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Antimicrobial resistance (AMR) is a growing global health challenge, compromising bacterial infection treatments and necessitating robust surveillance and mitigation strategies. The overuse of antimicrobials in humans and farm animals has made them hotspots for AMR. However, the spread of AMR genes in wildlife and the environment represents an additional challenge, turning these areas into new AMR hotspots. Among the AMR bacteria considered to be of high concern for public health, Campylobacter has been the leading cause of foodborne infections in the European Union since 2005. This study examines the prevalence of AMR genes and virulence factors in Campylobacter isolates from wild birds and surface waters in Luxembourg. The findings reveal a significant prevalence of resistant Campylobacter strains, with 12% of C. jejuni from wild birds and 37% of C. coli from surface waters carrying resistance genes, mainly against key antibiotics like quinolones and tetracycline. This study underscores the crucial role of the environment in the spread of AMR bacteria and genes, highlighting the urgent need for enhanced surveillance and control measures to curb AMR in wildlife and environmental reservoirs and reduce transmission risks to humans. This research supports One Health approaches to tackling antimicrobial resistance and protecting human, animal, and environmental health.

Wastewater-based surveillance (WBS) has become a widespread method to monitor transmission of SARS-CoV-2 and other human pathogens in Europe. We conducted a survey about WBS systems' objectives, approaches, representativeness and usefulness in 10 invited European countries in 2023, i.e. Austria, Belgium, Denmark, Finland, Greece, Hungary, Italy, Luxembourg, the Netherlands and Norway. All countries completed the study questionnaire about their SARS-CoV-2 WBS systems, and shared information about WBS of other pathogens as deemed relevant. SARS-CoV-2 WBS systems primarily monitored national and subnational trends (population coverage: 25-99%), and a majority (8/10) also tracked variant distribution. Nine of 10 countries reported that their SARS-CoV-2 WBS systems were representative of their population and all countries remarked that the findings were valuable for public health decision-making. Results were shared with relevant public health authorities and published via dedicated websites and/or dashboards. WBS systems of other pathogens were mostly in the early stages, with some countries implementing pilots. Notable exceptions were the well-established poliovirus surveillance systems in Finland, Italy and the Netherlands. This study brings understanding the diverse landscape of WBS in Europe, offering insights for future developments and collaborations. Furthermore, it highlights the need for further integration of WBS into other European surveillance systems.

Campylobacter jejuni is the leading cause of bacterial gastroenteritis worldwide, but, unlike other foodborne pathogens, is not commonly reported as causing outbreaks. The population structure of the species is characterized by a high degree of genetic diversity, but the presence of stable clonally derived genotypes persisting in space and time, and potentially leading to diffuse outbreaks, has recently been identified. The spread of these recurring genotypes could be enhanced by wild birds, suspected to act as vectors for a wide range of microorganisms that can be transmissible to other animals or humans. This study assessed the genetic diversity of C. jejuni carriage in wild birds and surface waters to explore a potential link between these environments and the persistence over years of recurring lineages infecting humans in Luxembourg. These lineages corresponded to over 40 % of clinical isolates over a 4 year period from 2018 to 2021. While mainly exotic genotypes were recovered from environmental samples, 4 % of C. jejuni from wild birds corresponded to human recurring genotypes. Among them, a human clinical endemic lineage, occurring for over a decade in Luxembourg, was detected in one bird species, suggesting a possible contribution to the persistence of this clone and its multi-host feature. Whereas 27 % of wild birds were carriers of C. jejuni, confirming their role as spreader or reservoir, only three out of 59 genotypes overlapped with recurring human strains. While direct transmission of C. jejuni infection through wild birds remains questionable, they may play a key role in the environmental spreading of stable clones to livestock, and this issue merits further investigation.

Understanding the persistence of SARS-CoV-2 biomarkers in wastewater should guide wastewater-based epidemiology users in selecting best RNA biomarkers for reliable detection of the virus during current and future waves of the pandemic. In the present study, the persistence of endogenous SARS-CoV-2 were assessed during one month for six different RNA biomarkers and for the pepper mild mottle virus (PMMoV) at three different temperatures (4, 12 and 20 °C) in one wastewater sample. All SARS-CoV-2 RNA biomarkers were consistently detected during 6 days at 4° and differences in signal persistence among RNA biomarkers were mostly observed at 20 °C with N biomarkers being globally more persistent than RdRP, E and ORF1ab ones. SARS-CoV-2 signal persistence further decreased in a temperature dependent manner. At 12 and 20 °C, RNA biomarker losses of 1-log 10 occurred on average after 6 and 4 days, and led to a complete signal loss after 13 and 6 days, respectively. Besides the effect of temperature, SARS-CoV-2 RNA signals were more persistent in the particulate phase compared to the aqueous one. Finally, PMMoV RNA signal was highly persistent in both phases and significantly differed from that of SARS-CoV-2 biomarkers. We further provide a detailed overview of the latest literature on SARS-CoV-2 and PMMoV decay rates in sewage matrices., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

As the world's leading cause of human gastro-enteritis, the food- and waterborne pathogen Campylobacter needs to be intensively monitored through a One Health approach. Particularly, wild birds have been hypothesized to contribute to the spread of human clinical recurring C. jejuni genotypes across several countries. A major concern in studying epidemiological dynamics is resolving the large genomic diversity of strains circulating in the environment and various reservoirs, challenging to achieve with isolation techniques. Here, we applied a passive-filtration method to obtain isolates and in parallel recovered genotypes from metagenomic sequencing data from associated filter sweeps. For genotyping mixed strains, a reference-based computational workflow to predict allelic profiles of nine extended-MLST loci was utilized. We validated the pipeline by sequencing artificial mixtures of C. jejuni strains and observed the highest prediction accuracy when including obtained isolates as references. By analyzing metagenomic samples, we were able to detect over 20% additional genetic diversity and observed an over 50% increase in the potential to connect genotypes across wild-bird samples. With an optimized filtration method and a computational approach for genotyping strain mixtures, we provide the foundation for future studies assessing C. jejuni diversity in environmental and clinical settings at improved throughput and resolution.

Despite the ubiquity of viruses in soils, their diversity in soil water has not been explored, mainly due to the difficulty of collecting them. In hydrology, soil water is usually collected using porous candles. This study proposes using these porous candles as a new tool for sampling viruses in soil water to analyze their passage through the ceramic part of the candles. The recovery of the viruses was determined after filtration under laboratory conditions using three model bacteriophages (MS2, ΦX174, and Φ6) and Escherichia coli, at neutral and acidic pH. Then, a field experiment was carried out where soil water filtration and viral identification by metagenomic shotgun were performed. At neutral pH, all bacteriophages tested successfully passed through the porous candles during the filtration process, with reductions of 0.02 log, 0.16 log, and 0.55 log for MS2 ΦX174 and Φ6, respectively. At pH 4.4, the passage of MS2 was not affected while ΦX174 underwent a slight reduction in recovery, probably caused by adsorption onto the filter material. Regarding the application of the porous candles in the field, the results obtained allowed the successful recovery of viruses, exposing porous candles as a new method suitable for the collection of viruses from soil water in the context of the study of viral communities., (© 2022 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Bacteriophages participate in soil life by influencing bacterial community structure and function, biogeochemical cycling and horizontal gene transfer. Despite their great abundance, diversity, and importance in microbial processes, they remain little explored in environmental studies. The influence of abiotic factors on the persistence of bacteriophages is now recognized; however, it has been mainly studied under experimental conditions. This study aimed to determine whether the abiotic factors well-known to influence bacteriophage persistence also control the natural distribution of the known DNA bacteriophage populations. To this end, soil from eight study sites including forests and grasslands located in the Attert River basin (Grand Duchy of Luxembourg) were sampled, covering different soil and land cover characteristics. Shotgun metagenomics, reference-based bioinformatics and statistical analyses allowed characterising the diversity of known DNA bacteriophage and bacterial communities. After combining soil properties with the identified DNA bacteriophage populations, our in-situ study highlighted the influence of pH and calcium cations on the diversity of the known fraction of the soil DNA bacteriophages. More interestingly, significant relationships were established between bacteriophage and bacterial populations. This study provides new insights into the importance of abiotic and biotic factors in the distribution of DNA bacteriophages and the natural ecology of terrestrial bacteriophages.

