The feasibility of improved live-dead distinction in qPCR-based microbial source tracking.

PCR-based microbial source tracking (MST) has become a useful tool to identify dominant sources of fecal pollution in water. The method has previously been successfully combined with viability PCR (using propidium monoazide) allowing the preferential detection of membrane-intact bacteria. This study aimed at further improving the selectivity for intact cells when targeting host-specific markers in Bacteroidales bacteria. One approach was to increase amplicon sizes that had been shown to be useful for other applications of viability PCR. For this purpose, two different amplicon sizes were compared when targeting either the genus of Bacteroidales or subgroups thereof specifically associated with human and ruminant fecal material. When applied to different environmental samples, the proposed proportion of intact cells could drop by up to 38% (for sewage treatment effluent from 64 to 26%) when targeting longer sequences. Furthermore co-incubation of the viability dye with dimethylsulfoxide (DMSO) was found to be beneficial, although this observation is currently still empirical. When examining signal decay of artificially contaminated unfiltered river water over six weeks, the PMA treatment effect was observed from the beginning, but the ratio of intact and damaged cells remained constant over time with signals disappearing at the same rate independent of PMA treatment. In this instance the contribution of other factors to overall signal decay seemed more important than loss of membrane integrity.

The River Ruhr - an urban river under particular interest for recreational use and as a raw water source for drinking water: The collaborative research project "Safe Ruhr" - microbiological aspects.

Along the intense industrialization of the Ruhr valley (Germany), the River Ruhr became increasingly polluted. Over time, using it for recreational purposes became a serious health hazard and bathing was banned due to chemical and microbiological risks. The purpose of the collaborative project "Safe Ruhr" was to verify the current status and to provide a scientific basis for lifting the bathing ban. As the river also provides a raw water source for drinking water production, it was investigated how well the treatment procedures control possible hygienic risks. As study area, the barrier Lake Baldeney was chosen as it embraces earlier bathing sites and tributes to river bank filtration water for drinking water treatment plants. The hygienic condition of the river water was determined over 18 months by measuring general physical, chemical and microbiological water quality parameters including fecal indicators, bacterial obligate and facultative pathogens, parasitic protozoa, enteric viruses and schistosome parasites (Trichobilharzia). Samples were taken at eight locations including sites before and after receiving the discharge of stormwater and treated wastewater, potential future bathing sites and a raw water abstraction point for potable water production. In summary, for all investigated physico-chemical parameters no significant difference between the eight investigated sampling locations on a distinct sampling date were observed. This study focused on hygienically relevant bacteria and parasitic protozoa. Fecal indicators, Escherichia coli, intestinal enterococci and Clostridium perfringens as well as coliform bacteria were detected in 94-100% of the water samples. Enteric pathogens, including Campylobacter spp. and Salmonella enterica, were isolated from 33% and 28% of the samples, respectively, in relatively low concentrations. Among the environmental facultative pathogens, P. aeruginosa was detected at a high frequency of 82% of all samples, but in low numbers, while Aeromonas spp. were found in all water samples in relative high concentrations. The levels of all target organisms were not clearly associated with sources of pollution, with the exception of slightly enhanced numbers of coliform bacteria and E. coli downstream of a sewage discharge point from a wastewater treatment plant. Seasonal variations were observed with higher detection rates of Campylobacter spp. in winter and S. enterica in autumn and winter in contrast to the other bacterial groups, which showed no significant fluctuations throughout the year. Precipitation within two days prior to sampling resulted in a trend of enhanced numbers of coliform bacteria, E. coli, intestinal enterococci and Aeromonas. Sampling and analysis of parasitic protozoa was carried out in accordance to the European bathing water guideline and the ISO 15553 method. Characteristics of the river (flow, vegetation, birds protection zone, bathing of people, sewage etc.) were compared to the number of organisms detected. All in all 184 samples were investigated for Cryptosporidium spp. and Giardia spp. 80% of the samples were positive for Giardia spp. with a mean of 5cysts/100l (0.1-157.9). Highest values were achieved in autumn and winter, lowest values during the assumed bathing season. There seemed to be a trend to lower values in and after a reservoir in the river course, but with no statistical significance. A statistical significance could be shown for higher concentrations after heavy rainfall that led to discharge of combined sewage overflows in the city of Essen. Only 29% of the samples were positive for Cryptosporidium spp. with a single maximum value of 27.7 and all other concentrations below 5 oocysts/100l. On a low level there seemed to be slightly higher findings during summer and bathing season than in autumn and winter. No correlation to heavy rainfall could be found. The findings correspond to earlier results from the River Rhine (Germany). The influence of sewage on the water quality of the Ruhr could be shown from the correlation of Giardia load and activity of combined sewage overflows after heavy rainfall. The rare and low findings of Cryptosporidium spp. lead to the same conclusion, that microbial water quality in the investigation area is rather influenced from sewage water than from diffuse water sources into the River Ruhr.

