This study presents the first rigorous, multi-parameter evaluation of near-real-time flow cytometry (FCM) for automated on-site monitoring at a model alpine karst spring used as a drinking water resource. We tested automated FCM for its capacity to indicate microbial cell inputs from the catchment surface (SRI) and to indirectly indicate diffuse faecal contamination (DFC). A nested 10-year study design combined low-frequency monitoring with high-frequency event sampling. The instruments tested in the study performed well under challenging field conditions and reliably quantified microbial cells, as validated against established methods. FCM parameters robustly detected SRI and, indirectly, DFC, in a catchment with diffuse animal faecal contamination sources. The same performance was observed for the online physico-chemical parameters UV254 and turbidity. The combination of these biotic and abiotic indicators outperformed single parameters in early warning analyses, demonstrating their potential for water safety planning by enabling timely and specific responses. The various aspects of the automated on-site monitoring devices used are discussed in detail. While automated FCM already supports the indirect detection of diffuse faecal pollution, further technical advances are necessary to extend its scope toward the specific and sensitive detection of faecal contamination.

Die biologische Stabilität von Trinkwasser ist ein zentrales Qualitätsziel, da mikrobielles Wachstum im Verteilungsnetz sensorische, betriebliche und hygienische Beeinträchtigungen verursachen kann. Die mikrobiologische Bewertung stützte sich lange Zeit auf kultivierungsbasierte Indikatoren, welche primär Veränderungen der kultivierbaren Fraktion erfassen und dynamische Prozesse innerhalb der mikrobiellen Gemeinschaft nur eingeschränkt abbilden. Vor diesem Hintergrund gewinnen integrative, kultivierungsunabhängige Ansätze zur Beurteilung der mikrobiellen Dynamik in Trinkwassersystemen zunehmend an Bedeutung. In diesem Beitrag wird die Umsatzzeit der bakteriellen Gemeinschaft (Englisch: bacterial community turnover time, BaCTT) als neuer, kultivierungsunabhängiger In-situ-Parameter zur Bestimmung der biologischen Stabilität vorgestellt. BaCTT basiert auf der Kombination eines Biomasseparameters (Gesamtzellzahl bestimmt mittels Durchflusszytometrie) und eines Aktivitätsparameters (bakterielle Kohlenstoffproduktion bestimmt mittels In-situ-3H‑Leucin-Inkorporation) und beschreibt die Zeitspanne, die eine bakterielle Gemeinschaft benötigt, um ihre Biomasse zu verdoppeln. Die Eignung von BaCTT wurde über einen Zeitraum von sieben Monaten in einem großen, chlordesinfizierten urbanen Trinkwasserversorgungssystem im Dauerbetrieb untersucht. Dabei wurden sowohl die Trinkwasseraufbereitung als auch unterschiedliche Bereiche des Verteilungsnetzes einbezogen. Während kultivierungsbasierte Parameter keine klaren räumlichen oder saisonalen Muster zeigten und Zellzahlen überwiegend von der Rohwassercharakteristik beeinflusst waren, ermöglichte BaCTT eine Identifikation räumlicher und zeitlicher Hotspots erhöhter mikrobieller Dynamik. Die Ergebnisse zeigen, dass BaCTT einen praxisnahen, zeitnah verfügbaren und einfach interpretierbaren Parameter zur integrativen Bestimmung der biologischen Stabilität in Trinkwassersystemen darstellt. Der Artikel schließt mit einer Einordnung der Ergebnisse im Hinblick auf die Evaluierung von BaCTT und zeigt dessen Potenzial als Grundlage für weiterführende Anwendungen und zukünftige Untersuchungen in der Trinkwasserversorgung auf. Dieser Artikel ist eine adaptierte und etwas verkürzte deutsche Version der internationalen englischen Peer-review-Publikation „Introducing bacterial community turnover times to elucidate temporal and spatial hotspots of biological instability in a large Austrian drinking water distribution network“, die 2024 in der Fachzeitschrift Water Research erschienen ist (Campostrini et al. 2024). Dieses Thema wurde im Zuge der Forschungskooperation Vienna Water Resource Systems 2020+ (VIWA 20+) beim Mid-Term-Symposium 2025 behandelt.

