Back to the Roots: Agrobacterium-Specific Phages Show Potential to Disinfect Nutrient Solution from Hydroponic Greenhouses.

Agrobacterium biovar 1 is a soilborne plant pathogen with the ability to colonize the irrigation system of greenhouses, causing hairy root disease (HRD). Currently, management focuses on using hydrogen peroxide to disinfect the nutrient solution, but due to the emergence of resistant strains, its efficacy and sustainability are questioned. Using a relevant collection of pathogenic Agrobacterium biovar 1 strains, OLIVR1 to 6, six phages specific to this pathogen and belonging to three different genera were isolated from Agrobacterium biovar 1-infected greenhouses. All phages were named OLIVR, referring to their location of isolation, Onze-Lieve-Vrouwe-Waver, and were characterized by whole-genome analysis, confirming their strictly lytic lifestyle. They remained stable under greenhouse-relevant conditions. To assess the efficacy of the phages, their ability to disinfect greenhouse nutrient solution inoculated with agrobacteria was tested. Each of the phages infected their host, but their ability to decrease the bacterial concentration differed. For instance, OLIVR1 reduced the bacterial concentration with 4 log units without phage resistance emerging. While OLIVR4 and OLIVR5 were also infectious in nutrient solution, they did not always decrease the bacterial load below the limit of detection, and phage resistance emerged. Finally, the mutations causing phage resistance by receptor modification were identified. For OLIVR4-resistant Agrobacterium isolates, but not for OLIVR5-resistant isolates, motility decreased. Together, these data show the potential of some of these phages as disinfectant of nutrient solution, and they might be a valuable tool to tackle HRD. IMPORTANCE Hairy root disease, caused by rhizogenic Agrobacterium biovar 1 is a rapidly emerging bacterial disease worldwide. It affects tomatoes, cucumbers, eggplant, and bell pepper, causing high yield losses in hydroponic greenhouses. Recent findings suggest that the current management practices, mainly focusing on UV-C and hydrogen peroxide to disinfect contaminated water, have a questionable efficacy. Hence, we investigate the potential of phages as a biological means of preventing this disease. Using a diverse collection of Agrobacterium biovar 1, we isolated three different phage species that together infect 75% of the collection. Since these phages are strictly lytic, while remaining both stable and infectious under greenhouse-relevant conditions, they might be suitable candidates for biological control.

Isolation, characterization and genome analysis of an orphan phage FoX4 of the new Foxquatrovirus genus.

The growing interest in the therapeutic application of bacteriophages leads to a drastic increase in the number of sequenced genomes. Luckily, recent insights in phage taxonomy facilitate the classification of phages in a comprehensive and data-driven manner as recently proposed by the International Committee on Taxonomy of Viruses. In this research, we present the taxonomical classification of a novel, narrow host range Xanthomonas phage FoX4, isolated from a Brussels sprouts field in Belgium infested with Xanthomonas campestris pv. campestris. The phage has a limited ability to lyse a bacterial culture, yet adsorbs efficiently to its host. Based on its genome sequence and low similarity to previously described phages, the phage comprises the novel phage genus Foxquatrovirus.

Bacteriophage therapy as a treatment strategy for orthopaedic-device-related infections: where do we stand?

Antibiotic resistance represents a key challenge of the 21st century. Since the pipeline of new antibiotics in development is limited, the introduction of alternative antimicrobial strategies is urgently required. Bacteriophage therapy, the use of bacterial viruses to selectively kill bacterial pathogens, is re-emerging as a potential strategy to tackle difficult-to-treat and multidrug-resistant pathogens. The last decade has seen a surge in scientific investigation into bacteriophage therapy, including targeting orthopaedic-device-related infections (ODRIs) in several successful case studies. However, pharmacological data, knowledge on the interplay with the immune system and, especially in ODRIs, the optimal local application strategy and treatment outcomes remain scarce. The present review reports the state-of-the-art in bacteriophage therapy in ODRIs and addresses the hurdles in establishing bacteriophage therapy under good clinical practice guidelines. These hurdles include a lack of data concerning bacteriophage production, processing, administration and dosing, as well as follow-up clinical monitoring reports. To overcome these challenges, an integrated clinical approach is required, supported by comprehensive legislature to enable expansive and correctly implemented clinical trials.

