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Simkania negevensis is an obligate intracellular bacterium grouped into the order Chlamydiales. This new amoeba-resistant intracellular bacterium might represent a novel etiologic agent of bronchiolitis and community-acquired pneumonia and occurs in aquatic habitats such as drinking water and reclaimed wastewater. Another amoeba-related bacterium, Legionella pneumophila, is an etiologic agent of pneumonia transmitted by environmental aerosols or contaminated water/air cooling systems. These transmission pathways are important in the natural history of Legionellae infections and possibly other intracellular microorganisms such as Parachlamydiaceae; thus, understanding the feasibility of Simkania infection by these routes is relevant. In the present work, we investigated the prevalence of this newly identified pathogenic bacterium in cooling towers by quantitative PCR (qPCR) and its possible relationship with Legionella pneumophila co-infection. Our results show Simkania detection in 2 of 70 cooling towers analyzed. To our knowledge, this report is the first describing Simkania negevensis detection in this category of environmental water samples. Fil: Perez, Leonardo Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Química Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Química Rosario; Argentina. Universidad Politécnica de Catalunya; España Fil: Codony, Francesc. Universidad Politécnica de Catalunya; España Fil: Ríos, Karina. Universidad Politécnica de Catalunya; España. Universidad de Antioquia; Colombia Fil: Adrados, Bárbara. Universidad Politécnica de Catalunya; España Fil: Fittipaldi, Mariana. Universidad Politécnica de Catalunya; España Fil: De Dios, Gregori. Stenco Industrial S.L.; España Fil: Peñuela Mesa, Gustavo Antonio. Universidad de Antioquia; Colombia Fil: Morató Farreras, Jordi. Universidad Politécnica de Catalunya; España

Rural areas of developing countries require low-cost treatment systems to purify wastewater which is contaminated with pesticides and organic matter. This work evaluated for six months the simultaneous removal of chlorpyrifos and dissolved organic matter in water using four horizontal sub-surface flow constructed wetlands (SSFCW) at a pilot scale, that were planted with Phragmites australis at 20 ± 2 °C water temperature. In each wetland, three concentrations of chlorpyrifos and three of dissolved organic carbon (DOC) were tested by liquid chromatography and an organic carbon analyzer respectively. The pesticide and DOC were added to the wetlands in synthetic wastewater. For the experiments, four wetlands of equal dimensions were used, with granular material of igneous rocks, 3.9–6.4 mm in diameter and at a depth of 0.3 m with a layer of water 0.2 m deep. For each treatment, regular sampling was carried out for the influent and effluents. As a supporting feature NH4+, NO3− and PO43− were quantified and in situ measurements of dissolved oxygen (DO), pH, electrical conductivity, water temperature and redox potential were taken. The overall removal of the chlorpyrifos (92.6%) and DOC (93.2%) was high, as was DOC removal as a function of pesticide concentration in the influent. The minimum magnitude (92.0%) was reached with 425.6 μg L−1 of chlorpyrifos and, with the highest pesticide removal (96.8%). At lower concentrations of the agrochemical, DOC removal increased. The removals were possibly due to mineralization processes, biological decomposition and sorption in plants. These findings demonstrate that SSFCW are capable of simultaneously removing dissolved organic matter and organophosphate pesticides such as chlorpyrifos, which indicate that chlorpyrifos did not interfere with the removal of organic material.

Background: While Helicobacter pylori exists in a bacillary form in both the natural habitat and the human host, detrimental environmental circumstances have been observed to lead to the conversion of H. pylori from the bacillary to the coccoid form. However, the viability or nonviability of coccoid forms remains to be established in H. pylori. The aim of this study was to determine whether the quantitative PCR combined with propidium monoazide could be an alternative and good technique to determine H. pylori viability in environmental samples and, to contribute to understanding of the role of the H. pylori forms. Materials and Methods: Viability, morphological distribution, and the number of live H. pylori cells were determined using a propidium monoazide-based quantitative PCR method, at various time points. Results: Under adverse environmental conditions was observed the conversion of H. pylori from the bacillary to the coccoid form, and the decrease in amplification signal, in samples that were treated with propidium monoazide, over the time. Conclusions: Incorporation of propidium monoazide indicates that there is an increase in H. pylori cells with the damaged membrane over the study, leading to the manifestation of cellular degeneration and death. Consequently, quantitative PCR combined with propidium monoazide contributes to our understanding of the role of H. pylori cells, under adverse environmental conditions.

