Submitted by (edna.saturno@ufrpe.br) on 2016-10-07T13:54:35Z No. of bitstreams: 1 Fernanda Silva de Meirelles.pdf: 599414 bytes, checksum: be09ab548e388263d8d38ab7cabb0cf5 (MD5) / Made available in DSpace on 2016-10-07T13:54:35Z (GMT). No. of bitstreams: 1 Fernanda Silva de Meirelles.pdf: 599414 bytes, checksum: be09ab548e388263d8d38ab7cabb0cf5 (MD5) Previous issue date: 2010-02-26 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / In Brazil, it is considered that the annual production of tilapias overcomes 100 thousand tons, mainly due to the productive potential of the Northeast area that answered for 41% of the total production of 70.000 tons in 2004, generating income of US$ 200.000.000,00, due to your climatic conditions, technology readiness and consumption market, regional and national in expansion. The intensification of cultivations has as consequence the increase of organic matter, that it favors the multiplication of microorganisms, making possible him/it supplies of diseases in the occurrence of adverse situations to the fish. Tilapia (Oerochromis niloticus) of cultivations of the state of Pernambuco they were appraised with objective of determining the frequency of bacterial diseases. For so much, samples of several farms were analyzed, in the several aspects, with relevance for the bacterial agents. The tilapia were examined with relationship to the streptococcus presence, víbrios, coliform, pseudomonas and aeromonas. Clinical exams and autopsy were accomplished, for microscopic analysis of the lesions and better definition of the probable agent etiological. The analyses were accomplished at the Laboratory of Sanity of Aquatic Animals (LASAq) of the Rural Federal University of Pernambuco (UFRPE) and in the Center of Development and Diffusion of Technology in Aquaculture of the State University of Bahia (UNEB). The principal pathological agents were víbrios and aeromonas, that are pathogens responsible opportunists for significant losses, being identified the following species in 46 isolated: V. natriegens (1/46), V. metschnikovii (2/46), V. halioticoli (1/46), V. fischeri (2/46), V. mimicus (23/46), V. diabolicus (1/46), V. furnissi (1/46), V. cholerae o1 (1/46), V. scophthalmi (4/46), V. proteolyticus (3/46), V. argarivorans (1/46), V. ordalii (2/46) and Vibrio spp. ( 3/46). The isolated vibrios presented larger sensibility to the antimicrobianos enrofloxacina (100%) and florfenicol (98,18%) and in decreasing order, gentamicina (90,91%), cotrimoxazol (sulfametoxazol+trimetroprim) (76,36%), tetraciclin (67,27%), eritromicin (30,91%) and amoxilina (3,64%). The isolated ones tested they came 100% resistant the penicillin. In this study, 96,4% (53/55) of the víbrios they presented index multiple resistance to antibiotics (MAR), superior to 0,22, characterizing multiple resistance. In 35 isolated the identified aeromonas were: A.caviae (1/35), A. shubertii (11/35), A. media (4/35), A. popoffii (1/35), A. sobria (3/35), A. encheleia (4/35), A. veronii (4/35) e A. jandaei (4/35) Forty were tested isolated of aeromonas with relationship to the sensibility to 08 antimicrobial, of these they showed larger sensibility the florfenicol (100,0%), enrofloxacina(95,0%), gentamicina (95,0%) and in decreasing order, cotrimoxazol (sulfametoxazol+trimetroprima) (67,5%), tetraciclina (65,0%). There was smaller sensibility the eritromicina (20,0%), penicillin (5,0%) and the amoxilina (2,5%), confirming the resistance existence. The index of multiple resistance to antibiotics (MAR) for the isolated of aeromonas it varied in an interval from 0,12 to 0,62, of these 95% (38/40) they came superior to 0,22 characterizing multiple resistance. Most of the species of isolated víbrios is not considered pathogenic for the fish (environmental), but nevertheless they can represent risk for the health of the tilapia for subject