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Molecular Biology and DNA Microarray Technology for Microbial Quality Monitoring of Water

Posted on: Saturday, 11 September 2004, 06:00 CDT

Public concern over polluted water is a major environmental issue worldwide. Microbial contamination of water arguably represents the most significant risk to human health on a global scale. An important challenge in modern water microbial quality monitoring is the rapid, specific, and sensitive detection of microbial indicators and waterborne pathogens. Presently, microbial tests are based essentially on time-consuming culture methods. Rapid microbiological analyses and detection of rare events in water systems are important challenges in water safety assessment since culture methods present serious limitations from both quantitative and qualitative points of view. To circumvent lengthy culture methods, newer enzymatic, immunological, and genetic methods are being developed as an alternative. DNA microarray technology is a new and promising tool that allows the detection of several hundred or even thousands DNA sequences simultaneously. Recent advances in sample processing and DNA microarray technologies provide new perspectives to assess microbial water quality.

The aims of this review are to (1) summarize what is currently known about microbial indicators, (2) describe the most important waterborne pathogens, (3) present molecular methods used to monitor the presence of pathogens in water, and (4) show the potential of DNA microarrays in water quality monitoring.

Keywords Water Quality; Water Bourne Pathogens; Indicators; DNA; Molecular Detection

INTRODUCTION

Water is undoubtedly one of the major natural resources necessary to the maintenance and well being of human and animal life. Although the quality of drinking water is generally taken for granted in developed countries, health risks from polluted water remain a major public concern. The effects of water pollution include poisonous drinking water, unbalanced river and lake ecosystems that can no longer support full biological diversity, deforestation from acid rain, and many other effects specific to the various contaminants. Although risks to human health may result from exposure to toxic contaminants, nevertheless, pathogenic contamination of water arguably represents the most significant risk to human health globally, and there have been countless numbers of poisoning and disease outbreaks throughout history as a direct result of poorly treated or untreated water. Significant waterborne disease outbreaks have been reviewed worldwide in North America and Europe (Hrudey et al. 2002). A recent example occurred in May 2000 at Walkerton, Ontario (Canada), which resulted in 2300 illnesses and 7 deaths due to E. coli 0157:H7 and Campylobacter jejuni contamination in municipal drinking water (Hrudey et al. 2002). In March 1993, the largest recorded outbreak of Cryptosporidium occurred in a filtered and chlorinated surface supply at Milwaukee, Michigan (USA) and affected more than 400,000 people (MacKenzie et al. 1994). In 1992, 1400 cases of gastroenteritis of suspected viral etiology were diagnosed at Uggelose (Denmark) and linked to a filtered but not chlorinated water supply (Laursen et al. 1994).

Loading of contaminants in surface water, groundwater, sediments, and drinking water occurs via two primary routes: point source pollution (i.e., originates from discrete sources whose input into aquatic systems can often be defined in a spatially explicit manner) and non-point source pollution (i.e., originates from poorly defined diffuse sources that typically occur over broad geographical areas) (Ritter et al. 2002). Major potential sources of pathogens come from the excrement of humans and animals, waste materials that find their way into domestic sewage, and natural microbial flora in the source water (Ritter et al. 2002; Noguchi et al. 2002). Bacteria, viruses, protozoa, and helminths can be carried by water and transmitted to people by direct contact (i.e., water contact disease) or by ingestion (i.e., waterborne disease) (Straub & Chandler 2003; Theron & Cloete 2002; Grabow 1996; Moe 1997; Leclerc et al. 2002). In addition, sewages contribute numerous types of potentially infectious agents including Salmonella spp., E. coli, other coliforms, viruses, and protozoa to surface waters (Cooper & Danielson 1997; Goni-Urriza et al. 2000; Morinigo et al. 1992).

Currently, detection, isolation, and identification of water- borne pathogens are difficult, time-consuming, and hugely expensive if attempted on a regular basis (Straub & Chandler 2003). To avoid the necessity of undertaking such huge ventures, indicator microorganisms are commonly used to determine the relative risk from the possible presence of pathogenic microorganisms in a sample. As most of the microbial pathogens present in water and wastewater are of fecal origin, the detection of fecal contamination has been the main aim of water testing authorities. Historically, the thermotolerant coliform group, the enterococci and Clostridium perfringens have been the bacterial indicators of choice (Cooper & Danielson 1997; Goni-Urriza et al. 2000; Morinigo et al. 1992). In these cases, the indicator organisms are indigenous to feces, and thus their presence in the environment is indicative of fecal pollution. However, it is important to note that the presence of these indicators is not a confirmation of pathogen presence, but rather it implies the potential for pathogen presence because of the likelihood that infectious feces are present in wastewater or sludge. The greatest weakness of bacterial indicators as a public health monitoring tool for water and wastewater is their greater sensitivity to disinfection relative to that of viruses and protozoan cysts. In these instances, the absence of indicators is not a guarantee that more resistant microbial forms are absent (Lemarchand & Lebaron 2003). Due to these limitations, it is imperative to determine the presence or absence of microbial pathogens in wastewater used in reclamation projects. The ideal situation would be to monitor for the presence of all pathogens that might be present in a liquid or solid sample. Indeed, there are a number of instances where direct measurements of the presence and number of bacterial pathogens in natural samples are necessary. This approach is essential in epidemiological studies of waterborne diseases. It is also needed to understand the relationship between indicator numbers and specific pathogen concentrations required for standardizing measurement parameters, as well as in determining the efficacy of water/wastewater and solids treatment processes in the reduction of pathogens or in situations where the sanitary significance of high indicator numbers is in question. These pathogen detection and quantitation methods should be rapid, sensitive, highly accurate, easy to perform, and amenable to high- throughput analyses.

There are established culture and molecular methods for the detection of most microbial pathogens; however, most of these methods have important limitations, the majority of which are associated with the time taken to isolate and/or identify the pathogen and with the accuracy of the detection (Lemarchand et al. 2001). A large amount of research has been undertaken to develop methods that improve the detection of various microorganisms. These methods range from conventional culture methods on selective media, to immunological approaches, through to nucleic acid-based assays and DNA microarrays (Deisingh & Thompson 2002).

The aims of this review are (1) to summarize the current knowledge of wastewater fecal pollution monitoring and (2) to describe the potential role of DNA microarrays in the detection and identification of fecal pollutants in wastewater and environmental water samples.

WATER QUALITY INDICATORS

Pathogens are frequently found in low concentrations in water and thus cannot be easily detected and enumerated. As a consequence, it is extremely difficult, if not impossible, to test water samples for each specific pathogen to assure water quality. Instead, indicator microorganisms are employed for this purpose. Indicator microorganisms are organisms that have entered water by the same route, and at the same time, as pathogenic microorganisms; however, they are easier to detect and enumerate. Therefore, indicators are used indirectly to suggest the presence of pathogens in water. Presently, the World Health Organization (WHO) recognizes three different groups of indicators (Ashbolt et al. 2001):

* General (process) microbial indicators: A group of organisms that demonstrates the efficacy of a process, such as total heterotrophic bacteria or total coliforms during chlorine disinfection.

