Estudio del papel de las amebas de vida libre como reservorio de Helicobacter pylori y otras bacterias patógenas en aguas y alimentos mediante técnicas moleculares

En esta Tesis se estudia el posible papel de las FLA como reservorio de H. pylori y otras bacterias patógenas en aguas y alimentos mediante técnicas moleculares. En primer lugar se realizó un ensayo de cocultivo entre la bacteria H. pylori y la ameba Acanthamoeba castellanii. Se comprobó, mediante técnicas moleculares específicas para la detección de células viables, PMA-qPCR y DVC-FISH, que la ameba es capaz de internalizar a la bacteria y que esta última permanece viable, demostrando que H. pylori se comporta como una bacteria ARB. Seguidamente, se analizaron un total de 120 muestras ambientales, 100 de agua y 20 de vegetales para comprobar la presencia, tanto de FLA como de H. pylori internalizado en estas FLA. En el caso de las muestras de agua, se analizaron 69 muestras de agua residual y 31 de agua potable. Un total de 55 (79,7%) muestras de agua residual y 12 (38,7%) de agua potable resultaron positivas para la presencia de FLA. Mediante la técnica PMA-qPCR se demostró la presencia de H. pylori internalizado en las FLA presentes en 28 (50,9%) y 11 (91,7%) de las muestras de agua residual y potable analizadas, respectivamente. Mediante DVC-FISH se demostró que las células de H. pylori internalizadas dentro de las FLA presentes en las muestras eran viables en 16 (29,5%) y 5 (41,7%) de las muestras de agua residual y potable analizadas, respectivamente. Además, se consiguió recuperar formas viables cultivables de H. pylori procedente del interior de FLA en 10 (18,2%) de las muestras de agua residual analizadas. Las FLA aisladas e identificadas en las aguas residuales pertenecieron a los géneros Acanthamoeba, Naegleria, Vanellidae y a la familia Vahlkampfiidae. En el caso del agua potable, las FLA aisladas e identificadas pertenecieron a los géneros Acanthamoeba, Echinamoeba y Vermamoeba. En el caso de las muestras de vegetales, concretamente lechugas, todas ellas resultaron positivas para el aislamiento de FLA (100%). Mediante la técnica PMA-qPCR se demostró la presencia de H. pylori internalizado en las FLA en 11 (55,0%) de las muestras y, mediante DVC-FISH, se demostró que las células de H. pylori internalizadas dentro de las FLA eran viables en 5 (25,0%) de las muestras. En este caso no se recuperaron formas viables cultivables de la bacteria. Finalmente, mediante metagenómica de secuenciación dirigida, se analizó el microbioma de las FLA presentes en 20 de las muestras analizadas en esta Tesis (11 de agua residual, 3 de agua potable y 6 de lechugas). Para ello, se eligieron los iniciadores, se evaluaron in silico e in vitro y, una vez comprobada su idoneidad, se emplearon para la secuenciación de las muestras. En los tres tipos de muestras, la clase bacteriana más abundante fue la Gammaproteobacteria. Para los tres tipos de muestras, los filos más abundantes de las bacterias del microbioma de las FLA fueron Proteobacteria y Bacteroidetes y, en el caso del agua residual, también lo fue el filo Planctomycetes. H. pylori se detectó mediante esta técnica en los tres tipos de muestra. Además, como parte del microbioma de FLA de muestras ambientales, se detectaron otras bacterias de interés para la salud pública, tales como Aeromonas, Legionella, Mycobaterium o Pseudomonas. Los resultados obtenidos en esta Tesis demuestran la presencia de FLA patógenas en las muestras ambientales, así como el hecho de que, en algunos casos, estas son transportadoras de bacterias patógenas. Este trabajo también confirma que H. pylori se comporta como una bacteria ARB y que se encuentra viable en el interior de FLA presentes, tanto en aguas residuales y potables como en vegetales. De esta forma, se postula que un modo de transmisión de esta bacteria podría ser a través de las FLA presentes en agua o vegetales.In this Thesis, the possible role of FLA is studied as a reservoir of H. pylori and other pathogenic bacteria in waters and food by means of molecular techniques. Firstly, a coculture assay between the bacterium H. pylori and the amoeba Acanthamoeba castellanii was carried out. It was verified by means of molecular techniques specific for the detection of viable cells, PMA-qPCR and DVC-FISH, that the amoeba is capable of internalizing the bacterium and that the latter remains viable, demonstrating that H. pylori behaves as an ARB bacterium. Afterwards, a total of 120 environmental samples, 100 of water and 20 of vegetables, were analyzed to verify the presence FLA as well as internalized H. pylori into these FLA. In case of water samples, 69 samples of wastewater and 31 samples of drinking water were analyzed. A total of 55 (79,7 %) wastewater and 12 (38,7 %) of drinking water samples turned out to be positive for FLA's presence. By means of PMA-qPCR technique, the presence of FLA-internalized H. pylori was demonstrated in 28 (50,9 %) and 11 (91,7 %) of the wastewater and drinking water samples analyzed, respectively. By means of DVC-FISH it was demonstrated that the FLA-internalized H. pylori cells were viable in 16 (29,5 %) and 5 (41,7 %) of the wastewater and drinking water samples analyzed, respectively. In addition, viable cultivable forms of H. pylori coming from the inside of FLA were recovered from 10 (18,2 %) of the analyzed wastewater samples. The isolated and identified FLA from wastewater samples belonged to the genus Acanthamoeba, Naegleria, Vanellidae and to the family Vahlkampfiidae. In the case of drinking wáter, the isolated and identified FLA belonged to the genus Acanthamoeba, Echinamoeba and Vermamoeba. In the case of the vegetable samples, specifically lettuces, all of them turned out to be positive for FLA's