Developing an easy-to-apply model for identifying relevant pathogen pathways into surface waters used for recreational purposes.

Swimming in inner-city surface waters is popular in the warm season, but can have negative consequences such as gastro-intestinal, ear and skin infections. The pathogens causing these infections commonly enter surface waters via several point source discharges such as the effluents from wastewater treatment plants and sewer overflows, as well as through diffuse non-point sources such as surface runoff. Nonetheless, the recreational use of surface waters is attractive for residents. In order to save financial and organizational resources, local authorities need to estimate the most relevant pathways of pathogens into surface waters. In particular, when detailed data on a local scale are missing, this is quite difficult to achieve. For this reason, we have developed an easy-to-apply model using the example of Escherichia coli and intestinal enterococci as a first approach to the local situation, where missing data can be replaced by data from literature. The model was developed based on a case study of a river arm monitored in western Germany and will be generalized for future applications. Although the limits of the EU Bathing Water Directive are already fulfilled during dry weather days, we showed that the effluent of wastewater treatment plants and overland flow had the most relevant impact on the microbial surface water quality. On rainy weather days, combined sewer overflows are responsible for the highest microbial pollution loads. The results obtained in this study can help decision makers to focus on reducing the relevant pathogen sources within a catchment area.

Reducing pathogens in combined sewer overflows using ozonation or UV irradiation.

Fecal contamination of water resources is a major public health concern in densely populated areas since these water bodies are used for drinking water production or recreational purposes. A main source of this contamination originates from combined sewer overflows (CSOs) in regions with combined sewer systems. Thus, the treatment of CSO discharges is urgent. In this study, we explored whether ozonation or UV irradiation can efficiently reduce pathogenic bacteria, viruses, and protozoan parasites in CSOs. Experiments were carried out in parallel settings at the outflow of a stormwater settling tank in the Ruhr area, Germany. The results showed that both techniques reduce most hygienically relevant bacteria, parasites and viruses. Under the conditions tested, ozonation yielded lower outflow values for the majority of the tested parameters.

Occurrence and fate of the angiotensin II receptor antagonist transformation product valsartan acid in the water cycle--a comparative study with selected ß-blockers and the persistent anthropogenic wastewater indicators carbamazepine and acesulfame.

The substantial transformation of the angiotensin II receptor antagonist valsartan to the transformation product 2'-(2H-tetrazol-5-yl)-[1,1'-biphenyl]-4-carboxylic acid (referred to as valsartan acid) during the activated sludge process was demonstrated in the literature and confirmed in the here presented study. However, there was a severe lack of knowledge regarding the occurrence and fate of this compound in surface water and its behavior during drinking water treatment. In this work a comparative study on the occurrence and persistency of valsartan acid, three frequently used ß-blockers (metoprolol, atenolol, and sotalol), atenolol acid (one significant transformation product of atenolol and metoprolol), and the two widely distributed persistent anthropogenic wastewater indicators carbamazepine and acesulfame in raw sewage, treated wastewater, surface water, groundwater, and tap water is presented. Median concentrations of valsartan acid in the analyzed matrices were 101, 1,310, 69, <1.0, and 65 ng L(-1), respectively. Treated effluents from wastewater treatment plants were confirmed as significant source. Regarding concentration levels of pharmaceutical residues in surface waters valsartan acid was found just as relevant as the analyzed ß-blockers and the anticonvulsant carbamazepine. Regarding its persistency in surface waters it was comparable to carbamazepine and acesulfame. Furthermore, removal of valsartan acid during bank filtration was poor, which demonstrated the relevance of this compound for drinking water suppliers. Regarding drinking water treatment (Muelheim Process) the compound was resistant to ozonation but effectively eliminated (≥90%) by subsequent activated carbon filtration. However, without applying activated carbon filtration the compound may enter the drinking water distribution system as it was demonstrated for Berlin tap water.

