The purpose of our study was to describe the dynamics of E. coli in the rivers of the Seine watershed. This watershed is characterized by a poor microbiological water quality due to an important population density, industrial activities and intensive agriculture. The main objectives of our study were to obtain field microbiological data describing the system and to understand the underlying mechanisms in order to build a model able to describe and predict the fate of the fecal bacteria at the scale of the whole watershed. This model was built in order to help management of microbiological water quality. To be able to describe and modelize the fate of fecal bacteria at the scale of such a large and complex system, it was first necessary to identify and quantify the sources of contamination and to be able to describe the processes affecting these bacteria once discharged to the rivers. The quantification of E. coli in the natural aquatic systems through the traditional culture-based techniques has been strongly questioned during the last years because they have a long response delay (24 to 48 h) and they do not take into account the viable but not culturable bacteria (VBNC) which nevertheless could represent a health thread. For this reason we developed in this study two alternative methods: the direct (without passing through a cultivation step) measurement of the â-D-glucuronidase activity and a fluorescent in situ hybridization with an E. coli specific probe coupled with a viability test. The first has the main advangate to give a very rapid evaluation of the E. coli content of the water while the second one was able to enumerate VBNC E. coli. The study of the sources of contamination of the rivers revealed the predominance, in this anthropogenicised watershed, of the point sources (effluents from wastewater tretament plants). Diffuse sources soil leaching and surface runoff) may nevertheless be locally important and have also be quantified. The influence of the land use on these diffuse sources has been quantified demonstrating that soil leaching and surface runoff of grazed areas was mainly responsible of fecal contamination in rural areas. The attachment of E .coli to particles in the river waters was studied as it can influence the fate of this kind of bacteria in the aquatic systems. A linear regression was found between the percentage of particle-associated E. coli and the SM content of the water. Settling velocities of particle-associated E. coli were determined. The mortality rate of E. coli was analysed following various perspectives: the river order, the attachment of E. coli to particles, the importance of the cultivability loss in the disappearance of these bacteria and the importance of protozoan grazing. The results confirmed the importance of grazing and showed that the abondance of free living E. coli bacteria decreased two times more rapidly than particle-associated E. coli. In the Seine river, we showed that the net loss of culturable E. coli was 1.5 time higher than the net loss of viable E. coli. Finally, no relationship was found between mortality rate and river order. These results have been used to build two models CF-SENEQUE and CF-SiAM-3D which describe E. coli dynamics respectively for the whole Seine river watershed and for the Seine for the estuary. Comparisons model calcultaions with field data of fecal coliforms abundances showed that these models correctly simulated the longitudinal distribution of fecal colifoms in the main rivers of the Seine watershed and in the estuary. These models were used to test the impact on the microbiological water quality of various scenarii of wastewater management .

En esta Tesis se estudia el posible papel de las FLA como reservorio de H. pylori y otras bacterias patógenas en aguas y alimentos mediante técnicas moleculares. En primer lugar se realizó un ensayo de cocultivo entre la bacteria H. pylori y la ameba Acanthamoeba castellanii. Se comprobó, mediante técnicas moleculares específicas para la detección de células viables, PMA-qPCR y DVC-FISH, que la ameba es capaz de internalizar a la bacteria y que esta última permanece viable, demostrando que H. pylori se comporta como una bacteria ARB. Seguidamente, se analizaron un total de 120 muestras ambientales, 100 de agua y 20 de vegetales para comprobar la presencia, tanto de FLA como de H. pylori internalizado en estas FLA. En el caso de las muestras de agua, se analizaron 69 muestras de agua residual y 31 de agua potable. Un total de 55 (79,7%) muestras de agua residual y 12 (38,7%) de agua potable resultaron positivas para la presencia de FLA. Mediante la técnica PMA-qPCR se demostró la presencia de H. pylori internalizado en