Continuous surveillance of COVID-19 diffusion remains crucial to control its diffusion and to anticipate infection waves. Detecting viral RNA load in wastewater samples has been suggested as an effective approach for epidemic monitoring and the development of an effective warning system. However, its quantitative link to the epidemic status and the stages of outbreak is still elusive. Modelling is thus crucial to address these challenges. In this study, we present a novel mechanistic model-based approach to reconstruct the complete epidemic dynamics from SARS-CoV-2 viral load in wastewater. Our approach integrates noisy wastewater data and daily case numbers into a dynamical epidemiological model. As demonstrated for various regions and sampling protocols, it quantifies the case numbers, provides epidemic indicators and accurately infers future epidemic trends. Following its quantitative analysis, we also provide recommendations for wastewater data standards and for their use as warning indicators against new infection waves. In situations of reduced testing capacity, our modelling approach can enhance the surveillance of wastewater for early epidemic prediction and robust and cost-effective real-time monitoring of local COVID-19 dynamics., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Atte Aalto reports financial support was provided by Luxembourg National Research Fund (FNR). Daniele Proverbio reports financial support was provided by Luxembourg National Research Fund (FNR). Francoise Kemp reports financial support was provided by Luxembourg National Research Fund (FNR). Stefano Magni reports financial support was provided by Luxembourg National Research Fund (FNR). Leslie Ogorzaly reports financial support was provided by Luxembourg National Research Fund (FNR). Henri-Michel Cauchie reports financial support was provided by Luxembourg National Research Fund (FNR). Jorge Goncalves reports financial support was provided by 111 Project on Computational Intelligence and Intelligent Control., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

While MALDI-TOF mass spectrometry (MS) is widely considered as the reference method for the rapid and inexpensive identification of microorganisms in routine laboratories, less attention has been addressed to its ability for detection of antimicrobial resistance (AMR). Recently, some studies assessed its potential application together with machine learning for the detection of AMR in clinical pathogens. The scope of this study was to investigate MALDI-TOF MS protein mass spectra combined with a prediction approach as an AMR screening tool for relevant foodborne pathogens, such as Campylobacter coli and Campylobacter jejuni . A One-Health panel of 224 C. jejuni and 116 C. coli strains was phenotypically tested for seven antimicrobial resistances, i.e., ciprofloxacin, erythromycin, tetracycline, gentamycin, kanamycin, streptomycin, and ampicillin, independently, and were submitted, after an on- and off-plate protein extraction, to MALDI Biotyper analysis, which yielded one average spectra per isolate and type of extraction. Overall, high performance was observed for classifiers detecting susceptible as well as ciprofloxacin- and tetracycline-resistant isolates. A maximum sensitivity and a precision of 92.3 and 81.2%, respectively, were reached. No significant prediction performance differences were observed between on- and off-plate types of protein extractions. Finally, three putative AMR biomarkers for fluoroquinolones, tetracyclines, and aminoglycosides were identified during the current study. Combination of MALDI-TOF MS and machine learning could be an efficient and inexpensive tool to swiftly screen certain AMR in foodborne pathogens, which may enable a rapid initiation of a precise, targeted antibiotic treatment., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Feucherolles, Nennig, Becker, Martiny, Losch, Penny, Cauchie and Ragimbeau.)

There is a need for active molecular surveillance of human and veterinary Campylobacter infections. However, sequencing of all isolates is associated with high costs and a considerable workload. Thus, there is a need for a straightforward complementary tool to prioritize isolates to sequence. In this study, we proposed to investigate the ability of MALDI-TOF MS to pre-screen C. jejuni genetic diversity in comparison to MLST and cgMLST. A panel of 126 isolates, with 10 clonal complexes (CC), 21 sequence types (ST) and 42 different complex types (CT) determined by the SeqSphere+ cgMLST, were analysed by a MALDI Biotyper, resulting into one average spectra per isolate. Concordance and discriminating ability were evaluated based on protein profiles and different cut-offs. A random forest algorithm was trained to predict STs. With a 94% similarity cut-off, an AWC of 1.000, 0.933 and 0.851 was obtained for MLST CC , MLST ST and cgMLST profile, respectively. The random forest classifier showed a sensitivity and specificity up to 97.5% to predict four different STs. Protein profiles allowed to predict C. jejuni CCs, STs and CTs at 100%, 93% and 85%, respectively. Machine learning and MALDI-TOF MS could be a fast and inexpensive complementary tool to give an early signal of recurrent C. jejuni on a routine basis.

During a study on the prevalence and diversity of members of the genus Campylobacte r in a shellfish-harvesting area and its catchment in Brittany, France, six urease-positive isolates of members of the genus Campylobacter were recovered from surface water samples, as well as three isolates from stools of humans displaying enteric infection in the same period. These strains were initially identified as members of the Campylobacter lari group by MALDI-TOF mass spectrometry and placed into a distinct group in the genus Campylobacter , following atpA gene sequence analysis based on whole-genome sequencing data. This taxonomic position was confirmed by phylogenetic analysis of the 16S rRNA, rpoB and hsp60 ( groEL ) loci, and an analysis of the core genome that provided an improved phylogenetic resolution. The average nucleotide identity between the representative strain CA656 T (CCUG 73571 T =CIP 111675 T ) and the type strain of the most closely related species Campylobacter ornithocola WBE38 T was 88.5 %. The strains were found to be microaerobic and anaerobic, motile, non-spore-forming, Gram-stain-negative, spiral-shaped bacteria that exhibit catalase, oxidase and urease activities but not nitrate reduction. This study demonstrates clearly that the nine isolates represent a novel species within the C. lari group, for which the name Campylobacter armoricus is proposed. Here, we present phenotypic and morphological features of the nine strains and the description of their genome sequences. The proposed type strain CA656 T has a 1.589 Mbp chromosome with a DNA G+C content of 28.5 mol% and encodes 1588 predicted coding sequences, 38 tRNAs, and 3 rRNA operons.

Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) is today the reference method for direct identification of microorganisms in diagnostic laboratories, as it is notably time- and cost-efficient. In the context of increasing cases of enteric diseases with emerging multi-drug resistance patterns, there is an urgent need to adopt an efficient workflow to characterize antimicrobial resistance (AMR). Current approaches, such as antibiograms, are time-consuming and directly impact the "patient-physician" workflow. Through this mini-review, we summarize how the detection of specific patterns by MALDI-TOF MS, as well as bioinformatics, become more and more essential in research, and how these approaches will help diagnostics in the future. Along the same lines, the idea to export more precise biomarker identification steps by MALDI-TOF(/TOF) MS data towards AMR identification pipelines is discussed. The study also critically points out that there is currently still a lack of research data and knowledge on different foodborne pathogens as well as several antibiotics families such as macrolides and quinolones, and many questions are still remaining. Finally, the innovative combination of whole-genome sequencing and MALDI-TOF MS could be soon the future for diagnosis of antimicrobial resistance in foodborne pathogens., Competing Interests: The authors declare no conflict of interest.

