Improved traceability of Shiga-toxin-producing <Emphasis Type="Italic">Escherichia coli</Emphasis> using CRISPRs for detection and typing

Among strains of Shiga-toxin-producing Escherichia coli (STEC), seven serogroups (O26, O45, O103, O111, O121, O145, and O157) are frequently associated with severe clinical illness in humans. The development of methods for their reliable detection from complex samples such as food has been challenging thus far, and is currently based on the PCR detection of the major virulence genes stx1, stx2, and eae, and O-serogroup-specific genes. However, this approach lacks resolution. Moreover, new STEC serotypes are continuously emerging worldwide. For example, in May 2011, strains belonging to the hitherto rarely detected STEC serotype O104:H4 were identified as causative agents of one of the world’s largest outbreak of disease with a high incidence of hemorrhagic colitis and hemolytic uremic syndrome in the infected patients. Discriminant typing of pathogens is crucial for epidemiological surveillance and investigations of outbreaks, and especially for tracking and tracing in case of accidental and deliberate contamination of food and water samples. Clustered regularly interspaced short palindromic repeats (CRISPRs) are composed of short, highly conserved DNA repeats separated by unique sequences of similar length. This distinctive sequence signature of CRISPRs can be used for strain typing in several bacterial species including STEC. This review discusses how CRISPRs have recently been used for STEC identification and typing.     

Gefahrstoff-Substitution — Innovation durch erfolgreiches Zusammenwirken von Wirtschaft, Staat und Zivilgesellschaft

Ohne Zusammenfassung     

Endocrine disruption of water and sediment extracts in a non-radioactive dot blot/RNAse protection-assay using isolated hepatocytes of rainbow trout

Goal, Scope and Background In order to evaluate the estrogenic activity of sediments and XAD water extracts of selected sites of the catchment area of the River Neckar, a river system in Southern Germany, an integrative assessment approach was used to assess the ecological hazard potential of endocrine-disrupting compounds in sediment and water. Methods The approach is based on estrogen receptor-mediated vitellogenin synthesis induced in isolated hepatocytes of rainbow trout and quantified in a non-radioactive dot blot/RNAse protection-assay in parallel to comprehensive chemical analyses of estrogenic substances. Results and Discussion Numerous investigated extracts revealed an estrogen activity comparable to that of the positive control (1 nM 17?-estradiol corresponding to 270 ng/L in the test medium). Based on a concentration factor of 30 in the extracts and a recovery of XAD resins of approximately 80 %, 17?-estradiol equivalent concentrations between 20 and 26.7 ng/L could be calculated downstream of a sewage treatment plant (< 0.1 ng/L for a reference site). A comparison of the bioassay-derived Bio-TEQs (toxicity equivalents) and the Chem-TEQs revealed a high correlation with a Pearson coefficient of 0.85, indicating that the same ranking of the samples could be obtained with respect to the endocrine disrupting potential with both chemical and bioanalytical analysis. However, the TEQ concentrations computed from chemical analyses were significantly lower than the bioassay-derived TEQ concentrations. In fact, in none of the samples, more than 14 % of the vitellogenin-inducing potency could be attributed to the substances (steroids, alkylphenols, bisphenol A, diethylstilbestrol) analyzed. A comparison of the endocrine disrupting potential of sediments extracted by the solvents acetone and methanol revealed lower biological effects for acetone-extracted samples. Possible reasons may be a masking of endocrine effects in acetone extracts by cytotoxicity, a low extraction efficiency of the solvent acetone, or anti-estrogen potencies of some extracted sediment compounds. Using a mass balance approach, the contribution of the compounds analyzed chemically (Chem-TEQs) to the total endocrine activity (Bio-TEQs) was calculated. Based on the very low detection limits, particularly of the steroids with their high TEF factors, results revealed that a calculation of the Chem-TEQs is associated with considerable scale inaccuracy: Whereas only 7-15 % of the biological effectiveness (Bio-TEQs) could be explained by endocrine substances identified above the detection limits, the assumption of concentrations slightly below the given detection limits would result in a significant over estimation (137-197 %) of the Bio-TEQs. Even the interassay variation of the dot blot assay with different fish donors for primary hepatocyte (factor 2 - 2.5) is relatively low, when compared to the large range of the Chem-TEQ concentrations (factor 20) obtained when applying different modes of calculation. Conclusions and Outlook Overall, only a minor portion of the endocrine activity detected by bioassays could be linked to compounds identified by chemical analysis. In vitro assays for assessment of endocrine activities are useful as sensitive integrating methods that provide quantitative estimates of the total activity of particular receptor-mediated responses. Although discrepancies may also result from different bioanalytical approaches, it is overall likely that bioanalytical and not chemical analytical approaches give the correct estimate of endocrine disrupting potencies in environmental samples. As a conclusion, assessment of endocrine disruption based on chemical analysis alone does not appear sufficient and further research into the spectrum of substances with potential endocrine activity as well as into additive or even synergistic effects in complex environmental samples is urgently needed.     

A phenomenological approach shows a high coherence of warming patterns in dimictic aquatic systems across latitude

To predict the coherence in local responses to large-scale climatic forcing among aquatic systems, we developed a generalized approach to compare long-term data of dimictic water bodies based on phenomenologically defined hydrographic events. These climate-sensitive phases (inverse stratification, spring overturn, early thermal stratification, summer stagnation) were classified in a dual code (cold/warm) based on threshold temperatures. Accounting for a latitudinal gradient in seasonal timing of phases derived from gradients in cumulative irradiation (2.2?days per degree latitude), we found a high spatial and temporal coherence in warm–cold patterns for six lakes (84?%) and the Baltic Sea (78?%), even when using the same thresholds for all sites. Similarity to CW-codes for the North Sea still was up to 72?%. The approach allows prediction of phase-specific warming trends and resulting instantaneous or time-delayed ecological responses. Exemplarily, we show that warming during early thermal stratification controls food-web-mediated effects on key species during summer.     

Mikrobielle Sanierung von 2,4,6-Trinitrotoluol (TNT) kontaminierten Böden

Within the limits of a feasability study abouton-site bioremediation methods for TNT-contaminated soils, composting was chosen as a very promising and cheap method. This method was critically compared with those described in the literature and was primarily rated under ecotoxicological aspects. The investigated location is the former munition plant «Tanne» in the aerea of Clausthal-Zellerfeld in Lower Saxony, Germany. To estimate the autochtonic microflora, we assessed the number of aerobic heterotrophic bacteria and determined their respiration activity in soils. In addition, we isolated bacteria and examined their capacity to metabolize TNTin vitro. Both the amount of autochtonic microrganisms (4.7×10⁸ to 1.2×10¹⁰ colony forming units (cfu)/kg dryweight) as well as their respiration activity did not correlate with the concentrations of nitrotoluenes in the soils. With high contaminated soil (20 g TNT/kg dry weight) we carried out a small compost in the range of 10 liters. During 28 days of composting TNT-concentration decrease over 90% and only minor amounts of monoaminodinitrotoluenes were generated. However, an acidic pretreatment of the compost material at the end of the reaction showed that TNT could be partially resolved under these extreme conditions and that an ecotoxicological risk may still exist. Possible changes in the realization of the composting process in order to make sure that the contaminants are savely bound to the humin matrix are discussed.