Implementation and integration of microbial source tracking in a river watershed monitoring plan

Fecal pollution of water bodies poses a serious threat for public health and ecosystems. Microbial source tracking (MST) is used to track the source of this pollution facilitating better management of pollution at the source. In this study we tested 12 MST markers to track human, ruminant, sheep, horse, pig and gull pollution to assess their usefulness as an effective management tool of water quality. First, the potential of the selected markers to track the source was evaluated using fresh fecal samples. Subsequently, we evaluated their performance in a catchment with different impacts, considering land use and environmental conditions. All MST markers showed high sensitivity and specificity, although none achieved 100% for both. Although some of the MST markers were detected in hosts other than the intended ones, their abundance in the target group was always several orders of magnitude higher than in the non-target hosts, demonstrating their suitability to distinguish between sources of pollution. The MST analysis matched the land use in the watershed allowing an accurate assessment of the main sources of pollution, in this case mainly human and ruminant pollution. Correlating environmental parameters including temperature and rainfall with MST markers provided insight into the dynamics of the pollution in the catchment. The levels of the human marker showed a significant negative correlation with rainfall in human polluted areas suggesting a dilution of the pollution, whereas at agricultural areas the ruminant marker increased with rainfall. There were no seasonal differences in the levels of human marker, indicating human pollution as a constant pressure throughout the year, whereas the levels of the ruminant marker was influenced by the seasons, being more abundant in summer and autumn. MST analysis integrated with land use and environmental data can improve the management of fecal polluted areas and set up best practice

Identification of sapovirus GV.2, astrovirus VA3 and novel anelloviruses in serum from patients with acute hepatitis of unknown aetiology.

Hepatitis is a general term meaning inflammation of the liver, which can be caused by a variety of viruses. However, a substantial number of cases remain with unknown aetiology. We analysed the serum of patients with clinical signs of hepatitis using a metagenomics approach to characterize their viral species composition. Four pools of patients with hepatitis without identified aetiological agents were evaluated. Additionally, one pool of patients with hepatitis E (HEV) and pools of healthy volunteers were included as controls. A high diversity of anelloviruses, including novel sequences, was found in pools from patients with hepatitis of unknown aetiology. Moreover, viruses recently associated with gastroenteritis as sapovirus GV.2 and astrovirus VA3 were also detected only in those pools. Besides, most of the HEV genome was recovered from the HEV pool. Finally, GB virus C and human endogenous retrovirus were found in the HEV and healthy pools. Our study provides an overview of the virome in serum from hepatitis patients suggesting a potential role of these viruses not previously described in cases of hepatitis. However, further epidemiologic studies are necessary to confirm their contribution to the development of hepatitis

Evaluation of methods for the concentration and extraction of viruses from sewage in the context of metagenomic sequencing

Viral sewage metagenomics is a novel field of study used for surveillance, epidemiological studies, and evaluation of waste water treatment efficiency. In raw sewage human waste is mixed with household, industrial and drainage water, and virus particles are, therefore, only found in low concentrations. This necessitates a step of sample concentration to allow for sensitive virus detection. Additionally, viruses harbor a large diversity of both surface and genome structures, which makes universal viral genomic extraction difficult. Current studies have tackled these challenges in many different ways employing a wide range of viral concentration and extraction procedures. However, there is limited knowledge of the efficacy and inherent biases associated with these methods in respect to viral sewage metagenomics, hampering the development of this field. By the use of next generation sequencing this study aimed to evaluate the efficiency of four commonly applied viral concentrations techniques (precipitation with polyethylene glycol, organic flocculation with skim milk, monolithic adsorption filtration and glass wool filtration) and extraction methods (Nucleospin RNA XS, QIAamp Viral RNA Mini Kit, NucliSENS® miniMAG®, or PowerViral® Environmental RNA/DNA Isolation Kit) to determine the viriome in a sewage sample. We found a significant influence of concentration and extraction protocols on the detected viriome. The viral richness was largest in samples extracted with QIAamp Viral RNA Mini Kit or PowerViral® Environmental RNA/DNA Isolation Kit. Highest viral specificity were found in samples concentrated by precipitation with polyethylene glycol or extracted with Nucleospin RNA XS. Detection of viral pathogens depended on the method used. These results contribute to the understanding of method associated biases, within the field of viral sewage metagenomics, making evaluation of the current literature easier and helping with the design of future studies

