Removal of indigenous coliphages and enteric viruses during riverbank filtration from highly polluted river water in Delhi (India).

Emerging countries frequently afflicted by waterborne diseases require safe and cost-efficient production of drinking water, a task that is becoming more challenging as many rivers carry a high degree of pollution. A study was conducted on the banks of the Yamuna River, Delhi, India, to ascertain if riverbank filtration (RBF) can significantly improve the quality of the highly polluted surface water in terms of virus removal (coliphages, enteric viruses). Human adenoviruses and noroviruses, both present in the Yamuna River in the range of 10(5) genomes/100 mL, were undetectable after 50 m infiltration and approximately 119 days of underground passage. Indigenous somatic coliphages, used as surrogates of human pathogenic viruses, underwent approximately 5 log10 removal after only 3.8 m of RBF. The initial removal after 1 m was 3.3 log10, and the removal between 1 and 2.4 m and between 2.4 and 3.8 m was 0.7 log10 each. RBF is therefore an excellent candidate to improve the water situation in emerging countries with respect to virus removal.

[Detection of viruses in raw water as a basic tool for risk assessment]. / Nachweis von Viren im Rohwasser als Grundlage einer Risikoabschätzung.

Human pathogenic viruses may end up in surface waters by fecal contamination. However, the German drinking water ordinance requests that pathogens in drinking water should not be present in concentrations constituting a potential danger to human health. Since many viruses do have a very low dose of infection, they have to be sufficiently eliminated in the process of drinking water purification. Waterborne virus outbreaks in Europe, over the last few decades, were mostly linked to noncompliance with the generally accepted codes of practice for drinking water production. The aimed level of protection of drinking water supplies in Germany, however, exceeds prevention of outbreaks by even protecting against sporadic virus infections. Documentation of such a high level of protection is not achieved by end product control alone but requires a process analysis with risk assessment. To do such an analysis, information regarding the presence of viruses in the raw water used for drinking water production, as well as data of virus elimination rates during purification processes, are of major importance. This paper presents suggestions for implementation of such a risk assessment, focusing on the evaluation of raw water quality.

Application of culture-independent methods to assess the bacteria removal efficiency of subsurface flow constructed wetlands.

The bacteriologic treatment efficiency of vertical and horizontal subsurface flow constructed wetlands (SFCWs) was analysed in two multistage wastewater treatment systems by culture dependent and independent methods. When assessed with standard cultivation procedures, bacteria removal efficiency of the vertical and horizontal SFCWs was similar. However, microscopic enumerations of the wastewater bacteria after DNA staining revealed a completely different removal pattern: bacteria removal efficiency of the horizontal SFCWs was in general low and erratic, whereas the vertical SFCWs displayed high bacteria removal rates. The discrepancies in the results obtained by bacteria enumeration and cultivation was due to a strong decrease in bacterial culturability after treatment by the horizontal SFCWs, leading to overestimation of the real bacterial concentrations in these effluents. Additionally, a PCR based approach for the detection of the enteropathogenic bacteria Campylobacter jejuni and Yersinia enterocolitica was tested in the wastewater samples. The methods were specific and reproducible in the analysed samples and could be carried out within 12 h, proving very adequate as an alternative to cultivation. This work recommends a review of the current standard methodology for wastewater quality surveillance, as well as of the design of SFCW.

[Implementation of the new EU Bathing Water Directive in Germany]. / Umsetzung der neuen EG-Badegewässerrichtlinie in Deutschland.

A revised version of the Bathing Water Directive 76/160/EWG has been elaborated to include scientific progress in risk assessment of bathing-related illness. The new Bathing Water Directive 2006/7/EC came into force on March 24, 2006, and will have to be implemented in the federal states within two years. The new bathing water directive contains several positive innovations which will improve a protection of the bathers namely i) health related indicators, ii) uniform detection methods, iii) requirements for active bathing water management, and iv) stricter standards for coastal waters. In Germany, the 16 federal states of the Federal Republic of Germany - the Länder - are responsible for monitoring bathing waters and for implementing the new bathing water directive into national law. A common master directive is being written by a joint working-group to ensure comparable implementation in all parts of Germany. An immediate application of the new directive is not possible since the parameter "intestinal enterococci" is currently not routinely monitored. It was decided to start monitoring according to the new directive in Germany in 2008. This will allow the first classification of bathing waters according to the new directive in 2011.

[The EU Bathing Water Directive. Risk assessment and standards]. / Die Novelle der EU-Badegewässerrichtlinie. Aspekte der Risikobewertung bei der Grenzwertsetzung.

