Se efectuó un estudio en Lima – Perú, para determinar Aspectos clínicos epidemiológicos de infección urinaria en pacientes hospitalizados en el servicio de pediatría del Hospital María Auxiliadora entre 2011 a 2014. Objetivo: Determinar los aspectos epidemiológicos de la infección urinaria en pacientes hospitalizados en el servicio de pediatría, así mismo evaluar el campo clínico de la presentación, con el fin de ampliar el conocimiento para una mejor identificación, manejo. Materiales y Métodos: Fue un estudio observacional descriptivo transversal; realizado en el Hospital General María Auxiliadora del distrito de San Juan de Miraflores del departamento de Lima. La población conformada por 168 pacientes pediátricos, una muestra de 103 pacientes conforme a los criterios de inclusión y exclusión. Los datos se recolectaron mediante fichas de historia clínica que incluía datos epidemiológicos y clínicos de los pacientes pediátricos. Resultados: Las infecciones de tracto urinario se presentaron más frecuentemente entre el grupo de lactantes. Además se encontró el género femenino con mayor número de casos respecto al masculino. En los síntomas de ingreso de los pacientes, se halló la fiebre como el principal, el cual se presentó en más del 80% de los casos. Los días de estancia hospitalaria en promedio se encontraban en 5 días, pero oscilaban en un mínimo y máximo, entre 1 y 16 días respectivamente. Respecto a los urocultivo se halló que en más del 80 % de los casos, Escherichia Coli. En los años 2012 y 2014 se encontró al 100 %. El segundo germen en frecuencia fue klebsiella con 12.5 %. En la terapia antibiótica, el más usado fue amikacina.

Magíster en Ciencias de la Ingeniería, Mención Química / Ingeniero Civil en Biotecnología / Durante las últimas décadas, una rama de la investigación biotecnológica se ha enfocado en desarrollar y optimizar procesos de producción de proteínas recombinantes orientadas a aplicaciones terapéuticas. En particular, los péptidos terapéuticos ofrecen actualmente un nuevo potencial comercial para la industria farmacéutica y biotecnológica al generar estrategias efectivas en el tratamiento de diversas patologías como cáncer y alteraciones metabólicas entre otras. Estudios previos han demostrado la capacidad supresora de tumores de péptidos con blanco intracelular como p53p-Ant y PNC-27. Estos corresponden a regiones derivadas de la proteína p53, factor de regulación transcripcional que inhibe la progresión del ciclo celular e induce reparación celular o apoptosis, en caso de estrés genotóxico. Ambos péptidos contienen en su secuencia, un péptido de penetración celular, el cual les entrega la capacidad de atravesar la membrana plasmática. Así, bajo distintos mecanismos, con p53p-Ant y PNC-27 se ha reportado apoptosis o bien necrosis de manera selectiva en ciertos tipos de células tumorales. El presente trabajo tuvo por objetivo expresar en E. coli los péptidos p53p-Ant y PNC-27, y purificarlos mediante el sistema IMPACT. Éste es un mecanismo de expresión y purificación mediado por inteínas que permite purificar a través de una columna de afinidad, proteínas recombinantes con su secuencia nativa sin el uso de proteasas y minimizando pasos cromatográficos. Se logró producir ambos péptidos en forma soluble. La expresión soluble del péptido PNC-27 se consiguió a partir de los dos vectores de expresión proporcionados por el sistema IMPACT (pTXB1 y pTYB11), esta expresión resultó ser fuertemente dependiente de las condiciones de cultivo ya que se requiere de una inducción a baja temperatura (12 °C). Por su parte, la expresión soluble de p53p-Ant depende tanto del sistema de expresión como de las condiciones de cultivo. El diseño de las construcciones genéticas, en particular las que incluyen el vector de expresión pTYB11, permitió una efectiva purificación de los péptidos utilizando un único paso cromatográfico. Más aún, los niveles de rendimiento (0,4 1,2 mg L-1) superan los reportados para péptidos con el mismo sistema y los niveles de pureza obtenidos permitirían desarrollar investigación avanzada con ensayos biológicos in vivo o in vitro.

