Kolistin är ett gammalt polypeptidantibiotikum och används som sista utväg för behandling av allvarliga infektioner orsakad av multiresistenta gramnegativa bakterier. Nya studier har påvisat kolistinresistensgener, mcr (mobil colistin resistance), hos extended spectrum beta-lactamase (ESBL)-producerande Enterobacteriaceae. Mcr-genen ligger i plasmider som kan överföras mellan bakterier, vilket innebär att det är mycket svårare att behandla människor och djur vid infektion orsakad av patogen som erhållit denna resistens. Syfte med detta projekt var att utveckla en PCR-baserad metod för detektion av mcr-1 och mcr-3 gener. I denna studie optimerades en PCR-metod och sedan screenades 60 ESBL-isolat från Kristianstads vattenrike. Därefter undersöktes PCR-produkt med hjälp av agarosgelselektrofores. Resultatet visade att 51oC är den optimala annealingtemperaturen vid multiplex-PCR för detektion av mcr-1 och mcr-3. Ingen av mcr-generna kunde detekteras i de 60 ESBL-isolaten. Positiva kontrollstammar med specifika primers kunde detekteras i PCR-analyser som genomfördes i denna studie, vilket tyder på att den optimerade PCR metoden kan vara pålitlig för detektion av mcr-1 och mcr-3 generna.

Metabolic differences in the strategies used for pyrimidine base and nucleoside salvage were studied in the pseudomonads and enteric bacteria. Fluoro--analogs were used to select mutant strains of E. coli, S. typhimurium, P. putida, and P. aeruginosa blocked in one or more of the uracil and uridine salvage enzymes. HPLC analysis of cell-free extracts from wild-type and mutant strains examined the effectiveness of the selections. Evidence was found for cytidine kinase in Pseudomonas and for an activity that converted uracil compounds to cytosine compounds. Using media supplemented with 150 μg of orotic acid per ml, P. putida SOC 1, a Pyr, upp mutant which utilizes orotic acid as a pyrimidine source was isolated for the first time in any study.

This investigation has for its aim the determination, in a measure, of the degree of personal sanitation existent among students at North Texas State Teachers College, Denton, Texas.

Unidad de práctica para optar al título de Químico Farmacéutico / Autor no autoriza el acceso a texto completo de su documento / El propósito de este trabajo fue evaluar la capacidad del método de filtración por membrana en la determinación de coliformes totales y fecales utilizando un medio de cultivo “Chromocult® Coliform Agar ES diluido”, en muestras de agua potable a las cuales previamente se les neutralizó el cloro residual y fueron fortificadas con cepas de Escherichia coli (coliforme fecal), Enterobacter aerogenes (coliforme total) y Pseudomonas aeruginosa (no coliforme) en distintas diluciones; y de esta forma, poder validar su utilidad en los análisis de agua realizados por la sección Ecología y Medio Ambiente del Laboratorio de Criminalística (LACRIM) de la Policía de Investigaciones de Chile (PDI). Para llevar a cabo este proceso, se prepararon los inóculos de las cepas antes mencionadas para la realización de las pruebas estandarizadas, realizando diluciones hasta obtener un recuento cercano a 100 UFC (unidades formadoras de colonias) por placa, en el medio de cultivo estudiado y un medio de cultivo de referencia (Plate Count Agar), tanto para las cepas puras (siembras simples), así como para mezclas de ellas (cepas combinadas). Se determinó el porcentaje de recuperación del medio de prueba respecto del medio de cultivo de referencia no selectivo, obteniéndose porcentajes de 90,43% para E. coli; 102,94% para E. aerogenes y 0,78% para P. aeruginosa, valores que cumplen con los requisitos de la promoción de crecimiento para medios de cultivo en el rango de 50 y 200% para las cepas coliformes, así como una inhibición del crecimiento de la cepa no coliforme. Se determinó además, la robustez de la prueba, en términos de la concentración de microorganismos por inóculo puro y con cepas combinadas, no demostrando diferencias significativas entre ellas. Se concluyó que el método estudiado es de utilidad para el LACRIM, para su utilización como prueba de rutina en la determinación de coliformes totales y fecales en agua potable, siendo validado y aprobado internamente a través del presente trabajo. Finalmente, para ampliar las posibilidades de uso de la prueba, se recomienda validar el método de filtración por membrana con el medio Chromocult® ES diluido en matrices distintas, tales como aguas superficiales y agua de mar; además de la utilización de otras cepas, tanto coliformes totales, fecales y no coliformes; con el propósito de confirmar la utilidad del método en estas matrices y así como su robustez frente a una mayor variedad de cepas bacterianas

