5
New Biological Measurement Opportunities

INTRODUCTION

Recent and forecasted advances in microbiology, molecular biology, and analytical chemistry make it timely to reassess the long-standing paradigm of relying primarily or exclusively on traditional microbial (predominantly bacterial) indicators for waterborne pathogens in order to make public health decisions regarding the microbiological quality of water. This chapter provides an overview and discusses various issues and methods for making biological measurements. It underscores some of the key issues in making measurements both generically and specifically for pathogens and indicators of waterborne pathogens. The methods are evaluated critically in terms of their attributes, including potential applicability for measuring indicators and pathogens, as well as their limitations. The issues of standardization and validation of methods are then discussed, followed by a look toward the future that describes how new and emerging technologies and science will facilitate waterborne pathogen and indicator measurements. The chapter closes with a summary of its conclusions and recommendations.

Spatial and Temporal Granularity

As discussed in Chapter 4 and illustrated in Figure 5-1, the spatial and temporal scales (i.e., the “granularity”) at which indicators and indicator organisms are employed may differ widely among applications. Small spatial and short temporal scales (area A) are of particular interest in beach monitoring programs and,



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Indicators for Waterborne Pathogens 5 New Biological Measurement Opportunities INTRODUCTION Recent and forecasted advances in microbiology, molecular biology, and analytical chemistry make it timely to reassess the long-standing paradigm of relying primarily or exclusively on traditional microbial (predominantly bacterial) indicators for waterborne pathogens in order to make public health decisions regarding the microbiological quality of water. This chapter provides an overview and discusses various issues and methods for making biological measurements. It underscores some of the key issues in making measurements both generically and specifically for pathogens and indicators of waterborne pathogens. The methods are evaluated critically in terms of their attributes, including potential applicability for measuring indicators and pathogens, as well as their limitations. The issues of standardization and validation of methods are then discussed, followed by a look toward the future that describes how new and emerging technologies and science will facilitate waterborne pathogen and indicator measurements. The chapter closes with a summary of its conclusions and recommendations. Spatial and Temporal Granularity As discussed in Chapter 4 and illustrated in Figure 5-1, the spatial and temporal scales (i.e., the “granularity”) at which indicators and indicator organisms are employed may differ widely among applications. Small spatial and short temporal scales (area A) are of particular interest in beach monitoring programs and,

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Indicators for Waterborne Pathogens FIGURE 5-1 Spatial and temporal scales of indicators for various applications. potentially, to transient contamination of groundwater. Larger spatial scales and longer temporal scales (area B) are of importance in understanding overall sources of microbial loadings to a watershed (that may serve as a water supply) or in studying the contamination of an aquifer or well. Small spatial scales but long temporal scales (area C) may be useful in understanding “typical” conditions at a water supply intake on a river system for the purposes of developing treatment configurations to meet drinking water standards for finished water. Large spatial scales (area D) but short temporal scales may be useful in understanding the occurrence of contamination over a large recreational area under outbreak conditions or from a storm event. The temporal and spatial requirements for each particular application largely dictate the types of indicators or indicator approaches employed and the methods for measuring these indicators. As discussed throughout this report, particularly in Chapter 6, what is needed is a phased monitoring approach that makes use of a flexible “tool box” in which a variety of indicator methods and approaches are available for measuring a given indicator or pathogen for differing applications and circumstances. In many indicator applications, the level of perceived public health threat will determine the method or methods employed, as well as the spatiotemporal granularity. The indicator method, frequency, and spatial coverage of sampling will have to be “adaptive” in the sense that more frequent samples

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Indicators for Waterborne Pathogens taken over larger areas with more sensitive methods will be required when the threat level is high (e.g., following high rainfall events) than when the threat is low. In some cases, the number and type of indicators measured may also differ with changing environmental conditions. Classical Methods and Their Limitations Most of the indicator applications described in previous chapters rely on biological measurements of bacteria. The classical laboratory techniques presently used for those measurements are primarily culture based, involving quantification of a metabolic or growth response after a suitable incubation period in an appropriate substrate. As reviewed in Chapter 1, culture based methods have been used for more than 100 years in water and related areas of environmental microbiology and have been considered adequate to provide quantification of indicator organism (predominantly bacteria) concentrations. Culture methods may be limited by their incubation period since most require 24 hours or longer, during which time the public is potentially exposed to a health risk (see Chapter 4 for further information). The current choices of detection methods for indicator bacterial species or groups were motivated by the associated technical difficulties in culturing many types of waterborne pathogens. However, it is now possible to detect the growth of some specific pathogenic as well as indicator bacteria and also some viruses and parasites in as little as a few hours. For example, in clinical diagnostic and food microbiology bacteriology, automated bacteria culture detection and identification can be achieved in four to six hours (Fung, 2002; Lammerding et al., 2001; Murray et al., 1999); however, these and other advanced methods for rapid culture detection have not been well developed for or adapted to the rapid detection of indicator or pathogenic bacteria in water and other environmental samples. One reason why rapid culture-based detection works well in clinical diagnostic microbiology is that clinical specimens often contain high concentrations of the bacteria of interest, thereby allowing them to be cultured to even higher concentrations in only a few generations. In contrast, water and other environmental samples often contain very few bacteria of interest and therefore, many generations of bacterial growth are needed before these bacteria are readily detected by culture methods. Besides bacteria, coliphages—which are bacterial viruses infecting Escherichia coli (E. coli) that have been shown to be useful microbial indicators of fecal contamination and predictors of human health effects from recreational water exposures (see also Chapters 3 and 4)—can be cultured and detected in as little as six to eight hours by some methods (Lee et al., 1997; Sobsey et al., 1990). As discussed in Chapter 3, many types of pathogenic and indicator bacteria present in the environment are in various states of physiological health and fitness, depending on their origin, properties, and how long they have been in the