Objetivo: Evaluar la prueba del Ácido Glutámico Descarboxilasa (GAD) modificado para la rápida identificación de Escherichia coli aisladas de urocultivos. Métodos: Se procesó un total de 257 aislamientos bacterianos a partir de urocultivos procedentes del laboratorio de microbiología del Hospital San Bartolomé. Como controles internos, también se evaluaron aislamientos de diferentes muestras clínicas y cepas ATCC. Para obtener la sensibilidad, especificidad y la exactitud de la prueba GAD modificado, se utilizó la identificación bioquímica convencional como pruebaGold Standard. Resultados: Todas las Escherichia coli mostraron una reacción positiva a la prueba GAD modificado, incluyendo aislados lactosa negativa, dando una sensibilidad del 100%. Otras enterobacterias, que incluyeron a Salmonella grupo C, Morganella morganii, Enterobacter cloacae y Enterobacter aerogenes y Proteus vulgaris, dieron un resultado negativo a la prueba GAD modificado, aún cuando fueron incubadas por un tiempo adicional de 24 horas, dando una especificidad del 100%. Sin embargo, la prueba GAD modificado presentó reacción positiva con aislamientos de Shigella flexneri (2) y Shigella sonnei (2). Conclusiones: La prueba GAD modificado permite identificar a Escherichia coli a partir de aislamientos de urocultivos con alta sensibilidad, especificidad y exactitud, y puede ser utilizado en cualquier laboratorio porsu rapidez y su bajo costo. Palabras clave: Escherichia coli;GADmodificado;Glutamato Monosódico;Urocultivo. / --- Objetive: To evaluate the Glutamic Acid Decarboxylase (GAD) modified Testforrapid identification of Escherichia coli fromurine cultures. Methods: A total of 257 bacterial isolatesfrom urocultures were tested. As controls, bacterial isolates from other clinical samples and ATCC strains were also tested. In order to determinate the sensibility, specificity and the exactitude of the GAD test, the conventional biochemical identification for enterobacteria were used as Gold Standard. Results: All isolated strains of Escherichia coli were positive by themodifiedGAD test, even the lactose‐negative E. coli strains, giving a sensitivity of 100%. Other enterobacteria strains were negative by the modified GAD, even when the period of incubation was 24 hours, giving a specificity of 100%. However, Shigella flexneri and Shigella sonnei strains showed positive results when it was assayed on the modified GADtest. Conclusion: The modified GAD test can identify correctly any isolate of E. coli from urocultures with a high sensitivity and specificity, and is suitable for carrying out in any laboratory. Keywords: Escherichia coli; modified GAD test; Monosodium Glutamate; Urine culture.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

In this Thesis I present simulation- and theory-based studies of pattern formation and growth in collections of micro-organisms, in particular bacterial colonies. The aim of these studies is to introduce simple models of the 'micro-scale' behaviour of bacterial cells in order to study the emergent behaviour of large collections of them. To do this, computer simulations and theoretical techniques from statistical physics, and in particular non-equilibrium statistical physics, were used, as the systems under study are far from thermodynamic equilibrium, in common with most biological systems. Since the elements making up these sytems - the micro-organisms - are active, constantly transducing energy from their environment in order to move and grow, they can be viewed as `active matter' systems. First, I describe my work on a generalization of an archetypal model of active matter - the Vicsek model of flocking behaviour - in which the speed of motion of active particles depends on the local density of particles. Such an interaction had previously been shown to be responsible for some forms of pattern formation in bacterial colonies grown on agar plates in the laboratory. Simulations and theory demonstrated a variety of pattern formation in this system, and these results may be relevant to explaining behaviour observed in experiments done on collections of molecular motors and actin fibres. I then go on to describe work on modelling pattern formation and growth in bacterial biofilms - dense colonies of cells growing on top of solid surfaces. I introduce a simple simulation model for the growth of non-motile cells on a flat surface, whereby they move only by growing and pushing on each other as they grow. Such colonies have previously been observed experimentally to demonstrate a transition from round to 'branched' colonies, with a pattern similar to diffusion-limited aggregation. From these simulations and analytical modelling, a theory of the growth of such colonies is developed which is quite different from previous theories. For example, I find that the colony cannot grow at a constant speed if the cells are not compressible. Finally, I present some results on genetic drift and evolution in growing bacterial colonies. Genetic drift is greatly enhanced in colonies which are expanding in space, as only a few individuals at the edge of the population are able to pass on their genes onto their progeny. The individual-based simulations of biofilms described above are used to analyse which factors - such as the shape of the colony, the thickness of the growing layer of cells, and the interactions between the cells - affect the rate of genetic drift and the probability of fixation of beneficial mutations. This has implications, for example, for the evolution of antibiotic resistance in such colonies.