Mètodes computacionals per a la identificació de virus emergents analitzats per seqüenciació en massa en aigua i aliments

[cat] Segons l'Organització Mundial de la Salut (OMS), les malalties d'origen alimentari són un problema de salut pública en expansió. Actualment l'aigua es considera un recurs limitat, afectat per les variacions extremes en les condicions climàtiques, fent necessari en algunes regions realitzar polítiques de canvi en el seu ús que poden haver generat noves vies de transmissió de patògens vírics fins ara no presents. Un d'aquests canvis és la utilització d'aigües regenerades com a font d'aigua i nutrients que poden reaprofitar-se en les indústries, recàrrega de aqüífers, rec de jardins i rec de productes frescos, com per exemple les hortalisses. La població humana i els animals excreten una gran diversitat de virus patògens a les seves orines i femtes; com a resultat, l'aigua residual que es genera representa un dels principals vehicles per a la disseminació de patògens a les aigües superficials, subterrànies o costaneres, i com a conseqüència en aliments. La contaminació del medi ambient suposa un risc greu de salut pública. . Tot i les mesures destinades a millorar la qualitat de l'aigua i la seguretat alimentària, a nivell mundial s'identifiquen de manera recurrent brots causats per virus transmesos per aigua o aliments. També a escala global, les malalties transmeses per l'aigua o aliments constitueixen un problema que en els darrers 20 anys, lluny de disminuir per la millora de les condicions higièniques de molts països, s'ha complicat considerablement. Entre els principals factors responsables d'aquesta tendència podem destacar els següents: creixement i envelliment de la població humana; comerç internacional de vegetals, carns, aliments exòtics i animals de granja entre països amb estàndards microbiològics diferents; canvis en certes pràctiques agrícoles per abaratir costos; i com a rerefons el canvi climàtic. Per poder tenir una visualització global de la diversitat viral present en les diferents mostres ambientals es van realitzar estudis de metagenòmica de genomes complets, amb la tecnologia Miseq de Illumina. Més concretament es van analitzar 3 tipus de matrius diferents, aigua residual d’entrada de la planta de tractament, sèrum humà de pacients positius per hepatitis però sense agent etiològic identificat i finalment julivert regat amb aigua de riu la quals també es va analitzar. El objectiu de la tesis va ser treballar les seqüències obtingudes amb la seqüenciació eliminant seqüencies de baixa qualitat, redundants i de baixa complexitat associades a seqüències repetitives . Posteriorment s’ha realitzat l’ensamblat amb els programes CLCBio i Velvet-MetaVelvet. Posteriorment a l’ensamblat es va passar a caracteritzar i anotar taxonòmicament els diferents conjunts de seqüències obtingudes realitzant cerques amb el blast utilitzant 3 bases de dades diferents de genomes virals complets, seqüències virals i proteïnes virals. Per la visualització dels resultats de la seqüenciació d’una manera intuïtiva i integrada s’han desenvolupat taules estàtiques, taules dinàmiques i krones, dins d’un entorn web per poder gestionar-ho tot. En les mostres d’aigua residual urbana s’ha identificat més de 36 famílies entre les que cal destacar algunes d'elles que poden afectar a l'home com Adenoviridae, Hepeviridae, Circoviridae, Astroviridae, Picobirnaviridae, Polyomaviridae, Astroviridae i Anelloviridae En els anàlisis realitzats en ostres de sèrum de pacients amb hepatitis aguada sense agent causal identificat s’ha detectat virus pertanyents a les famílies Calicivirus, Astrovirus i Anellovirus que podrien ser els causants. I finalment en les mostres de riu i julivert regat amb aigua de riu s’han identificat patògens humans , mostrant el risc elevat que representa la utilització d’aigua amb contaminació viral en irrigació de productes frescos que es consumeixen en crus.[eng] Treated wastewater is increasingly recognized as a resource of water nutrients and is reused in industry, for landscape irrigation, aquifer recharge and, especially in Mediterranean Europe, also for irrigation of fresh produce for human consumption. Wastewater contains many potential well known pathogenic bacteria and viruses; but also other potential emerging bacterial and viral pathogens largely unknown. Although ICTV recognized 2243 viral species to date, the most recent estimates determine that we have identified and characterized less than 0.1% of the viruses present on this planet. However this number itself is likely a gross underestimate. For instance,approximately 40% of all diarrhea, being the third leading infectious cause of death worldwide, cases are unknown etiology. In this thesis was to study three diferent samples, wastewater, serum samples from paciens with hepatitis without identified aetiological agents and parsley irrigated with river water. All the sampes are sequenced with the tecnology of illumina MiSeq. The aim of the thesis was to work the sequences obtained with the sequencing deleting the sequences with low quality, redundant and low complexity associated to repetitive sequences. The next step is the assembly with use two differents programs CLCBio and Velvet-MetaVelvet. After the assembly, we proceeded to characterize and annotate the different sets of sequences obtained using Blast with 3 databases one of complete viral genomes, another of viral sequences and the last of viral proteins. For visualization of the results of the sequencing in an intuitive and integrated way we have developed static tables, dynamic tables and Krones, within a web environment to be able to manage everything. In urban wastewater samples, more than 36 families have been identified, including a few that may affect humans such as Adenoviridae, Hepeviridae, circoviridae, astroviridae, Picobirnaviridae, Polyomaviridae, astroviridae and Anelloviridae In serum samples has detected virus belonging to the families Calicivirus, Astrovirus and anellovirus that could be the cause. And finally, in the samples of river and parsley watered with river water, humasn pathogens have been identified, showing the high risk represented by the use of water with viral contamination in irrigation of fresh products consumed in the ra