of opportunism and of the consumer, mainly if consumed raw. The isolated and identified movable aeromonas in this study are commonly considered as environmental, however many of the analyzed fish presented compatible symptomatology with the illness caused by these agents opportunists. / No Brasil, estima-se que a produção anual de tilápias supere 100 mil toneladas, principalmente devido ao potencial produtivo da região Nordeste que em 2004 respondeu por 41 % da produção total de 70.000 toneladas, gerando renda de US$ 200.000.000,00, decorrente de suas condições climáticas, disponibilidade de tecnologia e mercado de consumo, regional e nacional em expansão. A intensificação de cultivos tem como conseqüência o aumento de matéria orgânica, que favorece a multiplicação de microrganismos, possibilitando o surto de doenças na ocorrência de situações adversas aos peixes. Tilápias (Oerochromis niloticus) de cultivos do estado de Pernambuco foram avaliadas com objetivo de determinar a freqüência de doenças bacterianas. Para tanto, analisaram-se amostras de diversas fazendas, nos diversos aspectos, com relevância para os agentes bacterianos. As tilápias foram examinadas quanto à presença de estreptococos, víbrios, coliformes, pseudomonas e aeromonas. Foram realizados exames clínicos e necropsias, para análise microscópica das lesões e melhor definição do provável agente etiológico. As análises foram realizadas no Laboratório de Sanidade de Animais Aquáticos (LASAq) da Universidade Federal Rural de Pernambuco (UFRPE) e no Centro de Desenvolvimento e Difusão de Tecnologia em Aqüicultura da Universidade Estadual da Bahia (UNEB). Os principais agentes patológicos foram víbrios e aeromonas, que são patógenos oportunistas responsáveis por perdas significantes, sendo identificadas as seguintes espécies em 46 isolados: Vibrio natriegens (1/46), V. metschnikovii (2/46), V. halioticoli (1/46), V. fischeri (2/46), V. mimicus (23/46), V. diabolicus (1/46), V. furnissi (1/46), V. cholerae O1 (1/46), V. scophthalmi (4/46), V. proteolyticus (3/46), V. argarivorans (1/46), V. ordalii (2/46) e Vibrio spp.(3/46). Os vibrios isolados apresentaram maior sensibilidade aos antimicrobianos enrofloxacina (100%) e florfenicol (98,18%) e em ordem decrescente, gentamicina (90,91%), cotrimoxazol (sulfametoxazol+trimetroprima) (76,36%), tetraciclina (67,27%), eritromicina (30,91%) e amoxilina (3,64%). Os isolados testados apresentaram-se 100% resistentes a penicilina. Neste estudo, 96,4% (53/55) dos víbrios apresentaram índice múltipla resistência a antibióticos (MAR), superior a 0,22, caracterizando múltipla resistência. Em 35 isolados as aeromonas identificadas foram: Aeromonas caviae (1/35), A. shubertii (11/35), A. media (4/35), A. popoffii (1/35), A. sobria (3/35), A. encheleia (4/35), A. veronii (4/35) e A. jandaei (4/35). Foram testados 40 isolados de aeromonas quanto à sensibilidade a oito antimicrobianos, tendo mostrado maior sensibilidade a florfenicol (100,0%), enrofloxacina(95,0%), gentamicina (95,0%), cotrimoxazol (sulfametoxazol+trimetroprima) (67,5%), tetraciclina (65,0%). Houve menor sensibilidade a eritromicina (20,0%), penicilina (5,0%) e a amoxilina (2,5%), confirmando a existência de resistência. O índice MAR para os isolados de aeromonas variou de 0,12 a 0,62, dos quais 95% (38/40) apresentaram índices superiores a 0,22, caracterizando multiresistência. A maioria das espécies de víbrios isoladas não é considerada patogênica para os peixes (ambientais), mas ainda assim podem representar risco para a saúde das tilápias por questão de oportunismo e do consumidor, principalmente se consumido cru. As aeromonas móveis isoladas e identificadas neste estudo são comumente consideradas como ambientais, porém muitos dos peixes analisados apresentaram sintomatologia compatível com a enfermidade causada por estes agentes oportunistas.

