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Sunday, 05 April 2009 17:00

Traditionally, assessment of microbial water quality does not target individual pathogens, but the presence of so-called indicator organisms. Indicator organisms are of fecal origin like the majority of waterborne pathogens. Water can be considered the transmission vehicle via the fecal-oral route. The presence of indicator organisms is typically analyzed by cultivation with plate counts providing quantitative data. The more indicator organisms are found in a water source, the higher the associated risk of simultaneous presence of pathogenic organisms. However, there are numerous examples where pathogen peaks do not correlate with indicator peaks or correlations are sometimes very weak ( Dorner et al. 2007; Hörman et al. 2004). In other cases pathogen peaks were reported to precede indicator peaks ( Dorner et al. 2007). During an event pathogen levels then decrease to undetectable levels.

Although the majority of the population in developed nations seems to consider clean water a granted fact, the following examples of studies highlight the high prevalence of waterborne pathogens:

Example 1: A study examining infectious disease outbreaks in Canada between 1974 and 2001 concluded that 288 outbreaks were linked to a drinking water source. In more detail, 99 outbreaks were related to public water systems, 138 to semi-public systems, and 51 to private systems ( Schuster et al. 2005). Severe weather with heavy rainfall, close proximity to animal populations, treatment system malfunctions, poor maintenance and insufficient treatment were considered as main factors responsible for disease outbreaks.

Example 2: Surface water samples from 7 lakes and 15 rivers in southwestern Finland were tested over five consecutive seasons (fall 2000 to fall 2001; Hörman et al. 2004). Testing by cultivation-based or molecular methods was performed for various enteropathogens (Campylobacter spp., Giardia spp., Cryptosporidium spp., and noroviruses) and fecal indicators (thermotolerant coliforms, E. coli, Clostridium perfringens, and F-RNA bacteriophages). Out of 139 samples, 41% tested positive for at least one of the pathogens. No significant correlation between pathogen and indicator levels were found underlining that monitoring of indicators is not sufficient for risk assessment. Also a Swedish study evaluating the occurrence of pathogens in a river near Göteborg and relating it to the risk for waterborne disease found that about half of the samples were positive for at least one of the following pathogens: Cryptosporidium, Giardia, norovirus, enterovirus, Campylobacter, and E. coli O157:H7 ( Aström et al. 2007). As short-term peaks in pathogen concentrations (e.g. after heavy rainfall) may considerably increase the risk of disease, the authors suggested a better regulation of surface water intake for drinking water production.

The sometimes occurring discrepancy between presence of indicators and the epidemiological risk by waterborne pathogens together with the increasing risk of intentional water contamination (with no link to fecal sources) stress the importance of direct detection and monitoring of waterborne pathogens. Enrichment and plating on selective media followed by biochemical and serological characterization is currently the most widely applied method for detection of waterborne pathogens. The time and labor demand and lack of quantitative information, however, makes this approach not suitable for routine monitoring of water quality. Results obtained days or even weeks after a possible contamination event are of little benefit in term of taking preventive measures. Although the coming years will see a profound change in monitoring methods, cultivation-based methods will remain important for verification and characterization of isolates. The current water quality assessment based on cultivation can be seen as a valuable tool to judge overall system performance allowing retro-perspective assessment of possible contamination in the case of illnesses caused by water consumption or exposure. However, prevention of outbreaks can only be achieved with faster and quantitative methods. In this respect, molecular methods seem to provide the technology of choice promising rapid and specific detection. Although molecular methods also suffer from technical limitations and biases, they are becoming increasingly used and validated. The development of advanced tools for pathogen detection and their application to water can supplement cultivation-based methods and has produced a significant amount of data especially regarding pathogen abundance in both source and finished water. This website puts a special focus on nucleic acid-based detection methods as these technologies have undergone a rapid development in the recent past. These advancements raise hopes that in the not too distant future multiple pathogens can be monitored at strategic water distribution sites.