As antibiotic resistant bacterial strains are becoming more prevalent in healthcare settings, it is necessary to find alternative methods of detecting and treating these infections. One of the antibiotic resistant strains of interest is the carbapenem-resistant Enterobacteriaceae (CRE). CREs have the ability to evade some of the most potent antibiotics currently in use and employ carbapenemases to negate the effect of antibiotics. The three most common carbapenemase genes, found in carbapenem-resistant Enterobacteriaceae along with a gene found only in Escherichia coli were chosen to create a qPCR assay for rapid detection of resistant infections. The carbapenemase genes are KPC, VIM and NDM and the E. coli gene is uidA, a β-glucuronidase gene. Consensus sequences were obtained from each of the genes to account for the many variants of each gene. We were able to triplex the assay and test it against a library for twenty isolates varying by which gene they contain. Additional research has been conducted on the library of carbapenem-resistant Enterobacteriaceae using bacteriophages or phage. The Phage Hunters class isolated and identified twenty phage that infect K. pneumoniae. Out of the twenty phage, seven phage were able to effectively infect carbapenem-resistant K. pneumoniae.

The intestinal tract of virtually any metazoan, including mammals, is colonized with a complex microbial community to which we refer as intestinal or gut microbiota. One of the roles of the healthy intestinal microbiota is to protect the host against gut colonization with pathogenic bacteria through a phenomenon known as colonization resistance (CR). Dysbiosis of the intestinal microbiota, usually as a result of an antibiotic treatment, may lead to the disruption of the CR, and subsequent colonization with bacterial pathogens. However, and despite the importance, the role of the microbiota dysbiosis on the gut colonization by many bacterial pathogens, such as multidrug resistant Enterobacteriaceae, has not been elucidated: the members of the microbiota that confer CR and factors that promote colonization remain mostly unknown. The general aim of this thesis has been to improve the understanding of the role of the microbiota dysbiosis in gut colonization by bacterial pathogens. For this purpose, 3 projects have been established. In the first project we tried to elucidate the role of the microbiota dysbiosis on colonization by multidrug resistant Enterobacteriaceae (MRE) in mice. In the second project we investigated the risk factors and members of the microbiota associated with the MRE colonization in hospitalized patients. MRE infections represent a great threat for hospitalized patients. Specifically, acute leukemia patients are often colonized with MRE, probably due to the impaired CR as a result of intensive antibiotic treatments these patients receive. In the third project we studied the role of the microbiota dysbiosis on the development of Epizootic Rabbit Enteropathy (ERE). ERE is a severe gastrointestinal disease with a high percentage of mortality that occurs in young rabbits during first weeks post-weaning. ERE rabbits have been shown to suffer microbiota dysbiosis during the development of the disease. Moreover, the disease could be reproduced by contact between healthy and sick animals and by administration of cecal contents from ERE rabbits to healthy rabbits, suggesting that a pathogenic agent may be involved in the development of this intestinal pathology, although no causative agent has been identified until now. / El tracto intestinal de prácticamente cualquier metazoo, incluidos los mamíferos, está colonizado por una compleja comunidad microbiana a la que nos referimos como microbiota intestinal. Uno de los papeles de la microbiota intestinal es proteger al huésped contra la colonización intestinal con bacterias patógenas a través de un fenómeno conocido como resistencia a la colonización (RC). La disbiosis de la microbiota intestinal, a menudo como resultado de un tratamiento antibiótico, puede conducir a la alteración de la RC y posterior colonización por patógenos bacterianos. Sin embargo, y pese a su importancia, el papel de la disbiosis de la microbiota en la colonización intestinal por muchos patógenos bacterianos, como son las Enterobacterias multirresistentes, no se ha esclarecido: los miembros de la microbiota que confieren RC y