Une partie conséquente de mon travail a été d'effectuer un crible génétique en utilisant une bibliothèque de mutants générés par insertion aléatoire de Tn5-Sm, un minitransposon bactérien. Le crible a été réalisé dans un contexte défini: celui de mouches-hôtes auxquelles manquait le gène Eater, lequel code un récepteur de phagocytose (Kocks et al., 2005). Dans ces mouches, l'infection n'est plus contrôlée dans l'hémocoele par les hémocytes et les drosophiles mutantes succombent rapidement à une bactériémie. Plusieurs phénotypes bactériens étaient attendus à l'issue de ce crible. Une première catégorie de phénotype prévisible était une virulence accrue, par exemple si les bactéries mutantes devenaientcapables de traverser plus rapidement ou efficacement la barrière intestinale conséquemment à la perte d'un régulateur négatif. Un deuxième type de phénotype attendu était une virulence atténuée pouvant s'expliquer de plusieurs manières: 1- perte de résistance à l'environnement existant dans le lumen intestinal (enzymes digestives et lysozyme, radicaux libres et peptides antimicrobiens induits au niveau de l'épithélium intestinal dans le cadre d'une réponse immunitaire locale de l'hôte); 2- incapacité à traverser la matrice péritrophique; 3-incapacité à envahir les cellules épithéliales (adhésion, pénétration); 4- incapacité à résister aux défenses intracellulaires potentielles; 5- incapacité à sortir du côté basal des entérocytes 6- incapacité à proliférer dans l'hémolymphe ou perte de la résistance à l'action de la réponse immunitairesystémique qui est, quant à elle, fortement induite en l'absence de phagocytose, laquelle empêche chez les mouches sauvages la prolifération des bactéries ayant traversé la paroi intestinale. [...] Dans le cadre d'une infection intestinale, les mouches sauvages (et imd) succombaient en six jours alors que, de manière surprenante, les mouches mutantes de la voie Toll périssaient plus lentement, une situation opposée à celle du modèle de la piqûre septique. Quelques bactéries sont capables de traverser la paroi intestinale mais sont incapables de proliférer à moins que la réponse cellulaire ait été préalablement bloquée. L'épithélium intestinal apparaissait normal à la dissection et la presque totalité des bactéries ingérées étaient tuées dans l'intestin. Après avoir exclu l'hypothèse d'une toxine sécrétée dans le surnageant des bactéries adsorbées sur le filtre sur lequel viennent se nourrir les mouches, nous avons testé l'hypothèse qu'une suractivation de la réponse immunitaire était à l'origine du décès des mouches. La génétique mettant hors de cause les peptides antimicrobiens, la voie Toll n'étant apparemment pas activée dans l'épithélium intestinal, nous avons alors étudié laréponse oxydative induite par l'ingestion de bactéries (Ha et al., 2009), laquelle est capable de tuer les mouches lorsqu'elle n'est pas régulée correctement. Là-aussi, le résultat s'est avéré négatif. En fin de compte, j'ai pu établir que la mort des mouches était due à un état de famine, confirmé par des mesures des réserves métaboliques. Mes travaux ont permis d'établir un nouveau rôle de la voie Toll dans la résistance à la famine, en présence ou absence d'infection, qui sera peut-être à mettre en relation avec un rôle métabolique de la voie Toll consistant à bloquer la voie de réponse à l'insuline lors d'une infection. En conclusion, mes travaux permettent de mieux comprendre les relations hôte-pathogènequi s'établissent lors d'une infection intestinale.

