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Identifying Future Drinking Water Contaminants (1999)

Chapter: 9 Methods to Identify and Detect Microbial Contaminants in Drinking Water

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Suggested Citation:"9 Methods to Identify and Detect Microbial Contaminants in Drinking Water." National Research Council. 1999. Identifying Future Drinking Water Contaminants. Washington, DC: The National Academies Press. doi: 10.17226/9595.
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9
Methods to Identify and Detect Microbial Contaminants in Drinking Water

Mark D. Sobsey

The transmission of infectious diseases via contaminated water continues to be a risk to public health in the United States and throughout the rest of the. world. Source and finished drinking waters are vulnerable to microbial pathogen contamination from a variety of sources of human and animal fecal wastes and from the introduction and proliferation of nonfecal pathogenic microbes. Throughout most of the modem history of drinking water supply, concerns about pathogenic microbes have focused on enteric bacteria of human fecal origin. These concerns led to the development of criteria and standards for bacteriological quality intended to protect against excessive risks from enteric bacterial pathogens such as Salmonella typhi and other nontyphoid Salmonella spp., Shigella spp., and Fibrio cholerae. The infectious disease risks in drinking water supplies from enteric viruses (such as hepatitis A virus), enteric parasites (such as Entamoeba histolytica and Giardia lamblia), and nonfecal bacterial pathogens (such as Legionella spp.) were not recognized until more recent times. These risks were recognized initially by the occurrence of waterborne outbreaks of disease mused by these pathogens.

Until recently there were no formal, legally required processes to identify or consider new or emerging water. me pathogens in the United States. It was only with reauthorization of the Safe Drinking Water Act in 1996 that the U.S. Environmental Protection Agency (EPA) was required to identify through a structured process candidate microbial pathogens for possible regulation in drinking water supplies. Prior to this the agency used an informal and largely reactive process to recognize, identify, and prioritize microbial pathogens for possible regulation. The new requirement for a proactive rational process to identify and consider microbial pathogens for possible regulation in drinking water is the essential motivation for this paper. As part of this process it is necessary to detect and quantify microbial pathogens in drinking water and its sources; to establish dose-response relationships as an essential step in health effects characterization for waterborne pathogens; and to identify, characterize, and quantify the virulence properties of these pathogens that influence their human health effects.

Suggested Citation:"9 Methods to Identify and Detect Microbial Contaminants in Drinking Water." National Research Council. 1999. Identifying Future Drinking Water Contaminants. Washington, DC: The National Academies Press. doi: 10.17226/9595.
×

Purpose

The purpose of this report is to consider and address the following questions: How should microbial contaminants for possible regulation in drinking water be identified, characterized, and quantified with respect to their risks to public health? What should be the essential elements of the process for waterborne microbial pathogen identification and characterization? What should be the basis for prioritizing, ranking, or choosing among the many potential drinking water pathogens for possible regulation? How should the microbial pathogen identification and selection process be integrated into the overall process of improving drinking water quality and reducing health risks through drinking water regulations? How should analytical methods for detection, characterization, and quantification of microbial contaminants be applied to the process of identifying, characterizing, and quantifying the risks from waterborne pathogens being considered for regulation? What. analytical methods are available for use in identifying, quantifying, and characterizing microbial pathogens in drinking water for possible regulation?

Analysis

Need for a Risk-Based Approach

The recognition, identification, prioritization, and characterization of microbial pathogens in drinking water should be risk based and should consider the relationships and interactions of the microbes, their hosts, and the environment. The microbial world consists of a wide variety of different types or classes of microbial agents potentially present in water. Viruses, bacteria, protozoans, fungi, and algae are widespread in soil, sediments, water, air, and food and on objects and surfaces with which humans have contact ("fomites"). Most of these microbes are not pathogenic (harmless) and are incapable of infecting or colonizing immunocompetent persons unless they somehow gain access to sterile internal sites in the body (such as the bloodstream and various organs) through trauma, surgery, or other such means. However, persons with immunodeficiencies are at risk of infection, colonization, and illness from microbes considered nonpathogenic for immunocompetent persons. Therefore, recognition and identification of a possible waterborne pathogen depends in part on the susceptibilities of the population to infection, colonization, and illness from a microorganism. Some pathogens are always potentially pathogenic and are often referred to as "frank" pathogens. Other pathogens are never or rarely pathogenic for immunocompetent and otherwise "healthy" people. However, these microbes can sometimes cause infection, colonization, and illness in persons who have an immune deficiency, have other conditions that make them susceptible, or because they encounter the microbe in an unusual or atypical way. Such microbes are sometimes referred to as "conditional" or "opportunistic" pathogens. As previously noted, there are nonpathogenic microbes in the environment that are capable of infecting, colonizing, and causing illness in humans only if they are able to dramatically breach the body's natural barriers

Suggested Citation:"9 Methods to Identify and Detect Microbial Contaminants in Drinking Water." National Research Council. 1999. Identifying Future Drinking Water Contaminants. Washington, DC: The National Academies Press. doi: 10.17226/9595.
×

and proliferate in normally sterile sites within the body. These nonpathogenic or "saprophytic" microbes are common in aquatic and other environments.

A waterborne pathogen may emerge or acquire increased public health importance because of changes in host susceptibility to infection. Factors influencing host susceptibility in the population include increases in the number of immunocompromised persons, increased use of immunosuppressive agents (among persons receiving cancer chemotherapy or undergoing organ transplants), increases in the elderly segment of the population, and poor nutrition. In identifying and prioritizing emerging waterborne pathogens the susceptibilities of these higher-risk population subgroups to specific infectious diseases is an important consideration (Morris and Potter, 1997).

The relationships between waterborne microbes and their human hosts are complex and are influenced by a variety of factors involving the characteristics and conditions of the microbe, the human and in some cases animal hosts, and the environment. Therefore, it seems necessary to identify, characterize, and quantify these relationships in order to determine if a potentially waterborne microbe should be considered or classified as a drinking water contaminant for possible regulation. Furthermore, the need to prioritize or otherwise determine the importance of a microbe for possible regulation in water suggests that a structured and quantitative approach must be used for such an evaluation or assessment.

Adapting Quantitative Risk Assessment to Recognize, Identify, Prioritize, and Characterize Drinking Water Pathogens

Over the past two decades considerable progress has been made in quantitative risk assessment (QRA) for making management decisions about waterborne pathogens. Initially, the National Academy of Sciences/National Research Council risk assessment strategy was adapted to assess microbial risks (Regli et al., 1991; Rose and Gerba, 1991; Rose et al., 1991; Sobsey et al., 1993). This process consists of hazard identification, exposure assessment, effects assessment, and risk characterization. Using this approach, quantitative risk assessments were done initially for several recognized waterborne pathogens, such as Giardia lamblia and rotaviruses. More recently, the process of quantitative risk assessment for pathogens in water was reconsidered and revised through a consensus-building International Life Sciences Institute (ILSI) workshop process (ILSI Risk Science Institute Pathogen Risk Assessment Working Group, 1996). This effort resulted in a modified quantitative risk assessment system that specifies the criteria, information needs, and analytical approaches for quantitative risk assessment for waterborne microbes (See Figure 9-1).

Presently, the EPA/ILSI system for quantitative risk assessment of microbes in water is being applied by several research groups to determine its utility, and other researchers will peer review the QRA products for utility,

Suggested Citation:"9 Methods to Identify and Detect Microbial Contaminants in Drinking Water." National Research Council. 1999. Identifying Future Drinking Water Contaminants. Washington, DC: The National Academies Press. doi: 10.17226/9595.
×

Figure 9-1

Risk assessment for waterborne microbial pathogens:  EPA/ILSI paradigm.

clarity, and transparency. The EPA/ILSI QRA approach was not intended for the purpose of selecting microbial contaminants for possible regulation in drinking water. Furthermore, it certainly is not the only way to identify, prioritize, and assess the risks from microbes in drinking water. However, this microbial QRA system does specify information needs and analytical methods that can be readily adapted to the recognition, identification, prioritization, and initial characterization of risks from a possible waterborne microbe. Considering that the EPA and many of the nation's scientists in the areas of water microbiology, infectious diseases, water treatment, epidemiology, and risk assessment invested much effort and time in the development of this system, it seems appropriate to interface it with the process for microbial contaminant selection. Furthermore, it can be said that the elements of the EPA/ILSI QRA system for microbes rely on the same types and sources of data and at least some of the same analytical methods that would be used in any structured process to identify, select, characterize, and prioritize candidate microbial contaminants for possible regulation in drinking water (See Figure 9-2).

Human Pathogens in Water: Classification, Sources, and Properties

Known and potential human pathogens in water include the spectrum of agents ranging, in order of increasing complexity, from prions, to viruses, to bacteria, and other prokaryotes, and the microbial eukaryotes (the protists), including protozoans, fungi, and algae (Moe, 1997). Prions have not been implicated in waterborne disease, but recent evidence for human spongiform encephalopathies from ingestion of beef contaminated with bovine spongiform agents suggests that vehicles such as food and possibly water contaminated with prions pose a risk of exposure (Ironside, 1998; Knight and Stewart, 1998). Furthermore, these agents are very small compared to other microbes, which makes them difficult to remove by physical-chemical processes, and they are extremely resistant to virtually all physical and chemical agents, which makes them persistent in the environment and resistant to virtually all drinking water disinfectants.

