La formation de biofilm des Escherichia coli producteurs de Shiga-toxines : caractérisation et rôle du régulon Pho

Shiga-toxin producing Escherichia coli (STEC) are food-borne pathogens that cause diarrhea, hemorrhagic colitis, and hemolytic-uremic syndrome, which may result in death. A major priority for the food industry is to avoid the presence of STEC in the environment and the food production chain. In the latter sector, biofilm formed by bacteria represents a real problem because in addition to contaminating the facilities, they are more resistant to traditional cleaning and disinfection protocols. Therefore, it is necessary to better characterize the biofilms formed by STEC and to identify the factors that contribute to their development. The objective of the first part of this thesis was to evaluate the biofilm formation of human STEC isolates representing the most pathogenic seropathotypes, to characterize the matrix composition of the strongest STEC biofilms and to evaluate their tolerance to sanitizers. The results showed that biofilm formation by STEC strains was highly variable. We also identified that proteins were important for the biofilm integrity. Moreover, seropathotype A isolates (O157:H7 and O157:NM), which are associated to the highest relative incidence of human infection, had a greater ability to form biofilms than seropathotype B and C isolates. Treatment with sanitizers reduced the viability of STEC but did not completely remove the biofilm matrix. Overall, our data indicate that biofilm formation in the environment could contribute to the persistence of STEC and specifically seropathotype A isolates in the environment. In the second part of this thesis, we studied how the stress of phosphate (Pi) depletion affected the biofilm formation of O157:H7 EDL933. E. coli O157:H7 can survive for prolonged periods under nutrient-deprived environmental conditions. Biofilms are thought to participate in this environmental lifestyle. When O157:H7 strain EDL933 was grown under Pi starvation conditions, its Pho regulon was induced by the regulator PhoB. The Pi-specific transport (Pst) system is the high-affinity Pi transporter. In its isogenic Δpst mutant, PhoB was constitutively activated. We identified that PhoB activation lead to the increase of O157:H7 biofilm formation. We have shown that when it was activated, the regulator PhoB was directly controlling the expression of two genes, waaH and ycgVEDL933, encoding respectively a glycosyltransferase and an autotransporter. These two genes were both shown to be involved in the biofilm increase in response to Pi-starvation. We also identified that in the Δpst mutant, lipopolysaccharides (LPS) vi were modified and that this could influence the biofilm composition. Taken together our results show that biofilm formation contributes to the persistence of STEC in the environment and that seropathotype A isolates have an increased ability to form biofilm. In response to environmental stress such as Pi deficiency, STEC adapt and transit from a free state to a sessile state. The acquired knowledge during this thesis could allow the exploration of two new applications to fight against the biofilm formation of STEC, including the use of protease to degrade the biofilm and the use of surface covered with phosphate ions to prevent biofilm formation.

Keyphrases: Biofilm, STEC, Phosphate, LPS, Pho regulon