isolation (100 %). By means of the PMA-qPCR technique, the presence of FLA-internalized H. pylori was demonstrated in 11 (55,0 %) of the samples and, by means of DVC-FISH, it was demonstrated that FLA-internalized H. pylori cells were viable in 5 (25,0 %) of the samples. In this case, viable cultivable forms of the bacterium could not be recovered. Finally, by means of amplicon-based metagenomics, the FLA microbiome of 20 previously analyzed samples in this Thesis (11 wastewater, 3 drinking water and 6 lettuce samples) was analyzed. To do so, a pair of primers were selected and evaluated in silco and in vitro and, once checked its suitability, they were used to perform the samples' sequencing. In the three types of samples, the most abundant bacterial class was the Gammaproteobacteria. For the three types of samples, the most abundant bacterial phylum of the FLA microbiome were Proteobacteria and Bacteroidetes and, in case of the wastewater, it was also the phylum Planctomycetes. H. pylori was detected by means of this technology in the three types of samples. In addition, as part of FLA's microbiome of environmental samples, other bacteria of public health interest were detected, such as Aeromonas, Legionella, Mycobacterium or Pseudomonas. The results obtained in this Thesis demonstrate the presence of pathogenic FLA in the environmental samples, as well as the fact that, in some cases, these they are carriers of pathogenic bacteria. This work also confirms that H. pylori behaves as an ARB bacterium and that it is viable inside the present FLA in wastewater as well as in drinking water and in vegetables. This way, it is postulated that a way of transmission of this bacterium might be through the FLA present in water or vegetables.En esta Tesi s'estudia el possible paper de les FLA com a reservori de H. pylori i altres bacteris patògens en aigües i aliments per mitjà de tècniques moleculars. En primer lloc es va realitzar un assaig de cocultiu entre el bacteri H. pylori i l'ameba Acanthamoeba castellanii. Es va comprovar per mitjà de tècniques moleculars específiques per a la detecció de cèl¿lules viables, PMA-qPCR i DVC-FISH, que l'ameba és capaç d'internalizar al bacteri i que esta última roman viable, demostrant que H. pylori es comporta com un bacteri ARB. A continuació, es van analitzar un total de 120 mostres ambientals, 100 d'aigua i 20 de vegetals per a comprovar la presència tant de FLA com de H. pylori internalitzat en estes FLA. En el cas de les mostres d'aigua, es van analitzar 69 mostres d'aigua residual i 31 d'aigua potable. Un total de 55 (79,7%) mostres d'aigua residual i 12 (38,7%) d'aigua potable van resultar positives per a la presència de FLA. Per mitjà de la tècnica PMA-qPCR es va demostrar la presència d'H. pylori internalitzat en les FLA presents en 28 (50,9%) i 11 (91,7%) de les mostres d'aigua residual i potable analitzades, respectivament. Per mitjà de DVC-FISH es va demostrar que les cèl¿lules d'H. pylori internalitzades dins les FLA presents en les mostres eren viables en 16 (29,5%) i 5 (41,7%) de les mostres d'aigua residual i potable analitzades, respectivament. A més, es va aconseguir recuperar formes viables cultivables d'H. pylori procedent de l'interior de FLA en 10 (18,2%) de les mostres d'aigua residual analitzades. Les FLA aïllades i identificades en les aigües residuals van pertànyer als gèneres Acanthamoeba, Naegleria, Vanellidae i a la família Vahlkampfiidae. En el cas de l'aigua potable, les FLA aïllades i identificades van pertànyer als gèneres Acanthamoeba, Echinamoeba i Vermamoeba. En el cas de les mostres de vegetals, concretament encisams, totes elles van resultar positives per a l'aïllament de FLA (100%). Per mitjà de la tècnica PMA-qPCR es va demostrar la presència d'H. pylori internalitzat en les FLA en 11 (55,0%) de les mostres i, per mitjà de DVC-FISH, es va demostrar que les cèl¿lules d'H. pylori internalitzades dins les FLA eren viables en 5 (25,0%) de les mostres. En este cas no es van recuperar formes viables cultivables del bacteri. Finalment, per mitjà de metagenómica de seqüenciació dirigida, es va analitzar el microbioma de les FLA presents en 20 de les mostres analitzades en esta Tesi (11 d'aigua residual, 3 d'aigua potable i 6 d'encisams). Per tal de fer això, es van triar els iniciadors, es van avaluar in silico i in vitro i, una vegada comprovada la seua idoneïtat, es van emprar per a la seqüenciació de les mostres. En els tres tipus de mostres, la classe bacteriana més abundant va ser la Gammaproteobacteria. Per als tres tipus de mostres, els filos més abundants dels bacteris del microbioma de les FLA van ser Proteobacteria i Bacteroidetes i, en el cas de l'aigua residual, també ho va ser el filo Planctomycetes. H. pylori es va detectar per mitjà d'esta tècnica en els tres tipus de mostra. A més, com a part del microbioma de FLA de mostres ambientals, es van detectar altres bacteris d'interés per a la salut pública, com ara Aeromonas, Legionella, Mycobaterium o Pseudomonas. Els resultats obtinguts en esta Tesi demostren la presència de FLA patògenes en les mostres ambientals, així com el fet de que, en alguns casos, estes són transportadores de bacteris patògens. Este treball també confirma que H. pylori es comporta com un bacteri ARB i que es troba viable en l'interior de FLA presents, tant en aigües residuals i potables com en vegetals. D'esta manera, es postula que una manera de transmissió d'este bacteri podria ser a través de les FLA presents en aigua o vegetals.Moreno Mesonero, L. (2018). Estudio del papel de las amebas de vida libre como reservorio de Helicobacter pylori y otras bacterias patógenas en aguas y alimentos mediante técnicas moleculares [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/111952TESI

