Memoria para optar al Título Profesional de Médico Veterinario / Staphylococcus spp, forman parte de la microbiota de piel y mucosas de caninos, pero son además patógenos oportunistas principalmente las especies S. intermedius y en menor medida S. schleiferi subesp. coagulans y S. aureus, produciendo pioderma y otitis externa. La terapia de este tipo de patologías por lo general es empírica y prolongada, pudiendo provocar resistencia antimicrobiana. El objetivo de este estudio fue caracterizar, a través de la detección de genes, las bases moleculares de resistencia a antimicrobianos como macrólidos (ermB), tetraciclinas (tetM), β-lactámicos (mecA) y detectar la mutación de los genes gyrA y grlA que determinan la resistencia a quinolonas. De un total de 170 cepas aisladas desde otitis externa (53) y pioderma (117), 1 cepa presentó resistencia fenotípica a oxacilina, 16 a tetraciclinas, 18 a macrólidos y 15 a quinolonas. La detección del gen mecA fue de un 100%, tetM un 81,25% (13) y ermB 94% (17). La mutación responsable de la resistencia en los genes gyrA y grlA fue detectada en el 100% de las cepas que se enviaron a secuenciar, encontrándose en la posición 84 en el caso de gyrA reemplazando el aminoácido serina por una leucina y en la posición 80 para grlA sustituyendo una serina por una isoleucina. Si bien la prevalencia de resistencia en Chile es aun menor en comparación a otros países, este es el primer estudio a nivel nacional que ayuda a conocer los mecanismos de resistencia en las bacterias de las mascotas, destacando además por primera vez en país, la presencia del gen mecA en Staphylococcus intermedius aislado de un canino / Proyecto MULT-O6/03-2

Memoria para optar al Título Profesional de Médico Veterinario / El presente trabajo corresponde a un estudio transversal que incluyó cepas de Staphylococcus spp. coagulasa positivo de gatos con diversas patologías dermatológicas atendidos en el Hospital Clínico Veterinario de la Universidad de Chile entre marzo a diciembre del año 2010. Se ingresaron 68 muestras de 68 pacientes con afecciones dermatológicas, obteniéndose 30 cepas (44%) de Staphylococcus spp. coagulasa positiva de 30 pacientes, las cuales se identificaron mediante el kit BBL Crystal TM para Gram positivas y se les realizó el estudio de sensibilidad antimicrobiana mediante el método de difusión en placa de Kirby-Bauer, según las normas de Clinical and Laboratory Standards Institute (CLSI, 2008). Los antimicrobianos en estudio fueron: oxacilina, amoxicilina con ácido clavulánico, ampicilina, clindamicina, eritromocina, cefadroxilo, doxiciclina, tetraciclina, sulfametoxazol-trimetoprim, vancomicina, ciprofloxacino y enrofloxacino. Staphylococcus intermedius fue la especie más frecuentemente aislada alcanzando el 67% (20 cepas), seguido por Staphylococcus aureus con un 33% (10 cepas). El 10% del total de cepas en estudio (3 cepas) fueron sensibles a todos los antimicrobianos utilizados, el 37% (11 cepas) fueron resistentes a un antimicrobiano y un 53% (16 cepas) fueron multirresistentes. El 13,3% del total de cepas (4 cepas) fueron resistentes a meticilina, siendo todas identificadas como S. intermedius, todas éstas fueron resistentes a fluoroquinolonas, lincosamidas, macrólidos y sulfonamidas. Ninguna cepa fue resistente a vancomicina / FIV 2009 Código: 12101401.9102.008