Suggested Citation:"9 Methods to Identify and Detect Microbial Contaminants in Drinking Water." National Research Council. 1999. Identifying Future Drinking Water Contaminants. Washington, DC: The National Academies Press. doi: 10.17226/9595.
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A. ELEMENTS CONSIDERED IN PATHOGEN OCCURRENCE

  Spatial distribution (clumping, particle-association, clustering)

  Concentrations in environmental vehicles and foods

  Seasonality and climatic effects

  Temporal distribution, duration, and frequency

  Niche (potential to multiply or survive in specific media)

  Amplification, die-off, persistence

  Indicators/surrogates predictive of pathogen

B. ELEMENTS CONSIDERED IN EXPOSURE ANALYSIS

  Identification of water and other media

  Unit of exposure

  Temporal nature of exposure (single or multiple; intervals)

  Route of exposure and transmission potential

  Demographics of exposed population

  Size of exposed population

  Behavior of exposed population

C. ELEMENTS CONSIDERED IN PATHOGEN CHARACTERIZATION

  Virulence and pathogenicity of the microorganism

  Pathological characteristics and diseases caused

  Survival and multiplication of the microorganism

  Resistance to environmental control measures

  Host specificity

  Infection mechanism and route; portal of entry

  Potential for secondary spread

  Taxonomy and strain variation

D. ELEMENTS CONSIDERED IN HOST CHARACTERIZATION

  Demographics of the exposed population (age, density, etc.)

  Immune status

  Pregnancy

  Concurrent illness or infirmity

  Nutritional status

  Genetic background

  Behavioral and social factors

E. ELEMENTS CONSIDERED IN HEALTH EFFECTS

  Morbidity, mortality, sequelae of illness

  Severity of illness

  Duration of illness

  Chronicity or recurrence

  Potential for secondary spread

  Quality of life

FIGURE 9-2 Microbial contaminant identification, selection and characterization using elements of the EPA/ILSI risk-based approach: Description of the information needs.

Suggested Citation:"9 Methods to Identify and Detect Microbial Contaminants in Drinking Water." National Research Council. 1999. Identifying Future Drinking Water Contaminants. Washington, DC: The National Academies Press. doi: 10.17226/9595.
×
Viruses

A variety of enteric and respiratory viruses of humans and in some cases other animals as well are potential agents of waterborne disease. For many of these viruses the role of water has been clearly established because of documented waterborne outbreaks, or it is strongly suspected because the viruses have been detected in drinking water or its sources. Some of these viruses are shown in Table 9-1, but other viruses and virus groups may also pose risks from exposure via drinking water. A notable feature of all of these viruses except the coronaviruses and picobirnaviruses is that they are nonenveloped (consisting only of a nucleic acid surrounded by an outer protein coat or capsid).

Nonenveloped viruses tend to be more resistant to various physical and chemical agents and more stable in the environment than the enveloped viruses, which probably contributes to their potential to cause waterborne disease. Another important feature of some of these viruses, as well as many of the bacterial and parasitic pathogens of concern in drinking water, is that they have known or suspected animal hosts and therefore are transmissible directly or indirectly from other animals to humans. The potential for animal-to-human transmission creates concerns about contamination of drinking water supplies with animal wastes containing these pathogens. As previously noted, similar concerns also apply to many bacterial and parasitic pathogens.

Bacteria

Many enteric and respiratory bacteria infect and cause morbidity and mortality in humans via the water route. Some of these bacteria also infect other

TABLE 9-1

Important Viral Contaminants of Drinking Water

Virus or Virus Group

Outbreak or Detected in Water

Animal Sources

Enteroviruses:

(polios, echos2, coxsackies2, etc.)

Yes

No

Hepatitis A virus2

Yes

No

Hepatitis E virus

Yes

Pigs?

Reoviruses

Yes

Yes

Rotaviruses

Yes

Yesb

Adenoviruses2

Yes

Yesb

Caliciviruses2:

Norwalk, Snow Mountain, etc.

Yes

No for some; maybe for others

Astroviruses

Yes

No

Parvoviruses; picornaviruses

Maybe

Uncertain

Coronaviruses

No

Uncertain

Picobirnaviruses and picotrirnaviruses

Unknown

Maybe

2 On EPA's microbial contaminants priority list.

b Humans and animals are usually infected by different ones but not always.

Suggested Citation:"9 Methods to Identify and Detect Microbial Contaminants in Drinking Water." National Research Council. 1999. Identifying Future Drinking Water Contaminants. Washington, DC: The National Academies Press. doi: 10.17226/9595.
×

animals, often asymptomatically, and some, such as the Legionella spp., the Mycobacteria spp., and the heterotrophic plate count bacteria, have abiotic environmental reservoirs. For many of these bacteria the role of waterborne transmission has been documented by waterborne outbreaks, or it is strongly suspected because the bacteria have been detected in drinking water and its sources (see Table 9-2). In the case of some of these waterborne bacteria their risks to human health from ingestion or inhalation of water or contact with water are uncertain because they have not been conclusively documented by outbreaks or other epidemiological evidence of waterborne disease. However, their presence in drinking water and the uncertainty of their risks to human health from drinking water exposure suggest the need for further investigation and analyses.

The risks posed by various bacteria potentially present in drinking water differ among the various genera and species as well as within the same genus and species of a bacterium. These differences in risks to human health pose considerable challenges to the detection and identification of these bacteria in water. Similar concerns apply to the protozoan parasites, algae, and fungi. Strains or variants of the same genus and species of bacterium can differ dramatically in their ability to cause disease because this ability is largely dependent on the presence of virulence factors or properties.

In some cases the virulence factors or properties of the bacterium responsible for disease are essential constituents of the cell. This appears to be the case for Salmonella typhi, the causative agent of typhoid fever, whose essential virulence properties are the O antigen (the lipopolysaccharide outer membrane of the cell wall; an endotoxin) and the Vi antigen (a capsule polysaccharide) (Salyers and Whitt, 1994; Levine, 1998). For many other bacteria, such as strains of Escherichia coli, Aeromonas hydrophila, and Yersinia enterocolitica, the ability to be a pathogen and cause disease is clearly associated with the presence of specific virulence properties that may or may not be present in specific strains or types. These virulence factors are often transmissible from one cell to another via transmissible plasmids or bacterial viruses (bacteriophages). Plasmids are extrachromosomal, small, circular DNA molecules that replicate separately from the bacterial chromosome and can move from one cell to another by a process called conjugation. Bacteriophages also can transmit virulence factors from one host cell to another, especially if the infecting bacteriophages do not kill the cell and instead integrate their DNA into the bacterial chromosome. Strains of a species bacterium possessing no virulence factors generally are not pathogenic and do not produce disease. Strains of the same species of bacterium possessing one or more specific virulence factors are pathogenic and capable of producing disease. Furthermore, the pathology and clinical features of the disease depend on the properties and activities of these virulence factors. For example, strains of E. coli lacking virulence properties generally are not pathogenic, and most humans and other mammals harbor about a billion E. coli cells per gram in their lower intestinal

Suggested Citation:"9 Methods to Identify and Detect Microbial Contaminants in Drinking Water." National Research Council. 1999. Identifying Future Drinking Water Contaminants. Washington, DC: The National Academies Press. doi: 10.17226/9595.
×

TABLE 9-2

Some Important Bacterial Contaminants of Drinking Water

Bacterium/Group

Outbreak or Detected in Water

Animal Feces

Non-Fecal Sources

Salmonella spp. (except S. typhi)

Yes

Yes

No

Salmonella typhi

Yes

No

No

Campylobacter spp.

Yes

Yes

No

Escherichia cell

Yes

Yes

Maybe

Helicobacter pyloria

Yes

Unknown

Unknown

Heromonas hydrophilaa

Yes

Yes

Yes

Yersinia enterocolitica

Yes

Yes

Yes

Fibrio cholerae and some other Fibrio sp.

Yes

Yes

Yes

Shigella spp.

Yes

No

No

Legionella sppb.

Yes

No

Yes

Mycobacterium avium-intracellularea and other Mycobacterium spp.

Yes

Yes

Yes

Leptospira spp.

Yes

Yes

Yes

Heterotrophic plate

Yes

Yes

Yes

Count bacteria

Yes

Yes

Yes

a On EPA's microbial contaminants priority list.

b On EPA's microbial contaminants priority list in groundwater.

tracts with no adverse effects. However, E. cell strains possessing one or more virulence properties are capable of causing different diseases. For example, enterohemorrhagic strains of E. cell strains possess the Shiga-like toxin (SLT). They are infectious at relatively low doses (< one thousand cells) and cause a bloody diarrhea without fever that can advance to life-threatening hemolytic-uremic syndrome, a form of hemolytic anemia and kidney failure, especially in young children. Other strains of E. coli possess a heat-stable toxin (ST) and/or heat labile toxin (LT; similar to the cholera toxin of Fibrio cholerae). These enterotoxigenic strains of E. coli are infectious at higher doses (> one million cells) and cause disease by increasing adenylate cyclase (LT) and guanylate cyclase (ST) activities in the small intestine that lead to watery diarrhea, electrolyte imbalance, and dehydration.

The roles of human and animal hosts as well as the environment in the selection for and emergence of new strains of virulent bacteria are becoming increasingly appreciated. For example, there is growing evidence that cattle and other agricultural (livestock) animals are major reservoirs of such waterborne and foodborne bacterial pathogens as enterohemorrhagic E. coli, Salmonella spp., and Yersinia enterocolitica (Tauxe, 1997).

The role of the aquatic environment as a reservoir for and source of emergence of new virulent strains of bacteria is becoming increasingly recognized in the case of some bacteria. For example, the genes coding for the cholera toxin of Fibrio cholerae are borne on and can be infectiously transmitted

Suggested Citation:"9 Methods to Identify and Detect Microbial Contaminants in Drinking Water." National Research Council. 1999. Identifying Future Drinking Water Contaminants. Washington, DC: The National Academies Press. doi: 10.17226/9595.
×

by a filamentous bacteriophage (Faruque et al., 1995). The natural history of V. cholerae, including the mechanisms and evolution of virulence and pathogenicity, as well as its taxonomy, appear to be intimately linked to the aquatic environment and the interactions of humans with this environment. Molecular epidemiological studies reveal clonal diversity among toxigenic V. cholerae strains. The continual emergence of new epidemic clones may be taking place in aquatic ecosystems through interaction of the phages bearing the cholera toxin with different strains or antigenic types of V. cholerae . These new strains may then be selected for during epidemics in human populations. This appears to be an example of the evolution of new toxigenic strains of a human pathogen in natural aquatic ecosystems systems and its selection during outbreaks in human hosts. Within the aquatic ecosystem, interactions of the genetic elements of the microbes and their host reservoirs mediate the transfer of virulence genes, thereby resulting in the creation and the subsequent selection in humans of these new pathogenic strains. The extent to which such evolution and selection occurs for other human pathogens in aquatic ecosystems is unknown and deserves further investigation.