* Fecal indicators: A group of organisms that indicates the presence of fecal contamination (e.g., thermotolerant coliforms or E. coli).

* Index organisms and model organisms: A group or species indicative of pathogen presence and behavior, respectively, such as E. coli as an index for Salmonella and F-RNA coliphages as models of human enteric viruses.

The most important criteria for an indicator of water quality are as follows (Toranzos & McFeters 1997):

* The indicator should be consistently present in feces and at higher concentrations than the pathogen(s).

* It should be absent in uncontaminated waters.

* It should not multiply in the environment.

* Its resistance to environmental conditions and disinfecti\on should equal or exceed that of the contaminating pathogens.

* It can be assayed by means of a simple and reliable test.

* Its concentration in water should correlate with concentration of feces-borne pathogens or with a measurable health hazard.

Commonly Used Indicators

Total Coliforms

The coliform group of bacteria describes gram-negative, nonspore- forming, facultative anaerobic, rod-shaped bacteria, that ferment lactose to acid and gas within 24-48 h at 36 1C. These criteria are not taxonomic; coliform bacteria belong to the family Enterobacteriaceae and include several genera such as Escherichia, Klebsiella, Enterobacter, and Citrobacter. The definition of coliform bacteria has classically been translated into biochemical reactions or the appearance of characteristic colonies on commonly used selective culture media (Venkateswaran Murakoshi & Satake 1996). The more recent advent of enzyme-specific media and tests has seen the addition of cytochrome oxidase and [beta]-galactosidase as additional criteria for the coliform group (Toranzos and McFeters 1997).

These bacteria are not indicators of fecal pollution. It is widely accepted that they can be considered as normal inhabitants of many soil and water environments that have not been impacted by pollution. In addition, these bacteria are able to grow in biofilms within drinking water distribution systems and are occasionally absent in water supplies during outbreaks of waterborne disease. Also, the persistence of these bacteria in aquatic systems is comparable to that of some bacterial pathogens but less than that observed for enteric viruses and protozoa. Due to these limitations, the quantitation of total coliform group of bacteria is a poor parameter for measuring the fecal contamination of waters. Consequently, it is very difficult to interpret the sanitary significance of their presence or have confidence in water quality in their absence.

Thermotolerant Coliforms (Fecal Coliforms)

Fecal coliform bacteria are the most commonly used indicators of fecal pollution in water and food. They are considered as an indicator of homeothermic fecal contamination of water. In addition to all criteria used to define total coliforms, they grow and ferment lactose with the production of gas and acid at 44.5 0.2C within 24 2 h. This latter criterion constitutes a thermotolerant adaptation of proteins to, and their stability at, the temperatures found in the enteric tracts of animals that are both constant and higher than temperatures in most aquatic and terrestrial environments. However, Klebsiella pneumoniae, a fecal coliform bacteria, has been isolated from environmental samples in the absence of fecal pollution. Such observations have been made on water receiving high levels of carbohydrate-rich industrial effluent and in contact with plant materials.

Escherichia coli represents more than 90% of the fecal coliform group. It has additional characteristics that make it a useful indicator of water quality. For example, it has been demonstrated to be a more specific indicator for the presence of human fecal contamination than the fecal coliform group of bacteria. In addition, E. coli conforms to taxonomic, as well as functional identification criteria, and is enzymatically distinguished by the absence of urease and the presence of [beta]-glucuronidase (in 90% of environmental E. coli) (Ashbolt et al. 2001). One possible disadvantage of this organism as an indicator in water is that it has been consistently found in pristine rain forest aquatic and plant systems and so may not be a reliable signal of fecal contamination in those environments (Toranzos & McFeters 1997). However, E. coli has been effectively used in Europe and US drinking water regulations as a specific indicator of fecal contamination.

Enterococci and Fecal Streptococci

Fecal streptococci are gram-positive, catalase-negative cocci that cleave esculin and are not inhibited by bile salts. They belong to the genera Enterococcus and Streptococcus possessing the Lancefield group D antigen (Ashbolt et al. 2001). Enterococci are a subset of fecal streptococci that can be differentiated by their ability to grow at 10 and 45C, at pH 9.6, and in a medium with 6.5% NaCl (Harwood et al. 2000). Like fecal coliforms, enterococci are found in the feces of all warm-blooded animals. Several studies have suggested that Enterococcux spp. have a better survival in water than fecal coliforms (McFeters et al. 1974). In addition, their survival rate through wastewater processes is higher than that of fecal coliforms and these organisms have persistence patterns that are similar to those of a range of potential waterborne pathogenic bacteria.

Total coliforms, fecal coliforms, and enterococci often do not indicate the persistence of pathogens in surface waters and wastewaters. Human viral and protozoan pathogens are more persistent than these indicators and are not inactivated as efficiently by water treatment processes, such as chlorination (Griffin et al. 1999) or aerobic wastewater treatment (Chauret et al. 1999). The simultaneous monitoring of water samples for alternate indicators enables a better assessment of fecal contamination.

Clostridium perfringens and Sulfite-Reducing Clostridia (SRC)

SRC are gram-positive, spore-forming, non-motile, strictly anaerobic rods that reduce sulfite to H^sub 2^S. The presence of these microorganisms in the feces of all warm-blooded animals is the basis for considering them as indicators of fecal pollution (Toranzos and McFeters 1997).

Clostridium perfringens is considered as a promising alternative bacterial indicator because:

* It is consistently present in moderate concentrations in human feces as well as in sewage.

* Its spores are more persistent in environmental systems than most enteric pathogens.

* It has been successfully used to monitor sewagecontaminated streams, ocean, and surface water (Morinigo et al. 1992; Ferguson et al. 1996; Griffin et al. 1999; Payment et al. 2000).

These microorganisms have been used in different studies to evaluate the relationship between pathogens and indicator organisms in various kinds of waters (Morinigo et al. 1992; Ferguson et al. 1996; Griffin et al. 1999; Payment et al. 2000).

C. perfringens was identified as the most useful indicator of fecal pollution and was the only indicator significantly correlated to the presence of pathogenic protozoan Giardia and the opportunistic bacterial genus Aeromonas (Ferguson et al. 1996). The two limitations for the use of these microorganisms as fecal indicators are (1) the extreme stability of the spores to environmental conditions (Davies et al. 1995) with the possibility that their detection indicates a pollution event that occurred a long time ago and (2) the obligate anaerobic physiology of these bacteria making their cultivation somewhat difficult. Because these spores may survive in the environment much longer than most pathogens, sulfite-reducing clostridia could be considered indicators of remote fecal pollution.

