Martin Strathmann

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.

Isolation and biochemical characterization of extracellular polymeric substances from Pseudomonas aeruginosa.

Publisher Summary This chapter discusses that Biofilms of Pseudomonas aeruginosa have been intensively studied during the last decade. Pseudomonas aeruginosa offers several advantages as a model organism in biofilm research. This gram-negative bacterium is well characterized with respect to its molecular genetics, biochemistry, and physiology; it is also of hygienic relevance as an opportunistic pathogen, because biofilms harboring this bacterium in technical water systems and on medical devices may be a source of human infections. The extracellular polymeric substances (EPS) of P. aeruginosa predominantly consist of different polysaccharides and proteins. Mucoid variants of P. aeruginosa are characterized by an overproduction of the viscous exopolysaccharide alginate, resulting in the production of large slimy colonies, when the bacteria are cultivated overnight on common agar-based media. Alginates from P. aeruginosa are high molecular weight unbranched copolymers consisting of (1→ 4)-linked uronic acid residues of β- D -mannuronate and α- L -guluronate. The formation of biofilms is regarded as part of a natural “life cycle” of mucoid P. aeruginosa. Alginates represent major components of the EPS of mucoid P. aeruginosa and have been implicated in the development as well as the maintenance of the mechanical stability of biofilms formed by P aeruginosa on living and abiotic surfaces. In this chapter, methods of isolation and biochemical characterization are described for both whole EPS and alginate from mucoid strains of P. aeruginosa grown as a confluent bacterial lawn on agar media as simple in vitro model systems for bacterial biofilms.

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

ABSTRACT 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.

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.

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.

Influence of Biofilms on Colloid Mobility in the Subsurface

Transport processes in subsurface environments are determined by complex interactions between the soil matrix and dissolved as well as particulate substances. Biofilms play an important role in the transport of colloids in the subsurface, since biofilms cover the solid soil matrix and hence influence the interaction of colloids with the soil matrix. Consequently, biofilms can influence the mobility of colloids and colloid-bound contaminants either by deposition of colloids within the biofilm matrix, by remobilization of bound colloids, and/or by co-elution of colloids together with detaching biofilm compartments. Further, biofilm organisms can take part in the degradation of colloids or colloid-bound contaminants as well as in colloid generation processes.

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.

A new concept for placing coupons and sensors for decontamination verification

Sentinel coupon devices may be installed in water distribution systems for decontamination surveillance. They give the possibility to extract samples and perform off-line laboratory analysis. The objective of this research work is to develop a concept for positioning sentinel coupons and sensors at representative and indicative locations in the water distribution system. A new integer linear programming formulation is proposed that is a weighted maximum coverage problem with weights that favor places where contaminant concentration is higher and the shear stress is lower. Extended period simulation models as contamination events are used inside a Monte Carlo simulation to calculate the averaged weights and the coverage matrix. The method was validated on a case study from the literature and applied on larger complex and detailed networks. The solution looks to favor sink nodes with long water paths. Finally, it was found that this new optimal placement problem yields solutions that are significantly different from earlywarning designs.