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Indicators for Waterborne Pathogens environment. The state of the microbes is influenced by the extent to which they have been exposed to various environmental stresses such as extreme temperatures and pH levels, hypo- or hypertonic salts, aerobic or anaerobic conditions, UV radiation, heavy metals, and various other antimicrobial chemicals, including chemical disinfectants such as chlorine (Hurst, 1977; McFeters and Camper, 1983; McFeters et al., 1986a,b). Therefore, enteric bacteria and many other bacteria in aquatic environments that are stressed, injured, and physiologically altered, may or may not be detected by various culture methods (Edwards, 2000). Typical culture methods for pathogen and indicator bacteria in water and other environmental samples greatly underestimate the true concentrations of viable and potentially infectious cells—sometimes by as much as a thousandfold (Colwell and Grimes, 2000; Ray, 1989). For example, the anaerobic enteric bacteria that are so plentiful in the human and animal gastrointestinal tract, such as Bifidobacteria and Bacteroides (see also Chapter 4), are very difficult to culture from water and other environmental media because they are highly sensitive to very low concentrations of oxygen. While these bacteria would appear to be attractive candidate indicators of fecal contamination, the inability to culture them efficiently from water and other environmental media has been a major impediment to their potential use as fecal indicator microbes. However, the advent of nucleic acid based molecular methods to detect these bacteria now makes it more plausible and practical to consider them as fecal indicators (Barnhard and Field, 2000). The underestimation of bacteria concentrations also results in part because the differential and selective media used to culture many types of waterborne pathogens and indicators contain inhibitory agents intended to suppress the growth of nontarget bacteria. Such agents also suppress the growth of injured or stressed target bacteria. In addition, other culture conditions, such as elevated incubation temperatures, may contribute to the lack of growth of target bacteria. Because bacteria injury is induced by the chemical disinfection and other treatment processes applied to water and wastewater, McFeters and colleagues (1986a,b) greatly improved the detection of injured coliform bacteria in water (by more than 10-fold) by the use of a medium that contained fewer inhibitory ingredients. According to some authorities, such bacteria can become viable but nonculturable (VBNC), as discussed in Chapter 3 and below. Whether the VBNC pathogenic and indicator bacteria in water are infectious for human and other hosts and, in the case of the pathogens, pose health risks, remains uncertain and is quite controversial (Bogosian and Bourneuf, 2001; Bogosian et al., 1998; Kell et al., 1998). Some studies have reported that bacteria in the VBNC state have the ability to infect humans or animals (Colwell et al., 1996; Jones et al., 1991). Other investigators have not been able to infect animal hosts with so-called VBNC bacteria or have reported evidence that a few culturable bacteria within a large population of non-culturable bacteria could be responsible for the observed infections (Hald et al., 1991; Medema et al., 1992;

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Indicators for Waterborne Pathogens Smith et al., 2002). Because of the lack of scientific agreement of the public health significance of VBNC bacteria and the objections of some authorities even to the use of this terminology, this report does not attempt to address the VBNC issue in the context of microbial indicators of pathogens and human health risks from waterborne pathogens. However, the report does address issues related to the detection of stressed, injured, and otherwise physiologically compromised bacteria in water, the roles and appropriateness of both culture and non-culture methods to detect and quantify bacteria and other waterborne microbes, and the quantitative relationships between bacteria concentrations in water and the human health effects from exposure to water by ingestion and other routes. The advent of increasingly sophisticated and powerful molecular biology techniques provide new opportunities and alternative approaches to improve upon present indicators and pathogens by both culture and non-culture methods. Molecular methods do not require incubation to culture bacteria because they can directly quantify existing cellular or subcellular structural properties. Therefore, these methods have the potential to be more rapid than culture methods, providing results in as little as minutes to a few hours rather than the typical overnight incubation time for culture methods. Some of these nucleic acid-based methods employ amplification schemes in which a small amount of indicator genetic material is replicated up to a billionfold for easy detection. They also have the potential to be less expensive, making direct measurement of pathogens more economically feasible. Much of the rest of this chapter is devoted to describing the types of molecular methods that are presently under development and have the potential to replace, supplement, or greatly improve the quality of information of classical (largely bacterial) culture-based methods in the future. It is important to mention that Appendix C (Detection Technologies) supplements the discussion (both generally and specifically) of these and other methods by describing them in more detail. Furthermore, molecular methods can be coupled with or linked to microbial culture methods in ways that can increase sensitivity, decrease detection time, and provide conclusive and rapid confirmation of identity and infectivity (e.g., Reynolds et al., 1996). Targets and Opportunities Several different analytes can be measured in microorganisms. For purposes of this discussion, microbes can be divided broadly into cells and viruses. Cells can be detected by the following categories of analytes, as summarized in Figure 5-2. Nucleic Acids Deoxyribonucleic acids and ribonucleic acids have unique sequences of nucleotide bases (adenine, thymidine [uracil in RNA], cytosine, and guanine) that

