Estudo epidemiológico, genotípico e fenotípico de estirpes de Staphylococcus aureus produtoras de biofilmes isoladas do ambiente de ordenha e de casos de mastite bovina

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Clonagem e expressão de fragmentos de anticorpos (scFV) contra o vírus da leucemia felina (FeLV) por phage display

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Phage-displayed peptides as capture antigens in an innovative assay for Taenia saginata-infected cattle

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Caracterização de Bacteriófagos Infectando Isolados Clínicos de Pseudomonas aeruginosa Armazenados em uma Coleção de Cultura

To isolate, to concentrate and to purify bacteriophages from isolates of P. aeruginosa; To observe the capacity of bacteriophages to infect isolates of P. aeruginosa susceptible and multiresitant to antimicrobial; To caractherize bacteriphages by electronic microscopy techniques. 10 isolates of Pseudomonas aeruginosa from LEMC culture collection were submitted to the experiments of ideal temperature for the lyse region appearance in the MaConkey culture plate and 2 extraction methods for the concentration of the phages, clorophorm (Silankorva) and filtration plus centrifugation (Bergan). Three infected clinical isolates of multiresistant P. aeruginosa an one susceptible isolate ( PA01) were evaluated by 3 transmission electron microscopy techniques to caractherize phages morphologically (“on grid”, “on drop” and direct extraction from the lyse region of the culture plate). The ideal temperature to obtain lyses region was 37°C. The stock solutions, obtained through the methodologies of Sillankorva and Bergan, had satisfactory results in infecting the multiresistant isolate and the negative control. Among the 3 techniques of electronic microscopy tested the direct from the lyse plate was the best to obtain the micrography of the phages

Resistência de bactérias ácido-láticas a bacteriófagos provenientes de unidades de processamento de queijo Coalho

Este trabalho teve como objetivos isolar bacteriófagos de amostras de leite, soro e queijo de Coalho e avaliar a resistência de cepas de Lactobacillus paracasei, pertencentes à Coleção de Micro-organismos de Interesse para a Agroindústria Tropical da Embrapa Agroindústria Tropical, aos fagos isolados. Posteriormente, a resistência destas cepas a fagos específicos para L. paracasei, da Coleção do Instituto de Lactología Industrial - INLAIN (Santa Fe, Argentina), também foi avaliada. As amostras para isolamento dos fagos foram obtidas em quatro unidades de processamento de queijo de Coalho, sendo duas artesanais e duas industriais, localizadas no Estado do Ceará. Para o isolamento dos bacteriófagos, foi empregado o teste de lise celular (spot), enquanto que a resistência das culturas aos fagos foi avaliada pelos testes de capacidade de produção de ácido e avaliação da turbidez. As cepas avaliadas foram resistentes aos bacteriófagos provenientes das unidades de processamento de queijo de Coalho e aos bacteriófagos da Coleção do INLAIN. Os resultados obtidos indicaram que as culturas láticas testadas, resistentes aos bacteriófagos, podem ser utilizadas na composição de fermento lático destinado à elaboração de queijo de Coalho, a partir de leite pasteurizado.