Chitinases have been shown to be potential agents for biological control of the plant diseases caused by various phytopathogenic fungi and insect pests, because fungal cell walls and insect exoskeletons contain chitin as a major structural component. Chitinase has also been found to increase the efficacy and potency of Bacillus thuringiensis crystal (Cry) proteins toxic to larvae of insect pests. The reason of this synergy is the presence of chitin in the structure of the outer membrane of larval midgut. In this study, the gene encoding chitinase A (chiA) from Serratia marcescens Bn10, a local isolate of Trabzon province was amplified by PCR and cloned into the E.coli/Bacillus shuttle vectors, pNW33N and pHT315. For the expression in B. thuringiensis, the promoter region of cry3Aa11 gene of B. thuringiensis Mm2 was placed at the upstream of chiA. The vectors carrying both chiA and promoter site of cry3Aa11 was first introduced into E. coli and then into Bacillus subtilis 168 which were used as intermediate hosts in this study. pHT315PC carying chiA was then introduced into Coleoptera-specific B. thuringiensis cells (strain 3023) and the specific chitinase activity of the recombinant B. thuringiensis was measured as 5056 U/min/mg which was 6.3 fold higher than that of the parental strain. The specific activity corresponded to about one third of that produced by S. marcescens Bn10. The chiA gene was next sequenced and characterized. The sequence was submitted to GeneBank (Accession No. DQ165083). Chitinase A of S. marcescens Bn10 was found to be a 563 residue protein with a calculated molecular mass of 60.9 kDa. The mean G+C content of the gene is 58.75%. The deduced amino acid sequence was 99.3&ndash / 91.5% identical to those of known chitinases from S. marcescens, Burkholderia cepacia and Enterobacter sp. It was found that the chitinase of S. marcescens Bn10 has six amino acids difference from the consensus sequence of aligned chitinases. The production of chitinase by the local isolate S. marcescens Bn10 in different cultural conditions was also investigated. Optimum temperature and pH for chitinase production was found to be 30 oC and 7.5, respectively. Varying the concentration of colloidal chitin and the inclusion of NAG into the medium had no effect on chitinase production. The effect of different parameters such as temperature, pH, substrate concentration and certain inhibitory elements on enzyme activity were next assayed. The highest activity was obtained at 45 oC and in a pH range of 4.0 to 9.0. Activity of chitinase increased with increasing substrate concentration up to 35 mg/mL. Ca2+, Co2+, Cu2+, EDTA, Fe2+, Mg2+, Mn2+ and Zn2+ were tested for their effects on the activity of enzyme. The enzyme was inhibited by only 4% in the presence of 10 mM EDTA, whereas 10 mM Co2+ included in the assay mixture increased the activity by 20%.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv. / Härtill 5 uppsatser.

Memoria para optar al título profesional de: Ingeniero Agrónomo Mención: Sanidad Vegetal / La pudrición gris, causada por el hongo Botrytis cinerea, corresponde al principal problema fitopatológico que enfrentan los productores de uva de mesa, puesto que limita su producción y exportación al desarrollarse pudriciones incluso en almacenaje. Para su control, se integran diferentes medidas entre las cuales el uso de fungicidas específicos es la base de los programas utilizados. Actualmente, la creciente preocupación por la presencia de residuos de fungicidas en la fruta y el riesgo ambiental asociado a su uso, además del desarrollo de resistencias en Botrytis a estas moléculas, han complicado su empleo como estrategia de control. Ante el cual se han aplicado métodos alternativos como la intensificación de prácticas culturales que disminuyan las condiciones predisponentes de la enfermedad, junto a herramientas naturales que incluyen el uso de extractos de cítricos y controladores biológicos como Trichoderma. Por otro lado, recientemente se ha elaborado un biofungicida con la bacteria antagonista Serratia plymuthica cepa CCGG2742, un controlador biológico de B. cinerea, que en este ensayo se ha probado su sensibilidad frente a fungicidas de uso común en vid, con el objeto de conocer su compatibilidad en un posible programa de control que integre estas estrategias. Se evaluaron los ingredientes activos boscalid, ciprodinil + fludioxonil, fenhexamida, iprodione, kresoxim methyl, pyrimethanil, tebuconazole y extracto de cítrico en concentraciones que fluctuaron entre 0 y 5.000 µg i.a. ∙ mL-1 y se calculó la EC50. Los valores de EC50 obtenidos fueron: 6,00∙104 ; 3,18∙107 ; 7,04∙1012 y 1,38∙1018 µg i.a. ∙ mL-1 , para fenhexamida, extracto de cítrico, tebuconazole y boscalid respectivamente, mientras que con iprodione, kresoxim methyl, pyrimethanil y la mezcla de c+f, no se obtuvieron valores de EC50 positivos, ni indicios de inhibición in vitro. De acuerdo a estos resultados, el uso de dosis comerciales de fungicidas no altera el desarrollo de la cepa CCGG2742. / Gray mold induced by Botrytis cinerea, is the most important disease of table grapes that causes significant losses to grape growers. Its ability to attack in the orchard and its ability to develop under conditions prevailing during storage, shipment and marketing make its control a challenge. Control programs have relied mainly on chemical strategies using specific fungicides. However, the growing public concern about fungicide residues in fruit, and the environmental risk associated to fungicide use, in addition to pathogen’s resistance development, have created a complicated situation for the continuous use of fungicides. Therefore, alternative methods have been developed for non-chemical control, such as cultural practices, and the use of natural products including citrus extracts and biological controls agents, as Trichoderma. Serratia plymuthica, antagonistic to B. cinerea, is a new biocontrol agent, and in this study the sensitivity of the strain CCGG2742 to fungicides commonly used in vineyards was tested, in order to know their compatibility for possible control programs that integrate these tools. Boscalid, ciprodinil + fludioxonil, fenhexamida, iprodione, kresoxim methyl, pyrimethanil, tebuconazole and citric extract were tested at concentrations ranging between 0 and 5,000 µg i.a. ∙ mL-1 . EC50 values obtained ranged between 5.48∙104 and 1.38∙1018 µg i.a. ∙ mL-1 . According to these results, commercial rates of the fungicides tested do not affect the development of S. plymuthica strain CCGG2742.

