Etude de facteurs biotiques et abiotiques qui contrôlent l'implantation des biofilms de Pseudomonas aeruginosa dans les réseaux de distribution d'eau thermale

Natural mineral waters are distinguished from drinking water bytheir content in minerals and trace elements. Their use for therapeuticpurposes under medical control is performed in spas. Management ofdistribution networks and the microbiological quality of water are subjectto regulations. Despite the establishment of specific procedures, spas areregularly confronted with microbiological contamination mainly due to P.aeruginosa. This opportunistic pathogen has substantial adaptive capacity,resistance and persistence in the environment, under biofilm.The aim of this thesis work is to bring understanding on the P.aeruginosa behaviour in the natural mineral water distribution networksand validate the effectiveness of treatment products.The influence of the mineral composition of 3 waters on biofilmformation capacity of 9 P. aeruginosa strains of various origins could bedemonstrated. It appears that the amount of biofilm produced after 24 hare less important for the strong mineralization. Modulation of theproduction of virulence factors depending on the water quality iscorrelated to the specific action of certain ions and iron bioavailability. Inhigh mineralization conditions it has been shown that organic materialspromote biofilm formation of P. aeruginosa while metallic materials tendto disadvantage it. The efficiency of a treatment sequence of a patient pointof use has been validated on the 24 h biofilm model and on all materials.Les eaux minérales naturelles se distinguent de l’eau potable par leur contenu en sels minéraux et en éléments traces. Leur utilisation à des fins thérapeutiques s’effectue sous contrôle médical dans des établissements thermaux. La gestion des réseaux de distribution ainsi que la qualité microbiologique de l’eau font l’objet de réglementations. Malgré la mise en place de procédures spécifiques, les établissements thermaux sont régulièrement confrontés à des contaminations microbiologiques majoritairement dues à P. aeruginosa. Ce pathogène opportuniste possède d’importantes capacités d’adaptation, de résistance et de persistance dans l’environnement, notamment sous forme de biofilms. L’objectif de ces travaux de thèse est d’apporter des éléments de compréhension sur le comportement de P. aeruginosa dans les réseaux de distribution d’eau minérale naturelle et de valider l’efficacité de produits de traitement. L’influence de la composition minérale de 3 eaux sur les capacités à former du biofilm de 9 souches de P. aeruginosa d’origines diverses a pu être démontrée. Il apparait que les quantités de biofilms produites au bout de 24 h sont moins importantes pour la minéralisation forte. La modulation de la production de facteurs de virulence en fonction de la qualité de l’eau est corrélée à l’action spécifique de certains ions et à la biodisponibilité du fer. En conditions de minéralisation forte il a été montré que les matériaux organiques favorisent la formation de biofilm de P. aeruginosa tandis que les matériaux métalliques ont tendance à la défavoriser. L’efficacité d’une séquence de traitement de postes de soins a pu être validée sur un modèle de biofilm âgé de 24 h et sur tous les matériaux

Study of biotic and abiotic factors that control the implementation of Pseudomonas aeruginosa biofilms in thermal water distribution networks