DVC-FISH to identify potentially pathogenic Legionella inside free-living amoebae from water sources

[EN] Despite all safety efforts, drinking and wastewater can still be contaminated by Legionella and free-living amoebae (FLA) since these microorganisms are capable of resisting disinfection treatments. An amoebae cyst harboring pathogenic Legionella spp. can be a transporter of this organism, protecting it and enhancing its infection abilities. Therefore, the aim of this work is to identify by DVC-FISH viable Legionella spp and Legionella pneumophila cells inside FLA from water sources in a specific and rapid way with the aim of assessing the real risk of these waters. A total of 55 water samples were processed, 30 reclaimed wastewater and 25 drinking water. FLA presence was detected in 52.7% of the total processed water samples. When DVC-FISH technique was applied, the presence of viable internalized Legionella spp. cells was identified in 69.0% of the total FLA-positive samples, concretely in 70.0% and 66.7% of wastewater and drinking water samples, respectively. L. pneumophila was simultaneously identified in 48.3% of the total FLA-positive samples, specifically in 50.0% and 44.4% of wastewater and drinking water samples, respectively. By culture, potentially pathogenic Legionella cells were recovered in 27.6% of the total FLA-positive bacteria, particularly in 35.0% and 11.1% of wastewater and drinking water samples, respectively. These findings demonstrate that FLA may promote resistance of bacteria to the performed disinfection treatments for drinking as well as for wastewater. So, in addition to the risk for the presence of pathogenic FLA in water it is necessary to take into account that these can be transporters of the pathogenic bacteria Legionella, which are able to survive inside them. The DVC-FISH method described here has been proved to be a rapid and specific tool to identify pathogenic Legionella spp. and L. penumophila viable cells harboured by FLA in these water sources, posing particular public health concern.This study has been supported by First research projects funding (PAID-06-18), Vice-Rectorate for Research, Innovation and Transfer of Universitat Politècnica de València" (UPV), València, Spain.Moreno Trigos, MY.; Moreno-Mesonero, L.; García Hernández, J. (2019). DVC-FISH to identify potentially pathogenic Legionella inside free-living amoebae from water sources. Environmental Research. 176:1-7. https://doi.org/10.1016/j.envres.2019.06.002S1717