Monogonont rotifers constitute, depending on the moment of the year, most of the zooplankton in many freshwater ecosystems. Sexual reproduction is essential in the development cycle of these organisms as it enables them to constitute stocks of cysts which can withstand adverse environmental conditions and hatch when favorable conditions return. However, endocrine disrupting compounds (EDCs) can interfere with the reproduction of organisms. The present work aimed to investigate the effects of cyproterone acetate (CPA, anti-androgen and progestogen synthetic steroid) at 0.5 mg L -1 , on the sexual reproduction of Brachionus calyciflorus in a cross-mating experiment. Results show no impact on mixis whereas the fertilization rate and resting egg production were higher in females exposed to CPA (from embryogenesis to adult stage), regardless of the treatment applied to the males with which they were mating (i.e. males hatched from CPA-treated females or from control females). Moreover, neonate females which mothers has been exposed to 0.5 mg L -1 CPA had more oocytes in their germarium than control neonates. Our results suggest that the effects of CPA observed are not related to toxicity but rather are consistent with an endocrine disruption-related impact, probably through disturbance of the mate recognition protein (MRP) production and through interference with a steroid receptor. Moreover, the absence of effect on mixis rate indicates that mixis induction on the one hand and mating process and resting production on the other hand are not controlled by the same hormonal pathways.

Lack of wastewater treatment efficiency causes receiving seawaters and bivalve molluscan shellfish to become contaminated, which can lead to public health issues. Six wastewater samples, five seawater samples and three batches of giant clams from Tahiti (French Polynesia) were investigated for the presence of enteric viruses, but also if present, for the diversity, infectivity and integrity of human adenoviruses (HAdV). Enteroviruses (EV), sapoviruses (SaV) and human polyomaviruses (HPyV) were detected in all wastewater samples. In decreasing frequency, noroviruses (NoV) GII and HAdV, rotaviruses (RoV), astroviruses (AsV), NoV GI and finally hepatitis E viruses (HEV) were also observed. Nine types of infectious HAdV were identified. HPyV and EV were found in 80% of seawater samples, NoV GII in 60%, HAdV and SaV in 40% and AsV and RoV in 20%. NoV GI and HEV were not detected in seawater. Intact and infectious HAdV-41 were detected in one of the two seawater samples that gave a positive qPCR result. Hepatitis A viruses were never detected in any water types. Analysis of transcriptomic data from giant clams revealed homologues of fucosyltransferases (FUT genes) involved in ligand biosynthesis that strongly bind to certain NoV strains, supporting the giant clams ability to bioaccumulate NoV. This was confirmed by the presence of NoV GII in one of the three batches of giant clams placed in a contaminated marine area. Overall, all sample types were positive for at least one type of virus, some of which were infectious and therefore likely to cause public health concerns.

Riverbed sediment is commonly described as an enteric virus reservoir and thought to play an important role in water column contamination, especially during rainfall events. Although the occurrence and fate of faecal-derived viruses are fairly well characterized in water, little information is available on their presence as their interactions with sediment. This study aimed at determining the main environmental factors responsible for the presence of enteric viruses in riverbed sediment. Using a combination of microbiological and physico-chemical analyses of freshly field-sampled sediments, we demonstrated their contamination by faecal phages. The in situ spatial distribution of phages in sediment was mainly driven by sediment composition. A preferential phage accumulation occurred along one bank of the river, where the quantity of fine sands and clay particles smaller than 0.2 mm was the highest. Additionally, a mineralogical analysis revealed the influence of the heterogeneous presence of virus sorbents such as quartz, calcite, carbonates and iron-bearing phases (goethite) on the phage spatial pattern. A more precise knowledge of the composition of riverbed sediment is therefore useful for predicting preferential areas of enteric virus accumulation and should allow more accurate microbial risk assessment when using surface water for drinking water production or recreational activities.

The assumption that cellulose degradation and assimilation can only be carried out by heterotrophic organisms was shattered in 2012 when it was discovered that the unicellular green alga, Chlamydomonas reinhardtii (Cr), can utilize cellulose for growth under CO₂-limiting conditions. Publications of genomes/transcriptomes of the colonial microalgae, Gonium pectorale (Gp) and Volvox carteri (Vc), between 2010⁻2016 prompted us to look for cellulase genes in these algae and to compare them to cellulases from bacteria, fungi, lower/higher plants, and invertebrate metazoans. Interestingly, algal catalytic domains (CDs), belonging to the family GH9, clustered separately and showed the highest (33⁻42%) and lowest (17⁻36%) sequence identity with respect to cellulases from invertebrate metazoans and bacteria, respectively, whereas the identity with cellulases from plants was only 27⁻33%. Based on comparative multiple alignments and homology models, the domain arrangement and active-site architecture of algal cellulases are described in detail. It was found that all algal cellulases are modular, consisting of putative novel cysteine-rich carbohydrate-binding modules (CBMs) and proline/serine-(PS) rich linkers. Two genes were found to encode a protein with a putative Ig-like domain and a cellulase with an unknown domain, respectively. A feature observed in one cellulase homolog from Gp and shared by a spinach cellulase is the existence of two CDs separated by linkers and with a C-terminal CBM. Dockerin and Fn-3-like domains, typically found in bacterial cellulases, are absent in algal enzymes. The targeted gene expression analysis shows that two Gp cellulases consisting, respectively, of a single and two CDs were upregulated upon filter paper addition to the medium.

The occurrence and propagation of enteric viruses in rivers constitute a major public health issue. However, little information is available on the in situ transport and spread of viruses in surface water. In this study, an original in situ experimental approach using the residence time of the river water mass was developed to accurately follow the propagation of F-specific RNA bacteriophages (FRNAPHs) along a 3-km studied river. Surface water and sediment of 9 sampling campaigns were collected and analyzed using both infectivity and RT-qPCR assays. In parallel, some physico-chemical variables such as flow rate, water temperature, conductivity and total suspended solids were measured to investigate the impact of environmental conditions on phage propagation. For campaigns with low flow rate and high temperature, the results highlight a decrease of infectious phage concentration along the river, which was successfully modelled according to a first-order negative exponential decay. The monitoring of infectious FRNAPHs belonging mainly to the genogroup II was confirmed with direct phage genotyping and total phage particle quantification. Reported k decay coefficients according to exponential models allowed for the determination of the actual in situ distance and time necessary for removing 90 % of infectious phage particles. This present work provides a new way to assess the true in situ viral propagation along a small river. These findings can be highly useful in water quality and risk assessment studies to determine the viral contamination spread from a point contamination source to the nearest recreational areas.

The faecal indicator Escherichia coli plays a central role in water quality assessment and monitoring. It is therefore essential to understand its fate under various environmental constraints such as predation by bacterivorous zooplankton. Whereas most studies have examined how protozooplankton communities (heterotrophic nanoflagellates and ciliates) affect the fate of E. coli in water, the capacity of metazooplankton to control the faecal indicator remains poorly understood. In this study, we investigated how the common filter-feeding cladoceran, Daphnia pulex, affects the fate of E. coli under different experimental conditions. Daphnia ingested E. coli and increased its loss rates in water, but the latter rates decreased from 1.65 d-1 to 0.62 d-1 after a 1,000-fold reduction in E. coli initial concentrations, due to lower probability of encounter between Daphnia and E. coli. The combined use of culture and PMA qPCR (viability-qPCR) demonstrated that exposure to Daphnia did not result into the formation of viable but non-culturable E. coli cells. In lake water, a significant part of E. coli population loss was associated with matrix-related factors, most likely due to predation by other bacterivorous biota and/or bacterial competition. However, when exposing E. coli to a D. pulex gradient (from 0 to 65 ind.L-1), we observed an increasing impact of Daphnia on E. coli loss rates, which reached 0.47 d-1 in presence of 65 ind.L-1. Our results suggest that the filter-feeder can exert a non-negligible predation pressure on E. coli, especially during seasonal Daphnia population peaks. Similar trials using other Daphnia species as well as stressed E. coli cells will increase our knowledge on the capacity of this widespread zooplankter to control E. coli in freshwater resources. Based on our results, we strongly advocate the use of natural matrices to study these biotic interactions in order to avoid overestimation of Daphnia impact., Competing Interests: The authors have declared that no competing interests exist.