las FLA presentes en 28 (50,9%) y 11 (91,7%) de las muestras de agua residual y potable analizadas, respectivamente. Mediante DVC-FISH se demostró que las células de H. pylori internalizadas dentro de las FLA presentes en las muestras eran viables en 16 (29,5%) y 5 (41,7%) de las muestras de agua residual y potable analizadas, respectivamente. Además, se consiguió recuperar formas viables cultivables de H. pylori procedente del interior de FLA en 10 (18,2%) de las muestras de agua residual analizadas. Las FLA aisladas e identificadas en las aguas residuales pertenecieron a los géneros Acanthamoeba, Naegleria, Vanellidae y a la familia Vahlkampfiidae. En el caso del agua potable, las FLA aisladas e identificadas pertenecieron a los géneros Acanthamoeba, Echinamoeba y Vermamoeba. En el caso de las muestras de vegetales, concretamente lechugas, todas ellas resultaron positivas para el aislamiento de FLA (100%). Mediante la técnica PMA-qPCR se demostró la presencia de H. pylori internalizado en las FLA en 11 (55,0%) de las muestras y, mediante DVC-FISH, se demostró que las células de H. pylori internalizadas dentro de las FLA eran viables en 5 (25,0%) de las muestras. En este caso no se recuperaron formas viables cultivables de la bacteria. Finalmente, mediante metagenómica de secuenciación dirigida, se analizó el microbioma de las FLA presentes en 20 de las muestras analizadas en esta Tesis (11 de agua residual, 3 de agua potable y 6 de lechugas). Para ello, se eligieron los iniciadores, se evaluaron in silico e in vitro y, una vez comprobada su idoneidad, se emplearon para la secuenciación de las muestras. En los tres tipos de muestras, la clase bacteriana más abundante fue la Gammaproteobacteria. Para los tres tipos de muestras, los filos más abundantes de las bacterias del microbioma de las FLA fueron Proteobacteria y Bacteroidetes y, en el caso del agua residual, también lo fue el filo Planctomycetes. H. pylori se detectó mediante esta técnica en los tres tipos de muestra. Además, como parte del microbioma de FLA de muestras ambientales, se detectaron otras bacterias de interés para la salud pública, tales como Aeromonas, Legionella, Mycobaterium o Pseudomonas. Los resultados obtenidos en esta Tesis demuestran la presencia de FLA patógenas en las muestras ambientales, así como el hecho de que, en algunos casos, estas son transportadoras de bacterias patógenas. Este trabajo también confirma que H. pylori se comporta como una bacteria ARB y que se encuentra viable en el interior de FLA presentes, tanto en aguas residuales y potables como en vegetales. De esta forma, se postula que un modo de transmisión de esta bacteria podría ser a través de las FLA presentes en agua o vegetales. / In this Thesis, the possible role of FLA is studied as a reservoir of H. pylori and other pathogenic bacteria in waters and food by means of molecular techniques. Firstly, a coculture assay between the bacterium H. pylori and the amoeba Acanthamoeba castellanii was carried out. It was verified by means of molecular techniques specific for the detection of viable cells, PMA-qPCR and DVC-FISH, that the amoeba is capable of internalizing the bacterium and that the latter remains viable, demonstrating that H. pylori behaves as an ARB bacterium. Afterwards, a total of 120 environmental samples, 100 of water and 20 of vegetables, were analyzed to verify the presence FLA as well as internalized H. pylori into these FLA. In case of water samples, 69 samples of wastewater and 31 samples of drinking water were analyzed. A total of 55 (79,7 %) wastewater and 12 (38,7 %) of drinking water samples turned out to be positive for FLA's presence. By means of PMA-qPCR technique, the presence of FLA-internalized H. pylori was demonstrated in 28 (50,9 %) and 11 (91,7 %) of the wastewater and drinking water samples analyzed, respectively. By means of DVC-FISH it was demonstrated that the FLA-internalized H. pylori cells were viable in 16 (29,5 %) and 5 (41,7 %) of the wastewater and drinking water samples analyzed, respectively. In addition, viable cultivable forms of H. pylori coming from the inside of FLA were recovered from 10 (18,2 %) of the analyzed wastewater samples. The isolated and identified FLA from wastewater samples belonged to the genus Acanthamoeba, Naegleria, Vanellidae and to the family Vahlkampfiidae. In the case of drinking wáter, the isolated and identified FLA belonged to the genus Acanthamoeba, Echinamoeba and Vermamoeba. In the case of the vegetable samples, specifically lettuces, all of them turned out to be positive for FLA's isolation (100 %). By means of the PMA-qPCR technique, the presence of FLA-internalized H. pylori was demonstrated in 11 (55,0 %) of the samples