Aims: It is difficult to determine the effects of bactericidal compounds against bacteria in a biofilm because classical procedures for determining cell viability require several working days, multiple complicated steps and are frequently only applicable to cells in suspension. We attempt to develop a compact, inexpensive and versatile system to measure directly the extent of biofilm formation from water systems and to determine the viability of respiring bacteria in high surface biofilms. Methods and Results: It has been reported that the reduction of tetrazolium sodium salts, such as XTT (sodium 3,3′-[1-[(phenylamino)carbonyl]-3,4-tetrazolium]Bis(4-methoxy)-6-nitro)benzene sulfonic acid hydrate), during active bacterial metabolism can be incorporated into a colorimetric method for quantifying cell viability. XTT is reduced to a soluble formazan compound during bacterial aerobic metabolism such that the amount of formazan generated is proportional to the bacterial biomass. Conclusions: We show here, for the first time, that this colorimetric approach can be used to determine the metabolic activity of adherent aerobic bacteria in a biofilm as a measure of cell viability. This technique has been used to estimate viability and proliferation of bacteria in suspension, but this is the first application to microbial communities in a real undisturbed biofilm. Significance and Impact of the Study: This simple new system can be used to evaluate the complex biofilm community without separating the bacteria from their support. Thus, the results obtained by this practice may be more representative of the circumstances in a natural system, opening the possibility to multiple potential applications.

The synthesis, characterization and comparative evaluation of properties of two series of cationic PBS copolyesters bearing respectively ammonium and tributylphosphonium side groups, are reported. The copolyesters with contents in ionic units up to 50 mole-% as well as the fully ionic homopolyesters were prepared by polycondensation in the melt catalyzed by CALB or TBT. Their Mn ranged between 20,000 and 5000 g mol-1 depending of composition and the type of ionic group that is involved. All the copolyesters were non-water soluble and showed good thermal stability. They were semicrystalline with melting temperatures and enthalpies decreasing with the ionic contents. The interactions interplayed by the ionic groups restricted largely the molecular mobility and caused a significant increase in the melt viscosity and glass transition temperature of PBS and a decrease in crystallization rate. Both ammonium and phosphonium containing PBS copolyesters were able to be coupled with sulfonated PBS to generate ionic polymer blends with modified crystallizability. The presence of both ammoniums and phosphoniums provided PBS with remarkable antimicrobial activity against gram-positive and gram-negative bacteria.

En España, debido a las prácticas agrícolas, el 13% del territorio ha sido declarado vulnerable a la contaminación del agua por nitratos. Para mitigar los impactos ambientales generados por el nitrógeno, se ha construido un sistema híbrido de humedales artificiales gracias al proyecto europeo LIFE REAGRITECH. El prototipo ha sido pensado como un sistema compacto, modular y móvil, automatizado y operado de forma remota. El monitoreo del sistema producirá conocimientos valiosos para la administración pública, los agricultores, investigadores y universidades, y proporcionará información útil para mejorar la toma de decisiones con el fin de preservar los recursos hídricos y el medio ambiente. El proyecto REAGRITECH tiene grandes perspectivas de futuro en el campo de la agricultura en Europa y en Colombia. Due to the agricultural practices in Spain, 13% of the entire territory has been declared as vulnerable to nitrate-polluted water. In order to mitigate the environmental impact caused by nitrogen, a hybrid system of artificial wetlands has been engineered thanks to the European project known as LIFE REAGRITECH. The prototype has been thought as a compact, modular, and portable system, automated and operated in a remote manner. Monitoring of the system will result in valuable knowledge for public management, farmers, researchers, and universities, and it will provide useful information intended to improve the decision-making process as a way of preserving water resources and environment. Project REAGRITECH offers significant future perspectives in the field of agriculture in Europe and in Colombia as well.