Ratification vote on taxonomic proposals to the International Committee on Taxonomy of Viruses (2015).

Changes to virus taxonomy approved and ratified by the International Committee on Taxonomy of Viruses in February 2015 are listed.

AN OUTBREAK OF EXTENSIVELY DRUG-RESISTANT ACINETOBACTER BAUMANNII IN A BELGIAN TERTIARY BURN WOUND CENTER.

The burn intensive care unit (ICU) of the Queen Astrid Military Hospital experienced an outbreak with an extensively drug-resistant Acinetobacter baumannii (XDR-Ab) strain, which began when all burn wound patients from all over Belgium were sent there as part of the national COVID-19 action plan. The purpose of this study is to report on the investigation and strategies that were implemented to contain the outbreak. Between October 2020 and May 2021, five of the 72 patients admitted to the ICU met the acute outbreak case definition (attack rate 7%). Their median age was 46 years and their median total body surface area burned was 39%. All patients developed at least one XDR-Ab infection, with in total three pulmonary, three bloodstream and five burn wound infections. One patient died. All XDR-Ab isolates were only susceptible to colistin. Whole genome sequencing of the isolates from the first two patients revealed an identical A. baumannii ST2 genotype, suggesting an outbreak. XDR-Ab-positive patients were cohorted with dedicated staff. The infection control team intensified its training on hand hygiene, excreta management and bio-cleaning procedures. Concurrently, 30 environmental samples were collected, which proved negative for XDR-Ab. Spatio-temporal associations were found for all XDR-Ab-positive patients, suggesting cross-transmission via staff's hands. We describe an XDR-Ab outbreak in a burn ICU over a seven-month period, in a context of increased workload. This series underlines the importance of a correct staff-to-patient ratio, especially in outbreak situations.

L'unité de soins intensifs (USI) pour brûlés de l'Hôpital Militaire Reine Astrid a connu une épidémie avec une souche d'Acinetobacter baumannii extrêmement résistante aux antibiotiques (XDR-Ab), qui a commencé pendant la période où tous les patients brûlés de Belgique y étaient référés à la suite du plan national COVID-19. Le but de cette étude est de décrire l'enquête épidémiologique et les stratégies utilisées pour contenir l'épidémie. Entre octobre 2020 et mai 2021, cinq des 72 patients admis à l'USI ont répondu à la définition de cas (taux d'attaque 7%). L'âge médian était de 46 ans, la surface corporelle brûlée médiane de 39%. Tous les patients ont développé au moins une infection par XDR-Ab : trois pneumonies, trois bactériémies et cinq infections de brûlures. Un patient est décédé. Tous les isolats XDR-Ab n'étaient sensibles qu'à la colistine. Le séquençage du génome entier des isolats des deux premiers patients a révélé un génotype identique d'A. baumannii ST2, suggérant une épidémie. Les patients XDR-Ab positifs ont été cohortés avec du personnel dédié. L'équipe d'hygiène hospitalière a intensifié sa formation sur l'hygiène des mains, la gestion des excréta et les procédures de bio-nettoyage. Simultanément, 30 échantillons environnementaux ont été collectés, qui étaient négatifs pour XDR-Ab. Des liens spatio-temporels ont été trouvés pour tous les patients XDR-Ab positifs, suggérant une transmission croisée manuportée. Nous décrivons une épidémie de XDR-Ab dans une USI pour brûlés sur une période de sept mois, dans un contexte de charge de travail accrue. Cette série souligne l'importance d'un ratio personnel-patients approprié, en particulier dans les situations d'épidémie.

Complete genome sequence of the novel Escherichia coli phage phAPEC8.

