Aim of this study was to select environmental E. coli isolates that produced colicins against the swine pathogen E. coli K88+. In initial evaluation using a modified plate method with 18 colicinogenic E. coli constructs, colicins E3, E4, E5, E9, Ia, K and N were found to possess inhibitory activity against 12 ETEC K88+ strains. A total of 463 environmental isolates from cattle rumen, cattle feces, pig feces and hog manure-amended soil were screened for colicin production by a modified plate test. Further, colicinogenic isolates were screened for five toxin genes LT, STa, STb, VT1 and VT2 as well as K88 (F4) fimbriae using PCR reactions. Fourteen non-pathogenic isolates were subjected to characterization of colicin genes by PCR using 9 new primer sequences, antibiotic susceptibilities and substrate utilization. Two potential probiotic strains of E. coli, UM-2 and UM-7 which produced colicins that could utilize potato starch and inulin were selected for in-vitro competition with E. coli K88+ strain 2-12. In vitro competition between the synbiotics and E. coli K88+ revealed inhibition of E. coli K88+. Based on the present in vitro studies it could be concluded that carefully selected potential synbiotics should be further studied for their role in protecting piglets from post-weaning diarrhea without antibiotics.

Aim of this study was to select environmental E. coli isolates that produced colicins against the swine pathogen E. coli K88+. In initial evaluation using a modified plate method with 18 colicinogenic E. coli constructs, colicins E3, E4, E5, E9, Ia, K and N were found to possess inhibitory activity against 12 ETEC K88+ strains. A total of 463 environmental isolates from cattle rumen, cattle feces, pig feces and hog manure-amended soil were screened for colicin production by a modified plate test. Further, colicinogenic isolates were screened for five toxin genes LT, STa, STb, VT1 and VT2 as well as K88 (F4) fimbriae using PCR reactions. Fourteen non-pathogenic isolates were subjected to characterization of colicin genes by PCR using 9 new primer sequences, antibiotic susceptibilities and substrate utilization. Two potential probiotic strains of E. coli, UM-2 and UM-7 which produced colicins that could utilize potato starch and inulin were selected for in-vitro competition with E. coli K88+ strain 2-12. In vitro competition between the synbiotics and E. coli K88+ revealed inhibition of E. coli K88+. Based on the present in vitro studies it could be concluded that carefully selected potential synbiotics should be further studied for their role in protecting piglets from post-weaning diarrhea without antibiotics.

The influence of the viable but non-culturable (VBNC) state on specific phenotypic traits of Escherichia coli O157:H7 as well as its transport behaviour in porous media was examined in this study. E.coli O157:H7 is a human pathogen capable of entering a VBNC state following exposure to sublethal stress. In the VBNC state, E.coli O157:H7 is not detectable by culture assays; yet, is able to retain its ability to cause human illness. This study examined specific transport-related properties of culturable and VBNC E.coli O157:H7 cells. As well, transport behaviors of the two cellular states were compared using sand-packed columns under steady-state flow. When E.coli O157:H7 cells entered a VBNC state, significant decreases in the hydrophobicity and lengths/widths of the cells, and a significant increase in extracellular polymeric substances on the cell surfaces were measured. Transport experiments indicated significantly (p<0.05) greater mass transport of VBNC cells through unwashed sand compared to culturable cells. This research contributes to the current knowledge describing VBNC E.coli O157:H7 cells, raises questions concerning the accuracy of culture-based E.coli O157:H7 identification protocols, and suggests that bacteria transport in the subsurface is a truly dynamic process. / Soil Science

The purpose of this thesis is to explore the interaction between antibiotics at sub-lethal doses, and E.coli. Initially we focussed on pairwise antibiotic interaction, and the potential to exploit these interactions to minimise antibiotic resistance. In testing the hypothesis that antagonism will slow adaptation by reducing selection for resistance we determined that there are conditions in which this fails to be the case. We furthermore caution against treating drug interactions as anything other than a dynamic property of the bacteria-drug interaction, by showing that the relationship between two drugs may be both synergistic and antagonistic depending on a variety of factors. Whilst exploring the adaptive response to drug combinations we discovered a highly unusual effect of Doxycycline to act as a growth stimulant to E.coli AG100. Chapter 3 and 4 are devoted to determining the nature and mechanism of this stimulation, and analysing any potential genomic changes using whole genome re-sequencing. Having shown that dose response is not always a monotone function of increasing drug dose, in chapter 5 we also look at the dose response in a diffusive context, using a custom built imaging system to show the common non-monotonicity of disk diffusion type assays, that manifest themselves as bullseye patterns of growth. We use a mathematical model to explore the ecological and adaptive reasons for such patterns. Finally in chapter 6 we look at the coevolutionary history of phage and E.coli REL606 strains, by determining trade-offs caused by lambda phage and the sole carbon source maltotriose both utilising the same porin (lamB) for cell entry.