los factores que promueven la colonización siguen siendo desconocidos. El objetivo general de esta tesis ha sido mejorar la comprensión del papel de disbiosis de la microbiota en la colonización intestinal por patógenos bacterianos. Para ello se han establecido tres proyectos. En el primer proyecto investigamos el papel de disbiosis de la microbiota intestinal en la colonización por Enterobacterias multiresistentes (MRE) en ratones. En el segundo proyecto investigamos los factores de riesgo y los miembros de la microbiota asociados con la colonización por MRE en pacientes hospitalizados. Las infecciones por MRE representan una gran amenaza para los pacientes hospitalizados. Específicamente, MRE a menudo colonizan los pacientes con leucemia aguda, probablemente debido a que la RC está alterada como resultado de tratamientos antibióticos intensivos recibidos por estos pacientes. En el tercer proyecto investigamos el papel de la disbiosis microbiana en desarollo de Enteropatía Epizoótica de Conejo (ERE). ERE es una enfermedad gastrointestinal severa con un alto porcentaje de mortalidad que ocurre en conejos jóvenes durante las primeras semanas después del destete. Se ha demostrado que los conejos con ERE sufren disbiosis microbiana después del inicio de la enfermedad, aunque no está claro el papel de la disbiosis en el desarollo de la enfermedad. Además, la enfermedad puede ser reproducida por contacto entre animales sanos y enfermos y por la administración del contenido cecal de conejos con ERE a conejos sanos, lo que sugiere que un agente patógeno podría estar implicado en el desarrollo de esta patología intestinal, aunque hasta ahora no se ha logrado identificar ningún agente causal. / El tracte intestinal de pràcticament qualsevol metazoo, inclosos els mamífers, està colonitzat per una complexa comunitat microbiana a la qual ens referim com microbiota intestinal. Un dels papers de la microbiota intestinal és protegir a l'hoste contra la colonització intestinal amb bacteris patògens a través d'un fenomen conegut com a resistència a la colonització (RC). La disbiosis de la microbiota intestinal, frecuentment com a resultat d'un tractament antibiòtic, pot conduir a l'alteració de la RC i posterior colonització per patògens bacterians. No obstant això, i malgrat la seva importància, el paper de la disbiosis de la microbiota en la colonització intestinal per molts patògens bacterians, com són les Enterobacteries multirresistentes, no s'ha esclarit: els membres de la microbiota que confereixen RC i els factors que promouen la colonització segueixen sent desconeguts. L'objectiu general d'aquesta tesi ha estat millorar la comprensió del paper de la disbiosis de la microbiota en la colonització intestinal per patògens bacterians. Per a això s'han establert tres projectes. En el primer projecte vam investigar el paper de la disbiosis de la microbiota intestinal en la colonització per Enterobacteries multiresistentes (MRE) en ratolins. En el segon projecte, investiguem els factors de risc i els membres de la microbiota associats amb la colonització per MRE en pacients hospitalitzats. Les infeccions per MRE representen una gran amenaça per als pacients hospitalitzats. Específicament, MRE sovint colonitza els pacients amb leucèmia aguda, probablement a causa de que la RC està alterada com a resultat de tractaments antibiòtics intensius rebuts per aquests pacients. En el tercer projecte, vam investigar el paper de la disbiosis microbiana en desenvolupament de l'Enteropatía Epizoótica de Conill (ERE). ERE és una malaltia gastrointestinal severa amb un alt percentatge de mortalitat que ocorre en conills joves durant les primeres setmanes després del deslleti. S'ha demostrat que els conills amb ERE sofreixen disbiosis microbiana després de l'inici de la malaltia, encara que no és clar el paper de la disbiosis en el desenvolupament de la malaltia. A més, la malaltia pot ser reproduïda per contacte entre animals sans i malalts i per l'administració del contingut cecal de conills amb ERE a conills sans, la qual cosa suggereix que un agent patogen podria estar implicat en el desenvolupament d'aquesta patologia intestinal, encara que fins ara no s'ha aconseguit identificar cap agent causal. / Djukovic, A. (2017). Role of intestinal dysbiosis on gut colonization by bacterial pathogens [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/90415 / TESIS