Aeromonas são bactérias Gram-negativas aquáticas. Algumas espécies deste gênero são patógenos oportunistas de muitos animais aquáticos e terrestres, inclusive o homem. Vários fatores de virulência, tais como enterotoxinas, hemolisinas e outras enzimas, tem sido associados com a sua patogenicidade. As proteases extracelulares devem ser vistas como importantes enzimas envolvidas na versatilidade metabólica que possibilitam as Aeromonas persistir nos ambientes aquáticos, interagir com outros organismos, e causar doenças. Neste contexto, o objetivo deste trabalho foi avaliar: as diferenças nas atividades de proteases extracelulares e o perfil eletroforético entre espécies e isolados de Aeromonas, a especificidade por substratos de poteases de Aeromonas, e a clonagem e expressão heteróloga da elastase de Aeromonas. Os resultados mostraram que A. hydrophila apresenta maior atividade proteolítica, sem, entretanto exibir diferenças significativas entre isolados clínicos e ambientais. Zimogramas proteolíticos dos isolados de Aeromonas demonstraram diferentes perfis com 0 a 5 bandas, correspondendo a metalo, serino ou outras proteases. Duas proteases (56K Da e 22 KDa) apresentaram atividade sobre um grande espectro de substratos. A atividade caseinolítica de A. hydrophila IBAer 109 mostra um típico comportamento de "quorum sensing", sendo induzida por gelatina e reprimida por glicose, citrato e amônio. O gene da elastatse (ahyB) de A. hydrophila ATCC7966 foi clonado e expresso em E. coli sob controle do promotor lac. O transformante apresentou bandas proteolíticas de 63 e 38 KDa no espaço periplasmático e extracelular, indicando maturação parcial e dificuldade de secreção desta enzima. / Aeromonas are ubiquitous water-borne Gram-negative bacteria. Some species of this genus are opportunistic pathogens of a variety of aquatic and terrestrial animals, including humans. Several virulence determinants, such as enterotoxins, hemolysins and hydrolytic enzymes, have been implicated in their ability to cause diseases. Extracellular proteases may be regarded as important enzymes in the mechanisms that confer the metabolic versatility that allow Aeromonas to persist in aquatic habitats, to interact with other organisms, and to cause diseases. In this context, the objective of this work was to assess the extent of differences in extracellular proteases activity and electrophoretic profiles among Aeromonas strains, the substrate specificity among Aeromonas proteases, and the cloning and heterologous expression of Aeromonas elastase. The results showed that A. hydrophila has the highest proteolytic activity with no significant difference between clinical and environmental isolates. Protease zymograms of Aeromonas isolates showed different profiles with 0 to 5 bands, corresponding to metallo, serine and other proteases. Two proteases (56 KDa and 22 KDa) showed activity against a large spectrum of substrates. Caseinolytic activity of A. hydrophila IBAer 109 showed a typical "quorum sensing" behavior, and was induced by gelatin and repressed by glucose, citrate and ammonium. The elastase gene (ahyB) of A. hydrophila ATCC7966 was cloned and expressed in E. coli under the control of lac promoter. The transformant showed both periplasmic and extracellular proteolytic bands of 63 and 38KDa, indicating partial maturation and secretion difficulties.