Serratia sp. ATCC 39006 (S39006) is a Gram-negative bacterium that is virulent in plant (potato) and animal (Caenorhabditis elegans) models. It produces two secondary metabolite antibiotics, prodigiosin and a carbapenem, and the plant cell wall degrading exoenzymes, pectate lyase and cellulase. A complex regulatory network controls production of prodigiosin, including a quorum sensing (QS) system, and the role of post-transcriptional regulation was investigated. It was hypothesized that Hfq-dependent small regulatory RNAs (sRNAs) might also play a role. Hfq is an RNA chaperone involved in post-transcriptional regulation that plays a key role in stress response and virulence in other bacterial species. An S39006 ∆hfq mutant was constructed and in the mutants production of prodigiosin and carbapenem was abolished, while production of the QS molecule, butanoyl homoserine lactone (BHL), was unaffected. Using transcriptional fusions, it was found that Hfq regulated the QS response regulators, SmaR and CarR. Additionally, exoenzyme production and swimming motility were decreased in the ∆hfq mutant, and virulence was attenuated in potato and C. elegans. It was also shown that the phenotype of an hfq mutant is independent of its role in regulating the stationary phase sigma factor, rpoS. In order to define the complete regulon of Hfq and identify relevant potential sRNAs, deep sequencing of strand-specific cDNAs (RNA-seq) was used to analyse the whole transcriptome of S39006 WT and the ∆hfq mutant. The regulon of another post-transcriptional regulator, RsmA, also involved in regulating prodigiosin production, was investigated by performing RNA-seq on an rsmA mutant. Moreover, global changes in the proteome of the hfq mutant was analysed using an LC-MS/MS approach with isobaric tags for relative and absolute quantification (iTRAQ). This study confirms a role for Hfq in pathogenesis and the regulation of antibiotic production in S39006, and begins to provide a systems-level understanding of Hfq and RsmA regulation using a combination of transcriptomics and proteomics.

Bats are crucial to ecological function and provide key ecosystem services to people but face a variety of significant threats. One current threat to North American bats is white-nose syndrome (WNS), a disease caused by the invasive fungal pathogen Pseudogymnoascus destructans (Pd) that has killed millions of hibernating bats across the continent. Remnant populations of affected bat species persist but are so depleted that they may now be highly vulnerable to new threats, or to the synergistic effects of multiple existing threats. The emergence of novel or opportunistic pathogens in bat hosts is a particular concern for the survival of these small, isolated colonies. Apart from studies of WNS and zoonotic pathogens of humans, however, bat diseases remain poorly understood. In this paper, I describe the pathology of a new, highly aggressive bat disease affecting hibernating little brown myotis (Myotis lucifugus) and identify candidate microbes as possible causative agents. The pathological signs that were observed diverged from those of WNS, and included blue fluorescence in the wings when trans-illuminated with ultraviolet light, and the rapid development of wing necroses and mortality within weeks of the onset of hibernation. Pathology, wing swab cultures, post-mortem analyses, and hemolysis testing identified an array of candidate species, but suggest that a possible cause is a polymicrobial infection involving two etiological agents – Trichosporon yeast and Serratia bacteria. Both species have been documented as part of the mycobiota and microbiota of healthy bats, and cave environments. They are also opportunistic pathogens, known to cause infection in other wild animals and immunocompromised humans. Opportunistic pathogens have been increasingly implicated as a cause of mass mortality events in wildlife. The disease identified here has, to my knowledge, not previously been described, and could represent a new threat to North American bats, compounding concerns for populations facing an already precarious situation. / Biology

<p dir="ltr"><i>Serratia marcescens </i>is a bacterium with a ubiquitous environmental distribution and the ability to cause opportunistic infections. This research explores three different group behaviors in <i>S. marcescen</i>s. These are biofilm formation, microbial hitchhiking, and responses to cannabinoid-induced stress. To study biofilm development, we used a crystal violet assay to measure biofilm and compared that to the bacterial growth of those cultures. We looked at the role of nutrients and temperature in biofilm produced by <i>S. marcescens</i> and tested four <i>S. marcescens</i> strains. We found that there were differences in the ratio of biofilm to growth when <i>S. marcescens</i> was grown in different media. The exact relationship between temperature and media composition requires more information than could be attained from these studies. Next, we wanted to investigate whether <i>S. marcescens</i> could also utilize movement of other, more highly motile species of bacteria through a process called microbial hitchhiking. <i>S. marcescens</i> was grown with a highly motile <i>Paenibacillus</i> sp. isolate. <i>S. marcescens</i> was tracked by the red pigment that it produces. It was observed that <i>S. marcescens</i> consistently spread farther across a surface when it was co-cultured with <i>Paenibacillus</i> sp. than when grown alone. This was repeated with three other <i>S. marcescens</i> strains and three different species of <i>Paenibacillus.