5.1 Taxa Profile-System und GENARAS GN4-Platte in der Diagnostik 85 von Burkholderia -Spezies 5.2 Taxa Profile-System als Testplatte und GENARS GN4-Platte 89 5.3 Ausblick und Empfehlungen 98 6 Zusammenfassung 101 7 Summary 102 8 Literaturverzeichnis 103 Anhang I-XL Danksagung Abkürzungsverzeichnis % Prozent (16) S °Svedberg § Paragraph °C Grad Celsius µg/ml Mikrogramm pro Milliliter µl Mikroliter µm Mikrometer α alpha ADH Arginin-Dihydrolase ara+ Arabinose-Fermentation positiv ARDRA amplified rDNA restriction analysis AST Arylsulfatase-Nachweisreagenz ATCC American Type Culture Collection B. Burkholderia BAnz Bundesanzeiger BGBl. Bundesgesetzblatt BgVV Bundesinstitut für gesundheitlichen Verbraucherschutz und Veterinärmedizin BMELV Bundesministerium für Ernährung, Landwirtschaft und Verbraucherschutz BmTierSSchV Binnenmarkt-Tierseuchenschutz-Verordnung B-Schutz Biologischer-Schutz Bv. Biovar bzw. beziehungsweise C- Kohlenstoff C. Chromobacterium ca. circa CASFM Comité de l`Antibiogramme de la Société Francaise de Microbiologie CCUG Culture Collection University Göteborg CF Cystische Fibrose CF-Test Complement Fixation Test CGD Chronische Granulomatöse Krankheiten cm Zentimeter D- rechtsdrehend d. h. das heisst dest. destiliert DIN Deutsches Institut für Normierung e. V. DL- rechts- und linksdrehend DNA Desoxyribonukleinsäure DSMZ Deutsche Stammsammlung für Mikroorganismen und Zellkulturen GmbH ELISA enzyme-linked immunosorbent assay EMEA European Agency for the Evaluation of Medical Products engl. englisch EPS Exopolysaccharid et al. et allii EU Europäische Union EWG Europäische Wirtschaftsgemeinschaft FLI Friedrich-Loeffler-Institut, Bundesforschungsinstitut für Tiergesundheit g Gramm GENARS German Network of Antimicrobial Resistance Surveillance h Stunde HIP Hippursäurehydrolyse-Nachweisreagenz i- iso- IBIZ Institut für bakterielle Infektionen und Zoonosen ID/IDLO Institute for Animal Science and Health, Lelystad, Niederlande IgG Immunglobulin der Klasse G IHA indirekte Hämagglutination IMB Institut für Mikrobiologie KBR Komplementbindungsreaktion L- linksdrehend L/S Erreger-Risikoklassen und Labor-Sicherheitsstufen nach der Weltgesundheitsorganisation (www.who.int) LDC Lysin-Decarboxylase LMG Laboratorium voor Microbiologie Universiteit Gent LPS Lipopolysaccharid M Molar mg/L Milligramm pro Liter MHK minimale Hemmkonzentration MIC Minmal Inhibiton Concentration ml Milliliter MLD mediane Letaldosis MLST multilocus sequence typing MLVA multilocus variance analysis mm Millimeter Mol moles N- Stickstoff n. nach NCBI National Center for Biotechnology Information NCCLS National Committee for Clinical Laboratory Standards NCTC National Collection of Type-Cultures, Colindale, London NIT Nitrit nm Nanometer OIE Office International des Epizooties ONPG ortho-Nitrophenyl-ß-D-Galactopyranosidase P. Pseudomonas PCA Pyrazinamidase-Nachweisreagenz PCB polychloriertes Biphenylen PCR Polymerase Chain Reaction pH H+-Ionenkonzentration PNPG β-Galactosidase Aktivität (4-Nitrophenyl-ß-D-Galactopyranosid) PPD purified protein derivative ppm parts per million Pv. Pathovar R resistent rDNA ribosomale Desoxyribonukleinsäure RFLP Restriktionsfragmentlängen-Polymorphismus RL Richtlinie rRNA ribosomale Ribonukleinsäure S sensibel S. Seite s. h. siehe hinten SLA Serumlangsamagglutination sog. sogenannt spp. Spezies ß beta TDA Tryptophandesamidase TierSG Tierseuchengesetz u. a. unter anderem UAE United Arabic Emirates UK United Kingdom USA United States of Amerika UV ultraviolettes Licht z. B. zum Beispiel 1 Einleitung Das Genus Burkholderia (B.) besteht momentan aus 36 Spezies und hat dadurch besondere Relevanz, da es zwei hochpathogene Erreger-Spezies, B. mallei und B. pseudomallei, enthält. In Europa und vielen anderen Ländern sind diese Bakterien als Erreger der Risikoklasse L/S 3 klassifiziert. B. mallei verursacht die Rotzerkrankung der Einhufer und stellt für den Menschen eine lebensbedrohliche Zoonose dar. Aufgrund steigender Rotzfälle bei Pferden muss diese in Westeuropa, Australien und Nordamerika als getilgt geltende Infektion als „re- emerging disease“, als wieder aufflammende Tierseuche, betrachtet werden. B. pseudomallei verursacht die Infektionskrankheit „Melioidose“. Diese potentiell fatale Krankheit tritt bei Tieren und Menschen auf und kann als „emerging disease“, als im Aufflammen begriffene Seuche, bezeichnet werden, die durch Migration von Menschen, Tourismus oder durch Tiertransporte rund um die Welt ihre endemischen Gebiete verlassen kann. Das Genus enthält ebenso für Mensch und Tier fakultativ pathogene Spezies (Burkholderia cepacia-Komplex, B. fungorum, B. gladioli und B. thailandensis), die in bestimmten Patientengruppen lebensbedrohliche Infektionen verursachen können. In diesem sehr uneinheitlichen, in ständigem Wandel begriffenen Genus mit sehr unterschiedlichen Verbreitungs- und Wirtsspektren, ist es essentiell, pathogene von oft sehr nahe verwandten apathogenen Stämmen abgrenzen zu können. Ein sicherer und eindeutiger Nachweis der Spezies ist deshalb von entscheidender Bedeutung. Ein routinetaugliches Identifizierungsverfahren für alle Burkholderia-Spezies existiert bisher nicht und auch die kommerziell erhältlichen biochemischen Identifizierungssysteme sind für eine ausreichende Differenzierung
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