Bacteroides and Bifidobacterium

Several researchers have suggested that the members of the genera Bacteroides and Bifidobacterium could be used as fecal indicator organisms (Resnick & Levin 1981; Fiksdal et al. 1985). Bifidobacteria are obligately anaerobic, non-acid-fast, nonsporogenous, non-motile, gram-positive bacilli and are one of the most numerous groups of bacteria in the feces of warm-blooded animals (Ashbolt et al. 2001 ). In addition, they are more abundant than Escherichia coli in the human intestinal tract, but because of their anaerobic status, they die rapidly when discharged into environmental waters. Members of the genus Bactemides are rod- shaped, gram-negative bacteria and are among the most numerous bacteria in human feces. They have similar rates of survival to coliforms in raw sewage but they die off more rapidly in water than Escherichia coli and Enterococcus faecalis (Fiksdal et al. 1985).

Bifidobacteria are one of the dominant groups of anaerobes in the gut of humans and are present in high numbers in the feces of humans and some animals (Resnick & Levin 1981). During their transfer into sewage, an initial rapid fall in numbers of bifidobacteria occurs, followed by a further reduction at a lower rate. They have been found in sewage and polluted waters, but not in unpolluted environments (Sinton et al. 1998).

The use of these indicators has been limited because strict anaerobes are often difficult to grow. This difficulty could be circumvented by using molecular genetic rather than culture-based methods (Bernhardt & Field 2000).

Bacteriophages

Bacterial viruses (bacteriophages) not indigenous to the polluted waters have been proposed as indicators of fecal pollution in surface waters (Goyal et al. 1980; Singh & Gerba 1983). The most commonly used are the somatic coliphages (a heterogeneous group that comprises Myoviridae, Siphoviridae, Padoviridae, and Microviridae), the F-RNA phages and the Bacteroides fragilis phages. Counts of viral plaques on various host cell lawns can be quite different, but standardization is being attained as ISO methods have been developed (Toranzos & McFeters 1997).

F-RNA Phages (Coliphages). F-RNA phages are a group of icosahedral phages that are morphologically similar to several important human enteric virus groups. These viruses have been correlated with enterovirus counts in freshwater (Havelaar et al. 1993) and used in an estuarine system to establish the relationship between indicators and pathogens. Coliphages are more stable in water (Sinton et al. 2002) and more resistant to disinfection than fecal coliform bacteria. Generally, the concentration of F-RNA phages in water is correlated with the level of the sewage contamination.

Bacteroides fragilis Phages. Due to their high host specificity, the detection of bacteriophages infecting Bacteroides fragilis strains has been proposed to \determine the source of environmental contamination (Puig et al. 1999). These bacteriophages are potentially important fecal indicators because:

* They multiply only in the intestinal tract and, therefore, share a major characteristic with human enteric viruses.

* They are absent in the feces of most animals and consequently, their presence is evidence of human and not animal fecal pollution.

* They survive well in the environment.

Rhodococcus coprophilus

Rhodococcus coprophilus is an aerobic bacterium, a non- cardioform actinomycete, forming a fungus-like mycelium that breaks up into bacteria-like elements. This organism is a natural inhabitant of the feces of domestic grazing farm animals, but it is not an active component of the rumen microbiota. R. coprophilus is recognized as a potential fecal indicator because of its presence in the feces of domesticated herbivores, in pasture run-off, and its association with contaminated waters and sediments. Interestingly, this organism is absent from human fecal wastes (Fiksdal et al. 1985).

Fecal Indicators That Can Differentiate Human from Animal Fecal Pollution

Human feces are generally perceived as constituting a greater human health risk than animal feces, because of their potential association with human pathogens. However, reliable epidemiological evidence in support of this theory is lacking. While none of the most commonly used fecal indicators-coliforms, fecal coliforms, Escherichia coli, and enterococci-are found exclusively in human fecal waste, their presence is indicative of the extent of the contamination in an area. In addition, by their very nature, microbial indicators of fecal pollution do not necessarily reflect the presence of all human enteric pathogens (Lipp et al. 2001). As the differentiation between human and animal pollution is a great challenge, potential indicators of fecal sources have been proposed to aid in this discrimination (Sinton et al. 1998). Investigations into the use of microorganisms for fecal source identification involve four basic approaches (Sinton et al. 1998):

* Speciation, based on findings that a particular species may be indicative of human versus animal sources.

* Biochemical reaction, whereby a simple biochemical test has been claimed to differentiate sources.

* Profiles and ratios, which have not been shown to be very effective (e.g., fecal coliform:fecal streptococci or FC:FS ratio).

* DNA profiles and genotyping, which are considered by some authors to be more reliable than phenotypic biochemical reaction.

Unfortunately, each approach possesses certain limitations. The first is that many microorganisms have multiple hosts and exhibit similar biochemical responses to their environment. In addition, inter-species gene transfer occurs, so even genomic differentiations have to be treated with caution. Moreover, the detection of some indicator microorganisms, which are in low numbers in the environment, can be difficult and requires cumbersome assays. Nevertheless, a wide range of microorganisms has been investigated as fecal source indicators.

Fecal Streptococci

The fecal streptococci are probably the group of microbes most intensively investigated as fecal source indicators. Streptococcal concentrations in human feces (ca. 10^sup 6^ cells per g) are generally less than fecal coliform concentrations. In sewage, these microorganisms are 10 to 100 times fewer in number than fecal coliforms. In contrast, they outnumber fecal coliforms in animal feces. In receiving waters, fecal streptococcal counts generally correlate with fecal coliform counts, although there is a continual change in the ratio of fecal streptococci versus fecal coliforms with time and distance from the fecal source (Sinton et al. 1998). In addition, these microorganisms are more persistent than fecal coliforms in water (greater amounts of solar radiation is required to inactivate enterococci in seawater compared to that required to inactivate coliforms) (Sinton et al. 2002; Gabutti et al. 2000). Streptococcal counts have been employed in different approaches to distinguish human from animal fecal contamination (i.e., species identification, Fecal Coliforms: Fecal Streptococci ratio and ratio shift) (Sinton et al. 1998). Nevertheless, these approaches are obsolete and current World Health Organization recommendations suggest the avoidance of statistical and mathematical estimations (Ashbolt et al. 2001 ) that are generally not reproducible in all environmental samples due to geographical and demographic variations.

Species Identification. The fecal streptococci group comprises enterococci species, E. faecium, E. faecalis, E. durans, E. aviun, and E. gallinarum, together with two non-enterococci species, S. bovis and S. equinus. The ratio of enterococci to non-enterococci species in feces is different among vertebrate species. Human feces, and consequently sewage pollution sources, are characterized by a predominance of enterococci, whereas animal sources contain significant numbers of non-enterococci. However, enterococci are indeed present in animal feces and are more persistent than other streptococci in the environment. In 1987, Rutkowski and Sjogren (1987) examined all members of the genus Streptococcus, including non-enteric species, from sewage treatment facilities and animal feces. Comparisons of streptococcal population distribution with samples from various sources revealed that human sources could be distinguished from other animal sources. Nonetheless, the streptococcal species identification approach to source identification is generally regarded as unreliable due to limitations in the use of enterococcal counts (Sinton et al. 1998).