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Indicators for Waterborne Pathogens FIGURE 5-2 Targets to measure on or in a cell. Note: ATP = adenosine 5′-triphosphate. enable the unequivocal identification of a particular organism. DNA and/or RNA is present in all cells and viruses. Cells contain both DNA and RNA, whereas viruses contain either DNA or RNA but never both. The choice of nucleic acids and the ways in which they are measured in microorganisms can provide different kinds of information with regard to microbial identification, viability, and infectivity or culturability. For example, some nucleic acid targets and the methods for their detection can provide very broad identification of a family or genus of microorganism, while other targets can provide very specific identification of species, strain, or subtype. Some nucleic acid targets can be taken as measures of viability or infectivity, such as messenger RNA (mRNA) of cellular microbes or mRNA production by viruses in infected cells. In some cases, mRNA targets are evidence of culturability or infectivity. In general, RNA correlates with viability because nucleases present in most biological samples destroy RNA rapidly. Therefore, both the presence and quality of RNA and the specific sequences present can provide a reasonable indication of viability (see more below). In developing methods to detect and quantify waterborne microorganisms and microbial indicators of pathogens, it is important to consider both the targets for detection and methods of detection with consideration of the value and interpretation of resulting data.

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Indicators for Waterborne Pathogens Surface Proteins Proteins present on the surface of a microbe and to a lesser extent those located within a microbe are often unique and offer a means to definitively identify a microorganism of interest. The most common method of analyzing such proteins is the use of immunoassays in which specific antibodies are raised against the proteins and used as binding reagents. Both monoclonal and polyclonal antibodies can be used. Polyclonal antibodies tend to be more broadly reactive, which makes them useful in detecting microbes as broad groups, such as genera. Monoclonal antibodies have greater specificity because they recognize and bind to a very specific epitope or functional group on or in the target microorganisms. The uniqueness of the epitope depends on its function within the microbe. Some epitopes are common to all members of a microorganism family, genus, or species (group or “common” antigens); others can be highly specific, appearing only in an individual strain, subtype, or variant. Other approaches to microbe identification based on proteins can employ non-antibody ligands, such as aptamers or phage display libraries, that will specifically recognize and bind to a particular protein or an epitope on it (Breaker, 2002). Such ligand binding probes to identify microorganisms, including bacterial spores (e.g., Bacillus anthracis; Zhen et al., 2002), are becoming more accessible because of the advances made in protein identification and mapping within microbes and the advances made in the synthesis of in vitro proteins, oligonucleotides, or oligopeptides. Certain proteins on the surface or in the interior of microbes can be detected by ligand binding assays. The presence of these markers on or in the cell can be evidence of microbe viability or infectivity. Certain proteins in cells and viruses may be present in a native state only when the microbe is intact and infectious. Therefore, the ability to specifically detect that molecule by a ligand-binding assay can be taken as a measure of viability or infectivity. Carbohydrates (Polysaccharides) Carbohydrates or polysaccharides present on the surface of a microbe or within a microbe also can offer a unique way to definitively identify a microorganism of interest. Many of these specific carbohydrates are oligosaccharides covalently bound to proteins to create glycoproteins. Such molecules on the surfaces of cells and viruses often have high specificity or uniqueness in identifying a microorganism. Immunoassays can be used to detect, identify, and quantify such polysaccharides or glycoproteins, again using specific polyclonal or monoclonal antibodies raised against the microbe or the specific target molecules. Like proteins, the specificity of polysaccharide epitopes depends on their function within the microbe, with some antigens common to all members of a microorganism family, genus, or species and others being highly specific for individual strains, subtypes, or variants. Non-antibody ligands also can be used to detect,

