Rainbow trout represents the most prominent species in freshwater farming in UK aquaculture. One of the common diseases constraining rainbow trout production and increasingly causing problems in Atlantic salmon (Salmo salar L.) hatcheries worldwide is rainbow trout fry syndrome (RTFS) or bacterial cold water disease (BCWD). During the last 20 years, the development of a commercial vaccine against RTFS has been hindered by the prevalence of a wide range of the fish pathogen F. psychrophilum, thus the current treatment of choice is the use of antibiotics. Studies involved in understanding the innate and adaptive immune response of vaccinated rainbow trout fry using inactivated whole cell are still lacking. Therefore, the aim of this thesis is to characterise the strain diversity and antibiotic susceptibility of UK F. psychrophilum isolates, evaluate the efficacy of a whole-cell formalin-killed polyvalent vaccine, which was developed based on the characterisation results of this study, and investigate the immune response in trout fry following the immersion vaccination via the changes in expression of relevant immune genes. A total of 315 F. psychrophilum isolates, 293 of which were collected within the UK, were characterised using four genotyping methods and a serotyping scheme. A high strain diversity was identified among the isolates with 54 pulsotypes, ten (GTG)5-PCR types, two 16S rRNA allele lineages, seven plasmid profiles and three serotypes. The predominant profile observed within the F. psychrophilum isolates examined was PFGE cluster II – (GTG)5-PCR type r1 – 16S rRNA lineage II – serotype Th (n= 70/156, 45%). The characterisation results not only revealed the wide distribution within the UK and the persistence within a site of predominant pulsotypes, but also the presence of unique genotypes in certain sites or countries. Co-existence of genetically and serologically heterogeneous isolates within each farm was detected, highlighting the reasons this disease is so difficult to control, especially by vaccination. The occurrence over time of F. psychrophilum pulsotypes within a site could provide important epidemiological data for farm management and the development of site-specific vaccines. The antimicrobial susceptibilities of 140 F. psychrophilum strains, 125 of which were from the UK, were evaluated by the broth microdilution (MIC) and disc diffusion methods. There was evidence of reduced susceptibilities to three of the main antimicrobials used in UK aquaculture. Broth microdilution testing showed that only 12% of 118 UK isolates tested were WT to oxolinic acid (MIC COWT 0.25 mg L-1), 42% were WT for oxytetracycline (MIC COWT 0.25 mg L-1), and 66% were WT for amoxicillin. In contrast, all the isolates tested were WT (MIC COWT 2 mg L-1) for florfenicol, the antimicrobial of choice for RTFS control in the UK. Despite the imprecision of disc diffusion-based COWT values due to high standard deviations, there was a high categorical agreement between the classification of the strains (into WT or NWT) by MIC and disc diffusion methods for florfenicol (100%), oxolinic acid (99%), amoxicillin (97%) and oxytetracycline (94%). In general, this study showed that the UK F. psychrophilum isolates examined remain susceptible to florfenicol and also stresses the importance of performing susceptibility testing using standardised methods and COWT values. Several statistically significant associations between genotypes and the reduced susceptibilities of F. psychrophilum strains were revealed. A whole-cell formalin killed polyvalent vaccine against RTFS/BCWD was developed by combining three genetically and serologically divergent strains, recently collected from UK farms. The efficacy of this polyvalent vaccine was evaluated after immersion vaccination in 5 g trout and bath challenge using hydrogen peroxide as a pre-stressor with a virulent heterologous isolate of F. psychrophilum strain. Significant protection was achieved with an RPS of 84%. The combination of exposure to hydrogen peroxide prior to bath challenge may be an alternative to an injection challenge with 12 g trout, although further standardisation and optimisation of the challenge model is required. Changes in the innate immune response of trout fry following the initial vaccination included the up-regulation of the interleukin 1 β (IL-1β) gene in head kidney at 4 h and the up-regulation of toll-like receptor-2 (TLR-2) in skin at day 2. While the expression levels of C3 was unchanged, the down regulation of CD8-α in head kidney and spleen and CD4-1 in spleen were documented. IgM and IgT transcripts were found to be up-regulated in hind-gut two days post-vaccination. Understanding the strain diversity and the antibiotic susceptibility of UK F. psychrophilum isolates could help improve the control strategies, such as preventing the spreading of pathogenic F. psychrophilum clones between fish farms, reducing the use of antibiotics in RTFS/BCWD treatment and monitoring the development of acquired antibiotic resistance mechanisms. Moreover, strain characterisation data of UK F. psychrophilum species has assisted in selecting suitable candidates for developing an effective RTFS vaccine.

