Orientador: Marcelo Brocchi / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-06T21:03:41Z (GMT). No. of bitstreams: 1 Gregoracci_GustavoBueno_M.pdf: 4620017 bytes, checksum: f7feaaf632175abbe340a9d82f379a0c (MD5) Previous issue date: 2006 / Resumo: Os bacteriófagos, vírus que infectam procariotos, são as entidades biológicas mais numerosas do globo. Sua abundância desencadeou revisões em diversos campos como genética, evolução e ecologia, e os fagos atualmente são reconhecidos como importantes vetores para o fluxo de matéria, energia e informação em ambientes naturais. Contudo, há uma enorme diferença entre a quantidade de bacteriófagos estimada no globo (mais de 1030 partículas virais) e a quantidade descrita na literatura (em torno de 5300 amostras). Esta amostragem restrita reflete complicações para estudos de diversidade viral e demonstra as limitações de nosso conhecimento sobre estes vírus. Além disso, a emergência de bactérias resistentes a múltiplos antibióticos implica em uma necessidade de novas práticas terapêuticas, e os fagos representam uma alternativa digna de estudos aprofundados. Assim sendo, este trabalho objetivou o isolamento, a partir de esgoto, e a caracterização biológica de bacteriófagos líticos contra patógenos humanos, visando ampliar a amostragem destes vírus e selecionar novos possíveis agentes terapêuticos. A caracterização envolveu análises morfológica, genética e de especificidade de hospedeiros. As metodologias adaptadas de isolamento e caracterização permitiram a análise de mais de 20 bacteriófagos, todos pertencentes à ordem Caudovirales, bem como de uma proposição para a classificação taxonômica dos isolados. Merece destaque especial o isolamento de três fagos contra Chromobacterium violaceum, feito inédito na literatura, bem como estudos sobre a suscetibilidade deste organismo a diversos bacteriófagos isolados. Por fim, ensaios biológicos sugerem estudos posteriores sobre a aplicação de um destes fagos, denominado Shfl1, para a terapia de Shigella flexneri em infecções humanas, mas são incertos quanto ao potencial do mesmo para contenção deste patógeno no processamento de esgoto comum / Abstract: Bacteriophages, viruses that infect prokaryotic hosts, are the most numerous biological entities of the world. Their abundance implied in reviews in several areas of knowledge, like genetics, evolution and ecology, and phages are now recognized as important vectors in the flux of matter, energy and information in natural environments. However, there is a huge difference between the estimated number of bacteriophages in the world (more than 1030 viral particles) and the quantity described in the literature (around 5300 samples). This restricted sampling reflects in complications to studies of viral diversity and demonstrates our limited knowledge regarding these viruses. Furthermore, the emergence of bacteria resistant to multiple antibiotics implies in a need for new therapeutical approaches, and phages represent an alternative worthy of additional studies. As being so, our purpose in this study was to isolate, from raw sewage, and to biologically characterize lytic bacteriophages, intending to extend these viruses¿ sampling and to select possible new therapeutic agents. Characterization involved morphological, genetic and host range analysis. The adapted protocols for isolation and characterization permitted the analysis of more than 20 bacteriophages, all belonging to the Caudovirales order, as well as a tentative taxonomic classification of the samples. Other distinctive results include the isolation of three bacteriophages against Chromobacterium violaceum, previously unknown in the literature, as well as the study about the susceptibility of this organism to several isolated phages. Moreover, biological assays suggested further studies about the application of one of these phages, named Shfl1, in the treatment of Shigella flexneri infections in humans, but were uncertain about the potential of the same virus to restrain this pathogen during sewage processing / Mestrado / Microbiologia / Mestre em Genética e Biologia Molecular

The production of biofuels and in particular bioethanol has increased rapidly since the early 1990’s. The advantages of biofuels include reduced CO2 production, a decrease in fuel importation for many nations (notably the US and Brazil), and comparatively simple blending with fossil fuels. The production of basic fuel ethanol (1st generation) involves the use of an energy crop feedstock (corn in US and sugar cane in Brazil). The feedstock is processed via simple mechanical methods to release the simple carbohydrates, mixed with water and fermented anaerobically via S. cerevisiae yeast into ethanol and CO2. Due to the low market value of fuel ethanol, profit margins are restrictive, and as a result sterilisation and aseptic techniques are not economically viable, and contamination by environmental organisms is commonplace. The current system of biocontrol involves the addition of antibiotics, primarily penicillin and virginiamycin, to the fermentation. While these antibiotics are broad spectrum and highly effective in reducing the impact of contamination, the negative environmental impacts of antibiotic usage are well known. In order to reduce the impact of contamination and reduce reliance on antibiotics an alternative system of biocontrol is required. In this thesis various biocontrol agents are assessed, including bacteriophage, hop acids, chitosan, onion oil extract, copper and silver ions. The effect of these agents on the growth of various contaminant bacteria and a strain of S. cerevisiae is assessed and fermentations are carried out under sterile and controlled contaminated conditions to generate data on the effect of the contaminant and the various methods of biocontrol. Other possibilities investigated include the insertion of plasmids containing heat shock proteins into S. cerevisiae to enhance thermo-tolerance.