Evidence of viable Helicobacter pylori and other bacteria of public health interest associated with free-living amoeba in lettuce samples by next generation sequencing and other molecular techniques

[EN] Vegetables are one of the sources from which Helicobacter pylori can be acquired. This bacterium infects > 50% of the global population and is a recognized type I human carcinogen. H. pylori enters into the viable but nonculturable state when it is in the environment, and therefore the use of molecular techniques is much convenient for its detection. Free-living amoebae (FLA) are protozoans found in vegetables. They are transmission vehicles for amoeba-resistant bacteria, among which H. pylori is included. The aim of this study is to study the occurrence and viability of H. pylori from lettuce samples, H. pylori internalized into FLA and the microbiome of FLA isolated from these samples. Special focus was pointed to human pathogenic bacteria. H. pylori was not directly detected in any lettuce sample by means of molecular techniques and neither by culture. However, intra-amoebic H. pylori DNA was detected by means of PMA-qPCR in 55% of the samples and viable intra-amoebic H. pylori cells in 25% of the samples by means of DVC-FISH technique. When FLA microbiome was studied, 21 bacterial genera were part of FLA microbiome in all samples. Helicobacter genus was detected as part of the FLA microbiome in two samples. Other bacteria of public health interest such as Aeromonas sp., Arcobacter sp., Legionella sp., Mycobacterium sp., Pseudomonas sp. and Salmonella sp. were detected as part of FLA microbiome along the analysed samples. This study demonstrates for the first time that H. pylori is internalized as well as alive inside FLA isolated from vegetables. Moreover, this study shows that FLA promote H. pylori detection in environmental samples. In addition, as far as we are aware, this is the first study which studies the microbiome of FLA isolated from vegetables. Among the FLA microbiome, bacteria of public health interest were detected, pointing out that FLA are carriers of these pathogens which can reach humans and cause a public health concern.This study has been supported by the Conselleria de Educacion, Investigacion, Cultura y Deporte, of the Community of Valencia, Spain, within the program of support for research under project AICO/2018/273. The author Laura Moreno-Mesonero is the recipient of a technician contract funded by the Consellerfa de Educacion, Investigacion, Cultura y Deporte, of the Community of Valencia, Spain, within the program of support for research under project AICO/2018/273.Moreno-Mesonero, L.; Hortelano, I.; Moreno Trigos, MY.; Ferrús Pérez, MA. (2020). Evidence of viable Helicobacter pylori and other bacteria of public health interest associated with free-living amoeba in lettuce samples by next generation sequencing and other molecular techniques. 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Comparative Genomic Analysis of the Class Epsilonproteobacteria and Proposed Reclassification to Epsilonbacteraeota (phyl. nov.). Frontiers in Microbiology, 8. doi:10.3389/fmicb.2017.00682White, C. I., Birtles, R. J., Wigley, P., & Jones, P. H. (2010). Mycobacterium avium subspecies paratuberculosis in free-living amoebae isolated from fields not used for grazing. Veterinary Record, 166(13), 401-402. doi:10.1136/vr.b4797Winiecka-Krusnell, J., Wreiber, K., Euler, A. von, Engstrand, L., & Linder, E. (2002). Free-living Amoebae Promote Growth and Survival of Helicobacter pylori. Scandinavian Journal of Infectious Diseases, 34(4), 253-256. doi:10.1080/00365540110080052Wu, D., Hugenholtz, P., Mavromatis, K., Pukall, R., Dalin, E., Ivanova, N. N., … Eisen, J. A. (2009). A phylogeny-driven genomic encyclopaedia of Bacteria and Archaea. Nature, 462(7276), 1056-1060. doi:10.1038/nature08656Yahaghi, E., Khamesipour, F., Mashayekhi, F., Safarpoor Dehkordi, F., Sakhaei, M. 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Natural antimicrobial compounds immobilised on silica microparticles as filtering materials: Impact on the metabolic activity and bacterial viability of waterborne microorganisms