</i><i> </i>Hitchhiking behavior was observed in all cases. To understand the mechanism by which <i>S. marcescens</i> hitchhikes on <i>Paenibacillus </i>spp., a <i>S. marcescens </i>flagellar mutant was used. Even in the absence of a flagellum, <i>S. marcescens</i> was still able to hitchhike implying that another mechanism must be involved. Finally, we evaluated the response of <i>S. marcescens </i>to cannabidiol (CBD) a phytocannabinoid with antimicrobial and antibiofilm properties, though it has limited potency against Gram-negative bacteria like <i>S. marcescens</i>. We found that CBD did not kill <i>S. marcescens </i>nor did it affect its biofilm development. Interestingly, <i>S. marcescens </i>cultures showed enhanced pigment production in response to high-dose CBD exposure compared to vehicle controls. This response was also observed with exposure to three additional phytocannabinoids. Results from these studies add to our understanding of how <i>S. marcescens</i> can access new areas and persist in a broad range of environments.</p>

Programa de Apoio aos Planos de Reestruturação e Expansão das Universidades Federais / The microbiota intestinal of Psittaciformes is mainly composed of Gram positive bacteria, is considered a sign of illness the presence of Gram negative bacteria. Wild birds are important to public health by harboring pathogens able of zoonotic transmission. The displacement of these birds, such as occurs in the trafficking of wild animals, is a propagation mechanism of new endemic foci of infections agents to long distances from where they were acquired. In this study we examined the presence of enterobacteria in psittacines using samples of organs of birds received for necropsy at the Laboratório Central de Diagnóstico de Patologias Aviárias (LCDPA) situate in the Departamento de Medicina Veterinária Preventiva (DMVP) at Centro de Ciências Rurais (CCR) of the Universidade Federal de Santa Maria (UFSM). We also collected samples using swabs of cloaca and crop, in live birds, kept in captivity in order to monitor the possible presence of pathogenic bacteria. Escherichia coli was considered the predominant bacteria in most samples, then after microbiological analysis and confirmation of E. coli isolates, we selected 28 colonies, which were tested for virulence factors iss, this gen is associated with the strain s ability to resist the lytic effects of serum, and iutA an outer membrane receptor of involved in the high affinity binding of iron-aerobactin. It is hoped that the data obtained in this research will help in establishing the health status of psittacines helping maintain the species and preventing risks to public health. / A microbiota intestinal de Psitaciformes é composta principalmente por bactérias Gram positivas, não se considerando saudável a presença de bactérias Gram negativas. As aves silvestres possuem importância para a saúde pública por albergarem patógenos passíveis de transmissão zoonótica. O deslocamento territorial dessas aves, como o que ocorre no tráfico de animais selvagens, constitui um mecanismo de propagação de novos focos endêmicos de agentes infecciosos a grandes distâncias dos locais onde foram adquiridos. Neste trabalho analisamos a presença de enterobactérias em psitacídeos utilizando amostras de órgãos de aves recebidas para necropsia na rotina do Laboratório Central de Diagnóstico de Patologias Aviárias (LCDPA) situado no Departamento de Medicina Veterinária Preventiva (DMVP) do Centro de Ciências Rurais (CCR) da Universidade Federal de Santa Maria (UFSM). Também foi realizada coleta de suabes cloacais e de inglúvio em aves vivas mantidas em cativeiro visando monitorar a presença de possíveis bactérias patogênicas. Escherichia coli foi considerada a bactéria predominante na maioria das amostras, então após análise microbiológica e confirmação dos isolados de E. coli, selecionamos 28 colônias, as quais foram testadas para os fatores de virulência iss, relacionado com a capacidade da cepa em resistir aos efeitos líticos do soro, e iutA, sendo este gene um receptor de membrana externa dos compostos sideróforos da aerobactina (genes iuc) que é um sistema de captação de ferro pelo qual E. coli expressa afinidade. Espera-se que os dados obtidos nessa pesquisa contribuam no estabelecimento do estado sanitário de psitaciformes auxiliando na manutenção da espécie e prevenindo riscos à saúde pública.