Bacteriophage phAPEC8 is an Escherichia coli-infecting myovirus, isolated on an avian pathogenic Escherichia coli (APEC) strain. APEC strains cause colibacillosis in poultry, resulting in high mortality levels and important economic losses. Genomic analysis of the 147,737-bp double-stranded DNA phAPEC8 genome revealed that 53% of the 269 encoded proteins are unique to this phage. Its closest relatives include the Salmonella phage PVP-SE1 and the coliphage rv5, with 19% and 18% similar proteins, respectively. As such, phAPEC8 represents a novel, phylogenetically distinct clade within the Myoviridae, with molecular properties suitable for phage therapy applications.

Complete genome sequence of the giant Pseudomonas phage Lu11.

The complete genome sequence of the giant Pseudomonas phage Lu11 was determined, comparing 454 and Sanger sequencing. The double-stranded DNA (dsDNA) genome is 280,538 bp long and encodes 391 open reading frames (ORFs) and no tRNAs. The closest relative is Ralstonia phage ϕRSL1, encoding 40 similar proteins. As such, Lu11 can be considered phylogenetically unique within the Myoviridae and indicates the diversity of the giant phages within this family.

Identification of genital tract markers in the human seminal plasma using an integrative genomics approach.

STUDY QUESTION: Can protein biomarkers of the male genital tract be identified in human seminal plasma? SUMMARY ANSWER: We identified potential biomarkers for each of the organs participating in the secretions of the human seminal plasma. WHAT IS KNOWN ALREADY: The seminal plasma fulfills critical functions for fertility by providing spermatozoa with a protective milieu, promoting their final maturation and modulating the immune responsiveness of the female reproductive tract. It is also considered to be a promising source of biomarkers of male infertility and/or pathologies of the male genital tract. STUDY DESIGN, SIZE, DURATION: This study combines proteomic analyses of normal seminal plasma together with transcriptomic gene expression profiling of human healthy tissues. MATERIALS, SETTING, METHODS: Non-liquefied seminal plasma proteins from a healthy donor were prefractionated using two sequential Proteominer™ libraries. Eight subproteome fractions were collected, trypsin digested and subjected to three successive mass spectrometry analyses for peptide characterization. The list of identified proteins was compared with and merged with other available data sets of the human seminal plasma proteome. The expression of corresponding genes was then investigated using tissue transcriptome profiles to determine where, along the male reproductive tract, these proteins were produced. Finally, tissue specificity of a selected subset of biomarker candidates was validated on human tissues. MAIN RESULTS AND THE ROLE OF CHANCE: We first performed a proteomic analysis of the human seminal plasma and identified 699 proteins. By comparing our protein list with other previous proteomic data sets, we found that 2545 unique proteins have been described so far in the human seminal plasma. We then profiled their expression at the gene level and identified 83 testis, 42 epididymis, 7 seminal vesicle and 17 prostate candidate protein markers. For a subset of testis-specific candidates, i.e. TKTL1, LDHC and PGK2, we further validated their germ cell expression and demonstrated that such markers could distinguish between semen from fertile and infertile men. LIMITATIONS, REASONS FOR CAUTION: While some of the markers we identified are well-known tissue-specific products, further dedicated studies to validate the biomarker status of new candidates will be required. Additionally, whether or not the abundance of these proteins is indeed decreased in some specific pathological situations remains to be determined. WIDER IMPLICATIONS OF THE FINDINGS: Using an integrative genomics approach, we identified biomarker candidates for each of the organs participating in the seminal plasma production. In this study, we essentially focused on germ cell markers and their potential application for the diagnosis of male infertility. Other types of markers also deserve a focused attention given their potential predictive value for various reproductive disorders, notably for prostate cancers. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Proteomics Core Facility at Biogenouest and was funded by Conseil Régional de Bretagne, IBiSA and Agence de la Biomédecine grants. The authors declare that there exists a competing financial interest in this work that is related to a patent application on the use of identified germ cell-specific proteins in an antibody-based assay (Fertichip™) to predict the successful testicular biopsy outcomes in human non-obstructive azoospermia.