Made available in DSpace on 2014-06-11T19:27:15Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-06-28Bitstream added on 2014-06-13T18:31:12Z : No. of bitstreams: 1 baldin_jc_me_jabo.pdf: 183312 bytes, checksum: f9e945ecca299b8ddcedf222e57ec9d5 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O gelo é um excelente meio de prolongar o frescor do peixe e na indústria alimentícia tem um dos mais importantes papéis, uma vez que retarda a multiplicação bacteriana e, se fabricado sob más condições sanitárias pode se tornar um fator de risco para os consumidores. Sendo assim, este estudo objetivou avaliar a qualidade microbiológica do gelo utilizado na conservação do pescado por meio da quantificação de microrganismos heterotróficos mesófilos e psicrotróficos viáveis, coliformes totais e termotolerantes, além de investigar a presença de Escherichia coli, Staphylococcus aureus e Salmonella spp. Para tal, foram colhidas 63 amostras de gelo de seis estabelecimentos, sendo cinco hipermercados e uma peixaria, situados no município de Ribeirão Preto/SP. Os resultados obtidos mostraram populações de microrganismos heterotróficos mesófilos variando entre 1,0 x 10 e 3,0 x 104 UFC.mL-1 e psicrotróficos variando entre 1,0 x 10 e 1,0 x 106 UFC/mL, sendo que 19,05% das amostras contendo microrganismos mesófilos encontravam-se com populações acima do permitido pela legislação vigente. Já 22,22% das amostras foram positivas para coliformes totais e 9,52% para coliformes termotolerantes. A presença de Staphylococcus aureus foi confirmada em três amostras, E. coli e Salmonella spp. não foram encontradas. Portanto, a avaliação da qualidade microbiológica do gelo, neste experimento, permitiu concluir que a água utilizada na fabricação de gelo utilizado para conservação de pescado comercializado nos estabelecimentos pode ser contaminada por manuseio inadequado e seu incorreto acondicionamento, o que é um fator de risco para a saúde do consumidor uma vez que o gelo em contato com o peixe pode contaminá-lo / The ice is an excellent way to prolong the freshness of the fish and in the food industry has one of the most important roles, since it slows the bacterial growth, and if it is manufactured under poor sanitary conditions could become a risk factor for consumers. So, this study had as objective to evaluate the microbiological quality of the ice used in the seafood conservation by counting of mesophilic and psychrotrophics, total and thermoresistant coliforms and to investigate the presence or not of Escherichia coli, Staphylococcus aureus e Salmonella spp. For such, sixty-three samples of ice were collected from five supermarkets and one fish market located in the municipality of Ribeirão Preto/SP. The results showed populations of mesophilic heterotrophic microorganisms ranging from 1,0 x 10 to 3,0 x 104 UFC/mL and psychrotrophics ranging from 1,0 x 10 to 1,0 x 106 UFC/mL, being 19,05% of the samples containing mesophilic microorganisms above the allowed by the current legislation. It was found that 22,22% of the samples were positive for total coliforms and 9,52% for thermoresistant. The presence of Staphylococcus aureus was confirmed in three samples and E. coli and Salmonella spp. were not found. So, the evaluation of the microbiological quality in this study permitted to conclude that though there is a specific legislation about water quality, the unpotable water still in use in the ice manufactured utilized in the seafood conservation commercialized in food stores, what is a risk factor to the consumers health since the ice in contact with the fish can contaminate it