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Indicators for Waterborne Pathogens identify, and quantify specific polysaccharide epitopes. As with microbial proteins, ligand-binding probes to microbial polysaccharides are becoming more accessible because of the advances made in functional polysaccharide identification and mapping and the advances made in ligand-binding chemistry. As is the case for certain nucleic acids and proteins, the detection of certain polysaccharides on or in microbes by ligand binding can be evidence of a microorganism’s viability or infectivity (Feng and Woo, 2001). Certain polysaccharides in cells and viruses are active receptors for attachment and infection and are present in the native state only when the microbe is intact and infectious. Therefore, detecting such molecules by a ligand-binding assay is a measure of viability or infectivity. Other Small Molecules Some microorganisms contain or release characteristic metabolites or products, such as sugars, polysaccharides, antibiotics, alkaloids, lipids, and (protein-based) enzymes and toxins into their environment or growth medium. These compounds may be products of either primary or secondary metabolism and can provide a distinct signature for the microorganism of interest. Many methods are available for analyzing such compounds including mass spectrometry, colorimetric assays, enzymatic assays, and various chromatographic methods. For example, adenosine 5′-triphosphate (ATP) is often measured as an indicator of viable and possibly infectious cells, because it is degraded rapidly when the cell dies (e.g., bioluminescence assays; Deininger and Lee, 2001). Special Considerations for Viruses Viruses are typically detected either by their DNA or RNA (for RNA viruses) and their surface proteins (either the capsid or the envelope; see Figure 5-3). Although many viruses do not contain small molecules or detectable amounts of internal protein, most animal viruses do. When present, these internal proteins can also be targets for detection, although often they are less accessible than surface proteins. Because viruses are inert outside their host cells, determining the infectivity of a virus often depends on culturing it in host cells. When they do infect host cells, viruses begin to produce new, virus-specific molecules that can be targeted for detection by molecular and other chemical methods as evidence of their presence, infectivity, and concentration. Virus-specific nucleic acids, such as mRNA and proteins, including both structural and nonstructural proteins, can be targeted for detection by chemical, immunochemical, and molecular methods. In addition, all viruses have specific functional groups or epitopes on their surfaces that are used for attachment to host cells. If the cell receptor or its functional ligand constituent can be identified, such a molecule can be used to detect and quantify viruses through a ligand-binding assay.

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Indicators for Waterborne Pathogens FIGURE 5-3 Targets to measure on or in a virus. Special Consideration for Protozoa Like bacteria, waterborne protozoa are single-celled organisms and consist of many of the same components. Unique to the enteric protozoa, however, is the formation of an (oo)cyst as part of its environmental and infectious stage (see Chapter 3 for further information). In most cases, this structure is currently detected by microscopy through the aid of stains and antibodies against the (oo)cyst cell wall. Enteric protozoa are obligate parasites and are similar to viruses in that they need a host organism to reproduce. Thus, determination of the potential viability of protozoa has been studied using vital dye inclusion-exclusion as a measure of the integrity of an (oo)cyst’s outer wall as well as its inner cytoplasmic and nuclear membranes. Huffman et al. (2000) showed, however, that vital dyes grossly overpredicted infectivity of Cryptosporidium under some circumstances. Cell culture methods are now being used and have been found to be statistically comparable to animal infectivity for the determination of infectious oocysts (Slifko et al., 2002). Methods continue to evolve, and as with other microorganisms, polymerase chain reaction (PCR) techniques to target the nucleic acid components as well as methods that combine cell culture and PCR are now being used for detecting protozoa in water (Quintero-Betancourt et al., 2002; see also Appendix C). For example, the free-living amoeba (e.g., Naegleria) can be isolated from water using a culture technique (i.e., their growth in the trophozoite

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Indicators for Waterborne Pathogens stage is responsive to a bacterial culture). In addition, PCR, probes, and culture methods are now being combined to identify those species and subtypes that are particularly lethal to humans (Kilvington and Beeching, 1995). ISSUES IN SAMPLING AND ANALYSIS The process of making a measurement consists of the four steps shown in Figure 5-4. A common misunderstanding is that measurement is the only critical step in the analysis process. FIGURE 5-4 Four steps involved in performing a measurement. However, as discussed below, all four components of the process must be considered to ensure accurate analysis of microbial water quality. Collection and Sampling Issues The first step in performing a measurement is collecting a sufficiently representative sample, and this remains one of the most challenging problems in water quality monitoring. By representative, it is meant that the sample will reliably portray the presence and concentrations of the analyte of interest (e.g., a microorganism or a chemical) in the water being evaluated or analyzed for its quality. Furthermore, it is important that the sample also be representative of human exposures that may lead to pathogen ingestion and any resulting infection and illness. As noted previously, it is important to recognize that the presence and concentrations of microorganisms and chemicals in water and other environmental media can be highly variable over time (at different times) and space (at different locations within the same body of water). Therefore, as described in Chapter 4, obtaining representative samples often requires taking multiple samples over an extended period (e.g., daily, weekly, monthly), sometimes from different locations within a body of water during the same time period. The importance of addressing variability in microorganism concentrations in water as related to human exposures to pathogens has been well documented in recreational water epidemiologic studies (Fleisher et al., 1993; Kay et al., 1994). The temporal variabil-