Anti-infective DNase I coatings on polydopamine functionalized titanium surfaces by alternating current electrophoretic deposition.

Implant-associated infections (IAIs) can cause serious problems due to the difficult-to-treat nature of the biofilms formed on the implant surface. In mature biofilms, the matrix, which consists of polysaccharides, proteins, lipids and extracellular DNA (eDNA), forms a protective environment for the residing bacteria, shielding them from antibiotics and host defenses. Recently, the indirect prevention of biofilm growth through the degradation of eDNA using an enzyme, such as deoxyribonuclease (DNase) I, has gained attention and is regarded as a promising strategy in the battle against IAIs. In this study, coatings of DNase I were applied on titanium implant materials and their anti-infective properties were investigated. First, the effectiveness of alternating current electrophoretic deposition (AC-EPD) as a novel processing route to apply DNase I on titanium was examined and compared with the commonly applied diffusion methodology (i.e. classic dipping). For the same processing time, the use of AC-EPD in combination with a polydopamine (PDA) coupling chemistry on the titanium electrode surface significantly increased the protein deposition yield as compared to classic dipping, thereby yielding homogeneous coatings with a thickness of 12.8 nm and an average surface roughness, Sa, of ∼20 nm. X-ray photoelectron spectroscopy confirmed the presence of peptide bonds on all DNase-coated substrates. Time-of-flight secondary ion mass spectrometry detected a more dense DNase I layer in the case of AC-EPD for electrodes coupled as anode during the high-amplitude half cycle of the AC signal. The enzyme activity, release kinetics, and shelf life of DNase I coatings were monitored in real-time using a quantitative qDNase assay. The activity of DNase I coatings produced using AC-EPD was three time higher than for coatings prepared by classic dipping. For both deposition methods, a high initial burst release was observed within the first 2 h, while some activity was still retained at the surface after 7 days. This can be explained by the stable attachment of a small fraction of DNase to the surface through covalent bonding to the PDA layer, while superimposing DNase deposits were only loosely bound and therefore released rapidly upon immersion in the medium. Interestingly, coatings prepared with AC-EPD exhibited a prolonged, gradual release of DNase activity. The AC-EPD DNase coatings significantly reduced biofilm formation of both Staphylococcus epidermidis and Pseudomonas aeruginosa up to 20 h, whereas DNase coatings prepared by short classic dipping only reduce S. epidermidis biofilm formation, and this to a lesser extent as compared to AC-EPD DNase coatings. Overall, this study indicates that AC-EPD allows to rapidly concentrate DNase I on PDA-functionalized titanium, while maintaining the enzyme activity and anti-infective ability. This highlights the potential of AC-EPD as a time-efficient coating strategy (as opposed to the much slower dip-coating methodologies) for bioactive molecules in a wide variety of biomedical applications.

Use of bacteriophage endolysin EL188 and outer membrane permeabilizers against Pseudomonas aeruginosa.

AIMS: To select and evaluate an appropriate outer membrane (OM) permeabilizer to use in combination with the highly muralytic bacteriophage endolysin EL188 to inactivate (multi-resistant) Pseudomonas aeruginosa. METHODS AND RESULTS: We tested the combination of endolysin EL188 and several OM permeabilizing compounds on three selected Ps. aeruginosa strains with varying antibiotic resistance. We analysed OM permeabilization using the hydrophobic probe N-phenylnaphtylamine and a recombinant fusion protein of a peptidoglycan binding domain and green fluorescent protein on the one hand and cell lysis assays on the other hand. Antibacterial assays showed that incubation of 10(6) Ps. aeruginosa cells ml(-1) in presence of 10 mmol l(-1) ethylene diamine tetraacetic acid disodium salt dihydrate (EDTA) and 50 µg ml(-1) endolysin EL188 led to a strain-dependent inactivation between 3·01 ± 0·17 and 4·27 ± 0·11 log units in 30 min. Increasing the EL188 concentration to 250 µg ml(-1) further increased the inactivation of the most antibiotic resistant strain Br667 (4·07 ± 0·09 log units). CONCLUSIONS: Ethylene diamine tetraacetic acid disodium salt dihydrate was selected as the most suitable component to combine with EL188 in order to reduce Ps. aeruginosa with up to 4 log units in a time interval of 30 min. SIGNIFICANCE AND IMPACT OF THE STUDY: This in vitro study demonstrates that the application range of bacteriophage encoded endolysins as 'enzybiotics' must not be limited to gram-positive pathogens.