This thesis investigates aspects of the diversity, abundance and biology of the fish species inhabiting Mnjoli Dam, an irrigation dam that is located in the rural lowveld region of Swaziland. Specific objectives for this thesis were: to determine the fish species present and select three principal species based on their abundance; to investigate the relative abundance and distribution of the three selected species; and to describe key population parameters, such as growth, maturity, reproductive seasonality and mortality. The three dominant species were selected on their fisheries potential. These were Marcusenius pongolensis, Oreochromis mossambicus and Schilbe intermedius. Marcusenius pongolensis was the most abundant species in terms of both catch per unit effort and mass. Catch rates were shown to vary according to habitat type. Age and growth parameters were obtained from sectioned otoliths. Marginal zone analysis showed that annulus formation occurred in winter for all three species. The maximum-recorded ages were 8, 6 and 8 for M. pongolensis, O. mossambicus and S. intermedius, respectively. Growth for the three species was best described by the three parameter von Bertalanffy growth model as Lt = 1.88.67(1- exp⁻¹·⁴⁸⁽t⁺°·⁶⁷⁾) mm FL for M. pongolensis; Lt = 226.83(1- exp⁻°·⁴⁽t⁺²·°²⁾) mm TL for O. mossambicus and Lt = 214.59(1- exp⁻°·⁶°⁽t⁺¹·²°⁾) mm FL for S. intermedius. Sexual maturity was estimated for male and female M. pongolensis at 134 mm FL and 119 mm FL, respectively. Oreochromis mossambicus matured at 239 mm TL and S. intermedius at 205 mm FL. Two spawning peaks for M. pongolensis and S. intermedius were observed, one at the onset of summer (November) and the second at the beginning of autumn (February-March). Oreochromis mossambicus also exhibited two spawning peaks, one in spring (September) and the second in autumn (February –March). Mean mortality rate (Z), estimated using catch curve analysis was 0.77 year⁻¹ for M. pongolensis, 0.49 year⁻¹ for O. mossambicus and 0.79 year⁻¹ for S. intermedius. Natural mortality was assumed to be equal to Z since there is no fishery activity at the dam. Size specific selectivity curves were developed for each species. Of all the mesh sizes used, the 44 mm mesh size net had the highest catch rates in terms of numbers for M. pongolensis and S. intermedius. The 75mm mesh caught the highest catch rates for O. mossambicus. . The 44mm and 75 mm meshes seem to be the most suitable mesh sizes for harvesting the candidate species. It is concluded that no commercial fishery should be established on the dam, yet a small subsistence fishery could be a possible option. Management options such as input controls, open and closed seasons and areas and minimum sizes are discussed.

The Streptococcus Anginosus/Milleri Group (SMG) is made up of three closely related but distinct bacterial species: Streptococcus intermedius, Streptococcus constellatus, and Streptococcus anginosus. The SMG are recovered from about one-third of healthy, asymptomatic individuals. Despite this, the SMG cause more incidences of invasive streptococcal disease than Group A and Group B Streptococcus combined. Members of this group are somehow able to live a dual lifestyle. Little work has been conducted on the molecular pathogenicity of the SMG and host factors that contribute to host susceptibility to this group have been under-investigated. My research works towards discovering how the host recognizes the SMG as well as what enables the SMG to evade clearance by the immune system. I hypothesize that: 1) recognition of the SMG by toll-like receptor 2 (TLR2) plays a key role in triggering a cytokine response by the innate immune branch (which coordinates the immune response to the SMG), 2) the expression of cytolysins and extracellular polysaccharides by members of the SMG enables evasion of innate immune recognition and cytokine responses. hTLR2 reporter and monocyte-like cell lines as well as human blood samples from healthy donors were used to investigate the host factors that contribute to SMG infection. Five clinical reference SMG strains and a transposon mutant library were used to probe the contributing bacterial factors. It was found that TLR2 activation plays an important role in the cytokine response to the SMG, but there is heterogeneity between strains in their ability to activate TLR2. It was also found that intermedilysin expression by S. intermedius strains enables evasion of recognition; however, different hosts display varying susceptibility to this cytolysin. This study reveals that investigation of both host and microbial factors is essential to build an understanding of the mechanisms of SMG transition from commensalism to pathogenicity. / Thesis / Master of Science (MSc) / The Streptococcus Miller/Anginosus Group (SMG) is a group of bacteria comprised of three species. Members of this group are recovered from roughly one-third of healthy individuals. However, the SMG are also found in samples collected from patients with invasive disease. It is not well understood why some human-SMG relationships are pathogenic and others are not. However, it is likely that the combination of both human and SMG factors determine the nature of the relationship formed between the two. In this study, the human and SMG factors that contribute to infection were investigated. The ways by which human cells recognize members of the SMG and defend themselves from damage was explored. Additionally, SMG factors that potentially contribute to infection were probed to discover their effect on human cells. By investigating both the bacterial and host factors that lead to infection, disease treatments and preventative strategies can be tailored to individual cases.