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Indicators for Waterborne Pathogens ity of microbial occurrence in groundwater has also been documented (EPA, 2000). Collecting representative samples requires careful consideration of the objectives or purpose of sampling in the context of the need to obtain a reliable estimate of microbial exposure in a timely fashion. Unfortunately, sample collection often involves simply “grabbing” a volume of water and placing it in a storage vessel. For many samples, it is important to preserve the sample, by refrigeration or chemical preservatives, to avoid degradation. All or a fraction of the sample is then taken to the analysis site for further processing. Typically, a sufficient sample volume is taken either to determine whether a microorganism or other analyte is present (i.e., presence-absence) or to estimate the concentration of microbes or other analytes in the water being analyzed (e.g., number of microbes per unit volume). For microorganisms of public health concern in water, both types of analysis (presence-absence and concentrations estimates) are now used for estimating exposures and making decisions regarding the acceptability of the water for beneficial use under the Clean Water Act (CWA; see also Chapter 1), such as drinking water supply. In some cases, the goal of the analysis is to document that samples of a certain volume (e.g., 100 mL) do not contain a particular microorganism the vast majority of the time (e.g., absence of total coliforms in 95 percent of successive 100-mL drinking water samples) or ever (e.g., absence of Escherichia coli in successive 100-mL volumes of drinking water all of the time). In other cases, the goal of the analysis is to document that samples of a certain volume contain a particular microorganism at concentrations below a threshold level considered indicative of an unacceptable health risk (e.g., maximum allowable concentrations of fecal indicator bacteria in recreational bathing waters). In water analysis based on either presence-absence or estimates of concentration, the variability of microbial concentrations is typically addressed by taking repeated samples from the body of water over time and determining both central tendency (e.g., mean or median) and dispersion (e.g., minimum-maximum values, interquartile range, 95 percent confidence limits). The focus of data analysis and interpretation is often on typical exposures that are portrayed by central tendencies and dampened extremes, such as 95 percent confidence limits, that are based on logarithmically transformed data. Recent evidence from food microbiology and foodborne disease outbreaks indicates that measures of central tendency and the use of logarithmic transformations of microbial concentration data for the purposes of calculating geometric means and corresponding logarithmic measures of dispersions may be inappropriate for extrapolating to higher exposures and estimating corresponding health risks (Paoli, 2002). Such transformations tend to suppress the effects of extreme values, including the high values on the upper end of a frequency distribution that represent the greatest levels of exposure and health risk. Characterizing the extremes of exposure is necessary because illnesses can result from combinations of rare

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Indicators for Waterborne Pathogens ment may not contain the species to be measured simply because they are not adequately representative of the bulk sample or because the detection method is not sensitive enough. This sampling problem is of particular concern for pathogens that can pose unacceptable health risks at very low concentrations. Thus, there is a need to address the sensitivity of miniaturized detection methods and ensure that sample collection, preprocessing or processing, concentration, and purification are given adequate attention. In other words, microorganisms of concern must either be removed from large-volume samples and presented to these miniaturized methods in small aliquots, or larger sample volumes must be passed by the sensor. Currently, this area represents one of the most important technological challenges to the analysis of pathogens and indicators in water and other environmental samples. Separation of target microorganisms from water during sample preparation before application to modern and sophisticated detection systems is an important area for further research. Specifically, elimination, reduction, and destruction of inhibitors, debris, food particles, lipids, proteins, organic and inorganic particles, cellular matter, and so forth, in samples are all important issues to be resolved. Cost and Technology Transfer Many modern diagnostic and detection systems utilize sophisticated instrumentation that may be excessively costly for most potential users. In fact, the average cost of an automated instrument can easily reach or surpass $30,000 and perhaps even $250,000 for a mass spectrometer (e.g., Fourier transform infrared spectrometer). Of course, if one performs a large number of tests regularly, the average cost per test will be low, but in many instances, smaller laboratories may find that the volume of tests does not justify the cost of the instrument. For example, a laboratory that routinely conducts less than 100 Salmonella tests per week has little or no need for a sophisticated, automated, and very expensive instrument that can perform thousands of tests per week. The committee recognizes the lack of technical, infrastructure, and financial resources required to implement water monitoring in many parts of the United States and recommends that efforts be made to support the development of inexpensive and rapid fieldable methods for testing microbial water quality. Finally, while many detection technologies exist that are applicable to the detection of waterborne pathogens and indicator organisms, they are primarily laboratory based. The need to develop rapid fieldable methods will require the concurrent development of reagents, methods, and the attendant portable instruments that can survive repeated transport and use in the less stringently controlled environments of the field.

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Indicators for Waterborne Pathogens Unrealistic Expectations and Resistance to Change New users of an automated measurement device often expect the system to operate perfectly and perform all their necessary tests immediately. Usually, such expectations are too high. Once it is discovered that a particular system does not satisfy all immediate measurement needs, some users will either discard the system totally or develop a negative impression of the system. Users have to understand that an instrument is designed and marketed after extensive testing for specific applications and that even a slight deviation from the specified protocol (e.g., putting acidic water into a sensor not designed to handle a low-pH sample) may result in unsatisfactory performance. No system is 100 percent perfect all the time. At present, there is no sensor that can be placed into a water sample and left alone to make an autonomous measurement without some level of attention. There is also an intrinsic resistance to change that pervades virtually every analytical community where certain well-established methods have been employed successfully for long periods. This innate conservatism is well founded in some cases where new methods have not been validated. A specific application can sometimes lead to errors or compromise existing long-term data sets. In such cases, it is important that new methods be tested side by side with well-established methods so that the user can acquire a degree of comfort with the new method. The ideal situation is to design “foolproof” systems so that no human error can interfere with the operation of the system from the point of sample application to the end results. Although the microbiological community is moving ever closer to that reality, it is not yet achievable. LOOK TO THE FUTURE Today’s measurement techniques are aimed at detecting viable organisms or specific components present in the organism of interest and correlating their presence to human health risk assessment. Sensors and biosensors are beginning to play a role in several application areas including clinical medicine, environmental science, and process control fields. Analyzers are designed to integrate the steps of sampling, preprocessing, and measurement into a single, functional device. In some cases, sampling is determined by sensor placement; no preprocessing is required due to the sensor’s specificity; signal transduction-detection is an integral function; and sensors offer the potential for real-time monitoring capability because they measure continuously. Further advances in sensors, including making them sufficiently robust for field deployment, will enable them to address some of the measurements discussed throughout this chapter. Another area that will have an important impact on microbial analysis is molecular recognition. The use of combinatorial methods such as phage display (Sidhu, 2000), aptamers (O’Sullivan, 2002), and combinatorial chemistry has