In light of an urgent need for improved antimicrobial diagnostics and therapeutics, understanding bacterial behaviour, and bacterial responses to treatments in particular, is one of the key objectives of modern medical research. While the molecular mode of action of antibiotics is usually well known, their effect on the cell at a "systems" level (on the regulatory networks, metabolism, etc.) is only beginning to be quantitatively understood. We address some of these response phenotypes in Escherichia coli testing different antibiotic classes and growth conditions. We study the short (< 15 s) time-scale fluctuation dynamics of fluorescently-tagged chromosomal loci and cytosolic aggregates, which report for the state of locus ”compaction” and the levels of macromolecular crowding of the cytosol, respectively. We improve the precision of those measurements developing a novel data treatment procedure and discover that sub-lethal doses of ciprofloxacin, rifampicin, and vancomycin as well as hyperosmotic shock conditions cause small but consistent changes (unique to each treatment agent) to the physical organisation of chromosomal Ori2 and Ter3 loci and the cytosol. We reveal, among other findings, strong correlations between the effects in different parts of the chromosome and between the chromosome and cytosol. In addition, we complement the marker dynamics work with single-cell level gene expression measurements during sub-lethal translation inhibition. Specifically, we compare responses to tetracycline and chloramphenicol from constitutive and ribosomal promoters in Ori3 and Ter3 chromosomal positions over long (7 h) treatment times in exponentially growing bacteria. We reveal, for the first time, the kinetics of cellular resource allocation and provide novel insights on globally regulated transcription, relevant to the three-component proteome partitioning model, gene-length dependent effects of the processivity of translation, and ”reversibility” of ribosome-binding antibiotics. In addition, we discover a strong correlation between the timing of responses from promoters in the Ori3 and Ter3 positions, and a small but consistent difference in the response magnitude between the two positions.

Made available in DSpace on 2014-06-12T15:53:47Z (GMT). No. of bitstreams: 2 arquivo4868_1.pdf: 953580 bytes, checksum: 30f8d7e6ee8c034d2c9fd4e760ecf3e3 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2006 / As beta-lactamases de espectro estendido estão em expansão mundial, conferindo em isolados clínicos importantes fenótipos de multi-resistencia em bactérias que abrigam estas enzimas. Pacientes com infecção urinária (ITU) são principalmente infetados pelas E. coli e K. pneumoniae, que são as principais produtoras das ESBLs (beta-lactamases de espectro estendido). Neste trabalho, nós analisamos a presença de ESBL produzidas pela E. coli e K. pneumoniae em isolados de ITU de origem nosocomial e em comunidade. Padrões de suscetibilidade e a presença de genes de ESBL para SHV, TEM e CTX foram estudados em amostras de ESBL positivas. Os resultados mostraram que as cepas produtoras de ESBL estavam presentes em 25% das amostras de ITU nosocomiais e 9.3% nas de comunidade. As ESBLs esteve presente em 12.2 e 18.2% dos isolados de E. coli e de K.pneumoniae, respectivamente. Foram descobertos genes de ESBL em isolados de origem nosocomial e em comunidade. Todos os isolados que carregam os genes de ESBL apresentaram fenótipos de multi-resistência que alertam à necessidade de vigilância contínua nas ITU

In this work, electrophotocatalytic disinfection on selected microorganisms was verified. The electrophotocatalytic system allows the application of electrical bias to the photoanode coated with a titanium dioxide layer. The disinfecting effect was observed on E.coli and C.glabrata in aqueous solution. The effect of radiation intensity on electrophotocatalysis and selected optimal conditions for further experiments was observed in the E. coli organism. Photocatalytic disinfection was carried out under suitable conditions on C.glabrata yeast and the effect of sodium sulfate electrolyte on electrophotocatalytic disinfection was observed in this case.

No description available.

Coccidiosis is a costly parasitic disease to the poultry industry with multiple prevention methods being explored to control its impact. One approach under development is the use of -glucans, which are carbohydrates from cell walls of various plant species. The first study evaluated the feeding effects of algae- derived -glucans on performance and responses of broilers during a coccidiosis challenge. Cobb 500 broilers (n=1280) were fed a control diet, control supplemented with 150 g/MT Algamune (BG), 100 g/MT Algamune ZPC (BGZn), or 0.01% Salinomycin (Sal). On d 15, challenged birds received mixed Eimeria inoculum. Measurements were taken on d 7, 14, 21, and 28, and lesion scores assessed on d 21. The challenge resulted in reduced BW, and higher feed conversion ratio (FCR) was observed in the challenged birds with Sal and BGZn. Escherichia coli (E. coli) is normally commensal to the gastrointestinal tract, but certain serotypes cause disease in domestic poultry. A subsequent study was conducted to evaluate the feeding effects of algae-derived glucan (1,3 -glucan) on performance of broiler chickens during an E. coli challenge. Cobb 500 broilers (n=900) were fed a control diet, control + 25 mg/kg of -glucan, or control + 100 mg/kg of -glucan. On d 0, litter was sprayed with E. coli inoculum. Measurements were taken on d 7, 14, 21, and 28. -glucan supplementation increased BW gain andlowered FCR. The results from these studies offer some insight to the effects of -glucans on poultry and their potential to offset negative effects caused by infectious challenges. / Master of Science

