Browsing by Subject "Escherichia coli"
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- PublicationOpen AccessAn ideal spacing is required for the control of Class II CRP-dependent promoters by the status of CRP K100(2020-11-11) Ecija Conesa, Ana; Gallego Jara, Julia; Lozano-Terol, Gema; Browning, Douglas F.; Busby, Steve J.W.; Wolfe, Alan J.; Cánovas Díaz, Manuel; De Diego Puente, María Teresa; Bioquímica y Biología Molecular B e InmunologíaTranscription activation by the Escherichia coli CRP at Class II promoters is dependent on direct interactions between RNA polymerase and CRP, therefore the spatial proximity between both proteins plays a significant role in the ability of CRP to activate transcription. Using both in vivo and in vitro techniques, here we demonstrate that the CRP K100 positive charge, adjacent to AR2, is required for full promoter activity when CRP is optimally positioned. Accordingly, K100 mediated activation is very position-dependent and our data confirm that the largest impact of the K100 status on transcription activation occurs when the spacing between the CRP binding site and the A2 of the -10 element is 22 bp. From the results of this study and the progress in the understanding about open complex DNA scrunching, we propose that CRP-dependent promoters should now be numbered by the distance from the centre of the DNA site for CRP and the most highly conserved base at position 2 of the -10 hexamer in bacterial promoters.
- PublicationOpen AccessAn ideal spacing is required for the control of Class II CRP-dependent promoters by the status of CRP K100.(Oxford Academic, 2020) Écija Conesa, Ana; Gallego Jara, Julia; Lozano Terol, Gema; Browning, Douglas F.; Busby, Steve J. W.; Wolfe, Alan J.; Canovas Diaz, Manuel; De Diego Puente, María Teresa; Bioquímica y Biología Molecular B e InmunologíaTranscription activation by the Escherichia coli CRP at Class II promoters is dependent on direct interactions between RNA polymerase and CRP, therefore the spatial proximity between both proteins plays a significant role in the ability of CRP to activate transcription. Using both in vivo and in vitro techniques, here we demonstrate that the CRP K100 positive charge, adjacent to AR2, is required for full promoter activity when CRP is optimally positioned. Accordingly, K100 mediated activation is very position-dependent and our data confirm that the largest impact of the K100 status on transcription activation occurs when the spacing between the CRP binding site and the A2 of the −10 element is 22 bp. From the results of this study and the progress in the understanding about open complex DNA scrunching, we propose that CRP-dependent promoters should now be numbered by the distance from the center of the DNA site for CRP and the most highly conserved base at position 2 of the −10 hexamer in bacterial promoters
- PublicationEmbargoBacteriemia por Escherichia coli: factores predictivos de presencia de bacterias productoras de betalactamasas de espectro extendido e influencia de la resistencia en la mortalidad de los pacientes(Elsevier Doyma, 2010-07-31) García-Hernández, Ana; García-Vázquez, Elisa; Gómez Gómez, Joaquín; Canteras, Manuel; Hernández-Torres, Alicia; Ruiz Gómez, Joaquín; MedicinaRESUMEN: Fundamento y objetivo: Analizar los factores predictivos de bacteriemia por E. coli productor de betalactamasa de espectro extendido (BLEE) frente a E. coli no productor de BLEE y su repercusión en la mortalidad. Pacientes y método: Estudio observacional de una cohorte de pacientes adultos ingresados con bacteriemia por E. coli. Resultados: Se incluyeron 153 bacteriemia por E. coli (22% eran BLEE). Los factores de riesgo para BLEE fueron: consumo previo de antibióticos (odds ratio [OR] 2,61; intervalo de confianza del 95% [IC 95%] 1,1–6,19), índice de Winston <_2 (OR 9,83, IC 95% 3,42–28,26) y adquisición asociada a cuidados sanitarios (OR 5,35; IC 95% 1,57–18,27). La mortalidad relacionada fue del 21%, siendo factores de riesgo: neoplasia de base (OR 4,02; IC 95% 1,08–14,82), gravedad de enfermedad de base (según índice de McCabe) (OR 7,69; IC 95% 1,96–30,09) y gravedad al diagnóstico (según índice de Winston) (OR 48,89; IC 95% 11,58–206,97). El tratamiento empírico inadecuado era más frecuente en pacientes con bacteriemia por E. coli BLEE (67%, p<0,05). Conclusiones: El tratamiento antibiótico previo, el índice de Winston <_2 y la adquisición relacionada con cuidados sanitarios se asocian a bacteriemia por E. coli productor de BLEE comparado con no productor. Ni la producción de BLEE ni el tratamiento empírico inadecuado se asociaban a mayor mortalidad, pero sí la existencia de neoplasia de base y la gravedad clínica al diagnóstico