The FC:FS Ratio (Fecal Coliforms: Fecal Streptococci). The FC:FS ratio has been used extensively during the last lew decades. This ratio has been reported as >4 in human feces and <0.7 in animal feces (Toranzos & McFeters 1997). Although it is theoretically possible to ascribe a human or animal source to fecal pollution based on the FC:FS value, the application of this ratio to environmental samples needs to be undertaken with care. The major weakness of this approach is that FC and FS die off at different rates. Consequently, the ratio will gradually change and thus will no longer reflect the original ratio in the fresh fecal material. Since it is not always possible to judge the age of pollution, the problem of differential die-off makes the FC:FS ratio an unreliable method of determining the source of pollution. If the FC:FS ratio is used in an attempt to provide information on possible sources, the following guidelines are recommended (Geldreich & Kenner 1969; Sargeant 1999):

* The pH range of waters being tested should be between 4.0 and 9.0 because fecal coliforms die off quicker than fecal streptococci in acid or alkaline water.

* Sampling should occur within 24 hours after waste deposition. The faster die-off rate of fecal streptococci will alter the ratio as time from contamination increases.

* Sample near the point of discharge or as close as possible to the pollution source. Pollution from several sources can alter the ratio and confuse the results.

* Ratios should not be used when fecal streptococcal counts are less than 100 CFU (colony-forming units) per 100 ml because it becomes difficult to distinguish fecal streptococci in wastes from those that occur naturally in soil and water.

* FC:FS ratios are of limited value in waters were re-growth can occur.

* The mean FC:FS ratio for a site is largely meaningless because the range of ratios is so great. Evaluating the frequency with which FC:FS ratios fall within certain indicative values is a more accurate predictor of fecal contamination source.

* A single sample has little diagnostic value. Numerous samples and thorough knowledge of the watershed are necessary.

Ratio Shift. This approach is linked to the differential dieoff of fecal coliforms and enterococci in natural environments. A predominantly human source-dominated by enterococci which are more persistent than fecal coliforms-should exhibit an initial ratio >4 which declines during storage. In contrast, a non-human source, dominated by S. bovis and S. equines, which are less persistent than fecal coliforms, should exhibit an initial ratio <0.7, which subsequently rises upon storage. Since this approach gave variable results, due to the many non-quantifiable factors involved in this environment, researchers generally recommend caution in using FC:FS ratio in receiving waters (Sinton et al. 1998).

Bifidobacteria

Bifidobacteria are detectable in human intestines within the first six days of neonatal life. They are one of the dominant bacterial groups in the gut of humans and can reach densities up to 1010 cells per g of feces. Bifidobacteria counts in human feces are generally 10 to 100 times greater than fecal coliforms counts (Resnick & Levin 1981). There is conflicting information in the literature as to the incidence of bifidobacteria in animals (partially attributable to the selective nature of the media used for their detection). In addition, some authors found no difference between the survival of bifidobacteria and coliforms in water (Gyllenberg 1960), whereas Resnick and Levin (1981) demonstrated that bifidobacteria did not survive as well as E. coll in fresh or marine waters. Early studies have been centered on the relationship between the source of the organism and the eleclrophoretic mobility of the enzyme phosphokelatase. Isolates from birds, cattle, and humans appeared to show distinct electrophoretic patterns (Scardovi et al. 1971), but Gavini et al. (1991) demonstrated that this approach did not correlate well with the phenotypic or genomic description of the species.

Phenotypic analysis revealed the presence of seven main groups of bifidobacteria: three from human feces, three from animal feces, and one from sewage (Gavini et al. 1991). Th\e main difference between these groups was that cells from animal feces were able to grow at 45C in trypticase phytone yeast broth (TPYB), whereas cells from human feces could not. In addition, Mara and Oragui (1983) isolated, on mannitol sorbitol agar (MSA), both mannitol-fermentative bifidobacteria strains (from both human and animal samples) and sorbitol-fermentative strains (from human feces only). This biochemical characteristic of the strains isolated from human feces prompted these authors to propose these sorbitol-fermentative strains of Bifidobacteriiim serve as specific indicators of human fecal pollution.

Bacteroides spp.

Like bifidobacteria, Bacteroides spp. are also one of the dominant groups of bacteria in the human intestinal tract and can represent up to 10^sup 9^ cells per gram of feces. They are present in 100-fold greater numbers than E. coll in human feces, whereas little or no cells are recovered from animal feces (Sinton et al. 1998; Puig et al. 1999). In 1995, Kreader (1995) examined the feces from nine humans and 70 non-humans to identify B. distasonis, B. thetaiotaomicron, and B. vulgatus by PCR amplification of 16S rDNA genes, followed by hybridization detection of specific PCR products. While target Bactemides species were detected either at high levels (six of the nine samples) or not at all (three of the nine samples) in human feces, only low levels of the three target bacteria were occasionally detected in nonhuman feces (Kreader 1995). To account for the absence of Bacteroides species in three human feces samples, the authors postulated that this could be due to their primer and probe design not recognizing strains of the three Bacteroides species (Kreader 1995). Because conventional methods for isolating, identifying and enumerating Bacteroides spp. are cumbersome and time- consuming, the use of these bacteria as fecal source indicators will probably utilize DNA-based techniques such as the one used by Kreader (1995) rather than culture techniques. However, one major limitation that may preclude the use of these bacteria as fecal source indicators is their rapid inactivation in water compared to fecal coliforms (Fiksdal et al. 1985).

Phages of Bacteroides fragilis. The basis underlying the use of Bacteroides fragilis HSP40 as fecal source indicators is their specificity for human hosts (Sinton et al. 1998). In 1986, Jofre et al. (1986) reported that phages infecting B. fragilis HSP40 have a survival rate similar to coliphage f2, poliovirus, and simian rotavirus in both freshwater and seawater and have a superior survival rate compared to F-RNA phages. In addition, these phages possess a relatively high resistance to treatment processes. Phages of B. fragilis HSP40 and B. fragilis RYC4023 are specific indicators of human pollution (Puig et al. 1999). The overall value of using these phages as indicators in environmental samples is in doubt because of low counts recorded in some parts of the world. The underlying reason for these low counts is uncertain, but it is known that a phage carrier state, or pseudolysogeny, may be found in many phage-host systems (Sinton et al. 1998).

F-RNA Phage Serogroups

F-RNA phages are uncommon in animal feces and rare in human feces. Paradoxically, they have been found in significant concentrations in human waste (Havelaar et al. 1990). In 1987, Furuse (1987) reported that four different serogroups of F-RNA phages could be distinguished:

* Serogroups II and III tend to be isolated from human feces and sewage.

* Serogroup I is usually isolated from feces from nonhuman mammals.

* Serogroup IV has been found present in feces of mixed origin (i.e., human and nonhuman).