Dinoflagellate hosts determine the community structure of marine Chytridiomycota: Demonstration of their prominent interactions

15 pages, 7 figures, 1 table, supporting Information https://doi.org/10.1111/1462-2920.16182.-- Data Availability Statement: DNA sequences data from cultures have been submitted to the GenBank databases under accession number ON081632-ON081642 (SSU rDNA) and ON113503-ON113504 (LSU rDNA Dinomyces arenysensis). Raw data used in the study are available in NCBI SRA PRJNA630546The interactions of parasitic fungi with their phytoplankton hosts in the marine environment are mostly unknown. In this study, we evaluated the diversity of Chytridiomycota in phytoplankton communities dominated by dinoflagellates at several coastal locations in the NW Mediterranean Sea and demonstrated the most prominent interactions of these parasites with their hosts. The protist community in seawater differed from that in sediment, with the latter characterized by a greater heterogeneity of putative hosts, such as dinoflagellates and diatoms, as well as a chytrid community more diverse in its composition and with a higher relative abundance. Chytrids accounted for 77 amplicon sequence variants, of which 70 were found exclusively among different blooming host species. The relative abundance of chytrids was highest in samples dominated by the dinoflagellate genera Ostreopsis and Alexandrium, clearly indicating the presence of specific chytrid communities. The establishment of parasitoid-host co-cultures of chytrids and dinoflagellates allowed the morphological identification and molecular characterization of three species of Chytridiomycota, including Dinomyces arenysensis, as one of the most abundant environmental sequences, and the discovery of two other species not yet describedThis study was funded by the Spanish MICINN Project SMART PID2020-112978GB-I00, the MINECO Project COPAS CTM2017-86121-R. The authors acknowledge the institutional support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S). Alan Denis Fernández-Valero is funded by the MICIU grant ‘Ayudas para contratos predoctorales para la formación de doctores 2018 (PRE2018-084893)’Peer reviewe

Diversity of parasitic chytrids associated with dinoflagellate blooms on the Catalan coast

19th International Conference on Harmful Algae (ICHA 2021), 10-15 october 2021, La Paz, Baja California Sur, MéxicoTo understand bloom dynamics, biological mechanisms play a key role in phytoplankton behavior. Factors such as parasitism and saprophytism can impair bloom development and enhance the decline of natural HAB populations, as well as allow the transfer of organic matter to other trophic levels. An example of this dynamics is the parasitic phytoplankton-fungi relationship. However, it is mostly unknown in the marine environment. For this reason, here, we evaluated the diversity of Chytridiomycota during blooms of 6 different dinoflagellate species occurring in several coastal locations in the NW Mediterranean Sea. For this purpose, we combined metabarcoding, microscopy observations and live cultivation of chytrids from seawater and sediment samples. Our results showed that chytrids represented an overall abundance of 1.4% in the eukaryotic community with a total of 77 amplicon sequence variants (ASVs). Furthermore, the chytrid community varied significantly among sites, mainly depending on the blooming host. The highest diversity and abundance of chytrids were found in Ostreopsis and Alexandrium blooms as well as having a higher diversity and abundance mainly in sediment compared to seawater. ASVs corresponding to the chytrid species Dinomyces arenysensis co-occurred with the dinoflagellate Ostreopsis sp. in all locations where present. Such relationship was also confirmed by lab experiments. Therefore, our results suggest a diverse and wide geographic distribution of chytrid communities during dinofagellate coastal bloom, in some cases, specific distribution of chytrids species is strongly dependent on its host distributio