Orientador: Oswaldo Durival Rossi Junior / Banca: Angela Cleusa de Fátima Banzatto de Carvalho / Banca: Laudicéia Giacometti Lopes / Resumo: O gelo é um excelente meio de prolongar o frescor do peixe e na indústria alimentícia tem um dos mais importantes papéis, uma vez que retarda a multiplicação bacteriana e, se fabricado sob más condições sanitárias pode se tornar um fator de risco para os consumidores. Sendo assim, este estudo objetivou avaliar a qualidade microbiológica do gelo utilizado na conservação do pescado por meio da quantificação de microrganismos heterotróficos mesófilos e psicrotróficos viáveis, coliformes totais e termotolerantes, além de investigar a presença de Escherichia coli, Staphylococcus aureus e Salmonella spp. Para tal, foram colhidas 63 amostras de gelo de seis estabelecimentos, sendo cinco hipermercados e uma peixaria, situados no município de Ribeirão Preto/SP. Os resultados obtidos mostraram populações de microrganismos heterotróficos mesófilos variando entre 1,0 x 10 e 3,0 x 104 UFC.mL-1 e psicrotróficos variando entre 1,0 x 10 e 1,0 x 106 UFC/mL, sendo que 19,05% das amostras contendo microrganismos mesófilos encontravam-se com populações acima do permitido pela legislação vigente. Já 22,22% das amostras foram positivas para coliformes totais e 9,52% para coliformes termotolerantes. A presença de Staphylococcus aureus foi confirmada em três amostras, E. coli e Salmonella spp. não foram encontradas. Portanto, a avaliação da qualidade microbiológica do gelo, neste experimento, permitiu concluir que a água utilizada na fabricação de gelo utilizado para conservação de pescado comercializado nos estabelecimentos pode ser contaminada por manuseio inadequado e seu incorreto acondicionamento, o que é um fator de risco para a saúde do consumidor uma vez que o gelo em contato com o peixe pode contaminá-lo / Abstract: The ice is an excellent way to prolong the freshness of the fish and in the food industry has one of the most important roles, since it slows the bacterial growth, and if it is manufactured under poor sanitary conditions could become a risk factor for consumers. So, this study had as objective to evaluate the microbiological quality of the ice used in the seafood conservation by counting of mesophilic and psychrotrophics, total and thermoresistant coliforms and to investigate the presence or not of Escherichia coli, Staphylococcus aureus e Salmonella spp. For such, sixty-three samples of ice were collected from five supermarkets and one fish market located in the municipality of Ribeirão Preto/SP. The results showed populations of mesophilic heterotrophic microorganisms ranging from 1,0 x 10 to 3,0 x 104 UFC/mL and psychrotrophics ranging from 1,0 x 10 to 1,0 x 106 UFC/mL, being 19,05% of the samples containing mesophilic microorganisms above the allowed by the current legislation. It was found that 22,22% of the samples were positive for total coliforms and 9,52% for thermoresistant. The presence of Staphylococcus aureus was confirmed in three samples and E. coli and Salmonella spp. were not found. So, the evaluation of the microbiological quality in this study permitted to conclude that though there is a specific legislation about water quality, the unpotable water still in use in the ice manufactured utilized in the seafood conservation commercialized in food stores, what is a risk factor to the consumers health since the ice in contact with the fish can contaminate it / Mestre

Orientador: Oswaldo Durival Rossi Junior / Banca: Angela Cleusa de Fatima Banzatto de Carvalho / Banca: Francisca Neide Costa / Resumo: Tem sido evidente o aumento no consumo de pescado, especialmente do salmão (Salmo salar) sob a forma "in natura", em pratos da cozinha oriental. Como conseqüência, tem havido maior preocupação quanto às suas características higiênico-sanitárias, tendo em vista a facilidade que microrganismos encontram para se desenvolverem em sua carne, o que pode expor os consumidores a agentes que causam desde uma simples gastrenterite até o óbito. Diante desta preocupação, desenvolveu-se