The association of viruses with settling particles is certainly a major process controlling the spread of viral pollution in surface water and sediment. To better understand the viral distribution in a river system, the behavior of F-specific RNA bacteriophages (FRNAPHs) was investigated in relationship with the suspended solids and sediment. The partitioning of phage particles (free or associated with solids) in surface water and the attachment capabilities of eight distinct strains of phages to sediment were studied in lab experiments. In situ observations were also performed with the genotyping of 166 individual plaques of FRNAPHs isolated from surface water and sediment. The results reported here demonstrate a variation of the status of infectious phages as a function of the hydro-climatological conditions. Phage-solid association seems to mainly occur during the peak of rainfall-runoff events but also to a certain extent during the recession phase compared to low flow conditions. The transfer of phages from the water column to sediment may occur at this time. Furthermore, the ability of FRNAPHs to interact with sediment was established for six strains out of eight, belonging to genogroups II, III and IV. A similar dynamic was observed for strains within a same genogroup despite different intensity of attachment and inactivation rates for strains of genogroups III and IV. The latter results match the in situ observations in the water and sediment compartments of the studied area. Infectious FRNAPH genogroup II was more abundant in sediment than in surface water. Its capability to sorb to sediment and its higher persistence in the environment compared to genogroups III and IV were the two main explanations. Together, lab and in situ experiments produce an overall vision of the mechanisms governing FRNAPH distribution among the water column and riverbed sediment., (Copyright © 2016 Office national des forêts. Published by Elsevier B.V. All rights reserved.)

Campylobacter is the most common causative agent of human bacterial gastroenteritis and is frequently found in surface water, where it indicates recent contamination with animal faeces, sewage effluent, and agricultural run-off. The contribution of different animal reservoirs to surface water contamination with Campylobacter is largely unknown. In the Netherlands, the massive poultry culling to control the 2003 avian influenza epidemic coincided with a 44-50% reduction in human campylobacteriosis cases in the culling areas, suggesting substantial environment-mediated spread of poultry-borne Campylobacter. We inferred the origin of surface water Campylobacter jejuni and Campylobacter coli strains in Luxembourg and the Netherlands, as defined by multilocus sequence typing, by comparison to strains from poultry, pigs, ruminants, and wild birds, using the asymmetric island model for source attribution. Most Luxembourgish water strains were attributed to wild birds (61.0%), followed by poultry (18.8%), ruminants (15.9%), and pigs (4.3%); whereas the Dutch water strains were mainly attributed to poultry (51.7%), wild birds (37.3%), ruminants (9.8%), and pigs (1.2%). Attributions varied over seasons and surface water types, and geographical variation in the relative contribution of poultry correlated with the magnitude of poultry production at either the national or provincial level, suggesting that environmental dissemination of Campylobacter from poultry farms and slaughterhouses can be substantial in poultry-rich regions., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

MALDI-TOF MS has been utilized as a reliable and rapid tool for microbial fingerprinting at the genus and species levels. Recently, there has been keen interest in using MALDI-TOF MS beyond the genus and species levels to rapidly identify antibiotic resistant strains of bacteria. The purpose of this study was to enhance strain level resolution for Campylobacter jejuni through the optimization of spectrum processing parameters using a series of designed experiments. A collection of 172 strains of C. jejuni were collected from Luxembourg, New Zealand, North America, and South Africa, consisting of four groups of antibiotic resistant isolates. The groups included: (1) 65 strains resistant to cefoperazone (2) 26 resistant to cefoperazone and beta-lactams (3) 5 strains resistant to cefoperazone, beta-lactams, and tetracycline, and (4) 76 strains resistant to cefoperazone, teicoplanin, amphotericin, B and cephalothin. Initially, a model set of 16 strains (three biological replicates and three technical replicates per isolate, yielding a total of 144 spectra) of C. jejuni was subjected to each designed experiment to enhance detection of antibiotic resistance. The most optimal parameters were applied to the larger collection of 172 isolates (two biological replicates and three technical replicates per isolate, yielding a total of 1,031 spectra). We observed an increase in antibiotic resistance detection whenever either a curve based similarity coefficient (Pearson or ranked Pearson) was applied rather than a peak based (Dice) and/or the optimized preprocessing parameters were applied. Increases in antimicrobial resistance detection were scored using the jackknife maximum similarity technique following cluster analysis. From the first four groups of antibiotic resistant isolates, the optimized preprocessing parameters increased detection respective to the aforementioned groups by: (1) 5% (2) 9% (3) 10%, and (4) 2%. An additional second categorization was created from the collection consisting of 31 strains resistant to beta-lactams and 141 strains sensitive to beta-lactams. Applying optimal preprocessing parameters, beta-lactam resistance detection was increased by 34%. These results suggest that spectrum processing parameters, which are rarely optimized or adjusted, affect the performance of MALDI-TOF MS-based detection of antibiotic resistance and can be fine-tuned to enhance screening performance.

Heavy rainfall events were previously reported to bring large amounts of microorganisms in surface water, including viruses. However, little information is available on the origin and transport of viral particles in water during such rain events. In this study, an integrative approach combining microbiological and hydrological measurements was investigated to appreciate the dynamics and origins of F-specific RNA bacteriophage fluxes during two distinct rainfall-runoff events. A high frequency sampling (automatic sampler) was set up to monitor the F-specific RNA bacteriophages fluxes at a fine temporal scale during the whole course of the rainfall-runoff events. A total of 276 rainfall-runoff samples were collected and analysed using both infectivity and RT-qPCR assays. The results highlight an increase of 2.5 log10 and 1.8 log10 of infectious F-specific RNA bacteriophage fluxes in parallel of an increase of the water flow levels for both events. Faecal pollution was characterised as being mainly from anthropic origin with a significant flux of phage particles belonging to the genogroup II. At the temporal scale, two successive distinct waves of phage pollution were established and identified through the hydrological measurements. The first arrival of phages in the water column was likely to be linked to the resuspension of riverbed sediments that was responsible for a high input of genogroup II. Surface runoff contributed further to the second input of phages, and more particularly of genogroup I. In addition, an important contribution of infectious phage particles has been highlighted. These findings imply the existence of a close relationship between the risk for human health and the viral contamination of flood water., (Copyright © 2016 Luxembourg institute of Science and Technology. Published by Elsevier Ltd.. All rights reserved.)