and, by means of DVC-FISH, it was demonstrated that FLA-internalized H. pylori cells were viable in 5 (25,0 %) of the samples. In this case, viable cultivable forms of the bacterium could not be recovered. Finally, by means of amplicon-based metagenomics, the FLA microbiome of 20 previously analyzed samples in this Thesis (11 wastewater, 3 drinking water and 6 lettuce samples) was analyzed. To do so, a pair of primers were selected and evaluated in silco and in vitro and, once checked its suitability, they were used to perform the samples' sequencing. In the three types of samples, the most abundant bacterial class was the Gammaproteobacteria. For the three types of samples, the most abundant bacterial phylum of the FLA microbiome were Proteobacteria and Bacteroidetes and, in case of the wastewater, it was also the phylum Planctomycetes. H. pylori was detected by means of this technology in the three types of samples. In addition, as part of FLA's microbiome of environmental samples, other bacteria of public health interest were detected, such as Aeromonas, Legionella, Mycobacterium or Pseudomonas. The results obtained in this Thesis demonstrate the presence of pathogenic FLA in the environmental samples, as well as the fact that, in some cases, these they are carriers of pathogenic bacteria. This work also confirms that H. pylori behaves as an ARB bacterium and that it is viable inside the present FLA in wastewater as well as in drinking water and in vegetables. This way, it is postulated that a way of transmission of this bacterium might be through the FLA present in water or vegetables. / En esta Tesi s'estudia el possible paper de les FLA com a reservori de H. pylori i altres bacteris patògens en aigües i aliments per mitjà de tècniques moleculars. En primer lloc es va realitzar un assaig de cocultiu entre el bacteri H. pylori i l'ameba Acanthamoeba castellanii. Es va comprovar per mitjà de tècniques moleculars específiques per a la detecció de cèl¿lules viables, PMA-qPCR i DVC-FISH, que l'ameba és capaç d'internalizar al bacteri i que esta última roman viable, demostrant que H. pylori es comporta com un bacteri ARB. A continuació, es van analitzar un total de 120 mostres ambientals, 100 d'aigua i 20 de vegetals per a comprovar la presència tant de FLA com de H. pylori internalitzat en estes FLA. En el cas de les mostres d'aigua, es van analitzar 69 mostres d'aigua residual i 31 d'aigua potable. Un total de 55 (79,7%) mostres d'aigua residual i 12 (38,7%) d'aigua potable van resultar positives per a la presència de FLA. Per mitjà de la tècnica PMA-qPCR es va demostrar la presència d'H. pylori internalitzat en les FLA presents en 28 (50,9%) i 11 (91,7%) de les mostres d'aigua residual i potable analitzades, respectivament. Per mitjà de DVC-FISH es va demostrar que les cèl¿lules d'H. pylori internalitzades dins les FLA presents en les mostres eren viables en 16 (29,5%) i 5 (41,7%) de les mostres d'aigua residual i potable analitzades, respectivament. A més, es va aconseguir recuperar formes viables cultivables d'H. pylori procedent de l'interior de FLA en 10 (18,2%) de les mostres d'aigua residual analitzades. Les FLA aïllades i identificades en les aigües residuals van pertànyer als gèneres Acanthamoeba, Naegleria, Vanellidae i a la família Vahlkampfiidae. En el cas de l'aigua potable, les FLA aïllades i identificades van pertànyer als gèneres Acanthamoeba, Echinamoeba i Vermamoeba. En el cas de les mostres de vegetals, concretament encisams, totes elles van resultar positives per a l'aïllament de FLA (100%). Per mitjà de la tècnica PMA-qPCR es va demostrar la presència d'H. pylori internalitzat en les FLA en 11 (55,0%) de les mostres i, per mitjà de DVC-FISH, es va demostrar que les cèl¿lules d'H. pylori internalitzades dins les FLA eren viables en 5 (25,0%) de les mostres. En este cas no es van recuperar formes viables cultivables del bacteri. Finalment, per mitjà de metagenómica de seqüenciació dirigida, es va analitzar el microbioma de les FLA presents en 20 de les mostres analitzades en esta Tesi (11 d'aigua residual, 3 d'aigua potable i 6 d'encisams). Per tal de fer això, es van triar els iniciadors, es van avaluar in silico i in vitro i, una vegada comprovada la seua idoneïtat, es van emprar per a la seqüenciació de les mostres. En els tres tipus de mostres, la classe bacteriana més abundant va ser la Gammaproteobacteria. Per als tres tipus de mostres, els filos més abundants dels bacteris del microbioma de les FLA van ser Proteobacteria i Bacteroidetes i, en el cas de l'aigua residual, també ho va ser el filo Planctomycetes. H. pylori es va detectar per mitjà d'esta tècnica en els tres tipus de mostra. A més, com a part del microbioma