Group I Introns and Homing Endonucleases in T-even-like Bacteriophages

Homing endonucleases are rare-cutting enzymes that cleave DNA at a site near their own location, preferentially in alleles lacking the homing endonuclease gene (HEG). By cleaving HEG-less alleles the homing endonuclease can mediate the transfer of its own gene to the cleaved site via a process called homing, involving double strand break repair. Via homing, HEGs are efficiently transferred into new genomes when horizontal exchange of DNA occurs between organisms. Group I introns are intervening sequences that can catalyse their own excision from the unprocessed transcript without the need of any proteins. They are widespread, occurring both in eukaryotes and prokaryotes and in their viruses. Many group I introns encode a HEG within them that confers mobility also to the intron and mediates the combined transfer of the intron/HEG to intronless alleles via homing. Bacteriophage T4 contains three such group I introns and at least 12 freestanding HEGs in its genome. The majority of phages besides T4 do not contain any introns, and freestanding HEGs are also scarcely represented among other phages. In the first paper we looked into why group I introns are so rare in phages related to T4 in spite of the fact that they can spread between phages via homing. We have identified the first phage besides T4 that contains all three T-even introns and also shown that homing of at least one of the introns has occurred recently between some of the phages in Nature. We also show that intron homing can be highly efficient between related phages if two phages infect the same bacterium but that there also exists counteracting mechanisms that can restrict the spread of introns between phages. In the second paper we have looked at how the presence of introns can affect gene expression in the phage. We find that the efficiency of splicing can be affected by variation of translation of the upstream exon for all three introns in T4. Furthermore, we find that splicing is also compromised upon infection of stationary-phase bacteria. This is the first time that the efficiency of self-splicing of group I introns has been coupled to environmental conditions and the potential effect of this on phage viability is discussed. In the third paper we have characterised two novel freestanding homing endonucleases that in some T-even-like phages replace two of the putative HEGs in T4. We also present a new theory on why it is a selective advantage for freestanding, phage homing endonucleases to cleave both HEG-containing and HEG-less genomes.

Viruses in the marine environment: community dynamics, phage-host interactions and genomic structure

[EN] There are an estimated 1030 viruses in the world oceans, the majority of which are phages (viruses that infect bacteria). Extensive research has demonstrated the significant influence of marine phages on microbial abundance, community structure, genetic exchange and global biogeochemical cycles. In this thesis, we contribute to increase the knowledge about the ecological role of viruses in marine systems, but also we aimed to provide a better understanding about the interactions between phages and their hosts and the genetic pool and biogeography of some the isolated phages genomes.

Phage therapy: A software system for phage quantification and kinetic model inference.

One of the most serious problems of the modern medicine is the growing emergence of antibiotic resistance among pathogenic bacteria. In this circumstance, different and innovative approaches for treating infections caused by multidrug-resistant bacteria are imperatively required. Bacteriophage Therapy is one among the fascinating approaches to be taken into account. This consists of the use of bacteriophages, viruses that infect bacteria, in order to defeat specific bacterial pathogens. Phage therapy is not an innovative idea, indeed, it was widely used around the world in the 1930s and 1940s, in order to treat various infection diseases, and it is still used in Eastern Europe and the former Soviet Union. Nevertheless, Western scientists mostly lost interest in further use and study of phage therapy and abandoned it after the discovery and the spread of antibiotics. The advancement of scientific knowledge of the last years, together with the encouraging results from recent animal studies using phages to treat bacterial infections, and above all the urgent need for novel and effective antimicrobials, have given a prompt for additional rigorous researches in this field. In particular, in the laboratory of synthetic biology of the department of Life Sciences at the University of Warwick, a novel approach was adopted, starting from the original concept of phage therapy, in order to study a concrete alternative to antibiotics. The innovative idea of the project consists in the development of experimental methodologies, which allow to engineer a programmable synthetic phage system using a combination of directed evolution, automation and microfluidics. The main aim is to make “the therapeutics of tomorrow individualized, specific, and self-regulated” (Jaramillo, 2015). In this context, one of the most important key points is the Bacteriophage Quantification. Therefore, in this research work, a mathematical model describing complex dynamics occurring in biological systems involving continuous growth of bacteriophages, modulated by the performance of the host organisms, was implemented as algorithms into a working software using MATLAB. The developed program is able to predict different unknown concentrations of phages much faster than the classical overnight Plaque Assay. What is more, it gives a meaning and an explanation to the obtained data, making inference about the parameter set of the model, that are representative of the bacteriophage-host interaction.