Les lésions iléales de patients atteints de maladie de Crohn (MC) sont anormalement colonisées par Escherichia coli adhérent et invasif (AIEC). Les mutations du gène NOD2 sont un facteur de risque pour la MC iléale et diminuent la clairance bactérienne. Le système immunitaire inné et la flore commensale sont indispensables au maintien de l’intégrité de la barrière intestinale. Le but de notre étude est d’évaluer l’impact d’un déséquilibre de la flore commensale induit par un traitement antibiotique sur la colonisation par AIEC chez des souris sauvages (WT) et NOD2 knock-out (NOD2KO), et les conséquences sur l'inflammation intestinale.MÉTHODE : Après un traitement antibiotique de 3 jours par voie orale, des souris WT et NOD2KO sont infectées avec 10^9 UFC d'AIEC (06362 ou LF82) une fois par jour pendant 2 jours. Les contrôles sont constitués de souris non infectées et de souris infectées sans traitement antibiotique. Les animaux sont sacrifiés à J1, J5 et J60 après l’infection par AIEC. En parallèle, des souris WT sont infectées par AIEC consécutivement à un traitement par dextran sodium sulfate (DSS) et sont sacrifiées 9 jours plus tard. Des échantillons de côlon, d’iléon, et tissus mésentériques sont prélevés pour 1) quantifier AIEC dans les muqueuses colique et iléale, 2) étudier la translocation bactérienne et 3) évaluer les signes d’inflammation histologiques et moléculaires. RÉSULTATS : Sans traitement antibiotique, AIEC n’est pas capable de coloniser les souris WT et NOD2KO. Comparé aux animaux non traités, le traitement antibiotique a permis une augmentation significative de la colonisation des flores adhérentes colique et iléale par AIEC chez les souris WT et NOD2KO. Une colonisation persistante par AIEC était encore observée à J5 uniquement chez les souris NOD2KO, mais n’était plus détectée à J60. La translocation mésentérique d’AIEC était observée uniquement chez les souris NOD2KO. De plus, on remarque une plus grande sensibilité des souris NOD2KO traitées par antibiotiques à la colonisation intestinale par des entérobactéries opportunistes potentiellement pathogènes. Aucun signe d’inflammation intestinale histologique ou moléculaire n’est observé chez les souris WT et NOD2KO traitées par antibiotiques et infectées par AIEC. Au cours de la colite induite par le DSS, une colonisation et une persistance d'AIEC ont été observés dans le côlon. De plus, une augmentation dramatique des paramètres clinique, histologique et moléculaire de la colite a été observé chez les souris infectées par AIEC mais pas chez les souris infectées par une souche d'E. coli commensale.CONCLUSION : Le traitement antibiotique était nécessaire à la colonisation de l'intestin et des tissus mésentériques par AIEC, et la persistance d'AIEC était dépendante de NOD2. AIEC a exacerbé une colite préexistante induite par le DSS chez les souris WT. / Ileal lesions of Crohn's disease (CD) patients are abnormally colonized by adherent-invasive Escherichia coli (AIEC). NOD2 gene mutations are risk factors for CD and impair bacterial clearance. Since innate immune system and commensal microbiota are critical to maintain intestinal barrier integrity, we evaluated the impact of commensal microbiota disruption induced by short term antibiotic treatment on AIEC colonization in wild type (WT) and NOD2 knock-out mice (NOD2KO), and the consequences on intestinal inflammation. Methods: After 3 days of oral antibiotic treatment, WT and NOD2KO mice were infected with 10^9 CFU AIEC once a day for 2 days. Controls are constitued by non infected mice and mice challenged with AIEC without antibiotic treatment. Animals were sacrificed 1, 5 and 60 days after AIEC administration. In parallel, mice were challenged with AIEC subsequent to a dextran sodium sulfate (DSS) treatment and sacrificed 9 days later. Ileum, colon, and mesenteric tissues were sampled for 1) AIEC quantification in ileal and colonic tissues, 2) bacterial translocation, and 3) evaluation of intestinal inflammation. Results: Without antibiotic treatment, AIEC was not able to colonize WT and NOD2KO mice. Compared with non-treated animals, antibiotic treatment led to a significant increase in ileal and colonic adherent AIEC colonization in both WT and NOD2KO mice. Persistent AIEC colonization was still observed until day 5 only in NOD2KO mice, disappearing at day 60. Mesenteric translocation of AIEC was observed only in NOD2KO mice. Morover, NOD2KO mice treated with antibiotics were more colonized by opportunistic enterobacteria. No inflammation was observed in WT and NOD2KO mice treated with antibiotics and infected with AIEC. During DSS-induced colitis, colonization and persistence of AIEC was observed in the colon. Moreover, a dramatic increase in clinical, histological, and molecular parameters of colitis was observed in mice infected with AIEC but not with a commensal E. coli strain.Conclusion: Antibiotic treatment was necessary for AIEC colonization of the gut and mesenteric tissues and persistence of AIEC was dependent on NOD2. AIEC exacerbated a preexisting DSS-induced colitis in WT mice.