Protozoan Parasites

In the past three decades, protozoan parasites have emerged as important waterborne pathogens (Marshall, 1998). Some of the important protozoan parasites infecting humans and found in water are listed in Table 9-3. The ameba Entamoeba histolytica, the cause of amebic dysentery, has long been recognized as a waterborne pathogen. However, outbreaks of waterborne amebic dysentery have not been reported for decades in the United States and there are no major nonhuman reservoirs of this parasite. It was only with the recognition in the 1960s and 1970s of Giardia lamblia as a waterborne pathogen having important animal reservoirs and considerable resistance to chlorination and other drinking water disinfection practices that serious attention began to focus on this agent and other human pathogenic protozoans in drinking water. Since then, Cryptosporidium parvum has become a high-priority pathogen for regulation in drinking water because of documented waterborne disease, many animal reservoirs, ubiquitous presence in drinking water sources, relatively small size, and resistance to chlorine and other drinking water disinfectants. Other protozoan parasites, including the free-living amebas (e.g., Acanthamoeba spp), the coccidians Cyclospora cayatenensis and Toxoplasma gondii, the microsporidia, and the ciliate Balantidium coli are now recognized as human pathogens that deserve consideration in drinking water. Some of these agents, such as the free-living amebas, have a natural aquatic habitat. Many of the others, including Giardia lamblia, Cryptosporidium parvum, Toxoplasma gondii, Balantidium coli, as well as the microsporidia have nonhuman animal reservoirs that contribute to their presence in drinking water supplies and sources. The microsporidia are among the most ubiquitous protozoan parasites of animals and

Suggested Citation:"9 Methods to Identify and Detect Microbial Contaminants in Drinking Water." National Research Council. 1999. Identifying Future Drinking Water Contaminants. Washington, DC: The National Academies Press. doi: 10.17226/9595.
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TABLE 9-3

Important Protozoan Parasite Contaminants of Drinking Water

Parasite

Outbreak or Detected in Water

Animal Feces

Non-fecal Sources

Acanthcanoeba spp.a

Yes

No

Yes

Cryptosporidiurn parvum

Yes

Yes

No

Cyclospora cayatenensisa

Yes

Unknown

Unknown

Giardia lamblia

Yes

Yes

No

Entarnoeba histolytica

Yes

Rare

No

Balantidium cell

Yes

Yes (pigs)

No

Microsporidiaa (Enterocytozoon and Septata)

Yes?

Yes

Maybe

Toxoplasma gondiia

Yes

Yes

No

a On EPA's microbial contaminants priority list.

infect hosts ranging from insects and other invertebrates to fish, birds, and mammals. Only a few species of human microsporidia have been recognized, and the Significance to human health of the many microsporidia of other animals is unknown at this time.

A particular challenge to the detection of protozoans of public health concern in drinking water is that many of the currently available and widely used analytical methods, especially the various microscopic techniques (such as brightfield, immunofluorescent, phase contrast, and differential interference contrast microscopy) cannot always distinguish the human pathogenic genera, species, and strains from the many others that are noninfectious and therefore harmless to humans (EPA, 1996, 1998). Furthermore, these microscopic methods cannot distinguish the infectious parasites posing a human health risk from the noninfectious inactivated ones no longer posing risks to human health. Even the latest of sensitive and specific molecular genetic methods, such as polymerase chain reaction (PCR) amplification and restriction fragment length polymorphism (RFLP) analyses, may not distinguish human pathogenic from nonhuman nonpathogenic strains of a parasite such as Cryptosporidium (Champliaud et al., 1998). The limitations of currently available detection methods in identifying species and strains of protozoans capable of causing human infection and disease also apply to the methods of detecting other classes of microbes in drinking water that pose risks to human health.

Methods for Detecting Pathogenic Microbes in Water

Introduction

The detection of pathogenic microbes in water typically involves three main steps: (1) recovery and concentration, (2) purification and separation, and (3) assay and characterization. In most cases the concentrations of pathogenic microbes in drinking water are so low that practical detection requires an initial

Suggested Citation:"9 Methods to Identify and Detect Microbial Contaminants in Drinking Water." National Research Council. 1999. Identifying Future Drinking Water Contaminants. Washington, DC: The National Academies Press. doi: 10.17226/9595.
×

step of concentration or enrichment from the water. Because many concentration and recovery procedures for pathogens also recover and concentrate other microbes and other constituents in the water sample, subsequent purification procedures are needed to separate the target pathogens from these other materials. Furthermore, the volumes of concentrated samples often are still too large for sensitive detection and analysis of the target pathogens, and therefore additional steps of concentration as well as purification are needed.

The physical and chemical properties of the microbes have an important influence on their ability to be recovered by the various physical and chemical separation methods available. The size, shape, and density of the microbes influence various physical methods of recovery, such as filtration, sedimentation, and flotation. The surface properties of the microbes, such as their hydrophilicity, surface charge, isoelectric point, hydrophobicity, permeability, and chemical reactivity, will influence chemical and physical-chemical methods of recovery and separation. Most microbes are hydrophilic and negatively charged near neutral pH, but most are also somewhat hydrophobic and their surface has both hydrophobic and hydrophilic domains. The environmentally stable stages or forms of some microbes, including bacterial spores and protozoan cysts, oocysts, and spores, have thick outer "walls." Some bacteria have other surface features influencing physical-chemical behavior, such as outer polysaccharide layers (capsules), and appendages such as flagella and pili (fimbrae). These many and varied physical and chemical properties must be considered in the development and application of methods to recover and concentrate microbes from water.

A variety of assay and characterization procedures can be applied to the detection and quantitation of target pathogens in drinking water. These include enumeration or quantal assays of total, viable, active, or infectious target microbes and their distinction from nontarget microbes based on identification or characterization of genus, species, type, strain, and virulence or other relevant properties. Recently, nucleic acid amplification methods such as PCR and nucleic acid identification and characterization methods, such as hybridization (gene probes), RFLP analysis, and nucleotide sequencing, have been applied to the detection of microbes in water. Despite the potential sensitivity and specificity of these methods, they are not always capable of reliably detecting and quantifying infectious or viable organisms because they often detect the nucleic acid of noninfectious inactivated microbes (Sobsey et al., 1998). There are similar concerns about the various immunochemical methods to detect and quantify microbes in water; these methods detect and quantify antigens that may still be present and reactive in noninfectious or inactivated microbes.

Suggested Citation:"9 Methods to Identify and Detect Microbial Contaminants in Drinking Water." National Research Council. 1999. Identifying Future Drinking Water Contaminants. Washington, DC: The National Academies Press. doi: 10.17226/9595.
×

Initial Recovery and Concentration of Pathogens from Water

Sedimentation by Centrifugation

For bacteria, parasites, and other cellular microbes, initial concentration and recovery are sometimes done by sedimenting the cells using centrifugation. Typically, bacteria and parasites can be sedimented from water and other aqueous samples at relative centrifugal forces (RCFs) of several thousand times gravity for several minutes to several tens of minutes. The supernatant water is removed, and the sedimented cells are resuspended in a small volume of water or other aqueous solution for subsequent analysis and characterization, with or without further purification or concentration. A modified centrifugation method recently applied to Cryptosporidium is the use of a blood cell separator (Borchardt and Spencer, 1998). Water is continuously centrifuged through the device at about 1,000x gravity, and Cryptosporidium oocysts and other particles are deposited in a separation channel. The deposited Cryptosporidium oocysts and other particles are then recovered from the separation channel and collected for microscopic examination.

Viruses also can be recovered and concentrated by centrifugation, but because of their small size this requires ultracentrifugation (Sobsoy, 1976). Typical ultracentrifugation conditions for viruses are RCFs of 50,000 to 100,000x gravity for periods of several hours. Ultracentrifugation is not widely used to concentrate and purify viruses from water because of the high cost and lack of portability of ultracentrifuges and the tendency for low levels of viruses to be recovered with poor and variable efficiency. Using simple centrifugation methods, other particles in the same size and density range of the target microbes also are recovered and concentrated. This lead of other nontarget particles often greatly exceeds the concentration of target microbes, and these excess nontarget particles can 'interfere with further separation, concentration, assay, and characterization of the target microbes.

Filtration

Microbes can be recovered and concentrated from water by a variety of filtration methods (Brock, 1983). The most widely used filtration method for recovering bacteria is membrane filtration using microporous membranes typically composed of cellulose esters. This method is the basis of the widely used membrane filtration methods for detecting indicator bacteria, including total and fetal coliforms, enterococci, and Clostridium perfringens (Eaton et al., 1995). These methods and modifications of them are also widely used for initial concentration and recovery of bacterial pathogens in water, including Salmonella, Shigella, and Campylobacter. The cells recovered on a membrane filter can be directly cultured on differential and selective broth (liquid) or agar (solid) media in order to detect and assay the recovered bacteria by enrichment (or presence-absence) or by the development of bacterial colonies. The enriched bacteria or bacterial colonies are further characterized to confirm their identity.

Alternative filtration methods have been used to recover and detect bacteria and parasites, including microporous filters composed of nylon,

Suggested Citation:"9 Methods to Identify and Detect Microbial Contaminants in Drinking Water." National Research Council. 1999. Identifying Future Drinking Water Contaminants. Washington, DC: The National Academies Press. doi: 10.17226/9595.
×

polycarbonate, fiberglass, porous ceramics, and other media. Track-etched polycarbonate and other membrane filters have been used to concentrate and recover bacteria and parasites for direct microscopic detection. These microscopic methods often employ immunofluorescence assays to facilitate identification, assays for determination of cellular activity as a measure of viability (e.g., inclusion or exclusion of vital dyes or other compounds), and infectivity assays, such as cultivation of bacteria on solid media for the development of microcolonies detectable by microscopy or fluorometry.

