Thèse Plasmaresist. Étude Biologique et Chimique à l'Échelle du Laboratoire de la Décontamination de l'Eau par Plasma Froid pour Lutter Contre l'Antibiorésistance. H/F Doctorat.Gouv.Fr

Établissement : Université de Toulouse École doctorale : GEETS - Génie Electrique Electronique,Télécommunications et Santé : du système au nanosystème Laboratoire de recherche : DPHE - Diagnostic des plasmas hors équilibre Direction de la thèse : Philippe GUILLOT ORCID 0000000151974735 Début de la thèse : 2027-09-01 Date limite de candidature : 2026-11-23T23:59:59 Les eaux usées hospitalières constituent une source majeure de contamination environnementale par les résidus d'antibiotiques et les gènes de résistance aux antibiotiques (ARGs), contribuant à la dissémination de l'antibiorésistance dans les milieux naturels. Les procédés de traitement conventionnels ne permettent pas toujours d'éliminer efficacement ces contaminants, ni de neutraliser le potentiel de transfert horizontal des gènes de résistance. Le plasma froid à pression atmosphérique constitue une technologie émergente et prometteuse pour ce type de traitement, grâce à sa production d'espèces réactives d'oxygène et d'azote (RONS) capables de dégrader les molécules organiques et d'inactiver les microorganismes.
Ce projet doctoral, mené dans le cadre d'une cotutelle entre l'Université de Toulouse (laboratoire Diagnostics des Plasmas Hors Équilibre, DPHE, INU Champollion, Albi, France), l'Université de Bucarest (Centre de recherche pour la protection de l'environnement et la gestion des déchets, PROTMED, Bucarest, Roumanie) et l'Institut national roumain de physique des lasers, du plasma et du rayonnement (INFLPR, Magurele, Roumanie), propose une approche interdisciplinaire combinant physique des plasmas, chimie analytique et microbiologie. Six chercheurs, dont les spécialités (physique, chimie, biologie) s'inscrivent dans l'interdisciplinarité du sujet, contribueront à l'encadrement de la thèse.
DPHE est un laboratoire de plasmas travaillant sur la conception, la caractérisation et l'optimisation de procédés par plasma pour des applications biologiques et chimiques. Il comprend une unité de microbiologie dédiée à la contamination et à la décontamination de cibles solides et liquides. PROTMED est une structure de recherche de la Faculté de biologie de l'Université de Bucarest. Elle se concentre sur les solutions durables de traitement de l'eau, en combinant de nouveaux catalyseurs, des matériaux adsorbants et des technologies hybrides gris-vert pour éliminer les polluants émergents, les métaux lourds et les excès de nutriments présents dans l'eau. INFLPR de Magurele est un institut de recherche roumain spécialisé en physique des lasers, des plasmas et du rayonnement, impliqué dans la recherche scientifique et le développement de procédés avancés, contribuant à différents domaines tels que l'élimination des contaminants des eaux usées grâce à des traitements innovants et des outils de contrôle analytique.
Le projet est structuré en quatre work packages (WP). Le WP1 établira les caractéristiques électriques, spectroscopiques et physico-chimiques du procédé plasma pulsé à pression atmosphérique, en définissant les conditions de traitement utilisées tout au long du projet. Le WP2 caractérisera les voies de dégradation des antibiotiques et leurs produits de transformation à l'aide de méthodes de chimie analytique. Le WP3 déterminera si, et dans quelles conditions, le traitement par plasma réduit la pression de sélection exercée par les antibiotiques résiduels ainsi que le potentiel de dissémination des gènes de résistance. Enfin, le WP4 validera les performances du traitement dans des matrices d'eau complexes et représentatives des conditions environnementales réelles des eaux usées hospitalières, en tenant compte de l'influence des métaux traces et de la matière organique naturelle sur la chimie du plasma et la réponse microbienne.
Ce projet, qui comprend une période de mobilité de 12 mois en Roumanie et de 24 mois en France, contribuera au développement de stratégies de traitement innovantes visant à limiter la dissémination environnementale de l'antibiorésistance, tout en offrant au doctorant une expérience de formation interdisciplinaire et internationale.
Antimicrobial resistance (AMR) is included by the World Health Organisation among the top 10 threats to public health. A recent report assessing the global burden of AMR over three decades estimated 4.71 million deaths associated with bacterial AMR in 2021, including 1.14 million deaths directly attributable to bacterial AMR (GBD 2021, 2024). Increasing mortality in the adult population during 1990-2021 was evidenced, with the most pronounced rise among the elderly who recorded over 80% AMR mortality increase, while projections for 2050 forecast an upward trend unless effective remediation measures are implemented.
While overuse and misuse of antibiotics in human and veterinary medicine remain the major drivers of resistance, increasing evidence highlights the contribution of the aquatic environment as a critical reservoir and transmission pathway for AMR. In this respect, the release of antimicrobial residues and resistant microorganisms or resistance genes into the environment through insufficiently or inefficiently treated wastewater represents a major challenge and is increasingly recognised within the One Health approach. Consequently, effective AMR mitigation should extend beyond the prudent use of antibiotics, and include interventions aimed at reducing their environmental release and limiting the dissemination of resistant microorganisms and antibiotic resistance genes (ARGs).
Among emerging advanced oxidation processes, cold plasma has proved promising as a green approach for wastewater treatment. Cold plasma is a partially ionised gas containing electrons, ions and radicalic species, characterised by strong non-equilibrium, with the electron energy much higher than the energy of the heavy species. Plasma generated in electrical discharges in contact with water emits UV radiation and produces a complex mixture of reactive oxygen and nitrogen species (RONS) that collectively enable the degradation of a broad range of organic contaminants, including pharmaceutical compounds (Bruggeman et al., 2016). While the removal of antibiotics belonging to different classes has received considerable attention and proved feasible (Magureanu et al., 2021), the formation and fate of transformation products remain less comprehensively characterized.
Beyond the removal of the parent compounds, the biological effects of the treatment itself also need to be considered. Decontamination efficacy depends not only on the discharge parameters but also on the physiological state of the bacterial cells exposed (Courti et al., 2021). Furthermore, it is useful to distinguish resistance - a heritable, genetically encoded decrease in antibiotic susceptibility - from tolerance, the phenotypic and generally reversible capacity of bacteria to survive an otherwise lethal exposure without a change in susceptibility (Brauner et al., 2016). Antibiotics occur in wastewater at sub-inhibitory concentrations that can already select for resistance; cold plasma treatment adds further pressures such as sub-lethal oxidative stress, and reactive transformation products whose own biological activity is largely uncharacterised, which may trigger adaptive stress responses, transiently increasing tolerance or selecting for resistant subpopulations (Courti et al., 2022). Whether cold plasma mitigates or inadvertently promotes resistance therefore remains an open question central to its safe application.
The effect of cold plasma treatment on the dissemination of ARGs is likewise insufficiently understood. Plasma-generated reactive species may induce oxidative damage and inactivate ARGs (Zhong et al. 2023). However, this is strongly influenced by the wastewater matrix and by the intracellular or extracellular localisation of the genes - since cell lysis can convert intracellular genes into free, potentially transferable extracellular DNA - and the extent to which plasma treatment eliminates the potential for horizontal gene transfer remains unknown. The composition of the receiving matrix further complicates this picture: hospital effluents in particular carry metals, such as copper from medical uses, that are known to co-select for and maintain antibiotic resistance, so that removing the antibiotic alone may not relieve the full selective pressure (Chaudhary et al., 2026; Ferrea et al., 2026).
These gaps indicate that the environmental performance of plasma-based wastewater can not be evaluated by the assessment of antibiotic removal efficacy alone. This project is built on the hypothesis that the biological outcome of the plasma treatment of wastewater is controlled by the interplay between reactive species produced, transformation products generated, the physiological state of bacteria and matrix's characteristics. Testing this hypothesis requires an approach combining plasma physics, analytical chemistry, microbiology and microbial ecology within an interdisciplinary supervision framework.
The overall objective of this thesis is to investigate plasma treatment of water as a strategy simultaneously targeting antibiotic resistance genes (ARGs) and micropollutants. The research will rely on a multidisciplinary experimental approach, integrating laboratory investigations to elucidate the mechanisms responsible for ARGs inactivation under the plasma action, and the degradation of micropollutants, with a particular focus on quinolone antibiotics. The general objectives are as follows :
OG.1 - To fully characterise the operation of the pulsed plasma source for effective water treatment by investigating its electrical and optical properties, the spatio-temporal plasma-liquid interaction, and the resulting reactive species transfer and physicochemical changes in the liquid.
OG.2 - To identify transformation products and elucidate degradation pathways, in correlation with the reactive species generated in the discharge, thus achieving a comprehensive mechanistic understanding of the plasma-induced removal of quinolone antibiotics.
OG.3 - To determine whether, and in what conditions, the plasma treatment reduces the selective pressure exerted by residual antibiotics and the functionality of antibiotic resistance genes.
OG.4 - To evaluate the effectiveness of cold plasma treatment under environmentally relevant wastewater conditions by investigating how complex water matrix constituents influence the degradation of antibiotic contaminants, the inactivation of antibiotic resistance determinants, and microbial responses.

WP1: Plasma source characterizations and plasma-liquid interactions
Specific objectives
OS.1.1 - To characterize the electrical power consumed by the process.
OS.1.2 - To identify the plasma emissions and the corresponding reactive species in the gas phase.