An experimental system has been developed that allows the monitoring of biofilm development on supports exposed to water of different characteristics. The system consists of a series of packed-bed reactors filled with glass beads, and by periodically removing biofilm attached to these beads for off-line analyses this provides a means for monitoring biofilm development. Despite its reduced dimensions (6.9 cm long and 1.58 cm in diameter), the experimental system used has a sampling surface of 90.3 cm2 (including only the surface of the glass beads). This allows reproducible and representative samples to be taken from different water systems, providing a reliable and economic method for evaluating in situ the formation of biofilms from different environments. The set-up of the entire experimental system was constructed to meet the demands of field experiments in a well-defined hydrodynamic environment and to allow easy removal of samples for biomass quantification and microscopic observation. Data obtained using this device can be used as an indicator of the risk of biofilm formation in different water systems. This indicator, "the biofilm accumulation potential", represents an effective and representative tool for the monitoring of biofilm development in an integrated antifouling strategy, in order to help keep biofouling, scaling and microbial risks under control. According to the experiments with the packed-bed reactors used with a high flow regime, the ratio TCN/HPC could provide an indication of the state of the biofilm, and lower ratios could indicate a higher biofilm accumulation potential.

This study investigates the effects of chlorothalonil (CLT) on chemical oxygen demand (COD) and dissolved organic carbon (DOC) in pilot-scale horizontal subsurface flow constructed wetlands (HSSFCW) planted with Phragmites australis. Physicochemical parameters of influent and effluent water samples, microbial population counting methods and statistical analysis were used to evaluate the influence of CLT on organic matter removal efficiency. The experiments were conducted on four planted replicate wetlands (HSSFCW-Pa) and one unplanted control wetland (HSSFCW-NPa). The wetlands exhibited high average organic matter removal efficiencies (HSSFCW-Pa: 80.6% DOC, 98.0% COD; HSSFCW-NPa: 93.2% DOC, 98.4% COD). The addition of CLT did not influence organic removal parameters. In all cases CLT concentrations in the effluent occurred in concentrations lower than the detection limit of the analytical method. Microbial population counts from HSSFCW-Pa showed significant correlations among different microbial groups and with different physicochemical variables. The apparent independence of organic matter removal and CLT inputs, along with the CLT depletion observed in effluent samples demonstrated that HSSFCW are a viable technology for the treatment of agricultural effluents contaminated with organo-chloride pesticides like CLT.

Waddlia chondrophila is an emerging pathogen considered as a potential agent of abortion in humans and bovines, and is related with human respiratory disease. Despite these findings, the infection source and transmission pathways have not been identified. The evidence of growth into amoeba suggests water as a possible environmental source. The presence of Waddlia chondrophila was determined in drinking and well water samples (n=70) by quantitative PCR (Q-PCR). Positive results were observed in 10 (25%) of the 40 well samples analyzed; therefore, well water could be a potential reservoir and possible infection source of Waddlia chondrophila in animals and humans.

