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

Les Escherichia coli producteurs de Shiga-toxines (STEC) sont des bactéries pathogènes d'origine alimentaire responsables de diarrhées, de colites hémorragiques et du syndrome hémolytique et urémique, pouvant entraîner la mort. Une des priorités de l’industrie agro-alimentaire est d’éviter la présence des STEC dans l’environnement et dans les chaînes de production. Dans ce secteur, les bactéries sous forme de biofilm représentent un réel problème car en plus de contaminer les installations des chaînes de production, elles sont plus résistantes aux protocoles de nettoyage et de désinfection. Il est donc nécessaire de mieux caractériser les biofilms formés par les STEC et d’identifier les facteurs qui contribuent à leur développement. Les objectifs de la première partie de cette thèse étaient de caractériser le potentiel de formation de biofilms de différents sérotypes STEC. Pour cela nous avons montré que la capacité de formation de biofilms des STEC est très variable. Nous avons identifié que les protéines jouent un rôle important dans l'intégrité des biofilms des STEC. De plus, les isolats du séropathotype A (O157:H7 et O157:NM), avaient un meilleur potentiel de formation de biofilm que les isolats du séropathotype B et C. Nous avons aussi montré que le traitement des biofilms STEC avec des désinfectants réduisait la viabilité mais n'éliminait pas complètement la matrice de biofilm. Nos données indiquent que la formation de biofilms dans l’environnement pourrait contribuer à la persistance des STEC et plus précisément des souches du séropathotype A dans l’environnement. Dans une deuxième partie nous avons étudier comment la carence en phosphate (Pi) affectait la formation de biofilm de la souche O157:H7 EDL933. Les souches E. coli O157:H7 peuvent survivre pendant de longues périodes de temps dans un environnement pauvre en nutriments. Les biofilms pourraient contribuer à surmonter les stress rencontrés dans l’environnement. Lorsqu’E. coli est cultivée en conditions pauvres en Pi, le régulon Pho est induit par PhoB. Le système de transport spécifique au Pi (Pst) est le transporteur de haute affinité du Pi. Dans le mutant Δpst, PhoB est activée de manière constitutive. Nous avons montré que l'activation de PhoB conduisait à l'augmentation de la formation de biofilm chez la souche O157:H7 EDL933. En réponse à la carence en Pi, l'activation de PhoB stimulait directement l'expression des gènes waaH et ycgVEDL933, codant respectivement pour une glycosyltransférase et pour un autotransporteur, tous deux impliqués dans l'augmentation du biofilm. Nous avons également identifié que, dans le mutant Δpst, les lipopolysaccharides (LPS) étaient modifiés et que cela pourrait influencer la composition du biofilm. Pris ensemble, nos résultats montrent que la formation de biofilms contribue à la persistance des STEC dans l'environnement et que les isolats de séropathotype A ont démontré une capacité accrue à former des biofilms. En réponse au stress environnemental tel que la carence en Pi, les STEC adaptent leur mode de vie et passent d'un état libre à un état sessile. Les connaissances acquises au cours de cette thèse pourrait permettrent l’exploration de deux nouvelles applications pour lutter contre la formation de biofilms des STEC, incluant l’utilisation de protéase pour dégrader le biofilm et l’utilisation de surface recouverte d’ions phosphate pour prévenir la formation de biofilm.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

Assessing airflow distribution in vents of a naturally ventilated test facility using reference air velocity measurements

Emission measurement in naturally ventilated buildings is a complex task because wind conditions can change quickly, inducing high spatial and temporal variations in the air velocity and pollutant concentration at the vent level. Simply taking the product of differential pollutant concentration and airflow rate may generate inaccurate results because the limited number of measurement locations usually fails to correctly reflect the velocity and concentration distributions in the vents. To assess the predictability of the airflow distribution in the vents of a naturally ventilated building, detailed measurements were conducted in the vents. Linear regression was applied to velocity measurements taken in the vents and at a 10 m mast (meteomast) located 20 m away. The detailed airflow measurements were used to validate statistical models. Results showed that the velocity distribution in the ridge vent could be modeled accurately and precisely for all wind directions (R-2 > 89%). Models for unidirectional airflows showed high predictability for the side vent (R-2 > 92%). Models for bidirectional airflows showed good predictability for the windward side when the air flowed in the same direction as the outside wind (R-2 > 88%) but showed less accurate results for the leeward side as well as for airflows moving in the opposite direction to the outside wind. For all models and wind directions, the most important input variable was the velocity component measured perpendicular to the vents at the meteomast. The importance of the velocity component measured parallel to the vents increased near the edges of the vent when the vent was on the windward side but did not reach the importance of the perpendicular component. The results confirmed the importance of using different models for unidirectional and bidirectional airflows to obtain accurate airflow assessments

Field test facility for the development of a reference method for ventilation rate and emission measurements in naturally ventilated pig houses

A test facility for field measurements of natural ventilation in livestock buildings was developed. Choice of used materials and methods were taken into account: ultrasonic wind anemometers, linear guiding systems for sensor movement and data loggers were carefully chosen. The true scale test facility makes it possible to measure and study air flows and related characteristics for long periods, under real conditions of natural ventilation

Development of a reference method for the measurement of the ventilation rate through rectangular ventilation openings using ultrasonic anemometers

A measurement method for the airflow rate through rectangular ducts was developed with the use of 2D and 3D ultrasonic anemometers. The measurements were carried out 0.15 duct diameters (Dd) upstream and 0.37 Dd downstream of the outlet of a 1 m wide rectangular duct (1.0 m x 0.5 m) and 0.29 Dd downstream of the outlet of a 3 m wide rectangular duct (3.0 m x 0.5 m). An automated sensor frame was developed to automatically measure consecutive sampling positions. Experiments under varying conditions of flow profile, airflow rate and duct size were carried out. These experiments are a first step towards the implementation of such a method in naturally ventilated systems

Interactions of Intestinal Bacteria with Components of the Intestinal Mucus

The human gut is colonized by a variety of large amounts of microbes that are collectively called intestinal microbiota. Most of these microbial residents will grow within the mucus layer that overlies the gut epithelium and will act as the first line of defense against both commensal and invading microbes. This mucus is essentially formed by mucins, a family of highly glycosylated protein that are secreted by specialize cells in the gut. In this Review, we examine how commensal members of the microbiota and pathogenic bacteria use mucus to their advantage to promote their growth, develop biofilms and colonize the intestine. We also discuss how mucus-derived components act as nutrient and chemical cues for adaptation and pathogenesis of bacteria and how bacteria can influence the composition of the mucus layer