No description available.

Meloiodgyne incognita, one of the most aggressive plant parasitic nematodes species on potato in South Africa, belongs to a group of plant parasitic nematodes commonly known as root knot nematodes (RKN). This group of nematodes is widely distributed throughout the world. Meloidogyne spp. cause major economic losses to important crops such as potato and therefore decrease their market value in many countries across the world. The second stage juveniles are the only mobile and infective phase of the root knot nematode. As they infect host roots, they create wounds that can be used by other plant pathogens to penetrate the host in large numbers. The most effective management strategy for root knot nematodes is the use of nematicides such as Temik and Methyl bromide. However, these have been banned due to adverse on the environment. Therefore, Meloidogyne spp. will inevitably become a big problem in the potato industry of many countries due to the lack of effective alternatives to banned chemicals. Pectobacterium carotovorum subsp. brasiliensis (Pcb) is one of the most important soft rot-causing agents in South Africa. This pathogen belongs to a group of pathogens commonly known as soft rot Enterobacteriaceae (SRE). Bacteria belonging to this group of pathogens are known to cause soft rot and blackleg diseases on potato and other crops. Pcb is known as an opportunistic pathogen that can only penetrate host root tissue through natural openings or wounds that result from a variety of agents. Post penetration, the bacteria will increase in number and cause soft rot and blackleg. As rotting plant tissue disintegrates the bacteria escapes into the soil where it serves as inoculum and can infect healthy hosts. Many interactions have been documented between Meloidogyne spp. and other plant pathogens but to our knowledge there are no interactions that have been reported between Meloidogyne spp. and Pectobacterium spp. Considering the life cycles of RKN and SRE, we hypothesised that there could be an interaction between the two pathogen groups. Since both RKN and SRE are potato pathogens, they share the same space in the rhizosphere. This likely can lead to synergies and complex formation between the two pathogens. Likely, the wounds created by RKN J2s as they penetrate plant tissue can potentially be used by opportunistic Pcb to infect various hosts. It is from these identified overlaps that the first part of this study focused on investigating the potential interaction between M. incognita and Pcb. The first objective was to determine whether Pcb can attach onto M. incognita J2s and, if this was the case, to determine whether the J2s can disseminate the bacteria as they move around in the environment. The second objective was to determine whether there is a synergistic interaction between RKN and SRE and the combined effect of the two pathogens on their host Solanum tuberosum cv Mondial. The results obtained in the first part of the study strongly suggested that Pcb can attach onto M. incognita J2s and can be disseminated as the J2s move around in the environment. Thus, this indicated that there is a synergistic interaction between M. incognita and Pcb as there was increased disease severity and incidence in plants inoculated with both pathogens compared to those inoculated with individual pathogens. Significantly higher Pcb concentrations were found in plants inoculated with both pathogens. There was no breakage of tolerance to Pcb-caused blackleg on an otherwise resistant cultivar, BP1. The second aim of this study was to determine whether the induction of natural resistance using environmentally friendly resistance inducing chemicals can potentially be used as an alternative to chemical control. To this end, the effect of three inducers at different concentrations, amongst DL-β-aminobutyric acid, Acibenzolar-s-methyl and Messenger on potato plants infected with RKN was compared. The most effective resistance inducer amongst the three was 20mM BABA as it was able to reduce the number of J2s that penetrated host tissue, the number of females in the roots and the rate of egg production. Furthermore, the galling index observed in potato roots was significantly lower when plants were treated with 20mM BABA. Additionally, the reduced rate of RKN infection in plants primed with 20mM led to a decrease in the rate of Pcb infection. / Dissertation (MSc)--University of Pretoria, 2013. / gm2013 / Microbiology and Plant Pathology / Unrestricted