Another filtration method used for recovery and concentration of bacteria as well as viruses, parasites, and other microbes is ultrafiltration. As the name implies, ultrafilters have much smaller pore sizes that are expressed as the molecular weight of the smallest retained particles or molecules (molecular weight cutoff or MWCO). Typically, this is in the range of several thousand to 100,000 MWCO. Ultrafiltration is often done using tangential flow systems in which the water is made to flow parallel to the membrane surface. This is done in order to keep the microbes and other particles suspended in the retained water (retentate) and prevent them from accumulating at the filter surface where they would cause clogging and reduce hydraulic flux. Tangential flow ultrafiltration systems include stirred cells, hollow fibers, spinning cartridges, and stacked sheets.

Because of the small size of viruses, they are recoverable from water by pore size exclusion filtration only with ultrafilters or even smaller pore size filters (nanofilters and reverse osmosis filters). Ultrafiltration has been used for virus concentration from water for decades, although the high costs of ultrafiltration hardware and the ultrafilters themselves have limited the use of these methods (Sobsey, 1976). Recently economical, disposable hollow fibers have been used to concentrate viruses as well as bacteria and the parasite Cryptosporidium parvum from raw source water and finished drinking water (Juliano and Sobsey, 1997).

Size exclusion filtration is widely used to concentrate parasites from water, with most of the historical and current focus on Giardia and Cryptosporidium. The filters initially and still widely used are yarn-wound, 10-inch-long, cartridge filters composed of polypropylene or other media, and having nominal pore sizes of one to several micrometers in diameter (EPA, 1996). A disadvantage of these filters is the need to remove them from the filter housing and manually cut them apart in order to recover the parasites and other retained particles by physically washing them from the filter medium using an aqueous detergent solution. Parasite cysts and oocysts in the recovered solution of several liters volume are further concentrated and recovered by centrifugation to sediment them. Because these depth filters have only nominal pore size ratings and the cartridges are typically pressure held in their plastic housings by flexible O-ring or gasket seals, Cryptosporidium oocysts have penetrated or bypassed the filters, resulting in appreciable losses. Furthermore, recoveries from the filters are highly variable, resulting in large coefficients of variation. Additionally, because the target sample volumes are 100 L or more, there are

Suggested Citation:"9 Methods to Identify and Detect Microbial Contaminants in Drinking Water." National Research Council. 1999. Identifying Future Drinking Water Contaminants. Washington, DC: The National Academies Press. doi: 10.17226/9595.
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high loads of other particles in the resulting pellets. These other particles can interfere with subsequent purification and microscopic examination of the parasite cysts and oocysts.

Other filters having absolute pore size ratings smaller than the size of the target cysts, oocysts, and spores are alternatives for concentrating parasites from water. These filters are preferred because they are expected to achieve absolute retention of the protozoan cysts, oocysts and spores and because their physical characteristics facilitate easier and more efficient recovery of the retained microorganisms by simpler elution methods than cutting apart and macerating the filter material. Formats for these filters include flat track-etched polycarbonate disks, cellulose acetate membranes (that are dissolved in acetone to recover Cryptosporidium oocysts), pleated capsule filters (1 um pore size polyether-sulphone filters in a polycarbonate housing), and ultrafilters (spinning cartridge and hollow fiber units). Such filters, as well as the smaller water sample volumes, are now recommended by the EPA, and some of them are specified in the recently developed Method 1622 (EPA, 1998). Another type of filter being used to concentrate Cryptosporidium from water is a compressible "sponge" filter. This filter is compressed into a water pipe to achieve a small pore size, and water is allowed to flow through the compressed filter for a period of time. The filter is recovered from the pipe, and the parasite cysts and oocysts are readily washed off of the now decompressed sponge-like filter medium for further processing and analysis.

The most widely used methods for initial concentration and recovery of viruses from water employ microporous filters that retain viruses primarily by adsorption to the filter medium (Sobsey, 1976; Sobsey, 1982; .De Leon and Sobsey, 1991). These filters retain viruses by both electrostatic and hydrophobic interactions between the surfaces of viruses and the filter media. Formats used for virus adsorbent filters include membranes, disks, and pleated cartridges. The media used initially as virus adsorbent filters were negatively charged cellulose esters, fiberglass, and other materials. These filters adsorb viruses efficiently only at lower (acidic) pH levels (pH of 3 to 6) and/or in the presence of multivalent cation salts, such as divalent calcium or magnesium or trivalent aluminum salts. Relatively large volumes of conditioned water are passed through the filter, and viruses adsorb to the filter medium surfaces. Subsequently, filters that are electropositive near neutral pH and adsorb viruses directly without acidifying or adding cations salts to the water were developed for virus concentration (De Leon and Sobsey, 1991).

Electropositive filter media are composed of charge-modified fiberglass sold commercially as disks or pleated cartridges, fiberglass filter disks that are coated with precipitated aluminum or iron salts, or positively charged, natural quartz fiberglass that one packs into a column to make an adsorbent filter. The current EPA-approved ICR method to detect culturable enteric viruses in drinking water supplies specifies use of commercially available, electropositive filter (EPA, 1996). Viruses adsorbed to both electronegative or electropositive filters are subsequently eluted and recovered by passing a relatively small volume of aqueous elution medium through the filter. Viruses in the resulting filter eluates are assayed directly or after further steps of concentration, purification, and extraction.

Suggested Citation:"9 Methods to Identify and Detect Microbial Contaminants in Drinking Water." National Research Council. 1999. Identifying Future Drinking Water Contaminants. Washington, DC: The National Academies Press. doi: 10.17226/9595.
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Initial Recovery and Concentration of Pathogens from Water by Chemical Precipitation Methods

Pathogens can be recovered and concentrated from water by chemical precipitation methods, and such methods have been used primarily for vital and protozoan pathogens. Chemical precipitation of viruses is done typically with either polyethylene glycol or cation salts (aluminum, iron, magnesium, etc.; Sobsey, 1976; Dobberkau et al., 1981), and for protozoans such as Cryptosporidium it is done primarily with calcium carbonate (Vesey et al., 1993). Virus concentration from water by aluminum hydroxide flocculation and Cryptosporidiurn concentration from water by calcium carbonate precipitation has been used for recoveries from sample volumes up to about 10 to 20 L. For virus recovery, aluminum sulfate is added to the water, the water is acidified, and after several hours of settling the supernatant is aspirated and the remaining floc or sediment is centrifuged to remove additional water. The resulting floc is dissolved in an acidic or other buffer, such as citric acid. Viruses in the dissolved floc are assayed directly or after further concentration and purification. For Cryptosporidium parvum oocyst recovery, water is supplemented with calcium chloride and sodium bicarbonate and brought to pH 10 with NaOH to precipitate calcium carbonate. After settling for several hours, the supernatant is removed and the remaining precipitate is dissolved in dilute sulfamic acid. The Cryptosporidiurn oocysts are recovered by centrifugation, resuspended, and microscopically enumerated after fluorescent antibody staining.

Other Primary Recovery and Concentration Methods

Other solid-phase or granular media, including minerals (such as iron oxide, talc and quartz sand), glass beads, and synthetic resins (ion exchange and adsorbent) have been used to concentrate microbes from water by adsorption, filtration, and related processes. These methods are not as widely used as the others described above because they are less effective, often cumbersome, and often not readily portable for field use. Furthermore, elution, desorption, or flushing of the target microbes from these media is often inefficient and cumbersome. Other filtration and adsorption media that are better defined, more portable, and more amenable to efficient microbial recovery are now preferred.

Purification Methods for Waterborne Pathogens

Purification, separation, and concentration of target microbes in primary samples or sample concentrates is intended to separate target microbes from other particles and solutes and reduce the sample size by further concentration. A variety of physical, chemical, and immunochemical methods are used for this purpose. Sedimentation and flotation using density solutions or gradients are

Suggested Citation:"9 Methods to Identify and Detect Microbial Contaminants in Drinking Water." National Research Council. 1999. Identifying Future Drinking Water Contaminants. Washington, DC: The National Academies Press. doi: 10.17226/9595.
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techniques used primarily for parasites, but they have also been applied to viruses, bacteria, and other eukaryotes besides protozoans. Chemical precipitation methods are used for viruses and parasites, and some of these are similar if not identical to those used for primary concentration of these same microbes from large volumes of water. Filtration methods are applied for purification and further concentration of all classes of microbes, and often they are similar to those filtration methods used for primary concentration of these same microbes from large volumes of water.

Immunomagnetic separation (IMS), sometimes referred to as immunocapture or antibody capture, is a method now being applied to all classes of microbes. The method uses paramagnetic synthetic beads, other magnetic or paramagnetic particles, or other solid surfaces (e.g., microcentrifuge tubes and the wells of microtiter plates) that have been coated with antibodies directed against the target microbes to recover the microbes from the sample by an antigen-antibody reactions. The retained microbes can be analyzed directly or after they or their components (e.g., their nucleic acids) have been subsequently released or extracted from the antibody and solid phase by various physical or chemical methods. IMS methods have the advantage of selecting, separating, and purifying specific target microbes from other microbes and particles of similar size and shape and as well as from solutes, based on the specificity of the antigen-antibody reaction. This is a powerful approach for recovering, enriching, purifying, and concentrating the target organisms from the sample matrix.

Other purification and concentration methods include ion exchange, adsorption, chelation, chromatography, and related chemical and physical-chemical techniques to remove or separate impurities from the sample containing the target microbes. For example, particle size exclusion chromatography using Sephadex gel has been used to separate enteric viruses from solutes in the sample matrix and achieve a high degree of purification for subsequent detection by cell culture or nucleic acid amplification-nucleic acid hybridization methods (Sobsey et al., 1996). Extraction methods using organic solvents, detergents, lytic enzymes, and other chemicals have been used to partition target microbes from impurities in the sample (phase partitioning) or to solubilize sample impurities and facilitate their physical separation from the microbes in the sample. However, care must be used in applying these chemical treatments in order to avoid injury or damage of the target microbes that would interfere with their detection by cultivation or other methods.