IntroductionNowadays, as in the past, our knowledge of microbiology islimited because of the capacity of available technologytools. In recent decades, molecular techniques are playinga central role in the understanding of the microbial world. Inthis sense, a complete scientific evaluation of biologicalsamples is not possible without using molecular biology.The most influential technique has probably been the poly-merasechainreaction(PCR),whichallowsustoworkbeyondclassicalmicrobiology.WithPCR,insomecases,theneedforculture may be avoided. Nevertheless, until the developmentof real-time PCR, our evaluation of the microbial complexityhas been merely qualitative.Real-time PCR is an evolution of the conventional end-point PCR wherein the amplification of a target gene and itsfluorescence detection occurs simultaneously during eachcycle. Different strategies based on the use of nonspecificDNA intercalating dyes or specific fluorescent probes existfor linking target amplification to fluorescence detection.Regardless of the fluorescence emission mechanisms in-volved, real-time PCR enables both detection and quantifica-tion of a genetic target by a continuous fluorescencemonitoring of DNA amplification (Higuchi et al. 1993).An advantage in real-time PCR isthat target quantificationsensitivity is independent of the copy number within thesample (Freeman et al. 1999). Moreover, the key aspect thatdifferentiates real-time PCR from previous semi-quantitativemethodologies is that target DNA concentration can be deter-mined from the fractional cycle at which a threshold amountof target DNA is produced (i.e., measured signal exceeds abackground level), set at a point where amplicon DNA justbecomesdetectablebutisstillwithintheexponentialphaseoftheamplification(Higuchietal.1993;Rasmussen2001).Thisquantificationcycle(Cq)(orcyclethreshold,Ct)areinverselyproportionaltotheamountoftargetnucleicacidinthesample(i.e., the lower the Cq value, the greater the amount of targetnucleic acid in the sample). This approach ensures that inter-fering factors associated with later stages of the amplificationare minimized and considerably improves the measurementprecision (Rasmussen 2001).One of the most important points is that real-time PCRallows quantitative measurements. In order to estimate atarget concentration in environmental samples, absolutequantification can be performed using a standard curveconstructed by amplifying known amounts of target DNA(Rutledge and Cote 2003). Nowadays, this approach is themost commonly used in environmental microbiology.Real-time PCR has many advantages over conventionalendpoint PCR including speed, broad dynamic range ofquantification, reduced risk of contamination, and goodsensitivity. However, despite all these objective strengths,real-time PCR sensitivity is not better than conventionalPCR if a protocol validation is not performed (Bastien etal. 2008).

Simkania negevensis is an obligate intracellular bacterium grouped into the order Chlamydiales. This new amoeba-resistant bacterium represents a novel aetiologic agent of bronchiolitis and community-acquired pneumonia in both adults and children. It has been suggested that Simkania could be an ubiquitous microorganism presented in water environments. In the natural history of infections with amoeba-related bacteria encountered in aquatic habitats, the transmissions by environmental aerosols or contaminated water/air systems have been extensively recognized. Therefore, understanding the feasibility of Simkania infection by these or similar routes is relevant. In the present work, we investigated the prevalence of this novel disease-associated microorganism in water samples from different sources by real-time PCR (qPCR). Our results show Simkania detection in 5 of 185 water analyzed samples (2.7%: 2 of 88 cooling towers and 3 of 8 waste water samples). However, no Simkania was detected in a drinking water.

Even though the advent of quantitative polymerase chain reaction (PCR) has improved the detection of pathogen microorganisms in most of areas of microbiology, a serious limitation of this method may arise from the inability to discriminate between viable and nonviable pathogens. To overcome it, the use of real-time PCR and selective nucleic acid intercalating dyes like propidium monoazide (PMA) have been effectively evaluated for different microorganisms. To assess whether PMA pretreatment can inhibit PCR amplification of nonviable amoeba DNA, Acanthamoeba castellani survival was measured using cell culture and real-time PCR with and without PMA pretreatment. Autoclave and contact lens disinfecting solutions were used to inactivate amoebae. After these inactivation treatments, the results indicated that the PMA pretreatment approach is appropriate for differentiating viable A. castellani, both trophozoites and cysts. Therefore, the PMA-PCR approach could be useful as a rapid and sensitive analytical tool for monitoring treatment and disease control, assessing effective disinfection treatments, and for a more reliable understanding of the factors that contribute to the interaction amoeba-pathogenic bacteria.

This work used subsurface flow constructed wetlands, planted with Phragmites australis, using 2 water depths and 2 sizes of granular material, in order to find the optimal conditions for the remova...

While Helicobacter pylori exists in a bacillary form in both the natural habitat and the human host, detrimental environmental circumstances have been observed to lead to the conversion of H. pylori from the bacillary to the coccoid form. However, the viability or nonviability of coccoid forms remains to be established in H. pylori. The aim of this study was to determine whether the quantitative PCR combined with propidium monoazide could be an alternative and good technique to determine H. pylori viability in environmental samples and, to contribute to understanding of the role of the H. pylori forms.Viability, morphological distribution, and the number of live H. pylori cells were determined using a propidium monoazide-based quantitative PCR method, at various time points.Under adverse environmental conditions was observed the conversion of H. pylori from the bacillary to the coccoid form, and the decrease in amplification signal, in samples that were treated with propidium monoazide, over the time.Incorporation of propidium monoazide indicates that there is an increase in H. pylori cells with the damaged membrane over the study, leading to the manifestation of cellular degeneration and death. Consequently, quantitative PCR combined with propidium monoazide contributes to our understanding of the role of H. pylori cells, under adverse environmental conditions.