Identifica grupos filogenéticos de Escherichia coli aislados a partir de hisopados rectales de neonatos alpacas (Vicugna pacos) con diarrea. El muestreo se realiza con hisopos estériles y es transportado en medio Stuard, el aislamiento en agar Mc Conkey y la identificación por pruebas bioquímicas convencionales. E. coli es aislada de 119 muestras de un total 150 colectadas. La determinación de los grupos filogenéticos se realiza mediante la utilización del método de PCR triplex descrito por Clermont et al. (2000), para clasificar a E. coli en 4 grupos filogenéticos A, B1, B2 y D, basándose en 3 marcadores genéticos chuA, yjaA y TSPE4.C2. Determina que los grupos filogenéticos B1 y D tienen mayor frecuencia con 65.55% y 19.33 %, respectivamente; el grupo filogenético menos frecuente es el B2 con 1.68%. Cabe resaltar que esta es la primera investigación realizada en el Perú para agrupar cepas patógenas de E. coli basado en la agrupación filogenética.

Doctor of Philosophy / Department of Animal Sciences and Industry / J. S. Drouillard / The first chapter of this dissertation reviews factors affecting E. coli O157:H7 prevalence in the gastrointestinal tracts of cattle. Chapter 2 assessed E. coli O157:H7 ability to use bovine intestinal mucus and its constituents as substrates for growth in vitro in the presence and absence of fecal inoculum and exogenous enzymes. Whole mucus supported the greatest pathogen growth (P < 0.05), but all components tested were able to sustain E. coli growth. Chapter 3 evaluated the impact of crude glycerin feeding on E. coli O157 fecal shedding by cattle fed growing and finishing feedlot diets with corn or a combination of corn, distiller’s grains, and soybean hulls. Increasing levels of crude glycerin decreased incidence of E. coli O157 in growing cattle (linear effect, P < 0.01) and tended to do so in finishing cattle fed corn-based diets (P < 0.06). No effect of glycerin was observed in finishing cattle fed the byproduct-based diets (P > 0.05), highlighting potential for glycerin use as a means for controlling fecal prevalence of E. coli O157 in cattle fed conventional grain-based diets. Chapter 4 evaluated transportation and lairage effects on fecal shedding of E. coli in feedlot cattle by mimicking transport to the abattoir. Shedding patterns were influenced by transportation, with significantly lower E. coli O157 prevalence in transported animals 4 hours after transit (P < 0.05). Additional post-transit samplings are, however, needed to confirm effects of transport stress on pathogen prevalence and shedding patterns. The experiment summarized in chapter 5 evaluated the potential for utilizing fecal long-chain fatty acid (LCFA) profiles as an indicator of E. coli O157 status. Out of 39 LCFA evaluated, only eicosapentaenoic acid (EPA) concentration was associated with presence of the pathogen (P < 0.02). The final chapter assessed the impact of dietary menthol, up to 0.3% of diet DM, on antimicrobial resistance in commensal E. coli. Menthol addition affected prevalence of tetracycline resistant E. coli, but contrary to our hypothesis, increased their occurrence after 30 days of treatment (P < 0.006). No hypothesis on mechanism responsible for this increase could be made from the present study.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Daniel U. Thomson / A total of 284,300 animals from 10 commercial feedyards in Nebraska and Colorado were used to evaluate the effectiveness of a siderophore-receptor/porin protein-based vaccine under commercial feedlot conditions. Individual feedlots were assigned to 1 of 2 treatments: 1) all incoming cattle injected with 2 ml of SRP E. coli O157:H7 vaccine subcutaneously at arrival and at time of re-implant (VAC) or 2) all incoming cattle were not vaccinated, and were used as negative controls (CON). Twenty freshly voided fecal samples were taken from 5 pen floors of market ready cattle at each feedyard once a month during May, June, July, and August of 2010. Pre-harvest blood samples were collected on 3 occasions throughout the summer (June, July, and August). For each sampling month, 1 lot of 5 animals representing each feedyard was sampled. Fecal and blood samples were shipped to Epitopix, LLC for subsequent microbiology and anti-SRP antibody testing. Samples were coded such that laboratory personnel were blinded to the location and treatment of samples. Cattle receiving VAC treatment had reduced prevalence of E.coli O157:H7 in their feces relative to the E. coli O157:H7 prevalence in the feces of CON cattle (12.83% vs. 20.25% for VAC and CON, respectively; P = 0.07). Anti-SRP antibody titer was higher in the serum from VAC cattle relative to the SRP titer levels in serum obtained from CON cattle (0.622 and 0.075 for VAC and CON, respectively; P < 0.001). These data suggest that vaccination of feedlot cattle with SRP upon arrival at the feedlot and again 70-100 days pre-harvest reduces shedding of E. coli O157:H7.