Hsu et al. (1995) and Beekwilder et al. (1996) have reported that F-RNA phage serogroups may have potential in future microbial pollution source identification studies. Although the association of serogroups II and III with human excreta and I and IV with animal excreta was statistically significant, the results reported by Schaper et al. (2002) suggest that this association cannot be used for absolute distinction between fecal pollution of human and animal origin. These results indicate that the association of specific serogroups with human or animal feces may not be as absolute as previously thought. In addition, the use of serotyping for routine analysis of water is difficult because serotyping is considered too expensive and time-consuming to allow high throughput screening. Nevertheless, It has been shown that, with a few exceptions, members of each of the serogroups belong to a genetically distinguishable genotype (Beekwilder et al. 1996). These distinct genotypes can, therefore, be used for fecal pollution source tracking purposes.

Rhodococcus coprophilus

R. coprophilus is consistently isolated from the dung of herbivores, from poultry reared in proximity to farm animals and from freshwater and wastewater polluted with animal fecal material. Rhodococcus counts correlated reasonably well with those of fecal streptococci, thereby, confirming its fecal origin. More importantly, this organism appears to survive considerably longer than E. coli and fecal streptococci in receiving waters and sediments. The presence of R. coprophilus in the dung of herbivores, but its apparent absence from human feces, suggests that this organism is an ideal grazing animal indicator in water contaminated by animal feces. The ratio of R. coprophilus to fecal coliforms also increases with increasing distance downstream of human settlements (Jagals et al. 1995). This organism may need to be used in conjunction with the relatively short-lived Streptococcus bovis to indicate the proximity of animal pollution. The long survival period of R. coprophilus means its presence alone points to contamination with animal fecal matter of remote or distant origin. Its presence together with S. bovis would confirm recent animal fecal pollution (Sinton et al. 1998).

Fecal Indicator Limitations

Frequently, a lack of correlation is observed between pathogen and indicator concentration in water systems. This can at least be partly explained by the fact that the transport and behavior of the different organisms is governed by several hydrodynamic, chemical, and/or biological factors (i.e., water flow, attachment to particles, sedimentation and resuspension, survival, etc.) (Ferguson et al. 1996). For instance, enterococci survive better than coliforms in surface waters, Cryptosporidium oocysts can sediment in quiet waters, whereas free-living bacteria remain planktonic and are more prone to migration. Consequently, these factors may contribute to different behaviors of these organisms, not only in natural waters, but also in wastewater treatment plant systems (Chauret et al. 1999). Although numerous enteric pathogens have similar reservoirs within watersheds (e.g., cattle, humans), fecal bacteria are not sufficient to indicate the presence of pathogenic protozoa such as Cryptosporidium (Lemarchand & Lebaron 2003).

In summary, a single, ideal universal fecal contamination indicator has yet to be adopted, since the validity of any indicator system is affected by the relative rates of removal and destruction of the indicator versus the target hazard (Ashbolt et al. 2001).

WATERBORNE PATHOGENS

Large quantities of pathogenic organisms can be introduced into the aquatic environment through fecal contamination from infected persons or animals, and discharged into sewers or unprotected waterways. When such fecal contamination mixes with unprotected or inadequately treated drinking water, large numbers of susceptible hosts can be exposed to numerous pathogenic agents and become infected either by ingestion, direct skin/mucus membrane contact, or inhalation of the contaminated water (see Table 1 ). There are five critical elements in the transmission of infectious agents through water (Moe 1997):

* Source of the infectious agent

* Specific water-related modes of transmission

* Specific attributes of the organism that allow it to survive and possibly spread and multiply within the aquatic environment

* Infectious dose and virulence factors

* Host susceptibility factors

Most microbial waterborne pathogens of concern originate in the intestinal tract of humans and animals and enter the aquatic environment via fecal contamination. The concentration of these pathogens in a community water supply will depend, in part, on the number of infected persons or animals in the community and the opportunity for feces to enter the water supply (Moe 1997). In recent years, several pathogenic microorganisms have become problematic in freshwater as well as in drinking water sources, production and distribution:

* Newly recognized pathogens from fecal sources: Campylobacter jejuni, enterohemorrhagic E. coli, Yersinia enterocolitica, new enteric viruses (e.g., rotavirus, calicivirus, Norwalk virus, and astrovirus), the parasites Toxoplasma, Giardia lamb lia, Cryptosporidiumparvum, and microsporidia.

* Indigenous aquatic organisms that are able to grow in drinking water distribution systems and only recently recognized as pathogens: Legionella spp., Aeromonas spp.,Mycobacterium spp., and Pseudomonas aeruginosa.

Some of these microorganisms may have been causing diseases for a long time but were not identified as drinking water pathogens, due to a lack of proper detection methods. This finding is essentially true for viruses and some other parasites that are difficult to grow on routine culture media. Presumably, none of these new pathogens are of recent origin with many of them having acquired new virulence factors or new antibiotic resistance genes during their evolution in the environment or within their respective hosts. The most prominent example of this phenomenon are pathogenic E. coli strains that, having acquired various virulence genes by horizontal transfer, resulted in a potent new pathotype, the enterohemo\rrhagic E. coli (EHEC). The occurrence of these allochtonous organisms and their survival in surface waters are dependent on environmental conditions (temperature, salinity, etc.) and on the presence of other organisms (protozoa, predators, etc.). Moreover, some pathogens, such as E. coli, Helicobacter pylori, Salmonella, and Campylobacter species are able to exist in an active but nonculturable stage (ANC) (Tholozan et al. 1999; Cho & Kim 1999; Wang & Doyle 1998; Velazquez & Feirtag 1999; Rompre et al. 2002).

In the United States, from 1982 to 1995, drinking water out- breaks due to bacterial agents have been due predominantly to Shigella species, followed by Campylobacter species. The majority of recreational outbreaks have been dermatitis infections caused by Pseudomonas species, followed by shigellosis and Legionella infections. A great proportion of waterborne outbreaks have been due to parasites, such as Cryptosporidium and Giardia species. The infectious dose varies considerably by type of organism. In general, enteric viruses and protozoa have low infectious doses, typically between one and 50 tissue culture infectious units, plaque-forming units (PFU), cysts, or oocysts, whereas bacterial pathogens tend to require a relatively large number of cells (100 to 10^sup 8^ CFU depending of the pathogen) to establish an infection (Moe 1997).

TABLE 1

Examples of illnesses acquired by ingestion or contact with contaminated water

Pathogens from Fecal Sources

Enteric Viruses

Enteric viruses are frequently found in fecally contaminated water. In recent years, more attention has been paid to the significance of viruses as the cause of gastrointestinal illnesses. One difficulty inherent in studying enteric viruses is that many are not culturable or poorly culturable. This is especially true for those responsible for waterborne outbreaks like rotaviruses, small round structured viruses such as Norwalk viruses, and caliciviruses. An increasing number of molecular methods, including RT-PCR (Reverse Transcriptase Polymerase Chain Reaction), followed by nucleic acid hybridization, are being developed for the detection and identification of viruses in water. Although enteric viruses cannot grow outside the human body, many of them are able to survive in water in an infectious state for months. Bacteriophages and enteric viruses have been found in drinking water biofilms (Szewzyk et al. 2000). Because several viruses are relatively chlorine resistant, contamination of the drinking water with low viral numbers may occur periodically.

