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Etude De Traitement Par Plasma Froid De Surfaces Contaminés Par Biofilms Zuzana Šipoldova

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Etude De Traitement Par Plasma Froid De Surfaces Contaminés Par Biofilms Zuzana Šipoldova Etude de traitement par plasma froid de surfaces contaminés par biofilms Zuzana Šipoldova To cite this version: Zuzana Šipoldova. Etude de traitement par plasma froid de surfaces contaminés par biofilms. Autre. Université Paris Saclay (COmUE); Univerzita Komenského (Bratislava), 2016. Français. NNT : 2016SACLC054. tel-01394379 HAL Id: tel-01394379 https://tel.archives-ouvertes.fr/tel-01394379 Submitted on 9 Nov 2016 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. NNT : 2016SACLC054 Thèse de doctorat de Comenius University in Bratislava et de l'Université Paris-Saclay préparée à CentraleSupélec École doctorale n◦575 Electrical, Optical, Bio-physics and Engineering Spécialité de doctorat : Physique par Mrs. Zuzana Kovalova Etude de traitement par plasma froid de surfaces contaminés par biolms Thèse présentée et soutenue à Gif-sur-Yvette, le 30 août 2016. Composition du Jury : M. Michael DuBow Professeur, Université Paris-Sud Président du jury M. Jean-Michel Pouvesle Directeur de recherche, Université d'Orléans Rapporteur M. Lluis Mir Directeur de recherche, Institute Gustave-Roussy Rapporteur M. David Graves Professeur, University of California Berkeley Rapporteur M. Martin Kopáni Professeur associé, Comenius University Examinateur M. Emmanuel Odic Professeur, CentraleSupélec Directeur de thèse M. Zdenko Machala Professeur associé, Comenius University Directeur de thèse M. Michael Kirkpatrick Docteur, CentraleSupélec CoDirecteur de thèse Acknowledgments My deepest thanks go to my supervisors for making this thesis possible. Thank you Zdenko Machala for contacting Emmanuel Odic and giving a good recommendation for me and supporting and believing in my work even when I was in doubts. Thank you Emmanuel for making my doctorate in France possible, for helping me with all the paperwork and finding an accommodation for me. I acknowledge with gratitude the help I have received from both of you during the last four years and for time you found to read this thesis before publication and valuable suggestions. I also wish to warmly thank Mike Kirkpatrick for all your help in laboratory, for English corrections in our publications and for all cool experiments, especially cloud chamber and plasma speaker, you showed me. I am indebted for a knowledge of bacterial bioflms and their cultivation to Magali Leroy, who spent with us at Supelec only one year, but it was a very fruitful and intense year. I would like to thank Carolyn Jacobos for the set up of the spectrometer at Supelec and all the help with time resolved spectroscopy. I also wish to thank Karol Hensel, you were practically my supervisor during the first year of my PhD, without you we would be lost during our trip in Japan, also thank you for all the help with FTIR measurements. Thanks to Mário Janda for building us the fluorescence spectrometer and all funny stories about your sons. Special thanks go to Dana Lajdová and Ľubomír Tomáška from The Department of Genetics of Faculty of Natural Sciences at Comenius University in Bratislava for CSLM analysis. I am grateful to Micheal DuBow, Christophe Laux and Filipa Lopes, who were at the beginning of this thesis and gave it direction. Many thanks to Baša, Katka, Braňo, both Florents, Alex, Dany and others, I had a great time and lots of fun with you during my PhD, and even before with some of you. Lastly I would like to thank my husband Michal for your patience and support during long months of my stay in France, my parents, brother and grandparents that you supported my decision to go study in France and were always happy to see me during our Skype calls on Sundays. ii Abstract In this PhD thesis, biomedical applications of low-temperature plasmas at atmospheric pres- sure are investigated. In particular, bio-decontamination of planktonic bacteria and bacte- rial biofilms on both flat and complex surfaces by direct current (DC) air corona discharges and by pulsed discharges in argon is considered. We first provide a theoretical overview of biofilms, plasmas, and the current state of knowledge on bio-decontamination by low- temperature plasma, as well as methods presently used for biofilm decontamination in the plasma medicine domain. We characterize three plasma sources for the decontamination of Escherichia coli bac- teria and biofilm. DC corona discharges in air - positive streamer corona and negative Trichel pulses were used for decontamination of planktonic bacteria and bacterial