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Indicators for Waterborne Pathogens expanded the ability to rapidly and efficiently generate and screen new molecular entities that may be helpful in producing new recognition elements that can be used as labels, purification reagents, and sensors. A technology area that will enable significant reductions in sample preparation and separation times is in the field of microfluidics and microelectromechanical systems (MEMS). Complete “lab-on-a-chip” devices are being created out of inexpensive materials such as glass or plastic. These chips contain fully integrated analytical systems with the ability to concentrate, separate, and detect a multitude of analytes including nucleic acids, proteins, and small molecules. Because the overall device sizes are small compared to most benchtop analytical systems, they can perform analysis in second-to-minute time frames. Further advances in chip design and detection schemes should facilitate more complex and sensitive analysis. One of the most exciting fields of current research in science and technology is the area of nanotechnology. In this area, defined as systems with features on the nanometer scale, functional devices and materials are being developed at an increasing rate. While the material costs associated with the technology may be high, the number of devices one can prepare from a small amount of material is enormous. For example, a gram of nanoparticles contains trillions of individual particles, each of which can, in principle, serve a particular function. With the advent of nanotechnology, and even microtechnology, materials costs will therefore actually decrease. The ability to pack functions, such as communications hardware, on-board processing, and signal transduction, into ever-smaller devices suggests that in the not-too-distant future it should be possible to create sensors with a high degree of measurement capability in an extremely small device. One of the more recent trends in sensing systems is array technology (see also Appendix C). In these systems, tens to thousands of sensor elements can be placed on a single substrate with overall dimensions of several square millimeters. The burgeoning area of DNA microarrays for genomics is driving advances in this area. Developments in protein and carbohydrate arrays will further advance the applicability of arrays to microbial analysis. Nanotechnology will cause feature sizes to shrink even further. The ability to place so many sensors on a single device raises the prospect of what has been referred to as a “universal sensor”—a system able to detect virtually anything of interest. Such systems can be built on chips in which a sensor is present for every analyte of potential interest. Alternatively, such arrays may be able to measure patterns of response in which signatures of various analytes signify the presence of various water quality conditions, organisms, or toxins. In this approach, pattern recognition algorithms, combined with prior training, could be used to assess water quality and identify potential hazards. One of the advantages of such a system is its ability to be anticipatory, such that new or difficult-to-culture pathogens could be detected by presenting a signature that is similar to known pathogens.

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Indicators for Waterborne Pathogens As described previously and elaborated further in the Appendix C, mass spectral techniques for performing microbial analysis using entire mass spectral patterns is in its infancy and should also have an impact, with the limitation that it is unlikely to be an inexpensive, portable field analytical tool. With the prospect for such an enormous amount of data to be collected from the many sensors disposed on arrays, the large numbers of sensor arrays deployed for water monitoring, and the continuous data streams coming from these sensor networks, attention must be paid to data analysis, intelligent decision making, and archiving. Ongoing research in the micro- and nanotechnology field, combined with efforts in array sensing and intelligent processing, should provide the tools for creating inexpensive, ubiquitous, universal sensing and detection systems beginning now and continuing over the next several decades. While many of the new and innovative molecular methods discussed in this chapter (and Appendix C) enhance the opportunity for direct measurement of pathogens, more effective use of direct pathogen measurement will require establishment of the correlation between pathogen concentration and health risk. There are presently no standards on which to base health risk decisions for most pathogens. Current epidemiologic studies (as reviewed elsewhere in this report), on which recreational water exposure standards are based, have been conducted almost exclusively for indicator bacteria such as fecal coliforms and enterococci. Even for presently used indicator bacteria, the relationship to health risk will have to be reestablished for the new molecular-based methods. Existing epidemiology studies have all been based on quantifying exposure using culture-based methods, which measure some aspect of metabolic activity. Some of the new indicator and pathogen methods quantify the presence of cellular structure, but many do not assess the ability to grow or to infect. As such, they have the potential to overestimate health risk relative to present standards. Consistent with its previous related recommendations, the committee recommends that epidemiologic studies should be designed and performed to both establish the correlation among indicator and pathogen concentrations and health risk, and reestablish the health risks associated with existing and new pathogen indicators for new (non-culture-based) detection methods. SUMMARY: CONCLUSIONS AND RECOMMENDATIONS Recent, emerging, and forecasted advances in microbiology, molecular biology, and analytical chemistry make it timely to reassess the long-standing paradigm of relying primarily or exclusively on traditional microbial (primarily bacterial) indicators for waterborne pathogens to support public health decision making regarding the microbiological quality of water. Although classic microbiological culture methods for detection of indicator microorganisms and pathogens have proved effective over many decades, they suffer from a number of