Simultaneous monitoring of biofilm growth, microbial activity, and inorganic deposits on surfaces with an in situ, online, real-time, non-destructive, optical sensor.

Deposits on surfaces in water-bearing systems, also known as 'fouling', can lead to substantial losses in the performance of industrial processes as well as a decreased product quality. Early detection and localization of such deposits can, to a considerable extent, save such losses. However, most of the surfaces that become fouled, for example, in process water pipes, membrane systems, power plants, and food and beverage industries, are difficult to access and analyses conducted on the water phase do not reveal the site or extent of deposits. Furthermore, it is of interest to distinguish biological from non-biological deposits. Although they usually occur together, different countermeasures are necessary. Therefore, sensors are required that indicate the development of surface fouling in real-time, non-destructively, and in situ, preferably allowing for discrimination between chemical and/or biological deposits. In this paper, an optical deposit sensor is presented which fulfills these requirements. Based on multiple fluorescence excitation emission matrix analysis, it detects autofluorescence of amino acids as indicators of biomass. Autofluorescence of nicotinamide adenine dinucleotide + hydrogen is interpreted as an indicator of biological activity, thus it acts as a viability marker, making the method suited for assessing the efficacy of disinfection treatments. Scattering signals from abiotic deposits such as calcium carbonate or corrosion products can clearly be distinguished from biotic substances and monitored separately. The sensor provides an early warning of fouling, allowing for timely countermeasures to be deployed. It also provides an assessment of the success of cleaning treatments and is a promising tool for integrated antifouling strategies.

Grafted glycopolymer-based receptor mimics on polymer support for selective adhesion of bacteria.

A sugar-containing monomer (2-lactobionamidoethyl methacrylate, LAMA) was grafted on a polypropylene (PP) microfiltration membrane surface by UV-induced graft copolymerization. The degree of grafting can be controlled by variation of monomer concentration, UV irradiation time, and photoinitiator concentration. Fourier transform infrared spectroscopy and scanning electron microscopy were employed to confirm the surface modification on the membranes. The water contact angle was used to evaluate the hydrophilicity change of the membrane surface before and after modification. Bacteria capture experiments showed that the membrane could selectively bind E. faecalis while adhesion of S. maltophilia was not influenced by the functionalization of PP with grafted poly(LAMA). The adhesion of E. faecalis onto poly(LAMA) grafted membrane could be inhibited by 200 mM galactose solution; however, glucose solution showed no inhibition effect. Moreover, occupying sugar residues on the membrane surface primarily by a galactose targeting lectin, peanut agglutinin, could significantly suppress the following adhesion of E. faecalis. All these results clearly demonstrate that this poly(LAMA) grafted PP membrane can selectively capture E. faecalis and that this selection is based on the interaction between galactose side groups on grafted flexible functional polymer chains on the membrane surface and galactose binding protein on the E. faecalis cell membrane.

Influence of biofilms on the movement of colloids in porous media. Implications for colloid facilitated transport in subsurface environments.

Colloid transport through porous media can be influenced by the presence of biofilms. Sterile and non-sterile sand columns were investigated using Laponite RD as model colloid and a highly mucoid strain of Pseudomonas aeruginosa as model biofilm former. Laponite RD was marked specifically by fluorescent complexes with rhodamine 6G. Breakthrough curves (BTCs) were used as parameters for determination of colloid transport characteristics. In the sterile columns, the colloid was mobile (collision efficiencies from 0.05 to 0.08) both after the presence of Na(+) and Ca(2+) ions followed by deionised water influent. In the biofilm-grown column, the same treatment did not result in colloid retention in the case of Na(+) exposure, but in altered or enhanced colloid transport. In the case of Ca(2+) ions exposure, colloid retention increased with biofilm age. After 3 weeks, almost complete retention was observed. Similar observations were made in columns packed with material from slow sand filtration units. These data reveal the complex interactions between biofilms, cations and colloid transport. Changes in the electrolyte composition of water percolating the subsurface can frequently occur and will result in different colloid transport characteristics with regard to the dominating species of ions and the relative abundance of microbial biofilms. This has to be considered when modelling colloid transport through the subsurface.

