Phage Selection Against Antibiotic Resistance or Virulence in Opportunistic Bacterial Pathogens

Bacteriophages (phages) are prolific, ubiquitous viruses that infect bacterial cells. Phages have proven to be invaluable tools of the biological sciences, enabling the discovery and description of some of the foundations of molecular biology and genetics; excellent systems for the study of evolutionary dynamics; and their seemingly limitless diversity promises to produce interesting new biology for many years to come. As natural predators of bacteria, phages might additionally be employed for phage therapy, the therapeutic use of phages to treat bacterial infections. Lytic phages impose strong selective pressure on bacteria to evolve resistance to phage infection. Under certain environmental conditions this selection pressure can result in the evolution of phage resistance at reduced fitness; this is an example evolutionary trade-off, or the evolution of a certain trait at the detriment of the fitness. In particular, it is possible that phage-imposed selection on bacterial surface expressed molecules may result in reduced antibiotic resistance and/or attenuated virulence of opportunistic bacterial pathogens. In this thesis, we will examine the idea of phage-imposed trade-offs on opportunistic bacterial pathogens. A published review paper serves as the first chapter and an introduction to phage biology and phage therapy. We present the history of the discovery of phages as well as the early reports of phage therapy. We highlight recent animal studies, case reports and clinical trials that have investigated the therapeutic use of phage. Finally, we discuss new approaches to phage therapy as well as identify interesting question and potential hurdles that will likely underscore a modern approach to phage therapy. In chapter 2, transposon insertional sequencing (INSeq) is used as a new high-throughput method to identify phage receptors to identify new phage that use antibiotic resistance or virulence factors as receptors. Preliminary experiments using characterized phages T2, T4, T6 and T7 demonstrate that the top results of INSeq screens with phage are involved in phage binding. These screens were extended to enable receptor identification for six newly isolated phages, R3, U115, EC35, EC14, 8S and P2. Adsorption assays and efficiency of plaquing assays to demonstrate phage binding and infection are used to validate the results of the INSeq screens with uncharacterized phage. In summary, this chapter establishes the use of INSeq screens as a high-throughput method to identify phage receptors which should allow for identification of phage that target virulence factors or molecules contributing to antibiotic resistance. Evolutionary dynamics between Pseudomonas aeruginosa and a lytic phage, OMKO1 that selects against antibiotic resistance, are examined in a short-term coevolution experiment in chapter 3. Time shift assays showed that coevolution of the three experimental populations followed arms race dynamic. Interestingly, only one experimental population demonstrated the predicted trade-off between phage resistance and antibiotic resistance. Whole genome sequencing of bacterial population allowed for identification of mutations underlying bacterial evolution and the trade-off. Results from this study demonstrate that evolution may not be as repeatable or predictable as previous experimental evolution studies have suggested. In chapter 4, we identify a new Shigella flexneri phage that selects for phage-resistant bacteria that are attenuated for virulence. We isolate and characterize a new Myoviridae phage, A1-1 which uses OmpA of S. flexneri as a receptor. Assays to interrogate membrane permeability including, live-dead staining, minimum inhibitory concentrations to various antibiotics and measurements of total lipopolysaccharide (LPS) quantity demonstrate that phage A1-1 selects for two different phenotypes of resistant mutants: OmpA deficient and altered LPS. Whole genome sequencing reveals that the five phage-resistant mutants examined in this study have mutations in either ompA or in genes involved in LPS biosynthesis genes. Using bacterial plaque assays, we show that all five resistant mutants are attenuated for intercellular spread, indicating that in the case of phage A1-1, phage resistance trades off with bacterial virulence. While the previous two chapter highlight phage selection resulting in a trade-off, the final chapter investigates a system in which phage selection results in a trade-up with antibiotic resistance. Tsx is a nucleoside porin in Escherichia coli which also serves as a receptor for phage T6, phage U115 and albicidin, a DNA gyrase inhibitor. We show that selection for resistance to any of these three antibacterials results in cross-resistance to the other two. Competition assays show resistance to these antibacterials does not result in a fitness cost relative to wild-type. In all 29 of the resistant mutants observed in this study, mutations in tsx were found. The trade-up between phage resistance and antibiotic resistance in this study highlights the need for rigorous studies of phage-bacteria interactions prior to deployment of phage therapy