Campylobacter spp.

Campylobacter spp. are gram-negative, slender, curved, microaerophilic, motile rods. During the last 20 years, Campylobacter jejuni has become one of the most common pathogenic bacteria involved in waterborne disease outbreaks in industrialized countries. The most important natural reservoir for pathogenic Campylobacter is birds, but they may occur in other warm-blooded animals. Environmental waters are thought to be a significant source of human contamination, and contaminated surface waters have been responsible for numerous C. jejuni infections (Salis et al. 2002). Campylobacter spp. have been found in high numbers in raw sewage (10- 10^sup 5^ CFU/100 ml), and they are also found in fecally contaminated surface waters (<10-10^sup 2^ CFU/100 ml) (Szewzyk et al. 2000; Stelzer & Jacob 1991; Brennhovd et al. 1992; Schaffter & Parriaux 2002). The use of unchlorinated surface water and the secondary contamination of drinking water in storage reservoirs are the main risks for infection with Campylobacter spp. Because Campylobacter is microaerophilic, close cooperation with other natural water bacteria (e.g., in biofilms) seems to promote its survival, as it was shown that Campylobacter isolates survived better in water in the presence of the autochthonous water flora and especially in biofilms (Szewzyk et al. 2000).

Pathogenic E. coli

E. coli cells are gram-negative, motile, facultatively anaerobic enteric bacilli. E. coli is responsible for three types of infections in humans: urinary tract infections (UTI), neonatal meningitis, and intestinal diseases (gastroenteritis). Five classes of E. coli that cause diarrheal diseases are now recognized: enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), enterohemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), and enteroaggregative E. coli (EAggEC) (Nataro & Kaper 1998).

E. coli O157:H7 is the most prominent representative of the EHEC, but almost 100 other serotypes of E. coli have been determined to produce Shiga toxin and to cause enterohemorrhagic disease. The infectious dose of E. coli O157:H7 is quite low (<100 organisms). The route of transmission of EHEC is mostly direct animal contact or the consumption of contaminated food. However, there is epidemiological evidence that waterborne infections can occur via recreational water, well water, or contaminated public water. Presently, little is known about the distribution of pathogenic E. coli in natural water environments, but they have occasionally been detected in surface and wastewater (Tamanai-Shacoori et al. 1994). From the physiological characteristics (e.g., survival in stressful conditions, adaptation, etc.) of pathogenic E. coli, it can be assumed that they have good survival in water, but more research is needed to improve the methods for the detection of these organisms in environmental samples in order to obtain more information on the behavior of these bacteria in natural habitats as well as during drinking water treatment and distribution.

Shigella spp.

The genus Shigella is composed of four species of gram-negative, non-motile nonsporogenous, facultatively anaerobic, rod-shaped bacteria (i.e. Shigella dysenteriae, Shigella boydii, Shigella sonnei, and Shigella flexneri). The infective dose of Shigella is very low and can vary from 10 to 10^sup 4^ organisms (Theron et al. 2001). All of the virulent Shigella strains harbor a 120 to 230 kb plasmid, aptly named the Virulence plasmid, which interestingly can also be found in EIEC strains (E. coli). Epidemiological studies have shown that various water sources (e.g., ponds, lakes, wells, rivers) can act as sources of Shigella spp. infection (Islam et al. 1993).

Salmonella spp.

Salmonella spp. are gram-negative, motile, facultatively anaerobic enteric bacilli. A characteristic feature of Salmonella spp. is the broad host spectrum of this genus, comprising most animal species in addition to humans. Hosts are mainly infected through food, drinking water or environmental sources. Individuals infected with Salmonella shed the organisms in their feces, which can enter domestic sewage and consequently contaminate drinking water sources (Waage et al. 1999a). In the environment, the concentration of Salmonella can be below 5 CFU/100 ml, consequently the detection of Salmonella cells in water requires sensitive and specific detection methods.

Yersinia enterocolitica

Strains of the gram-negative, variably motile, small rodshaped bacterium Yersinia enterocolitica involved in human disease belong to serotypes O:3, O:5,27, O:8, O:9, O:13a, 13b, O:20 and O:21, with swine serving as a major reservoir for strains of serotypes O:3 and O:9 (Waage et al. 1999b). The routes of infection have not been completely elucidated, but contaminated food and water are the most likely sources. Y. enterocolitica has an animal reservoir and has been isolated from both wild and domestic animals. Pathogenic strains of Y. enterocolitica have been identified in surface waters (Brennhovd et al. 1992; Schaffter & Parriaux 2002) but have rarely been isolated from drinking water, with only a few reported outbreaks of gastrointestinal illness associated with Y. enterocolitica-contaminated drinking water. Y. enterocolitica is able to survive for several weeks in natural river water where several factors make its detection difficult (e.g., growth inhibition from other bacteria, differentiation between pathogenic and non-pathogenic strains) (Kapperud 1991). As a consequence, underestimation of pathogenic strains in the environment is likely.

Vibrio cholerae and Vibrio parahaemolyticus

The genus Vibrio contains motile, gram-negative bacteria that are obligate aerobes. Vibrio cells have a recognizable curved shape (comma-like) and a single polar flagellum. Although Vibrio species are non-invasive pathogens, they cause some of the most serious cases of diarrhea. These waterborne organisms are transmitted to humans via infected water or through fecal transmission. V. cholerae is the causative agent of cholera, an infection characterized by severe diarrhea. After the ingestion of V. cholerae, the organisms bind to the epithelial cells of the intestinal tract and release their exotoxin. In 1998, Isaac-Mrquez and coworkers (1998), demonstrated that the water distribution system plays an important role as a transmission vehicle for non-O1 V. cholerae. Another species of Vibrio that causes diarrhea is V. parahaemolyticus. Raw seafood (e.g., sushi, oysters) is the general source of human infection. Along with severe diarrhea, patients can also experience cramps, nausea, and fever but the disease is self-limiting, lasting only about three days.

Helicobacter pylori

Helicobacterpylori is a motile, micraerophilic gram-negative bacterium involved in the pathogenesis of human active chronic gastritis, peptic and duodenal ulcer diseases and gastric cancer. This organism is fastidious to culture because it requires nutrient- rich media, an atmosphere enriched in CO2, high humidity (96-100%) and a pH near 7.0 (Velazquez and Feirtag 1999). The mode of transmission of Helicobacter pylori is still unknown, but foodborne and waterborne pathways have been speculated. The presence of Helicobacter spp. has been demonstrated in wastewater and drinking water by PCR (Enroth & Engstrand 1995; Hultenet al. 1998), but there is no evidence of \waterborne transmission.