The genus Legionella comprises more than 40 species and 64 serogroups with approximately half of those species associ ated with human diseases. Legionella pneumophila Serogroup 1 is the most common pathogenic species and is responsible for up to 80% of legionellosis cases in the world. Legionella levels in water are assessed routinely by culture on a selective medium, but its slow growth is a serious drawback, given that at least 10 days are required to obtain results. In an attempt to provide a simple screening method for Legionella pneumophila in water systems samples a real time PCR assay using SYBR Green was developed. A total of 50 samples from cooling towers and hot tap water systems were analysed by DNA amplification using 2 pairs of primers targeting the mip and dot genes. Legionella pneumophila Serogroup 1 (NCTC12821) was used as a reference strain and to evaluate real-time PCR performance. The assays were successful with both primer sets; good and similar amplification efficiencies were achieved. In addition, high sensitivity was obtained; the method proved to allow for the detection of fewer than 10 gene copies per reaction. Results of real-time PCR were compared to conventional analysis based on culture. Although no strong correlation was observed between both methods and consequently real-time PCR could not substitute for the reference method, it represents a powerful screening tool. The inexpensive, sensitive and rapid real-time PCR based in SYBR Green method is of interest in monitoring Legionella pneumophila contamina tion, especially in environmental samples, and should be economical for large-scale routine tests.

Selective nucleic acid intercalating dyes—ethidium monoazide (EMA) and propidium monoazide (PMA)— represent one of the most successful recent approaches to detect viable cells (as defined by an intact cell membrane) by PCR and have been effectively evaluated in different microorganisms. However, some practical limitations were found, especially in environmental samples. The aim of this work was to show that in the application of viable real-time PCR, there may be significant biases and to propose a strategy for overcoming some of these problems. We present an approach based on the combination of three real-time PCR amplifications for each sample that should provide an improved estimation of the number of viable cells. This approach could be useful especially when it is difficult to determine a priori how to optimize methods using PMA or EMA. Although further studies are required to improve viable real-time PCR methods, the concept as outlined here presents an interesting future research direction.

The advent of quantitative PCR has improved the detection of human viral pathogens in the environment. However, a serious limitation of this method may arise from the inability to discriminate between viruses that are infectious and viruses that have been inactivated and do not represent a human health hazard. To assess whether propidium monoazide (PMA) pre-treatment is a good approach to inhibiting DNA amplification from non-infectious viruses, bacteriophage T4 survival was measured using cell culture titration and real-time PCR with and without PMA pre-treatment. Heat (85 degrees C) and proteolysis methods were carried out. After these inactivation treatments, the results indicated that the PMA pre-treatment approach is not appropriate for differentiating infectious viruses. However, when a heat treatment at 110 degrees C was undertaken, PMA pre-treatment did allow differentiation of non-infectious from infectious viruses. In this case, effective binding of PMA to bacteriophage T4 DNA could be taken to indicate capsid damage. Therefore, PMA pre-treatment may be appropriate for assessing effective disinfection treatments and for a more reliable understanding of the factors that contribute to viral inactivation through capsid damage monitoring. The PMA-PCR approach could be useful as a rapid and inexpensive analytical tool for screening and evaluation of the efficacy of disinfectants.

Helicobacter pylori are ubiquitous Gram-negative bacteria with a high estimated level of infection in the world populations, but a majority of the infected persons are asymptomatic. This pathogen has been classified by the World Health Organization as a class I carcinogen and recognized as the causal agent of most peptic ulcers and chronic gastritis that might lead to stomach cancer. Although not all the transmission pathways of these bacteria into humans have been properly identified, enough data have suggested that the oral-oral or fecal-oral ones are the main infection routes. Helicobacter pylori have been detected in non-treated water and in drinking water, which suggested that water might be an important infection source. As childhood is the critical period of infection, the aim of the present work was to examine the presence of Helicobacter pylori in soil samples from public playing areas of Spanish parks.

