Bacterial persistence is considered as one of the primary reason for antibiotic tolerance besides genetically acquired antibiotic resistance. Persisters are the subpopulation of a clonal bacterial population, which can survive environmental extremes and become invulnerable to stresses due to limited metabolic activities and physiological functions. Cognate toxin and antitoxin (TA) pairs, which are transcribed simultaneously from the same or different operons within the bacterial chromosomes or plasmids, play an important role for bacterial survival during stressful growth environments. Pseudomonas aeruginosa PAO1 is one of the most versatile microorganisms in the environment. Despite its ubiquitous presence, no studies have shown the differential expression pattern of its toxin-antitoxins, and persistence related genes. The purpose of the following study is to analyze differential expression of P. aeruginosa PAO1 type II toxin-antitoxins and persistence related genes under different growth conditions and to show how their stoichiometric ratio changes during different growth conditions. Differential expression analysis indicated that the toxins and antitoxin pairs behave differently under different growth conditions. In addition, the genes related to persistence presented relatively consistent differential expression pattern under different growth environment. / Master of Science / Bacterial persistence is one of the main reason for antibiotic tolerance and recurrent infections. Toxin-antitoxin molecules play an important role during bacterial persistence. Change in the expression of toxin, antitoxins, and persistence related genes and the ratio of the toxin to antitoxin mRNA molecules are important for bacterial survival in stressful environments. Pseudomonas aeruginosa PAO1 is one of most ubiquitous bacteria and responsible for recurrent infection in patients with weaker and compromised immunity. This mRNA sequence (RNA-Seq) analysis study of P. aeruginosa PAO1 showed different expression levels of toxin, antitoxin, and persistence related genes in various stressful growth conditions. This expression also showed the different ratios of the toxin to antitoxin mRNA molecules under different stress conditions. These implicate the different hypothetical roles of these toxin and antitoxin molecules in different growth conditions.

Les systèmes bactériens sont complexes et adaptatifs. Soumis à des perturbations, telles qu’un traitement antibiotique, ils survivent, se régénèrent et évoluent. Ceci est d’autant plus vrai pour les biofilms, capables de surmonter des traitements létaux à des bactéries planctoniques. La capacité des systèmes à retrouver leur équilibre initial, certaines fonctions ou compositions après un choc est appelée résilience. La résilience est souvent considérée comme complémentaire à la résistance en écologie. Pourtant, la résilience aux antibiotiques reçoit peu de considération en comparaison de la résistance aux antibiotiques en bactériologie. L’une des raisons de ce désintérêt est que ce concept est souvent mal défini et ambigu. Dans cette thèse, nous proposons tout d’abord une base conceptuelle de la résilience aux antibiotiques. A partir de l’analyse de différentes définitions existantes de la résilience, nous fournissons une démarche pour formaliser le concept de résilience dans le contexte d’une population bactérienne soumise à des traitements antibiotiques. De cette première analyse, le mécanisme biologique de persistance bactérienne est ressorti comme important dans la résilience aux antibiotiques. Ce phénomène repose sur la formation de cellules tolérantes aux antibiotiques, les persisters, dont la formation est influencée par les conditions environnementales. Afin de relier la formation des persisters aux conditions environnementales, nous avons développé des modèles mathématiques de transition phénotypique entre cellules sensibles et persisters que nous avons calibrés et testés à l’aide de données expérimentales. Enfin, nous avons étudié l’effet de la persistance bactérienne sur la résilience aux antibiotiques des biofilms. Pour cela, nous avons développé un modèle individu-centré de biofilm intégrant des transitions entre cellules sensibles et persisters. Différentes stratégies de transition ont été reliées à la capacité des biofilms à croître, survivre et se régénérer après un choc antibiotique. La mise en place d’expériences capables de fournir des données à comparer aux simulations est proposée dans la discussion de cette thèse. Cette thèse contribue à la clarification du concept de résilience aux antibiotiques et à la compréhension du phénomène de persistance bactérienne dans les biofilms. Elle ouvre des perspectives sur l’utilisation du concept de résilience en bactériologie clinique et souligne l’importance de l’hétérogénéité des populations bactériennes dans leur capacité à confronter les perturbations et évoluer. / Bacterial systems are complex and adaptive. When faced with disturbances, such as antibiotic treatments, they survive, recover and evolve. This is particularly true for biofilms, which survive treatments that planktonic cells cannot overcome. The capacity of systems to recover their initial state, some of their functions or composition after a disturbance is called resilience. The resilience concept is often considered complementary to resistance in ecology. However, antibiotic resilience has received little attention compared to antibiotic resistance. One reason of this lack of interest comes from the fact that the resilience concept is often poorly defined and