Cryptosporidium spp. and Giardia spp. (Protozoa)

Cryptosporidium and Giardia spp. are protozoan parasites that can infect humans and livestock. Recent studies of cryptosporidiosis cases showed that two Cryptosporidium species can infect humans: Cryptosporidium hominis (Morgan-Ryan et al. 2002) and Cryptosporidium parvum Genotype 1 that has been isolated almost exclusively from humans and associated mainly with human-to-human transmission cycles (Fayer et al. 2000). Aside from C. parvum nine other Cryptosporidium species are recognized: C. baileyi (bird), C. meleagridis (bird), C. felis (cat), C. muris (rodent), C. wrairi (guinea pig), C. serpentis (reptile), C. saurophilum (lizard), C. andersoni (cattle), and C. nasorum (fish) (Payer et al. 2000; Ong et al. 2002). Although previously thought to be host-specific, some of these species (i.e. C. felis, C. meleagridis, C. canis, and C. muris) have been implicated in a few reports concerning human infections (Ong et al. 2002; Akiyoshi et al. 2003; Caccio et al. 2002; Guyot et al. 2001).

Cryptosporidium and Giardia are obligate parasites that are able to multiply only within their respective hosts. Their resistant forms (i.e., oocysts and cysts) are shed in feces and are very resistant to environmental stress conditions, allowing them to survive for weeks or months in natural systems. Cysts are present in high numbers in raw sewage (10^sup 3^-10^sup 5^/100 liters), but there are seasonal and geographic differences in concentrations. They can also be found in a high percentage of surface waters contaminated by sewage or manure (1-10^sup 5^/100 liters) (Rose 1997). Moreover, these resistant forms have been detected in fully treated drinking water (0.5-20/100 liters) but in reality, little is known about the persistence of Giardia and Cryptosporidium spp. in water distribution systems. In 1997, Olson and co-workers (1997) undertook a study on Canadian farms animals (cattle, sheep, pigs, and horses) to investigate the prevalence of these parasites. They showed that both Giardia and Cryptosporidium are not localized to certain geographical areas and are common in most farm animals in Canada. Moreover, Payment et al. (2000) demonstrated that these two parasites were present along the Saint-Lawrence River (Quebec, Canada) at a wide range of values (Cryptosporidium: <0.04-1.02 oocysts/liter; Giardia: <0.04-9.58 cysts/liter). In addition, a recent survey of the Tech river watershed (France) demonstrated the presence of fewer than 0.04-7.3 Cryptosporidium oocysts per liter in the watersheds (Lemarchand & Lebaron 2003).

Microsporidia

Microsporidia is a non-taxonomic name used to describe protozoan parasites belonging to the phylum Microspora. This phylum includes very small (0.5-1.2 m), obligate intracellular parasites of vertebrates and invertebrates. The genera that infect humans include Encephalitozoon, Nosema, and Enterocytozoon (Szewzyk et al. 2000). Little information is available about the occurrence and distribution of human pathogenic microsporidia in the environment, especially about the effects of routine disinfection on microsporidian spores viability. In 1998, Dowd and coworkers (1998) have developed a new method, based on a PCR amplification of the small-subunit ribosomal DNA, which allowed them to detect human pathogenic microsporidia in different kinds of water (i.e., raw sewage, tertiary effluent, surface water, and groundwater). In 2000, Wolk and coworkers used a spore counting method combined with cell culture to study the effect of chlorine disinfection on Encephalitozoon syn. Septata intestinalis spores and showed that, in vitro, this treatment may prove to be a reasonable procedure for inactivation of E. intestinalis in water.

Environmental Pathogens

Several pathogenic organisms are indigenous aquatic organisms and are introduced from the surface water into the drinking water system usually in low numbers. As they are able to multiply in water or in biofilms, their number may increase in the distribution system and constitute a risk for public health. Natural water bacteria have been shown to play an important role in the survival and growth of pathogenic bacteria.

Legionella spp.

Legionella spp. are gram-negative aerobic rods, difficult to grow in vitro. Since 1976, more than 40 new species with approximately 60 serotypes of the genus Legionella have been identified. L. pneumophila serotype 1 is responsible for most of the infections, but 17 other species have been associated with disease (e.g., L. longbeachae, L. micdadei, and L. bozemani). Legionella species are natural inhabitants of freshwater and soil. The only natural waters considered as potential sources for outbreaks of Legionnaires disease are natural warm waters with temperatures ranging from 30C to 40C and occasionally up to 60C. Legionella species are introduced into drinking water from the source water and are able to grow under favorable conditions in cold and hot water distribution systems, heaters, pools, and spas. Growth also occurs in cooling towers. L. pneumophila is able to survive in sterile drinking water for months or years, and its survival is increased by the presence of other bacteria. Furthermore, L. pneumophila was shown to grow intracellularly in amoebae, like Acanthamoeba, Hartmannella, Valkampfia, Naegleria spp., and in other protozoa, like ciliates of the Tetrahymena pyriformis group. In addition to providing nutrients for Legionella organisms, these hosts also protect them from adverse environmental conditions. For example, it has been reported that Legionella spp. can be enclosed in cysts of amoebae and, thereby, protected from chlorination levels as high as 50 mg chlorine/liter. Because Legionella species live in close association with other bacteria and protozoa, they are considered typical biofilm organisms (Szewzyk et al. 2000).

Pseudomonas aeruginosa

Pseudomonas spp. are gram-negative, aerobic, nonsporogenous, motile by polar flagella bacteria. Today, P. aeruginosa is among the most important opportunistic pathogens causing nosocomial infections in immunodepressed patients and patients with underlying conditions such as wounds, urinary tract infections, and pneumonia (Szewzyk et al. 2000). In natural habitats, P. aeruginosa can be found in water exposed to fecal pollution, such as surface waters influenced by wastewater discharge, and in soil. This bacterium was not found in well-protected groundwater and was only occasionally found in non- polluted surface waters. P. aeruginosa is a typical biofilm organism that grows on materials that release nutrients, like certain fittings, and shower heads, as well as in sinks and sink drains with standing water (Szewzyk et al. 2000).