In an attempt to reduce the risk of infection in natural bathing waters the European Union is in the process of improving the Bathing Water Directive 76/160/EWG, which regulates the safety of such waters. The proposal contains several positive innovations which will improve the protection of the bathers: (1) health-related indicators, (2) harmonized detection methods, (3) requirements for active bathing water management, and (4) stricter standards for coastal waters. One of the most salient features of the current draft is the introduction of bacterial standards that are more stringent for coastal than for fresh waters. This decision on different standards seems unjustified: it was taken solely on the grounds that in two epidemiological studies-one carried out in coastal, the other in fresh waters-the maximum excess rate of gastroenteritis among bathers in coastal waters was higher than among bathers in fresh waters. However, it was not taken into account that the concentrations of bacterial indicators at which the gastroenteritis rate began to increase was nearly identical in both studies. The ratio between the standard concentrations of E. coli and intestinal enterococci in the draft was set at 2.5. This value does not correspond to the ratio found in German surface waters with low pollution levels, with ratios ranging from 2.7 to 4.0, and to the even higher ratios found in raw and treated sewage effluents. As a consequence in a majority of cases the non-compliance of bathing waters in Germany would be caused exclusively by a violation of E. coli standards. In assessing risks of infection it must also be taken into account that the adequacy of E. coli and intestinal enterococci for signaling the presence of viruses in water is far from optimal. The decay of viruses in water-estimated by the decay of bacteriophages-was found to be substantially slower than the die-off of indicator bacteria.

[Environmental Survey for Children-- the environmental module of KiGGS. I. Design and research program]. / Kinder-Umwelt-Survey--das Umweltmodul im KiGGS. Teil 1: Konzeption und Untersuchungsprogramm.

The German Environmental Survey for Children (GerES IV) is the environment-oriented module of the National Health Interview and Examination Survey for Children and Adolescents (KiGGS) which is being performed nationwide in Germany. From 2003 to 2006, a random subsample of 1800 children aged 3-14 years is being studied with regard to their body burden and health impairments linked to housing conditions and the personal environment- and health-relevant behaviour. The basic study programme includes the analysis of blood, urine, tap water and house dust as well as the application of an extensive questionnaire. The data gained from this population sample, which is representative for Germany's children, are the basis for deriving reference values to characterise the background exposure of children aged 3-14 years. Trends over time can be detected and the success of environmental policies verified by comparing the data with those of the German Environmental Survey 1990/92 (GerES II), also conducted in close cooperation with the National Health Survey, which included children aged 6-14 years. By linking the data from the Environmental and the Health Surveys, health-relevant environmental exposures can be detected and different scientific hypotheses can be tested. The main subjects that are being dealt with using subcollectives of GerES IV are 'VOC and eye and nasopharynx irritation', 'indoor allergens and allergic diseases of the respiratory system', 'chromium, nickel, fragrances and contact allergens', and 'noise, hearing capacity and stress hormones'.

Contribution of the colmation layer to the elimination of coliphages by slow sand filtration.

River bank or slow sand filtration is a major procedure for processing surface water to drinking water in central europe. In order to model the performance of river bank and slow sand filtration plants, we are studying the different mechanisms by which the elimination of pathogens is realized. An important question concerning the mode of action of slow sand filters and river bank filtration units is the role of the colmation layer or "schmutzdecke" on the elimination of human pathogens. The schmutzdecke is an organic layer which develops at the surface of the sand filter short after the onset of operation. We have inoculated a pilot plant for slow sand filtration with coliphages and determined their rate of breakthrough and their final elimination. In the first experiment, with a colmation layer still missing, the breakthrough of the coliphages in the 80 cm mighty sandy bed amounted to ca. 40 %. In contrast, less than 1 % of coliphages escaped from the filter as the same experiment was repeated two months later, when a substantial colmation layer had developed. Our preliminary conclusions are that the colmation layer is extremely efficient in eliminating of viruses.

Microbiological safety of drinking water.

Emerging pathogens in drinking water have become increasingly important during the decade. These include newly-recognized pathogens from fecal sources such as Cryptosporidium parvum, Campylobacter spp., and rotavirus, as well as pathogens that are able to grow in water distribution systems, like Legionella spp., mycobacteria, and aeromonads. To perform a risk analysis for the pathogens in drinking water, it is necessary to understand the ecology of these organisms. The ecology of the drinking-water distribution system has to be evaluated in detail, especially the diversity and physiological properties of water bacteria. The interactions between water bacteria and (potential) pathogens in such diverse habitats as free water and biofilms are essential for the survival or growth of hygienically relevant organisms in drinking water. Results of epidemiological studies together with ecological data are the basis for effective resource protection, water treatment, and risk assessment.