[EN] The aim of this work was to assess the capability of filtering materials based on silica microparticles functionalised with essential oil components (EOCs) to remove waterborne bacteria from water, and to elucidate the mechanism of action of the inhibitory effect of the filtering materials on the metabolic activity and viability of the studied pathogens. Different silica microparticles (25, 50, 75, 200 or 375 mu m) were functionalised with carvacrol, eugenol, thymol and vanillin to obtain filtering materials which removal capability was evaluated using distilled water inoculated with Escherichia coli, Helicobacter pylori, Legionella pneumophila or Pseudomonas aeruginosa (10(4)-10(7) cells/mL). Water samples were filtered through different layer thicknesses (0.5, 1 or 1.5 cm) of the filtering materials and the microbial load retained was determined by plate count. In addition, fluorescent viability staining, determination of cellular ATP content, direct viable count-fluorescent in situ hybridisation (DVC-FISH) and propidium monoazide-quantitative polymerase chain reaction (PMA-qPCR) analyses were performed to prove the materials' antimicrobial properties. The results exhibited that EOC-functionalised supports were capable of eliminating waterborne microorganisms from water with log reduction values falling within the 3-5 range, whereas the non-functionalised materials did not present relevant inhibitory capacity. The irreversible effect of the EOC-functionalised supports on the viability and metabolic activity of treated bacteria was confirmed by fluorescent staining (absence or red stained cells) and DVC-FISH (no elongated cells). Cellular ATP content was significantly reduced after filtering the inoculated water samples through the EOC-functionalised supports (cATP values below 10 pg/mL). Similarly, the concentration of viable bacteria determined by PMA-qPCR showed the inhibitory effect of the developed materials with negative quantification values for H. pylori and values of 7.98 . 10(1)-6.07 .10(3) GU/mL for L. pneumophila water samples filtered with the EOC-functionalised supports. Thus, the use of the functionalised filtering materials led to loss of bacterial viability of the treated microorganisms with irreversible morphological and metabolic alterations, which confirms their potential use as filtering aids with additional properties for the biological control of water. (C) 2020 Elsevier B.V. All rights reserved.The authors gratefully acknowledge the financial support from the Ministerio de Ciencia, Innovacion y Universidades and FEDER-EU (Project RTC-2017-6100-2). M.R.R. acknowledges the Generalitat Valenciana for her Postdoctoral Fellowship (APOSTD/2019/118). Emivasa Company is acknowledged for their participation in the project and technical supportRibes Llop, S.; Ruiz Rico, M.; Moreno-Mesonero, L.; Moreno Trigos, MY.; Barat Baviera, JM. (2021). Natural antimicrobial compounds immobilised on silica microparticles as filtering materials: Impact on the metabolic activity and bacterial viability of waterborne microorganisms. Environmental Technology & Innovation. 21:1-14. https://doi.org/10.1016/j.eti.2020.101219S1142

Characterization of the efficiency and uncertainty of skimmed milk flocculation for the simultaneous concentration and quantification of water-borne viruses, bacteria and protozoa

[EN] In this study, the use of skimmed milk flocculation (SMF) to simultaneously concentrate viruses, bacteria and protozoa was evaluated. We selected strains of faecal indicator bacteria and pathogens, such as Escherichia coli and Helicobacter pylori. The viruses selected were adenovirus (HAdV 35), rotavirus (RoV SA-11), the bacteriophage MS2 and bovine viral diarrhoea virus (BVDV). The protozoa tested were Acanthamoeba, Giardia and cryptosporidium. The mean recoveries with q(RT)PCR were 66% (HAdV 35), 24% (MS2), 28% (RoV SA-11), 15% (BVDV), 60% (E. coli), 30% (H. pylori) and 21% (Acanthamoeba castellanii). When testing the infectivity, the mean recoveries were 59% (HAdV 35), 12% (MS2), 26% (RoV SA-11) and 0.7% (BVDV). The protozoa Giardia lamblia and Cryptosporidium parvum were studied by immunofluorescence with recoveries of 18% and 13%, respectively. Although q(RT)PCR consistently showed higher quantification values (as expected), q(RT)PCR and the infectivity assays showed similar recoveries for HAdV 35 and RoV SA-11. Additionally, we investigated modelling the variability and uncertainty of the recovery with this method to extrapolate the quantification obtained by q(RT)PCR and estimate the real concentration. The 95% prediction intervals of the real concentration of the microorganisms inoculated were calculated using a general non-parametric bootstrap procedure adapted in our context to estimate the technical error of the measurements. SMF shows recoveries with a low variability that permits the use of a mathematical approximation to predict the concentration of the pathogen and indicator with acceptable low intervals. The values of uncertainty may be used for a quantitative microbial risk analysis or diagnostic purposes. (C) 2017 The Author(s). Published by Elsevier B.V.This study was funded by the Water Challenges for a Changing World Joint Programming Initiative (W2013-095-C03-01), the Spanish Ministry of Economy and Competitiveness (MINECO - AGL2014-55081-R) and the Grup de Recerca Consolidat: Virus, bacteris i protozous d'interes en salut publica, aigua y aliments (Generalitat de Catalunya, Virbap - 2014-SGR-914). Eloy Gonzales-Gustavson would like to acknowledge the Presidente de la República scholarship from the Peruvian Government, which supported a PhD grant.Gonzales-Gustavson, E.; Cárdenas-Youngs, Y.; Calvo, M.; Figueira, M.; Hundesa, A.; Amoros, I.; Moreno Trigos, MY.... (2017). Characterization of the efficiency and uncertainty of skimmed milk flocculation for the simultaneous concentration and quantification of water-borne viruses, bacteria and protozoa. Journal of Microbiological Methods. 134:46-53. https://doi.org/10.1016/j.mimet.2017.01.006S465313