Campylobacteriosis has increased markedly in Luxembourg during recent years. We sought to determine which Campylobacter genotypes infect humans, where they may originate from, and how they may infect humans. Multilocus sequence typing was performed on 1153 Campylobacter jejuni and 136 C. coli human strains to be attributed to three putative animal reservoirs (poultry, ruminants, pigs) and to environmental water using the asymmetric island model. A nationwide case-control study (2010-2013) for domestic campylobacteriosis was also conducted, including 367 C. jejuni and 48 C. coli cases, and 624 controls. Risk factors were investigated by Campylobacter species, and for strains attributed to different sources using a combined case-control and source attribution analysis. 282 sequence types (STs) were identified: ST-21, ST-48, ST-572, ST-50 and ST-257 were prevailing. Most cases were attributed to poultry (61.2%) and ruminants (33.3%). Consuming chicken outside the home was the dominant risk factor for both Campylobacter species. Newly identified risk factors included contact with garden soil for either species, and consuming beef specifically for C. coli. Poultry-associated campylobacteriosis was linked to poultry consumption in wintertime, and ruminant-associated campylobacteriosis to tap-water provider type. Besides confirming chicken as campylobacteriosis primary source, additional evidence was found for other reservoirs and transmission routes.

Background: The occurrence of faecal pathogens in drinking water resources constitutes a threat to the supply of safe drinking water, even in industrialized nations. To efficiently assess and monitor the risk posed by these pathogens, sampling deserves careful design, based on preliminary knowledge on their distribution dynamics in water. For the protozoan pathogens Cryptosporidium and Giardia, only little is known about their spatial distribution within drinking water supplies, especially at fine scale., Methods: Two-dimensional distribution maps were generated by sampling cross-sections at meter resolution in two different zones of a drinking water reservoir. Samples were analysed for protozoan pathogens as well as for E. coli, turbidity and physico-chemical parameters., Results: Parasites displayed heterogeneous distribution patterns, as reflected by significant (oo)cyst density gradients along reservoir depth. Spatial correlations between parasites and E. coli were observed near the reservoir inlet but were absent in the downstream lacustrine zone. Measurements of surface and subsurface flow velocities suggest a role of local hydrodynamics on these spatial patterns., Conclusion: This fine-scale spatial study emphasizes the importance of sampling design (site, depth and position on the reservoir) for the acquisition of representative parasite data and for optimization of microbial risk assessment and monitoring. Such spatial information should prove useful to the modelling of pathogen transport dynamics in drinking water supplies.

The prokaryotic community of a groundwater aquifer exposed to high concentrations of tetrachloromethane (CCl₄) for more than three decades was followed by terminal restriction fragment length polymorphism (T-RFLP) during pump-and-treat remediation at the contamination source. Bacterial enrichments and isolates were obtained under selective anoxic conditions, and degraded 10 mg·L(-1) CCl₄, with less than 10% transient formation of chloroform. Dichloromethane and chloromethane were not detected. Several tetrachloromethane-degrading strains were isolated from these enrichments, including bacteria from the Klebsiella and Clostridium genera closely related to previously described CCl₄ degrading bacteria, and strain TM1, assigned to the genus Pelosinus, for which this property was not yet described. Pelosinus sp. TM1, an oxygen-tolerant, Gram-positive bacterium with strictly anaerobic metabolism, excreted a thermostable metabolite into the culture medium that allowed extracellular CCl₄ transformation. As estimated by T-RFLP, phylotypes of CCl₄-degrading enrichment cultures represented less than 7%, and archaeal and Pelosinus strains less than 0.5% of the total prokaryotic groundwater community.

This study aims to establish a straightforward and original workflow for high-throughput typing of human adenoviruses (HAdVs) in environmental samples. Occurrence of HAdVs in water is well documented worldwide, but data on diversity of HAdV types circulating in water are scarcely available. Here, the characterisation of viral particles was performed by determination of amplicon sequences using a next-generation sequencing (NGS) approach. Adenoviral DNA was either directly isolated from wastewater or river water concentrates or after a cell culture passage. Genome amplification targeted a hyper variable region of the hexon gene, allowing the discrimination of the 54 human adenoviral types described until now. After read generation on the benchtop MiSeq platform (Illumina), data were analysed using the Mothur software for identification of all HAdV species and types simultaneously present in a unique sample. NGS results showed a relatively wide HAdV diversity of up to six types in one sample, whereas Sanger sequencing always only retrieved the dominant one. Detected types included HAdV-1, HAdV-2, HAdV-3, HAdV-6, HAdV-12, HAdV-31, HAdV-40 and HAdV-41, HAdV-41 being the most abundant in tested samples. In addition, the influence of the cell line (A549 vs 293A cells) on the infectious HAdV typing results was clearly determined. The 293A appeared to be the most suitable cell line allowing the detection of a larger diversity of infectious HAdVs and reflecting a more realistic initial species distribution than using the A549 cells. These findings demonstrated the feasibility of amplicon sequencing NGS approach to identify viruses in complex environmental water samples.

A reliable characterization of cladocerans' growth kinetic on their substrates is crucial for the estimation of their biochemical conversion rate in pond models. Although many studies reported cladocerans' growth inhibitions by high chlorophyceae contents, their growth kinetics had continued to be described in many pond system models by Monod-type kinetic, which describes growth saturation by high substrate contents, but fails to explain the disappearance of cladocerans observed during chlorophyceae's bloom periods. This study aimed to develop a methodology and assess whether growth-inhibition-type models used to describe microbial growth kinetics can be applicable to cladocerans. Experiments were carried out using Daphnia pulex populations and Scenedesmus sp. First, biomass of D. pulex was measured through digital image processing (DIP) during growth experiments. Then, three candidate models (i.e., Andrews, Edward and Haldane models), along with the Monod model, were fitted to the observed data and compared. The results showed that the DIP technique provided reliable results for estimating the biomass of D. pulex. Our findings show that the candidate growth inhibition-type models satisfactorily described D. pulex's growth kinetic (86% variance accounted for). Scenesdemus sp. were not strong inhibitors of the growth of D. pulex (high inhibition constant and low half-saturation constant found).

The detection of low virus concentrations in biological matrices, especially stool samples, is facing significant limitations as far as common diagnostic methods (enzyme-linked-immunosorbent assay (ELISA) or quantitative real-time PCR (qPCR)) are considered. Here the development of a new immuno real-time PCR (iPCR) is described and its performance in the detection of human adenoviruses (HAdVs) in spiked stools is compared with those of ELISA and qPCR assays. For the iPCR, detection of the sandwich formed by the complexation of capture antibody-antigen-detection antibody was performed by qPCR thanks to the substitution of peroxydase by a chimeric DNA. This modification increased the detection sensitivity 200-fold compared to ELISA. The direct qPCR results revealed that only 0.3-9.5% of the spiked HAdV were detectable, resulting from important losses of DNA occurring at the extraction step. This step was not necessary in the iPCR workflow, avoiding this drawback. The losses of viral particles occurred at the elution step from the stool only. The recovery rate of the iPCR was thus better and ranged between 21 and 54%. As a result, iPCR enabled the detection of lower virus concentrations in stool samples compared to those detected by ELISA and qPCR. The iPCR could be considered as a 'hyper sensitive ELISA' for early detection of HAdV infections, especially in the case of immunocompromised patients after haematopoietic stem cell transplant., (© 2014 The Authors Clinical Microbiology and Infection © 2014 European Society of Clinical Microbiology and Infectious Diseases.)

