Hazard Analysis and Critical Control Points (HACCP) was applied for identifying and controlling exposure to pathogenic microorganisms encountered during normal sludge and wastewater handling at a 12,500 m3/d treatment plant utilising tertiary wastewater treatment and mesophilic sludge digestion. The hazardous scenarios considered were human exposure during treatment, handling, soil application and crop consumption, and exposure via water at the wetland-area and recreational swimming. A quantitative microbial risk assessment (QMRA), including rotavirus, adenovirus, haemorrhagic E. coli, Salmonella, Giardia and Cryptosporidium, was performed in order to prioritise pathogen hazards for control purposes. Human exposures were treated as individual risks but also related to the endemic situation in the general population. The highest individual health risk from a single exposure was via aerosols for workers at the belt press for sludge dewatering (virus infection risk = 1). The largest impact on the community would arise if children ingested sludge at the unprotected storage site, although in the worst-case situation the largest number of infections would arise through vegetables fertilised with sludge and eaten raw (not allowed in Sweden). Acceptable risk for various hazardous scenarios, treatment and/or reuse strategies could be tested in the model.

Free-living amoebae and Acanthamoebae are known to harbour a range of opportunistic microbial pathogens such as Legionellae, sequestering them from antimicrobial agents as well as environmental stresses. Less is known however of the interaction between the thermotolerant free-living amoebae and Legionellae. In the current study, such phenomena were investigated between an environmental and clinical thermotolerant Acanthamoebae isolate and 6 Legionellae; L. anisa, L. birminghamiensis, L. bozemanii, L. dumoffii, L. erythra and L. pneumophila. All Legionellae could be located within either Acanthamoeba isolate, with L. erythra, and L. pneumophila found located within vacuoles. At concentrations exceeding 2 mg/l, free chlorine was a better disinfectant than combined chlorine against Acanthamoebae-bound Legionellae, though thermal treatment was the most effective of the treatment types investigated. While the interaction with free-living Acanthamoebae increased the resistance of Legionellae to thermal treatment, it increased the sensitivity of Legionellae to free and combined chlorine. Interaction with biofilms did not affect the sensitivity of sessile and intracellular Legionellae to disinfection, caused in part by the thin coverage of biofilm on coupon surfaces. Acanthamoebae cysts remained viable after treatment with 100 mg/l chlorine (free and combined) for 10 min, as well as 80 degrees C, implying that conventional hyper-disinfection may be insufficient for long-term control of Acanthamoebae-bound Legionellae in water distribution systems.

Free-water-phase and surface-associated microorganisms from drinking water were detected and roughly identified by hybridization with fluorescence-labeled oligonucleotide probes complementary to regions of 16S and 23S rRNA characteristic for the domains Bacteria, Archaea, and Eucarya and the beta and gamma subclasses ofProteobacteria. Samples of glass-attached biofilms and plankton were taken from a Robbins device installed in a water distribution system. More than 70% of the surface-associated cells and less than 40o of the planktonic cells visualized by 4',6-diamidino-2-phenylindole staining bound detectable amounts of rRNAtargeted probes. These findings are an indication for higher average rRNA content and consequently higher physiological activity of the attached microbial cells compared with the free-living cells. All detectable cells hybridized with the bacterial probe, whereas noArchaea and no Eucarya cells could be detected. Simultaneous hybridization with probes specific for the beta and gamma subclasses of Proteobacteria revealed that microcolonies already consisted of mixed populations in early stages with fewer than 50 cells. These observations provide further evidence that the coexistence and interaction of bacteria in drinking water biofilms may be an integral part of their growth and survival strategies.

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The results of an independent evaluation of 60 case studies of water and sanitation infrastructure projects in India, Mexico, and South Africa, most of them implemented since 2000, demonstrate an ongoing problem of failing infrastructure even in economically advanced developing countries. This paper presents a meta-analysis of those project case study results and analyses whether the design of existing policies or other factors contribute to failures. It concludes that the observed failures are due to well-known reasons and recommends how the implementation of the Dublin-Rio Principles can be improved. (They were introduced twenty years ago to avoid such failures by means of more sustainable planning.).

Bacteria of the family Legionellaceae form a monophyletic group within the y-subclass of Proteobacteria. Based on comparative sequence analysis we constructed two oligonucleotide probes complementary to regions of 165 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 O/ O 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, immunof luorescence and the probe assay were in good accordance for routine environmental and clinical isolates. L. pneumophila suspended in drinking water at approximately 103-104 c.f.u. ml-l 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 165 rRNA-targeted oligonucleotides could, in the future, complement established techniques like immunof luorescence and PCR in ecological and epidemiological studies of Legionellaceae.

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The adhesion of Salmonella typhimurium to the mineral particles quartz, albite, feldspar, and magnetite was shown to correlate with the hydrophobicity of the cell surface as measured by hydrophobic interaction chromatography. The same effects were also seen for seven other selected test strains, including Streptococcus faecalis, Streptococcus faecium, Escherichia coli, Citrobacter freundii, Shigella sonnei, and Shigella boydii. When the test strain of Salmonella typhimurium, was repeatedly cultivated in Luria broth, thus selecting for different degrees of fimbriation and roughness of the cell surface, varied cell hydrophobicity but constant negative and positive charge values were obtained. High hydrophobicity values always coincided with enhanced adhesion to the mineral particles. The negative charge of the bacterial surface as measured by electrostatic interaction chromatography appeared to play no role in the adhesion event. However, the positive charges on the cell surface contributed to the adhesion process. This was especially evident for cells exhibiting a high degree of hydrophobicity. Alteration of the pH between 4 and 9 did not significantly affect the adhesion process.