<p>The Streptococcus Milleri/Anginosus group (SMG) includes Streptococcus anginosus, Streptococcus constellatus and Streptococcus intermedius. The SMG is found in healthy individuals but these bacteria are most known clinically for being associated with invasive disease and more recently, airway infections including cystic fibrosis (CF). The SMG like many other streptococci are naturally competent, being able to actively bind, uptake and integrate extracellular DNA. Competence regulation involves a competence-stimulating peptide (CSP) derived from the ComC precursor and a two- component signaling system (a histidine kinase ComD and its response regulator ComE). In this study, I examined the distribution of CSP/ComD sequences and competence in 170 SMG clinical isolates from CF airways and invasive disease. Five predicted CSP sequences were observed; one represented a newly predicted CSP and two arose from frameshift mutations in comC and appeared to be non-functional. The three CSPs fall into two functional groups that do not cross-activate due to receptor specificity. In addition, I observed that the Streptococcus constellatus subspecies pharyngis strains could not be transformed. However, I demonstrated that the pharyngis strains possess a functional ComCDE pathway, suggesting that the CSP regulates genes other than those involved in natural transformation. For many strains, I observed high endogenous competence levels that were only marginally induced by added peptide. These strains appear to be constitutively competent during exponential growth. The high basal level of expression and the heterogeneity in the SMG competence systems could impact how the SMG evolve during colonization and infections and specifically acquire antibiotic resistance and virulence genes.</p> / Master of Science (MSc)

Seed dispersal by caching rodents is a context-dependent mutualism in many systems. Plants benefit when seed remaining in shallow caches germinates before being eaten, often gaining protection from beetles and a favorable microsite in the process. Caching in highly unfavorable microsites, conversely, could undermine the dispersal benefit for the plant. Plant invasions could disrupt dispersal benefits of seed caching by attracting rodents to the protection of a dense invasive canopy which inhibits the establishment of native seedlings beneath it. To determine whether rodents disproportionately cache seed under the dense canopy of an invasive grass in southeastern Arizona, we used nontoxic fluorescent powder and ultraviolet light to locate caches of seed offered to rodents in the field. We fitted a general habitat-use model, which showed that disproportionate use of plant cover by caching rodents (principally Chaetodipus spp.) increased with moonlight. Across all moon phases, when rodents cached under plants, they cached under the invasive grass disproportionately to its relative cover. A greenhouse experiment showed that proximity to the invasive grass reduced the growth and survival of seedlings of a common native tree (Parkinsonia microphylla) whose seeds are dispersed by caching rodents. Biased dispersal of native seed to the base of an invasive grass could magnify the competitive effect of this grass on native plants, further reducing their recruitment and magnifying the effect of the invasion.

In this thesis, an analytical platform was designed and applied to various in vitro bacterial and eukaryotic cell cultures. An extraction and an analytical protocol were developed for comprehensive and simultaneous analysis of both lipid and polar metabolites for intra- and extracellular metabolomics using HILIC-LC-TOF-MS. This analytical platform was applied to four diverse research questions such as the effect of oxygen environment on growth, the interplay between gene expression and metabolism, metabolic changes that occur with age, and PAH toxicity. Specifically: (i) the effect of oxygen on the growth, physiology and metabolism of the Gram positive Streptococcus intermedius were investigated by comprehensive intra- and extracellular metabolomes and transcriptome. (ii) Metabolic insights into the role of the multipartite genome of the Gram negative bacteria Sinorhizobium meliloti and its metabolic preferences in a nutritionally complex environment. (iii) Age-associated metabolic dysregulation in murine bone marrow-derived macrophages during bacterial lipopolysaccharide-induced inflammation. (iv) Comprehensive intracellular metabolomic profiles of Sinorhizobium meliloti to sub-lethal exposure of individual or mixtures of polycyclic aromatic hydrocarbon revealed additive and dose-dependent effects. This thesis has demonstrated the versatility of the designed analytical platform and its use for diverse research in cell biology. / Thesis / Doctor of Philosophy (PhD)

The purposes of the present study were to resolve a number of major inconsistencies found in the literature on the structure of the quadriceps femoris muscle and to extend knowledge of its structure using descriptive, qualitative methodology. The quadriceps femoris muscle was investigated in 41 cats, and the findings were confirmed in 6 human cadavers. Two aponeuroses with major biomechanical functions (rectus-vastus and vastus aponeurosis), neither of which had been previously described in the literature, were characterized in both species. The study also resolved many major inconsistencies in the literature: The muscle sometimes described as vastus intermedius (VI) was found to be the articularis genu, the muscle sometimes described as vastus medialis (VM) was found to be the VI, the rectus femoris head was found to have an additional proximal nerve branch not previously recognized, no anomalous 5th head was ever found, and the distal VM were not found to have 2 heads (in either cats or humans). The authors’ anatomical descriptions and bimechanical models of the muscles, tendons, and neurovascular should provide a helpful foundation for future studies on the quadriceps. Two general recommendations are made: 1) that the feline model be considered a viable model to elucidate human knee pathomechanics; and 2) that regardless of the anatomical structure of interest, orthopedic nurses, orthopedic surgeons, and research investigators should routinely use the research literature for anatomical guidance instead of standard anatomical textbooks. © 2002, Sage Publications. All rights reserved.