Contribution à l'étude de plasmas froids à pression atmosphérique pour le traitement des substrats biologiques

par Chih-min Chen

Projet de thèse en Génie électrique

Sous la direction de Emmanuel Odic.

  • Résumé

    Etude des mécanismes de Dégradation des Biofilms par Plasma froid à pression atmosphérique

  • Titre traduit

    Contribution to the study of atmospheric pressure non-thermal plasma for the treatment of biological substrates

  • Résumé

    THE MECHANISMS OF BIOFILM DEGRADATION INDUCED BY ATMOSPHERIC PRESSURE NON-THERMAL PLASMA DECONTAMINATION keywords: microbiology, plasma physics, plasma chemistry, biofilms, decontamination 1. Motivation & objectives Biofilms, the dominant mode of bacterial life, are an assemblage of surface-associated microbial cells enclosed in an exopolymer matrix composed of polysaccharides, proteins and nucleic acids. Biofilms can form on almost any type of surface, ranging from living tissues to inert materials, including medical devices, such as catheters, endoscopes, and implants. These have been shown to harbor biofilms that can cause device-associated infections that can then become chronic. Nosocomial infections are the fourth leading cause of death in the U.S., with 2 million cases annually, and about 60-70% of nosocomial infections are associated with some type of implanted medical device . Biofilm formation can also cause problems in many industries, such as in air conditioning systems and industrial processes that may cause hygiene and health related problems. Disinfection treatments are used in industrial and medical environments to control and eliminate surface bio-contamination. However, some microorganisms may survive and give rise to health-risk problems. In addition, many currently available methods (e.g. wet heat treatment) may be inappropriate for heat-sensitive medical devices for which chemical treatment is the common alternative but which lacks efficacy against all microorganisms or that can cause problems such as material incompatibility and severe toxicity . Furthermore, bacteria in biofilms are known to be 100-1000 times more resistant to antimicrobial agents compared with their planktonic counterparts . It is thus clear that biofilm resistance to disinfectants can be a multifactorial process resulting from different decontamination response mechanisms and that new control strategy and technolo gies are needed to overcome these limitations. Non thermal plasmas (NTPs) can represent an alternative technology to conventional disinfection and sterilization methods, and its' mechanisms remain to be examined under different conditions. Non-thermal plasmas (NTPs) can efficiently kill microorganisms and free-living (planktonic) cells, such as bacteria and spores, including potential bio-terrorism agents. Electrical discharges are NTP sources which can be used for bio-decontamination and sterilization of surfaces, medical instruments, water, air, food containers, etc. However, as mentioned above, most microorganisms live within biofilms, with concomitant increased resistance to antimicrobial agents compared with their planktonic counterparts. Over the course of the past 15 years, the use of NTPs for biomedical applications has been increasingly investigated . In particular, the biological decontamination of surfaces has been the subject of numerous studies involving planktonic mi croorganisms . However, few studies have investigated the effect of plasma on biofilm decontamination , . A deeper understanding of the elementary mechanisms of plasma/biofilm interaction, the potential synergies of different mechanisms (UV irradiation and oxidative species), as well as knowledge of how microorganisms are killed and/or develop resistance to plasma-active agents due to genetic and biofilm characteristics, is necessary in order to develop efficient applications of plasma decontamination in the fields of environment and health. A multidisciplinary approach joining together plasma physicists/chemists and microbiologists is thus necessary in this area. 2. Project In non-equilibrium electrical discharges, partially ionized gas results from inelastic collisions between accelerated electrons (1-18 eV) and a neutral gas. These collisions take place in electronic avalanche mechanisms leading to a multiplication of the charged particles (electrons and ions) and also to th e formation of excited species. Relaxation to the ground state occurs through both radiation (line spectrum emission) and collision (lifespan of the excited species depending on the gas pressure) mechanisms. In the specific case of molecular gases, molecule dissociation induced by direct electron impact leads to the production of highly