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No. of bitstreams: 2 Dissertação - Kezia Gomes de Oliveira - 2016.pdf: 1770872 bytes, checksum: de67c9847e819d3256098ae1514be2d0 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-10-07 / Outro / The several advantages of miniaturization of DNA amplification and coupling with sample preparation and detection steps on the same chip are well known. Currently, most miniaturized systems for nucleic acids analysis are based on polymerase chain reaction (PCR). PCR amplification requires precise temperature control, switching between heating and cooling solution in three specific temperatures. Therefore, the adaptation of PCR for microchip is relatively complex and presents some limitations particularly for use in remote locations. Without the need for heating cycles, isothermal microsystems for DNA amplification can be designed to be simple and low energy consumption and hence can overlap the portable PCR detection systems. The loop-mediated isothermal amplification (LAMP) is a novel technique which has emerged as a simple and fast tool for DNA amplification which can be used for the detection and identification of several pathogens. The LAMP using Bst DNA polymerase enzyme which is an enzyme having strand displacement activity and uses a set of four primers designed from six individual segments of the sequence to be amplified. In this study, we developed a simple and rapid LAMP reaction for the E. coli malB gene amplification in the reaction was thermally controlled with a thermoblock for 60 minutes at 66 ° C. The PeT microdevices demonstrated compatibility with all reagents used in the LAMP and the success of the isothermal amplification was observed by agarose gel electrophoresis, yielding detectable amount amplicons as few as starting with 1 copy of DNA. Moreover, the success of the nucleic acid amplification reaction was evaluated by visual detection of the amplicons in the microchip by the use of fluorescent DNA intercalators, which yielded fluorescence in positive reactions. The LAMP in PeT microdevice is a simple and inexpensive method, that allowed a rapid detection (62 minutes) of E. coli. Because of simple operation and without the need for sophisticated instrumentation, LAMP held in microchip PeT has proven to be a valuable tool for molecular diagnostics, with great potential for applications in point-of-care. / As diversas vantagens da miniaturização das reações de amplificação de DNA e o acoplamento com as etapas de preparo da amostra e de detecção no mesmo chip já são bem conhecidas. Até o presente momento, a maioria dos sistemas miniaturizados para a análise de ácidos nucleicos são baseados na reação em cadeia da polimerase (PCR). A PCR necessita de controle preciso de temperatura, alternando entre aquecimento e resfriamento da solução em três temperaturas específicas. Desta forma, a adaptação da PCR em microchips é relativamente complexa apresentando algumas limitações relacionadas principalmente a utilização em lugares remotos. Sem a necessidade de ciclos de aquecimento, os microssistemas isotérmicos podem ser projetados para serem simples e de baixo consumo de energia e, portanto, pode sobrepor a PCR em sistemas de detecção portáteis. A amplificação isotérmica mediada por loop (LAMP) é uma técnica recente e inovadora que surgiu como uma ferramenta simples e rápida de amplificação de DNA que pode ser utilizada para detecção e identificação de diversos patógenos. A LAMP utiliza a enzima Bst DNA polimerase que é uma enzima com atividade de deslocamento de fita e utiliza um conjunto de quatro iniciadores desenhados a partir de seis segmentos específicos da sequência a ser amplificada. Neste trabalho foi desenvolvida uma metodologia simples e rápida para detecção de E.coli através da amplificação isotérmica do gene malB em dispositivos descartáveis de poliéster-toner (PeT) contendo um microcâmara com capacidade para 5 μL, e a reação foi incubada a 66 ºC em um termobloco por 60 minutos. Os microchips de PeT demonstraram compatibilidade com todos os reagentes utilizados na LAMP e o sucesso da amplificação isotérmica foi observado por eletroforese em gel de agarose, obtendo quantidade de amplicons detectáveis no gel em reações que partiram de 1 cópia de DNA. Além disso, o sucesso da reação de amplificação do ácido nucleico também foi avaliado através da detecção visual dos produtos amplificados no microchip através do uso de intercaladores fluorescentes de DNA, que produziram fluorescência nas reações positivas. A LAMP realizada em microdispositivos de PeT representa um método simples e de baixo custo, que permitiu a detecção rápida (62 minutos) da E.coli. Devido a simples operação, e sem a necessidade de instrumentação sofisticada, a LAMP realizada no microchip de PeT demonstrou ser uma ferramenta valiosa para diagnósticos moleculares, apresentando grande potencial para aplicações no point-of-care.