Escherichia coli (E. coli) is a universally accepted indicator of faecal pollution in water, and can be divided into several groups of which one group would be commensal E. coli (generally the indicator organism), and five diarrhoeagenic E. coli types. Pathogenic E. coli offers attractive possibilities to model the pathogenicity of polluted water that people drink. To date, techniques used for the detection of these pathogens include standard culturing techniques, the use of molecular probes directed against known pathogens and polymerase chain reaction (PCR) for the amplification of known virulence genes, usually after a pre-culturing step. There is however a need to speed up the process and to accurately quantify (for risk purposes) the type and number of E. coli (commensal and pathogenic) present in any given water sample. The use of more recently developed techniques such as real-time PCR and competitive PCR (c-PCR) offers us a way of quantifying the target organism and has been successfully applied in various parts of the world. The aim of this study was to adapt and use developed methodologies that employs both c-PCR and multiplex PCR (m-PCR) to quantify total E. coli, detect and distinguish between the diarrhoeagenic E. coli patho-types present in selected South African water samples without prior culture or enrichment. Using E. coli as a model pathogen, a technique was developed for the concentration of bacteria from water samples, isolation of DNA from bacteria and performing PCR’s on the extracted DNA. Three m-PCR’s were developed directed towards the housekeeping (Mdh) and virulence (eaeA, Stx1, Stx2, ST, LT, Ial and Eagg) genes associated with entero-pathogenic E. coli. c-PCR was performed using the Mdh housekeeping gene, with our modified version of the PCR product used as competitor DNA. Results obtained lead to a protocol consisting of the filtration of 100 mℓ environmental water, DNA extraction directly from the membranes, followed by quantitative c-PCR to screen for PCR inhibition as well as to quantify isolated DNA, thereafter screening of the DNA for the presence of virulence genes with m-PCR. Initial testing with known pathogens showed that this methodology was a viable option. DNA could be recovered from the filters, yielding PCR-ready templates. A total of 49 water samples were collected, these samples included household containers from rural areas, river water from three provinces in South Africa and wastewater from 4 different wastewater treatment plants around Johannesburg (Gauteng province). These water samples were subjected to the above-mentioned protocol with 100 % (49/49) of the samples testing positive for presence of the E. coli Mdh house keeping gene. Of the samples tested, 57 % (28/49) tested positive for EAEC, 0 % (0/49) tested positive for EIEC, 92 % (45/49) tested positive for ETEC, 2 % (1/49) tested positive for atypical EHEC and 0 % (0/49) tested positive for atypical EPEC. 38 percent of the water samples could be successfully quantified by c-PCR and were able to detect as low as 3 cells/mℓ. However, the remaining 62 percent DNA samples (isolated from water samples) was diluted to overcome PCR inhibition but failed to be quantified by c-PCR because the level of target DNA was too low to detect which allowed over competition of the Mdh competitor DNA. In conclusion, this method could successfully isolate E. coli DNA from various water samples. The isolated DNA could be used as PCR template and PCR inhibition could be overcome in all the samples by either diluting the sample or adding PCR facilitators. The PCR was able to amplify low levels of isolated E. coli DNA from most of the water samples and the presence of pathogens could be detected in the water with the m-PCR. Molecular quantification could be used to quantify DNA isolated from the water samples, but one limitation is the detection of very low numbers of E. coli DNA due to the nature of c-PCR, i.e. co-amplification of the target and competitor DNA. / Prof. Paul Jagals Dr. T.G. Barnard Mr. K. Maclean