- PublicationOpen AccessEngineering protein production by rationally choosing a carbon and nitrogen source using E. coli BL21 acetate metabolism knockout strains.(Springer Nature, 2019-09-04) Lozano Terol, Gema; Gallego Jara, Julia; Sola Martinez, Rosa Alba; Canovas Diaz, Manuel; De Diego Puente, María Teresa; Bioquímica y Biología Molecular B e InmunologíaBackground: Escherichia coli (E. coli) is a bacteria that is widely employed in many industries for the production of high interest bio-products such as recombinant proteins. Nevertheless, the use of E. coli for recombinant protein production may entail some disadvantages such as acetate overfow. Acetate is accumulated under some culture conditions, involves a decrease in biomass and recombinant protein production, and its metabolism is related to protein lysine acetylation. Thereby, the carbon and nitrogen sources employed are relevant factors in cell host metabolism, and the study of the central metabolism of E. coli and its regulation is essential for optimizing the production of biomass and recombinant proteins. In this study, our aim was to fnd the most favourable conditions for carrying out recombinant protein production in E. coli BL21 using two diferent approaches, namely, manipulation of the culture media composition and the deletion of genes involved in acetate metabolism and Nε-lysine acetylation. Results: We evaluated protein overexpression in E. coli BL21 wt and fve mutant strains involved in acetate metabolism (Δacs, ΔackA and Δpta) and lysine acetylation (ΔpatZ and ΔcobB) grown in minimal medium M9 (inorganic ammonium nitrogen source) and in complex TB7 medium (peptide-based nitrogen source) supplemented with glucose (PTS carbon source) or glycerol (non-PTS carbon source). We observed a dependence of recombinant protein production on acetate metabolism and the carbon and nitrogen source employed. The use of complex medium supplemented with glycerol as a carbon source entails an increase in protein production and an efcient use of resources, since is a sub-product of biodiesel synthesis. Furthermore, the deletion of the ackA gene results in a fvefold increase in protein production with respect to the wt strain and a reduction in acetate accumulation. Conclusion: The results showed that the use of diverse carbon and nitrogen sources and acetate metabolism knockout strains can redirect E. coli carbon fuxes to diferent pathways and afect the fnal yield of the recombinant protein bioprocess. Thereby, we obtained a fvefold increase in protein production and an efcient use of the resources employing the most suitable strain and culture conditions.
- PublicationOpen AccessImpact of the Expression System on Recombinant Protein Production in Escherichia coli BL21(Frontiers Media, 2021-06-21) Lozano Terol, Gema; Gallego Jara, Julia; Sola Martinez, Rosa Alba; Martinez Vivancos, Adrián; Cánovas Diaz, Manuel; De Diego Puente, María Teresa; Bioquímica y Biología Molecular B e InmunologíaRecombinant protein production for medical, academic, or industrial applications is essential for our current life. Recombinant proteins are obtained mainly through microbial fermentation, with Escherichia coli being the host most used. In spite of that, some problems are associated with the production of recombinant proteins in E. coli, such as the formation of inclusion bodies, the metabolic burden, or the inefficient translocation/transport system of expressed proteins. Optimizing transcription of heterologous genes is essential to avoid these drawbacks and develop competitive biotechnological processes. Here, expression of YFP reporter protein is evaluated under the control of four promoters of different strength (PT7lac, Ptrc, Ptac, and PBAD) and two different replication origins (high copy number pMB10 and low copy number p15A). In addition, the study has been carried out with the E. coli BL21 wt and the ackA mutant strain growing in a rich medium with glucose or glycerol as carbon sources. Results showed that metabolic burden associated with transcription and translation of foreign genes involves a decrease in recombinant protein expression. It is necessary to find a balance between plasmid copy number and promoter strength to maximize soluble recombinant protein expression. The results obtained represent an important advance on the most suitable expression system to improve both the quantity and quality of recombinant proteins in bioproduction engineering.