TRA-932: PLANNING FOR GOODS MOVEMENT: ONTARIO\u27S FREIGHT-SUPPORTIVE GUIDELINES & OFF PEAK DELIVERIES PILOT

As communities grow and change it has become increasingly important to understand, plan and design for the movement of freight in order to maintain goods movement efficiency and the economic competitiveness of communities, while integrating and balancing the needs of other transportation system users and the compatibility of surrounding land uses. The Ontario Ministry of Transportation has developed Freight-Supportive Guidelines to assist municipalities, planners, engineers, developers and other practitioners in creating safe and efficient freight-supportive communities. The Guidelines provide land use planning, site design, road design and operational best practices, examples and implementation tools that are applicable to a wide range of communities and municipalities across Canada. Transportation demand management strategies can also be used to improve the efficiency of urban freight movement. During the Toronto 2015 Pan Am and Parapan Am Games, the Ontario Ministry of Transportation conducted a pilot to explore the potential of using off-peak deliveries as an urban freight transportation demand management strategy. The pilot allowed businesses and municipalities to explore the suitability and potential benefits and challenges of off-peak deliveries

Soft x-ray resonant magneto-optical Kerr effect as a depth-sensitive probe of magnetic heterogeneity: Its application to resolve helical spin structures using linear p polarization

We have calculated the soft x-ray resonant Kerr intensities as a function of the incident grazing angle of linearly p-polarized waves from the model spin structures, where the chirality (handedness) of the spin spirals (twist in depth) in a magnetic layer and the periodicity of a unit spiral are designed to vary. Variations in the chirality and the periodicity lead to noticeable changes in the Kerr intensity versus the grazing angle, which is due not only to a large sensitivity of the Kerr intensity of the linear p polarization to both the magnitude and direction of the transverse components of magnetizations, but also to a large dependence of the depth sensitivity on the grazing angle at the resonance regions. The measurement and analysis of the specular Kerr intensity are relatively straightforward in determining the inhomogeneous spin structures in depth, compared to those of the Kerr rotation and ellipticity. This is proven to be a convenient and useful probe to determine the handedness of spin spiral structures, as well as to resolve the detailed magnetic heterostructures in depth in ultrathin-layered films.open4

Atomic-scale depth selectivity of soft x-ray resonant Kerr effect

A study was performed to demonstrate that soft x-ray Kerr rotation, ??K, versus incident grazing angle, ??, and energy, hv, measurements provide an extremely large depth selectivity on the atomic scales even in an ultrathin single layer, simply by choosing appropriate ?? and hv around the resonant regions. Both the experimental and simulation results of ?? vs ??K measurements were considered for depth-varying magnetization reversals in a 3.5-nm-thick Co layer of NiFe/FeMn/Co/Pd films.open161

Soft x-ray resonant magneto-optical Kerr effect as a depth-sensitive probe of magnetic heterogeneity: A simulation approach

We report a noticeable depth sensitivity of soft x-ray resonant magneto-optical Kerr effect able to resolve depth-varying magnetic heterostructures in ultrathin multilayer films. For various models of depth-varying magnetization orientations in an ultrathin Co layer of realistic complex layered structures, we have calculated the Kerr rotation, ellipticity, intensity spectra versus grazing incidence angle ??, and their hysteresis loops at different values of ?? for various photon energies ?? 's near the Co resonance regions. It is found from the simulation results that the Kerr effect has a much improved depth sensitivity and that its sensitivity varies remarkably with ?? and ?? in the vicinity of the resonance regions. These properties originate from a rich variety of wave interference effects superimposed with noticeable features of the refractive and absorptive optical effects near the resonance regions. Consequently, these allow us to resolve depth-varying magnetizations and their reversals varying with depth in a single magnetic layer and allow us to distinguish interface magnetism from the bulk properties in multilayer films. In this paper, the depth sensitivity of the Kerr effect with an atomic-scale resolution is demonstrated and discussed in details in several manners with the help of model simulations for various depth-varying spin configurations.open9