This website is actually an extended scientific review about waterborne pathogens, which was put on the web instead of being published in a journal or book. The reason for this was to make the information easily accessible and updatable. Despite the currently limited scope of the site we would like to understand this website as a first step in providing a comprehensive knowledge platform for people involved in water microbiology. In contrast to classical reviews, which typically present highly condensed conclusions in a generalized form, we chose to stick more closely to the original data and to present selected examples of different scientific studies in more detail. This path was chosen as the relevance of data strongly depends on the analysis methods and experimental conditions. Factors like temperature, water source, cell concentrations, presence or absence of microbial flora etc. strongly impact the obtained results, their knowledge is therefore crucial for validating and interpreting the reported observations. An example for the biological complexity when studying microbial pathogens in water is that the susceptibility towards disinfection can strongly depend on previous growth conditions. This was observed when studying the susceptibilities of Yersinia enterocolitica and Klebsiella pneumoniae to chlorine dioxide ( Harakeh et al. 1985). Bacterial populations grown under conditions that more closely resembled natural aquatic environments (i.e. low temperatures and nutrient limitation) showed higher resistance than 'spoilt' lab cultures. This and many more examples tell us that generalizations are often misleading and thorough knowledge about experimental conditions is needed when interpreting scientific conclusions.This webpage tries to include as much detailed information as possible which the authors considered relevant.
A section about prevalence and abundance was included for all pathogens covered on this website. It shall provide an indication of the range of pathogen concentrations that were reported in different water types in the current literature. The increasing accumulation of quantitative data on 'typical' pathogen concentrations is indispensable for assessment of public health risks. Nevertheless it has to be considered that the given numbers greatly depend on the detection method applied. Pathogen numbers based on detection of nucleic acids can vary greatly from numbers based on cultivation or microscopic visualization due to intrinsic biases of the methods or due to differences in the viability status of the targeted organisms. All data has therefore to be seen relative and only serves to provide a general overview.
Knowledge about the efficiency of different disinfection methods is critical for eliminating the risk of disease. Susceptibilities to commonly used disinfection methods vary greatly between different waterborne pathogens. Especially protozoan cysts and oocysts can be highly resistant. Monitoring disinfection efficacy is traditionally based on cultivation, which does, however, not allow the detection of sublethally damaged and non-culturable cells. Molecular methods can therefore be of great advantage in this respect. However, one has to be careful when interpreting a specific experiment, a thorough knowledge of technical aspects is needed. A typical problem for detection of either DNA (for bacteria, protozoa or DNA viruses) or RNA (for RNA viruses) is that the presence of these nucleic acids does not correlate with their viability or infectivity. When studying the effect of different disinfection methods on animal caliciviruses, Duizer et al. 2004 showed for most methods that at levels where infectivity was no longer detected, viral RNA remained detectable. Harsher conditions (higher disinfectant concentrations, longer exposure times or lower/higher pH) were needed to reduce PCR-detectable units (PDUs) compared to eliminating infectivity. Interestingly, when comparing the reduction in PDUs by conventional end-point PCR and quantitative PCR, the authors observed greater reduction of detectability by end-point PCR than with qPCR. For example, when exposing feline caliciviruses to pH 2, qPCR suggested a reduction of less than 2 log10 units, whereas end-point PCR suggested a reduction of more than 7 log10 units. The difference was suspected to be caused by the fact that the end-point PCR amplicons were much bigger than the qPCR amplicons. Damage and loss of amplifiability occurs in a longer stretch of template with higher probability compared to a shorter sequence. Assuming both approaches have the same sensitivity, a conclusion from this would be that qPCR results are more conservative than end-point PCR results for judging PDU reduction.
Whereas the overall prevalence and abundance of pathogens in water is a matter of the load of contamination (normally of fecal origin), the concentration of infectious particles depends on the survival characteristics of the specific pathogen in an aqueous environment. Survival of pathogens in water is influenced by many factors including temperature, nutrient availability, light exposure, oxygen levels, the concentration of minerals and inorganic substances, pH, the surrounding microbial flora, surface availability and presence of biofilms, and predation. Growth is normally of less importance (with exceptions) and water is generally considered as a mode of transmission ( Thomas et al. 1999). As survival is very species- and even strain-dependent, the presentation of selected studies investigating persistence in different water types received substantial attention in this document. Of special value are reports correlating pathogen persistence with the persistence of indicator organisms. The use of indicator organisms only makes sense if these indicators persist longer in an aqueous environment than the pathogens. In the case of bacteria, risk evaluation is however complicated by the fact that cells can enter a state where they have lost culturability, but still display metabolic activity. The latter is often measured as the ability to reduce CTC or other viability dyes. This physiological state is typically referred to as non-culturable (NC), viable-but non culturable (VBNC), or active-but non culturable (ABNC). Due to the undefined nature of the NC state it remains to be elucidated what epidemiological risk is associated with NC pathogens. The interpretation of data on total counts, viable counts, PCR-detectable units (PDUs), or other parameters in terms of health risk is essential for regulatory purposes and will be a major challenge for the years to come. The most informative data comes from infectious dose studies with animal models, but the performance of this type of monitoring is prohibitively expensive, time-consuming, and ethically questionable. Better correlations between measured quantitative parameters and epidemiological risk are needed for including novel methods into monitoring programs. The distinction between viable and non-viable pathogens will be critical in this respect.
Many pathogens show low infectious doses. The latter especially applies to protozoan parasites, but also to viruses and some bacteria. In general, data on infectious doses are difficult to obtain and partly originate from animal experiments (leading to a probability of illness which can only be viewed as a rough indication of human risk) or spectacular self-experiments like in the example of Helicobacter pylori which was ingested by its discoverer Barry Marshall.  It is obvious that the infectious dose does not only depend on the number of ingested organisms, but also on the viability and infectivity status of the pathogen, the health status of the person, and the resistance of the person to the organism or toxin ( AWWA Committee Report 1999). Typically, the most susceptible persons are children, the elderly and immuno-compromised people. Pathogens can also reveal significant strain variation. All these difficulties explain the scarcity of information about dose-response relationships for many pathogens.
The challenge for waterborne pathogen monitoring and disease prevention consists in the facts that most of these organisms occur in water in very low concentrations. Molecular detection methods have an advantage in this respect as they tend to be more sensitive compared to traditional detection methods. Compared to electron microscopy for astrovirus and norovirus detection, Logan et al. reported a tenfold and a fourfold increase in the rate of detection, respectively, when using quantitative PCR ( Logan et al. 2007). This example highlights the role of molecular assays for pathogen detection. One of the most positive aspects of molecular biology is its modular character, which allows for the versatile combination of different experimental units. However, despite the focus on molecular methods, this resource does not intend to neglect their downsides. The problems associated with culture are well known, but there are also considerable complications with molecular tools. One of them is the difficulty of many methods to distinguish between live infectious organisms and dead organisms. This problem in general applies to all assays that are not based on detection of labile mRNAs and leads to the question what does a molecular 'yes' or even a quantitative number mean in terms of health risk?  A PCR-positive result lacking information about viability and infectivity is difficult to correlate with the actual health risk. Epidemiological correlation currently only exists for culture-based monitoring of indicator organisms. Once a molecular assay has been agreed upon for routine monitoring, epidemiological guidelines have to be established. The correlation between CFUs and genome copies is sometimes poor. The number of genome copies tends to be significantly higher than the numbers obtained by plate counts. Reasons can lie in the higher sensitivity of molecular methods, but also the existence of dead cells, non-culturable (NC) cells, and non-infective pathogens. Differences in the physiological states can also in part explain the heterogeneity of test results comparing different molecular methods that measure different cellular parameters.