Another physical separation and purification method, as well as a detection method, becoming more widely applied to the purification, separation, and concentration of pathogens in water is flow cytometry. Flow cytometry is a laser-based technology to measure cells or other particles made to flow single file through a sensing area. The cells are measured by both forward and 90° angle laser light scatter as well as by fluorescence (when labeled with a fluorochrome such as fluorescent antibody). These systems can also sort the detected cells by electronically charging them when detected and then deflecting them into a separate liquid stream. Recently, flow cytometry has been applied to the detection, separation, and purification of Cryptosporidium parvum oocysts concentrated from water (Vesey et al., 1997). Despite the advances in applying flow cytometry to the concentration, purification, and detection of C. parvum and potentially other pathogens in water, there remain technical and other barriers to

Suggested Citation:"9 Methods to Identify and Detect Microbial Contaminants in Drinking Water." National Research Council. 1999. Identifying Future Drinking Water Contaminants. Washington, DC: The National Academies Press. doi: 10.17226/9595.
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the application of this technology. The instruments are expensive and require a skilled, dedicated analyst; infectious and non-infectious organisms can not be reliably distinguished and sample cross-contamination is a high risk because field samples and positive control (calibration) samples pass through the same chambers and channels.

Assay Methods for Waterborne Pathogens

Introduction

Assay methods include all of the approaches involving either propagation or other analyses of microbes. These assay methods include (1) culture or infectivity, (2) viability or activity measurements, (3) immunoassays, (4) nucleic acid assays, and (5) microscopic and other optical or imaging methods. Often, several of these assays are combined or used concurrently in order to provide more definitive information on the quantity, identity, characteristics and state of the target organisms. Detection of microbial pathogens by culture or infectivity assays is preferred because it demonstrates that the target microbe is alive and capable of multiplication or replication. From a public health and risk assessment standpoint, microbial pathogen assays based infectivity are the most relevant and interpretable ones.

Culture or Infectivity Assays for Bacteria

Culture of bacterial pathogens is widely used in clinical diagnostic microbiology, and, for many waterborne bacterial pathogens, culture methods are adapted from those initially developed for medical diagnosis. Typical approaches are culturing the target microbes from specified volumes of water by preenrichment and enrichment methods using broth media or filtering the organisms from specified volumes of water and placing the filters in broth or agar culture media. Using membrane filters, the bacteria are often cultured directly by placing the filter on differential and selective media and incubating at appropriate temperatures to allow the development of discrete colonies of the target pathogens. Usually, the identity of the cultured bacteria must be confirmed by one or more of several methods. These methods include (1) subculturing on other differential and selective media; (2) biochemical, metabolic and other phenotypic analyses (for substrate utilization or conversion, enzyme activity, oxidation and reduction reactions, antibiotic resistance, motility, etc.); (3) immunological analyses (e.g., serological, immunofluorescent, enzyme-immune, or radio-immune assays); and (4) nucleic acid or genetic analysis. The nucleic acid methods include hybridization (gene probe), nucleic acid amplification (by PCR and other methods), restriction enzyme fragment length analyses (restriction fragment length polymorphism; RFLP), and nucleotide sequencing.

Suggested Citation:"9 Methods to Identify and Detect Microbial Contaminants in Drinking Water." National Research Council. 1999. Identifying Future Drinking Water Contaminants. Washington, DC: The National Academies Press. doi: 10.17226/9595.
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Detection of bacterial pathogens in water continues to be of interest because of newly recognized, newly appreciated, and evolving agents. Despite the ability to culture many bacterial pathogens for more than a century, culturing them from water continues to be technologically underdeveloped and has not advanced greatly beyond the application of methods used routinely in clinical diagnostic microbiology (Eaton et al., 1995). While conventional culture and antibiotic sensitivity methods are often suitable for medical diagnostic microbiology applications, these methods are not always suitable for application to the detection of bacteria in water. This is because of the need for sensitive, specific, and efficient detection and quantitation of low levels of bacterial pathogens in water and the ability to distinguish them from nonpathogenic strains of the same or similar genera and species. For some of the recognized enteric bacterial pathogens such as various species of the Salmonella, Shigella, Campylobacter, and Vibrio genera, culture methods for their detection in clinical, food, and water samples have changed little beyond attempts to improve recoveries and provide more distinctive recognition using modified preenrichment and enrichment broths and differential and selective agars.

For some other enteric bacterial pathogens, such as the recently appreciated enterohemorrhagic strains of Escherichia coli (O157:H7), for example, culturing from water and other samples continues to be a challenge because of the relative abundance of other nonpathogenic strains of E. coli. Culturine the target pathogenic strains from water then becomes an exercise in attempting to select for their growth based on distinctive biochemical or other properties that would facilitate their separation from the other nontarget strains. In the case of E. coli O157:H7, for example, it can be separated from other E. coli strains by its inability to ferment sorbitol. Therefore, its detection is facilitated by using a modification of the standard MacConkey agar as the differential and selective medium by including sorbitol in it. On sorbitol MacConkey agar, E. coli O157:H7 colonies appear atypical because sorbitol is not fermented. However, such colonies must be further confirmed by serological or other methods to confirm their identity as E. coli O157:H7.

Other waterborne pathogenic bacteria for which culture methods remain underdeveloped and inadequate are those for Yersinia enterocolitica, Aeromonas hydrophila and other Aeromonas species, Helicobacter pylori, Legionella species, and Mycobacterium avium-intracellulare. These bacteria are still difficult to reliably culture using currently available media and methods because their growth is inefficient (low plating efficiency), growth rates are slow, and they are often overgrown by other nontarget bacteria. Efforts to culture some of these bacteria include the use of antibiotics as well as physical (heat) and chemical (acid) treatments to reduce or eliminate nontarget bacteria. Even when these bacteria are cultured, they often must be separated or distinguished from other, nontarget bacteria that were also cultured from the sample. In some cases, it is impossible to distinguish nonpathogenic strains from pathogenic strains of the same bacterial genus and species unless advanced immunochemical, nucleic acid, or bioassay methods are used to detect specific antigens, genes, or activities found only in the pathogenic strains of the bacterium. For example, in recent efforts to detect potentially pathogenic Aeromonas species in water and food, isolates were tested for cytotoxins by cell culture and PCR assays, enterotoxins by PCR assays, and invasiveness in cell cultures (Granum et al., 1998). All

Suggested Citation:"9 Methods to Identify and Detect Microbial Contaminants in Drinking Water." National Research Council. 1999. Identifying Future Drinking Water Contaminants. Washington, DC: The National Academies Press. doi: 10.17226/9595.
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Aeromonas hydrophila strains, as well as two A. trota strains and the single A. veronii isolate, produced and secreted cytotoxins at 37°C; one A. schubertii strain and one A. caviae strain were invasive. The ability to detect specific virulence factors in water isolates of Aeromonas species helps elucidate their possible role in waterborne disease.

Detection of Stressed, Injured, and Viable-But-Nonculturable (VBNC) Bacteria

Studies over the past several decades demonstrate that many waterborne bacterial pathogens and indicators are physiologically altered such that they are not efficiently cultured using standard selective and differential media (Ray, 1989; Colwell and Grimes, 1998). This results in considerable underestimation of the hue concentrations of these bacteria in water and therefore underestimation of their risks to human health. It is contended that stressed, injured, and VBNC bacteria are still fully infectious for humans and other animal hosts, although there is disagreement on this matter. Some studies report human and animal experimental infection by VBNC or injured bacteria, and other studies report no animal infectivity by such cells. Despite disagreement about the public health significance of VBNC, injured, and stressed bacteria, a number of experimental procedures clearly demonstrate that the number of culturable cells in a population of VBNC, injured, or stressed bacteria can be increased using modified assay methods. These methods include the use of nonselective media (yeast extract, nonselective broth, or agar media), less selective media (containing fewer or reduced concentrations of inhibitory agents), and other, less stressful culture conditions (such as, lower incubation temperatures, optimum pH levels, optimum concentrations of salts and nutrients, etc.). Despite evidence that such injury repair and resuscitation methods improve the detection of viable and potentially cultural bacteria, these methods are rarely used to detect pathogens in drinking water. Estimates of the occurrence and risks from pathogenic bacteria in water would be improved if injury repair and resuscitation methods were more widely used for detection in water.

Detection of Viral Pathogens by Culture

As obligate intracellular parasites, many enteric viruses can be propagated or cultured in susceptible hosts of either whole animals or mammalian cells grown in culture (Payment and Trudel, 1994; Payment, 1997). Some enteric viruses can be grown in experimental animals, such as mice (certain enteroviruses, adenoviruses, reoviruses), pigs (rotaviruses and hepatitis E virus), or subhuman primates (hepatitis A and E viruses and rotaviruses). However, experimental animals are almost never used for enteric virus detection in water because of high costs, technical demands, and ethical considerations of animal rights. Some enteric viruses are readily cultured in susceptible

Suggested Citation:"9 Methods to Identify and Detect Microbial Contaminants in Drinking Water." National Research Council. 1999. Identifying Future Drinking Water Contaminants. Washington, DC: The National Academies Press. doi: 10.17226/9595.
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mammalian host cells, and cell culture techniques for their cultivation have been available for nearly 50 years. Some viruses, such as certain enteroviruses, reoviruses, adenoviruses, and astroviruses, will propagate in susceptible host cell cultures and produce morphologically distinct cytopathogenic effects (CPEs). Other viruses, including some enteroviruses, reoviruses, adenoviruses, rotaviruses, astroviruses, and hepatitis A virus, grow poorly or slowly in cell cultures and produce little or no CPE. Detection of these viruses requires the use of additional analytical techniques directed at detecting viral antigens (immunofluorescence, immunoenzyme, and radioimmune assays) or nucleic acids (nucleic acid hybridization and amplification assays). Other viruses, such as certain enteroviruses, caliciviruses, parvoviruses, coronaviruses, picobirnaviruses, and hepatitis E virus, cannot be propagated in any known cell cultures. Such viruses will not be detected in water unless sensitive and specific analytical methods, such as nucleic acid amplification by PCR or reverse transcription (RT)-PCR, are applied directly to concentrated samples.