Les eaux minérales naturelles se distinguent de l’eau potable par leur contenu en sels minéraux et en éléments traces. Leur utilisation à des fins thérapeutiques s’effectue sous contrôle médical dans des établissements thermaux. La gestion des réseaux de distribution ainsi que la qualité microbiologique de l’eau font l’objet de réglementations. Malgré la mise en place de procédures spécifiques, les établissements thermaux sont régulièrement confrontés à des contaminations microbiologiques majoritairement dues à P. aeruginosa. Ce pathogène opportuniste possède d’importantes capacités d’adaptation, de résistance et de persistance dans l’environnement, notamment sous forme de biofilms. L’objectif de ces travaux de thèse est d’apporter des éléments de compréhension sur le comportement de P. aeruginosa dans les réseaux de distribution d’eau minérale naturelle et de valider l’efficacité de produits de traitement. L’influence de la composition minérale de 3 eaux sur les capacités à former du biofilm de 9 souches de P. aeruginosa d’origines diverses a pu être démontrée. Il apparait que les quantités de biofilms produites au bout de 24 h sont moins importantes pour la minéralisation forte. La modulation de la production de facteurs de virulence en fonction de la qualité de l’eau est corrélée à l’action spécifique de certains ions et à la biodisponibilité du fer. En conditions de minéralisation forte il a été montré que les matériaux organiques favorisent la formation de biofilm de P. aeruginosa tandis que les matériaux métalliques ont tendance à la défavoriser. L’efficacité d’une séquence de traitement de postes de soins a pu être validée sur un modèle de biofilm âgé de 24 h et sur tous les matériaux.Natural mineral waters are distinguished from drinking water bytheir content in minerals and trace elements. Their use for therapeuticpurposes under medical control is performed in spas. Management ofdistribution networks and the microbiological quality of water are subjectto regulations. Despite the establishment of specific procedures, spas areregularly confronted with microbiological contamination mainly due to P.aeruginosa. This opportunistic pathogen has substantial adaptive capacity,resistance and persistence in the environment, under biofilm.The aim of this thesis work is to bring understanding on the P.aeruginosa behaviour in the natural mineral water distribution networksand validate the effectiveness of treatment products.The influence of the mineral composition of 3 waters on biofilmformation capacity of 9 P. aeruginosa strains of various origins could bedemonstrated. It appears that the amount of biofilm produced after 24 hare less important for the strong mineralization. Modulation of theproduction of virulence factors depending on the water quality iscorrelated to the specific action of certain ions and iron bioavailability. Inhigh mineralization conditions it has been shown that organic materialspromote biofilm formation of P. aeruginosa while metallic materials tendto disadvantage it. The efficiency of a treatment sequence of a patient pointof use has been validated on the 24 h biofilm model and on all materials

Occurrence de

Pseudomonas aeruginosa is an opportunistic pathogen that naturally occurs in aquatic environments. This bacterium is adapted to oligotrophic environmental conditions and could be detected in drinking water systems. Although, water for human consumption is required to be free of any bacteria that might pose a health risk, P. aeruginosa is not included in parameters to be analyse and in a recent report the ANSES conclude that there is no proof that the normal use of contaminated drinking water can be a source of infection by oral route in the general population. This bacterium is mostly harmless for healthy people but must be regarded as a relevant opportunistic pathogen for sensitive human population especially for hospitalized people in intensive care unit. This bacterium is responsible for almost 10% of the hospital-borne infections and drinking water network inside the building is suspected to be a source and a reservoir of P. aeruginosa. Although the target level to achieve is the absence of P. aeruginosa at terminal point-of-use, several studies showed that 4.5 in 97% of water samples taken in intensive care units are contaminated by P. aeruginosa. The origin of patients’ contamination remained discussed but among exogen sources of contamination, tap water is often suspected to be a source and a reservoir for these opportunistic pathogens. Nevertheless control strategies are limited for drinking water and recurrent contamination of tap water remained unsolved. Water contamination could be explained by retrograde contamination and by colonization of taps. To better characterize the populations of P. aeruginosa present in biofilm drinking water networks, we developed a protocol to sample biofilm from taps. Assays were performed on biofilms established inside taps and on the surface of tap swirls. Mechanical action of glass beads was compared with swabbing and proved to have more efficiency in sampling biofilm on surface of tap swirls. The protocol was then adapted to sample biofilm in faucets. It consists in filling the tap to study with a mixture of sterile distilled water and borosilicate glass beads of 1 mm in diameter and then to perform a mechanical shaking of 3 minutes with a vortex. This protocol will be used to collect biofilm on terminal point-of-use and to compare occurrence of P. aeruginosa in this type of biofilm and in the biofilm established on plumbing material

Optimized tuning of TALEN specificity using non-conventional RVDs

A key feature when designing DNA targeting tools and especially nucleases is specificity. The ability to control and tune this important parameter represents an invaluable advance to the development of such molecular scissors. Here, we identified and characterized new non-conventional RVDs (ncRVDs) that possess novel intrinsic targeting specificity features. We further report a strategy to control TALEN targeting based on the exclusion capacities of ncRVDs (discrimination between different nucleotides). By implementing such ncRVDs, we demonstrated in living cells the possibility to efficiently promote TALEN-mediated processing of a target in the HBB locus and alleviate undesired off-site cleavage. We anticipate that this method can greatly benefit to designer nucleases, especially for therapeutic applications and synthetic biology