Optically transparent porous medium for nondestructive studies of microbial biofilm architecture and transport dynamics.

We describe a novel and noninvasive, microscopy-based method for visualizing the structure and dynamics of microbial biofilms, individual fluorescent microbial cells, and inorganic colloids within a model porous medium. Biofilms growing in flow cells packed with granules of an amorphous fluoropolymer could be visualized as a consequence of refractive index matching between the solid fluoropolymer grains and the aqueous immersion medium. In conjunction with the capabilities of confocal microscopy for nondestructive optical sectioning, the use of amorphous fluoropolymers as a solid matrix permits observation of organisms and dynamic processes to a depth of 2 to 3 mm, whereas sediment biofilms growing in sand-filled flow cells can only be visualized in the region adjacent to the flow cell wall. This method differs fundamentally from other refractive index-matching applications in that optical transparency was achieved by matching a solid phase to water (and not vice versa), thereby permitting real-time microscopic studies of particulate-containing, low-refractive-index media such as biological and chromatographic systems.

Alginate acetylation influences initial surface colonization by mucoid Pseudomonas aeruginosa.

Mucoid strains of Pseudomonas aeruginosa overproduce the exopolysaccharide alginate, which is substituted with O-acetyl groups. Under non-growing conditions in phosphate buffer, a mucoid clinical strain formed microcolonies on steel surfaces, while an acetylation-defective mutant was unable to form cell clusters. Enzymatic degradation of alginate by alginate lyase prevented microcolony formation of the mucoid parent strain. In a continuous-culture flow-cell system, using gluconate minimal medium, the mucoid strain with acetylated alginate formed microcolonies and grew into heterogenous biofilms, whereas the acetylation-defective mutant produced a thinner and more homogeneous biofilm. A lowered viscosity of extracellular material from the acetylation-defective mutant indicated a weakening of exopolymer interactions by loss of acetyl groups. These results suggest that acetyl substituents are necessary for the function of high-molecular-mass alginate to mediate cell aggregation into microcolonies in the early stages of biofilm development by mucoid P. aeruginosa, thereby determining the architecture of the mature biofilm.

Pseudomonas aeruginosa lectin LecB is located in the outer membrane and is involved in biofilm formation.

Pseudomonas aeruginosa is an opportunistic pathogen which causes a variety of diseases, including respiratory tract infections in patients suffering from cystic fibrosis. Therapeutic treatment of P. aeruginosa infections is still very difficult because the bacteria exhibit high intrinsic resistance against a variety of different antibiotics and, in addition, form stable biofilms, e.g. in the human lung. Several virulence factors are produced by P. aeruginosa, among them the two lectins LecA and LecB, which exert different cytotoxic effects on respiratory epithelial cells and presumably facilitate bacterial adhesion to the airway mucosa. Here, the physiology has been studied of the lectin LecB, which binds specifically to L-fucose. A LecB-deficient P. aeruginosa mutant was shown to be impaired in biofilm formation when compared with the wild-type strain, suggesting an important role for LecB in this process. This result prompted an investigation of the subcellular localization of LecB by cell fractionation and subsequent immunoblotting. The results show that LecB is abundantly present in the bacterial outer-membrane fraction. It is further demonstrated that LecB could be released specifically by treatment of the outer-membrane fraction with p-nitrophenyl alpha-L-fucose, whereas treatment with D-galactose had no effect. In contrast, a LecB protein carrying the mutation D104A, which results in a defective sugar-binding site, was no longer detectable in the membrane fraction, suggesting that LecB binds to specific carbohydrate ligands located at the bacterial cell surface. Staining of biofilm cells using fluorescently labelled LecB confirmed the presence of these ligands.

