Determinación de la contaminación fecal a través del gen uidA (Beta-D-glucuronidasa) en muestras de agua ambientales.
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2025
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Universidad de Concepción
Resumen
La determinación de la contaminación fecal de los cuerpos de agua es una preocupación a nivel mundial. Los métodos tradicionales como la fermentación en tubos múltiples (FTM), aunque ha demostrado ser efectivo, requieren tiempos extensivos (entre 48-96 horas) y su capacidad de respuesta es limitada ante situaciones de emergencias sanitarias. En búsqueda de soluciones, los métodos moleculares han sido clave por su rapidez y especificidad, por lo tanto, el presente estudio evaluó la utilidad del gen uidA, que codifica la enzima β-D glucuronidasa, como marcador molecular para la detección y cuantificación de Escherichia coli (principalmente) en muestras de agua ambientales, con énfasis en su aplicación en agua de mar.
En el presente trabajo, se estandarizó una técnica de PCR convencional y cuantitativo para la detección del gen uidA, mediante dos experimentos de microcosmos en agua de mar en condiciones controladas. Los experimentos fueron montados utilizando material fecal fresco de animales de cría. Posteriormente, con la técnica estandarizada, se analizaron muestras ambientales provenientes de agua de mar, dulce y agua subterránea de distintas localidades de la región del Biobío y Ñuble.
La estandarización de la técnica mediante PCR convencional permitió obtener un fragmento amplificado de aproximadamente 160 pares de bases, que corresponde al tamaño descrito en la literatura. La curva de calibración construida con este fragmento para el PCR cuantitativo tuvo un límite de detección de 1x102 copias/µL. Los resultados del experimento de microcosmos se ajustan a un modelo de regresión lineal (log-log), positivo y significativo (p<0.05), entre el número más probable de coliformes fecales (NMP/100mL) y el número de copias del gen uidA (copias del gen uidA/100mL), detectadas por qPCR. No obstante, el análisis estadístico de las muestras ambientales muestra que no existe una correlación significativa entre estas variables.
En conclusión, la detección de este gen correlaciona bien con la presencia de bacterias de origen fecal termotolerantes en material fresco, no así en muestras ambientales donde existe un ensamble microbiano con células vivas, muertas, trozos de ellas y otros microorganismos no termotolerantes. Sin embargo, ofrece una ventaja importante sobre la técnica de fermentación en tubos en situaciones que requieren una respuesta rápida, pudiendo obtener resultados dentro de 24 horas. Así también, su efectividad en agua subterránea es relevante, puesto que en ese caso la sola presencia de E. coli (vivas o muertas) indicaría la contaminación con material fecal de la napa freática y, por lo tanto, evidenciaría el riesgo sanitario al utilizar este tipo de agua para consumo humano o de animales de cría.
The determination of fecal contamination in water bodies is a global concern. Traditional methods such as multiple-tube fermentation (MTF), although proven effective, require extensive time (between 48-96 hours) and have limited responsiveness in sanitary emergency situations. In the search for solutions, molecular methods have been key due to their speed and specificity. Therefore, this study evaluated the utility of the uidA gene, which encodes the β-D-glucuronidase enzyme, as a molecular marker for the detection and quantification of Escherichia coli (mainly) in environmental water samples, with emphasis on its application in seawater. In this work, a conventional and quantitative PCR technique was standardized for the detection of the uidA gene, through two microcosm experiments in seawater under controlled conditions. The experiments were set up using fresh fecal material from farm animals. Subsequently, with the standardized technique, environmental samples from seawater, freshwater, and groundwater from various locations in the Biobío and Ñuble regions were analyzed. The standardization of the technique using conventional PCR allowed obtaining an amplified fragment of approximately 160 base pairs, which corresponds to the size described in the literature. The calibration curve constructed with this fragment for quantitative PCR had a detection limit of 1x102 copies/µL. The results of the microcosm experiment fit a linear regression model (log-log), positive and significant (p<0.05), between the most probable number of fecal coliforms (MPN/100mL) and the number of uidA gene copies (uidA gene copies/100mL), detected by qPCR. However, the statistical analysis of the environmental samples shows that there is no significant correlation between these variables. In conclusion, the detection of this gene correlates well with the presence of thermotolerant fecal bacteria in fresh material, but not in environmental samples where there is a microbial assemblage with live cells, dead cells, fragments of them, and other non-thermotolerant microorganisms. However, it offers a significant advantage over the multiple-tube fermentation technique in situations that require a rapid response, being able to obtain results within 24 hours. Also, its effectiveness in groundwater is relevant, since in that case the mere presence of E. coli (live or dead) would indicate contamination with fecal material from the water table and, therefore, would evidence the sanitary risk when using this type of water for human or farm animal consumption.
The determination of fecal contamination in water bodies is a global concern. Traditional methods such as multiple-tube fermentation (MTF), although proven effective, require extensive time (between 48-96 hours) and have limited responsiveness in sanitary emergency situations. In the search for solutions, molecular methods have been key due to their speed and specificity. Therefore, this study evaluated the utility of the uidA gene, which encodes the β-D-glucuronidase enzyme, as a molecular marker for the detection and quantification of Escherichia coli (mainly) in environmental water samples, with emphasis on its application in seawater. In this work, a conventional and quantitative PCR technique was standardized for the detection of the uidA gene, through two microcosm experiments in seawater under controlled conditions. The experiments were set up using fresh fecal material from farm animals. Subsequently, with the standardized technique, environmental samples from seawater, freshwater, and groundwater from various locations in the Biobío and Ñuble regions were analyzed. The standardization of the technique using conventional PCR allowed obtaining an amplified fragment of approximately 160 base pairs, which corresponds to the size described in the literature. The calibration curve constructed with this fragment for quantitative PCR had a detection limit of 1x102 copies/µL. The results of the microcosm experiment fit a linear regression model (log-log), positive and significant (p<0.05), between the most probable number of fecal coliforms (MPN/100mL) and the number of uidA gene copies (uidA gene copies/100mL), detected by qPCR. However, the statistical analysis of the environmental samples shows that there is no significant correlation between these variables. In conclusion, the detection of this gene correlates well with the presence of thermotolerant fecal bacteria in fresh material, but not in environmental samples where there is a microbial assemblage with live cells, dead cells, fragments of them, and other non-thermotolerant microorganisms. However, it offers a significant advantage over the multiple-tube fermentation technique in situations that require a rapid response, being able to obtain results within 24 hours. Also, its effectiveness in groundwater is relevant, since in that case the mere presence of E. coli (live or dead) would indicate contamination with fecal material from the water table and, therefore, would evidence the sanitary risk when using this type of water for human or farm animal consumption.
Descripción
Tesis presentada para optar al título de Ingeniero en Biotecnología Marina y Acuicultura
Palabras clave
Escherichia coli, Contaminación del agua, Agua Análisis