Classifying Drinking Water Contaminants for Regulatory Consideration (2001) / Chapter Skim
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6Virulence-Factor Activity Relationships INTRODUCTION The term "virulence-factor activity relationship," or VFAR (formerly referred to as virulence-activity relationship or VAR; NRC, 1999a) , is rooted in a recognition of the utility of using structure-activity relationships (SARs)
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Virulence can be defined as the quality of being poisonous or injurious to life (i.e., virulent)
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• Data of this kind can also be used to identify sources of, and thus exposures to, microorganisms through molecular "fingerprinting." • The functional genomics or bioinformatics expertise needed to establish a nationwide VFAR program already exists in the private and public sectors. Thus, the committee concludes that a VFAR concept, with many parallels to the SAR concept used for chemicals, would be a powerful approach to examining emerging waterborne pathogens, opportunistic microorganisms, and other newly identified microorganisms.
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dilemmas to be addressed is that current regulatory practice requires that methods to culture organisms of interest be developed before occurrence data can be gathered. Thus, a microorganism ordinarily must first be identified as a pathogen, and be capable of in vitro culture, before occurrence data are acquired.
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agents in environmental and health sciences. For example, chemical properties are amenable to prediction through use of structure-activity relationships, which can be distinguished from propertyactivity relationships (PARs)
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as being the relationship that ties specific descriptors to outcomes of concern (see Figure 6–1)
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measured or otherwise determined. When correlation of the response and descriptor variables yields a consistent relationship, the result can be used as a (quantitative)
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Descriptor Variables Descriptor variables, in this context, are those attributes of a microorganism that may prove useful in predicting their virulence. For example, the presence of toxins, adherence factors, adhesins, invasins, capsular components, fimbria, hemolysins, metabolic pathways, and antibiotic resistance could prove to be effective descriptors of microbial virulence.
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infects ferrets. Subsequent studies indicate the existence of an eighth genotype (Sulaiman et al., 2000)
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regard, the committee anticipates that the same rationale that exists for using genomics also exists for proteomics. For example, in a subsequent section of this chapter the committee discusses the use of DNA chips that act as sensors for finding the characteristic DNA elements that encode a particular virulence factor.
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TABLE 6–1 Examples of Microbial Genome Databases for Waterborne Pathogens Microorganism Size (Million Base Pairs) Campylobacter jejuni 1.7 Encephalitozoon cuniculi 2.9 Enterococcus faecalis 3.0 Escherichia coli 4.6 Giardia lamblia 12 Helicobacter pylori 1.66 Klebsiella pneumoniae Legionella pneumophila 4.0 Leptospira interrogans serovar icterohaemorrhagiae 4.8 Mycobacterium avium 4.7 Pseudomonas aeruginosa 5.9 Salmonella paratyphi A 4.5–4.8 Salmonella typhi Salmonella typhimurium Shigella flexneri 4.7 Vibrio cholerae 4.0 SOURCE: TIGR (see http://www.tigr.org)
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the Subcommittee on Biotechnology was to summarize the activities in microbial genetics of a number of federal agencies; identify each federal agency's areas of interests; and identify opportunities for, and limitations to, research in microbial genetics.1 One of the main findings of this report is that the current effort in microbial genomics in each federal agency is based on the mission of the specific agency, and, thus, there are clear research gaps -- or opportunities for cooperation -- in the area of microbial genomics. Notably, despite EPA's clear mandate for the surveillance of water supplies and developing a rational and transparent scheme for regulating pathogenic water contaminants to date, it has not yet participated in these interagency genome project efforts.
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factors -- in different organisms. As discussed later in this chapter, many wellrecognized waterborne pathogens share the same or very similar genes that encode virulence factors.
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bases (adenine, thymidine, cytosine, and guanine represented as A, T, C, and G, respectively)
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FIGURE 6–2 Illustration of two complementary strands of DNA forming A-T and G-C base pairs. Protozoa Several recent papers have described a variety of PCR-based protocols for the detection of waterborne pathogens, including Cryptosporidium (Champliaud et al., 1998; DiGiovanni et al., 1999; Kimbell et al., 1999; Kostrzynnska et al., 1999; Sulaiman et al., 1999; Xiao et al., 1999a,b)
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that there are extensive genetic differences among different Cryptosporidium species, as well as within C parvum (Sulaiman et al., 2000)
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of viability is β-tubulin mRNA (Widmer et al., 1999)
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and watch for damage to the cells (a "cytopathic effect")
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However, it is now apparent that even bacteria of very major public health concern are not always culturable. This important issue is also discussed at length later in this chapter under virulence factor "persistence." One of the best paradigms in this regard is the agent of cholera, Vibrio cholerae.