Background: Surveillance and field investigations of Campylobacter infections require molecular tools with genetic markers appropriate for tracing purposes, i.e. based on the principle that some Campylobacter lineages acquire a host signature under adaptive selection pressure. We developed a sequence-based method targeting the quinolone resistance determining region within the subunit A of DNA gyrase (gyrA). Host specificity was evaluated by characterizing two collections of Campylobacter jejuni (N = 430) and Campylobacter coli (N = 302) originating from surface waters, domestic mammals and poultry., Results: Based on nucleotide identity, a total of 80 gyrA alleles were observed. Thirty nine alleles assigned to C. coli encoding two peptides fell into three clades: two associated with surface waters and one associated with domestic mammals and poultry. The variability in GC content generated by synonymous mutations suggested that surface waters isolates originated from two distinct ecological niches. A total of 42 alleles were recorded from C. jejuni strains and encoded 8 peptides including one lying in a distinct lineage associated with wildlife. Seven of the 23 alleles encoding peptide #1 displayed the synonymous mutation G408A not identified in poultry isolates. By contrast, the substitution Ser22Gly observed in 4 different peptide groups was significantly associated with domestic birds (P = 0.001). The change in amino acid sequences Thr86Ile conferring resistance to quinolones was significantly associated with poultry (P < 0.001) in both C. jejuni and C. coli with 38.7% and 67.9% of quinolone-resistant strains, respectively., Conclusions: The gyrA typing method presented here is an informative tool as sequences appear to be predictive of particular ecological niches. Combined with multi-locus sequence typing, it could increase the resolution of source attribution, and combined with porA/flaA typing it could be suitable for detecting temporal clusters of human cases. All gyrA alleles identified were deposited in the freely accessible online database http://pubmlst.org/campylobacter.

Because of their significant public health impact, waterborne Cryptosporidium and Giardia have been monitored in surface water in order to assess microbial quality of water bodies used for drinking water production and/or for recreational purposes. In this context, sampling strategy is of key importance and should be representative enough to appropriately assess the related microbial risk. This, however, requires sound knowledge on the behaviour of both pathogens in water. In the present study, the spatial and temporal distribution of Cryptosporidium and Giardia was explored in the rural Upper-Sûre watershed used for drinking water production in Luxembourg. By subdividing it into three compartments including (i) sub-catchments, (ii) the Sûre River fed by the sub-catchments and (iii) the Upper-Sûre reservoir fed by the Sûre River, parasite distribution was assessed using sampling designs adapted to the hydro-dynamic characteristics of the respective compartments. Results highlighted the high spatial and temporal variability in parasite distribution at watershed scale, as well as the prevalence of Giardia over Cryptosporidium. Besides land use features and catchment characteristics, hydro-climatology appeared to be a major driver of parasite behaviour in the watershed. It introduced a seasonal trend in their occurrence, highest densities being detected during the wet season. Peaks of contamination triggered out by rainfall-induced runoff were further observed in the three compartments. In the Sûre River, Cryptosporidium and Giardia fluxes peaked at 10(9) and 10(10) (oo)cysts.d(-1), respectively, and were discharged into the drinking water reservoir, where they underwent a 2 to 3 log10 removal rate. Despite this, parasite fluxes entering the drinking water treatment plant were still high (10(6) to 10(7) (oo)cysts.d(-1)) and stressed on the need for improved watershed management upstream the water treatment barrier. The catchment-wide analysis described here constitutes a valuable tool for assessment of catchment microbial dynamics, especially within the framework of Water Safety Plans., (© 2013.)

Development of rapid, sensitive and specific methods for detection of infectious enteric viruses in water is challenging but crucial for gaining reliable information for risk assessment. An immunocapture real-time PCR (IC-qPCR) was designed to detect jointly the two major viral particle components, i.e. the capsid protein and the viral genome. Targeting both constituents helps circumventing the technical limits of cell culture approaches and the inability of PCR based methods to predict the infectious status. Two waterborne pathogenic virus models, human adenovirus types 2 and 41, were chosen for this study. IC-qPCR showed a detection limit of 10MPNCU/reaction with a dynamic range from 10(2) to 10(6)MPNCU/reaction. Sensitivity was thus 100-fold higher compared to ELISA-based capture employing the same anti-hexon antibodies. After optimisation, application on environmental water samples was validated, and specificity towards the targeted virus types was obtained through the qPCR step. Heat-treated pure samples as well as surface water samples brought evidence that this method achieves detection of encapsidated viral genomes while excluding free viral genome amplification. As a consequence, adenovirus concentrations estimated by IC-qPCR were below those calculated by direct qPCR. The results demonstrate that the IC-qPCR method is a sensitive and rapid tool for detecting, in a single-tube assay, structurally intact and thus potentially infectious viral particles in environmental samples., (Copyright © 2013 Elsevier B.V. All rights reserved.)

In order to provide a more suitable response to public health concerns, we improved the detection of infectious human adenoviruses in water by optimising the commonly used integrated cell culture-PCR method. Risk evaluation studies seek for rapid detection of infectious adenoviruses, including the enteric types 40 and 41 that are considered as the second most common agents of gastroenteritis in children next to rotaviruses. The here-employed 293A cell line used for infectious status assessment showed its ability to multiply adenoviruses including type 41. Two modifications were moreover applied to the workflow for viral detection. The first occurred at the nucleic acid extraction step performed directly on all infected cells, while the second was the application of real-time quantitative PCR as detection tool. All adaptations led to a 3-day reduction of the response delay and an improved sensitivity especially for the enteric adenoviral types. The infectious status of laboratory strain types 2 and 41 was demonstrated by a more than 2-log10 increase in genome quantity. These conclusions were confirmed and reinforced by the analysis of water samples applying the improved assay. Naturally occurring infectious adenoviruses were detected in wastewater and river water, within 2 days. Types belonging to the species human adenoviruses C and type 31 were observed, but the most frequently identified type was 41 (71 % of identified sequences, n = 34). This highlights the usefulness of our method for a wide range of types, and especially for the most prevalent and public health-relevant enteric adenoviruses.

Aims: Development of TaqMan MGB real-time PCR assays for quantitative typing of major cattle and human-pathogenic Cryptosporidium species., Methods and Results: Three specific TaqMan MGB real-time PCRs, based on the SSU rRNA gene, were directed towards livestock-restricted Cryptosporidium andersoni and Cryptosporidium bovis as well as both human-pathogenic Cryptosporidium parvum and Cryptosporidium hominis. A generic TaqMan assay further identified all known Cryptosporidium species and simultaneously monitored PCR inhibition through an external amplification control. The generic and specific assays were highly reproducible, and all displayed a detection limit of one oocyst per reaction. The specific TaqMan protocols also proved valuable for specifically detecting and quantifying target DNA in the presence of non-target DNA in environmental samples., Conclusions: All TaqMan MGB real-time PCR assays fulfilled the required specificity and sensitivity criteria, both on laboratory strains and on a surface water matrix., Significance and Impact of the Study: No molecular-based method was yet available for the quantitative detection of C. andersoni and the cluster formed by C. bovis, Cryptosporidium ryanae and Cryptosporidium xiaoi. This work provides a novel tool to evaluate the parasite load from domestic ruminants and humans, and to improve assessment and management of microbial risk through better appraisal of the origin and fate of faecal pollutions., (© 2012 The Society for Applied Microbiology.)

This study presents an original rDNA PCR and microscopic survey of pelagic freshwater fungal communities, and was designed to unveil the diversity of true Fungi (i.e. the kingdom Eumycota) in three contrasting lake ecosystems (Lakes Pavin, Aydat and Vassivière) located in the French Massif Central. Three clone libraries were constructed from samples collected in the euphotic layers of the lakes during spring 2007. Phylogenetic analysis of the combined data from the three lakes clustered our sequences into thee divisions: Chytridiomycota (50% of total sequences), Ascomycota (40%) and Basidiomycota (10% in Pavin and Aydat only). Several sequences were assigned to a novel Chytridiomycota clade first recovered in Lake Pavin in 2005. Most of the sequences retrieved in the investigated lakes were affiliated with known fungal species, most of which were apparently well adapted to thrive in the pelagic realm. Their main functions (i.e. parasitism and saprophytism), putatively inferred from the closest relatives of the retrieved molecular sequences, were confirmed by microscopic approaches and by enrichment experiments with pollen grains. The occurrence of three fungal forms (zoosporic, yeast and mycelial) was associated with different trophic modes, establishing fungi as strong potential competitors for various niches in pelagic ecosystems, primarily in relation to the processing of particulate organic matter and the production of propagule food sources for grazers. For the first time, this study provides insight into the diversity and the associated functions of all members of the Kingdom Eumycota investigated in the whole plankton fraction of aquatic ecosystems., (© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.)

