Microbiological contamination of conventional and reclaimed irrigation water: evaluation and management measures

The wide diversity of irrigation water sources (i.e., drinking water, groundwater, reservoir water, river water) includes reclaimed water as a requested measure for increasing water availability, but it is also a challenge as pathogen exposure may increase. This study evaluates the level of microbial contamination in different irrigation waters to improve the knowledge and analyses management measures for safety irrigation. Over a one-year period, the occurrence of a set of viruses, bacteria and protozoa, was quantified and the performance of a wetland system, producing reclaimed water intended for irrigation, was characterized. Human fecal pollution (HAdV) was found in most of the irrigation water types analysed. Hepatitis E virus (HEV), an emerging zoonotic pathogen, was present in groundwater where porcine contamination was identified (PAdV). The skin-carcinoma associated Merkel cell polyomavirus (MCPyV), was found occasionally in river water. Noroviruses were detected, as expected, in winter, in river water and reclaimed water. Groundwater, river water and reservoir water also harboured potential bacterial pathogens, like Helicobacter pylori, Legionella spp. and Aeromonas spp. that could be internalized and viable inside amoebas like Acanthamoeba castellanii, which was also detected. Neither Giardia cysts, nor any Cryptosporidium oocysts were detected. The wetland system removed 3 Log10 of viruses and 5 Log10 of bacteria, which resembled the river water quality. Irrigation waters were prone to variable contamination levels and according to the European guidance documents, the E. coli (EC) levels were not always acceptable. Sporadic detection of viral pathogens as NoV GII and HAdV was identified in water samples presenting lower EC than the established limit (100MNP/100 mL). When dealing with reclaimed water as a source of irrigation the analysis of some viral parameters, like HAdV during the peak irrigation period (summer and spring) or NoV during the coldest months, could complement existing water management tools based on bacterial indicators

Determinación de la contaminación microbiológica del agua de riego aplicando nuevas estrategias de análisis

[spa] Esta tesis doctoral tuvo como objetivo principal identificar y cuantificar la presencia de bacterias patógenas, mediante el uso de indicadores de contaminación fecal y Legionella en aguas de riego aplicando métodos moleculares. Para su desarrollo se abordaron los siguientes temas: 1. Puesta a punto de los métodos moleculares para la determinación de las bacterias presentes en el agua mediante qPCR. Para ello, se pusieron en marcha la qPCR de E. coli y de E. faecalis, se actualizaron la qPCR de Legionella spp. y la de Legionella pneumophila, y se utilizó la qPCR para bacterias totales. Para validar las qPCR de E. coli y de E. faecalis se compararon las cuantificaciones realizadas mediante cultivo en placa, citometría de flujo y qPCR. Se logró que todas las qPCRs tuviesen curvas estándares con aspectos como la pendiente de la curva, el intercepto con el eje Y, el coeficiente de determinación y el % de eficiencia dentro de los límites acordados como aceptables. 2. Implementación de métodos de concentración para bacterias: se utilizó la concentración por floculación orgánica con leche desnatada (SMF) para concentrar diferentes microorganismos, comparando dos métodos de extracción y de cuantificación. Se estudio la recuperación de células viables cultivables y de los genomas después del proceso de concentración SMF. También se comparó la concentración de diferentes microorganismos por SMF con la concentración mediante la columna monolítica de adsorción MAF. En estos estudios encontramos que el SMF es un método que reduce drásticamente la viabilidad de las células para ser cultivadas, reduciendo a menos del 1 % la recuperación de viables cultivables. Una vez concentradas las muestras de aguas por SMF, el método de extracción de ADN que mayor recuperación de genomas permite es el kit de extracción de ADN de Qiagen, y que SMF mostró valores de genomas recuperados levemente mayores que los recuperados por MAF. 3. Se monitoreo la calidad del agua de riego procedente de dos tipos de fuentes: las convencionales, y las no convencionales. Se pudo apreciar la calidad del agua de riego procedentes de acuíferos naturales y la de diferentes estaciones de muestreo dentro de una EDAR y ERA.[eng] The main objective of this doctoral thesis was to identify and quantify the presence of pathogenic bacteria, using indicators of faecal contamination and Legionella in irrigation waters using molecular methods. For its development the following subjects were approached: 1. Development of the molecular methods for the determination of the bacteria present in the water by qPCR. To do this, the qPCR of E. coli and E. faecalis were started, the qPCR of Legionella spp and Legionella pneumophila were updated, and qPCR was used for total bacteria. To validate the qPCR of E. coli and E. faecalis, the quantifications made by plaque culture, flow cytometry and qPCR were compared. It was achieved that all the qPCRs had standard curves with aspects such as the slope of the curve, the intercept with the Y axis, the coefficient of determination and the percentage of efficiency within the limits agreed as acceptable. 2. Implementation of concentration methods for bacteria: concentration by organic flocculation with skimmed milk (SMF) was used to concentrate different microorganisms, comparing two methods of extraction and quantification. The recovery of viable culturable cells and genomes after the SMF concentration process was studied. The concentration of different microorganisms by SMF was also compared with the concentration by the monolithic column of MAF adsorption. In these studies we found that the SMF is a method that drastically reduces the viability of the cells to be cultivated, reducing the recovery of viable crops to less than 1%. Once the water samples were concentrated by SMF, the DNA extraction method that allows greater genome recovery is the Qiagen DNA extraction kit, and that SMF showed values of recovered genomes slightly higher than those recovered by MAF. 3. Irrigation water quality was monitored from two types of sources: conventional, and unconventional. The quality of irrigation water from natural aquifers and that of different sampling stations within a WWTP and Regenerated Water Station could be appreciated