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Indicators for Waterborne Pathogens limitations that are discussed throughout this report. The advent of increasingly sophisticated and powerful molecular biology techniques provides new opportunities to improve upon present indicators and pathogens by both culture and non-culture methods. What is needed is a phased monitoring approach that makes use of a flexible tool box, in which a variety of indicator methods and approaches are available for measuring a given indicator or pathogen for different applications that considers spatial and temporal scale (granularity) issues. The need for such a phased monitoring approach and examples of its implementation are discussed in detail in Chapter 6. It is vital that all four components of the process of performing a measurement (i.e., collection, sample processing or preprocessing, measurement, and data processing; not just the measurement itself) be considered in order to make an accurate analysis of microbial water quality. The collection of representative samples requires careful consideration of the objectives or purpose of sampling in the context of the need to obtain a reliable estimate of microbial exposure in a timely fashion. Furthermore, the widespread use of logarithmic transformations and measure of central tendency and dispersion of log-transformed data to estimate exposures and health risks needs to be reconsidered in water microbiology, epidemiology, and health risk assessment. At present, most water quality measurement methods are single-parameter based. Ongoing research in the micro- and nanotechnology field, combined with efforts in array sensing and intelligent processing, should provide the tools for creating inexpensive, ubiquitous universal sensing and detection systems beginning now and over the next several decades. This development is essential because the committee recognizes the lack of technical, infrastructure, and financial resources required to implement advanced water quality monitoring methods in many parts of the United States. The microbiological community needs to develop and implement multiparameter approaches in which many technologies and methods are integrated to provide the best possible information. Similarly, the water monitoring community needs to be aware of new developments in these areas that can be brought to bear on microbiological water quality monitoring. Although evolving detection methods will be increasingly able to rapidly detect specific pathogens, the use of well-characterized (conventional) indicator approaches will continue to be necessary because our understanding of existing and emerging pathogens will never be complete. Regardless, more effective use of direct pathogen measurement discussed in this chapter will require establishment of the relationship between pathogen concentration and health risk (see also Chapters 2 and 4). Similarly, the relationship to health risk will have to be reestablished for presently used indicator bacteria and new (non-culture-based) methods. The funding of methods development has been relatively poor to date for many pathogens, for new and emerging methods, and for new and innovative indicators. Investigations into only a few pathogens, specifically those targeted

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Indicators for Waterborne Pathogens for regulations in drinking water such as Cryptosporidium and Giardia, have been substantially funded (largely by EPA and AWWARF) for the development of new and improved methods. Greater and more consistent efforts should be made to support methods development for new and emerging microbial detection technologies, for many more pathogens, and for new and improved candidate indicators of waterborne pathogens. Newer methods involving immunofluorescence techniques and nucleic acid analysis are proving their value, and novel microtechnologies are evolving rapidly, spurred in part by recent concerns about bioterrorism. Problems associated with sample concentration, purification, and efficient (quantitative) recovery remain and will require significant effort to be resolved. One technology area that will enable significant reductions in sample preparation and separations time is the field of microfluidics and MEMS. Thus, the introduction of molecular techniques for nucleic acid analysis is viewed by the committee as a growth opportunity for waterborne pathogen detection. With the prospect for such an enormous amount of data to be collected from the many sensors disposed on arrays, the potentially large numbers of sensor arrays deployed for water monitoring, and the continuous data streams coming from these sensor networks, greater attention must be paid to the fields of data analysis, intelligent decision making, and archiving. There is need for a database that compiles and serves as a clearinghouse for all microbiological methods that have been utilized and published for studying water quality. Research methods, in particular those that have great potential for evolving into conventional methods, will have to be documented. Recent developments in molecular and microbiology methods and their application to public health-related water microbiology have necessitated a new approach for rapid assessment, standardization, and validation of such methods. It is clear that a major effort is needed for accessible methods to examine microbial water quality for health decisions. To move new methods into the main-stream, a process is required that not only allows for standardization and validation but also facilitates widespread acceptance and implementation. In this regard, the committee concludes that the AOAC Peer-Verified approach or its equivalent may be the best way forward. However, a major program on methods development will need to be established with water research laboratories in academic institutions in collaboration with industry research and government research laboratories. Based on these conclusions, the committee makes the following recommendations: A specific program on promising research methodologies for waterborne microorganisms of public health concern should be supported by EPA and other organizations concerned with microbial water quality. Such methodologies need