de FLA de mostres ambientals, es van detectar altres bacteris d'interés per a la salut pública, com ara Aeromonas, Legionella, Mycobaterium o Pseudomonas. Els resultats obtinguts en esta Tesi demostren la presència de FLA patògenes en les mostres ambientals, així com el fet de que, en alguns casos, estes són transportadores de bacteris patògens. Este treball també confirma que H. pylori es comporta com un bacteri ARB i que es troba viable en l'interior de FLA presents, tant en aigües residuals i potables com en vegetals. D'esta manera, es postula que una manera de transmissió d'este bacteri podria ser a través de les FLA presents en aigua o vegetals. / Moreno Mesonero, L. (2018). Estudio del papel de las amebas de vida libre como reservorio de Helicobacter pylori y otras bacterias patógenas en aguas y alimentos mediante técnicas moleculares [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/111952 / TESIS

The purpose of our study was to describe the dynamics of E. coli in the rivers of the Seine watershed. This watershed is characterized by a poor microbiological water quality due to an important population density, industrial activities and intensive agriculture. The main objectives of our study were to obtain field microbiological data describing the system and to understand the underlying mechanisms in order to build a model able to describe and predict the fate of the fecal bacteria at the scale of the whole watershed. This model was built in order to help management of microbiological water quality. <p>To be able to describe and modelize the fate of fecal bacteria at the scale of such a large and complex system, it was first necessary to identify and quantify the sources of contamination and to be able to describe the processes affecting these bacteria once discharged to the rivers. <p>The quantification of E. coli in the natural aquatic systems through the traditional culture-based techniques has been strongly questioned during the last years because they have a long response delay (24 to 48 h) and they do not take into account the viable but not culturable bacteria (VBNC) which nevertheless could represent a health thread. For this reason we developed in this study two alternative methods: the direct (without passing through a cultivation step) measurement of the â-D-glucuronidase activity and a fluorescent in situ hybridization with an E. coli specific probe coupled with a viability test. The first has the main advangate to give a very rapid evaluation of the E. coli content of the water while the second one was able to enumerate VBNC E. coli. <p>The study of the sources of contamination of the rivers revealed the predominance, in this anthropogenicised watershed, of the point sources (effluents from wastewater tretament plants). Diffuse sources soil leaching and surface runoff) may nevertheless be locally important and have also be quantified. The influence of the land use on these diffuse sources has been quantified demonstrating that soil leaching and surface runoff of grazed areas was mainly responsible of fecal contamination in rural areas.<p>The attachment of E .coli to particles in the river waters was studied as it can influence the fate of this kind of bacteria in the aquatic systems. A linear regression was found between the percentage of particle-associated E. coli and the SM content of the water. Settling velocities of particle-associated E. coli were determined. The mortality rate of E. coli was analysed following various perspectives: the river order, the attachment of E. coli to particles, the importance of the cultivability loss in the disappearance of these bacteria and the importance of protozoan grazing. The results confirmed the importance of grazing and showed that the abondance of free living E. coli bacteria decreased two times more rapidly than particle-associated E. coli. In the Seine river, we showed that the net loss of culturable E. coli was 1.5 time higher than the net loss of viable E. coli. Finally, no relationship was found between mortality rate and river order. <p>These results have been used to build two models CF-SENEQUE and CF-SiAM-3D which describe E. coli dynamics respectively for the whole Seine river watershed and for the Seine for the estuary. Comparisons model calcultaions with field data of fecal coliforms abundances showed that these models correctly simulated the longitudinal distribution of fecal colifoms in the main rivers of the Seine watershed and in the estuary. These models were used to test the impact on the microbiological water quality of various scenarii of wastewater management .<p> / Doctorat en sciences, Spécialisation biologie animale / info:eu-repo/semantics/nonPublished