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.

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.

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...

Orientador: Paola Castro Moraes / Resumo: As infecções causadas por organismos resistentes a múltiplos fármacos (multi-drug resistance – MDR) estão associadas à maior morbidade, mortalidade e aumento significativos nos custos com cuidados da saúde. Cocos Gram-positivos e bastonetes Gram-negativos são os grupos de bactérias mais frequentemente relacionadas a infecção do sítio cirúrgico em cães. Dentro do contexto de saúde única, é imperativo o aprimoramento contínuo de métodos de avaliação de contaminação e infecção bacteriana no ambiente hospitalar veterinário. O presente estudo objetivou avaliar o perfil de resistência de cocos Gram-positivos e bastonetes Gram-negativos isolados nas mãos do cirurgião, antes e após a desinquinação, no ambiente e sítio cirúrgico, durante o transoperatório de cirurgias limpas/limpas-contaminadas (G1;n=20) e cirurgias contaminadas (G2;n=10). Das 150 amostras coletadas, as do ambiente (n=30) foram obtidas pela exposição de placa com ágar Brain Heart Infusion na sala cirúrgica durante o procedimento, enquanto as do sítio cirúrgico (n=60) e as das mãos do cirurgião (n=60) foram coletadas por cotonete estéril e semeadas em ágar sangue e MacConkey. Todas as placas foram incubadas a 37ºC por 24h em estufa bacteriológica sob condições anaeróbias. Colônias sugestivas de cocos Grampositivos e bastonetes Gram-negativos foram submetidas a testes bioquímicos para identificação dos gêneros. Por fim, foi realizado o teste de susceptibilidade aos antimicrobianos por disco difusão em ágar Müller-Hinton... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Infections caused by multidrug-resistant (MDR) organisms are associated with increased morbidity, mortality and health care spending. Gram-positive cocci and Gram-negative bacilli are bacterial species frenquently related with surgical site infections in dogs. In the context of one health, become necessary the continuous improvement of evaluation methods of bacterial infection and contamination in the veterinary hospital environment. The present project aims to evaluate the antimicrobial profile of Gram-positive cocci and Gram-negative bacilli isolated of the surgical site and room during the intraoperative period and of the surgeon’s hands, before and after antisepsis. All samples were obtained during intraoperative of clean/cleancontaminated (G1) and contaminated surgery (G2). A total of 153 samples were collected, of which the environmental samples were obtained by exposure of Brain Heart Infusion agar plate in the surgical room during the procedure and samples from the surgical site and surgeon’s hands were collected by swabs and seeded on blood and Macconkey agar. The plates were incubated at 37ºC for 24h. Suggestive bacterial colonies for Gram-positive cocci and Gram-negative bacilli were submitted to biochemical test for genus differentiation. Finally, antimicrobial susceptibility testing was performed by disk diffusion method. Forty-three isolates with morphological and biochemical characteristics of Staphylococcus spp. and 13 of Gram-negative bacilli were obtained. W... (Complete abstract click electronic access below) / Mestre

Salmonella är en gramnegativ bakterie som tillhör gruppen Enterobacteriacea. Bakterien delas in i två arter S. enterica och S. bongori och totalt har mer än 2600 serotyper upptäckts. Dessa serotyper skiljs från varandra utifrån sina flagellära H-antigener och somatiska O-antigener. Salmonellos är ett exempel på ett sjukdomstillstånd som orsakas av olika salmonella typer. De flesta salmonellainfektioner hos människor sker genom konsumering av kontaminerade livsmedel med bakterien. Polymerase chain reaction, PCR, är en snabb metod som kan detektera bakterier i till exempel livsmedel. I metoden används omväxlande upphettning och nedkylning av PCR-proven. Reaktionen i PCR sker i ett antal steg som möjliggör bildning av många kopior av DNA efter bara 40 cykler. Syftet med denna studie var att validera PCR metoden genom att hitta olika koncentrationer av salmonella i 25 gram värmebehandlad kycklingprov. Metoden börjades med att inokulera kycklingproven med salmonella och de bestämda halterna var 100, 10 och 1 cfu/prov. Huvudfokus var att hitta 1 cfu/kycklingprov och för att uppnå målet gjordes därför totalt fyra försök för att kunna dra pålitliga resultat och slutsatser. Vid varje försök gjordes två metoder parallellt där den ena var PCR metoden medan den andra var referens till PCR och genomfördes bara genom odling på agarplattor. Positiva prover bekräftades genom konfirmering i ett antal steg och vid varje försök gjordes negativ kontroll för att undvika falsk positiva resultat. Resultatet visade att 100 cfu/prov och 10 cfu/prov i kycklingprov kunde detekteras med PCR. Det närmaste värdet till 1 cfu/prov som var målet, blev 0,8 cfu/prov och med denna bakteriehalt lyckades PCR detektera två positiva prover av totalt tre prover. Den låga bakteriehalten behöver därför upprepas flera gånger för att metoden ska kunna valideras.