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Bacillus Subtilis Spore Resistance Towards Low Pressure Plasma Sterilization = Bacillus Subtilis Sporenresistenz Gegenüber Plas

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Bacillus Subtilis Spore Resistance Towards Low Pressure Plasma Sterilization = Bacillus Subtilis Sporenresistenz Gegenüber Plas Bacillus subtilis Spore Resistance towards Low Pressure Plasma Sterilization Dissertation to obtain the degree Doctor Rerum Naturalium (Dr. rer. nat.) at the Faculty of Biology and Biotechnology Ruhr University Bochum International Graduate School of Biosciences Ruhr University Bochum (Chair of Microbiology) Submitted by Marina Raguse from KönigsWusterhausen, Germany Bochum, April 2016 First supervisor: Prof. Dr. Franz Narberhaus Second supervisor: Prof. Dr. Peter Awakowicz Bacillus subtilis Sporenresistenz gegenüber Plasmasterilisation im Niederdruck Dissertation zur Erlangung des Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) der Fakultät für Biologie und Biotechnologie der Ruhr-Universität Bochum Internationale Graduiertenschule für Biowissenschaften Ruhr-Universität Bochum (Lehrstuhl für Biologie der Mikroorganismen) Vorgelegt von Marina Raguse aus KönigsWusterhausen, Germany Bochum, April 2016 Erstbetreuer: Prof. Dr. Franz Narberhaus Zweitbetreuer: Prof. Dr. Peter Awakowicz This work was conducted externally at the German Aerospace Center, Institute for Aerospace Medicine, Department of Radiation Biology, Research Group Astrobiology/Space Microbiology, in 51147, Cologne, Germany, under the supervision of Dr. Ralf Möller from 01.12.2012 until 31.07.2016. Diese Arbeit wurde extern durchgeführt am Deutschen Zentrum für Luft- und Raumfahrt, Institut für Luft-und Raumfahrtmedizin, Abteilung Strahlenbiologie, Arbeitsgruppe Astrobiologie/Weltraummikrobiologie in 51147, Köln, unter der Betreuung von Dr. Ralf Möller im Zeitrahmen vom 01.12.2012 – 31.07.2016. Danksagung Zuerst möchte ich mich herzlich bei meinem Doktorvater Prof. Dr. Franz Narberhaus für die Unterstützung bedanken und für die Möglichkeit auch extern am Lehrstuhl für Biologie der Mikroorganismen der Ruhr-Universität Bochum zu promovieren. Mein großer Dank gilt ebenfalls meinem Korreferenten Prof. Dr. Peter Awakowicz, für sein allzeit großes Interesse an der Arbeit mit Sporen und die anregenden Diskussionen in den PlasmaDecon- Meetings. Ganz besonders danken möchte ich meinem Betreuer Dr. Ralf Möller, der mich während der gesamten Arbeit fortwährend unglaublich unterstützt und gepusht hat, immer ein offenes Ohr hatte, und mir tolle Möglichkeiten zur wissenschaftlichen und persönlichen Entwicklung geboten hat. Seine kreativen und wegweisenden Ideen haben wesentlich zum Gelingen dieser Arbeit beigetragen. Ich hoffe, er verrät mir jetzt endlich, warum er keine Schokolade mag. Ebenfalls danken möchte ich der Abteilung Strahlenbiologie des Deutschen Zentrums für Luft-und Raumfahrt, allen voran Dr. Günther Reitz, Dr. Christine Hellweg, und Dr. Petra Rettberg für die Ermöglichung der experimentellen Durchführung der Arbeit, sowie allen Kollegen für die gute Zusammenarbeit. Ein riesiges Dankeschön geht an meine Kollegen der Arbeitsgruppe Space Microbiology, Katja Nagler, Andrea Schröder, und Felix Fuchs, sowie meine ehemalige Bachelorstudentin Vanessa Neumann, die Freuden und Ängste gleichermaßen mit mir teilten, immer für eine ausgelassene Stimmung im Labor gesorgt haben, und mir über die Zeit sehr ans Herz gewachsen sind. Ohne Katharina Stapelmann, Marcel Fiebrandt, und dem Lehrstuhl AEPT wären die Plasmaexperimente und besonders die Interpretation der Daten nicht möglich gewesen. Ich danke euch sehr für eure Unterstützung und die unermüdliche und geduldige Erklärung der Plasmaphysik für „den Biologen“. Darüber hinaus danke ich meinen zahlreichen Kooperationspartnern, die mich auf Reisen stets überaus freundlich empfangen haben, mir die Möglichkeit gaben, spannende Daten zu generieren, und mir mit ihrer Expertise stets zur Seite standen. Besonderer Dank gilt hier Dr. Patrick Eichenberger von der New York University, Dr. Peter Eaton und Dr. Maria Feio von der Universität Porto, sowie Dr. Ryuichi Okayasu, Dr. Akira Fujimori, Dr. Hiroshi Fujisawa, und Dr. Hirokazu Hirakawa vom National Institute of Radiological Sciences. Desweiteren möchte ich mich bei Dr. Michael Laue und Dr. Kazimierz Madela vom Robert-Koch- Institut für die Anfertigung der Life-Cell-Imaging Daten und das rege Interesse an unserer Forschung bedanken. Mein großer Dank gilt ebenso Dr. Peter Setlow vom UConn Health Center, Dr. Juan Alonso vom Spanish National Research Council, und Dr. Joanne E. Thwaite vom Defence Science and Technology Laboratory für den regen Wissensaustausch und die tatkräftige Unterstützung bei der Manuskripterstellung. Ebenfalls bedanken möchte ich mich bei der Werkstatt ME, allen voran Christoph Steger, für die aktive Hilfe bei größeren und kleineren Problemen, sowie die unterhaltsamen Mittagspausen. Ein ganz besonderer Dank gilt meinen Eltern und Brüdern, meinem Verlobten Konrad, und meinen tollen Freunden, allen voran Dani und Nathalie, die immer an mich geglaubt haben und mir stets Mut zugesprochen haben. Ich danke euch aus ganzem Herzen für euren Beistand, die wunderbaren Ablenkungen, und das unglaubliche Video! Bei der Deutschen Forschungsgemeinschaft DFG bedanke ich mich für die Finanzierung im Rahmen des Projektes „DFG PlasmaDecon“ (PAK 728 to R.M. (MO 2023/2-1)). A. Introduction ........................................................................................................................ 