reactive radicals and atoms. The resultant plasma will present specific properties: electrostatic properties (mainly due to space charge), radiation (from UV to IR), thermal properties (limited heating of the gas in the case of non-thermal plasmas), acoustic emission / shock wave emissions (resulting from transient and local heating of the gas) and chemical properties (production of excited species, atoms and radicals). These properties, acting together or synergistically, are responsible for the decontamination effect of plasma treatment. In an exploratory project involving the same partners (GeePs and I2BC), direct exposure of the bio-contaminate d surface to the discharge plasma was examined. Plasma was initiated in a dielectric tube with argon flowing at atmospheric pressure and propagated (plasma plume) onto a bacterial contaminated surface. Bactericidal effects were examined for increasing exposure time using both bacterial colony forming abilities counting and confocal scanning laser microscopy imaging. A 106 fold reduction was observed for a 48 hour Escherichia coli biofilm exposed to a 20-minute plasma treatment. Microscopy revealed a 60% biofilm thickness reduction as well as the presence of viable but non-culturable cells. The proposed project is a continuation of this work and thus highly likely to be successful. Our goal is to obtain a better understanding of the mechanisms affecting bacteria and biofilm effects when exposed to plasmas and to eventually be able to propose an alternative plasma-based biofilm control strategy for industrial and biomedical applications. This collaborative project is interdisciplina ry in the domain of the life sciences, with strong interactions with plasma physics and its associated chemistry. Different non-thermal plasma sources will be used at atmospheric pressure, depending on the geometry of the surface bearing the biofilm (plasma “bullets” and plasma “plumes”). A physical diagnostic of the plasma will be performed using Optical Emission Spectroscopy to provide time-resolved monitoring of various key parameters such as the temperature of the gas, as well as the nature and concentration of important reactive species produced (O atoms, OH radicals). Finally, the overall level of UV radiation impinging on the biofilms is also a key parameter that will be studied. Quantitative measurements of total radiation will be performed with UV and visible emission spectroscopy. Stable atomic species produced by the plasma discharge (hydrogen peroxide) will be detected and quantified by means of infrared absorption spectroscopy (FTIR), UV absorption spectroscopy and co lorimetric methods. The mechanisms leading to reduced bacterial viability and biofilm degradation (structural effects) upon plasma treatment will be investigated. The influence of biofilm age, composition and thickness will be studied. Biofilms of three model bacterial species (Bacillus subtilis, a Gram positive spore-forming bacterium, Escherichia coli and Pseudomonas aeruginosa PAK, both Gram negative bacteria, will be studied. These bacteria were chosen because biofilms of E. coli and Bacillus species have been frequently reported on medical devices . These two bacteria have a different surface composition and structure that can be used to study mechanisms of resistance to decontamination agents . In addition, P. aeruginosa naturally produces an alginate-based exopolymer. Synthetic alginate-based biofilms from planktonic cells of the three different species at controlled parameters such as different cell densities, alginate concentrations and biofilm thicknesses. These syntheti c biofilms will be subjected to plasma treatments and examined as stated above. The role played by the exopolymer will be investigated, as will cell physiology. Furthermore, natural and synthetic biofilms will be prepared with different E. coli single mutant strains in order to identify the main stress factors induced by the plasma treatment: katE (catalase – negative and thus greater sensitivity to oxidative stress), uvrA (Uvr A - negative and thus more sensitive selective to UV exposure), rpoS (S – negative and thus more sensitive to many different stresses), recA (Rec A – negative and thus more sensitive selective to mutagens), lexAIND – (non-inducible SOS pathway and thus much more sensitive to mutagens). To measure the plasma bio-decontamination efficiency on these biofilms, bacterial viability will be assessed by a variety of complementary techniques, including standard microbiology cultures, fluorescence assays (e.g. DAPI) and confocal imagery, as stated above. The to tal number of bacteria in the biofilm will also be enumerated by fluorescence microscopy with a nonspecific DNA stain (DAPI, 4',6-diamidino-2-phenylindole). Confocal Scanning Laser Microscopy (CSLM) imaging will also be used to study the effect of plasma treatment on biofilm architecture. Furthermore, bacteria viability distribution with biofilm depth will be studied by CSLM using physiological indicator stains (e.g. BacLight LIVE/DEAD) for the determination of the maximum depth of plasma inactivation efficiency. In this manner, broad and publishable fundamental and applied interdisciplinary results will be obtained by the doctoral student, who will thus have an exceptional start to their future career in science. 