Virulent Aeromonas hydrophila (vAh) is a causative agent of motile Aeromonas septicemia (MAS) in catfish. There are limitations in the current therapeutic and preventative strategies against vAh. The pathogenesis of MAS as well as the immune response of catfish to vAh infection are poorly understood. The aim of this study is to: 1) develop a dual live attenuated vaccine against MAS and enteric septicemia of catfish caused by Edwardsiella ictaluri; and 2) evaluate the vAh bacterial load and gene expression patterns in catfish tissues following vAh infection. Previously, six recombinant vAh proteins (outer membrane protein, TonB-dependent receptor, three fimbrial proteins, and an ATPase) were identified to have vaccine efficacy against MAS, and live attenuated E. ictaluri vaccine strain ESC-NDKL1 was identified as an effective vector for expressing vAh antigens. A total of 29 recombinant ESC-NDKL1 strains have been constructed with the integration of one, two, or three genes encoding vAh antigens into the ESC-NDKL1 chromosome. Vaccine efficacy of the constructed strains was evaluated in channel catfish fingerlings. Four recombinant ESC-NDKL1 strains expressing two vAh antigens (ESC-NDKL1::atpase::fimMrfG, ESC-NDKL1::fim::fimMrfG, ESC-NDKL1::tdr::fimMrfG, and ESC-NDKL1::fim::ompA) showed significant protection against MAS with relative percent of survival (RPS) values of 55.72%, 60.18%, 61.74%, and 54.81%. Four triple recombinant ESC-NDKL1 strains (ESC-NDKL1::fimMrfG::ompA::fimA, ESC-NDKL1::atpase::fimMrfG::ompA, ESC-NDKL1::fim::fimMrfG::ompA and ESC-NDKL1::atpase::tdr::fim) showed the best protection with RPS values of 77.93%, 63.18%, 67.74%, and 82.35%. To gain a better understanding of vAh pathogenesis, catfish fingerlings were intraperitoneally injected with vAh strain ML09-119. The anterior kidney, liver, and spleen were collected for determination of vAh distribution and expression of thirteen pro-inflammatory, innate, and adaptive immune-related genes using real-time PCR. Results revealed that vAh spread into catfish tissues within 2 hours and peaked at 12 hours post-infection. vAh infection initiated a strong inflammatory response in catfish tissues. Additionally, our research revealed that surviving catfish were able to develop a primary immune response and possibly generation of memory B cells against MAS. Such information will facilitate the development of vaccines and therapeutic drugs for preventing and treating MAS outbreaks in catfish aquaculture.

Channel catfish farming is the largest aquaculture industry in the U.S., and virulent Aeromonas hydrophila is an important re-emerging bacterial pathogen of farmed catfish. A. hydrophila is ubiquitous in the aquatic environment and causes motile Aeromonas septicemia (MAS) in farmed catfish. A. hydrophila infection is challenging to treat due to rapidly progressive mass mortalities. Although florfenicol has been used in managing outbreaks, acute onset of anorexia, drug cost, and emergence of antimicrobial-resistant strains are significant concerns. Prevention of MAS by vaccination could be a promising approach, but a commercial vaccine is not available. Thus, it is critical to develop effective vaccines against A. hydrophila to prevent losses in the catfish industry. The overall objective of this study is to develop effective live attenuated A. hydrophila vaccines and understand macrophage-A. hydrophila interactions. To achieve this, potential virulence-related genes from T6SS, secretion pathway, tat pathway, and flagellar system were deleted by in-frame, and mutants’ virulence and protection were evaluated in channel catfish. Also, macrophage-A. hydrophila interactions were explored by studying global gene expression to understand macrophage responses to A. hydrophila and A. hydrophila virulence mechanisms

No description available.