The Catabacteriaceae is a new bacterial family with a unique member: Catabacter hongkongensis is a strictly anaerobic, non-sporulating, Gram-positive coccobacillus that is phylogenetically related to some clostridial clusters. Little is known of its epidemiology and environmental distribution, but the inclusion of its 16S rRNA gene sequence in GenBank has allowed it to be detected qualitatively. As a first approach for prospective surveys, a real-time polymerase chain reaction (PCR) procedure to identify C. hongkongensis has been developed. The presence of Catabacteriaceae in 29 water bodies subjected to possible human or animal impact has been investigated. Four of them were positive. The results confirm that highly polluted water can contain C. hongkongensis.

La aparicion de depositos indeseados en cualquier sistema de distribucion de agua potable es inevitable en condiciones normales de operacion. Conocer la naturaleza de tales depositos ayudara a establecer las causas de su formacion y, en consecuencia, a mantener altos niveles de calidad del agua. En este trabajo se presentan los resultados de un amplio estudio realizado en el sistema de distribucion de agua de una ciudad situada en una zona tropical. La caracterizacion de los depositos obtenidos en diferentes sitios a lo largo del sistema se realizo mediante Espectroscopia Infrarroja (IR), Difraccion de Rayos X (DRX), Energias Dispersivas de Rayos X (EDS) y Microscopia Electronica de Barrido (MEB). El analisis de las muestras indica la presencia de tres tipos predominantes de depositos: depositos color marron, tuberculos y depositos blancos. Se hallo que los primeros estan constituidos principalmente por aluminosilicatos y acidos humicos. Los tuberculos se componen principalmente de mezclas de magnetita, goethita y, en algunos casos, lepidocrocita. Por su parte, los depositos blancos estan constituidos por calcita, aluminosilicatos y cuarzo. Ademas, se hallaron contenidos de materia organica y solidos volatiles de 14.0 ± 5.0% para los depositos marron y de 11.2 ± 2.0% para los tuberculos.

Microbial quality in water distribution systems is strongly affected by the development of microbial biofilms. Production and release of microbial cells by the biofilm affect microbial levels in the water column and in some cases this fact constitutes a public health concern. In this study, we attempt to analyze in which way the existence of different episodes of chlorine depletion affects both biofilm formation and microbial load of an artificial laboratory system. The work was carried out using two parallel packed bed reactors both supplied with running tap water. One of the reactors was used as a control and was permanently exposed to the action of chlorine. In the other reactor, chlorine was neutralized at selected times during the experiment and for periods of variable length. During the experiment the concentration of total and viable cells from the effluent was monitored at the exit of each of the reactors. The data obtained were used to estimate microbial production from the biofilms. As an average, release of microbial cells to the water phase increased tenfold in the absence of chlorine. The results also indicate that disinfectant efficiency against the biofilm was not recovered when chlorine returned to normal levels after each event of chlorine neutralization. Cell viability in the water phase in the presence of chlorine was low at the beginning of the experiment but increased 4 orders of magnitude after five neutralization periods. Therefore, subsequent episodes of chlorine depletion may accelerate the development of microbial communities with reduced susceptibility to disinfection in real drinking water systems.

The disinfectant properties of chlorine have been known for centuries but in the last few years water chlorination has attracted some criticism due to its secondary effects and the increased resistance of bacterial strains to chlorine inactivation. In this paper the kinetics of inactivation by chlorine of different Gram-positive and Gram-negative bacterial strains isolated from chlorinated water is studied. The Gram-positive strains were more resistant to chlorine and the behaviour of some of them in the presence of chloramphenicol suggests either the synthesis of unique proteins or aggregation of the bacteria as mechanisms of resistance to inactivation. The concept of Ki, the inactivation rate constant, by comparison with Ks in Michaelis-Menten enzyme kinetics (considering enzymic saturation), or with Ks in Monod growth kinetics (considering limiting rates of transport and metabolism of substrates), may be an interesting parameter to define microbial resistance to disinfectants and toxics.