- PublicationOpen AccessImpact of the Expression System on Recombinant Protein Production in Escherichia coli BL21(2021-06-21) Lozano Terol, Gema; Gallego Jara, Julia; Sola Martinez, Rosa Alba; Martinez Vivancos, Adrián; Canovas Diaz, Manuel; De Diego Puente, María Teresa; Bioquímica y Biología Molecular B e InmunologíaRecombinant protein production for medical, academic, or industrial applications is essential for our current life. Recombinant proteins are obtained mainly through microbial fermentation, with Escherichia coli being the host most used. In spite of that, some problems are associated with the production of recombinant proteins in E. coli, such as the formation of inclusion bodies, the metabolic burden, or the inefficient translocation/transport system of expressed proteins. Optimizing transcription of heterologous genes is essential to avoid these drawbacks and develop competitive biotechnological processes. Here, expression of YFP reporter protein is evaluated under the control of four promoters of different strength (PT7lac, Ptrc, Ptac, and PBAD) and two different replication origins (high copy number pMB10 and low copy number p15A). In addition, the study has been carried out with the E. coli BL21 wt and the ackA mutant strain growing in a rich medium with glucose or glycerol as carbon sources. Results showed that metabolic burden associated with transcription and translation of foreign genes involves a decrease in recombinant protein expression. It is necessary to find a balance between plasmid copy number and promoter strength to maximize soluble recombinant protein expression. The results obtained represent an important advance on the most suitable expression system to improve both the quantity and quality of recombinant proteins in bioproduction engineering.
- PublicationOpen AccessMedium- and long-term Immune responses in the small intestine in piglets from oral vaccination against Escherichia coli(MDPI, 2024-09-26) Miralles, Aida; Ramis, Guillermo; Pallarés, Francisco J.; Párraga Ros, Ester; Seva Alcaraz, Juan; Anatomía y Anatomía Patológica ComparadaPost-weaning stress, together with Escherichia coli, are two of the key factors in the occurrence of post-weaning diarrhea. There are different commercial vaccines that induce immunity at the local or systemic level, improving farm health and avoiding economic losses in the pork industry. That is why the objective of this study was to evaluate the effect of an oral enterotoxigenic E. coli F4/F18 vaccine on immunity and intestinal integrity in the middle and long term after inoculation. The gene expression of the biomarkers indicative of cellular infiltration (calprotectin, CAL), tight junction proteins (occludin, OCL; zonulin, ZON; and claudin, CLA) and a panel of proinflammatory (interleukins, IL: IL1α, IL1β, IL6, IL8, IL12p35 and IL12p40; interferons, IFN: IFNα and IFNγ; and tumoral necrosis factor, TNF: TNFα) and anti-inflammatory mediator cytokines (TGFβ and IL10) were analyzed, as well as histomorphology in jejunum and ileum, the cell density of goblet cells, intraepithelial lymphocytes and IgA-producing cells. Differences were observed in ZON, CLA and CAL, with greater gene expression in observed in vaccinated piglets at 42 days post vaccination (dpv) in the ileum. Regarding the expression of cytokines, the vaccinated animals showed significant differences in IL1α, IL6, IL12p35, IL12p40, IFNα, IFNγ, TNFα and TGFβ at 42 dpv in the jejunum or ileum. The villi showed greater height in the vaccinated piglets and the ratio between villus height and crypt depth was significantly greater in the vaccinated group in the jejunum at 84 dpv. The count of IgA-producing cells shows higher values for the unvaccinated group in the ileum, while intraepithelial lymphocytes show a significant increase in both jejunum and ileum in vaccinated piglets. We can conclude that oral vaccination against E. coli produces an evident effect, which manifests itself even in the middle and long term after the challenge, including immune response, decrease in antimicrobials usage, better histological structure in intestine and the improvement of performance.