Aeromonas salmonicida ssp. salmonicida est un agent pathogène aquatique causant la furonculose chez les salmonidés, particulièrement les poissons d’élevage. L'antibiothérapie est la méthode couramment utilisée pour traiter cette maladie dans le monde entier. Toutefois, son efficacité est menacée par l’apparition de souches résistantes, voire multirésistantes, aux antibiotiques. L’étude de ces souches bactériennes devient donc nécessaire afin d’identifier les éléments génétiques responsables de ces résistances et comprendre comment ils contribuent à la propagation de l’antibiorésistance. C’est dans cette démarche que se positionne la présente étude qui a pour objectif de caractériser de nouveaux éléments génétiques porteurs de résistances aux antibiotiques chez A. salmonicida ssp. salmonicida à l’aide du séquençage à longues lectures. Plus spécifiquement, au centre de ce projet se trouve la souche SHY16-3432 dont l’étude a permis d’identifier trois nouveaux variants de plasmides nommés pAsa9b, pAsa5-3432 et pRAS3-3432, où ces deux derniers confèrent les résistances aux antibiotiques observées. L’analyse des séquences de ces trois variants a révélé qu’ils se distinguent tous de leur contrepartie classique par leur contenu en éléments génétiques mobiles. Le plasmide pAsa5-3432 possède une nouvelle région de multirésistances composée de plusieurs éléments génétiques mobiles. Celle-ci semble avoir été acquise à la suite d’une recombinaison entre deux plasmides. De son côté, pRAS3-3432 porte un nouvel élément inséré qui a uniquement été identifié chez l’agent pathogène porcin Chlamydia suis. Quant à pAsa9b, il se différencie du plasmide de référence par l’absence d’une séquence d’insertion. Ces découvertes soulignent ainsi l’importance des éléments mobiles sur la plasticité génomique d’A. salmonicida ssp. salmonicida ainsi que sa grande capacité d’interactions avec d’autres bactéries incluant des agents pathogènes animaux. En outre, les données obtenues dans ce projet suggèrent qu’il est nécessaire d’utiliser le séquençage à lectures longues pour caractériser complètement le génome de bactéries au plasmidome complexe comme A. salmonicida ssp. salmonicida. / Aeromonas salmonicida subsp. salmonicida is an aquatic pathogen that causes furunculosis to salmonids, especially in fish farms. Antibiotherapy is a common method used to treat this worldwide disease. Unfortunately, its effectiveness is becoming limited due to the presence of drug-resistant strains and even multidrug-resistance strains of the bacterium. The study of these bacterial strains becomes necessary in order to identify the genetic elements responsible for this resistance and to understand how they contribute to the spread of antibiotic resistance. In this study, we focused on the A. salmonicida subsp. salmonicida strain SHY16-3432 to characterize novel genetic elements conferring resistance to antibiotics using long-read sequencing technologies. It allowed us to identify three novel plasmid variants named pAsa9b, pAsa5-3432 and pRAS3-3432, the latter two being responsible for the observed antibiotic resistance. The sequence analysis of these three variants revealed that they all differ from their classical counterparts through the presence or absence of mobile genetic elements. The plasmid pAsa5-3432 carries a new multi-drug resistance region composed of numerous mobile genetics elements that appears to have been acquired through plasmid recombination. As for pRAS3-3432, it contains a new inserted element that has only been reported in the swine pathogen Chlamydia suis. Lastly, the only variation between pAsa9b and the reference plasmid is the absence of an insertion sequence in pAsa9b. Overall, these discoveries highlight the significant implication of mobile genetics elements in the genomic plasticity of A. salmonicida subsp. salmonicida and suggest that this aquatic bacterium has a high capacity to interact with other bacteria, including animal pathogens. Furthermore, the data obtained suggest that the use of long-read sequencing technologies is required to fully decipher the genome of bacteria possessing complex plasmid repertoire, such as A. salmonicida subsp. salmonicida.