3. Project partners The project joins two research teams having complementary expertise in the domains of microbiology and in particular the study of biofilms (I2BC), with that of a team studying plasma physics and diagnostics associated chemistry (GeePs). Cooperati on between the research teams, resulting in joint publications, has been ongoing for close to a decade7, , . The partners are: Emmanuel Odic is currently Professor at CentraleSupélec and with the Group of Electrical Engineering of Paris (GeePs – UMR 8507). His research activities are mainly focused on electrical discharge physics, partial discharges and atmospheric pressure non-thermal plasmas for: Fundamental studies of water vapor dissociation Flue gas treatment and VOC removal (advanced oxidation) with and without coupling with catalyst devices Surface bio-decontamination, including protein and DNA degradation Publications 3 book chapters, 31 peer reviewed journal articles, 5 patents, 4 invited lectures, 50 communications. Among these publications, 1 book chapter, 10 peer reviewed journal articles, 3 patents, 3 invited lectures and 15 communications are directly related to bio-decontamination by means of atmospheric pressure non-thermal plasmas. Selected recent publ ications – Odic: F. Sainct, D Lacoste, M. J. Kirkpatrick, E. Odic C. Laux “Temporal evolution of temperature and OH density produced by nanosecond repetitively pulsed discharges in water vapour at atmospheric pressure” J. Phys. D: Appl. Phys. 47 (2014) 075204 (8p) doi:10.1088/0022-3727/47/7/075204 S. Limam, E. Odic, M.J. Kirkpatrick, A-M. Pointu, “Bacterial decontamination of the inner wall of narrow tube by a nitrogen afterglow at atmospheric pressure and its relation to local atomic nitrogen concentration” Plasma Process. Polym. (2013) Vol. 10, Issue 8, pp. 679–685 DOI: 10.1002/ppap.201200170 F.P. Sainct, D.A. Lacoste, M.J. Kirkpatrick, E. Odic, C.O. Laux, “Experimental study of nanosecond repetitively pulsed discharges in water vapor” Intern. J. of Plasma Environ. Sci. & Technol., Vol. 6, n°2, September 2012, pp. 125-129 M. J. Kirkpatrick, E. Odic, S. Zinola, J. Lavy, “Plasma assisted heterogeneous catalytic oxidation: HCCI Diesel engine investigations” Applied Catalysis B: Environmental 117– 118 (2012) 1– 9 A-M. Pointu, A. Ricard, E. Odic, M. Ganciu 'Nitrogen atmospheric pressure post discharges for biological decontamination of inside small diameter tubes', Plasma Process. Polym. (2008), 5, 559–568 Michael S. DuBow is currently a Professor at Université Paris-Sud in Orsay. He is the head of the ”Genomics and Microbial Biodiversity of Biofilms” group of the Institute for Integrative Biology of the Cell (I2BC) (UMR 9198). His research activities are mainly focused on medical and environmental biofilms using genomic approaches. He is a Fellow of the American Academy of Microbiology (AAM). Publications 2 books, 4 book chapters, more than 100 peer reviewed journal articles, 6 patents, 20 invited lectures and short courses, more than 200 communications. Selected recent publications – DuBow: An S, Sin HH, and DuBow MS. Modification of atmospheric sand-associated bacterial communities during Asian sandstorms in China and South Korea. Heredity: 114: 460-467 (2015). Kovalova Z, Leroy M, Jacobs C, Kirkpatrick MJ, Machala Z, Lopes F, Laux CO, DuBow MS and Odic E. Atmospheric pressure argon surface discharges propagated in long tubes: physical characterization and application to bio-decontamination. J. Physics D: Applied Physics 48: 464003 (2015). Winter C, Garcia JA, Weinbauer MG, DuBow MS, Herndl GJ. Comparison of deep-water viromes from the Atlantic Ocean and the Mediterranean Sea. PloS One. 9: e100600. (2014). An S, Couteau C, Luo F, Neveu J and DuBow MS. Bacterial diversity of surface sand samples from the Gobi and Taklamaken deserts. Microbial Ecology. 66: 850-860 (2013). Jakubek D, Le Brun M, Leblon G, DuBow MS and Binet, M. The impact of monochloramine on the diversity and dynamics of Legionella pneumophila subpopulations in a nuclear power plant cooling circuit. FEMS Microbiology Ecology. 85: 302-312 (2013).