In aquatic environments some pollutants are present in water and sediments and organisms possess cellular detoxification systems to face up these xenobiotics. The gudgeon, Gobio gobio, is a freshwater benthopelagic fish that appears particularly adequate for an ecotoxicological assessment of rivers. The aim of this study was the identification of GST and GPx genes in this organism in order to develop new indicators of early exposure to xenobiotics in aquatic environments. Reverse-transcription PCR using degenerate primers and RACE-PCR allowed us to identify a selenium-dependent glutathione peroxidase (Se-GPx), belonging to the class one (GPx-1), and a pi-class glutathione S-transferase (pi-GST) cDNAs. These sequences encoded for 191 and 208 amino acids proteins respectively, they exhibit high identities and similarities with corresponding proteins in other fish and the residues essential to the enzymatic functions are highly conserved. The expression patterns of these two mRNA were established by real-time PCR in five gudgeon tissues: liver, gills, kidney, spleen and muscle. The mRNA levels of these genes were highest in the liver and their expression in the other tissues exhibited some differences. Gudgeons exposed to PCB 77 in the food exhibited an increase in pi-GST mRNA and a decrease in GPx-1 transcripts levels in the liver. However, no modification of the enzymatic activities was observed. The present study provides the first transcriptional data regarding pi-GST and GPx-1 in the gudgeon Gobio gobio., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Starting in 2006, a monitoring of Giardia lamblia and Cryptosporidium parvum occurrence was conducted for 2 years in the largest drinking water reservoir of Luxembourg (Esch-sur-Sûre reservoir) using microscopy and qPCR techniques. Parasite analyses were performed on water samples collected from three sites: site A located at the inlet of the reservoir, site B located 18 km downstream site A, at the inlet of the drinking water treatment plant near the dam of the reservoir and site C where the finished drinking water is injected in the distribution network. Results show that both parasites are present in the reservoir throughout the year with a higher occurrence of G. lamblia cysts compared to C. parvum oocysts. According to our results, only 25% of the samples positive by microscopy were confirmed by qPCR. (Oo)cyst concentrations were 10 to 100 times higher at site A compared to site B and they were positively correlated to the water turbidity and negatively correlated to the temperature. Highest (oo)cyst concentrations were observed in winter. In contrast, no relationship between the concentrations of (oo)cysts in the reservoir and rain events could be established. Though a correlation has been observed between both parasites and faecal indicators in the reservoir, some discrepancies highlight that the latter do not represent a reliable tool to predict the presence/absence of these pathogenic protozoa. In summer 2007, the maximal risk of parasite infection per exposure event for swimmers in the reservoir was estimated to be 0.0015% for C. parvum and 0.56% for G. lamblia. Finally, no (oo)cysts could be detected in large volumes of finished drinking water.

Noroviruses (NoV) in 78 wastewater samples from Luxembourg were quantified, cloned, and sequenced in 2008-2009. The concentrations of NoV genogroup II and the relative occurrences of certain genotypes changed significantly during the winter season. NoV genogroup I was frequently detected by real-time reverse transcription-PCR (RT-PCR), albeit at 30-fold lower concentrations than for genogroup II, hampering attempts to assess overall genetic diversity by the cloning/sequencing approach.

The genetic diversity of norovirus strains obtained from gastroenteritis outbreaks, sporadic case surveillance and wastewater plants was compared in Luxembourg from October 2008 until June 2009. Except for GI.6 and GIV.1 strains detected exclusively in wastewater, all other genotypes were also found in human samples. Of the nine NoV genotypes detected, only three (GII.4, GIIb/II.3 and GIIc/II.12) were associated with institutional outbreaks. The majority of sequences from all sources belonged to genotype GII.4, including two potentially new sub-variants. Strains collected in the context of outbreaks may significantly under-represent the overall genetic diversity of NoVs circulating in a country., (© 2011 The Authors. Clinical Microbiology and Infection © 2011 European Society of Clinical Microbiology and Infectious Diseases.)

Contamination levels of PCBs, and of the heavy metals cadmium (Cd), lead (Pb) and mercury (Hg) were analyzed in four fish species from seven rivers in the North of Luxembourg. During August and September 2007, 85 samples of fish were collected belonging to four species: the stone loach (Barbatula barbatula, n=12 pools), the chub (Squalius cephalus, n=36), the barbel (Barbus barbus, n=23) and eel (Anguilla anguilla, n=14). The concentration of seven indicator PCBs ( summation operator(7)PCBs) reached a mean of 39ngg(-1) and varied between 4.0 and 346.2ngg(-1) (wet wt) depending on the site and species. Fish collected at Wallendorf on the Our River and sites on the Wiltz and the Clerve rivers showed the highest concentrations for PCBs. In comparison with 1994, PCB levels in fish decreased strongly during the last decade in these rivers. Lead was detected at low levels (0-181.4ngg(-1) wet wt). Mercury concentrations ranged between 10.3 and 534.5ngg(-1) (wet wt) exceeding maximum tolerable levels for human consumption of 500ngg(-1) in two fish out of 85. Chubs and eels from the Sûre River were the most contaminated by mercury. Cadmium levels varied between 4.0 and 103.9ngg(-1) (wet wt). In addition to mercury in fish, cadmium was the most problematic pollutant on the Our, the Wiltz, the Clerve and the Troine Rivers, because values found in 20% of fish exceeded the threshold of about 10-50ngg(-1) (wet wt) recommended for human health. The total PCB level predicted to accumulate in livers from otter potentially feeding on these fish based on a previously published mathematical model is 37.7microgg(-1) (lipid wt), which is between a proposed "safe level" and a "critical level" for otters. Rivers in the North of Luxembourg are thus to some extent polluted, and the establishment of otter populations could be affected by current levels of contamination., (2009 Elsevier Ltd. All rights reserved.)

Enteroviruses and noroviruses are pathogenic viruses excreted by infected individuals. Discharged in wastewaters, some of these viruses can be captured by biofilms. In the present study, we assessed the occurrence and persistence of these viruses in wastewaters and in corresponding biofilms. Natural wastewaters and biofilms were analyzed monthly from January to July using real-time RT-PCR. Enterovirus RNA was detected in wastewater in June while norovirus RNA was detected from January to March. In contrast, biofilm analysis revealed the presence of both enterovirus and norovirus genomes throughout the study period. For instance, enterovirus and norovirus genogroups (GG) I and II were detected in 50, 46 and 37% of the biofilm samples, respectively (n=24). In a laboratory experiment, persistence of norovirus GGI RNA (quantified using molecular techniques) and F-specific bacteriophages (quantified using both culture and molecular techniques) was assessed in wastewater and corresponding naturally-contaminated biofilms at both 4 and 20 degrees C. The concentrations of viral genomes (norovirus GGI and F-specific RNA phage) were very stable in biofilms. Indeed, no significant decrease was observed during the persistence experiment that lasted 49 days. Furthermore, regardless of our experimental conditions, viral genome and infectious F-specific bacteriophages persisted longer in biofilm than in wastewater. According to our results, wastewater biofilms may contribute to the persistence and dispersal of pathogenic viruses outside of epidemic periods.