Prevalence of Cryptosporidium oocysts and Giardia cysts in raw and treated sewage sludges

Treated sludge from wastewater treatment plants (WWTPs) is commonly used in agriculture as fertilizers and to amend soils. The most significant health hazard for sewage sludge relates to the wide range of pathogenic microorganisms such as protozoa parasites.The objective of this study was to collect quantitative data on Cryptosporidium oocysts and Giardia cysts in the treated sludge in wastewater treatment facilities in Spain. Sludge from five WWTPs with different stabilization processes has been analysed for the presence of Cryptosporidium and Giardia in the raw sludge and after the sludge treatment. A composting plant (CP) has also been assessed. After a sedimentation step, sludge samples were processed and (oo)cysts were isolated by immunomagnetic separation (IMS) and detected by immunofluorescence assay (IFA). Results obtained in this study showed that Cryptosporidium oocysts and Giardia cysts were present in 26 of the 30 samples (86.6%) of raw sludge samples. In treated sludge samples, (oo)cysts have been observed in all WWTP's analysed (25 samples) with different stabilization treatment (83.3%). Only in samples from the CP no (oo)cysts were detected. This study provides evidence that (oo)cysts are present in sewage sludge-end products from wastewater treatment processes with the negative consequences for public health.We appreciate the financial support provided by Entidad Publica Saneamiento Aguas (EPSAR).Amoros Muñoz, I.; Moreno Trigos, MY.; Reyes-Sosa, MB.; Moreno-Mesonero, L.; Alonso Molina, JL. (2016). Prevalence of Cryptosporidium oocysts and Giardia cysts in raw and treated sewage sludges. Environmental Technology. 37(22):2898-2904. doi:10.1080/09593330.2016.1168486S28982904372

Metagenomic analysis of viruses, bacteria and protozoa in irrigation water

[EN] Viruses (e.g., noroviruses and hepatitis A and E virus), bacteria (e.g., Salmonella spp. and pathogenic Escherichia coli) and protozoa (e.g., Cryptosporidium parvum and Giardia intestinalis) are well-known contributors to food-borne illnesses linked to contaminated fresh produce. As agricultural irrigation increases the total amount of water used annually, reclaimed water is a good alternative to reduce dependency on conventional irrigation water sources. European guidelines have established acceptable concentrations of certain pathogens and/or indicators in irrigation water, depending on the irrigation system used and the irrigated crop. However, the incidences of food-borne infections are known to be underestimated and all the different pathogens contributing to these infections are not known. Next-generation sequencing (NGS) enables the determination of the viral, bacterial and protozoan populations present in a water sample, providing an opportunity to detect emerging pathogens and develop improved tools for monitoring the quality of irrigation water. This is a descriptive study of the virome, bacteriome and parasitome present in different irrigation water sources. We applied the same concentration method for all the studied samples and specific metagenomic approaches to characterize both DNA and RNA viruses, bacteria and protozoa. In general, most of the known viral species corresponded to plant viruses and bacteriophages. Viral diversity in river water varied over the year, with higher bacteriophage prevalences during the autumn and winter. Reservoir water contained Enterobacter cloacae, an opportunistic human pathogen and an indicator of fecal contamination, as well as Naegleria australiensis and Naegleria clarki. Hepatitis E virus and Naegleria fowleri, emerging human pathogens, were detected in groundwater. Reclaimed water produced in a constructed wetland system presented a virome and bacteriome that resembled those of freshwater samples (river and reservoir water). Viral, bacterial and protozoan pathogens were occasionally detected in the different irrigation water sources included in this study, justifying the use of improved NGS techniques to get a comprehensive evaluation of microbial species and potential environmental health hazards associated to irrigation water.This work was supported through a grant funded by the Spanish Ministry of Economy and Competitiveness (MINECO) in the frame of the collaborative international consortium JPIW2013-095-C03-01, JPIW2013-095-C03-02 and JPIW2013-095-C03-03 of the Water Challenges for a Changing World Joint Programming Initiative (Water JPI) Pilot Call and partially by AGL2017-86797-C2-1-R. Silvia Bofill-Mas is a Serra-Hunter fellow at the University of Barcelona.Rusiñol, M.; Martinez-Puchol, S.; Timoneda, N.; Fernandez-Cassi, X.; Pérez-Cataluña, A.; Fernández-Bravo, A.; Moreno-Mesonero, L.... (2020). Metagenomic analysis of viruses, bacteria and protozoa in irrigation water. International Journal of Hygiene and Environmental Health. 224. https://doi.org/10.1016/j.ijheh.2019.113440S22

