Assessment of human health impacts from emerging microbial pathogens in drinking water by molecular and epidemiological studies
The development and validation of molecular detection technologies is urgently needed in order to support comprehensive safety concepts for European drinking water, and is a main focus of the HEALTHY-WATER project, in addition to the epidemiology of waterborne diseases. Molecular methods for screening, detection and quantification of pathogens and the assessment of the physiological state of drinking water bacteria are developed, validated and applied within the HEALTHY-WATER project. An example, demonstrating the applicability of the molecular technology for whole drinking water supplies is given below.
Currently, hygienic quality and potential health risk from the consumption of drinking water is assessed by the cultivation of indicator bacteria (Directive 98/83/EC). This classical microbiological methodology relies on the cultivation of specific bacteria, e.g. plate counts of coliforms, and has a variety of serious drawbacks, like no correlation to many microbial pathogens and no valid identification of the pathogens. Therefore, this methodology is completely inappropriate for the detection of emerging pathogens in drinking water. For this reason, state-of-the-art detection methodology is a main focus of this project.
Advanced methodology for environmental detection of microorganisms depends on several factors: (1) the type of microorganism; (2) the level of taxonomic resolution; (3) the detection limit; and (4) the price per analysed sample. The universal approach currently pursued is to analyse nucleic acids, extracted directly out of drinking water, with a suite of molecular methods ranging from polymerase chain reaction (PCR) and DNA array-based techniques. Additionally, viruses in drinking water can be detected and quantified by PCR techniques, such as real-time PCR, without the need to grow them in host cells.
The overall aim of the project is to advance our knowledge on pathogenesis of emergent microbial pathogens in drinking water to understand their transmission to humans. The project will focus on all major types of pathogens, i.e. viruses, bacteria and protozoa, and will concentrate on a representative set of European drinking water supply systems and source waters of specific sensitivity to human health. To reach the overall goal, the following detailed objectives are approached:
An integrated research approach will be pursued to achieve these objectives by combining molecular and classical detection, activity assessment and epidemiological understanding of emerging pathogens in a specific set of drinking water systems from different European regions.
For the detection of the targeted microbial pathogens by molecular methods, the necessary primer pairs were designed, tested, validated and/or improved in silico. Using these validated primers, a first survey for the targeted pathogenic bacteria was performed on a subset of drinking water samples from France, Spain and Germany. The first molecular screening of these samples resulted in several positive hits for the detection of members of the genus Helicobacter in some samples of drinking water supply systems. These samples were then subjected to high resolution electrophoresis using single strand conformation polymorphism (SSCP) analysis followed by sequencing of the single major bands. Some of the bands were identified as belonging to the genus Helicobacter and even the species could be determined for these Helicobacter-positive samples.
A specific effort was made to quantify all the major targeted pathogens using real-time PCR. For this purpose the validated primers were tested with reference nucleic acids (DNA/ RNA) in the real-time PCR to confirm the performance in comparison to normal PCR. Also, spiking experiments were conducted using the respective bacterial cells or viral particles to validate the concentration and extraction methodology developed. These spiking experiments resulted in the determination of detection limits and the validation of the laboratory procedures for all major targeted pathogens, such as hepatitis A virus (HAV), Norovirus, Helicobacter spp., Campylobacter spp., Legionella spp., pathogenic Escherichia coli, Cyrtosporidium spp. and Gardia spp.
In addition to the real-time PCR for the quantification of the targeted pathogens, a DNA-chip was also validated for the massive parallel molecular screening of most of the targeted pathogens in raw and drinking water samples using this type of spiking assays. Different staining methods giving insight into the physiological state of drinking water bacteria were also evaluated (see Figure 1). These results demonstrate that the developed screening and detection technology is in place for the next round of molecular analysis, i.e. the broad-scale, in-depth, molecular survey of the raw and drinking water samples.
HEALTHY-WATER will generate validated detection technologies for the targeted waterborne pathogens and reveal possible routes of transmission to humans via drinking water consumption. This new knowledge will provide guidance for improving the hygienic quality of European drinking water supplies and reduce the burden of waterborne infections for those living in Europe.