[Genome instability of Pseudomonas aeruginosa phages of the EL species: examination of virulent mutants].

The article continues a study of pseudolysogeny in Pseudominas aeruginosa infected with phiKZ-like phages of the EL species. Analysis was performed for several newly isolated virulent mutants of EL phages (EL and RU) that were virulent (capable of causing lysis of bacteria infected with the wild-type phage) and a lower extent of opalescence of negative colonies (NCs). Wile-type recombinants were detected in crosses of virulent mutants of phages EL and RU to confirm the polygenic control of virulence. Since a deletion mutation was found in one of the virulent EL mutants and high genetic instability was characteristic of another mutant, a mobile genetic element was assumed to play a role in mutagenesis. Pseudolysogeny of bacteria provides for horizontal gene transfer between different bacterial strains. Hence, sequencing of the phage genome and demonstration of the lack of toxic gene products are insufficient for the phage to be included into a therapeutic mixture. To use live phages, it is essential to study in detail the possible consequences of their interaction with host bacteria.

A VersaTile-driven platform for rapid hit-to-lead development of engineered lysins.

Health care authorities are calling for new antibacterial therapies to cope with the global emergence of antibiotic-resistant bacteria. Bacteriophage-encoded lysins are a unique class of antibacterials with promising (pre)clinical progress. Custom engineering of lysins allows for the creation of variants against potentially any bacterial pathogen. We here present a high-throughput hit-to-lead development platform for engineered lysins. The platform is driven by VersaTile, a new DNA assembly method for the rapid construction of combinatorial libraries of engineered lysins. We constructed approximately 10,000 lysin variants. Using an iterative screening procedure, we identified a lead variant with high antibacterial activity against Acinetobacter baumannii in human serum and an ex vivo pig burn wound model. This generic platform could offer new opportunities to populate the preclinical pipeline with engineered lysins for diverse (therapeutic) applications.

In vitro and in vivo assessment of phage therapy against Staphylococcus aureus causing bovine mastitis.

OBJECTIVE: The aim of this study was to assess the efficacy of lytic bacteriophages on Staphylococcus aureus causing bovine mastitis, by in vitro and in vivo assays using Galleria mellonella and murine mastitis models. METHODS: Between May and December 2016, ten S. aureus (five methicillin-resistant and five methicillin-sensitive) isolates were isolated from milk samples of cattle with mastitis in Belgium and Norway. The isolates were assessed in vitro for their susceptibility to four lytic bacteriophages (Romulus, Remus, ISP and DSM105264) and subsequently in vivo in G. mellonella larvae and in murine mastitis model. RESULTS: Romulus, Remus and ISP showed a lytic activity against the S. aureus isolates in vitro. A larvae survival rate below 50% was observed at 4 days post-inoculation (DPI) in the groups infected with a methicillin-sensitive S. aureus isolate and treated with these three phages in vivo. An incomplete recovery of the mouse mastitis was observed at 48h post-inoculation (HPI) in the groups infected and treated with the ISP phage in vivo. CONCLUSIONS: The observations are much more pronounced statistically between the infected- phosphate buffered saline (PBS)-treated and infected-phage-treated groups in G. mellonella and the murine mastitis model demonstrating an effect of the phages against S. aureus associated with bovine mastitis.

Dip-a-Dee-Doo-Dah: Bacteriophage-Mediated Rescoring of a Harmoniously Orchestrated RNA Metabolism.