Fil: Adrados, B. Universitat Politècnica de Catalunya. Health and Environmental Microbiology Laboratory; España Fil: Adrados, B. Unesco Chair on Sustainability; España Fil: Arias, C. A. Aarhus University. Department of Bioscience. Dinamarca Fil: Pérez, Leonardo Martín. Pontificia Universidad Católica Argentina. Facultad de Química e Ingeniería de Rosario. Departamento de Investigación Institucional; Argentina Fil: Pérez, Leonardo Martín. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Pérez, Leonardo Martín. Universitat Politècnica de Catalunya. Health and Environmental Microbiology Laboratory; España Fil: Pérez, Leonardo Martín. Unesco Chair on Sustainability; España Fil: Codony, F. Laboratori Municipal de Mataró; España Fil: Bécares, E. University of León. Faculty of Environmental and Biological Sciences. Department of Biodiversity and Environmental Management; España Fil: Brix, H. Aarhus University. Department of Bioscience. Dinamarca Fil: Morató, J. Universitat Politècnica de Catalunya. Health and Environmental Microbiology Laboratory; España Fil: Morató, J. Unesco Chair on Sustainability; España Abstract: The aim of the present work was to evaluate and compare the performance in the removal of pathogenic microbes in four different types of decentralized wastewater treatment systems, namely: horizontal flow constructed wetlands (HFCW), vertical flow constructed wetlands (VFCW), biological sand filters (BSF) and biofilters (BF). All the systems analyzed are located in Jutland, Denmark. Water sampling took place during a three months period that covered from winter to spring. Conventional microbial indicators such as Escherichia coli, total coliforms (TC), intestinal enterococci and sulphite-reducing clostridia were quantified using traditional microbiological culture methods, whereas Bacteroides spp. determination was performed by quantitative PCR (qPCR). Other water quality parameters such as dissolved oxygen, biological oxygen demand (BOD5), total suspended solids (TSS), pH, temperature, ammonium concentration and conductivity of influent and effluent water samples were also analyzed. The results showed that bacterial indicators significantly reduced in all the systems analyzed. In general, BF showed the best performance in the removal of microbes for all bacteria studied, while BSF demonstrated an improved capacity to eliminate E. coli and TC. Contrarily, VFCW seems to be more effective reducing the amount of intestinal enterococci, sulphite-reducing clostridia, and Bacteroides spp. In the present study, HFCW were the less efficient wastewater treatment system for the elimination of the evaluated pathogens. However, the performance in the removal of microbes was still significant considering that such systems were the oldest under operation (with over 20 years of continuous task).

Constructed wetlands constitute an interesting option for wastewater reuse since high concentrations of contaminants and pathogenic microorganisms can be removed with these natural treatment systems. In this work, the role of key design factors which could affect microbial removal and wetland performance, such as granular media, water depth and season effect was evaluated in a pilot system consisting of eight parallel horizontal subsurface flow (HSSF) constructed wetlands treating urban wastewater from Les Franqueses del Vallès (Barcelona, Spain). Gravel biofilm as well as influent and effluent water samples of these systems were taken in order to detect the presence of bacterial indicators such as total coliforms (TC), Escherichia coli, fecal enterococci (FE), Clostridium perfringens, and other microbial groups such as Pseudomonas and Aeromonas. The overall microbial inactivation ratio ranged between 1.4 and 2.9 log-units for heterotrophic plate counts (HPC), from 1.2 to 2.2 log units for total coliforms (TC) and from 1.4 to 2.3 log units for E. coli. The presence of fine granulometry strongly influenced the removal of all the bacterial groups analyzed. This effect was significant for TC (p = 0.009), E. coli (p = 0.004), and FE (p = 0.012). Shallow HSSF constructed wetlands were more effective for removing Clostridium spores (p = 0.039), and were also more efficient for removing TC (p = 0.011) and E. coli (p = 0.013) when fine granulometry was used. On the other hand, changes in the total bacterial community from gravel biofilm were examined by using denaturing gradient gel electrophoresis (DGGE) and sequencing of polymerase chain reaction (PCR)-amplified fragments of the 16S rRNA gene recovered from DGGE bands. Cluster analysis of the DGGE banding pattern from the different wetlands showed that microbial assemblages separated according to water depth, and sequences of different phylogenetic groups, such as Alpha, Beta and Delta-Proteobacteria, Nitrospirae, Bacteroidetes, Acidobacteria, Firmicutes, Synergistetes and Deferribacteres could be retrieved from DGGE bands Fil: Morató, Jordi. Universidad Politecnica de Catalunya; España Fil: Codony, Francesc. Universidad Politecnica de Catalunya; España Fil: Sánchez, Olga. Universidad Autonoma de Barcelona. Departamento de Genética y Microbiología; España Fil: Perez, Leonardo Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Química Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Química Rosario; Argentina. Universidad Politecnica de Catalunya; España Fil: García, Joan. Universidad Politecnica de Catalunya; España Fil: Mas, Jordi. Universidad Autonoma de Barcelona. Departamento de Genética y Microbiología; España