Multi-parasite community in coastal dinoflagellate blooms

XIV Reunião Ibérica sobre Microalgas Nocivas e Biotoxinas Marinhas - XIV Reunión Ibérica sobre Microalbas Nocivas y Biotoxinas Marinas, a 3 de Junho 2022, Algés, LisboaMarine protist communities are not exempt from parasitic infections and saprophytic interactions, especially by highly diverse groups, which include Apicomplexa, Perkinsozoa, Syndiniales, and Chytridiomycota among others. Parasites, parasitoids and saprophytes take part in complex networks of interactions and multi-host–multiparasite interactions which determine coexistence of a large number of species, microbial web chain length and ecosystem properties. In their host interactions, parasites can be generalists, infecting a multitude of hosts, or specialists, infecting only one or a limited range of hosts. Infections by either type of parasites or by both, whether sequentially or simultaneously, may have additive, antagonistic, neutral, or synergistic effects on their hosts. Furthermore, the order of infection by different parasites and the time lag between exposures may fundamentally shape competition and the progression among competent host and non-host populations within a community as well as host and parasite fitness. Here, we faced the complexity of multi-host and multi-parasite systems in costal planktonic communities of protists disentangle their parasites associated and the impact on their hostsPeer reviewe

Vertical and temporal distribution of chytrids infecting diatoms in the Gulf of Naples (Italy, Mediterranean Sea)

16 pages, 7 figures, 1 table, supporting information https://doi.org/10.1111/maec.12726.-- Data Availability Statement: Data generated in this study including environmental parameters, phytoplankton counts, metabarcoding final datasets and results, images and videos generated from incubation experiments are deposited in the public repository DIGITAL.CSIC, accessible by the DOI 10.20350/digitalCSIC/14744The presence of phytoplankton parasites along the water column was explored at the Long-Term Ecological Station MareChiara (LTER-MC) in the Gulf of Naples (Mediterranean Sea) in October 2019. Microscopy analyses showed diatoms dominating the phytoplankton community in the upper layers (0–20 m). Metabarcoding data from the water column showed the presence of Chytridiomycota predominantly in the upper layers coinciding with the vertical distribution of diatoms. Laboratory incubations of natural samples enriched with different diatom cultures confirmed parasitic interactions of some of those chytrids—including members of Kappamyces—with diatom taxa. The temporal dynamics of diatoms and chytrids was also explored in a 3-year metabarcoding time-series (2011–2013) from surface waters of the study area and in sediment samples. Chytrids were recurrently present at low relative abundances, and some taxa found to infect diatoms in the incubation experiments were also identified in the ASV time-series. However, co-occurrence analyses did not show any clear or recurrent pairing patterns for chytrid and diatom taxa along the 3 years. The chytrid community in the sediments showed a clearly different species composition compared with that recorded in the water column samples, with higher diversity and relative abundance. The combination of observations, incubations, and metabarcoding confirmed that parasites are a common component of marine protist communities at LTER-MC. Host–parasite interactions must be determined and quantified to understand their role and the impact they have on phytoplankton dynamicsThe research leading to these results received funding from the European Union's Horizon 2020 Research and Innovation Program under grant agreement No 730984, ASSEMBLE Plus Project. AR and EG were funded by the Spanish MICINN Project SMART (PID2020-112978GB-I00), and thank the institutional support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019–000928-S). The research program LTER-MC is funded by the Stazione Zoologica Anton DohrnPeer reviewe

NGS Techniques Reveal a High Diversity of RNA Viral Pathogens and Papillomaviruses in Fresh Produce and Irrigation Water