Assays of viruses in cell cultures can be quantified using quantal or enumerative methods. In quantal methods different volumes (or dilutions) of sample are inoculated into replicate hosts (cell cultures or animals) and the numbers of infected (positive) and negative (uninfected) hosts for each volume are scored to calculate a most probable number (MPN), 50 percent tissue culture infectious dose (TCID50), or other expression of concentration. Enumerative methods are typically done by plaque assays in cell cultures where virus infection of inoculated cells is confined by the presence of solidified (agar-containing) medium so that the viruses can infect only adjacent cells. This results in the formation of localized areas or lesions of infection and host cell lysis called plaques, and each plaque is assumed to have originated from a single infectious virus. Virus concentrations are expressed as plaque-forming units, analogous to colony-forming units for bacteria on solid media.

Detection of Protozoan Parasites by Culture

The environmental forms of some protozoan parasites, such as spores, cysts and oocysts, can be cultured on susceptible host cells. Cysts of the free-living ameba, such as Naegleria spp. and Acanthamoeba spp., can be excysted and cultured on lawns of host bacteria, such as E. coli, on nonnutrient agar, on which they form local lesions that can be directly counted. Spores of some of the important human microsporidia, such as Encephalitozoon intestinalis and Enterocytozoon bieneusi, and oocysts of Cryptosporidium parvum can be cultured on mammalian host cells where spores germinate or oocysts excyst and active stages of the organisms can proliferate in the cells (Arrowood et al., 1994; Upton et al., 1994; Visvesvara et al., 1995a,b). The living stages can be detected (after immunofluorescent or other staining) and quantified by scoring positive and negative microscope fields or cell areas (slide wells) or by counting the numbers of discrete living stages of groups (loci) of them. Concentrations can be expressed as an MPN or in some other unit, such as numbers of live stages. Detection is also possible by PCR, immunoblotting, and electron microscopy. For other waterborne parasites, such as Giardia lamblia and Cyclospora

Suggested Citation:"9 Methods to Identify and Detect Microbial Contaminants in Drinking Water." National Research Council. 1999. Identifying Future Drinking Water Contaminants. Washington, DC: The National Academies Press. doi: 10.17226/9595.
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cayatenensis, culture from the environmental stage (the cyst or oocyst, respectively) recovered in a water sample is still not possible.

Combined Cell Culture and Nucleic Acid Detection and Amplification of Waterborne Pathogens

For some enteric viruses and protozoan parasites, detection and quantitation of the infectious pathogens are improved by using the combined techniques of cell culture and then nucleic acid hybridization and/or amplification. The sample is inoculated into susceptible host cell cultures, and the cultures are incubated to allow the viruses or parasites to infect the cells and proliferate. After an incubation period sufficient to produce enough nucleic acid for direct detection or further amplification, the nucleic acid is denatured and fixed either in situ or after extraction. Then the target nucleic acid is detected by hybridization with a gene probe either directly or after further amplification by PCR or RT-PCR. These methods facilitate the detection of infectious but noncytopathogenic viral and protozoan pathogens that are capable of proliferating in cell cultures. Combining cell culture and PCR or RT-PCR also reduces the incubation time to detect pathogen nucleic acid, because even small amounts of the target nucleic acid produced in culture can be rapidly and specifically amplified in vitro using these techniques. Combined cell culture and nucleic acid detection has been used to detect HAV and other fastidious enteric viruses in water (Shieh et al., 1991; Reynolds et al., 1996; Sobsey et al., 1996). Recently, a combination of cell culture and PCR was used successfully to detect Cryptosporidium parvum recovered from water using centrifugation and immunomagnetic separation methods (Di Giovanni et al., 1998).

Detection of Waterborne Pathogens by Viability or Activity Assays

Bacterial pathogens concentrated and purified from water can be assayed for viability or activity by combining microscopic examination with chemical treatments to detect activity or "viability." These chemical treatments include measurements of enzymatic activities, such as dehydrogenase, esterase, protease, lipase, and amylase. An example is tetrazolium dye reduction by bacteria, such as reduction of 2-( p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride, which measures dehydrogenase activity. Reduction of tetrazolium dye leads to precipitation of reduced products in the bacterial cells that are seen microscopically as dark crystals. Another assay of viable gram-negative bacteria is the cell elongation assay using nalidixic acid ("Kogure" method). Nalidixic acid inhibits RNA synthesis in live gram-negative cells, thereby causing them to elongate and be distinguished from dead cells.

Application of these methods to pathogens in water often involves combinations of methods, such as combining an activity measurement with an

Suggested Citation:"9 Methods to Identify and Detect Microbial Contaminants in Drinking Water." National Research Council. 1999. Identifying Future Drinking Water Contaminants. Washington, DC: The National Academies Press. doi: 10.17226/9595.
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immunochemical assay (for specific bacteria). An example of this approach combines fluorescent antibody (FA) with tetrazolium dye reduction and involves looking for reduced crystals in cells specifically stained with fluorescent antibodies. For example, Pyle et al. (1995) combined incubation using cyanoditolyl tetrazolium chloride (CTC) to detect respiratory activity with a modified FA technique, for the enumeration of viable E. coli O157:H7 in water in three to four hours. Bacteria were captured by filtration on nonfluorescent polycarbonate membranes, incubated on absorbent pads saturated with CTC medium, and reacted with a specific antibody conjugated with fluorescein isothiocyanate. The membrane filters were examined by epifluorescence microscopy with optical filters permitting concurrent visualization of fluorescent red-orange CTC-formazan crystals in respiring cells that were also stained fluorescent apple-green with specific FA.

"Viability" or activity assays for protozoan cysts and oocysts include reaction with DAPI (the fluorogenic stain 4',6-diamidino-2-phenylindole), which is taken up by live oocysts and propidium iodide (PI), which is taken up by dead oocysts. Viable Cryptosporidium oocysts are DAPI positive and PI negative, and nonviable oocysts are DAPI negative and PI positive. Other fluorogenic compounds that react specifically with targets in "viable" cells also have been used to detect bacteria cells and protozoan cysts and oocysts. Another activity or viability approach is based on detecting a nucleic acid target consistent with viability in the ribosomal RNA, messenger RNA, or genomic RNA of the pathogen. Detection of pathogen nucleic acid by fluorescent in situ hybridization has been applied to detecting bacteria, protozoan cysts, and oocysts, as well as viruses in infected cell cultures. Nucleic acid methods for pathogen detection, quantitation and characterization in water are described in the next section.

Detection of Waterborne Pathogens by Nucleic Acid Methods

Methods for nucleic acid cloning, synthesis, hybridization, sequencing, and other analyses now make it possible to detect pathogens in environmental samples. However, the application of direct nucleic acid hybridization using cDNA or RNA ("gene") probes to detect and quantify environmental pathogens is inadequate owing to (1) high detection limits (about 100 to 1000 generate targets), (2) large sample volumes that are impractical for most hybridization protocols without further pathogen concentration, (3) hybridization reaction failures (false negatives) and ambiguities (false positives) due to sample-related interferences and nonspecific reactions, and (4) uncertainties about whether positive reactions are truly indicative of infectious pathogens.

Some of the limitations of direct nucleic acid hybridization for pathogen detection in environmental samples are overcome by first culturing bacteria and by inoculating viruses and protozoans into cell cultures for replication prior to gene probing. This approach, which was introduced in a previous section of this paper, has several advantages. Allowing pathogens to multiply amplifies target viral nucleic acids prior to extraction and gene probing and thereby facilitates detection. Culturing also helps to accommodate environmental sample or sample concentrate volumes, which are much larger than the volumes accommodated by direct gene probing. In addition, inoculating samples for culture dilutes and

Suggested Citation:"9 Methods to Identify and Detect Microbial Contaminants in Drinking Water." National Research Council. 1999. Identifying Future Drinking Water Contaminants. Washington, DC: The National Academies Press. doi: 10.17226/9595.
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facilitates the removal of sample-related interfering components. Furthermore, because the pathogens have the opportunity to multiply, it becomes possible to relate gene probe detection to pathogen infectivity. Despite these advantages, this approach still relies on culturing and so it cannot be applied to noncultivable pathogens.

Nucleic Acid Amplification

With the recent development of PCR and other in vitro enzymatic amplification techniques for target gene sequences, the direct detection of low levels of human pathogens in environmental samples becomes more plausible, practical, and economically feasible than ever before (Persing et al., 1993; Dieffenbach and Dveksler, 1995; Persing, 1996; Sobsey et al., 1996). For example, methods to detect enteric viruses in water by nucleic acid amplification using PCR and RT-PCR have advanced in recent years to the point where they have been successfully applied to investigating waterborne outbreaks caused by nonculturable human caliciviruses viruses (Bellar et al., 1997) and surveying for enteric viruses in drinking water sources (Abbaszadegan et al., 1998). Despite these advances and successes, a variety of strategic issues and technical problems must be further addressed in order for PCR and related nucleic acid amplification and detection methods to become practical and reliable for the direct detection of emerging pathogens in environmental samples.

There are several essential steps in the development and application of PCR, other related enzymatic amplification techniques, and nucleic acid hybridization techniques such as oligonucleotide probing for successful detection of human pathogens in environmental samples. These key steps are (1) identification and selection of oligonucleotide primers and hybridization probes for target genomic sequences; (2) testing of selected primers and probes for sensitivity, specificity and selectivity; (3) further purification and concentration of the pathogens in environmental sample concentrates to enable efficient and reliable enzymatic amplification of low numbers of target genomic sequences; (4) testing of the methods for their applicability to natural pathogen strains and actual field samples; and (5) verifying that PCR amplification and oligonucleotide probing detect human pathogens that are potentially infectious and therefore pose a risk to human health.