- PublicationOpen AccessPatotipos de Escherichia coli detectados en muestras cárnicas mediante un programa de vigilancia sanitaria de la Consejería de salud de la Región de Murcia.(Universidad de Murcia. Servicio de publicaciones, 2024) Arcenillas Hernández, Irene; Ortega Hernández, Nieves; Villa López, María Inés; Caro Vergara, María RosaEscherichia coli es un microorganismo con amplia capacidad para sobrevivir en diferentes medios y ecosistemas. Sin embargo, su aparición en alimentos es indicadora de contaminación, siendo algunas cepas de esta bacteria dañinas para la salud humana. Por ello, el objetivo de este trabajo fue estudiar la presencia de determinados patotipos de E. coli, considerados de riesgo para la población: STEC, EAEC y AIEC. Para ello se analizaron muestras cárnicas procedentes de establecimientos y mataderos de la Región de Murcia. Las muestras tenían distinto origen animal (avícola, porcino, ovino, bovino o caprino), y podían ser frescas o procesadas. Estas muestras se cultivaron para aislar colonias de E. coli, de las que posteriormente se realizó la extracción de ADN. Ese ADN se analizó para detectar la presencia de los genes asociados a los patotipos objeto del estudio a través de la técnica de PCR. Los resultados revelaron que el 86.3 % de las muestras cárnicas analizadas tenían presencia de E. coli, de las cuales, obtuvieron resultados positivos a los genes de virulencia el 15.2 %. El patotipo más frecuente fue AIEC (66.6 %), seguido de STEC (40.4 %) y EAEC (2.4 %). A pesar de que cualquiera de los orígenes animales obtuvo resultados positivos para alguno de los patotipos, fueron las muestras de origen porcino las que mayor número de casos reportaron. Aunque los porcentajes globales no son elevados (AIEC 8.7 %, EAEC 5.3 % y STEC 0.3 %), los resultados revelan la presencia de estos genes de virulencia tanto en muestras recogidas directamente en matadero, como en productos cárnicos procesados y distribuidos en el área de la Región de Murcia. Nuestros resultados demuestran la importancia que tiene la instauración de programas de vigilancia para la detección de agentes patógenos emergentes o re-emergentes, como los patotipos patógenos de E. coli, con el fin de evitar la aparición de toxinfecciones alimentarias.
- PublicationOpen AccessRelative impact of three growth conditions on the Escherichia coli protein acetylome(Cell Press, 2024-02-16) Lozano- Terol, Gema; Zenezini Chiozzi, Riccardo; Gallego-Jara, Julia; Sola-Martínez, Rosa Alba; Martínez Vivancos, Adrián; Ortega, Álvaro; Heck, Albert J.R.; Cánovas Díaz, Manuel; De Diego Puente, María Teresa; Bioquímica y Biología Molecular B e InmunologíaNε-lysine acetylation is a common posttranslational modification observed in Escherichia coli. In the present study, integrative analysis of the proteome and acetylome was performed using label-free quantitative mass spectrometry to analyze the relative influence of three factors affecting growth. The results revealed differences in the proteome, mainly owing to the type of culture medium used (defined or complex). In the acetylome, 7482 unique acetylation sites were identified. Acetylation is directly related to the abundance of proteins, and the level of acetylation in each type of culture is associated with extracellular acetate concentration. Furthermore, most acetylated lysines in the exponential phase remained in the stationary phase without dynamic turnover. Interestingly, unique acetylation sites were detected in proteins whose presence or abundance was linked to the type of culture medium. Finally, the biological function of the acetylation changes was demonstrated for three central metabolic proteins (GapA, Mdh, and AceA).
- PublicationOpen AccessResistencia de Escherichia coli a quinolonas y factores asociados en pacientes hospitalizados por pielonefritis aguda(Universidad de Murcia, 2022-04-05) Mira Bleda, Enrique; Enrique Bernal Morell; Rosa Salazar, Vladimir; Bartolomé García Pérez; Escuela Internacional de DoctoradoIntroducción. La pielonefritis aguda (PNA) es una infección del parénquima renal que afecta con mayor frecuencia a mujeres. A pesar de que se trata de una patología habitualmente benigna se pueden producir complicaciones, algunas graves, presenta una elevada prevalencia y supone un gran consumo de recursos tanto asistenciales como económicos. Suelen estar producidas por enterobacterias, fundamentalmente, constituyendo Escherichia coli (E. coli) el principal microorganismo aislado. En los últimos años, el empleo indiscriminado de las quinolonas ha conllevado un aumento marcado de las resistencias, erigiéndose las cefalosporinas como una de las principales alternativas. Objetivos. Analizar la resistencia de E. coli a quinolonas y cefalosporinas e identificar factores de riesgo asociados. Estudiar las principales causas de