Special issue Next-Generation Sequencing and Emerging Technologies for the Identification and Control of Microbial Contaminants in the Food Chain.-- 21 pages, 2 figures, 5 tables, supplementary materials https://www.mdpi.com/article/10.3390/foods10081820/s1.-- Data Availability Statement: The datasets generated during the current study are available in Zenodo under the DOI number https://doi.org/10.5281/zenodo.4722797Fresh fruits and vegetables are susceptible to microbial contamination at every stage of the food production chain, and as a potential source of pathogens, irrigation water quality is a critical factor. Next-generation sequencing (NGS) techniques have been flourishing and expanding to a wide variety of fields. However, their application in food safety remains insufficiently explored, and their sensitivity requires improvement. In this study, quantitative polymerase chain reaction (qPCR) assays showed low but frequent contamination of common circulating viral pathogens, which were found in 46.9% of samples of fresh produce: 6/12 lettuce samples, 4/12 strawberries samples, and 5/8 parsley samples. Furthermore, the application of two different NGS approaches, target enrichment sequencing (TES) for detecting viruses that infect vertebrates and amplicon deep sequencing (ADS), revealed a high diversity of viral pathogens, especially Norovirus (NoV) and Human Papillomavirus (HPV), in fresh produce and irrigation water. All NoV and HPV types found in fresh fruit and vegetable samples were also detected in irrigation water sources, indicating that these viruses are common circulating pathogens in the population and that irrigation water may be the most probable source of viral pathogens in food samplesThis work was partially funded by a grant from the Spanish Ministry of Economy and Competitiveness (MINECO) AGL2017-86797-C2-1-R. IDEA-CSIC is a Centre of Excellence Severo Ochoa (Spanish Ministry of Science and Innovation (MICINN), Project CEX2018-000794-S)With the institutional support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S)Peer reviewe

VERDI [Dataset]

File1: VERDI_samples_parameters.xlsx - Physico-chemical variables obtained from CTD profile - Inorganic nutrients concentrations - Chlorophyll-a concentrations - Organic carbon and nitrogen concentrations - Phytoplankton abundances - Detections of chytrids File 2: VERDI_asv_table.tbl: ASV abundances from natural samples and incubations File 3: VERDI_tax_table.tbl: Taxonomic assignments of ASVs File 4: VERDI_asv_seqs.fa: Sequences of ASVs File 5: VERDI_incubations_images.zip - Compilation of images taken during incubations with diatomsThe presence of phytoplankton parasites along the water column was explored at the Long Term Ecological Station MareChiara (LTER-MC) in the Gulf of Naples (Mediterranean Sea) in October 2019. Microscopy analyses showed diatoms dominating the phytoplankton community in the upper layers (0-20 m). Metabarcoding data from the water column showed the presence of Chytridiomycota predominantly in the upper layers coinciding with the vertical distribution of diatoms. Laboratory incubations of natural samples enriched with different diatom cultures confirmed parasitic interactions of some of those chytrids – including members of Kappamyces – with diatom taxa. The temporal dynamics of diatoms and chytrids was also explored in a three-year metabarcoding time-series (2011-2013) from surface waters of the study area and in sediment samples. Chytrids were recurrently present at low relative abundances, and some taxa found to infect diatoms in the incubation experiments were also identified in the ASV time-series. However, co-occurrence analyses did not show any clear or recurrent pairing patterns for chytrid and diatom taxa along the three years. The chytrid community in the sediments showed a clearly different species composition compared to the recorded in the water column samples, with higher diversity and relative abundance. The combination of observations, incubations and metabarcoding confirmed that parasites are a common component of marine protist communities at LTER-MC. Host-parasite interactions must be determined and quantified to understand their role and the impact they have on phytoplankton dynamics- European Union’s Horizon 2020 research and innovation programme under grant agreement No 730984, ASSEMBLE Plus project. - Spanish MICINN Project SMART (PID2020-112978GB-I00) - The research program LTER-MC is funded by the Stazione Zoologica Anton Dohrn- Physico-chemical variables obtained from CTD profile - Inorganic nutrients concentrations - Chlorophyll-a concentrations - Organic carbon and nitrogen concentrations - Phytoplankton abundances - Detections of chytrids - Metabarcoding ASV abundances from natural samples and incubations - Metabarcoding Taxonomic assignments of ASVs - Metabarcoding Sequences of ASVs - Compilation of images taken during incubations with diatomsPeer reviewe