Notre travail a consisté à rechercher des éléments génétiques nouveaux associés aux souchesde Streptococcus agalactiae (SGB) responsables de méningites néonatales (MNN).L'amplification au hasard des ADN génomiques a identifié 3 fragments prophagiquesassociés aux souches à haut risque de MNN. Trente-six phages ont été isolés à partir desouches invasives et non invasives de SGB. La caractérisation moléculaire a défini sixgroupes moléculaires. L'étude du spectre lytique a montré des particularités pour chacun dessix groupes moléculaires. L'étude des capacités métaboliques de souches de SGB a montréque les souches lysogènes appartenant aux lignées phylogénétiques impliquées dans les MNNet présentent des pertes cataboliques. Les transferts génétiques horizontaux qui marquent lesclones des MNN ne semblent pas jouer un rôle dans la résistance aux macrolides. Desmécanismes de transduction ont joué un rôle important dans l'émergence de lignéesphylogénétiques impliquées dans les MNN. / We tried to identify new genetic elements in the genome of Streptotoccus agalactiae (SGB)strains responsible for neonatal meningitis (MNN). Three prophage-related DNA elementswere identified by randomly amplified polymorphie DNA analysis significantly associatedwith SGB strains presented a high risk of causing MNN. Thirty-six phages were isolated from invasive and non invasive SGB strains. Molecular characterization of phage DNA identified six distantly related molecular groups. The various phage groups had specifie lytic activities.The lysogenic strains belonging to phylogenetic lineages most involved in MNN presented catabolic losses. The horizontal gene transfers that mediate mobile genetic elementsrecognized as markers of highly virulent clones do not seem to be have played a role in theemergence of macrolide resistance. Ali our results indicate that transduction may have play arole in the emergence of phylogenetic lineages implicated in MNN.

An array of Bacillus licheniformis strains were isolated from a commercial powdered milk process. Bacteriophages exhibiting activity against B. licheniformis were isolated from cattle manure and effluent samples destined for a lagoon at a dairy farm. After sequencing, 8 of the 10 phages were found to be novel and genetically differentiated. Transmission electron scanning microscopy (TSEM) was performed. All bacteriophages were of the family Herelleviridae with contractile tail sheaths ranging from 80µm to 150µm and, surprisingly, survived a common fluid milk processing treatment used to inactivate vegetative cells. The survival of the phage after high temperature short time pasteurization of 73℃ for 20 s shows that the use of bacteriophages in milk to control B. licheniformis could be applied as a potential quality control, retarding the germination of spores and reduction of final spore counts in products with long run times such as dairy powders.

Master thesis deals with the use of bacteriophage as a theranostic drug nanocarrier. The term theranostics is used in recent years for systems that allow connecting of diagnostics, targeted drug delivery and monitoring of patient’s response to administered treatment in a single modality. These systems are very suitable especially with heterogeneous diseases, such as cancer. Nowadays, the treatment of cancer has often severe side effects to the patient’s body, which lowers his capability to fight the disease. Theoretical part of this work is focused on the properties of viral capsids, proteins and inorganic materials as drug nanocarriers. In practical part of this work, different methods for cultivation of bacteriophage are compared, both in liquid and solid medium and with different concentrations of the maltose, trough whose receptors bacteriophage is able to enter the host cell. Optimal was cultivation with 0.2% maltose in solid medium. Practical part is focused mainly on the use of bacteriophage as a nanocarrier for cytotoxic drug doxorubicin. Bacteriophage was able to encapsulate all applied concentrations of doxorubicin (0; 12.5; 25; 50; 100 and 200 g/ml), which was proved using fluorescent detection. Different times of encapsulation (2; 4; 8 and 12 hours) were studied. Optimal time was 2 hours. Encapsulation properties of bacteriophage were compared to apoferritin. Bacteriophage was able to encapsulate 4× higher concentrations of doxorubicin and its release during rinsing with water was 10× lower compared to apoferritin. This work concludes that bacteriophage is a very suitable platform for targeted drug delivery in theranostics.

