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Introducción: Las biopelículas constituyen un factor clave en el desarrollo de enfermedades infecciosas y la resistencia a fármacos para su control. Nuevas estrategias terapéuticas incluyen productos naturales como agentes antibiopelículas. Sin embargo, la comparación de los resultados suele ser difícil debido a la falta de homogeneidad y estandarización en los métodos empleados para estudiar la formación de biopelículas in vitro. Objetivo: Estandarizar un ensayo de adhesión en microplaca de Escherichia coli para su uso en el cribado de potenciales agentes antibiopelículas. Métodos: Se utilizó el método de adherencia en microplaca y la tinción con violeta cristal. Se evaluó la influencia de condiciones experimentales como la concentración bacteriana, el medio de cultivo y el tiempo de incubación. Resultados: Se identificaron como condiciones óptimas para la formación de biopelículas: el medio Luria Bertani (LB), la concentración bacteriana a 105 UFC/mL y un tiempo de incubación de 24 h. Conclusiones: Los resultados mostraron que las condiciones de cultivo influyen en la formación de biopelículas. Se determinaron las condiciones de cultivo óptimas para la formación de biopelículas de E. coli, que podrían emplearse en futuros estudios acerca del efecto de productos naturales sobre la inhibición o destrucción de biopelículas.
Introduction: Biofilms are a key factor in the development of infectious diseases and drug resistance for their control. New therapeutic strategies include natural products as anti-biofilm agents. However, comparing results is often difficult due to the lack of homogeneity and standardization of the methods used to study biofilm formation in vitro. Objective: To standardize an Escherichia coli microplate adhesion assay for potential anti-biofilm agents screening. Methods: The microplate adhesion method and crystal violet staining were used. The influence of experimental conditions such as bacterial concentration, culture medium, and incubation time were evaluated. Results: Optimal conditions for biofilm formation included: Luria Bertani (LB) medium, bacterial concentration at 105 CFU/mL, and an incubation time of 24 hours. Conclusions: The results showed that culture conditions influence biofilm formation. Optimal culture conditions for the formation of E. coli biofilms were determined, which could be used in further studies on the effect of natural products on the inhibition or destruction of biofilms.
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HumanosRESUMEN
INTRODUCTION: In inflammatory respiratory diseases, the imbalance between proteases and endogenous protease inhibitors leads to an exacerbated activity of human neutrophil elastase (a protease that destroys the extracellular matrix and stimulates proinflammatory cytokine release). Elastase is considered a target in the search for therapeutic treatments for inflammatory respiratory diseases. Pulmonary surfactant is a promising product for this purpose, because in addition to its biophysical function, it has anti-inflammatory properties. OBJECTIVE: Evaluate effect of the Cuban porcine pulmonary surfactant (Surfacen), the rCmPI-II elastase inhibitor, and the Surfacen/rCmPI-II combination on activated neutrophil elastase activity in vitro, and determine if Surfacen's interface property changes in the presence of the inhibitor. METHODS: The anti-elastase effect of Surfacen, rCmPI-II and the Surfacen/rCmPI-II combination was evaluated in an in vitro model of activated neutrophils, previously purified from the blood of healthy subjects. The cells were stimulated with LPS/fMLP and were incubated with different concentrations of Surfacen, rCmPI-II and the Surfacen/rCmPI-II combination. Elastase activity was measured. The interface property was determined on a Langmuir surface balance. The new index, called the abdominal adipose deposit index, was obtained by multiplying the subcutaneous fat thickness by visceral fat thickness, both measured by ultrasound. A cutoff point was established that facilitated discernment of an unhealthy phenotype: normal weight but metabolically obese, a cardiometabolic risk factor. RESULTS: Surfacen at 10 mg/mL inhibited 71% of stimulated neutrophil elastase activity. rCmPI-II at 0.1 µM reduced 20% of elastase activity; at 200 µM-the maximum concentration evaluated-inhibition was 68%. Both products had a dose-dependent effect. The Surfacen/inhibitor combination (0.5 mg/mL/80 µM) did not affect the surfactant interface property or the inhibitory activity of rCmPI-II against human neutrophil elastase. CONCLUSIONS: Surfacen and the rCmPI-II inhibitor have an anti-elastase effect on an activated neutrophil model. rCmPI-II does not affect Surfacen's interface property and, therefore, both can be evaluated for combined use in treating inflammatory lung diseases.