Cryptosporidium-Oozysten stellen aufgrund ihrer hohen Resistenz gegenüber chemischen Desinfektionsmitteln und ihrer Rolle bei bedeutenden wasser-assoziierten Krankheitsausbrüchen nach wie vor eine Herausforderung für die Trinkwassersicherheit dar. Bei der Trinkwasseraufbereitung ist die Sandfiltration ein wichtiger Baustein im Multi-Barrieren-System. Eine zuverlässige Bewertung des Rückhaltevermögens der Sandfiltration ist daher sowohl für die Planung von Aufbereitungsanlagen als auch für die betriebliche Qualitätssicherung unerlässlich. Da die Arbeit mit lebenden Oozysten komplex und kostspielig ist, werden in der Forschung und bei Pilotversuchen häufig Ersatzpartikel, sogenannte Surrogate eingesetzt. Dennoch ist die Eignung der gängigen Surrogate in der wasserwirtschaftlichen Praxis noch nicht ausreichend erforscht. In dieser Studie untersuchen wir vier potenzielle Surrogate unter Bedingungen, die für Trinkwasser und die Schnellsandfiltration relevant sind: Bakterien-Sporen (Bacillus subtilis), unbeschichtete gelb-grüne Mikrosphären, unbeschichtete gelb-orange Mikrosphären und Glykoprotein-beschichtete gelb-orange Mikrosphären. Die Ergebnisse zeigen, dass die Eignung der Surrogate als Modelle für Cryptosporidien-Oocysten stark von physikochemischen Eigenschaften wie Partikelgröße, Oberflächenladung, Hydrophobizität und vom Vorhandensein von Oberflächen-Makromolekülen abhängt. Die Bewertung der Rückhalteleistung der Oozysten und Surrogate in den Sandfilter-Experimenten ergab, dass Bakteriensporen und die gelb-grünen Mikrosphären die Rückhaltung der Oozysten deutlich unterschätzen, also zu konservativ sind, die unbeschichteten gelb-orangen Mikrosphären die Retention überschätzen, wodurch ein potenzielles Sicherheitsrisiko entstehen kann, und dass die Glykoprotein-beschichteten Mikrosphären die höchste Übereinstimmung mit dem Verhalten von Cryptosporidium-Oozysten zeigten. Die Studie belegt, dass eine sorgfältige Auswahl der Surrogate entscheidend für eine effektive Dimensionierung von Sandfiltern sowie für die Validierung und die Darstellung der Reinigungsleistung ist. Der Einsatz präzise abgestimmter Surrogate bewirkt, das Vertrauen in die Sandfiltration zu stärken und vermeidet unnötige Überdimensionierungen, wobei Sicherheitsmargen und Kosteneffizienz gegeneinander abgewogen werden müssen. Diese Arbeit wurde im Zuge der Forschungskooperation Vienna Water Resource Systems 2020+ erstellt und stellt eine für die Österreichische Abfall- und Wasserwirtschaft abgestimmte Version der im Journal of Water Process Engineering im Jahr 2025 publizierten Originalarbeit dar (Stevenson et al. 2025).

Die zunehmende Verbreitung antibiotikaresistenter Bakterien mit klinischer Relevanz stellt eine Gefahr für die globale Gesundheit dar. Klinisch relevante Resistenzen finden sich in zunehmendem Maße auch in der Umwelt. Aquatische Ökosysteme fungieren dabei als potenziell wichtige Ausbreitungswege. In dieser Studie wurden Antibiotikaresistenzen in Escherichia coli Populationen in zwei großen, extensiv bewirtschafteten österreichischen Karstquelleinzugsgebieten in den nördlichen Kalkalpen untersucht, die auch Grundwasserressourcen für die Trinkwassergewinnung darstellen. E. coli sind nicht nur klinisch relevante Krankheitserreger, sondern werden auch als standardisierte mikrobiologische Fäkalindikatoren für das Auftreten von Antibiotikaresistenzen in der Umwelt verwendet. E. coli konnten in den Quellwässern nur während der Sommermonate nachgewiesen werden, und zeigten einen Zusammenhang mit der Präsenz von Wiederkäuern in den Einzugsgebieten, welcher durch eine Korrelation mit Wiederkäuer-assoziierten mikrobiellen genetischen Source-Tracking-Markern bestätigt werden konnte. Durch die Untersuchung einer repräsentativen Anzahl von 551 E.-coli-Isolaten von 13 Standorten in den zwei Einzugsgebieten konnte gezeigt werden, dass die Prävalenz von Antibiotikaresistenzen in diesem Untersuchungsgebiet gering ist. In 3,4

Die Kenntnis über Persistenz bzw. den Zerfall genetischer fäkaler Marker ist eine grundlegende Voraussetzung für ihre Nutzung als Indikatoren, da ihr Verhalten in der Umwelt – im Gegensatz zu dem vieler chemischer Stoffe – von verschiedenen Prozessen beeinflusst wird. Obwohl PCR-basierte Diagnostik weltweit zunehmend zur Quantifizierung fäkaler Verunreinigungen in Wasserressourcen eingesetzt wird, ist das Wissen über die Stabilität genetischer fäkaler Marker bislang nur begrenzt verfügbar. Hier wird die – nach unserem Kenntnisstand – erste vergleichende Mikrokosmen-Studie vorgestellt, in der die Persistenz häufig verwendeter qPCR-basierter und primär human-assoziierter, genetischer fäkaler Marker (crAssphage bzw. HF183/BacR287 und BacHum) in einem großen europäischen Flusssystem untersucht wurde. Weiters wurde das Persistenzverhalten der kulturbasierten Fäkalindikator-Organismen (FIO) Escherichia coli, intestinale Enterokokken, Clostridium-perfringens-Sporen und somatische Coliphagen mit standardisierten Methoden bestimmt, um einen direkten Vergleich zwischen neuen molekularen und den auf Anzucht etablierten Verfahren zu ermöglichen. Für die Mikrokosmen wurde Flusswasser mit unbehandeltem Abwasser aus zwei kommunalen Mischwasserkanalisationen versetzt, und bei 8 °C bzw. 22 °C für bis zu 66 Tage gelagert. Zusätzlich wurden zwei Filtrationsmethoden für die Aufkonzentrierung der Mikroorganismen aus den Wasserproben getestet: ein Cellulose-Mischester-Filter (MCE), der für Viren/Phagen und Bakterien entwickelt wurde, und ein Polycarbonatfilter (PC), der ausschließlich Bakterien erfasst. Als Maß für den Abbau eines Markers oder für die Inaktivierung eines FIO wurden T90-Werte – die Zeit bis zum Absinken der Konzentration eines Markers/FIO auf 10

Microplastics originating from pellets, paints, fragmented macroplastics, textiles, tire wear, and personal care products enter sewage and can aggregate with viruses during treatment. After sludge application, these aggregates may infiltrate soils and enter groundwater, yet their aggregation mechanisms and effects on virus survival and transport remain unclear. To address this gap, we conducted a laboratory study to examine the effect of microplastics on virus persistence and transport in groundwater. Batch experiments were conducted by mixing the PRD1 bacteriophage (a surrogate for adenovirus) with microplastics in groundwater at various temperatures. Column experiments using saturated quartz sand were performed to evaluate the effect of microplastics on virus transport in groundwater. Microplastic particles were quantified using solid-phase cytometry, while PRD1 was enumerated using both molecular and culture-based methods. Experimental data were analyzed using the HYDRUS-1D 2-site attachment-detachment model and colloid filtration theory. Our experimental findings suggest that microplastics significantly influence viral stability and transport in groundwater. Batch experiments revealed a marked decrease in the persistence of infective viruses in the presence of microplastics, reducing viral longevity in the environment. On the other hand, co-transport experiments showed that microplastics promoted the transport of both total and infective viruses through saturated quartz sand. This dual role underscores the complex influence of microplastics in groundwater: while they may reduce virus viability, their ability to enhance viral mobility represents a significant pathway for virus dissemination. These findings highlight the need to consider microplastics not only as pollutants but also as vectors that can potentially increase the risk of waterborne transmission and compromise groundwater safety.

Dem Vorkommen von Antibiotikaresistenzen in Oberflächengewässern und Abwasser wird zunehmend Augenmerk geschenkt. Neben einer wachsenden Anzahl an wissenschaftlichen Publikationen wurde auch in der neuen Version der kommunalen Abwasserrichtlinie der EU ein Antibiotikaresistenz-Monitoring von Kläranlagen mit einem Einzugsgebiet > 100.000 EGW aufgenommen. Um die Datenlage zum Vorkommen von Antibiotikaresistenzen in österreichischen Flüssen zu erweitern, wurden umfassende quantitative Informationen hierzu in Wasserproben von vier niederösterreichischen Flüssen erhoben. Die Probenahmestellen reichten von eher unberührten Flussoberläufen bis zu Stellen flussabwärts von Kläranlagen, mit teilweise klinischen Abwässern im Einzugsgebiet. Für eine robuste Analyse der Antibiotikaresistenzen wurde ein dualer methodischer Ansatz gewählt. Dabei wurde sowohl das Vorkommen von Schlüssel-Resistenzgenen im gesamten Mikrobiom (das „Resistom“) mittels direkter molekularbiologisch/genetischer Diagnostik als auch die Häufigkeit phänotypischer Resistenzen (kultivierungsbasiert) in einem klinisch hoch relevanten Indikatorbakterium (Escherichia coli) berücksichtigt. Die Konzentration von neun genetischen Antibiotikaresistenzmarkern und die phänotypischen Resistenzen von 2736 E.-coli-Isolaten gegen 20 Antibiotika wurden zusätzlich mit modernen genetischen Fäkalmarkern verknüpft, die auch eine genetische Rückverfolgung der Verschmutzungsquellen umfasste (mikrobiologische Herkunftsbestimmung). Abwassereinleitungen hatten einen deutlichen Einfluss auf die Antibiotikaresistenzsituation. Phänotypische und genetische Resistenzwerte waren flussabwärts von Kläranlagen deutlich höher, und an diesen Standorten wurden klinisch hoch relevante Resistenzgene wie blaKPC oder blaOXA48 sowie phänotypische Resistenzen gegen Imipenem und Colistin gefunden. Auf Basis dieser Daten liefert die Studie ein erstes quantitatives Referenz- oder Bezugsniveau der Antibiotikaresistenzen für einen Teil des Donaueinzugsgebiets in Niederösterreich. Im Vergleich zu Flüssen in Europa und anderen Teilen der Welt befindet sich die Antibiotikaresistenzsituation in den untersuchten niederösterreichischen Flüssen auf einem niedrigen bis moderaten Niveau. Dennoch ist aufgrund der durch den Klimawandel bedingten Zunahme von Starkregenereignissen mit einem Anstieg von Mischwasserüberläufen sowie von diffusen Einträgen von Antibiotikaresistenzen in den Flüssen zu rechnen. Gleichzeitig ist zu erwarten, dass die in der neuen EU-Abwasserrichtlinie festgelegten Fortschritte bei der Abwasserbehandlung und der Infrastruktur zu einer Verringerung der Eintragsmengen von Antibiotikaresistenzen aus Punktquellen in die Umwelt führen werden.

BackgroundA world without antibiotics is hard to conceive. They have revolutionized the treatment landscape for bacterial infections, reducing mortality rates and enabling complex medical procedures. However, their widespread use has fueled the rise of antimicrobial resistance, a growing global health threat that demands new antibacterial therapies and strategies to preserve the efficacy of existing treatments. Among promising candidates, antimicrobial compounds (AMCs) offer broad-spectrum antimicrobial activity with a lower risk of resistance development. Recent studies suggest that unfractionated heparin, a commonly used anticoagulant, reduces the antibacterial and endotoxin-neutralizing activity of blood-derived AMCs, likely through ionic interactions.MethodsGiven the prevalence of negatively charged anticoagulants in clinical settings, we aimed to explore the effects of unfractionated heparin, low molecular weight heparin, and fondaparinux on the antibacterial activity of AMCs and antibiotics (colistin, daptomycin, gentamicin, imipenem, ofloxacin, and vancomycin).ResultsOur results revealed that both unfractionated and low molecular weight heparin markedly impaired the antibacterial activity of AMCs and positively charged antibiotics, whereas fondaparinux showed no such effect. For instance, exposure to 2.5 IU/mL of unfractionated and low molecular weight heparin led to a significant increase in the minimal inhibitory and minimal bactericidal concentrations of colistin and gentamicin.ConclusionsThese findings support our hypothesis that specific heparin-based anticoagulants interfere with the activity of blood-derived AMCs and positively charged antibiotics, reducing their efficacy in vitro. Our research aims to provide a foundation for future studies focused on optimizing anticoagulant use in clinical settings, ultimately improving patient outcomes in the ongoing fight against multidrug-resistant bacteria.

Iron and manganese (Fe/Mn) often lead to aesthetic quality issues in water supply. Strong and problematic black-brown particle formation was persistently observed in an alluvial drinking water well, even though oxygen enrichment probes, intended for in situ i.e., subsurface iron/manganese removal, were installed. To investigate the cause of the problem, a comparative and multiparametric approach was undertaken at the problematic well, seven additional wells (with 0.3 to 70 km distance to the affected well) and all the adjacent surface waters. Via a time-series investigation of up to 2.5 years, microbiological analysis (high-throughput 16S rRNA gene amplicon sequencing, total cell count) and chemical analysis (high-resolution elemental analysis using inductively coupled mass spectrometry and others) of the water samples were performed. Results revealed previously unreported, extremely dynamic, and seasonally recurring patterns of genus Crenothrix (a sheathed, filamentous bacterial population) in water samples obtained from the particle-affected well. Crenothrix spp. dominated the microbial community in summer months (up to 82 % relative abundance), being virtually absent in winter. Explanatory models for the high dynamics and association with bio-geochemical processes were established. These included methane formation and manganese mobilization in relation to riverbank filtration in the summer months, as well as changing aerobic and anaerobic conditions in the aquifer. Dominance of Crenothrix spp. in the affected well, low abundance in weak particle-affected wells, and total absence in non-affected wells was observed. This led to the suggestion of Crenothrix spp. as a technical indicator for Fe/Mn treatment failure for alluvial groundwater (e.g., genetic marker quantification by q/dPCR), to be evaluated in future studies regarding their applicability across a broader geographic context. Despite being first described in association with drinking water deterioration 150 years ago, this is the first study reporting seasonally recurring dominant patterns of Crenothrix spp. in association with operational/aesthetic issues for drinking water production.

Understanding bacterial dynamics in large river systems is crucial for predicting continental-scale ecological functioning under anthropogenic pressures. Here, two consecutive surveys 6-years apart along the 2600 km Danube River found that carbon incorporation per cell and hour decreased by 5000 atoms every kilometer and that cells multiplied five times during their travel down the entire river. Resolving these cell turnovers taxonomically revealed taxa with a hundredfold difference from these average numbers. Bacterial community turnover was due to replacement (phylotype turnover) and could be linked to species sorting. This was despite an overall decrease in diversity richness downstream. Using linear models, we were able to relate carbon, cell, phylotype and diversity turnover rates to water residence time and discharge with outliers associated with human impacts. As such the reproduceable macroecological models predict microbial changes from anthropogenic and climate alterations along a continental drainage system providing insights into their ecological consequences. ### Competing Interest Statement The authors have declared no competing interest. FWF Austrian Science Fund, , W1219-N22, P25817-B22, P32464-B City of Vienna, , Groundwater Resource Systems Vienna Swedish Foundation for Strategic Research, , ICA10-0015 Swedish Research Council, , VR2012-4592 Uninett Sigma2 AS, , nn9744k

Modeling bacterial dynamics in large river systems is crucial for predicting continental-scale ecosystem functioning under anthropogenic pressures. Although the River Continuum and Metacommunity concepts have provided theoretical frameworks, quantitative parameters necessary for microbial macroecological models remain scarce. Here, we present results from two whole-river surveys, conducted six years apart along 2600 km of the Danube River. Using bacterial secondary production, cell counts, and 16S ribosomal RNA (rRNA) gene amplicon sequencing, we quantified carbon, cell, phylotype, and diversity turnover along the river. Carbon incorporation per cell declined with water travel time by 6000-21 000 atoms per hour. Bacterial cells multiplied every eight days, resulting in four to six doublings during downstream transport. Growth responses at the level of individual phylotypes differed up to a hundredfold from these bulk community estimates. Bacterial diversity dynamics were dominated by phylotype turnover rather than phylotype loss. Turnover ranged from 0.92 to 0.96 along the river, indicating an almost complete replacement of phylotypes with 2%-11% of headwater-associated amplicon sequence variants (ASVs) persisting under base-flow conditions. Richness declined gradually downstream at a rate of ~0.13 ASVs per hour. Variations in bacterial secondary production, cell abundance, and observed ASVs were best explained by models combining hydrological and water quality parameters, whereas beta diversity followed a gradual development primarily structured by water travel time. Together, these results identify water travel time as the key integrative parameter governing microbial macroecological dynamics along large rivers, with environmental conditions fine-tuning local responses. These models can help predict changes in microbial diversity and functioning under anthropogenic alterations.

The spread of antibiotic resistance (ABR) via surface waters is of increasing concern. Large-scale studies investigating ABR drivers in different water bodies and habitats with uniform quantitative methods are largely missing. Here, we present a comprehensive investigation on ABR occurrence and drivers in water and biofilms of four Austrian rivers over a one-year-cycle using a harmonised quantitative tool-box and study-design. At the bacterial community level, human faecal pollution was a main factor driving the aquatic riverine resistome. Despite relatively low concentrations, also antibiotics and metals showed significant correlations, however to a different extent in the different rivers. At the organismic level, a decoupling of the Escherichia coli resistome from the bacterial community resistomes was observed. In biofilms, the relationships with anthropogenic pollution factors were heterogeneous and markedly dampened. Our results clearly show that general conclusions about the role of biofilms, the influence of pollution or the prevalence of resistance genes or phenotypic resistances must be drawn with caution. Results are dependent on the river and local situation of the sampling sites due to the large environmental heterogeneity. International harmonisation of the methodology and general awareness of this problem shall contribute to better understand environmental ABR to develop effective mitigation strategies.

Comparative information on decay characteristics of genetic faecal markers including crAssphage is very limited for water resources, although PCR-based indicator-diagnostics are increasingly used worldwide. To our knowledge, we performed the first comparative microcosm experiments to assess the decay behaviour of commonly used qPCR-based genetic faecal markers (crAssphages, HF183/BacR287, BacHum) in a large European river. In addition, cultivation-based standards (E. coli, intestinal enterococci, Clostridium perfringens spores, somatic coliphages) were enumerated to allow for a cross-comparison between novel and established targets. River water was spiked with raw sewage from two combined municipal sewer systems and incubated at 8 °C and 20 °C for up to 66 days. The focus of the experimental work was put on genetic faecal markers. Two different filtration methods targeting either virus/phages and bacteria (using mixed cellulose esters, MCE) or solely bacteria (using polycarbonate filters, PC) were also evaluated. Results indicated biphasic decay for all targets (i.e., initial fast followed by secondary slow decay), with Bacteroides markers showing lower persistence under all conditions when compared to crAssphage. In addition, a tendency for higher long-term persistence of crAssphage based on MCE filtration as compared to PC filtration was observed. Finally, a meta-data analysis was conducted, comparing the results obtained here with those from recent case studies. This analysis further substantiated the comparatively higher persistence and lower decay of crAssphage relative to Bacteroides markers and established FIO, with the sole exception of C. perfringens spores.

Ensuring biological stability in drinking water distribution systems (DWDSs) is important to reduce the risk of aesthetic, operational and hygienic impairments of the distributed water. Drinking water after treatment often changes in quality during transport due to interactions with pipe-associated biofilms, temperature increases and disinfectant residual decay leading to potential biological instability. To comprehensively assess the potential for biological instability in a large chlorinated DWDS, a tool-box of bacterial biomass and activity parameters was applied, introducing bacterial community turnover times (BaCTT) as a direct, sensitive and easy-to-interpret quantitative parameter based on the combination of 3H-leucine incorporation with bacterial biomass. Using BaCTT, hotspots and periods of bacterial growth and potential biological instability could be identified in the DWDS that is fed by water with high bacterial growth potential. A de-coupling of biomass from activity parameters was observed, suggesting that bacterial biomass parameters depict seasonally fluctuating raw water quality rather than processes related to biological stability of the finished water in the DWDS. BaCTT, on the other hand, were significantly correlated to water age, disinfectant residual, temperature and a seasonal factor, indicating a higher potential of biological instability at more distant sampling sites and later in the year. As demonstrated, BaCTT is suggested as a novel, sensitive and very useful parameter for assessing the biological instability potential. However, additional studies in other DWDSs are needed to investigate the general applicability of BaCTT depending on water source, applied treatment processes, biofilm growth potential on different pipe materials, or size, age and complexity of the DWDS.

Microplastics in urban runoff undergo rapid fragmentation and accumulate in the soil, potentially endangering shallow groundwater. To improve the understanding of microplastic transport in groundwater, column experiments were performed to compare the transport behavior of fragmented microplastics (FMPs ∼1-µm diameter) and spherical microplastics (SMPs ∼1-, 10-, and 20-µm diameter) in natural gravel (medium and fine) and quartz sand (coarse and medium). Polystyrene microspheres were physically abraded with glass beads to mimic the rapid fragmentation process. The experiments were conducted at a constant flow rate of 1.50 m day-1 by injecting two pore volumes of SMPs and FMPs. Key findings indicate that SMPs showed higher breakthrough, compared to FMPs in natural gravel, possibly due to size exclusion of the larger SMPs. Interestingly, FMPs exhibited higher breakthrough in quartz sand, likely due to tumbling and their tendency to align with flow paths, while both sizes (larger and smaller relative to FMPs) of SMPs exhibited higher removal in quartz sand. Therefore, an effect due to shape and size was observed.

Acting through a combination of direct and indirect pathogen clearance mechanisms, blood-derived antimicrobial compounds (AMCs) play a pivotal role in innate immunity, safeguarding the host against invading microorganisms. Besides their antimicrobial activity, some AMCs can neutralize endotoxins, preventing their interaction with immune cells and avoiding an excessive inflammatory response. In this study, we aimed to investigate the influence of unfractionated heparin, a polyanionic drug clinically used as anticoagulant, on the endotoxin-neutralizing and antibacterial activity of blood-derived AMCs. Serum samples from healthy donors were pre-incubated with increasing concentrations of heparin for different time periods and tested against pathogenic bacteria (Acinetobacter baumannii, Enterococcus faecium, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus) and endotoxins from E. coli, K. pneumoniae, and P. aeruginosa. Heparin dose-dependently decreased the activity of blood-derived AMCs. Consequently, pre-incubation with heparin led to increased activity of LPS and higher values of the pro-inflammatory cytokines tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6). Accordingly, higher concentrations of A. baumannii, E. coli, K. pneumoniae, and P. aeruginosa were observed as well. These findings underscore the neutralizing effect of unfractionated heparin on blood-derived AMCs in vitro and may lead to alternative affinity techniques for isolating and characterizing novel AMCs with the potential for clinical translation.

The Danube River is, at 2857 km, the second longest river in Europe and the most international river in the world with 19 countries in its catchment. Along the entire river, faecal pollution levels are mainly influenced by point-source emissions from treated and untreated sewage of municipal origin under base-flow conditions. In the past 2 decades, large investments in wastewater collection and treatment infrastructure were made in the European Union (EU) Member States located in the Danube River Basin (DRB). Overall, the share of population equivalents with appropriately biologically treated wastewater (without disinfection) has increased from 69

Running cold and hot water in buildings is a widely established commodity. However, interests regarding hygiene and microbiological aspects had so far been focussed on cold water. Little attention has been given to the microbiology of domestic hot-water installations (DHWIs), except for aspects of pathogenic Legionella. World-wide, regulations consider hot (or warm) water as 'heated drinking water' that must comply (cold) drinking water (DW) standards. However, the few reports that exist indicate presence and growth of microbial flora in DHWIs, even when supplied with water with disinfectant residual. Using flow cytometric (FCM) total cell counting (TCC), FCM-fingerprinting, and 16S rRNA-gene-based metagenomic analysis, the characteristics and composition of bacterial communities in cold drinking water (DW) and hot water from associated boilers (operating at 50 - 60 °C) was studied in 14 selected inhouse DW installations located in Switzerland and Austria. A sampling strategy was applied that ensured access to the bulk water phase of both, supplied cold DW and produced hot boiler water. Generally, 1.3- to 8-fold enhanced TCCs were recorded in hot water compared to those in the supplied cold DW. FCM-fingerprints of cold and corresponding hot water from individual buildings indicated different composition of cold- and hot-water microbial floras. Also, hot waters from each of the boilers sampled had its own individual FCM-fingerprint. 16S rRNA-gene-based metagenomic analysis confirmed the marked differences in composition of microbiomes. E.g., in three neighbouring houses supplied from the same public network pipe each hot-water boiler contained its own thermophilic bacterial flora. Generally, bacterial diversity in cold DW was broad, that in hot water was restricted, with mostly thermophilic strains from the families Hydrogenophilaceae, Nitrosomonadaceae and Thermaceae dominating. Batch growth assays, consisting of cold DW heated up to 50 - 60 °C and inoculated with hot water, resulted in immediate cell growth with doubling times between 5 and 10 h. When cold DW was used as an inoculum no significant growth was observed. Even boilers supplied with UVC-treated cold DW contained an actively growing microbial flora, suggesting such hot-water systems as autonomously operating, thermophilic bioreactors. The generation of assimilable organic carbon from dissolved organic carbon due to heating appears to be the driver for growth of thermophilic microbial communities. Our report suggests that a man-made microbial ecosystem, very close to us all and of potential hygienic importance, may have been overlooked so far. Despite consumers having been exposed to microbial hot-water flora for a long time, with no major pathogens so far been associated specifically with hot-water usage (except for Legionella), the role of harmless thermophiles and their interaction with potential human pathogens able to grow at elevated temperatures in DHWIs remains to be investigated.

The global spread of antimicrobial resistance (AMR) in the environment is a growing health threat. Large rivers are of particular concern as they are highly impacted by wastewater discharge while being vital lifelines serving various human needs. A comprehensive understanding of occurrence, spread and key drivers of AMR along whole river courses is largely lacking. We provide a holistic approach by studying spatiotemporal patterns and hotspots of antibiotic resistance genes (ARGs) along 2311 km of the navigable Danube River, combining a longitudinal and temporal monitoring campaign. The integration of advanced faecal pollution diagnostics and environmental and chemical key parameters allowed linking ARG concentrations to the major pollution sources and explaining the observed patterns. Nine AMR markers, including genes conferring resistance to five different antibiotic classes of clinical and environmental relevance, and one integrase gene were determined by probe-based qPCR. All AMR targets could be quantified in Danube River water, with intI1 and sul1 being ubiquitously abundant, qnrS, tetM, blaTEM with intermediate abundance and blaOXA-48like, blaCTX-M-1 group, blaCTX-M-9 group and blaKPC genes with rare occurrence. Human faecal pollution from municipal wastewater discharges was the dominant factor shaping ARG patterns along the Danube River. Other significant correlations of specific ARGs were observed with discharge, certain metals and pesticides. In contrast, intI1 was not associated with wastewater but was already established in the water microbiome. Animal contamination was detected only sporadically and was correlated with ARGs only in the temporal sampling set. During temporal monitoring, an extraordinary hotspot was identified emphasizing the variability within natural waters. This study provides the first comprehensive baseline concentrations of ARGs in the Danube River and lays the foundation for monitoring future trends and evaluating potential reduction measures. The applided holistic approach proved to be a valuable methodological contribution towards a better understanding of the environmental occurrence of AMR.