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Indicators for Waterborne Pathogens not be microorganism specific, but should be application specific, focusing on the desirable attributes of the method. Ongoing research should be supported and expanded to develop and validate rapid, sensitive, and robust methods for detection and measurement of all classes of waterborne pathogens and their indicators. Such expanded research should go beyond pathogenic bacteria and indicators, to include improved methods for the detection of pathogenic viruses and protozoa. Additional research is needed to develop improved methods for rapid sample concentration and effective, reproducible microbial recovery. Specifically, elimination, reduction, and destruction of inhibitors, debris, food particles, lipids, proteins, organic and inorganic particles, cellular matter, and so forth, in samples are important issues. Research should be funded to develop approaches to the detection of infectious or viable microbes by nucleic acid detection methods, including the use of ligand-binding steps in microbial recovery from samples to select for intact and infectious microbes. The adoption of new molecular techniques should be accelerated for waterborne pathogen detection. New methods undergoing validation should be tested using whole microorganisms, rather than just extracted DNA or RNA targets, to perform tests for sensitivity and linearity. Focused efforts should be made to support the development of inexpensive and rapid fieldable methods for testing microbial water quality. This will require the concurrent development of reagents, methods, and the attendant portable instruments that can survive repeated transport and use in the field. There is a need to address the sensitivity of miniaturized detection methods and ensure that sample collection, preprocessing or processing, concentration, and purification are given adequate attention to achieve representativeness and have the ability to detect microbial concentrations posing unacceptable health risks. This represents one of the most important technological challenges to the analysis of pathogens and indicators in water and other environmental samples and will become more important with the introduction of micro- and nanotechnologies. EPA should reinvigorate its role with standard-setting organizations (including ASTM, AOAC International, and ISO) to facilitate microbial methods development that focuses particularly on new and innovative methods. In addition, regular and ongoing involvement of professional organizations such as the American Society for Microbiology will bring credible, independent, third-party input. EPA should support the design, development, and maintenance of a nationwide database that compiles and serves as a clearinghouse for all microbiological methods that have been utilized and published for studying water quality. Guidance on the appropriate data needed for methods studies should be included

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Indicators for Waterborne Pathogens in this database. Finally, a means for iterative development of consensus methods on-line should be provided. The committee recommends that epidemiologic studies should be designed and performed to both establish the correlation among indicator and pathogen concentrations and health risk, and reestablish the health risks associated with existing and new pathogen indicators for new (non-culture-based) detection methods. REFERENCES APHA (American Public Health Association). 1998. Standard Methods for the Examination of Water and Wastewater, 20th Edition. Washington, D.C. Armitage, P., and C.C. Spicer. 1956. The detection of variation in host susceptibility in dilution counting experiments. Journal of Hygiene 54: 401-414. Barnhard, A.E., and K.G. Field. 2000. Identification of nonpoint sources of fecal pollution in coastal waters by using host-specific 16S ribosomal DNA genetic markers from fecal anaerobes. Applied and Environmental Microbiology 66(4): 1587-1594. Blackmer, F., K.A. Reynolds, C.P. Gerba, and I.L. Pepper. 2000. Use of integrated cell culture-PCR to evaluate the effectiveness of poliovirus inactivation by chlorine. Applied and Environmental Microbiology 66(5): 2267-2268. Bogosian, G., and E.V. Bourneuf. 2001. A matter of bacteria life and death. EMBO Reports 2(9): 770-774. Bogosian, G., P.J. Morris, and J.P. O’Neil. 1998. A mixed culture recovery method indicates that enteric bacteria do not enter the viable but nonculturable state. Applied and Environmental Microbiology 64(5): 1736-1742. Breaker, R.R. 2002. Engineered allosteric ribozymes as biosensor components. Current Opinion in Biotechnology 13(1): 31-39. Call, J.L., M. Arrowood, L.T. Xie, K. Hancock, and V.C.W. Tsang. 2001. Immunoassay for viable Cryptosporidium parvum oocysts in turbid environmental water samples. Journal of Parasitology 87: 203-210. Chandler, D.P., J. Brown, D.R. Call, J.W. Grate, D.A. Holman, L. Olson, M.S. Stottlmyre, and C.J. Bruckner-Lea. 2001. Continuous, automated immunomagnetic separation and microarray detection of E. coli O157:H7 from poultry carcass rinse. International Journal of Food Microbiology 70: 143-154. Clark, J.A., and A.H. el-Shaarawi. 1993. Evaluation of commercial presence-absence test kits for detection of total coliforms, Escherichia coli, and other indicator bacteria. Applied and Environmental Microbiology 59: 380-388. Collier, J. L., and L. Campbell. 1999. Flow cytometry in molecular aquatic ecology. Hydrobiologia 401: 33-53. Colwell, R.R., and D.J. Grimes, eds. 2000. Nonculturable Microorganisms in the Environment. Washington, D.C.: American Society for Microbiology Press. Colwell, R.R., P. Brayton, A. Huq, B. Tall, P. Harrington, and M. Levine. 1996. Viable but nonculturable Vibrio cholerae O1 revert to a culturable state in the human intestine. World Journal of Microbiology and Biotechnology 12: 28-31. Cook, N. 2003. The use of NASBA for the detection of microbial pathogens in food and environmental samples. Journal of Microbiological Methods 53(2): 165-174. Deininger, R.A., and J. Lee. 2001. Rapid determination of bacteria in drinking water using an ATP assay. Field Analytical Chemistry and Technology 5(4): 185-189.

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