este estudo com objetivos de avaliar características microbiológicas do salmão por meio da quantificação de microrganismos heterotróficos mesófilos, coliformes totais e termotolerantes, o perigo de veiculação de Vibrio parahaemolyticus, Staphylococcus aureus, Salmonella sp., Escherichia coli e Aeromonas sp. através da carne e contribuir com subsídios técnicos para criar uma legislação brasileira com padrões microbiológicos específicos para o pescado consumido cru. Foram colhidas 31 amostras de salmão, 16 refrigeradas e 15 congeladas, no comércio varejista de cidades da região nordeste do estado de São Paulo. Os resultados obtidos mostram populações de microrganismos heterotróficos mesófilos variando entre 1,0 x 10 e 3,9 x 106 UFC/g, coliformes totais e termotolerantes em, respectivamente, 32,24% e 19,33% das amostras e Aeromonas sp. em 35,48% das amostras com variação populacional de 2,0 x 102 a 8,0 x 103 UFC/g. Ainda houve a presença de Staphylococcus aureus em uma amostra e ausência de Vibrio parahaemolyticus, Salmonella sp. e Escherichia coli. Os resultados obtidos podem servir de parâmetro para a criação de um padrão microbiológico específico para o pescado consumido cru e servem também de alerta para os consumidores do produto tendo em vista a veiculação de microrganismos potencialmente patogênicos. / Abstract: The increasing of seafood consumption has become evident especially in the use of salmon (Salmo salar) consumed raw in oriental dishes. Consequently, it has risen up the concern related to their hygienic-sanitary characteristics due to the facility that microorganisms multiply in the meat which can expose consumers to the causative agents of a mild gastroenteritis until the death. Regarding such informations, this study was aimed to evaluate microbiological characteristics of salmon by quantifying microrganisms heterotrophic mesophiles, total coliforms and thermotolerant. It was also evaluated the danger of transmission of Vibrio parahaemolyticus, Staphylococcus aureus, Salmonella sp., Escherichia coli and Aeromonas sp. on the fish muscle and contributed to technical informations to create a Brazilian regulations about specific microbiological standards for consumption of raw seafood. Thirty-one samples of salmon were collected, 16 chilled and 15 frozen, from the retail market in cities of the northeast region of São Paulo State. The results show populations of mesophilic heterotrophic microorganisms ranging from 1.0 x 10 and 3.9 x 106 CFU/g, in total and fecal coliforms, respectively, 32.24% and 19.33% of samples and Aeromonas sp. in 35.48% of samples ranging population of 2.0 x 102 to 8.0 x 103 CFU/g. Staphylococcus aureus was present in one sample and were not found Vibrio parahaemolyticus, Salmonella sp. and Escherichia coli. The results may serve as a parameter for the establishment of a microbiological standard for the consumption of raw seafood and also as a warning to consumers of the product for the presence of potentially pathogenic microorganisms. / Mestre

Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-10-09T20:37:27Z No. of bitstreams: 1 tamiryssilvabarino.pdf: 1193174 bytes, checksum: 4ad4ca2cfdc016bc7f65fda94a5fb98d (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-10-10T12:20:20Z (GMT) No. of bitstreams: 1 tamiryssilvabarino.pdf: 1193174 bytes, checksum: 4ad4ca2cfdc016bc7f65fda94a5fb98d (MD5) / Made available in DSpace on 2017-10-10T12:20:20Z (GMT). No. of bitstreams: 1 tamiryssilvabarino.pdf: 1193174 bytes, checksum: 4ad4ca2cfdc016bc7f65fda94a5fb98d (MD5) Previous issue date: 2017-02-13 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Microrganismos patogênicos são responsáveis pelo desenvolvimento de diversas infecções, e em ambientes hospitalares, levam muitos pacientes ao óbito. Modificar esse quadro é necessário, todavia as bactérias