Coagulase negative staphylococci (CoNS) are the most prevalent bacterial contaminants of platelet concentrates (PCs), and have been implicated in severe and fatal transfusion reactions. Of this group, Staphylococcus epidermidis is most frequently identified. The preliminary objective of this thesis was to confirm that S. epidermidis could form biofilms under platelet storage conditions. This was achieved using a modified crystal violet staining assay to detect plastic-adherent bacterial cells and examination of attachment processes by scanning electron microscopy. A collection of CoNS isolated from PCs obtained from reportedly healthy donors was then assessed for biofilm-forming potential at the genetic and phenotypic level. Despite the presumable commensal origin of these isolates, a high proportion of S. epidermidis strains displayed a biofilm positive phenotype. The threat of S. epidermidis biofilm formation during platelet storage identified herein signifies that any alterations made to platelet storage protocols should be evaluated with consideration of this risk. The advent of platelet additive solutions (PASs) as an alternative to plasma for PC storage provides a relevant example, since little is known about the effect of PAS on contaminant bacteria, and vice versa. Growth and biofilm formation by S. epidermidis and the Gram-negative bacterium Serratia liquefaciens were measured in PAS- or plasma-PCs over 5 days, simulating standard platelet storage conditions, after initial inoculation with low, clinically relevant bacterial concentrations. Assays for platelet quality were performed simultaneously. Only S. liquefaciens exhibited a slower doubling time in plasma-PCs than in PAS-PCs. Biofilm formation by both species was reduced during storage in PAS-PCs, increasing bacteria availability for detection. Although S. liquefaciens adversely affected platelet quality in both media, S. epidermidis contamination did not. Ultimately, culture-based detection remains the earliest indicator of bacterial presence in PAS-PCs. Lastly, since formation of platelet-bacteria aggregates is largely based on receptor-ligand interactions, it was postulated that biofilm formation by contaminant bacteria could be abrogated by receptor shielding. Methoxypoly(ethylene glycol) was applied to covalently modify the platelet surface using a process termed ‘PEGylation’. It is herein demonstrated that PEGylation of PCs inoculated with S. epidermidis results in significantly reduced bacterial binding and biofilm formation during platelet storage.

Coagulase negative staphylococci (CoNS) are the most prevalent bacterial contaminants of platelet concentrates (PCs), and have been implicated in severe and fatal transfusion reactions. Of this group, Staphylococcus epidermidis is most frequently identified. The preliminary objective of this thesis was to confirm that S. epidermidis could form biofilms under platelet storage conditions. This was achieved using a modified crystal violet staining assay to detect plastic-adherent bacterial cells and examination of attachment processes by scanning electron microscopy. A collection of CoNS isolated from PCs obtained from reportedly healthy donors was then assessed for biofilm-forming potential at the genetic and phenotypic level. Despite the presumable commensal origin of these isolates, a high proportion of S. epidermidis strains displayed a biofilm positive phenotype. The threat of S. epidermidis biofilm formation during platelet storage identified herein signifies that any alterations made to platelet storage protocols should be evaluated with consideration of this risk. The advent of platelet additive solutions (PASs) as an alternative to plasma for PC storage provides a relevant example, since little is known about the effect of PAS on contaminant bacteria, and vice versa. Growth and biofilm formation by S. epidermidis and the Gram-negative bacterium Serratia liquefaciens were measured in PAS- or plasma-PCs over 5 days, simulating standard platelet storage conditions, after initial inoculation with low, clinically relevant bacterial concentrations. Assays for platelet quality were performed simultaneously. Only S. liquefaciens exhibited a slower doubling time in plasma-PCs than in PAS-PCs. Biofilm formation by both species was reduced during storage in PAS-PCs, increasing bacteria availability for detection. Although S. liquefaciens adversely affected platelet quality in both media, S. epidermidis contamination did not. Ultimately, culture-based detection remains the earliest indicator of bacterial presence in PAS-PCs. Lastly, since formation of platelet-bacteria aggregates is largely based on receptor-ligand interactions, it was postulated that biofilm formation by contaminant bacteria could be abrogated by receptor shielding. Methoxypoly(ethylene glycol) was applied to covalently modify the platelet surface using a process termed ‘PEGylation’. It is herein demonstrated that PEGylation of PCs inoculated with S. epidermidis results in significantly reduced bacterial binding and biofilm formation during platelet storage.