Depuis la découverte de la pénicilline par Sir Alexander Fleming, les antibiotiques ont joué un rôle primordial et incontestable en médecine moderne en aidant à combattre les infections bactériennes. Cependant, les bactéries ont la capacité de se protéger par différents moyens des molécules antibiotiques. La surutilisation de ces molécules a accéléré le phénomène de résistance aux antibiotiques, rendant difficile, voire impossible, le traitement de certaines maladies infectieuses par cette approche. La résistance aux antibiotiques est une problématique d’envergure mondiale qui touche aussi négativement l’aquaculture, où les infections bactériennes peuvent causer d’importantes pertes économiques. L’une de ces bactéries est Aeromonas salmonicida subsp. salmonicida, l’agent étiologique de la furonculose. Bien qu’il fût déjà connu que plusieurs souches de cette bactérie étaient porteuses de plasmides conférant des résistances aux antibiotiques, l’ampleur de la problématique était encore inconnue. Les bactériophages (phages) sont des virus infectant spécifiquement les bactéries. Cette capacité à lyser les bactéries leur a valu d’être utilisés dans un contexte thérapeutique presque dès leur découverte au début du 20e siècle. Cependant, l’avènement des antibiotiques a fait en sorte que la thérapie par les phages a été oubliée dans plusieurs pays occidentaux. Maintenant que la résistance aux antibiotiques est devenue une inquiétude pour la pérennité de notre société, plusieurs études suggèrent que la thérapie par les phages pourrait être une alternative ou un complément aux traitements par antibiotiques. La présente thèse avait comme objectifs : (1) d’explorer la diversité génomique causant une résistance aux antibiotiques chez A. salmonicida subsp. salmonicida et (2) d’investiguer le potentiel d’un traitement par les phages pour contrer les infections causées par cette bactérie. Il a été possible de mettre à jour et de caractériser cinq nouveaux plasmides avec des gènes de résistance aux antibiotiques chez A. salmonicida subsp. salmonicida. De plus, la présence de deux de ces plasmides (pAB5S9b et pSN254b) causent une résistance à tous les antibiotiques approuvés par le gouvernement canadien pour une utilisation par l’industrie piscicole. Avant d’investiguer la diversité des phages infectant A. salmonicida subsp. salmonicida, il était crucial de mieux connaître la bactérie d’intérêt. Plusieurs phages sont connus pour avoir un spectre lytique étroit, n’infectant ainsi que certaines souches ou certaines sousespèces d’une bactérie. Or, la structure intra-espèce d’A. salmonicida était encore mal définie. De plus, l’une des sous-espèces d’A. salmonicida, pectinolytica, est considérée comme mésophile avec la capacité de croître à 37°C, alors que les autres sous-espèces, comme salmonicida, sont limitées à des températures d’environ 20°C et sont par conséquent qualifiées de psychrophiles. En caractérisant de nouvelles souches mésophiles, mes travaux ont mis en lumière que les séquences d’insertion peuvent être une raison pour expliquer cette dichotomie. De plus, il a été possible de démontrer une grande diversité génétique chez les souches mésophiles, comparativement à celles psychrophiles. Afin de vérifier le potentiel d’un traitement par les phages contre la furonculose, trois phages spécifiques à A. salmonicida subsp. salmonicida ont été isolés de l’environnement. L'ADN de ces phages, en plus de celui de neuf autres disponibles à la collection Félix d’Hérelle, a été séquencé à haut-débit sur un appareil MiSeq d’Illumina. En comparant ces séquences génomiques à celles déjà disponibles publiquement, il a été possible de déterminer six groupes génomiques de phages contre A. salmonicida subsp. salmonicida. Les 12 phages disponibles pour la présente étude ont été testés sur 65 souches d’A. salmonicida (incluant des sous-espèces autres que salmonicida), permettant de dresser un portrait de la capacité lytique de chacun de ces virus. Cette analyse a mis en lumière trois groupes de phages ayant des capacités lytiques variables. De plus, il a été possible de montrer que d’autres sous-espèces d’A. salmonicida psychrophiles peuvent être infectées par les phages isolés à partir de la sous-espèce salmonicida. Cependant, les souches mésophiles d’A. salmonicida sont insensibles à ces phages. Cette étude doctorale a montré que la résistance aux antibiotiques est un problème