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Caractérisations transcriptionnelle et phénotypique de bactéries dispersées de biofilm à Klebsiella pneumoniae

Abstract : Biofilm development is a complex process involving several steps. The bacterial adhesion to the surface is followed by formation of microcolonies and synthesis of extracellular matrix, which encased bacteria giving rise to mature biofilm. The last step, probably the most poorly described, corresponds to biofilm dispersal. This process is genetically controlled and triggered in response to a wide range of physical and chemical signals, such as temperature, nutrients availability or the accumulation of signal molecules (quorum sensing autoinducers, nitric oxide...). In response to these signals, sessile bacteria synthesize various effectors, which allow a subset of cells to leave the biofilm and colonize new environments. In the context of biofilm-related infections, the biofilm dispersal process is a key mechanism for the release of bacteria from biofilms formed on invasive medicals devices. Little is known about the properties of the resulting biofilm-dispersed bacteria, but they probably play a major role in the subsequent colonization and infection processes within the host. The objective of this work was to characterize the transcriptome and the phenotype of biofilm-dispersed bacteria of the opportunist pathogen Klebsiella pneumoniae comparatively to the planktonic and sessile forms. A transcriptional analysis was carried out using planktonic (both exponential and stationary forms), sessile and biofilm-dispersed bacteria by combining a flow-cell experimental model with global RNA sequencing (RNAseq). Results indicated that biofilm- dispersed bacteria displayed a unique transcriptional pattern in the bacterial lifecycle. Furthermore, analysis of the whole transcriptome of planktonic, sessile and biofilm-dispersed bacteria allowed to emphasize the transcriptional changes occurring in the course of K. pneumoniae lifestyle transitions and to select transcriptional signatures genes for the five bacterial physiological states. The unique transcriptional pattern of biofilm-dispersed bacteria suggests that they display specific physiological characteristics required to colonize new environments. The second part of this work consisted in analyzing the properties of biofilm-dispersed bacteria, by focusing on key mechanisms involved in the physiopathology of K. pneumoniae: adhesion, colonization and virulence. Phenotypic profiling showed that biofilm-dispersed bacteria colonized significantly more biotic and abiotic surfaces than their planktonic counterparts. This increased colonization capacity was not due to an enhanced adhesion or a higher metabolic activity but rather to the intrinsic capacity of the biofilm-dispersed bacteria to form rapidly microcolonies. Besides, biofilm-dispersed bacteria were more pathogenic than their planktonic counterparts showing a better resistance to the bactericidal activity of macrophages. The development of a murine pulmonary infection model is in progress and will enable to assess the virulence of biofilm-dispersed bacteria. Our results indicate that biofilm-dispersed bacteria are placed in a unique state in the bacterial lifecycle, being transcriptionally different from other bacterial forms, and with mixed properties, approaching both the planktonic form (elevated metabolic activity and growth rate) and the sessile form (strong colonization power and high resistance to phagocytosis). These properties certainly play a decisive role in the initiation of biofilm-related infections.
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Cyril Guilhen. Caractérisations transcriptionnelle et phénotypique de bactéries dispersées de biofilm à Klebsiella pneumoniae. Médecine humaine et pathologie. Université Clermont Auvergne, 2017. Français. ⟨NNT : 2017CLFAS015⟩. ⟨tel-02886428⟩

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