ambiguous. In this thesis, we firstly developed a conceptual framework of antibiotic resilience and applied this framework to the case of a bacterial population faced with antibiotics. This analysis highlighted the importance of the biological mechanism of bacterial persistence. This phenomenon is based on the formation of sub-populations of antibiotic tolerant cells, the persisters, which is influenced by environmental conditions. To relate persister formation to environmental conditions, we developed mathematical models of phenotypic switches between susceptible and persister cells and calibrated and tested them with experimental data. Lastly, we studied the influence of bacterial persistence on biofilm antibiotic resilience. For this purpose, we developed an individual-based model of biofilm with phenotypic switches between susceptible and persister cells. Different strategies of phenotypic switches were related to the dynamics of growth, survival and recovery of bacterial biofilms faced with antibiotic shocks. The setting up of experimentations to obtain data to compare to simulations is presented in the discussion of this thesis. Globally, this thesis contributes to the clarification of the concept of antibiotic resilience and to the understanding of bacterial persistence in biofilms. It gives new perspectives on the use of the resilience concept in clinical bacteriology and emphasizes the importance of the heterogeneity of bacterial populations in their capacity to face disturbances and evolve.

Together with the current antibiotic resistance crisis, bacterial persistence appears to play an increasingly important role in the frequent failure of antibiotic treatments. Persister cells are rare bacteria that transiently become drug tolerant, allowing them to survive lethal concentrations of bactericidal antibiotics. Upon antibiotic removal, persister cells are able to resume growth and give rise to a new bacterial population as sensitive to the antibiotic as the original population. Interest in persister cells seriously increased in the past few years as these phenotypic variants were shown to be involved in the recalcitrance of chronic infections, such as tuberculosis and pneumonia and in the well-known biofilm tolerance to antibiotics. Persistence has therefore been extensively studied throughout the last decade, which led to the discovery of large variety of different molecular mechanisms involved in persisters formation. However, the specific physiology of bacterial persisters remains elusive up to now, mainly because of the transient nature and the low frequencies of persister cells in growing bacterial cultures. This work aims to gain a better understanding of the physiology of Escherichia coli persisters by combining population analyses with single-cell observations.In the first part of this thesis, we developed an experimental method allowing for measuring persistence with increased reproducibility. The method was further refined, which allowed us to observe four distinct phases in the ofloxacin time-kill curve, suggesting the existence of a tolerance continuum at the population level at treatment time. Characterization of these four phases notably revealed that the growth rate and the intrinsic antibiotic susceptibility of the strain define the number of surviving cells at the onset of the persistence phase, while persister cells survival mainly relies on active stress responses (SOS and stringent responses in particular).We next investigated the molecular mechanisms underlying the well-known correlation between persistence and the growth rate. Interestingly, we showed that the growth rate determines the number of survival cells at the onset of the persistent phase, whereas it does not affect the death rate of persister cells during antibiotic treatment. Furthermore, slow growth was shown to influence survival to ofloxacin independently of the replication rate, thereby suggesting that target inactivation solely cannot explain this correlation. However, our preliminary data indicate that ppGpp induction upon ofloxacin exposure substantially increases in slow growing bacterial populations, supporting a model in which slow growth would allow bacteria to respond faster to the antibiotic treatment, thereby generating more persisters than fast growing bacterial populations.Finally, both population and single-cell analyses were performed to assess the influence of the SOS response on persistence to ofloxacin. Firstly, population analyses revealed that the SOS response is required for survival of both sensitive and persister cells, but only during recovery, after ofloxacin removal, presumably allowing cells to induce SOS-dependent DNA repair pathways, required to deal with the accumulated ofloxacin-induced DNA lesions. The SOS response therefore appears as a good target for anti-persisters strategies, as shown by the 100-fold decrease in persistence upon co-treatment of a bacterial population with an SOS-inhibitor and ofloxacin. Secondly, single-cell analyses revealed that persister cells sustain similar DNA damages than sensitive cells upon ofloxacin treatment and induce SulA- and SOS-independent filamentation upon antibiotic removal, probably reflecting the presence of remaining cleaved complexes, formed during ofloxacin exposure. Importantly, we showed filamentation to occur in persister cells upon ampicillin treatment as well, thereby suggesting these filaments to be part of a more general survival pathway, which molecular basis remains unknown. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished