The neutralizing effect of heparin on blood-derived antimicrobial compounds: impact on antibacterial activity and inflammatory response.

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.

A comparative study on antibiotic resistant Escherichia coli isolates from Austrian patients and wastewater-influenced Danube River water and biofilms.

Antimicrobial resistance (AMR) poses a major threat to human health worldwide. AMR can be introduced into natural aquatic ecosystems, for example, from clinical facilities via wastewater emissions. Understanding AMR patterns in environmental populations of bacterial pathogens is important to elucidate propagation routes and develop mitigation strategies. In this study, AMR patterns of Escherichia coli isolates from urinary tract infections and colonised urinary catheters of inpatients and outpatients were compared to isolates from the Danube River within the same catchment in Austria to potentially link environmental with clinical resistance patterns. Susceptibility to 20 antibiotics was tested for 697 patient, 489 water and 440 biofilm isolates. The resistance ratios in patient isolates were significantly higher than in the environmental isolates and higher resistance ratios were found in biofilm in comparison to water isolates. The role of the biofilm as potential sink of resistances was reflected by two extended-spectrum beta-lactamase (ESBL) producing isolates in the biofilm while none were found in water, and by higher amoxicillin/clavulanic acid resistance ratios in biofilm compared to patient isolates. Although, resistances to last-line antibiotics such as carbapenems and tigecycline were found in the patient and in the environmental isolates, they still occurred at low frequency.

Domestic hot-water boilers harbour active thermophilic bacterial communities distinctly different from those in the cold-water supply.

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.

Introducing bacterial community turnover times to elucidate temporal and spatial hotspots of biological instability in a large Austrian drinking water distribution network.

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.

Epidemiological cut-off values for non-O1/ non-O139 Vibrio cholerae disc diffusion data generated by standardised methods.

This work generates the data needed to set epidemiological cut-off values for disc-diffusion zone measurements of Vibrio cholerae. The susceptibility of 147 European isolates of non-O1/non-O139 V. cholerae to 19 antibiotics was established using a standardised disc diffusion method which specified incubation of Mueller Hinton agar plates at 35°C. Epidemiological cut-off values were calculated by analysis of the zone size data with the statistically based normalised resistance interpretation method. Cut-off values for 17 agents were calculated by analysis of the aggregated data from all 4 laboratories participating in this study. The cut-off values calculated were ≥18 mm for amoxicillin/clavulanate, ≥18 mm for amikacin, ≥19 mm for ampicillin, ≥27 mm for cefepime, ≥31 mm for cefotaxime, ≥24 mm for ceftazidime, ≥24 mm for chloramphenicol, ≥31 mm for ciprofloxacin, ≥16 mm for erythromycin, ≥ 27 mm for florfenicol, ≥16 mm for gentamicin, ≥23 mm for imipenem, ≥25 mm for meropenem, ≥29 mm for nalidixic acid, ≥28 mm for norfloxacin, ≥13 mm for streptomycin and ≥23 mm for tetracycline. For the other 2 agents the data from 1 laboratory was excluded from the censored aggregation because the data from that laboratory was considered excessively imprecise. The cut-off values for these 2 agents calculated for the aggregation of the data from 3 laboratories were ≥23 mm for trimethoprim and ≥24 mm for trimethoprim/sulfamethoxazole. These zone size data will be submitted to the Clinical Laboratory Standards Institute (CLSI) and European Committee for Antimicrobial Susceptibility Testing (EUCAST) for their consideration in setting international consensus epidemiological cut-off values for non O1/non-O139 V. cholerae.

Short-term impacts of a large cultural event on the microbial pollution status of a pre-alpine river.

Rivers are impacted by microbial faecal pollution from various sources. We report on a short-term faecal pollution event at the pre-alpine Austrian river Traisen caused by the large cultural event FM4 Frequency music festival, with around 200,000 visitors over 4 days. We observed a massive increase of the faecal indicator bacteria (FIB) intestinal enterococci during the event, while Escherichia coli concentrations were only slightly elevated. This increase poses a significant potential health threat to visitors and people recreating downstream of the festival area. A plausible explanation for the uncoupling of the two FIBs may have been a differential persistence caused by a combination of factors including water temperature, solar radiation, and the excessive presence of personal care products (PCPs) in the river water. However, a potential impact of PCPs on FIB assay performance cannot be ruled out. Our observations are relevant for other intensively used bathing sites; detailed investigations on persistence and assay performance of the FIB in response to different ingredients of PCPs are highly recommended. We conclude that for future festivals at this river or other festivals taking place under similar settings, a more effective management is necessary to reduce deterioration in water quality and minimise health risks.

Bacterial bioindicators enable biological status classification along the continental Danube river.

Despite the importance of bacteria in aquatic ecosystems and their predictable diversity patterns across space and time, biomonitoring tools for status assessment relying on these organisms are widely lacking. This is partly due to insufficient data and models to identify reliable microbial predictors. Here, we show metabarcoding in combination with multivariate statistics and machine learning allows to identify bacterial bioindicators for existing biological status classification systems. Bacterial beta-diversity dynamics follow environmental gradients and the observed associations highlight potential bioindicators for ecological outcomes. Spatio-temporal links spanning the microbial communities along the river allow accurate prediction of downstream biological status from upstream information. Network analysis on amplicon sequence veariants identify as good indicators genera Fluviicola, Acinetobacter, Flavobacterium, and Rhodoluna, and reveal informational redundancy among taxa, which coincides with taxonomic relatedness. The redundancy among bacterial bioindicators reveals mutually exclusive taxa, which allow accurate biological status modeling using as few as 2-3 amplicon sequence variants. As such our models show that using a few bacterial amplicon sequence variants from globally distributed genera allows for biological status assessment along river systems.

Vibrio cholerae-An emerging pathogen in Austrian bathing waters?

Vibrio cholerae, an important human pathogen, is naturally occurring in specific aquatic ecosystems. With very few exceptions, only the cholera-toxigenic strains belonging to the serogroups O1 and O139 are responsible for severe cholera outbreaks with epidemic or pandemic potential. All other nontoxigenic, non-O1/non-O139 V. cholerae (NTVC) strains may cause various other diseases, such as mild to severe infections of the ears, of the gastrointestinal and urinary tracts as well as wound and bloodstream infections. Older, immunocompromised people and patients with specific preconditions have an elevated risk. In recent years, worldwide reports demonstrated that NTVC infections are on the rise, caused amongst others by elevated water temperatures due to global warming.The aim of this review is to summarize the knowledge gained during the past two decades on V. cholerae infections and its occurrence in bathing waters in Austria, with a special focus on the lake Neusiedler See. We investigated whether NTVC infections have increased and which specific environmental conditions favor the occurrence of NTVC. We present an overview of state of the art methods that are currently available for clinical and environmental diagnostics. A preliminary public health risk assessment concerning NTVC infections related to the Neusiedler See was established. In order to raise awareness of healthcare professionals for NTVC infections, typical symptoms, possible treatment options and the antibiotic resistance status of Austrian NTVC isolates are discussed.

Have genetic targets for faecal pollution diagnostics and source tracking revolutionized water quality analysis yet?

The impacts of nucleic acid-based methods - such as PCR and sequencing - to detect and analyze indicators, genetic markers or molecular signatures of microbial faecal pollution in health-related water quality research were assessed by rigorous literature analysis. A wide range of application areas and study designs has been identified since the first application more than 30 years ago (>1100 publications). Given the consistency of methods and assessment types, we suggest defining this emerging part of science as a new discipline: genetic faecal pollution diagnostics (GFPD) in health-related microbial water quality analysis. Undoubtedly, GFPD has already revolutionized faecal pollution detection (i.e., traditional or alternative general faecal indicator/marker analysis) and microbial source tracking (i.e., host-associated faecal indicator/marker analysis), the current core applications. GFPD is also expanding to many other research areas, including infection and health risk assessment, evaluation of microbial water treatment, and support of wastewater surveillance. In addition, storage of DNA extracts allows for biobanking, which opens up new perspectives. The tools of GFPD can be combined with cultivation-based standardized faecal indicator enumeration, pathogen detection, and various environmental data types, in an integrated data analysis approach. This comprehensive meta-analysis provides the scientific status quo of this field, including trend analyses and literature statistics, outlining identified application areas, and discusses the benefits and challenges of nucleic acid-based analysis in GFPD.

Low antimicrobial resistance in Escherichia coli isolates from two large Austrian alpine karstic spring catchments.

The increasing occurrence of antibiotic resistant bacteria poses a threat to global public health. Clinically relevant resistances also spread through the environment. Aquatic ecosystems in particular represent important dispersal pathways. In the past, pristine water resources have not been a study focus, although ingestion of resistant bacteria through water consumption constitutes a potentially important transmission route. This study assessed antibiotic resistances in Escherichia coli populations in two large well-protected and well-managed Austrian karstic spring catchments representing essential groundwater resources for water supply. E. coli were detected seasonally only during the summer period. By screening a representative number of 551 E. coli isolates from 13 sites in two catchments, it could be shown that the prevalence of antibiotic resistance in this study area is low. 3.4 % of the isolates showed resistances to one or two antibiotic classes, 0.5 % were resistant to three antibiotic classes. No resistances to critical and last-line antibiotics were detected. By integrating fecal pollution assessment and microbial source tracking, we could infer that ruminants were the main hosts for antibiotic resistant bacteria in the studied catchment areas. A comparison with other studies on antibiotic resistances in karstic or mountainous springs highlighted the low contamination status of the model catchments studied here, most likely due to the high protection and careful management while other, less pristine catchments showed much higher antibiotic resistances. We demonstrate that studying easily accessible karstic springs allows a holistic view on large catchments concerning the extent and origin of fecal pollution as well as antibiotic resistance. This representative monitoring approach is also in line with the proposed update of the EU Groundwater Directive (GWD).

First report on the occurrence of Vibrio cholerae nonO1/nonO139 in natural and artificial lakes and ponds in Serbia: Evidence for a long-distance transfer of strains and the presence of Vibrio paracholerae.

Vibrio cholerae are natural inhabitants of specific aquatic environments. Strains not belonging to serogroups O1 and O139 are usually unable to produce cholera toxin and cause cholera. However, non-toxigenic V. cholerae (NTVC) are able to cause a variety of mild-to-severe human infections (via seafood consumption or recreational activities). The number of unreported cases is considered substantial, as NTVC infections are not notifiable and physicians are mostly unaware of this pathogen. In the northern hemisphere, NTVC infections have been reported to increase due to global warming. In Eastern Europe, climatic and geological conditions favour the existence of inland water-bodies harbouring NTVC. We thus investigated the occurrence of NTVC in nine Serbian natural and artificial lakes and ponds, many of them used for fishing and bathing. With the exception of one highly saline lake, all investigated water-bodies harboured NTVC, ranging from 5.4 × 101 to 1.86 × 104  CFU and 4.5 × 102 to 5.6 × 106 genomic units per 100 ml. The maximum values observed were in the range of bathing waters in other countries, where infections have been reported. Interestingly, 7 out of 39 fully sequenced presumptive V. cholerae isolates were assigned as V. paracholerae, a recently described sister species of V. cholerae. Some clones and sublineages of both V. cholerae and V. paracholerae were shared by different environments indicating an exchange of strains over long distances. Important pathogenicity factors such as hlyA, toxR, and ompU were present in both species. Seasonal monitoring of ponds/lakes used for recreation in Serbia is thus recommended to be prepared for potential occurrence of infections promoted by climate change-induced rise in water temperatures.

Probabilistic fecal pollution source profiling and microbial source tracking for an urban river catchment.

We developed an innovative approach to estimate the occurrence and extent of fecal pollution sources for urban river catchments. The methodology consists of 1) catchment surveys complemented by literature data where needed for probabilistic estimates of daily produced fecal indicator (FIBs, E. coli, enterococci) and zoonotic reference pathogen numbers (Campylobacter, Cryptosporidium and Giardia) excreted by human and animal sources in a river catchment, 2) generating a hypothesis about the dominant sources of fecal pollution and selecting a source targeted monitoring design, and 3) verifying the results by comparing measured concentrations of the informed choice of parameters (i.e. chemical tracers, C. perfringensspores, and host-associated genetic microbial source tracking (MST) markers) in the river, and by multi-parametric correlation analysis. We tested the approach at a study area in Vienna, Austria. The daily produced microbial particle numbers according to the probabilistic estimates indicated that, for the dry weather scenario, the discharge of treated wastewater (WWTP) was the primary contributor to fecal pollution. For the wet weather scenario, 80-99 % of the daily produced FIBs and pathogens resulted from combined sewer overflows (CSOs) according to the probabilistic estimates. When testing our hypothesis in the river, the measured concentrations of the human genetic fecal marker were log10 4 higher than for selected animal genetic fecal markers. Our analyses showed for the first-time statistical relationships between C. perfringens spores (used as conservative microbial tracer for communal sewage) and a human genetic fecal marker (i.e. HF183/BacR287) with the reference pathogen Giardia in river water (Spearman rank correlation: 0.78-0.83, p < 0.05. The developed approach facilitates urban water safety management and provides a robust basis for microbial fate and transport models and microbial infection risk assessment.

Corrigendum: Genetic microbial source tracking support QMRA modeling for a riverine wetland drinking water resource.

[This corrects the article DOI: 10.3389/fmicb.2021.668778.].

Wastewater-based epidemiology in countries with poor wastewater treatment - Epidemiological indicator function of SARS-CoV-2 RNA in surface waters.

Wastewater-based epidemiology (WBE) surveillance of COVID-19 and other future outbreaks is a challenge for developing countries as most households are not connected to a sewerage system. In December 2019, SARS-CoV-2 RNA was detected in the Danube River at a site severely affected by wastewaters from Belgrade. Rivers are much more complex systems than wastewater systems, and efforts are needed to address all the factors influencing the adoption of WBE as an alternative to targeting raw wastewater. Our objective was to provide a more detailed insight into the potential of SARS-CoV-2 surveillance in Serbian surface waters for epidemiological purposes. Water samples were collected at 12 sites along the Sava and Danube rivers in Belgrade during the fourth COVID-19 wave in Serbia that started in late February 2021. RNA was concentrated using Amicon Ultra-15 centrifugal filters and quantified using RT-qPCR with primer sets targeting nucleocapsid (N1 and N2) and envelope (E) protein genes. Microbiological (faecal indicator bacteria and human and animal genetic faecal source tracking markers), epidemiological, physicochemical and hydromorphological parameters were analysed in parallel. From 44 samples, SARS-CoV-2 RNA was detected in 31, but only at 4 concentrations above the level of quantification (ranging from 8.47 × 103 to 2.07 × 104 gc/L). The results indicated that surveillance of SARS-CoV-2 RNA in surface waters as ultimate recipients could be used as an epidemiological early-warning tool in countries lacking wastewater treatment and proper sewerage infrastructure. The performance of the applied approach, including advanced sampling site characterization to trace and identify sites with significant raw sewage influence from human populations, could be further improved by adaptation of the methodology for processing higher volumes of samples and enrichment factors, which should provide the quantitative instead of qualitative data needed for WBE.

Identifying Inorganic Turbidity in Water Samples as Potential Loss Factor During Nucleic Acid Extraction: Implications for Molecular Fecal Pollution Diagnostics and Source Tracking.

Molecular diagnostic methods are increasingly applied for food and environmental analysis. Since several steps are involved in sample processing which can affect the outcome (e.g., adhesion of DNA to the sample matrix, inefficient precipitation of DNA, pipetting errors and (partial) loss of the DNA pellet during DNA isolation), quality control is essential at all processing levels. In soil microbiology, particular attention has been paid to the inorganic component of the sample matrix affecting DNA extractability. In water quality testing, however, this aspect has mostly been neglected so far, although it is conceivable that these mechanisms have a similar impact. The present study was therefore dedicated to investigate possible matrix effects on results of water quality analysis. Field testing in an aquatic environment with pronounced chemo-physical gradients [total suspended solids (TSS), inorganic turbidity, total organic carbon (TOC), and conductivity] indicated a negative association between DNA extractability (using a standard phenol/chloroform extraction procedure) and turbidity (spearman ρ = -0.72, p < 0.001, n = 21). Further detailed laboratory experiments on sediment suspensions confirmed the hypothesis of inorganic turbidity being the main driver for reduced DNA extractability. The observed effects, as known from soil samples, were also indicated to result from competitive effects for free charges on clay minerals, leading to adsorption of DNA to these inorganic particles. A protocol modification by supplementing the extraction buffer with salmon sperm DNA, to coat charged surfaces prior to cell lysis, was then applied on environmental water samples and compared to the standard protocol. At sites characterized by high inorganic turbidity, DNA extractability was significantly improved or made possible in the first place by applying the adapted protocol. This became apparent from intestinal enterococci and microbial source tracking (MST)-marker levels measured by quantitative polymerase chain reaction (qPCR) (100 to 10,000-fold median increase in target concentrations). The present study emphasizes the need to consider inorganic turbidity as a potential loss factor in DNA extraction from water-matrices. Negligence of these effects can lead to a massive bias, by up to several orders of magnitude, in the results of molecular MST and fecal pollution diagnostics.

Genetic Microbial Source Tracking Support QMRA Modeling for a Riverine Wetland Drinking Water Resource.

Riverine wetlands are important natural habitats and contain valuable drinking water resources. The transport of human- and animal-associated fecal pathogens into the surface water bodies poses potential risks to water safety. The aim of this study was to develop a new integrative modeling approach supported by microbial source tracking (MST) markers for quantifying the transport pathways of two important reference pathogens, Cryptosporidium and Giardia, from external (allochthonous) and internal (autochthonous) fecal sources in riverine wetlands considering safe drinking water production. The probabilistic-deterministic model QMRAcatch (v 1.1 python backwater) was modified and extended to account for short-time variations in flow and microbial transport at hourly time steps. As input to the model, we determined the discharge rates, volumes and inundated areas of the backwater channel based on 2-D hydrodynamic flow simulations. To test if we considered all relevant fecal pollution sources and transport pathways, we validated QMRAcatch using measured concentrations of human, ruminant, pig and bird associated MST markers as well as E. coli in a Danube wetland area from 2010 to 2015. For the model validation, we obtained MST marker decay rates in water from the literature, adjusted them within confidence limits, and simulated the MST marker concentrations in the backwater channel, resulting in mean absolute errors of < 0.7 log10 particles/L (Kruskal-Wallis p > 0.05). In the scenarios, we investigated (i) the impact of river discharges into the backwater channel (allochthonous sources), (ii) the resuspension of pathogens from animal fecal deposits in inundated areas, and (iii) the pathogen release from animal fecal deposits after rainfall (autochthonous sources). Autochthonous and allochthonous human and animal sources resulted in mean loads and concentrations of Cryptosporidium and Giardia (oo)cysts in the backwater channel of 3-13 × 109 particles/hour and 0.4-1.2 particles/L during floods and rainfall events, and in required pathogen treatment reductions to achieve safe drinking water of 5.0-6.2 log10. The integrative modeling approach supports the sustainable and proactive drinking water safety management of alluvial backwater areas.

Detection of SARS-CoV-2 RNA in the Danube River in Serbia associated with the discharge of untreated wastewaters.

In Serbia less than 13% of collected municipal wastewaters is being treated before their release in the environment. This includes all municipal wastewater discharges from Belgrade (capital city of Serbia; population 1,700,000). Previous research has identified the impacts of raw wastewater discharges from Belgrade on the Danube River, and this study investigated if such discharges also provided a pathway for SARS-CoV-2 RNA material. Samples were collected during the most critical circumstances that occurred so far within the COVID-19 pandemics in Serbia. Grab and composite samples were collected in December 2020, during the peak of the third wave (in terms of reported cases) at the site which receives the wastewater loads in Belgrade. Grab samples collected upstream and downstream of Belgrade were also analyzed. RNA was quantified using RT-qPCR with primer sets targeting nucleocapsid (N1 and N2) and envelope (E) protein genes. SARS-CoV-2 RNA (5.97 × 103 to 1.32 × 104 copies/L) was detected only in samples collected at the site strongly impacted by the wastewaters where all three applied primer sets gave positive signals. Determined concentrations correspond to those reported in wastewater influents sampled at treatment plants in other countries indicating an epidemiological indicator function of used approach for rivers with high pollution loads in countries with poor wastewater treatment.

Modelling the interplay of future changes and wastewater management measures on the microbiological river water quality considering safe drinking water production.

Rivers are important for drinking water supply worldwide. However, they are often impacted by pathogen discharges via wastewater treatment plants (WWTP) and combined sewer overflows (CSO). To date, accurate predictions of the effects of future changes and pollution control measures on the microbiological water quality of rivers considering safe drinking water production are hindered due to the uncertainty of the pathogen source and transport variables. The aim of this study was to test an integrative approach for an improved understanding of these effects, i.e. climate change and population growth as well as enhanced treatment at WWTPs and/or prevention of CSOs. We applied a significantly extended version of QMRAcatch (v1.0 Python), a probabilistic-deterministic model that combines fate and transport modelling with quantitative microbial infection risk assessment. The impact of climatic changes until the period 2035-2049 was investigated by a conceptual semi-distributed hydrological model, based on regional climate model outputs. QMRAcatch was calibrated and validated using site- and source-specific data (human-associated genetic microbial source tracking marker and enterovirus). The study showed that the degree to which future changes affect drinking water safety strongly depends on the type and magnitude of faecal pollution sources and are thus highly site- and scenario-specific. For example, if the load of pathogens from WWTPs is reduced through enhanced treatment, climate-change driven increases in CSOs had a considerable impact. Preventing CSOs and installing enhanced treatment at the WWTPs together had the most significant positive effect. The simultaneous consideration of source apportionment and concentrations of reference pathogens, focusing on human-specific viruses (enterovirus, norovirus) and cross-comparison with bacterial and protozoan pathogens (Campylobacter, Cryptosporidium), was found crucial to quantify these effects. While demonstrated here for a large, wastewater-impacted river, the approach is applicable at other catchments and pollution sources. It allows assessing future changes and selecting suitable pollution control measures for long-term water safety planning.

DNA aptamers against bacterial cells can be efficiently selected by a SELEX process using state-of-the art qPCR and ultra-deep sequencing.

DNA aptamers generated by cell-SELEX against bacterial cells have gained increased interest as novel and cost-effective affinity reagents for cell labelling, imaging and biosensing. Here we describe the selection and identification of DNA aptamers for bacterial cells using a combined approach based on cell-SELEX, state-of-the-art applications of quantitative real-time PCR (qPCR), next-generation sequencing (NGS) and bioinformatic data analysis. This approach is demonstrated on Enterococcus faecalis (E. faecalis), which served as target in eleven rounds of cell-SELEX with multiple subtractive counter-selections against non-target species. During the selection, we applied qPCR-based analyses to evaluate the ssDNA pool size and remelting curve analysis of qPCR amplicons to monitor changes in pool diversity and sequence enrichment. Based on NGS-derived data, we identified 16 aptamer candidates. Among these, aptamer EF508 exhibited high binding affinity to E. faecalis cells (KD-value: 37 nM) and successfully discriminated E. faecalis from 20 different Enterococcus and non-Enterococcus spp. Our results demonstrate that this combined approach enabled the rapid and efficient identification of an aptamer with both high affinity and high specificity. Furthermore, the applied monitoring and assessment techniques provide insight into the selection process and can be highly useful to study and improve experimental cell-SELEX designs to increase selection efficiency.