[Recent methods for the detection of airborne microorganisms and source identification]. / Neuere Methoden zum Nachweis luftgetragener Mikroorganismen und zur Quellenidentifikation.

The detection of airborne microorganisms including selected cell constituents (e.g. allergens or endotoxins) depends on suitable methods and instruments for their collection. Furthermore, microbiological methods are necessary for their quantification and qualification. In the past these methods were largely based on the classical cultivation dependent approach. Modern molecular methods, e.g. direct staining procedures, hybridization assays with nucleic acids including the PCR-technology or immunological assays are promising new tools for a more sophisticated detection of bioaerosols. They allow a better detection rate, a more precise identification of certain members of the aerosol including cell constituents. With respect to speed and lower costs they are an important alternative to established detection methods.

[Mass development of Stachybotrys chartarum on compostable plant pots made from recycled paper]. / Massenentwicklung von Stachybotrys chartarum auf kompostierbaren Pflanztöpfen aus Altpapier.

After handling plants grown in decomposable pots made of recycling paper, three women working in a big horticulture developed very painful inflammated efflorescences at the finger-tips, followed by scaling off the skin. The pots appeared to be very mouldy. Black masses of conidia of Stachybotrys chartarum and perithecia of Chaetomium globosum were identified on almost every pot. Apart from various other fungal genera, Trichoderma und Acremonium were frequently detected. Considering the observed symptoms, special attention was payed to the mycotoxin producing species St. chartarum. To evaluate the inhalative spore load, air sampling was performed. The detection of St. chartarum in the air was only possible with the spore trap (sampling of particles) but not with the Andersen sampler (detection of colony forming units). Without moving the pots, measurements yielded values of 30-100 St. chartarum conidia per m3 of air. The concentration of air-borne conidia increased drastically by handling the pots, thus attaining up to 7,500 conidia per m3 of air for St. chartarum only. The occurrence of St. chartarum in such amounts is alarming because of possible toxin production. In addition, the allergenic stress by fungal spores has to be emphasized. The results are discussed with regard to general medical-mycological aspects related to the degradation of environmentally-friendly decomposable materials.

Molecular epidemiological studies of Mycobacterium bovis infections in humans and animals in Sweden.

Forty-nine isolates of Mycobacterium bovis from humans and animals in Sweden were analyzed by restriction fragment length polymorphism (RFLP) patterns probed by the insertion element IS6110. Most isolates had patterns indicating the presence of only one or two genomic copies of the IS6110 insertion element. This simple type of pattern was found in all human isolates. In contrast, isolates from M. bovis infections in five herds of farmed deer in Sweden showed a specific RFLP pattern with seven bands, indicating seven copies of the IS6110 sequence. In 1958, Sweden was declared free from M. bovis in cattle. However, in 1987, M. bovis was reintroduced with imported farmed deer, and since 1991, 11 outbreaks in deer herds, but not in other livestock or wildlife, have been diagnosed. Continued RFLP studies of the new Swedish M. bovis isolates can reveal possible transmission of this deer strain to other animals or humans.

In situ identification of Legionellaceae using 16S rRNA-targeted oligonucleotide probes and confocal laser scanning microscopy.

Bacteria of the family Legionellaceae form a monophyletic group within the gamma-subclass of Proteobacteria. Based on comparative sequence analysis we constructed two oligonucleotide probes complementary to regions of 16S rRNA characteristic for Legionellaceae. Probe specificities were tested by whole-cell or dot-blot hybridization against 14 serogroups of Legionella pneumophila, 22 different Legionella spp. and 72 non-legionellae reference strains. Using optimized conditions both probes hybridized to all tested strains of L. pneumophila. Probes LEG226 and LEG705 hybridized to 71% and 90% of the Legionella species tested, respectively. With the exception of Methylomonas alba none of the non-target strains showed complete sequence homology within the target molecule. In a preliminary evaluation the results of classical techniques employing selective media, immunofluorescence and the probe assay were in good accordance for routine environmental and clinical isolates. L. pneumophila suspended in drinking water at approximately 10(3)-10(4) c.f.u. ml-1 could be rapidly detected by a combination of membrane filtration on polycarbonate filters and whole-cell hybridization. Even after incubation for 1 year a proportion of the released cells was still detectable. In situ hybridization also facilitated visualization of Legionella spp, cells in model biofilms. A combination of in situ hybridization and confocal laser scanning microscopy (CLSM) was used to analyse the three-dimensional arrangement of L. pneumophila within cells of the ciliated protozoan Tetrahymena pyriformis. Whole-cell probing with 16S rRNA-targeted oligonucleotides could, in the future, complement established techniques like immunofluorescence and PCR in ecological and epidemiological studies of Legionellaceae.