OS.1.3 - To visualize the spatio-temporal evolution of the interactions between plasma and liquid surface.
OS.1.4 - To examine the evolution of the liquid's physicochemical properties.
Tasks
T.1.1. Electrical characterization of the plasma-source
Voltage and current waveforms will be measured to determine the electrical power and follow its evolution over the course of treatment, correlating this evolution with the resulting changes in liquid conductivity.
T.1.2. Identification of gas-phase reactive species
Optical emission spectroscopy will identify the reactives species generated by the plasma in the gas phase, establishing the chemical basis for the degradation and inactivation mechanisms in WP2 and WP3.
T.1.3. Spatio-temporal characterization of plasma-liquid interactions
Fast ICCD imaging combined with optical filtering will resolve the propagation of the discharge and its interaction with the liquid surface, linking the gas-phase species in T1.2 to their transfer into the liquid and to the dose experienced by the antibiotics and microorganisms in WP2 and WP3.
T.1.4. Quantification of plasma-induced changes in liquid properties
The physicochemical properties of the treated liquid (temperature, pH, conductivity) and the concentration of reactives species transferred to the liquid phase (notably H2O2) will be measured to establish the exposure metrics later challenged by the complex matrix constituents (metals, natural organic matter) studied in WP4.

WP2: Mechanistic investigation of plasma-driven degradation of antibiotic contaminants
Specific objectives
OS.2.1 - To assess the plasma treatment performance for quinolone antibiotics removal.
OS.2.2 - To identify the intermediate transformation products and establish degradation pathways.
OS.2.3 - To correlate the degradation pathways with plasma characteristics.
Tasks
T.2.1. Investigation of plasma-induced degradation kinetics of quinolone antibiotics in water
The degradation kinetics of the selected quinolone antibiotics will be systematically evaluated under various plasma operating conditions and solution characteristics. HPLC will be used to quantify the target compounds' concentrations. The removal efficiency and the degradation rate constants thus determined will allow identification of plasma operating parameters that maximize contaminant degradation.
T.2.2. Chemical characterization of the plasma-treated aqueous solutions
The intermediate transformation products formed during plasma-driven degradation of quinolone antibiotics will be identified by HPLC-QToF analysis (Bilea et al, 2024). Their temporal evolution will be continuously monitored throughout the plasma treatment, while the mineralization degree will be determined by total organic carbon analysis. These investigations will provide the basis for elucidating the main degradation pathways of the target contaminants under the action of plasma.
T.2.3. Establishing the relationship between degradation pathways and plasma characteristics
The removal kinetics, product distribution and degradation pathways will be correlated with plasma diagnostics to identify the key reactive species and mechanisms governing antibiotic degradation, providing a mechanistic framework for process optimization.

WP3: Microbial response, resistance/tolerance and ARGs fate
Specific objectives
OS.3.1 - To quantify the residual antimicrobial activity of plasma-treated antibiotic solutions and determine the plasma exposure required to reduce selective pressure.
OS.3.2 - To establish whether plasma treatment destroys resistance genes or releases them, and to what extent the resulting extracellular DNA remains functionally transformable.
OS.3.3 - To determine whether carrying resistance genes confers tolerance to plasma exposure, and whether sub-lethal exposure modifies the susceptibility of surviving bacteria.
Tasks
T.3.1. Quantifying the gap between chemical and biological abatement. Antibiotic solutions treated under a range of plasma conditions will be assayed for residual antimicrobial activity against an isogenic susceptible/resistant Escherichia coli pair, combining growth inhibition and survival measurements.
T.3.2. Discriminating ARG degradation from functional inactivation. Plasma treatment may reduce the number of resistance genes detected, but a lower count does not by itself mean the genes have been destroyed: cell lysis can release them intact into the surrounding medium, where they remain available for uptake by other bacteria. This task therefore follows antibiotic resistance genes in two compartments. For extracellular ARGs, their degree of fragmentation and their capacity to still transfer resistance to recipient bacteria will be assessed. For intracellular ARGs, gene counts will be compared with cell viability and with retention of the carrying plasmid, to establish whether the determinants are destroyed or released.
T.3.3. Assessing cold plasma-driven selection and induction of resistance
Bacteria carrying resistance genes may withstand decontamination processes better than susceptible ones, a cross-tolerance already reported for several biocides (Garratt et al., 2021). This hypothesis will be tested for cold plasma treatment by exposing the isogenic pair directly to plasma and comparing how the two strains survive across increasing doses. Furthermore, exposure that does not kill the bacteria may nonetheless modify the survivors' susceptibility to the antibiotic. To assess this sub-lethal effect, susceptibility will be measured both immediately after exposure and again after the bacteria have recovered without stress.