Anaerobic membrane bioreactors (AnMBR) have been established as an efficient method of wastewater treatment to obtain high-quality effluent with low energy consumption. However, membrane fouling leading to flux reduction and an increase in operational costs can negate potential benefits associated with AnMBR. Today’s conventional membrane cleaning process includes physical and chemical approaches, both of which have their own drawback. For this reason, the biological approach was proposed as an alternative to dangerous, energy-consuming, and environmentally unsafe treatment techniques. The combination of UV-C and bacteriophage offers an alternative chemical-free approach for biofouling control. This dissertation aims to test the different order of using UV-C and bacteriophage to clean anaerobic membrane. This dissertation also demonstrates a proof-of-concept to achieve semi-online cleaning using UV-C and bacteriophage, thus increasing the feasibility of described technology. As a result of this work, it was shown that preliminary UV exposure enhances bacteriophage propagation into thick biofilms, and that the bacteriophages are able to affect total cell number and extracellular polymeric substances (EPS) compared to the control. Compared to the control, the semi-online cleaning strategy also resulted in a membrane that took a longer time for the transmembrane pressure to increase in the next operation cycle after cleaning.

Since phages use the host resources to replicate themselves after infection, the different sizes of the phage genome should influence the replication rate. We, therefore, hypothesized that the smaller genomes should burst the cell faster than the larger ones. As well, the shorter genomes would have greater burst sizes because they should replicate faster. Here, we obtained 16 phages of various genome length. All phages were isolated on Streptomyces griseus and available in our phage bank at the University of North Texas. We performed one-step growth studies for the 16 phages, as well as determined the host doubling time from its growth curve. The results show that S. griseus grown in nutrient broth has a doubling time of 5 hours and 22 minutes. This doubling time is used as a guideline for the phage growth studies. Because the filamentous nature of the host caused several difficulties during the experiment, we isolated single cells by sonication and centrifugation. After the cell number was determined by viable cell count, the cells were infected with each type of phage using a multiplicity of infection (MOI) of 0.5. The results show that phages' burst times range between 45 (±0, standard error) and 420 (±30) minutes and burst sizes from 12 (±0) to 1500 (±60) The statistical analyses show that there is no correlation between either genome size and burst time (R= -0.01800, P=0.97894) or genome size and burst size (R= -0.32678, P=0.21670). We further performed the comparative genomics studies to investigate whether the phages with similar burst times and burst sizes show similar genome structures. The studies show that Eddasa and Lorelei have similar burst times of 45 to 60 minutes and share 52 homologs. For burst size, only Tribute and Blueeyedbeauty that have similar burst sizes of 21-30, and they are genetically related because of the 48 shared homologs. Although this study did not find any correlation between genome size and burst time/burst size, it provides a foundation for further studies to determine what regulates these two traits.

Deoxyribonucleic acid isolated from bacteriophage lambda can infect Escherichia coli K12 in the presence of adsorbed helper phage (Kaiser and Hogness, 1960; Kaiser, 1962). The manner in which lambda DNA enters the cell and the possible role of helper phage in the penetration process is still not clear. Kinetic studies conducted in this laboratory during the initial stages of infection by lambda DNA demonstrate a requirement for helper function during the penetration of an infectious molecule. Further investigation into this problem is needed and was the major objective of this study.

Silicification of organisms in silica-depositing environments can impact both their ecology and their presence in the fossil record. Although microbes have been silicified under laboratory and environmental conditions, viruses had not been, prior to this work. Bacteriophage T4 was successfully silicified under laboratory conditions that closely simulated those found in silica-depositing hot springs. Virus morphology was maintained during the short period of silicification (48 hours), and a clear elemental signature of silicon and phosphorus was detected by energy-dispersive X-ray spectrophotometry (EDX). However, the EDX signature of silicified virus was not sufficiently distinct from that of cell membrane or phosphate minerals for that technique to be used to discover viral remains in hot spring mineral deposits. Having shown that bacteriophage T4 can be silicified, it was then determined that the impact of silica exposure on infectivity varied widely between different viruses. The effect on infectivity did not appear to be related to virus size or morphology. In addition, the impact on infectivity was at least partially reversible, indicating that it was caused, at least in part, by occluding infection-related structures on the virus, rather than destruction or denaturation of the virus. Those viruses which showed a decline in infectivity with silica exposure also showed increased resistance to desiccation after being exposed to silica, which has implication for long-range virus dispersal. The desiccation resistance was proportional to the degree that silicification reduced infectivity in that virus. Desiccation resistance also declined with prolonged exposure to drying, suggesting that the mechanism was due to the silica coating helping to retain water rather than replacing the hydrogen bonding of water. Virus dispersal is critical for both the spread of disease and the diverse roles that viruses play in Earth ecology. However, the mechanisms of host-independent virus dispersal are poorly understood and hotly debated. These experiments showed that, under mild conditions, diverse viruses can be coated in silica and that silica coating provides some, if not most, viruses with remarkable desiccation tolerance. Virus silicification thus provides a potential mechanism for global dispersal of viruses that could not otherwise tolerate the desiccation of wind-borne transportation.

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