This study presents a depth-related survey of virioplankton abundance in Lake Pavin (Massif Central, France), in relation to the abundances of heterotrophic prokaryotes, picocyanobacteria (Pcy), autotrophic picoeukaryotes (Peu), and of autotrophic (ANF) and heterotrophic (HNF) nanoflagellates. The sampling strategy was designed to be representative of the physico-chemical gradients of the whole water column of the lake, and the seasonal variability as well. In mixolimnic surface waters, all communities were present and viral abundance peaked in summer and autumn. Viral abundance was significantly correlated (p < 0.001) with Pcy, Peu, and ANF, indicating that cyanophages and perhaps other phytoplankton viruses represent a significant pool of viral standing stocks in the mixolimnion of Lake Pavin. Microautotrophs were absent in the deep monimolimnic water masses, where viruses and heterotrophic prokaryotes exhibited highest seasonal abundances in summer and/or autumn and were significantly correlated (p < 0.001) to each other. This indicates that the anoxic monimolimnion of Lake Pavin is an exclusive habitat for viruses and heterotrophic prokaryotes. We conclude that in this habitat, host availability is prevalent over other factors (temperature, oxygen, nutrients, grazers) in favoring viral proliferation.

The aim of the present study was to evaluate the potential of transcript quantification as an indicator of Giardia cyst viability. The variations of beta-giardin, EF1A and ADHE mRNAs were quantified during excystation by real-time RT-PCR assays and compared with the percentages of viability estimated using propidium iodide staining and in vitro excystation. The first experiments were performed with purified G. duodenalis assemblage B cysts. When 55% of excysting protozoa were observed, the increase of the selected transcripts ranged from 0.40+/-0.13 to 0.97+/-0.11 log10 after 1h of incubation in excystation medium. Purified cysts were also stored at 4 degrees C for up to 56 days and analysed at several sampling times. Significant correlations were observed between the variations of the selected mRNAs and the percentages of viability estimated with staining and excystation methods. Among the three transcripts, beta-giardin appeared to be the most appropriate to study the viability of Giardia cysts concentrated from wastewater samples.

The objective of this study was to investigate the uptake of deltamethrin, an insecticide, by Daphnia magna neonates by SIMS and to compare these findings with results based on established toxicity tests. Young daphnids (aged <24 h) were exposed to 0, 50 and 200 microg L(-1) (ppb) deltamethrin. Mobile, immobile and dead animals were enumerated after 24 and 48 h following OECD 202 [OECD 202, 2004. Daphnia sp., acute immobilisation test, guideline for testing of chemicals] guidelines. The animals were embedded in epoxy resin, cut into semi-thin sections (500 nm) and placed on silicon supporters. NanoSIMS 50 (Cameca) images were made from tissues of the intestine for carbon, nitrogen (measured as CN), phosphorus and bromine. To distinguish between relative concentrations of bromine in the guts from different exposure concentrations of deltamethrin, a carbon normalization method was carried out. Both deltamethrin concentrations and time showed a significant effect on immobilization and mortality of the daphnids (P<0.0001). Bromine from deltamethrin could be visualized by NanoSIMS in all exposed gut tissues (gut wall, microvilli layer, perithropic membrane). Highest deltamethrin concentrations following (12)C normalization were found in animals exposed to 200 microg L(-1) deltamethrin, followed by 50 microg L(-1) and the control. NanoSIMS 50 was successfully used as a supplemental technique for elucidating the relation between the uptake and localization of deltamethrin and its toxicity to D. magna. These results highlight the potential usefulness of NanoSIMS to detect marker elements of xenobiotic compounds within exposed organisms, to compare relative exposure concentrations, and to locate these compounds at their original tissue location.

Biofilms colonizing surfaces inside drinking water distribution networks may provide a habitat and shelter to pathogenic viruses and parasites. If released from biofilms, these pathogens may disseminate in the water distribution system and cause waterborne diseases. Our study aimed to investigate the interactions of protozoan parasites (Cryptosporidium parvum and Giardia lamblia [oo]cysts) and viruses (vaccinal poliovirus type 1, phiX174, and MS2) with two contrasting biofilms. First, attachment, persistence, and detachment of the protozoan parasites and the viruses were assessed with a drinking water biofilm. This biofilm was allowed to develop inside a rotating annular reactor fed with tap water for 7 months prior to the inoculation. Our results show that viable parasites and infectious viruses attached to the drinking water biofilm within 1 h and persisted within the biofilm. Indeed, infectious viruses were detected in the drinking water biofilm up to 6 days after the inoculation, while viral genome and viable parasites were still detected at day 34, corresponding to the last day of the monitoring period. Since viral genome was detected much longer than infectious particles, our results raise the question of the significance of detecting viral genomes in biofilms. A transfer of viable parasites and viruses from the biofilm to the water phase was observed after the flow velocity was increased but also with a constant laminar flow rate. Similar results regarding parasite and virus attachment and detachment were obtained using a treated wastewater biofilm, suggesting that our observations might be extrapolated to a wide range of environmental biofilms and confirming that biofilms can be considered a potential secondary source of contamination.

We have described the use of Polyethylene glycol (PEG) for the precipitation of natural communities of aquatic viruses, and its comparison with the usual concentration method based on ultracentrifugation. Experimental samples were obtained from different freshwater ecosystems whose trophic status varied. Based on transmission electron microscope observations and counting of phage-shaped particles, our results showed that the greatest recovery efficiency for all ecosystems was obtained when we used the PEG protocol. On average, this protocol allowed the recovery of >2-fold more viruses, compared to ultracentrifugation. In addition, the diversity of virioplankton, based on genomic size profiling using pulsed field gel electrophoresis, was higher and better discriminated when we used the PEG method. We conclude that pegylation offers a valid, simple and cheaper alternative method to ultracentrifugation, for the concentration and the purification of pelagic viruses.

Biofilms within wastewater treatment plants can capture enteric microorganisms initially present in the water phase immobilising them either definitively or temporarily. Consequently, fates of microorganisms may totally change depending on whether they interact or not with biofilms. In this study, we assessed the stability of wastewater biofilms comparing the evolution of the concentrations of bacteria (heterotrophic plate count [HPC], thermotolerant coliforms [TC]) and viral (somatic coliphages [SC] and F-specific phages [F +]) indicators in the biofilms and in the corresponding wastewaters at 4 and 20 dgrees C. Additionally, we assessed the monthly occurrence of these bacterial and viral indicators as well as of pathogenic protozoa (Cryptosporidium oocysts and Giardia cysts) in three native wastewater biofilms for four months. Our results show that viral indicators (SC and F + ) persist longer in biofilms than in the corresponding wastewaters at 4 degrees C as well as at 20 degrees C. In contrast, persistence of bacterial indicators (TC and HPC) depends on both the temperature and the matrix. Differences between viral and bacterial persistence are discussed. Monthly analysis of native wastewater biofilms shows that bacterial and viral indicators, as well as Cryptosporidium oocysts and Giardia cysts, attach to wastewater biofilms to a concentration that remains stable in time, probably as a result of a dynamic equilibrium between attachment and detachment processes.

High-resolution vertical sampling and determination of viral and prokaryotic parameters in a deep volcanic lake shows that in the absence of thermal stratification but within light, oxygen, and chlorophyll gradients, host availability empirically is prevalent over the physical and chemical environments and favors lytic over lysogenic "viral life cycles."