apresentam distintos mecanismos de resistência após mutações em seu DNA. Além disso, o uso indiscriminado de antibióticos gera continuamente a seleção natural e propagação de cepas mais resistentes fazendo com que os antibióticos percam sua eficácia com o passar dos anos. Diante desse panorama, a elaboração de um nanocarreador contendo peptídeos antimicrobianos (PAMs ou AMPs do inglês Antimicrobial Peptides) tornase uma interessante alternativa contra bactérias patogênicas. O objetivo deste trabalho foi sintetizar nanoesferas de alginato de sódio pelo método de Gelificação Ionotrópica para a entrega de AMPs Lrot3 às células bacterianas de Escherichia Coli 25922 e Staphylococcus epidermidis 12228. A atividade antimicrobiana dos peptídeos livres e nanoestruturados (128, 64, 32, 16, 8 e 4µg/mL), foi avaliada por meio da concentração mínima inibitória (MIC), e o potencial citotóxico investigado em células HEK 293 humanas utilizando o ensaio de MTT (Tetrazólio de azul de tiazolilo). Os dados foram avaliados por ANOVA e as médias comparada pelo teste de Tukey. Para a bactéria E. coli 25922, o peptídeo livre na concentração de 64 µg/mL destacou-se por ser a única concentração com atividade antimicrobiana significativa (P˂0,01), apresentando ação similar ao do cloranfenicol (128 µg/mL e 64 µg/mL). As nanopartículas contendo AMPs, por sua vez, mostraram-se eficazes em todas as concentrações (P˂0,01). Diferentemente se deu no S. epidermidis 12228, onde observou-se maior resistência da bactéria a todos tratamentos. Os resultados do MTT sugerem que as nanopartículas de alginato não apresentam citotoxicidade in vitro, motivando a continuidade deste estudo. Concluindo, os AMPs nanoestruturados tem potencial para serem utilizados no combate a infecções ocasionadas por E. coli. / Pathogenic microorganisms are responsible for the development of various infections, particularly in hospital environment, leading to death in many patients. It is therefore necessary to change this scenario, it is difficult, however, because the bacteria have very distinct resistance mechanisms after mutations in their DNA. Furthermore, the indiscriminate use of antibiotics continuously generates the selection and propagation of more resistant strains, causing loss of effectiveness of antibiotics over the years. In this case, the development of a nanocarrier containing Antimicrobial Peptides (AMPs) becomes an interesting alternative against pathogenic bacteria. This paper aims to synthesize the delivery of AMPs to bacterial cells of Escherichia coli 25922 and Staphylococcus epidermidis 12228 through the use of nanospheres of sodium alginate by ionotropic gelation method. Alginate stands out for being a polyanionic, biodegradable and biocompatible polymer, which show interaction with Lrot3 cationic AMP. The antimicrobial activity of free and nanostructured peptides (128, 64, 32, 16, 8 e 4 µg/mL) was evaluated by the minimum inhibitory concentration (MIC) and the cytotoxic potential investigated in human HEK 293 cells using the MTT Assay (Tetrazolium thiazolyl blue). The data were evaluated by ANOVA and the means compared by Tukey test. For Gramnegative E. coli 25922 bacteria, the free peptide at concentration of 64 µg/mL stood out for being the only concentration with significant antimicrobial activity (P˂0,01), showing similar action to the chloramphenicol, at concentrations of 128 µg/mL and 64 µg/mL, respectively. NPs, in turn, showed inhibitory activity at all concentrations (P˂0,01). Unlike occurred in S. epidermidis 12228, which was observed more bacterial resistance to all treatments. The MTT data suggest that alginate nanoparticles do not exhibit in vitro cytotoxicity, encouraging the continuation of this study. In conclusion, the nanostructured AMPs has the potential to be used to fight infections caused by E. coli.

Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(工学) / 甲第16388号 / 工博第3469号 / 新制||工||1524(附属図書館) / 29019 / 京都大学大学院工学研究科都市環境工学専攻 / (主査)教授 田中 宏明, 教授 清水 芳久, 教授 藤井 滋穂 / 学位規則第4条第1項該当

M.Tech. (Biomedical Technology) / Drinking water in the greater Johannesburg area is produced by Rand Water and is transported to local Johannesburg Water where it is stored in reservoirs for distribution. At any point during the production, distribution and storage of the water, contamination with free-living amoebae, potentially containing amoeba resistant bacteria, may occur. Free-living amoebae are often resistant to the biocides used by water treatment industries and may thus be transmitted to public facilities, consumers’ homes and informal settlements through water distribution systems and during storage in small containers. The aim of our study was to analyse the water quality around Johannesburg with regard to free-living amoebae and amoeba resistant bacteria. A total of 182 tap and 5 storage tank water samples, collected from Hillbrow, Bertrams, Riverlea, Braamfischerville and Hospital Hill, were analysed for amoebae, indicator organisms, Legionellae, environmental mycobacteria, Shigella, Salmonella and Vibrio species using amoebal enrichment method. Direct microscopy indicated the presence of amoebae in 96.1% of samples. Acanthamoeba cysts were present in 69.0% of the samples. In 55.0% of these samples visibly active intracellular bacteria were observed within the sample suspensions. In the 46 samples analysed by polymerase chain reaction, the presence of Acanthamoeba species was confirmed in 65.2%, and the intracellular bacteria such as Legionella pneumophila and Mycobacterium avium was confirmed in 23.9% and 73.9% respectively. All samples indicated the presence of Shigella species while one sample contained Salmonella species on xylose lysine desoxycholate agar after amoebal enrichment processing. Vibrio species was not confirmed in the samples. Our results indicated a high risk of transmission of amoeba resistant bacteria through drinking water to people living in these areas.

La pathogénicité des bactéries a toujours été attribuée à des facteurs de virulence et les bactéries pathogènes sont considérées comme étant mieux armées, comparé à des bactéries ne provoquant pas de maladies. Selon les premières études génomiques, le fait de supprimer un certain nombre de gènes des bactéries pathogènes, limiterait leur capacité à infecter leurs hôtes. Au contraire, des études de génomique comparatives récentes, démontrent que la spécialisation des bactéries dans les cellules eucaryotes est associée à une perte de gènes massive, en particulier pour les endosymbiontes allopatriques qui sont isolés depuis longtemps dans une niche intracellulaire. En effet, les bactéries sympatriques, extracellulaires, ont souvent des génomes plus grands et présentent une résistance et une plasticité plus importante. Ces bactéries constituent, de fait, plutôt des complexes d’espèces que de vraies espèces. Certaines bactéries spécialistes, comme les bactéries pathogènes, arrivent à s’échapper de ces complexes et à coloniser une niche, bénéficiant alors d’un nom d’espèce. Leur spécialisation leur permet de devenir allopatriques et leurs pertes de gènes favorisent une évolution réductive. Ces observations nous ont conduits à réaliser une étude afin de quantifier le taux de perte de gènes lors de l’évolution de ces bactéries extracellulaires vers celle de bactéries spécialistes intracellulaires. Notre objectif était de vérifier que ce qui caractérise l’évolution des bactéries intracellulaires est bien la réduction génomique, en prenant en compte tous les événements possibles de gains de gènes. Par ailleurs, dans une étude neutre comparant les 12 espèces pandémiques les plus dangereuses pour l’homme avec les espèces non-épidémiques les plus proches, nous avons voulu identifier des spécificités génomiques associées à la capacité virulente de bactéries pathogènes et démontrer que, à part les toxines et les modules toxine-antitoxine, ce qui caractérise ces espèces ce ne sont pas les facteurs de virulence, mais la perte des gènes de régulation. Au final, les bactéries pathogènes ont un répertoire virulent dans lequel les gènes absents sont aussi importants que les gènes présents. / The virulence of pathogenic bacteria has been attributed to virulence factors and pathogenic bacteria are considered to have more genes compared to bacteria that do not cause disease. According to the first genomic studies, removing a certain number of genes from pathogenic bacteria impairs their capacity to infect hosts. However, more recent studies have demonstrated that the specialization of bacteria in eukaryotic cells is associated with massive gene loss, especially for allopatric endosymbionts that have been isolated for a long time in an intracellular niche. Indeed, bacteria living in sympatry often have bigger genomes and exhibit greater resistance and plasticity and constitute species complexes rather than true species. Specialists, including specific pathogenic bacteria, escape these bacterial complexes and colonize a niche; thereby gaining a species name. Their specialization allows them to adopt allopatric lifestyle and experience reductive genome evolution. These observations led us to design a study to quantify the rate of gene losses during the evolution of free-living bacteria to intracellular specialists. Our objective was to verify that what characterizes the evolution of intracellular bacteria is genomic reduction, taking under consideration all possible gene gain events. Furthermore, in another neutral study comparing the 12 most dangerous pandemic bacteria to Humans to their closest non-epidemic species, we wished to identify any genomic specificities associated to the virulent capacity of pathogenic bacteria and demonstrate that, besides toxins and surprisingly, toxin-antitoxin modules, pathogenic bacteria are not characterized by more virulence factors, but rather by a loss of regulatory genes. Finally, virulent bacteria exhibit a genomic repertoire in which absent genes are as important as present ones.