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

Estudio de los problemas de bioensuciamiento de membranas y espumas de origen biológico en sistemas MBR de tratamiento de lixiviados

Alonso Molina, JL.; Moreno Trigos, MY.; Zuriaga Agusti, E.; Moreno-Mesonero, L.; Amoros, I.; Fernández-Navarro, J.; Mendoza Roca, JA.... (2016). Estudio de los problemas de bioensuciamiento de membranas y espumas de origen biológico en sistemas MBR de tratamiento de lixiviados. Retema Medio Ambiente. 193:18-26. http://hdl.handle.net/10251/98045S182619

Detailed stratified GWAS analysis for severe COVID-19 in four European populations

Given the highly variable clinical phenotype of Coronavirus disease 2019 (COVID-19), a deeper analysis of the host genetic contribution to severe COVID-19 is important to improve our understanding of underlying disease mechanisms. Here, we describe an extended genome-wide association meta-analysis of a well-characterized cohort of 3255 COVID-19 patients with respiratory failure and 12 488 population controls from Italy, Spain, Norway and Germany/Austria, including stratified analyses based on age, sex and disease severity, as well as targeted analyses of chromosome Y haplotypes, the human leukocyte antigen region and the SARS-CoV-2 peptidome. By inversion imputation, we traced a reported association at 17q21.31 to a ~0.9-Mb inversion polymorphism that creates two highly differentiated haplotypes and characterized the potential effects of the inversion in detail. Our data, together with the 5th release of summary statistics from the COVID-19 Host Genetics Initiative including non-Caucasian individuals, also identified a new locus at 19q13.33, including NAPSA, a gene which is expressed primarily in alveolar cells responsible for gas exchange in the lung.S.E.H. and C.A.S. partially supported genotyping through a philanthropic donation. A.F. and D.E. were supported by a grant from the German Federal Ministry of Education and COVID-19 grant Research (BMBF; ID:01KI20197); A.F., D.E. and F.D. were supported by the Deutsche Forschungsgemeinschaft Cluster of Excellence ‘Precision Medicine in Chronic Inflammation’ (EXC2167). D.E. was supported by the German Federal Ministry of Education and Research (BMBF) within the framework of the Computational Life Sciences funding concept (CompLS grant 031L0165). D.E., K.B. and S.B. acknowledge the Novo Nordisk Foundation (NNF14CC0001 and NNF17OC0027594). T.L.L., A.T. and O.Ö. were funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation), project numbers 279645989; 433116033; 437857095. M.W. and H.E. are supported by the German Research Foundation (DFG) through the Research Training Group 1743, ‘Genes, Environment and Inflammation’. L.V. received funding from: Ricerca Finalizzata Ministero della Salute (RF-2016-02364358), Italian Ministry of Health ‘CV PREVITAL’—strategie di prevenzione primaria cardiovascolare primaria nella popolazione italiana; The European Union (EU) Programme Horizon 2020 (under grant agreement No. 777377) for the project LITMUS- and for the project ‘REVEAL’; Fondazione IRCCS Ca’ Granda ‘Ricerca corrente’, Fondazione Sviluppo Ca’ Granda ‘Liver-BIBLE’ (PR-0391), Fondazione IRCCS Ca’ Granda ‘5permille’ ‘COVID-19 Biobank’ (RC100017A). A.B. was supported by a grant from Fondazione Cariplo to Fondazione Tettamanti: ‘Bio-banking of Covid-19 patient samples to support national and international research (Covid-Bank). This research was partly funded by an MIUR grant to the Department of Medical Sciences, under the program ‘Dipartimenti di Eccellenza 2018–2022’. This study makes use of data generated by the GCAT-Genomes for Life. Cohort study of the Genomes of Catalonia, Fundació IGTP (The Institute for Health Science Research Germans Trias i Pujol) IGTP is part of the CERCA Program/Generalitat de Catalunya. GCAT is supported by Acción de Dinamización del ISCIII-MINECO and the Ministry of Health of the Generalitat of Catalunya (ADE 10/00026); the Agència de Gestió d’Ajuts Universitaris i de Recerca (AGAUR) (2017-SGR 529). M.M. received research funding from grant PI19/00335 Acción Estratégica en Salud, integrated in the Spanish National RDI Plan and financed by ISCIII-Subdirección