Selection of oligonucleotide primers and probes for target pathogens requires that sequence data be available. Such data are now available for some waterborne pathogens, and the database is increasing for a variety of emerging waterborne pathogens. However, these data are not comprehensive for all pathogens of concern, and they are still limited for some of the epidemiologically most important pathogens, such as the human caliciviruses and Cryptosporidium parvum. Proper selection of PCR primers and oligonucleotide probes requires detailed knowledge of the genomic organization and function and the nucleotide sequences of the target pathogens. Of particular importance are the type and

Suggested Citation:"9 Methods to Identify and Detect Microbial Contaminants in Drinking Water." National Research Council. 1999. Identifying Future Drinking Water Contaminants. Washington, DC: The National Academies Press. doi: 10.17226/9595.
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function of nucleic acid target (genome, chromosomal gene, extrachromosomal gene, rRNA, mRNA, etc.), its length (size), and location, and the extent to which its sequence is related to that of other, nontarget but genetically related microbes. It is essential to select oligonucleotide primers and probes having the following characteristics: (1) appropriate length, (2) desired sequence composition for specificity and selectivity, and (3) appropriate melting and annealing temperatures and other physical characteristics to prevent the formation of undesirable secondary structures, primer dimers, other primer interactions, and other artifacts that would interfere with RT, PCR, or nucleic acid hybridization.

Because of the large number of different waterborne pathogens, efforts have been made to amplify as many as possible using a single primer pair for those belonging to a genetically related taxonomic group. For the human enteric viruses, pan-specific primers and oligonucleotide probes have been developed for the human enteroviruses, group A rotaviruses, human caliciviruses, and adenoviruses. However, additional studies are needed to verify that these primers and probes do not amplify or detect similar or identical nucleotide sequences in the genomes of nonhuman animal viruses belonging to the same taxonomic groups. If so, alternative genomic sequences having greater specificity for the human pathogenic strains of these taxonomic groups must be selected. If this is not possible, additional analytical methods, such as RFLP or nucleotide sequencing, must be applied to the amplicons in order to conclusively identify them as being from the strain or type target microbe that infects and poses a health risk to humans.

PCR and RT-PCR detection of specific pathogens in a broader taxonomic group is achievable by several different methods. One method involves the use of additional primers internal to the group-specific primers that would amplify subsequently from the initially amplified cDNA ("nested amplification"). Another approach is hybridization using highly specific oligoprobes that would hybridize only with amplicons from a single pathogen type or strain. Selected oligonucleotide primers and probes must be tested for specificity and selectivity. It must be verified that primers have the ability to amplify a DNA of correct molecular weight and that the amplicon will hybridize with a specific nucleic acid probe (e.g., oligoprobe). Furthermore, it must be verified that the probes are nonreactive with the nucleic acid of nontarget microbes.

The sensitivities or lower detection limits of PCR or other nucleic acid amplification methods for target pathogens can be tested by determining the greatest dilution of a known quantified pathogen suspension that can be successfully amplified. The same sample is also quantified by infectivity, microscopic enumeration of the numbers of microbes or other methods so that the endpoint PCR titer can be compared to these other titers. Other approaches to quantifying nucleic acid amplification by PCR and RT-PCR have been described by Freeman et al. (1999). The quantification methods should include determining if there are materials in the sample interfering with PCR amplification and should estimate the concentration of target microbes in the sample. This can be done by measuring or quantifying the amount of amplicon (DNA product) produced under defined PCR or RT-PCR conditions using electrochemoluminescence, immunoassays, fluorescence signal increase (using a fluorescent primer that

Suggested Citation:"9 Methods to Identify and Detect Microbial Contaminants in Drinking Water." National Research Council. 1999. Identifying Future Drinking Water Contaminants. Washington, DC: The National Academies Press. doi: 10.17226/9595.
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incorporates into the amplicon), fluorescent signal decrease (by loss of fluorescent signal upon incorporation of the fluorescent primer into the amplicon), or other chemical methods. Also, the amount of target DNA amplified in the sample can be compared to the amount of target DNA amplified under the same conditions from a positive control sample containing a known amount of nucleic acid from the same target microbe suspended in a noninhibitory solution. Using this approach, however, it is not possible to determine if lack of or low amplification of the target is due to inhibition by sample constituents, to few target nucleic acids being present, or a combination of both.

Another approach to quantifying PCR or RT-PCR and determining the effects of sample-related inhibitors is to add a known amount of an internal nucleic acid standard as a positive control into the sample. Adding specific amounts of positive control nucleic acid sequences in the reaction mixture and determining the extent of amplification of this target (which differs from the true target) makes it possible to quantify both sample inhibition and the amount of actual target nucleic acid in the sample. This approach has been applied to the detection of human caliciviruses by RT-PCR (Schwab et al., 1997, 1998).

Sample Preparation for Pathogen Detection in Water by Nucleic Acid Methods

Typical environmental sample concentrates for pathogens are too large in volume (10 to 50 ml) and too contaminated with extraneous interfering constituents for reliable and sensitive enzymatic amplification of target pathogen genome sequences, especially at the low levels (low target nucleic acid numbers) typically found in most water samples. Therefore, in some previous studies target pathogen nucleic acid has been isolated from environmental samples by standard techniques of nucleic acid extraction, purification, and concentration, such as proteinase K digestion, phenol-chloroform extraction, and ethanol precipitation. However, these methods are cumbersome, laborious, and often inefficient, thus leading to poor recoveries and inadequate detection limits. Furthermore, disruption of pathogens to liberate target nucleic acid at an early stage of sample concentration and purification makes it impossible to compare pathogen detection by nucleic acid amplification to pathogen detection by other methods, such as infectivity or particle count.

Preferred sample cleanup and concentration strategies maintain pathogen integrity as long as possible prior to enzymatic amplification. The goal is to reduce the sample volume to <100 mL, remove interfering and inhibitory substances (e.g., particulates, salts, proteins, lipids, carbohydrates, and natural organic matter) and also place the pathogens in an aqueous medium compatible with enzymatic amplification as well as the other quantitation procedures, such as infectivity or particle count. For PCR or RT-PCR, the target nucleic acid is then liberated from the purified and concentrated pathogens by one or more of several physical methods, such as heat, freeze-thaw, sonication and bead beating, and/or

Suggested Citation:"9 Methods to Identify and Detect Microbial Contaminants in Drinking Water." National Research Council. 1999. Identifying Future Drinking Water Contaminants. Washington, DC: The National Academies Press. doi: 10.17226/9595.
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by other, chemical extraction methods as a final or near-final treatment step. This makes it possible to compare PCR endpoint titers with infectivity assay or particle count titers without intervening or additional purification and concentration steps that could cause differential losses.

Candidate chemical purification and concentration methods for subsequent nucleic acid amplification and some other pathogen detection methods include precipitation (e.g., polyethylene glycol for viruses); chromatography (e.g., Sephadex gels in spin-columns); chelation of metals (using EDTA, EGTA, "Chelex," etc.); ultrafiltration using high (e.g., 100,000 daltons) molecular weight cutoff filters; organic solvent (e.g., chloroform, fluorocarbon) extraction; detergent (e.g., Tweens and other nonionic detergents) treatment; enzymatic digestion (e.g., proteases, amylases, lipases, and nucleases); and antibody (immunoaffinity) capture on paramagnetic beads (immunomagnetic separation). Immunoaffinity capture and purification of pathogens as antigens is a cleanup and concentration option that has been successfully applied to some viruses, bacteria, and parasites. However, it is not applicable to some pathogens because of the lack of reagent quality antisera or the antigenic diversity of a large pathogen group lacking a common antigen (and hence requiring many antisera).

Overall, nucleic acid amplification by PCR or RT-PCR and hybridization using oligonucleotide probes is a specific, selective, and sensitive approach to the detection of pathogens in environmental samples. Oligonucleotide primers and probes can be selected to detect broad pathogen groups, such as the enteroviruses and Salmonella or specific pathogens, such as hepatitis A virus. In particular these methods can be used to detect fastidious pathogens, such as human caliciviruses and Cyclospora cayatenensis. Under optimized conditions method sensitivity or detection limit is less than one infectious unit and in principle as little as one target gene sequence. Methods have been developed and evaluated to concentrate and purify target pathogens from environmental samples such as water for successful detection by nucleic acid amplification and oligoprobe hybridization. These methods have been successfully applied to the detection of viral, bacterial, and protozoan pathogens in field samples of water. However, the inability of most of these nucleic acid methods to conclusively detect only the infectious pathogens is a limitation that remains to be overcome.

Microscopic and Analytical Imaging Methods to Detect Waterborne Pathogens

Microscopic methods include ordinary (light) microscopy (bright-field and dark-field), phase contrast, differential contrast, fluorescence, laser scanning, video, and other forms of microscopic and image analysis (Lawrence et al., 1997). These methods are not effective for viruses or other very small (ultra-small) agents, and some are not effective for very transparent agents unless the microbes are stained. The detection of internal and external structural features (e.g., organelles and reproductive units) is improved by various colored stains, fluorescent stains, advanced optical systems (phase contrast, Nomarski interference contrast, differential interference, and laser scanning), and the use of imaging by sensitive charge-coupled device cameras. For example, phase contrast and differential interference contrast microscopy are used to visualize

Suggested Citation:"9 Methods to Identify and Detect Microbial Contaminants in Drinking Water." National Research Council. 1999. Identifying Future Drinking Water Contaminants. Washington, DC: The National Academies Press. doi: 10.17226/9595.
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the internal structures of Giardia lamblia and Cryptosporidium parvum isolated from water. This helps distinguish them from algae and other particles of similar size and shape. Fluorescent (ultraviolet light) microscopy methods are useful for transparent cells or other cells or organelles that are difficult to detect. The cells are reacted with a fluorochrome that interacts with a cellular components or macromolecules, which are then detected by ultraviolet light microscopy. Capturing microscopic images by digital methods for image analysis using computers has greatly advanced the capabilities to detect, quantify, and characterize pathogens and other microbes in environmental samples. For example, computer-assisted laser scanning and video microscopy have been applied to the analysis of Cryptosporidium parvum oocysts in environmental samples (Anguish and Ghiorse, 1997). Another technology employing advanced image analyses to detect, quantify and characterize pathogens in water is laser-based flow cytometry plus fluorescent cell sorting, which are methods that have been previously described above.