d’envergure dont l’ampleur était insoupçonnée chez A. salmonicida subsp. salmonicida. Elle a aussi permis d’investiguer le potentiel de la thérapie par les phages. / Since the discovery of penicillin by Sir Alexander Fleming, antibiotics have played a paramount and indisputable role in modern medicine in helping to treat bacterial infections. However, bacteria have the ability to protect themselves against antibiotics by various mechanisms. The overuse of these molecules has accelerated the phenomenon of antibiotic resistance, making it difficult, if not impossible, to treat certain bacterial infections. Antibiotic resistance is a global problem that also negatively affects aquaculture, where bacterial infections can cause significant economic losses. One of these bacteria is Aeromonas salmonicida subsp. salmonicida, the etiologic agent of furunculosis. Although it was already known that several strains of this bacterium were carriers of plasmids conferring resistance to antibiotics, the extent of the problem was still unknown before this study. Bacteriophages (phages) are viruses specifically infecting bacteria. Their ability to lyse bacteria has been used in a therapeutic context almost as soon as they were discovered at the beginning of the 20th century. However, the advent of antibiotics has meant that phage therapy was forgotten in several Western countries. Now that antibiotic resistance has become a significant concern for the sustainability of our society, several studies suggest that phage therapy could be an alternative or supplement to antibiotic treatments. The objectives of this thesis were: (1) to explore the genomic diversity causing resistance to antibiotics in A. salmonicida subsp. salmonicida and (2) to investigate the potential of phage therapy to treat infections caused by this bacterium. Five new plasmids conferring antibiotic resistance to A. salmonicida subsp. salmonicida were discovered and characterized. Two of these plasmids, pAB5S9b and pSN254b, cause resistance to all antibiotics approved by the Canadian government for use in the fish industry. Before investigating the diversity of phages infecting A. salmonicida subsp. salmonicida, it was crucial to better know the bacterium of interest. Several phages are known to have a narrow host spectrum, infecting certain strains or subspecies. Until the present doctoral study, the intra-species structure of A. salmonicida was poorly defined. In addition, one of the subspecies of A. salmonicida, pectinolytica, is considered mesophilic with the ability to grow at 37°C, while other subspecies, such as salmonicida, are limited to growth temperatures around 20°C and are therefore considered psychrophilic. By characterizing new mesophilic strains, we found that insertion sequences may be a reason for this dichotomy. In addition, it was possible to demonstrate a high genetic diversity in mesophilic strains compared to psychrophilic strains. In order to verify the potential of phage treatment against furunculosis, three phages specific to A. salmonicida subsp. salmonicida were isolated from the environment. The genomic DNA of these phages, in addition to that of nine other phages available at the Felix d'Hérelle collection, was sequenced on an Illumina MiSeq device. By comparing these genomic sequences to those already available publicly, it was possible to determine six genomic groups of phages infecting A. salmonicida subsp. salmonicida. The 12 phages available were tested on 65 strains of A. salmonicida (including subspecies other than salmonicida), providing the host range of each virus. This analysis revealed three groups of phages with variable lytic capacities. In addition, it was possible to show that other psychrophilic subspecies of A. salmonicida can be infected by phages isolated from the subspecies salmonicida. However, the mesophilic strains of A. salmonicida are insensitive to these phages. This doctoral study showed that resistance to antibiotics is a large-scale problem in A. salmonicida subsp. salmonicida, and that phage therapy may represent one of the solutions to the growing concern

Yes / Here, we present the draft genome sequence of Aeromonas caviae strain L12, which shows quorum-sensing activity. The availability of this genome sequence is important to the research of the quorum-sensing regulatory system in this isolate. / High Impact Research Grants from the University of Malaya (A000001-50001; UM-MOHE HIR Grant UM C/625/1/HIR/MOHE/CHAN/14/1, H-50001-A000027)