This is the author's version of a work that was accepted for publication in Water research. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Water researc, Vol. 55 (May 2014), p. 304-312. DOI 10.1016/j.watres.2014.02.011 The prokaryotic microbial communities (Bacteria and Archaea) of three different systems operating in Denmark for the treatment of domestic wastewater (horizontal flow constructed wetlands (HFCW), vertical flow constructed wetlands (VFCW) and biofilters (BF)) was analysed using endpoint PCR followed by Denaturing Gradient Gel Electrophoresis (DGGE). Further sequencing of the most representative bacterial bands revealed that diverse and distinct bacterial communities were found in each system unit, being γ-Proteobacteria and Bacteroidetes present mainly in all of them, while Firmicutes was observed in HFCW and BF. Members of the Actinobacteria group, although found in HFCW and VFCW, seemed to be more abundant in BF units. Finally, some representatives of α, β and δ-Proteobacteria, Acidobacteria and Chloroflexi were also retrieved from some samples. On the other hand, a lower archaeal diversity was found in comparison with the bacterial population. Cluster analysis of the DGGE bacterial band patterns showed that community structure was related to the design of the treatment system and the organic matter load, while no clear relation was established between the microbial assemblage and the wastewater influent.

The influence of chlorine levels, the concentration of dissolved organic carbon and the abundance of bacteria in suspension, on the formation of biofilms on experimental glass surfaces were evaluated. Twelve reactors, packed with glass spheres, were continuously perfused with tap water. The properties of water were altered in different ways: chlorine was neutralized by the addition of thiosulfate, the levels of assimilable organic carbon were increased through the addition of acetate, and the bacterial load was modified by means of the continuous inoculation of a growing active culture of Pseudomonas aeruginosa. Continuous addition of bacteria to water containing 0.5 mg/l of free chlorine, did not result in the formation of detectable biofilms even after one month. When bacteria were added simultaneously with thiosulfate as a chlorine neutralizer, a community of attached bacteria appeared in less than 24 hours. Addition of acetate with the presence of 0.5 mg/l of chlorine did not stimulate the formation of biofilms. On the contrary, neutralization of chlorine with thiosulfate allowed the formation of biofilms with 10(6) cfu/cm(2) in about two weeks.

Helicobacter pylori infection is a risk factor for chronic active gastritis, peptic ulcers, gastric carcinoma and lymphoma. Although the infection may be acquired through different transmission routes, the presence and viability of H. pylori in water sources are not well known. Therefore, the aim of our study was to analyse the viability of H. pylori cells in urban surface waters collected at the Vallparadís public park in Terrassa, Barcelona, Spain. The water samples were analysed by viability quantitative polymerase chain reaction (qPCR) using propidium monoazide and specific primers for the H. pylori vacuolating cytotoxin (vacA gene). Viable H. pylori were found in 91.3% of the samples analysed, with an average concentration of 3.46 ± 1.06 log cell 100 mL−1. Our work proves a quick and simple procedure for evaluating viable H. pylori cells in environmental samples by qPCR. Furthermore, the results provide evidence that urban surface waters may contain considerable levels of viable H. pylori cells, thus indicating they are a potential source of infection, which represents a public health concern.