Apart from the problem of obtaining information about the viability status of the detected pathogens and the epidemiological relevance, molecular methods which employ the amplification of nucleic acids suffer from the problem of inhibition. This greatly limits the water volume which can be subjected to analysis. The unsolved problem of PCR-inhibition is caused by co-purified inhibitory substances that are often found in environmental samples. The integration of methods based on amplification of nucleic acids will not happen on a broad scale as long as problem of inhibition is not overcome. Maybe the most promising approach in this respect is the design of engineered polymerases that are less susceptible to inhibition or the use of different enzymes (as seen in isothermal amplification methods).

This resource tries to cover methods which were validated with water samples or methods which appear promising for testing water samples. Information about molecular targets, detection sensitivity and other data considered useful was incorporated whenever available. Regarding detection methodology, a selection had to be made due to the wealth of published information for every single pathogen. The selected examples do, however, not exclude the potential of other methods which have not been considered in this review. This particularly applies to commercially available kits which cannot be extensively covered. It also has to be pointed out that the molecular downstream detection always has to be seen in correlation with the upstream concentration method. Although the inclusion of the upstream sample processing would have been beyond the scope of this website, it is a highly important part of the overall detection methodology for waterborne pathogens which are often found in very low numbers. A detailed study of different extraction technologies and their efficiencies for concentrating different types of waterborne pathogens has been undertaken recently by Vince Hill et al. ( Water Research Foundation RFP 3108).

The future of this website will greatly depend on funding. The vast majority of hours that went into this site have not been covered by a paycheck. The driving factor of the people contributing to this page has been their belief that clean water is one of the most eminent challenges for this and the coming generations and that access to existing knowledge about waterborne pathogens is crucial to the scientific community. We are therefore very grateful to companies and organizations that are willing to contribute to make this website sustainable and to develop it into a knowledge platform for waterborne pathogens. Updates are planned on a regular basis and, based on our capabilities, the site can be extended with various attractive features. The editors can be contacted at This e-mail address is being protected from spambots. You need JavaScript enabled to view it .

Last Updated on Saturday, 31 October 2009 04:14
 

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