Interactions between laponite and microbial biofilms in porous media: implications for colloid transport and biofilm stability.

Quartz sand columns and sand-filled microscope flow cells were used to investigate the transport characteristics of the clay colloid laponite, and a biofilm-forming bacterium, Pseudomonas aeruginosa SG81. Separate experiments were performed with each particle to determine their individual transport characteristics in clean sand columns. In a second set of experiments, bacterial biofilms were formed prior to introduction of the clay colloids. In the independent transport experiments, bacteria and laponite each conformed to known physicochemical principles. A sodium chloride concentration of 7 x 10(-2) M caused complete retention of the laponite within the sand columns. P. aeruginosa SG81 was generally less influenced by ionic strength effects; it showed relatively low mobility at all ionic strengths tested and some (albeit reduced) mobility when introduced to the columns in 1M NaCl, the highest concentration tested, but nevertheless showed reproducible trends. Under conditions favourable to laponite retention and biofilm stability (7 x 10(-2) MNaCl), laponite suspensions were able to remobilise a portion of the attached bacterial biomass. At low ionic strength, the profile of laponite elution was also altered in the presence of a P. aeruginosa biofilm. These observations suggest that while a reduction in ionic strength has a dominant influence on the mobilisation of biological and inorganic colloids, the presence of laponite and biomass can have a distinct influence on the mobility of both types of colloids. Since these events are likely to occur in subsurface environments, our results suggest that colloid-biofilm interactions will have implications for colloid-bound contaminant transport and the remobilisation of pathogens.

Use of an oxonol dye in combination with confocal laser scanning microscopy to monitor damage to Staphylococcus aureus cells during colonisation of silver-coated vascular grafts.

The antimicrobial silver-coating of medical prostheses is regarded as a means to reduce the risk of bacterial colonisation after implantation. The effect of a silver-coating of vascular grafts on biofilm formation was assessed in batch cultures of Staphylococcus aureus, using confocal laser scanning microscopy. Total cells in biofilms were analysed by staining with the DNA-binding fluorochrome SYTO 62 and the proportion of damaged cells was quantified with the membrane potential-sensitive dye bis-(1,3-dibutylbarbituric acid) trimethine oxonol. Both the extent of biofilm formation and the proportion of viable biofilm cells were significantly diminished on the surface of the silver-coated vascular grafts compared with uncoated controls, probably due to the antimicrobial activity of silver ions released from the silver-coated graft surface.

Application of fluorescently labelled lectins for the visualization and biochemical characterization of polysaccharides in biofilms of Pseudomonas aeruginosa.

Fluorescently labelled lectins were used in combination with epifluorescence microscopy and confocal laser scanning microscopy to allow the visualization and characterization of carbohydrate-containing extracellular polymeric substances (EPS) in biofilms of Pseudomonas aeruginosa. A mucoid strain characterized by an overproduction of the exopolysaccharide alginate, and an isogenic, non-mucoid strain were used. Model biofilms grown on polycarbonate filters were treated with lectins concanavalin A (ConA) and wheat germ agglutinin (WGA) that were fluorescently labelled with fluorescein isothiocyanate or tetramethyl rhodamine isothiocyanate. Fluorescently labelled ConA yielded cloud-like regions that were heterogeneously distributed within mucoid biofilms, whereas these structures were only rarely present in biofilms of the non-mucoid strain. The bacteria visualized with the fluorochrome SYTO 9 were localized both within and between the ConA-stained regions. In WGA-treated biofilms, the lectin was predominantly associated with bacterial cells. Alginate seemed to be involved in the interaction of ConA with the EPS matrix, since (i) pre-treatment of biofilms with an alginate lyase resulted in a loss of ConA biofilm staining, and (ii) using an enzyme-linked lectinsorbent assay (ELLA), ConA was shown to bind to purified alginate, but not to alginate that was degraded by alginate lyase. The application of fluorescently labelled lectins in combination with ELLA was found to be useful for the visualization and characterization of extracellular polysaccharide structures in P. aeruginosa biofilms.