Immunoassays to Detect Pathogens in Water

Immunofluorescent detection by microscopy or other methods is a specific and potentially powerful way to detect pathogens and other microbes in water if there are enough target pathogens in the sample for detection (McDermott, 1997). Antibodies directed against antigens of the target pathogen can be labeled (conjugated) with a fluorochrome or fluorescent dye (e.g., fluorescein isothiocyanate or another fluorochrome) for direct immunofluorescence. These fluorescent antibodies are reacted with the target microbe, and then the microbe preparation is washed to remove meted fluorescent antibody. The sample is then examined for the target microbe by ultraviolet light microscopy or another analytical method to detect the immunofluorescent signal (e.g., fluorometry). Alternatively, secondary fluorochrome-labeled antibodies directed against the primary antibodies (now serving as antigens) of the species of animal in which the antibodies against the microbe were raised can be used in an indirect immunofluorescence assay. The target microbial antigen is reacted initially with a specific antibody, and then the resulting antigen-antibody complex is reacted with fluorochrome-labeled antispecies antibody to provide the signal for immunofluorescent detection.

Enzyme immunoassays employ enzyme-conjugated antibodies directed against target pathogens. The antigen-antibody complex is detected and quantified by the ability of the enzyme to react with a substrate that typically produces either a colored product for colorimetry or emits light for luminometry. Enzyme immunoassays often are done on a solid phase to which the pathogen antigens have been applied, such as a membrane filter or the bottom of a microtiter plate well. To provide increased specificity and to facilitate separation of the target microbial antigen from other particles and solutes in the sample, the target antigen can be captured on the solid phase using a specific antibody

Suggested Citation:"9 Methods to Identify and Detect Microbial Contaminants in Drinking Water." National Research Council. 1999. Identifying Future Drinking Water Contaminants. Washington, DC: The National Academies Press. doi: 10.17226/9595.
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("sandwich" or immunocapture assay format). As with other immunoassays, enzyme immunoassays can be direct (enzyme-conjugated, primary antibody against antigens of the target microbe) or indirect (enzyme-conjugated, antispecies secondary antibody). Studies have repeatedly shown that solid-phase enzyme immunoassays generally are too insensitive for direct detection of microbial pathogens in water, as they require a minimum of 10,000 to 100,000 target microbes (or their antigens) for detection. In most situations drinking water and its sources rarely contain high enough levels of most target pathogens for direct immnoenzymatic detection. However, enzyme immunoassays also have been combined with methods to propagate target pathogens by various culture methods, thereby increasing their numbers for immnoenzymatic detection. For example, cell culture infectivity has been used to propagate noncytopathogenic viral pathogens and thereby enhance their detection (Payment, 1997).

Agglutination methods are used to detect pathogens by combining dispersed cells, viruses, or other forms of pathogen antigens with antibodies (on a slide, for example) and allowing for antigen-antibody reactions to produce agglutination (clumping) that can be scored as negative or various degrees of positive (strong, medium, or weak). One modification is latex bead agglutination in which antibodies against a specific microbial antigen (especially nonparticulate or "soluble" antigen) are attached to latex beads. The beads are reacted with the sample. If the sample contains the specific antigen, agglutination occurs by the reaction of antigens with antibodies on the beads resulting in the beads clumping together (agglutinating). As with enzyme immunoassays, agglutination tests are too insensitive to directly detect and quantify most waterborne pathogens in drinking water and other aquatic samples. The target microbes must first be propagated in order to obtain a sufficient number of them or a sufficient amount of antigen to detect and antigenically characterize them by agglutination methods.

Signature Biolipid and Other Biochemical Detection Methods

Some pathogens are detectable became they contain distinctive macromolecules or biochemicals that aid in their identification and detection. These include cell wall component assays for lipopolysaccharides, muramic acids, and assays for signature biolipids (White, 1995). Signature lipid biomarker analysis is based on the use of techniques such as liquid extraction and thin layer chromatography to separate and purify the microbial lipids from the microbes in the environmental sample. This is followed by quantitative analysis using gas chromatography/mass spectrometry, infrared spectroscopy, and nuclear magnetic resonance spectroscopy. Using these techniques it has been shown that Cryptosporidium parvum contains a characteristic phosphatidyl-ethanolamine, that may make it possible to detect this parasite in environmental samples (Schrum et al., 1997).

Suggested Citation:"9 Methods to Identify and Detect Microbial Contaminants in Drinking Water." National Research Council. 1999. Identifying Future Drinking Water Contaminants. Washington, DC: The National Academies Press. doi: 10.17226/9595.
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Summary and Conclusions

The identification and detection of microbial contaminants in drinking water must continue to be a high priority for assessing the risks from and managing the microbial quality of drinking water supplies. The essential information needs for quantitative microbial risk assessment can be applied to detecting, identifying, and characterizing microbial contaminants in drinking water supplies. The recognition of new waterborne pathogens as well as re-emerging ones posing increased risks due to newly acquired virulence properties and other traits requires further improvement of waterborne pathogen detection methods and better use of the advanced analytical methods now available. There must be continued efforts to improve and apply the methods for waterborne pathogen recovery, concentration, purification, separation from interfering materials, detection, isolation, assay, and characterization.

Methods for waterborne pathogen recovery and concentration by centrifugation, filtration and precipitation can be further improved and newly developed methods can be more widely applied. Purification and separation methods, such as immunomagnetic capture, chromatography (by ion exchange, adsorption, chelation, and size exclusion) and chemical treatment (with enzymes, detergents, and organic solvents) are advancing and becoming more widely used for waterborne pathogens.

Methods to assay, quantify, and characterize waterborne pathogens are advancing, but greater efforts are needed to determine pathogen infectivity and viability. Culture methods for viruses, bacteria, and parasites are preferred because they detect and quantify infectivity, which is the most relevant unit of measure in terms of health risk. For bacterial pathogens in water, better methods to detect infectivity are needed and existing methods to detect infectious bacteria must be more widely used. The detection of stressed, injured, and viable but nonculturable bacteria in water continues to be a technological challenge, and available methods to detect such bacterial pathogens in water continue to be underutilized. Detection and quantitation of waterborne vital pathogens by cell culture infectivity is possible for some of them, but many of the important waterborne enteric viruses are still not culturable. The combined use of cell culture and nucleic acid amplification (PCR, RT-PCR, etc.) and hybridization has improved the detection of some of the more fastidious waterborne viral pathogens, but these methods must become more widely used. Recently, it has become possible to detect and quantify the infectivity of some of the important waterborne parasites (Cryptosporidium parvum and some microsporidia) by cell culture methods. However, these methods require further refinement and verification and they have yet to be extensively applied in occurrence studies or exposure assessments.

Waterborne pathogen detection and quantitation by nucleic acid amplification and hybridization methods has advanced greatly in the last decade, has been successfully applied to pathogen detection in field samples, and appears to be a most promising technology for the future. However, further

Suggested Citation:"9 Methods to Identify and Detect Microbial Contaminants in Drinking Water." National Research Council. 1999. Identifying Future Drinking Water Contaminants. Washington, DC: The National Academies Press. doi: 10.17226/9595.
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improvements in these nucleic acid amplification and hybridization methods are needed to reliably distinguish infectious from non-infectious waterborne pathogens. The combination of these nucleic acid methods with culture methods appears to be the most promising approach for sensitive and specific detection and quantitation of infectious waterborne pathogens. The methods to purify and concentrate the target microbes and their nucleic acids from water by various physical and chemical means have advanced greatly in recent years. However, these methods need further improvement, consolidation, and simplification in order to achieve reliable, sensitive, and specific detection and quantitation of the target pathogens recovered and concentrated from water.

Viability and activity methods to detect and quantify waterborne pathogens also continue to improve, and they have been applied to field samples of water on a limited basis. However, there continues to be uncertainty about the ability of many of these methods to detect truly infectious waterborne pathogens, and many of the existing methods are cumbersome, tedious, and unable to detect low concentrations of target pathogens in water. Similarly, microscopy, analytical imaging, immunoassays, and biochemical analyses continue to advance as methods to detect, identify, and characterize pathogens in water. However, these methods lack sensitivity for direct detection and may not distinguish infectious from non-infectious organisms. The usefulness of these methods is improved when they are applied to pathogens that have been concentrated and purified from water or have been enriched in numbers by culture methods. Advances continue to be made in immunological, biochemical, nucleic acid, and bioassay methods to characterize potentially waterborne pathogens. The bases for and mechanisms of virulence and pathogenicity of these pathogens are becoming better understood at the biochemical, immunological, and genetic level. Such advances provide the needed tools for improved detection and identification of these microbes in water, understanding the role of the aquatic environment in their persistence, evolution, and selection, and better characterization of the human health risks from these waterborne pathogens.

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With an increasing population, use of new and diverse chemicals that can enter the water supply, and emergence of new microbial pathogens, the U.S. federal government is faced with a regulatory dilemma: Where should it focus its attention and limited resources to ensure safe drinking water supplies for the future?

Identifying Future Drinking Water Contaminants is based on a 1998 workshop on emerging drinking water contaminants. It includes a dozen papers that were presented on new and emerging microbiological and chemical drinking water contaminants, associated analytical and water treatment methods for their detection and removal, and existing and proposed environmental databases to assist in their proactive identification and regulation.

The papers are preceded by a conceptual approach and related recommendations to EPA for the periodic creation of future Drinking Water Contaminant Candidate Lists (CCLs--produced every five years--include currently unregulated chemical and microbiological substances that are known or anticipated to occur in public water systems and that may pose health risks).

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