Target genes for virulence assessment of Escherichia coli isolates from water, food and the environment

The widespread species Escherichia coli includes a broad variety of different types, ranging from highly pathogenic strains causing worldwide outbreaks of severe disease to avirulent isolates which are part of the normal intestinal flora or which are well characterized and safe laboratory strains. The pathogenicity of a given E. coli strain is mainly determined by specific virulence factors which include adhesins, invasins, toxins and capsule. They are often organized in large genetic blocks either on the chromosome ('pathogenicity islands'), on large plasmids or on phages and can be transmitted horizontally between strains. In this review we summarize the current knowledge of the virulence attributes which determine the pathogenic potential of E. coli strains and the methodology available to assess the virulence of E. coli isolates. We also focus on a recently developed procedure based on a broad-range detection system for E. coli-specific virulence genes that makes it possible to determine the potential pathogenicity and its nature in E. coli strains from various sources. This makes it possible to determine the pathotype of E. coli strains in medical diagnostics, to assess the virulence and health risks of E. coli contaminating water, food and the environment and to study potential reservoirs of virulence genes which might contribute to the emergence of new forms of pathogenic E. coli.

Proceedings of the Twenty-First Annual Biochemical Engineering Symposium

The 21st Annual Biochemical Engineering Symposium was held at Colorado State University on April 20, 1991. The primary goals of this symposium series are to provide an opportunity for students to present and publish their research work and to promote informal discussions on biochemical engineering research. Contents High Density Fed-Batch Cultivation and Energy Metabolism of Bacillus thuringtensis; W.-M. Liu, V. Bihari, M. Starzak, and R.K. Bajpai Influences of Medium Composition and Cultivation Conditions on Recombinant Protein Production by Bacillus subtilis; K. Park, P.M. Linzmaier, and K.F. Reardon Characterization of a Foreign Gene Expression in a Recombinant T7 Expression System Infected with λ Phages; F. Miao and D.S. Kompala Simulation of an Enzymatic Membrane System with Forced Periodic Supply of Substrate; N. Nakaiwa, M. Yashima, L.T. Fan, and T. Ohmori Batch Extraction of Dilut Acids in a Hollow Fiber Module; D.G. O'Brien and C.E. Glatz Evaluation of a New Electrophoretic Device for Protein Purification; M.-J. Juang and R.G. Harrison Crossflow Microfiltration and Membrane Fouling for Yeast Cell Suspension; S. Redkar and R. Davis Interaction of MBP-β-Galactosidase Fusion Protein with Starch; L. Taladriz and Z. Nikolov Predicting the Solubility of Recombinant Proteins in Escherichia coli; D.L. Wilkinson and R.G. Harrison Evolution of a Phase-Separated, Gravity-Independent Bioractor; P.E. Villeneuve and E.H. Dunlop A Strategy for the Decontamination of Soils Containing Elevated Levels of PCP; S. Ghoshal, S. K. Banelji, and RK. Bajpai Practical Considerations for Implementation of a Field Scale In-Situ Bioremediation Project; J.P. McDonald, CA Baldwin, and L.E. Erickson Parametric Sensitivity Studies of Rhizopus oligosporus Solid Substrate Fermentation; J. Sargantanis, M.N. Karim, and V.G. Murphy, and RP. Tengerdy Production of Acetyl-Xylan Esterase from Aspergillus niger; M.R Samara and J.C. Linden Biological and Latex Particle Partitioning in Aqueous Two-Phase Systems; D.T.L. Hawker, RH. Davis, P.W. Todd, and R Lawson Novel Bioreactor /Separator for Microbial Desulfurization of Coal; H. Gecol, RH. Davis, and J .R Mattoon Effect of Plants and Trees on the Fate, Transport and Biodegradation of Contaminants in the Soil and Ground Water; W. Huang, E. Lee, J.F. Shimp, L.C. Davis, L.E. Erickson, and J.C. Tracy Sound Production by Interfacial Effects in Airlift Reactors; J. Hua, T.-Y. Yiin, LA Glasgow, and L.E. Erickson Soy Yogurt Fermentation of Rapid Hydration Hydrothermal Cooked Soy Milk; P. Tuitemwong, L.E. Erickson, and D.Y.C. Fung Influence of Carbon Source on Pentachlorophenol Degradation by Phanerochaete chrysosportum in Soil; C.-Y.M. Hsieh, RK. Bajpai, and S.K. Banerji Cellular Responses of Insect Cells Spodopiera frugiperda -9 to Hydrodynamic Stresses; P.L.-H. Yeh and RK. Bajpa1 A Mathematical Model for Ripening of Cheddar Cheese; J. Kim, M. Starzak, G.W. Preckshoi, and R.K. Bajpai