La qualité de l'eau est une préoccupation majeure pour la santé publique et la préservation de l'environnement. Nous proposons d’utiliser les phages comme des biosenseurs pour détecter les pathogènes humains ou animaux présents dans les eaux de surface, un outil nommé Phagosensor.La mise au point d’un phagosensor prototype a permis d’optimiser la détection des bactéries par cytométrie en flux. Les résultats montrent que cet outil est extrêmement rapide, sensible, et très spécifique. L’outil phagosensor permet de détecter des bactéries cibles présentes dans un environnement complexe tel que l’eau de mer.Le prototype fonctionnel a servi de base à la construction de phagosensors spécifiques de bactéries pathogènes. Les résultats montrent que la stratégie développée à partir du prototype peut être rapidement transposée à la détection de pathogènes tel que Salmonella. Le séquençage de novo et l’annotation des génomes de trois phages isolés de l’environnement permettront la construction de nouveaux phagosensors spécifiques de souches d’E. coli pathogènes.Cette stratégie a été adaptée à la détection d’un signal luminescent post-infection en utilisant les phages recombinants portant l’opéron luxCDABE. Les bactéries infectées sont rapidement détectables.L’ensemble de ces résultats démontre que la stratégie développée est applicable à la construction de phagosensor sur demande pour une détection rapide, sensible et spécifique des bactéries d’intérêt que ce soit en fluorescence ou en luminescence. La détection dans l’eau de mer suggère, qu’à terme, des outils pourront être conçus pour la détection de bactéries pathogènes dans d’autres matrices telles que le sang ou les aliments. / Water quality is a major concern for public health and natural environment preservation. We propose to use phages to develop biosensor tools able to detect human and animal pathogens present in water. The construction of a phagosensor prototype using an optimized genetic engineering strategy, infection and detection conditions, allowed the specific detection of bacteria. The results show that detection is fast, specific and highly sensitive. Moreover, the phagosensor tool detects target bacteria in a complex environment such as seawater. Phagosensors specific of pathogenic bacteria were constructed following the strategy developed for the prototype. Results show that the strategy we designed can be successfully transposed to detect pathogens such as Salmonella. De novo sequencing and genomes annotation of three phages isolated from the environment were carried out to develop phagosensors that are specific of pathogenic E. coli. This technology was then adapted to detect a luminescent signal arising post-infection using genetically modified phages carrying the entire luxCDABE operon. The bacteria infected with the lux recombinant phages were rapidly detected by luminescence emission. Together, these results demonstrate that our technology can be applied to construct various phagosensors adapted to the detection of different bacterial species of interest and using at least two output signals. These tools allow a rapid, specific and highly sensitive detection that are close to the European guideline. Efficient bacterial detection in seawater suggests that phagosensors could be developed to detect pathogenic bacteria in other matrices such as blood or food.

This study was conducted from March 3, through June 2, 1951, in order to determine to what extent pathogenic bacteria were entering the Trinity River between East Fort Worth, Texas and South Dallas, Texas, from municipal sewage disposal plants.