Yes / Aeromonas hydrophila has emerged worldwide as a human pathogen. Here, we report the draft whole-genome sequence of a freshwater isolate from Malaysia, A. hydrophila strain M062, and its N-acylhomoserine lactone genes are also reported here. / University of Malaya via High-Impact Research Grants (UM C/625/1/HIR/MOHE/CHAN/01, no. A-000001- 50001), and aUM-MOHEHIR grant (UM C/625/1/HIR/MOHE/CHAN/ 14/1, no. H-50001-A000027)

Eine effektive Strategie zur Verhinderung der Ausbreitung von Infektionskrankheiten in der Aquakultur ist die Anwendung geeigneter Präventionsmaßnahmen, insbesondere die Impfung von Fischen. Das effektivste Impfverfahren stellt die individuelle Injektion des Impfstoffes dar. Bei Milliarden von Fischen jährlich ist dies jedoch sehr zeit- und kostenintensiv. Bei Säugetieren gilt Niederfrequenz-Sonophorese (LFS) als eine der fortschrittlichsten Technologien zur transdermalen Verabreichung von Wirkstoffen. So entstand die Idee, bei Fischen die Aufnahme von Wirkstoffen während einer Tauchbadbehandlung mit Hilfe von LFS zu verbessern. Die vorliegende Arbeit zeigt, dass mit einer Beschallungsintensität von etwa 60 mW/cm^2 die Aufnahme eines Bakterienimpfstoffs in das Kiemengewebe von Regenbogenforellen um den Faktor 240 erhöht werden konnte. Bei dieser Intensität traten geringe oder keine Nebenwirkungen auf. Bei höheren Beschallungsintensitäten wurde eine noch höhere Aufnahme des Bakterienimpfstoffes, aber auch schädliche Nebenwirkungen beobachtet. Darüber hinaus zeigte LFS eine durch eine lokale Entzündungsreaktion und Aktivierung von T-Helferzellen in den Kiemen charakterisierte, Adjuvans-ähnliche Wirkung. Ein Impfversuch mit Koi Karpfen und einem inaktivierten Impfstoff gegen das Koi-Herpesvirus zeigte, dass LFS das Potential hat, den mit einer Tauchbadimpfung erzielbaren Immunschutz zu verbessern. LFS konnte auch für die Verabreichung anderer Substanzen wie Antibiotika verwendet und die Aufnahme von Oxytetracyclin um den Faktor fünf erhöht werden. Zusammenfassend lässt sich sagen, dass die Effektivität einer Tauchbadimpfung von Fischen mit LFS durch die gesteigerte Impfstoffaufnahme und dessen Adjuvans-ähnlichen Wirkung verbessert werden kann. Darüber hinaus könnte mithilfe LFS die therapeutische Dosis von Antibiotika bei Badbehandlungen verringert werden. Es erfordert jedoch noch weitere Studien, um diese Technologie aus dem Labor in die Praxis zu übertragen. / In aquaculture, the use of prevention methods, such as vaccination of fish, is an effective strategy to avoid infectious diseases. However, the most effective route of vaccination for fish is the one-by-one intraperitoneal injection, which that is very laborious and expensive to apply for billions of fish every year. Low Frequency Sonophoresis (LFS) has been recognized as one of the most advanced technologies in transdermal delivery of substances in mammals. Thus, it has been suggested to use LFS to enhance the uptake of substances in fish during bath treatments. The present study shows that a low sonication intensity of ca. 60 mW/cm^2 at 37 kHz increased the uptake of a bacterin into the gill tissue of rainbow trout by up to a factor of 240. At this intensity, no or only minimal side effects occurred. At higher sonication intensities, an even higher bacterin uptake but also deleterious side effects were observed. In addition, LFS showed an adjuvant-like effect characterized by a local inflammatory response and T-helper cell activation in the gills. A vaccination trial with koi carp and an inactivated vaccine against the Koi Herpes Virus (KHV) showed that LFS has the potential to enhance the immune protection achieved by immersion vaccination. In addition LFS can also be used for the administration of other substances, such as antibiotics, here we showed that the uptake of Oxytetracycline could be increased by factor five. In summary, the efficacy of the immersion vaccination of fish could be improved by low-frequency ultrasound due, to the increased vaccine uptake along with its adjuvant-like effect. Furthermore, LFS could also reduce the required therapeutic dose of antibiotics in bath treatments, making them more effective, cheaper and environmentally friendly. However, further practical studies will be required to transfer this technology from the lab to the field.