General de Evaluación and the Fondo Europeo de Desarrollo Regional (European Regional Development Fund (FEDER)-Una manera de hacer Europa’). B.C. is supported by national grants PI18/01512. X.F. is supported by the VEIS project (001-P-001647) (co-funded by the European Regional Development Fund (ERDF), ‘A way to build Europe’). Additional data included in this study were obtained in part by the COVICAT Study Group (Cohort Covid de Catalunya) supported by IsGlobal and IGTP, European Institute of Innovation & Technology (EIT), a body of the European Union, COVID-19 Rapid Response activity 73A and SR20-01024 La Caixa Foundation. A.J. and S.M. were supported by the Spanish Ministry of Economy and Competitiveness (grant numbers: PSE-010000-2006-6 and IPT-010000-2010-36). A.J. was also supported by national grant PI17/00019 from the Acción Estratégica en Salud (ISCIII) and the European Regional Development Fund (FEDER). The Basque Biobank, a hospital-related platform that also involves all Osakidetza health centres, the Basque government’s Department of Health and Onkologikoa, is operated by the Basque Foundation for Health Innovation and Research-BIOEF. M.C. received Grants BFU2016-77244-R and PID2019-107836RB-I00 funded by the Agencia Estatal de Investigación (AEI, Spain) and the European Regional Development Fund (FEDER, EU). M.R.G., J.A.H., R.G.D. and D.M.M. are supported by the ‘Spanish Ministry of Economy, Innovation and Competition, the Instituto de Salud Carlos III’ (PI19/01404, PI16/01842, PI19/00589, PI17/00535 and GLD19/00100) and by the Andalussian government (Proyectos Estratégicos-Fondos Feder PE-0451-2018, COVID-Premed, COVID GWAs). The position held by Itziar de Rojas Salarich is funded by grant FI20/00215, PFIS Contratos Predoctorales de Formación en Investigación en Salud. Enrique Calderón’s team is supported by CIBER of Epidemiology and Public Health (CIBERESP), ‘Instituto de Salud Carlos III’. J.C.H. reports grants from Research Council of Norway grant no 312780 during the conduct of the study. E.S. reports grants from Research Council of Norway grant no. 312769. The BioMaterialBank Nord is supported by the German Center for Lung Research (DZL), Airway Research Center North (ARCN). The BioMaterialBank Nord is member of popgen 2.0 network (P2N). P.K. Bergisch Gladbach, Germany and the Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases, University of Cologne, Cologne, Germany. He is supported by the German Federal Ministry of Education and Research (BMBF). O.A.C. is supported by the German Federal Ministry of Research and Education and is funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany’s Excellence Strategy—CECAD, EXC 2030–390661388. The COMRI cohort is funded by Technical University of Munich, Munich, Germany. This work was supported by grants of the Rolf M. Schwiete Stiftung, the Saarland University, BMBF and The States of Saarland and Lower Saxony. K.U.L. is supported by the German Research Foundation (DFG, LU-1944/3-1). Genotyping for the BoSCO study is funded by the Institute of Human Genetics, University Hospital Bonn. F.H. was supported by the Bavarian State Ministry for Science and Arts. Part of the genotyping was supported by a grant to A.R. from the German Federal Ministry of Education and Research (BMBF, grant: 01ED1619A, European Alzheimer DNA BioBank, EADB) within the context of the EU Joint Programme—Neurodegenerative Disease Research (JPND). Additional funding was derived from the German Research Foundation (DFG) grant: RA 1971/6-1 to A.R. P.R. is supported by the DFG (CCGA Sequencing Centre and DFG ExC2167 PMI and by SH state funds for COVID19 research). F.T. is supported by the Clinician Scientist Program of the Deutsche Forschungsgemeinschaft Cluster of Excellence ‘Precision Medicine in Chronic Inflammation’ (EXC2167). C.L. and J.H. are supported by the German Center for Infection Research (DZIF). T.B., M.M.B., O.W. und A.H. are supported by the Stiftung Universitätsmedizin Essen. M.A.-H. was supported by Juan de la Cierva Incorporacion program, grant IJC2018-035131-I funded by MCIN/AEI/10.13039/501100011033. E.C.S. is supported by the Deutsche Forschungsgemeinschaft (DFG; SCHU 2419/2-1).Peer reviewe