1 1. Nonthermal Plasmas for Sterilization .................................................................................. 1 1.1 Motivation for Search of Alternative Sterilization Methods ........................................ 1 1.2 Plasma – the 4th State of Matter .................................................................................... 1 1.3 Fields of Application for Non-thermal Plasmas ........................................................... 3 1.3.1 Application of Low Pressure Plasma for Sterilization .......................................... 4 1.3.2 Plasma as an Alternative Sterilization Method for Space Hardware .................... 6 1.4 Bacillus Spores as Bioindicators for Sterilization Processes ........................................ 7 1.5 Plasma Interaction with Living Biological Matter ....................................................... 8 2. Experimental Setup ............................................................................................................ 11 2.1 Very High Frequency-Capacitively Coupled Plasma Reactor ................................... 11 2.2 Double Inductively Coupled Plasma Reactor ............................................................. 13 3. Bacillus subtilis - the model organism ............................................................................... 14 3.1 Regulation of Bacillus subtilis Endospore Formation and Dormancy ....................... 14 3.1.1 Spore Coat Morphogenesis ................................................................................ 16 3.2 Bacillus Spore Structure and Role in Spore Resistance ............................................. 18 3.2.1 Exosporium ......................................................................................................... 19 3.2.2 Spore Coat Layers ............................................................................................... 19 3.2.3 Outer Membrane ................................................................................................. 20 3.2.4 Cortex .................................................................................................................. 20 3.2.5 Germ Cell Wall ................................................................................................... 21 3.2.6 Spore Inner Membrane ........................................................................................ 21 3.2.7 Spore Core ........................................................................................................... 21 3.3 Time Line of Spore Revival ....................................................................................... 23 3.3.1 Spore Germination .............................................................................................. 23 3.3.2 Early and Late Outgrowth Phase ......................................................................... 24 3.4 Bacillus subtilis Spore Resistance .............................................................................. 26 3.4.1 Ionizing Radiation Resistance ............................................................................. 26 3.4.2 UV Resistance ..................................................................................................... 27 3.4.3 Heat Resistance ................................................................................................... 29 3.4.4 Chemical Resistance ........................................................................................... 30 3.4.5 DNA Repair in B. subtilis Spore Revival ............................................................ 31 3.4.5.1 Spore Photoproduct Lyase…………………………………...……….…… 32 3.4.5.2 Excision Repair………………………………………………..……..…… 32 3.4.5.3 DNA Double strand Break Repair………………………………….…...... 33 4. Objective ........................................................................................................................ 37 B. Utilization of low-pressure plasma to inactivate bacterial spores on stainless steel screws.. ...................................................................................................................................... 39 C. Resistance of Bacillus subtilis spore DNA to lethal ionizing radiation damage relies primarily on spore core components and DNA repair, with minor effects of oxygen radical
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