Consensus report: E. coli O104:H4 (HUSEC041) and the potential threat to European water supplies.

Among the 3rd Seminar for PhD students working on Water and Health which was held in Cannes on 27­29 June 2011, experts from a number of universities and research institutes took the opportunity to discuss the emergence of Escherichia coli O104:H4 in Europe. Especially, possible threats for European water suppliers were considered. The consensus is summarized in this report. The main conclusion was that E. coli O104:H4 would not pose a substantial risk to well managed water supplies, especially where regular monitoring of indicator E. coli is negative. However, this may not apply for small and very small water systems which are quite common in Europe. New strategies like the Water Safety Plan approach are needed to protect also small scale drinking water systems and private wells in Europe. Water used in the processing of foods likely to be eaten raw, especially sprouts, should be of drinking water quality.

Direct coupling of high-performance thin-layer chromatography with UV spectroscopy and IR-MALDI orthogonal TOF MS for the analysis of cyanobacterial toxins.

Cyanobacteria are pathogenic prokaryotes and known for producing a high variety of cyclic hepatotoxic peptides in fresh and brackish water. Prominent members of these toxins are microcystin LR (MC LR) and nodularin (Nod), which are under suspicion to cause cancer. Various analytical methods have been reported for the detection of these cyclopeptides, and these are mainly based on liquid chromatography combined with mass spectrometric techniques. Here, we introduce a new approach based on the direct coupling of high-performance thin-layer chromatography (HPTLC) with infrared matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometry (IR-MALDI-o-TOF MS) using the liquid matrix glycerol. The analysis of the cyclopeptides involves the application of three complementary methods: (i) HPTLC separation of MC LR and Nod, (ii) their detection and quantification by UV spectroscopy at lambda = 232 nm, and (iii) direct identification of separated analytes on the HPTLC plate by IR-MALDI-o-TOF MS. Calibration curves exhibited a linear relationship of amount of analyte applied for HPTLC and UV absorption (R(2) > 0.99). The limits of detection were 5 ng for UV spectroscopy and 3 ng for mass spectrometric analysis of individual peptides. This novel protocol greatly improves the sensitive determination of toxins from pathogenic cyanobacteria in complex water samples. It was successfully applied to the detection and quantification of MC LR and Nod in a spiked, processed environmental water sample.

Occurrence of Legionella in hot water systems of single-family residences in suburbs of two German cities with special reference to solar and district heating.

A total of 452 samples from hot water systems of randomly selected single family residences in the suburbs of two German cities were analysed for the occurrence of Legionella. Technical data were documented using a standardized questionnaire to evaluate possible factors promoting the growth of the bacterium in these small plumbing systems. All houses were supplied with treated groundwater from public water works. Drinking water quality was within the limits specified in the German regulations for drinking water and the water was not chlorinated. The results showed that plumbing systems in private houses that provided hot water from instantaneous water heaters were free of Legionella compared with a prevalence of 12% in houses with storage tanks and recirculating hot water where maximum counts of Legionella reached 100,000 CFU/100ml. The presence of L. pneumophila accounted for 93.9% of all Legionella positive specimens of which 71.8% belonged to serogroup 1. The volume of the storage tank, interrupting circulation for several hours daily and intermittently raising hot water temperatures to >60 degrees C had no influence on Legionella counts. Plumbing systems with copper pipes were more frequently contaminated than those made of synthetic materials or galvanized steel. An inhibitory effect due to copper was not present. Newly constructed systems (<2 years) were not colonized. The type of hot water preparation had a marked influence. More than 50% of all houses using district heating systems were colonized by Legionella. Their significantly lower hot water temperature is thought to be the key factor leading to intensified growth of Legionella. Although hot water systems using solar energy to supplement conventional hot water supplies operate at temperatures 3 degrees C lower than conventional systems, this technique does not seem to promote proliferation of the bacterium. Our data show convincingly that the temperature of the hot water is probably the most important or perhaps the only determinant factor for multiplication of Legionella. Water with a temperature below 46 degrees C was most frequently colonized and contained the highest concentrations of legionellae. It is evident that the same factors affecting colonization by Legionella in large buildings also exist in small residential water systems. If temperatures are low there is no difference between large and small systems and Legionella counts are high in both. Since private residences are an important source of community-acquired legionellosis, these findings emphasize the need for preventive control measures in small residential buildings. In some situations it may be necessary to install filtration devices at the point-of-use.

Distribution of the urease gene cluster among and urease activities of enterohemorrhagic Escherichia coli O157 isolates from humans.

Enterohemorrhagic Escherichia coli (EHEC) O157 strains belong to two closely related major groups, which are differentiated by their sorbitol fermentation phenotypes. Here we studied the conservation of urease genes and their expression in sorbitol-fermenting (SF) and non-SF EHEC O157 isolates. PCR targeting ure genes (ureA, -B, -C, -D, -E, -F, and -G) demonstrated that each of these genes was present in 58 of 59 EHEC O157:H7 isolates. In contrast, none of 82 SF EHEC O157:NM (nonmotile) isolates contained any of the ure genes. Hence, the absence of the urease genes distinguishes SF EHEC O157:NM strains from EHEC O157:H7, but this absence demonstrates that the urease genes are not useful genetic targets for the detection of EHEC strains, because SF EHEC O157:NM strains are missed by such a strategy. When examined for urease activity on Christensen agar and in the API 20E system, only one O157:H7 strain displayed urease activity and produced elevated levels of ammonia, which was subsequently confirmed by ammonia electrode measurement. Because the ure genes were absent from each of nine strains of E. coli O55:H7, the proposed progenitor of EHEC O157, we hypothesize that EHEC O157:H7 diverged from the evolutionary pathway at an early stage and then acquired the O islands carrying the ure gene cluster.

Phenotypic and molecular analysis of tellurite resistance among enterohemorrhagic Escherichia coli O157:H7 and sorbitol-fermenting O157:NM clinical isolates.

A total of 66 (98.5%) of 67 enterohemorrhagic Escherichia coli (EHEC) O157:H7 strains had increased potassium tellurite (Te) MICs (32 to 1,024 microg/ml), grew on Te-containing media, and possessed Te resistance (ter) genes, whereas 83 (96.5%) of 86 sorbitol-fermenting (SF) EHEC O157:NM strains had Te MICs of

Analysis of microcystins in freshwater samples using high performance liquid chromatography and an enzyme-linked immunosorbent assay.

Blooms of non-toxin producing and toxin producing cyanobacteria are often observed in lakes, reservoirs and slowly flowing eutrophic freshwater. Microcystin congeners are the most important group of cyanobacterial toxins and determination of the microcystin concentration is necessary in order to assess the health risk when such waters are used for recreational purposes. The most widely used standard method for analysis of microcystins is high-performance liquid chromatography (HPLC). But this technique is time-consuming and needs a special expensive equipement. Therefore a commercially available microcystin enzyme-linked immunosorbent assay (ELISA) could be an alternative. However, when performed as recommended by the manufacturer, the ELISA is not suitable for a valid quantitative analysis. We have therefore modified the microcystin extraction method and the ELISA protocol and have been able to use the ELISA for a quantitative analysis comparable to that of the widely recommended HPLC method. The modified ELISA provides a relatively simple and highly sensitive approach for the quantitative analysis of microcystins in freshwater. Results can be obtained within two to three days after sampling.