Identification of freshwater diatoms in environmental samples to assess water quality using oligonucleotide probes and microarrays

Monitoring the quality of water is of paramount importance for public health. According to the Water Framework Directive 2000/60/EC, ''water is not a commercial product but a heritage that must be protected, defended and treated as such''. In Europe, about 40% of the drinking water is derived from surface waters and pathogenic organisms occurring in lakes and rivers represent a particularly serious health-hazard. In fact, approximately 100 million Europeans lack safe and reliable water supplies and microbial pathogens in drinking water are causing significant morbidity and mortality with major170.000 estimated cases of water-related disease in Europe (UN News Centre, Protocol on Water and Health). The potential threat of water contamination and the spread of waterborne diseases are becoming more serious than ever before. This is the result of a number of factors, including the increase in population, globalisation and the movement of more people across borders and between continents, and the effects of global warming. Furthermore, both abundance and distribution of all microbes are expected to change as a result of global climate change that will cause, for instance, increased or decreased rainfall, temperature and radiation. As the nature of the diatom communities reflects environmental conditions and since they react rapidly and sensitively to water quality changes, these microorganisms have become widely used over the last 50 years as biomarkers for water quality assessment. Moreover, diatom doubling time is one of the quickest among various biological indicators and rapidly reveals a change in water quality. Diatoms are excellent bioindicators [...]. Traditional methods for diatom classification are based on microscopic frustule morphology but are laborious, time-consuming and not suitable for routine monitoring programs. Molecular biological tools have now greatly enhanced our ability to investigate biodiversity by identifying species and estimating gene flow and distribution of species. Therefore, there is a strong incentive to explore and develop alternative methods that are faster, less expensive, reliable and efficient for identification of diatoms in complex environmental samples. Microarrays have been the subject of several EU projects, like EU PICODIV and MICROPAD. Microarrays were developed for the detection of algae and protozoa, and results from chip hybridization were favourably compared to other measurements of diversity, (i.e., direct cell counts and clone libraries). In these two projects, the microarrays were in early stages of development and proof of principle was the major outcome, because it was discovered that probes previously made for fluorescence in situ hybridisation (FISH) could not be directly transferred to a microarray chip format. With a few exceptions, nearly every probe had to be modified for successful use in the microarray chip format. Problems with transferring FISH probes to a microarray chip format led workers in the EU project MIDICHIP to modify their probes and microarrays, especially those designed for detection of cyanobacteria. The FP6 project FISH AND CHIPS made use of prototype findings to develop a microarray chip for phytoplankton at the class level and field data were analysed over three years with rRNA as the preferred target molecule [...] In the EU project AQUACHIP, pathogenic bacteria were the target of interest. This project developed a chip for five bacteria but they were not widely tested with environmental samples. In addition, the detection system developed for this chip was based on a microtiter plate system with detection under a fluorescent microscope. This, however, is not a standard protocol that can be used in a commercial microarray chip reader. The results obtained in the earlier European Union (EU) projects were promising and contributed to the success of an application for an EU-funded grant. μAQUA is the acronym for the EU research project "Universal microarrays for the evaluation of fresh-water quality based on detection of pathogens and their toxins" funded by the 7th Framework Programme of the European Commission and coordinated by Dr. Claudio Gualerzi and Dr. Roberto Spurio of the University of Camerino (Italy). μAQUA aims to design and develop a universal microarray chip for the high-throughput detection in freshwater of known and emerging pathogens (bacteria, viruses, protozoa and cyanobacteria) that cause human diseases and to assess the water quality by monitoring the presence of select bioindicators (diatoms). Existing techniques are laborious and time-consuming, requiring labor-intensive cultivation and microscopic examination of potential pathogens from water samples. The innovative molecular biological techniques being investigated by the μAQUA, by contrast, should enable the rapid - and more reliable - detection of pathogens and diatoms in large volumes of water. The design of probes specifically targeting rDNA or rRNA from higher rank down to species level is made possible by the steadily growing number of rDNA-sequences available (e.g. in the Ribosomal Database Project) [...]. The rDNAspecific probes are used to analyse phytoplankton communities with detection by flow cytometry, epifluorescence microscopy [...] or other methods that take advantage of the specificity ensured by nucleic acid hybridisation. However, a major drawback of these methods is that they are very time-consuming vis-a' -vis the complexity of a microbial sample insofar as they can only be used to identify one or few organisms at a time [...]. By contrast, the DNA-microarray chip technology, which allows the rapid and simultaneous analysis of up to 200,000 probes without a cultivation step [...], has enormous potential as a method of choice for the analysis of complex environmental samples.