biofilms. In some experiments water was electrosprayed onto samples from a high voltage (HV) elec- trode. Stable reactive species were identified in the gas phase by Fourier transform infrared spectroscopy (FTIR) and the concentration of reactive oxygen and nitrogen species was mea- sured in the electrosprayed water. Bio-decontamination of bacterial biofilms was carried out on glass cover slides. Within 15 min of the plasma treatment, most of the bacteria were rendered uncultivable. Some of the uncultivable bacteria remained viable, only bacteria in the top layers of the biofilm were killed, as demonstrated by a confocal laser scanning mi- croscopy (CLSM) of biofilms stained by live/dead viability kit. Water electrospray through the corona discharges significantly improves the biofilm inactivation and disintegrates the polymeric protective matrix of the biofilm. The second plasma source investigated was the pulsed corona discharge propagated inside long narrow quartz tubes, in which dry argon or argon with water vapor were flowing at atmospheric pressure. This type of discharge has a potential application in decontamination of inner surfaces of catheters or other long tubular devices, and could be able to deliver low-temperature plasma over longer distances inside the human body. This pulsed corona discharge was characterized by its electrical parameters, such as the discharge propagation velocity and the mean electric field in the plasma channel. Optical emission spectroscopy of such plasma identified UV B emission form excited hydroxyl radicals, especially with humid argon working gas. The effect of this UV B was tested on planktonic bacteria sensitive to UV damage (recA-), and was found to cause a substantial damage even to bacteria placed far downstream in the tube or on the outer surface of the tube. The third plasma source tested was an argon jet based on cylindrical dielectric barrier discharge with dry, humid or water saturated argon as a working gases. This discharge was used for biofilm decontamination, where we obtained similar results as with DC corona discharges – strong inactivation of biofilm bacteria determined from the cultivability tests and biofilm thickness shrinkage shown by CLSM. We used this discharge to test the effect iv of vacuum UV on bacterial survival, but the effect was neither confirmed nor disproved. Keywords: Corona discharges, argon plasma jet, Escherichia coli, planktonic bacteria, bacterial biofilm v Abstrakt V predkladanej dizertačnej práci sú rozoberané aplikácie nízkoteplotnej plazmy pri atmos- férickom tlaku na bio-dekontamináciu planktonických baktérií a bakteriálnych biofilmov na rovných a zložitých povrchoch pomocou korónových výbojov vo vzduchu napájaných jednos- merným napätím, pulzných korónových výbojov v argóne a pomocou argónovej trysky. V práci sú zhrnuté teoretické poznatky o biofilmoch, plazme a súčasný stav vedomostí o bio- logickej dekontaminácii nízkoteplotnou plazmou a metódach, ktoré sú používané v štúdiách dekontaminácie biofilmov a v plazmovej medicíne. V práci sú charakterizované tri zdroje plazmy, ktoré sú použité na dekontamináciu bak- térií a biofilmov Escherichia coli. Korónové výboje vo vzduchu – pozitívna streamerová ko- róna a negatívne Trichelove pulzy boli použité pre dekontamináciu planktonických baktérií a bakteriálnych biofilmov. V niektorých experimentoch bola voda rozprašovaná na vzorky z vysokonapäťovej (VN) elektródy. Stabilné reaktívne častice boli identifikované v plynnej fáze pomocou infračervenej spektroskopie (FTIR) a koncentrácia aktívnych zložiek bola meraná v rozprašovanej vode. Bio-dekontaminácia bakteriálnych biofilmov bola vykonaná na skle- nených krycích sklíčkach. Po 15 minútach plazmového opracovania bola väčšina baktérií nekultivovateľná. Časť z týchto nekultivovateľných baktérií zostalo životaschopných, pričom boli zabité iba baktérie z vrchných vrstiev biofilmu, čo bolo preukázané pomocou konfokál- nej laserovej skenovacie mikroskopie (CLSM) biofilmov farbených live/dead viability kitom. Rozprašovanie vody v korónovom výboji výrazne zlepšuje inaktiváciu biofilmu a rozkladá polymérový ochranný matrix biofilmu. Druhým zdrojom plazmy bol pulzný korónový výboj v dlhej úzkej kremennej trubici, v ktorej prúdil suchý argón alebo argón s vodnou parou pri atmosférickom tlaku. Tento typ výboja má potenciálne uplatnenie pri dekontaminácii vnútorných povrchov katétrov alebo iných dlhých rúrkovitých zariadení. Tiež by mohol umožniť prevod nízkoteplotnej plazmy na väčšie vzdialenosti do vnútra ľudského tela. Najskôr bol tento zdroj nízkoteplotnej plazmy charakterizovaný jeho elektrickými parametrami, ako je rýchlosť šírenia
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