New aspects and strategies for methane mitigation from ruminants

The growing demand for sustainable animal production is compelling researchers to explore the potential approaches to reduce emissions of greenhouse gases from livestock that are mainly produced by enteric fermentation. Some potential solutions, for instance, the use of chemical inhibitors to reduce methanogenesis, are not feasible in routine use due to their toxicity to ruminants, inhibition of efficient rumen function or other transitory effects. Strategies, such as use of plant secondary metabolites and dietary manipulations have emerged to reduce the methane emission, but these still require extensive research before these can be recommended and deployed in the livestock industry sector. Furthermore, immunization vaccines for methanogens and phages are also under investigation for mitigation of enteric methanogenesis. The increasing knowledge of methanogenic diversity in rumen, DNA sequencing technologies and bioinformatics have paved the way for chemogenomic strategies by targeting methane producers. Chemogenomics will help in finding target enzymes and proteins, which will further assist in the screening of natural as well chemical inhibitors. The construction of a methanogenic gene catalogue through these approaches is an attainable objective. This will lead to understand the microbiome function, its relation with the host and feeds, and therefore, will form the basis of practically viable and eco-friendly methane mitigation approaches, while improving the ruminant productivity.

CTXφ immunity: Application in the development of cholera vaccines

CTXφ is a filamentous bacteriophage that encodes cholera toxin, the principal virulence factor of Vibrio cholerae. CTXφ is unusual among filamentous phages because it encodes a repressor and forms lysogens. CTXφ can infect the existing live-attenuated V. cholerae vaccine strains derived from either the El Tor or classical V. cholerae biotypes and result in vaccine reversion to toxinogenicity. Intraintestinal CTXφ transduction assays were used to demonstrate that El Tor biotype strains of V. cholerae are immune to infection with the El Tor-derived CTXφ, whereas classical strains are not. The El Tor CTXφ repressor, RstR, was sufficient to render classical strains immune to infection with the El Tor CTXφ. The DNA sequences of the classical and El Tor CTXφ repressors and their presumed cognate operators are highly diverged, whereas the sequences that surround this “immunity” region are nearly identical. Transcriptional fusion studies revealed that the El Tor RstR mediated repression of an El Tor rstA-lacZ fusion but did not repress a classical rstA-lacZ fusion. Likewise, the classical RstR only repressed a classical rstA-lacZ fusion. Thus, similar to the mechanistic basis for heteroimmunity among lambdoid phages, the specificity of CTXφ immunity is based on the divergence of the sequences of repressors and their operators. Expression of the El Tor rstR in either El Tor or classical live-attenuated V. cholerae vaccine strains effectively protected these vaccines from CTXφ infection. Introduction of rstR into V. cholerae vaccine strains should enhance their biosafety.

Sequence of a 189-kb segment of the chromosome of Rhodobacter capsulatus SB1003

Cosmids from the 1A3–1A10 region of the complete miniset were individually subcloned by using the vector M13 mp18. Sequences of each cosmid were assembled from about 400 DNA fragments generated from the ends of these phage subclones and merged into one 189-kb contig. About 160 ORFs identified by the CodonUse program were subjected to similarity searches. The biological functions of 80 ORFs could be assigned reliably by using the WIT and Magpie genome investigation tools. Eighty percent of these recognizable ORFs were organized in functional clusters, which simplified assignment decisions and increased the strength of the predictions. A set of 26 genes for cobalamin biosynthesis, genes for polyhydroxyalkanoic acid metabolism, DNA replication and recombination, and DNA gyrase were among those identified. Most of the ORFs lacking significant similarity with reference databases also were grouped. There are two large clusters of these ORFs, one located between 45 and 67 kb of the map, and the other between 150 and 183 kb. Nine of the loosely identified ORFs (of 15) of the first of these clusters match ORFs from phages or transposons. The other cluster also has four ORFs of possible phage origin.