Thermophilic, anaerobic bacteria isolated from a deep borehole in granite in Sweden.

A borehole drilled to a total depth of 6779 m in granitic rock in Gravberg, Sweden, was sampled and examined for the presence of anaerobic, thermophilic, fermenting bacteria and sulfate-reducing bacteria. Growth in enrichment cultures was obtained only from water samples collected from a specific sampling depth in the borehole (3500 m). The hole was cased down to a depth of 5278 m and open to the formation below that level. All the water below 2000 m in depth standing in the borehole at the time of sampling must have entered at the 5278-m level or below, during a prior pumping operation. A strong salinity stratification certifies that no major amount of vertical mixing had taken place. The depth from which bacteria could be enriched was that of a pronounced local minimum of salinity. Pure cultures of thermophilic, anaerobic, fermenting bacteria were obtained with the following substrates: glucose, starch, xylan, ethanol, and lactate. The morphology and physiology of the glucose- and starch-degrading strains indicate a relationship to Thermoanaerobacter and Thermoanaerobium species. All but one of the newly isolated strains differ however from those by lacking acetate as a fermentation product. The glucose-degrading strain Gluc1 is phylogenetically related to Clostridium thermohydrosulfuricum, with an evolutionary distance based upon rRNA sequence comparisons of 3%. No sulfate-reducing or methanogenic bacteria were found.

Growth and survival of Serratia marcescens under aerobic and anaerobic conditions in the presence of materials from blood bags.

Several patients receiving blood transfusions during the summer of 1991 developed bacteremia after the transfusion. In all cases, the infection was caused by Serratia marcescens. The same strain of Serratia marcescens was isolated from the patients and from the outer surface of unfilled blood bags. The transport containers for the blood bags were made anoxic by using a catalyst in order to prevent microbial growth. The survival and growth of S. marcescens K202, which was isolated from the blood bags, was studied at different oxygen concentrations in deionized water containing materials derived from the blood bags. The rate of survival and growth of S. marcescens was highest under anaerobic conditions, in which growth occurred with all materials and even in deionized water alone. In contrast, S. marcescens did not survive in control cultures under semi-anaerobic and aerobic conditions. Growth was observed, however, under both aerobic and semi-anaerobic conditions in the presence of each of the tested blood bag materials. These findings indicate that the conditions in the transport containers for the blood bags were favorable for the survival and growth of S. marcescens.

Improved recovery of Legionella from water samples by use of black membrane filters.

A higher recovery of Legionella bacteria from water samples was achieved using black filters that were directly placed on agar plates after filtration, compared to the traditional method using white filters that are shaken in buffer after filtration. Up to 100 times more Legionella colonies/100 ml were found using black filters compared to the traditional method. The two filtration methods revealed, however, no big differences with pure culture experiments. Treatment with acid solution, directly on the filters without dilution of the concentrated sample, effectively reduced the number of accompanying bacteria.

Characteristics of the adhesive determinants of Lactobacillus fermentum 104.

The adhesion of Lactobacillus fermentum 104-R and the variant strain 104-S to porcine gastric squamous epithelium was investigated. An epithelium-specific adhesion was detected for strain 104-S; however, strain 104-R expressed enhanced adhesion capacity to the control surfaces of polystyrene and bovine serum albumin. To characterize the adhesive determinants, the bacterial cells were exposed to various treatments. The adhesion pattern of bacterial cells in buffers of pH values ranging from 2 to 7 was determined. The adhesion of strain 104-S to epithelium was greater in a buffer with a higher pH value. On the other hand, adhesion of strain 104-R to the epithelium was rather unaffected by a change in pH. To the control surfaces of polystyrene or bovine serum albumin, the adhesion of both strains was greatest at pH 2 to 4. Treatment of strain 104-S with metaperiodate did not affect the adhesion to epithelium or polystyrene; however, protease treatment dramatically decreased the adhesion of both strains, thus suggesting that the determinants responsible for the adhesion were proteinaceous. Carbohydrates may be partially involved in the adhesion of 104-R because metaperiodate-treated cells adhered more poorly than control, iodate-treated cells. The adhesion-promoting components are most probably tightly bound to the cell wall, because washing with low-pH buffer (pH 1.2) or sodium dodecyl sulfate had no major effect on the adhesion.