El método didáctico como variable en los procesos de composición de los alumnos de duodécimo grado : estudio de casos

El titulo revela el problema que se investiga: como las estrategias utilizadas en el aula contribuyen o no al desarrollo de habitos y rutinas en los procesos de composicion de un grupo de alumnos de duodecimo grado en Puerto Rico. Existe una evidente preocupacion en la sociedad puertorriqueña por las limitaciones para la comunicacion escrita que demuestra un gran numero de estudiantes a su ingreso a las universidades del pais. Sin embargo, el problema de la enseñanza aprendizaje de la composicion en el vernaculo permanece sin investigar. Mediante este estudio de casos se ha explorado como los metodos didacticos de los profesores de todas las materias propician o inhiben el desarrollo de las habilidades necesarias para la composicion, desde el punto de vista de los alumnos. La metodologia empleada aborda la cuestion desde angulos y medios .

Wideband Reconfigurable Harmonically Tuned GaN SSPA for Cognitive Radios

The paper presents the architecture of a wideband reconfigurable harmonically-tuned Gallium Nitrate (GaN) Solid State Power Amplifier (SSPA) for cognitive radios. When interfaced with the physical layer of a cognitive communication system, this amplifier topology offers broadband high efficiency through the use of multiple tuned input/output matching networks. This feature enables the cognitive radio to reconfigure the operating frequency without sacrificing efficiency. This paper additionally presents as a proof-of-concept the design, fabrication, and test results for a GaN inverse class-F type amplifier operating at X-band (8.4 GHz) that achieves a maximum output power of 5.14-W, Power Added Efficiency (PAE) of 38.6, and Drain Efficiency (DE) of 48.9 under continuous wave (CW) operation

Soft X-ray resonant Kerr rotation measurement and simulation of element-resolved and interface-sensitive magnetization reversals in a NiFe/FeMn/Co trilayer structure

We report experimental observations of element- and buried interface-resolved magnetization reversals in an oppositely exchange-biased NiFeFeMnCo trilayer structure by soft x-ray resonant Kerr rotation measurements. Not only Co-, Ni-, Fe-specific exchange-biased loops but also interfacial uncompensated (UC) Fe reversal loops coupled to the individual Co and NiFe layers are separately observed. From the experimental results interpreted with the help of the model simulations of soft x-ray resonant Kerr rotation, the effective thicknesses of interfacial UC regions at the buried interfaces of both FeMnCo and NiFeFeMn are found to be tUC =13??2 A and 6??4 A, respectively. The depth sensitivity as well as element specificity of the x-ray resonant Kerr effect offer an elegant way into the investigations of element- and depth-resolved magnetization reversals of ferromagnetic ultrathin regions at buried interfaces in multicomponent multilayer films.open91

Multiband Reconfigurable Harmonically Tuned Gallium Nitride (GaN) Solid-State Power Amplifier (SSPA) for Cognitive Radios

The paper presents the architecture of a wideband reconfigurable harmonically-tuned Gallium Nitride (GaN) Solid State Power Amplifier (SSPA) for cognitive radios. When interfaced with the physical layer of a cognitive communication system, this amplifier topology offers broadband high efficiency through the use of multiple tuned input/output matching networks. This feature enables the cognitive radio to reconfigure the operating frequency without sacrificing efficiency. This paper additionally presents as a proof-of-concept the design, fabrication, and test results for a GaN inverse Class-F type amplifier operating at X-band (8.4 GHz) that achieves a maximum output power of 5.14-W, Power Added Efficiency (PAE) of 38.6 percent, and Drain Efficiency (DE) of 48.9 percent under continuous wave (CW) operation