RNA turnover and processing in bacteria are governed by the structurally divergent but functionally convergent RNA degradosome, and the mechanisms have been researched extensively in Gram-positive and Gram-negative bacteria. An emerging research field focuses on how bacterial viruses hijack all aspects of the bacterial metabolism, including the host machinery of RNA metabolism. This review addresses research on phage-based influence on RNA turnover, which can act either indirectly or via dedicated effector molecules that target degradosome assemblies. The structural divergence of host RNA turnover mechanisms likely explains the limited number of phage proteins directly targeting these specialized, host-specific complexes. The unique and nonconserved structure of DIP, a phage-encoded inhibitor of the Pseudomonas degradosome, illustrates this hypothesis. However, the natural occurrence of phage-encoded mechanisms regulating RNA turnover indicates a clear evolutionary benefit for this mode of host manipulation. Further exploration of the viral dark matter of unknown phage proteins may reveal more structurally novel interference strategies that, in turn, could be exploited for biotechnological applications.

[Comparison of new giant bacteriophages OBP and Lu11 of soil pseudomonads with bacteriophages of phiKZ-supergroup of Pseudomonas aeruginosa].

Study of two recently isolated giant bacteriophages Lu11 and OBP that are active on Pseudomonas putida var. Manila and Pseudomonas fluorescens, respectively, demonstrated their similarity in morphology, genome size, and size of phage particles, with giant bacteriophages of Pseudomonas aeruginosa assigned to the supergroup of phiKZ-like phages of the family Myoviridae designated in this manner according to the best studied phage phiKZ that belongs to the species of this group widely distributed in nature. Comparison of major polypeptide sizes of mature particles suggests the similarity of certain proteins in the phages examined. In OBP particles visualized with an electron microscope, an "inner body" was detected, which points to the specific DNA package intrinsic to phages of phiKZ group. In the meantime, phages Lul11 and OBP do not exhibit resemblance among themselves or with any of earlier described phiKZ-like phages in respect to other traits; particularly, they have no detectable DNA homology. Note that phage Lu11 of P. putida var. Manila exhibits very slight homology with phage Lin68 of the family of P. aeruginosa phiKZ-like phages detected only in blot hybridization. This suggests the possible involvement of these phages in interspecies recombination ("gene shuffling") between phages of various bacterial species. Results of partial sequencing of phage genomes confirmed the phylogenetic relatedness of phage OBP to phages of the phiKZ-supergroup, whereas phage Lu11 most probably belongs to a novel species that is not a member of supergroup phiKZ composition. The results of the study are discussed in terms of the evolution of these phages.

Characterization of the bacteriophage PhiKMV DNA ligase.

Gene 17 product (gp17) of the Pseudomonas aeruginosa-infecting bacteriophage phiKMV shows in silico similarity to T7 DNA ligase. In a semi-quantitative activity assay, it is shown that gp17 is a functional, ATP-dependent DNA ligase, in spite of some structural differences related to DNA-binding properties). Enzymatic activity of His6-based purified expression product was optimised (4 degrees C at 24h for sticky end double-stranded DNA fragments) and estimated at 0.5 Weiss U/microg.

[Comparison of the genome for phylogenetically related bacteriophages phiKZ and EL of Pseudomonas aeruginosa: evolutionary aspects and minimal genome size].

Bacteriophages of the family Myoviridae represent one of the most widespread domains of the biosphere substantially affecting the ecological balance of microorganisms. Interestingly, sequence analysis of genomic DNAs of large bacteriophages revealed many genes coding for proteins with unknown functions. A new approach is proposed to improve the functional identification of genes. This approach is based on comparing the genome sequence for phylogenetically and morphologically related phages showing no considerable homology at the level of genomic DNA. It is assumed that gene functions essential for the development of phages of a given family are conserved and that the corresponding genes code for similar orthologous proteins even when lacking sequence homology. The genome was sequenced and compared for two Pseudomonas aeruginosa giant bacteriophages, phiKZ and EL, which belong to a group of (phiKZ-related phages. A substantial difference in genome organization was observed, suggesting specific features of phage evolution. In addition, the problem of the minimal genome of the superfamily is discussed on the basis of the difference in size and structure between the phiKZ and EL genomes.