WP4: Evaluation of cold plasma treatment under environmentally relevant wastewater conditions
Specific objectives:
SO.4.1. To assess the performance of cold plasma treatment in complex wastewater matrices representative of hospital effluents (including synthetic formulations and real wastewater samples).
SO.4.2. To evaluate the influence of wastewater constituents, including dissolved organic matter and inorganic contaminants, on the plasma-induced suppression of antimicrobial activity, bacterial response to plasma exposure, and the persistence and functional inactivation of antibiotic resistance genes.
Tasks
T.4.1. Assessment of the influence of trace metals (e.g. Cu) and natural organic matter (NOM) loads on plasma-generated reactive species (RONS) and antibiotic degradation performance - Their potential catalytic or scavenging effects on plasma-induced oxidation processes will be evaluated in relation to antibiotic degradation efficiency.
T.4.2. The biological endpoints established in WP3 - residual antimicrobial activity, bacterial survival and tolerance to plasma exposure, and the persistence and functional inactivation of antibiotic resistance genes - will be examined in synthetic matrices representative of hospital wastewater and, where feasible, in real wastewater samples, with particular attention to the effect of trace metals (e.g., Cu) and NOM. Transfer potential is characterised on the same basis as in WP3. Given the recognized role of certain metals in the co-selection and maintenance of antimicrobial resistance, the experiments will also assess whether plasma treatment can effectively mitigate combined chemical and biological selective pressures in wastewater matrices.
The outcomes of this work package will provide critical knowledge regarding the applicability, limitations, and optimization of cold plasma technologies for wastewater treatment, contributing to the development of effective strategies for mitigating the environmental dissemination of antimicrobial resistance.
Concerning the plasma part, electrical diagnostics (voltage probes, current probes, digital oscilloscopes) will be used to measure the different signals and to determine the electrical power. Next, optical emission spectroscopy (PI-HRS-750 coupled with a PI-MAX4) will be conducted to identify the reactive species generated by plasma for various conditions. Additionally, an ICCD camera (PI-MAX1) will allow us to capture the plasma propagation and visualize the interactions with the liquid surface. Different optical filters will be applied to visualize the spatial distribution of specific species within the plasma. Finally, concerning the plasma-liquid interactions, physicochemical data from the liquid will be measured (temperature, pH and conductivity) and the presence of reactive species will be analyzed, mainly the concentrations of hydrogen peroxide in the treated water to further evaluate the system's effectiveness.
Quinolone antibiotics have been chosen as a model due to their frequent detection in the environment (Bekele et al., 2026) and their multivariant AMR risk, associated with high human and veterinary consumption, and multiple resistance mechanisms. The plasma-treated solutions will undergo comprehensive chemical and biological characterization. Advanced analytical techniques, including high-performance liquid chromatography (HPLC), liquid chromatography coupled with mass spectrometry (LC-QToF), and total organic carbon analysis, will be used to monitor the degradation kinetics of the antibiotics, identify and follow the temporal evolution of intermediate products and determine degradation pathways, and quantify mineralization.
In parallel, the plasma-treated solutions will undergo biological characterisation targeting the three endpoints of WP3. The residual antimicrobial activity of the treated solutions will be assessed against an isogenic susceptible/resistant Escherichia coli pair, combining growth-rate inhibition monitored by optical density in microplate cultures with survival measurements by colony counting, so as to capture both bacteriostatic and bactericidal effects. Bacterial tolerance to direct plasma exposure will be evaluated from dose-survival curves of the two strains, and any change in antibiotic susceptibility of the survivors following sub-lethal exposure will be determined by minimum inhibitory concentration (MIC) assays performed immediately after treatment and after recovery without stress.
The fate of antibiotic resistance genes will be followed by quantitative PCR (qPCR), distinguishing intracellular from extracellular DNA. Gene integrity will be estimated from the ratio of short- to long-amplicon amplification, used as a fragmentation index, and membrane integrity will be resolved by viability qPCR (PMA treatment) and compared with culturability and plasmid retention. The functional transferability of extracellular resistance genes will be tested by transformation of competent recipient cells, expressed as transformants per unit mass of DNA.
To assess the applicability of plasma treatment on real-world conditions, tests will be carried out in a progressive manner, starting with synthetic single-component solutions, followed by synthetic mixtures designed to simulate real wastewater, and finally real wastewater collected from sanitary units. The water matrix will be characterized in terms of organic matter through Total Organic Carbon (TOC) and Chemical Oxygen Demand (COD) analysis, and in terms of metal content through atomic absorption spectrometry (AAS).
Integrating plasma diagnostics with chemical and biological analyses will provide mechanistic understanding of the links between plasma operating conditions, reactive species production, organic contaminant degradation and ARG destruction, as well as valuable information on the safety of the treated water.