Environmental Mycobacteria

The genus Mycobacterium comprises the strictly pathogenic species M. tuberculosis, M. bovis, M. africanum, and M. leprae, which are not transmitted by water but have human or animal reservoirs only. It also comprises the so-called atypical mycobacteria, which may be pathogenic for humans. This latter group of mycobacteria is also called environmental mycobacteria because they possess, in contrast to the strictly pathogenic species, an environmental reservoir (e.g., water or soil). This group is composed of gram-positive, aerobic rods. The pathogenic mycobacteria have been known for a long time, whereas several of the environmental mycobacteria have been described only recently. Several species among the environmental mycobacteria are opportunistic pathogens, including M. avium, M. intracellulare, M. kansasii, M. chelonae, and M. fortuitum. Infections with environmental mycobacteria are not common (estimates are 2-4 cases/10^sup 5^ inhabitants) and are usually restricted to immunocompromised patients or persons with underlying diseases. Environmental mycobacteria are found in soil and water, with no correlation to fecal contamination. They have been isolated from all parts of the drinking water treatment and distribution system. For some species (e.g., M. gordonae, M. xenopi, and M. kansasii), tap water seems to be their natural environment. Drinking water has been suggested as a source of infection, but the epidemiological data are inconclusive. The occurrence of environmental mycobacteria in drinking water distribution systems is not correlated to fecal contamination. Further research is needed to understand the ecology of mycobacteria in drinking water, especially their interactions with other microorganisms, and to detect other possible environmental reservoirs and transmission routes (Szewzyk et al. 2000).

Aeromonas spp.

Aeromonas spp. are gram-negative, facultatively anaerobic, small rods. They are widespread in surface, fresh, and marine waters and in soil. They have been found in high numbers in raw sewage (10^sup 6^-10^sup 8^ CFU/ml) and in sewage effluents (10^sup 3^-10^sup 5^ CFU/ml). Reported densities in natural waters range from 10^sup 2^- 10^sup 3^ CFU/ml in river water to 1-100 CFU/liter in groundwater (Moe 1997). In temperate climates, they are able to grow in water, if sufficient nutrients are available. Their occurrence is, therefore, not necessarily an indicator of fecal pollution but of eutrophication in general. In the summer months, their number can easily reach 10^sup 4^ CFU/ml in nutrient-rich recreational waters. Like Legionella spp., Aeromonas spp. are introduced into drinking water in small numbers but, as they are able to grow under low nutrient conditions (in the range of 1 g/liter), regrowth occurs in most drinking water distribution systems. However, the amount of regrowth varies considerably, depending upon the availability of nutrients, residence time, and water temperature. Indeed, the amount of regrowth is a useful indicator of drinking water (potable) quality. It has been reported that the number of aeromonads in a drinking water distribution system correlated to th\e number of diarrheal cases from Aeromonas spp. in the connected population. These Aeromonas spp. in drinking water were shown to possess several virulence factors (e.g., production of toxins and ability to adhere to and invade epithelial cells) (Brandi et al. 1999). Furthermore, it has been shown that the growth of aeromonads is proportional to the biofilm formation potential of drinking water. Aeromonads have been found to occur in drinking water biofilms which in turn provide protection from disinfection with chlorine (Szewzyk et al. 2000).

Cyanobacterial Toxins

Cyanobacteria are known to produce a great variety of toxins, including neuro-toxins (i.e., anatoxins), hepatotoxins (i.e., microcystins), and skin irritants. The most commonly occurring toxin is microcystin-LR, a cyclic heptapeptide. Cyanobacteria that are often associated with toxin production include Microcystis, Planktothrix, Anabaena, Aphanizomenon, and Oscillatoria spp. Cyanobacteria dominate many lakes during the summer months. In several countries, including Australia, Brazil, Canada, China, and Sweden, cyanobacterial toxins have been linked to human illness from drinking water supplies that use surface water as a resource or that are contaminated with surface water containing high concentrations of Cyanobacteria (Szewzyk et al. 2000).

Vibrio spp. (except V. cholerae and V. parahaemolyticus)

Vibrio spp. are gram-negative, facultatively anaerobic, non- sporogenous rods. They are one of the most common bacterial organisms in surface waters of the world. They occur in both marine and freshwater habitats and in association with aquatic animals.

Vibrio vulnificus is a bacterium that occurs naturally in estuarine and seawaters (Hervio-Heath et al. 2002), residing in high numbers in filter-feeding shellfish (e.g., oysters, clams, mussels). This organism establishes human infections through ingestion (typically by eating raw oysters) or through a wound (typically a cut or puncture acquired while shucking oysters, peeling shrimp, cleaning fish, etc.). In Europe and North America, outbreaks have generally been associated with contaminated seafood (e.g., mussels, oysters) consumption. Fortunately, most healthy people are resistant to the infection. Unlike other Vibrio species, V. vulnificus is invasive and is able to enter the blood stream through the gut epithelium. These two Vibrio species may represent an important waterborne health risk in developing countries.

Impact of Biofilms on Water Quality

A biofilm is an assemblage of surface-associated microbial cells enclosed in an extracellular polymeric substance matrix (Donlan 2002). Noncellular materials such as mineral crystals, corrosion particles, or clay or silt particles, depending on the environment in which the biofilm has developed, may also be found in the biofilm matrix. Biofilms may form on a wide variety of surfaces, including living tissues, in-dwelling medical devices, industrial or potable water system piping, or natural aquatic systems. In drinking water systems, as well as in other aquatic systems, all existing interfaces are covered by biofilms, from the waterworks through the distribution system to domestic home installations.

For microorganisms to persist in biofilms, several of the following attributes are required (Cirillo 1999):

* Adherence to or colonization of surfaces (including other cell types).

* Resistance to bacteriolytic compounds.

* Avoidance of phagocytosis.

* Invasion through layers of other cells in the biofilm.

* Entry into and survival within other organisms.

The spatial structure and chemical composition of biofilms can vary widely depending on the environmental conditions and the population structure of the community. The inorganic depositions and encrustations represent new habitats with conditions completely different from those existing in the free water phase. Three factors play a major role in influencing the appearance of biofilms: temperature, oxygen availability, and redox potential. In these habitats, bacteria may survive and even grow, in spite of inhospitable conditions within the flowing water such as disinfectants or heat treatment. In drinking water systems, biofilms can be dominated by organic matter with only sporadic depositions of inorganic matter like quartz grains and iron particles. The polymeric substances excreted by the cells in the biofilm, often referred to as slime, not only cover the cells but also fill the space between them. As a consequence, this matrix acts as a stabilizing factor and a protective barrier against different chemical compounds and predators (Szewzyk et al. 2000). Several bacterial pathogens have been shown to associate with, and in some cases, grow within biofilms, including Legionella pneumophila, Campylobacter spp., Escherichia coli O157:H7, Vibrio cholerae, and Helicobacter pylori. These organisms have the ability to attach to surfaces but are not able to compete with indigenous microorganisms and consequently, are incapable of extensive growth in biofilms. Biofilms inside of potable water distribution systems have the potential to harbor enteric pathogens, Legionella pneumophila, nontuberculous mycobacteria, and possibly Helicobacter pylori (Donlan 2002). Presently, microbiological processes, in the fields of clinical, food, water, and environmental microbiology, need to be investigated from a biofilm perspective.

MOLECULAR METHODS USED TO DETECT FECAL POLLUTION IN WATERS

The primary goal of monitoring natural waters is to determine the presence or absence of pathogens rather than fecal indicators. To date, methods for the detection and enumeration of pathogens from wastewater and sludge have used a cumbersome approach that includes enrichment, isola

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