Impact of industrial and mine drainage wastes on the heavy metal distribution in the drainage basin and estuary of the Sado River (Portugal).

This paper presents results from a survey of the heavy metal distribution in sediments in the drainage basin and estuary of the Sado River (Portugal). In the drainage basin, heavy metals originate mostly from pyrite outcrop erosion and mining activities (Cd, Zn, Cu and locally Hg, Pg), and also from crust erosion (Sn, Ni, Ti, Zr). These sources are not correlated with the particulate organic carbon (POC) and so the metals are thought to be in inorganic forms in this area. Anthropogenic heavy metal sources (urban and industrial) are found in the lower estuary (Sn, Cd, Hg, Zn, Pb and Cu) along with high POC concentrations. In this zone, these metals are thought to be strongly adsorbed onto organic particles. Furthermore, organo-metallic species are likely to be present, as demonstrated in the case of Sn, since methyl- and butyl-tin species were detected in sediments from this area. This suggests the need for the detection of organo-metallic species to understand the heavy metal geochemical cycles. No long-term changes in metal concentrations are found in sediment cores, except in the middle estuary (Zn, Cu) due to the development of mining activities on an industrial scale in the 1860s.

Organo-tins in sediments and mussels from the Sado estuarine system (Portugal).

Analyses of methyl- and butyl-tin levels in freshwater, estuarine and marine sediments from the Sado estuarine system, and in mussels (Mytilus galloprovincialis) from its adjacent coast, have been performed in order to detect the contaminated areas. The main inputs of tributyl-tin (TBT), along with degradation products di- and monobutyl-tin (DBT and MBT), were detected in the estuarine zone, due to high discharge from shipyards located in this area. These levels are sometimes very high, ranging from 235 to 12,200 ng g(-1) total butyl-tins in sediments. Such inputs lead to higher bioconcentration values in mussels in the estuarine zone, as well as in a harbour located along the adjacent coast. The bioconcentration of organo-tins in mussel tissues could be enhanced in estuarine turbid waters, due to an ingestion of butyl-tins adsorbed onto fine particles, in comparison with non-turbid coastal waters. Debutylation processes occur in both sediments and mussel tissues; in organisms, these processes may lead to the formation of inorganic tin, which may be methylated differently according to the period of the year.

Reed beds: constructed wetlands for municipal wastewater treatment plant sludge dewatering.

Reed beds are an alternative technology wastewater treatment system that mimic the biogeochemical processes inherent in natural wetlands. The purpose of this project was to determine the effectiveness of a reed bed sludge treatment system (RBSTS) in southern New England after a six-year period of operation by examining the concentrations of selected metals in the reed bed sludge biomass and by determining the fate of solids and selected nutrients. Parameters assessed in both the reed bed influent and effluent: total suspended solids, biochemical oxygen demand, nitrate-nitrogen and total phosphorus. In addition, the following metals were studied in the reed bed influent, effluent and Phragmites plant tissue and the sludge core biomass: boron, cadmium, chromium, copper, iron, lead, manganese, molybdenum, nickel, and zinc. The removal efficiencies for sludge dewatering, total suspended solids and biochemical oxygen demand were all over 90%. Nitrate and total phosphorus removal rates were 90% and 80% respectively. Overall metals removal efficient was 87%. Copper was the only metal in the sludge biomass that exceeded the standards set by the Massachusetts Department of Environmental Protection for land disposal of sludge. The highest metal concentrations, for the most part, tended to be in the lower tier of the sludge profile. The exception was boron, which was more concentrated in the middle tier of the sludge profile. The data and results presented in this paper support the notion that reed bed sludge treatment systems and the use of reed beds provide an efficient and cost effective alternative for municipal sludge treatment.