The microbiological quality of drinking water from groundwater is monitored using various indicators. This tells us whether the water is faecally polluted, but not where such pollution comes from. Vitens and Deltares, with the collaboration of Wageningen University, have investigated whether DNA fingerprinting can detect faecal bacteria and identify the source. With that information, drinking water companies would be able to quickly take targeted measures.
Groundwater naturally contains few bacteria, making it in principle highly suited to the production of drinking water. However with some groundwater extraction, there is exchange with surface water. Such is the case for example if the production site is supplied through an infiltration basin, or if it is located close to a river and has a water inflow from the river. The infiltration of surface water to groundwater increases the chance of micro-organisms, including pathogenic or faecal ones, entering the pumped up water. For this reason the quality of the intake water and the wells is subject to enhanced monitoring. Culture-related methods test for several microbial indicators including E. coli, somatic coliphages and Clostridium perfringens bacteria.
For E. coli, the RT-PCR (reverse transcription polymerase chain reaction) screening method has been in use since 2019 alongside the classic culture methods. This method has been developed by the cooperating drinking water laboratories. The advantage of the RT-PCR method is that it shows relatively quickly whether there is faecal pollution present. However RT-PCR does not show where the pollution comes from. Does it come from the surface water, or is there another reason for its presence? Drinking water companies need more information on the potential sources of microbiological pollution in order to be able to take more targeted measures faster.
Is Next Generation Sequencing the answer?
Next Generation Sequencing (NGS) is a method for making a ‘DNA fingerprint’ of the drinking water, which gives a practically complete picture of the microbiological composition of the water that is tested. This makes it possible to monitor bacteria from the surface water throughout the entire drinking water production process and to flag up any changes in good time.
Does this also mean that NGS can be used to establish the origin and microbiological quality of the water used as source for producing drinking water? Is the method suitable for use with relatively pure water samples, and can the data be easily and correctly interpreted? The question is pertinent because with NGS huge volumes of data are collected and the question arises as to how they can be converted into information that can be used to give greater assurance regarding the microbiological safety of drinking water.
These questions were the focus of the two-year study ‘AlTeRnative indicAtor of the origins of miCrobially pollutEddrinking wateR’, or ‘TRACER’ for short.
Four testing sites
Four production sites were chosen for the TRACER study, all four of them influenced by surface water: Engelse Werk, Vechterweerd, Epe and Schalterberg. In the spring of 2019 and 2020 samples were taken from the clear water reservoirs (drinking water), from various wells (groundwater), and from the surface water located nearby. Two different NGS techniques were used to establish the microbiological composition of the samples, of which there were 54 in all. 16S rRNA amplicon sequencing and metagenomic sequencing. The former technique detects only 16S rRNA genes as markers. The latter detects all the DNA that is present, thus including all other living organisms in the water.
The data from the metagenomic sequencing were elaborated in collaboration with Wageningen University’s Microbiology Laboratory. Among other things this allowed us to make use of their Bio-IT pipeline in order to properly identify all DNA sequences and compare them with one another.
The marker genes for 16S rRNA look different for each type of bacterium. This makes them a suitable indicator for determining which bacteria are present in the samples and to what extent. The NGS results were first compared with 16S rRNA genes since bio-IT methods were already available for this. The results were shown in terms of genes in bar charts by location (illustration 1). The charts thus give a picture of the microbiological composition by location, by sample and by type of water.
Illustration 1: Microbiological DNA fingerprints of individual water samples based on the 16S rRNA gene. Water samples are GW = groundwater (well), DW = drinking water, OW = overground (surface) water. The colours reflect the relative contribution of different microbial species.
Principal component analysis (PCA) is then used to show the relationship between the types of sample for each location. As part of this process the fingerprints of the individual samples are compared with one another. For the Engelse Werk and Vechterweerd sites it can be clearly seen (illustration 2) that the samples are clustered according to their various origins. The origin (drinking water, surface water, groundwater) can thus be traced on the basis of the microbiological composition. To a lesser extent this also applies to the Epe site. However, for the Schalterberg site (surface water infiltration) the fingerprints of samples from different sources are far more similar to one another. The influence of the surface water on the drinking water produced is relatively great here in comparison with the other extraction sites. An appreciable number of the micro-organisms from the surface water were seen to have found their way into the drinking water at this location. This is striking, in that the picture at the Epe location is different, even though Epe also makes use of an infiltration basin. The fact that the microbiological composition of the drinking water produced in Epe is far more similar to that of the groundwater points to the filtration effect of the soil in Epe being better than it is in Schalterberg.
Illustration 2. PCA plot of the four locations studied, bringing together the information on the microbiological composition of samples from different sources and from two rounds of sampling.
Deriving a quick indicator
In establishing the bacteriological differences among surface water, groundwater and drinking water, only the 16S rRNA gene was used to fingerprint them. The differences based on this gene prove sufficient to be able to distinguish the types of water from one another. The total metagenome of the water sample of course contains many other genes, a number of which can potentially be used as a quick indicator. For example, algae could be a logical indicator for surface water.
A targeted analysis of genes that are present exclusively in a single type of water can provide a quick answer as to whether exchange takes place with that specific type of water. The relative presence of a hundred different genes was investigated for all samples (metagenomic analysis), and shown in a heat map. The heat map uses colours to indicate the relative presence of each gene by sample. The drinking water samples show a recognisable profile that is easily distinguishable from that of the groundwater and surface water samples. The psbV gene (cytochrome c of cyanobacteria), is particularly noticeable in drinking water samples and can thus be used as a quick indicator of the influence of surface water on groundwater. Meanwhile research has started on the feasibility of using this gene as an indicator.
What have we learnt?
NGS gives a good picture of the microbiological composition of the groundwater used as a source of drinking water and of the origin of the water. Contrary to expectations, it is not necessary to analyse a complete metagenome for this; the simpler 16S rRNA amplicon sequencing method provides the answer. This is a welcome finding, because the elaboration of the sequence data in particular is less complicated in the case of 16S rRNA amplicon sequencing. The elaboration of a metagenome analysis is rather specialized work.
In order to establish the presence of a given type of water, a metagenome analysis is useful if there is a specific indicator (for example, genes involved in the conversion of certain nutrients in groundwater, for which a targeted method of analysis such as qPCR can be developed.)
The TRACER project did not provide any information on the presence of specific pathogenic micro-organisms in the samples, even though the microbiological composition was shown. Identification of the micro-organisms present was mostly to class or family level, because the resolution of the DNA sequences is still too limited to be able to identify bacteria to species level.
The results of the TRACER study have led Vitens to explore the extent to which increased understanding of microbiological composition with the help of NGS can be used to establish stability of the water quality in groundwater extraction facilities. The MIKROWSEQ project that is meanwhile under way should provide some clarity on this. Over a period of one year this project uses NGS or qPCR to show the microbiological composition of the groundwater at four potentially vulnerable groundwater extraction sites every two months. At each extraction site this is done at several points of the flow path, so that the bacteria’s routes can be followed. By regularly evaluating the stability of the groundwater quality, where possible in combination with the composition of surrounding surface water, any changes in the groundwater quality can be detected at an early stage.
To establish its microbiological quality, drinking water is monitored for several indicators. The indicators show whether the water is faecally polluted, but not the origin of the pollution. This makes it difficult to take targeted measures.
This two-year study, called TRACER, shows that next generation sequencing (NGS) or DNA fingerprinting gives a more complete picture of the microbiological composition of water, in this case drinking water, and of where the pollution might originate. NGS can thus be used to investigate the origin of pathogenic microbiological contamination. On this basis, drinking water companies can take targeted measures to protect the quality of drinking water.