Background: Plastic stents used for the treatment of biliary obstruction will become occluded over time due to microbial colonization and formation of biofilms. Treatment of stent-associated cholangitis is often not effective because of inappropriate use of antimicrobial agents or antimicrobial resistance. We aimed to assess the current bacterial and fungal etiology of stentassociated biofilms, with particular emphasis on antimicrobial resistance. Methods: Patients with biliary strictures requiring endoscopic stent placement were prospectively enrolled. After the retrieval of stents, biofilms were disrupted by sonication, microorganisms were cultured, and isolates were identified by matrix-associated laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and/or biochemical typing. Finally, minimum inhibitory concentrations (MICs) were determined for various antimicrobial agents. Selected stents were further analyzed by fluorescence in situ hybridization (FISH). Results: Among 120 patients (62.5% males, median age 64 years) with biliary strictures (35% malignant, 65% benign), 113 double pigtail polyurethane and 100 straight polyethylene stents were analyzed after a median indwelling time of 63 days (range, 1–1274 days). The stent occlusion rate was 11.5%and 13%, respectively, being associated with a significantly increased risk of cholangitis (38.5% vs. 9.1%, P<0.001). Ninety-five different bacterial and 13 fungal species were detected; polymicrobial colonization predominated (95.8% vs. 4.2%, P<0.001). Enterococci (79.3%), Enterobacteriaceae (73.7%), and Candida spp. (55.9%) were the leading pathogens. Candida species were more frequent in patients previously receiving prolonged antibiotic therapy (63% vs. 46.7%, P = 0.023). Vancomycinresistant enterococci accounted for 13.7%, extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae with co-resistance to ciprofloxacin accounted for 13.9%, and azole-resistant Candida spp. accounted for 32.9% of the respective isolates. Conclusions: Enterococci and Candida species play an important role in the microbial colonization of biliary stents. Therefore, empirical antimicrobial treatment of stent-associated cholangitis should be guided toward enterococci, Enterobacteriaceae, streptococci, anaerobes, and Candida. To determine causative pathogens, an accurate microbiological analysis of the extracted stent(s) may be helpful.

Colistin is a last-resort antibiotic used to treat serious infections caused by Enterobacteriaceae and other multidrug resistant gram-negative bacteria. Recently discovered plasmid-borne colistin resistance, mediated by the mcr genes, poses a serious risk to colistin therapy. The aim of this diploma thesis was to map the occurrence of Enterobacteriaceae carrying the mcr-1 to 8 genes in hospitalized patients, travellers, prospective colistin-resistant clinical isolates and in a retrospective collection of Enterobacteriaceae using a combination of selective cultivation and qPCR. Isolates with a detected mcr gene were characterized by Whole-Genome Sequencing. The localization of mcr genes was determined and other resistance genes and plasmids were identified. Furthermore, the physiological profile of selected colistin- resistant Escherichia coli isolates was characterized. In the presence of a subinhibitory amount of colistin, a strain carrying the mcr-1 gene may be favored. Later, the mcr-9 gene was described and its occurence was subsequently tested retrospectively. Enterobacter spp. isolates carrying the mcr-9 gene were mostly colistin-sensitive but, in some cases, resistance was induced after exposure to sublethal doses of colistin. The results of the study show that the incidence of plasmid-mediated...