reingreso y reinfección. Definir criterios de mal pronóstico y mala evolución, y comprobar su prevalencia. Creación de un modelo para predecir la resistencia de E. coli a quinolonas y mal pronóstico. Material y métodos. Estudio observacional, retrospectivo, descriptivo y con componentes analíticos, de una cohorte de pacientes ingresados con el diagnóstico de PNA a lo largo de dos años en el Hospital Clínico Universitario Virgen de la Arrixaca (HCUVA). Se incluyeron datos epidemiológicos, analíticos, clínicos y microbiológicos. Para el análisis estadístico se utilizó el programa Statistical Package for the Social Sciences (SPSS) y para la creación de los modelos predictivos R-proyect. Resultados. La muestra la conformaron 404 pacientes de los que 87,1% fueron mujeres, con una edad media de 42,7 años. La fiebre supuso el síntoma más frecuente. Se observó una estancia hospitalaria media de 5,7 días, tratando la Unidad de Corta Estancia (UCE) al mayor número de enfermos (42,6%). Resultaron positivos 234 urocultivos (60,3%); en 177 se aisló E. coli (75,7%). La resistencia de E. coli a quinolonas fue del 18,8% y del 7,4% a cefalosporinas. Dentro de los factores de riesgo, destacaron el sexo masculino para la resistencia a quinolonas y la cirugía urinaria en las cefalosporinas. Las tasas de reinfección y reingreso fueron del 21,8% y del 16,3%, respectivamente. La reinfección se asoció, fundamentalmente, con las infecciones urinarias previas, mientras que el reingreso se precisó en pacientes renales principalmente. El 28,2% de los enfermos presentaron una mala evolución y el 49% mal pronóstico; un ingreso reciente y un Índice de Charlson alto se relacionaron con la mala evolución, y la resistencia a quinolonas y la bacteriemia en el caso del mal pronóstico; el tratamiento con cefalosporinas, por su parte, se demostró como un factor protector. Conclusiones. E. coli continúa siendo el principal agente responsable de las PNA. Actualmente, la resistencia de E. coli a quinolonas y cefalosporinas en nuestro medio se encuentra en descenso, por debajo de la media nacional y europea, aunque en el primer caso sigue siendo superior a la de otros países con un nivel de desarrollo sociosanitario similar. Sin embargo, se debe seguir evitando el uso de las quinolonas, sobre todo en varones; las cefalosporinas constituyen un tratamiento empírico adecuado para las PNA, con la excepción de pacientes sometidos a cirugías urológicas previamente. Con los modelos predictivos creados, se ha calculado una probabilidad del 60% de resistencia de E. coli a quinolonas y del 90% para mal pronóstico en pacientes que cumplen las tres condiciones: sexo masculino, ingreso en los dos meses previos e Índice de Charlson elevado.
- PublicationEmbargoSynergistic antimicrobial effect of photodynamic therapy and chitosan on the titanium-adherent biofilms of Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa: An in vitro study(Wiley, 2022) Camacho Alonso, Fabio; Salinas, Jesús; Sánchez Siles, Mariano; Pato Mourelo, Jesús; Cotrina Veizaga, Brian Davis; Ortega, Nieves; Dermatología, Estomatología, Radiología y Medicina Física; Sanidad AnimalBackground: To date, no studies on the combined use of photodynamic therapy (PDT) and chitosan against peri-implantitis have been published. The aim of this study was to evaluate the possible synergistic antimicrobial effect of PDT and chitosan on the titanium-adherent biofilms of Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. Methods: A total of 60 titanium discs were included in this study. The discs were randomized into three bacterial contaminations (n = 20 discs per bacterium). After being cultured (incubated for 48 hours) they were randomized again into four different disinfection modalities (n = 5 discs per treatment): control (without treatment), PDT, chitosan 3 mg/mL, and PDT + chitosan 3 mg/mL. After the treatments, the colony forming units (CFU) were measured to determine antimicrobial effects, and field emission scanning electron microscopy (FESEM) was used to study cell morphology and titanium adherence. Results: For all the evaluated bacteria and all the variables studied the order from highest to lowest antimicrobial effectiveness was: PDT + chitosan 3 mg/mL > chitosan 3 mg/mL > PDT > control. Although, all disinfection methods were significantly effective when compared to control, the combined treatment of PDT + chitosan 3 mg/mL had the greatest antimicrobial effect against the three studied bacteria. Conclusions: The combination of PDT and chitosan has a synergistic antimicrobial effect against the bacteria S. aureus, E. coli, and P. aeruginosa, all closely related to peri-implantitis. However, further in vivo studies are needed because this study provides data based on an in vitro scenario that might not be extrapolated to patients with peri-implantitis.