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clever aspect of this work by Kingombe et al.
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labeled (e.g., with fluorescent molecules) and detected using a variety of means such as spectroscopy.
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Viral Examples Influenza -- Mutation is Associated with Virulence Influenza can be considered as a prototypic emerging disease. Although it is not known to be waterborne, it is discussed here to support the contention that specific virulence attributes can be mapped to an important and well-understood pathogen that is widely recognized by the public.
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Influenza thus represents a virus whose surface molecules correlate with virulence, but researchers do not yet know why this is the case. Nonetheless, genetic recognition of the molecules associated with virulence can serve as an important public health tool.
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Thus, it is probable that modern molecular surveillance of U.S. drinking water could detect HEV of animal and potentially human origin.
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nervous system (and not just the intestine) is called neurotropism.
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been developed at the same time that similar PCR techniques have been evaluated and found to be cost-effective for the rapid diagnosis of enterovirus infections in humans (Nigrovic and Chiang, 2000)
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virulence -- the M and T antigens -- and actual adverse human outcomes. Rheumatic heart disease was once one of the leading causes of death in the United States, and it remains a major cause of morbidity and death in many developing countries (Stollerman, 1998)
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originally described from the bacterium Shigella dysenteriae type 1, an essentially identical set of toxins is made by Escherichia coli O157:H7, which is why this and other E coli strains that make the toxin are of such public health concern.
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host cell as they move into an adjacent cell and accomplish this by causing host proteins to jell behind them and force them forward like a jet-propelled object. These genes are shared between bacteria that are as unrelated as Shigella (Goosney et al., 1998)
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subsequent human exposure and health. The factors responsible for pathogen decay are reviewed below.
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al., 1987; Davies et al., 1995; Laliberte and Grimes, 1982; Matson et al., 1978; Palmer, 1988; Smith et al., 1978)
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well-characterized but may result from protection against biotic antagonists or from transport of inhibitory substances to the solid-associated microbe. Some microorganisms are able to enter dormant stages (e.g., bacterial spores)
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in detecting nonculturable bacteria rather than on persistence responses.) In some cases, however, the pathogenicity of the putative viable nonculturable organisms may be low, as reported by Caro et al.
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Determination of Viability For studies that measure decay rates in microorganisms, the particular assays used for assessment of viability become important. In most cases, but particularly for data obtained before the mid-1990s, the assessment of viability was based on culturing methods such as agar plate growth of bacteria and mostprobable-number assays for bacteria or viruses (in the form of the TCID50 determination [i.e., tissue culture infectious dose, or dose required to infect 50 percent of the tissue culture in which a sample is inoculated]
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Data for More Recent Pathogens There have been studies of additional microorganisms since the efforts summarized below in Table 6–3. Although a comprehensive review of such studies is beyond the objective of this report, a brief synopsis of findings for some emerging pathogens is appropriate.
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Robertson et al.
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• ability to survive and/or multiply within aquatic protozoa or other microbial hosts; • ability to survive freezing; • ability to survive desiccation; • ability to survive wastewater treatment and to reenter drinking water; and • ability to survive in anaerobic sediment. INTERPRETATION OF ISSUES Data Information and Management Issues Although the technology, methodology, and even the genetic databanks exist, the application of a VFAR approach to assess waterborne pathogens would require considerable effort and expenditure of resources by EPA in conjunction with the Centers for Disease Control and Prevention, National Institutes of Health, and other federal and state health organizations (NRC, 1999b)
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possibility of screening hundreds of water samples for thousands of key microbial hazards could be achieved through development of custom chip arrays. The use of appropriate data and sophisticated data management would be crucial to the development and validation of VFARs.
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affects the ability of the VFAR concept to be developed, used, or validated. Since these elements either are present or can reasonably be expected to be available in the near future, the committee strongly concludes that the development and use of VFARs is indeed feasible.
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regards as confirming its judgment of the adequacy, power, and affordability of the methodology. The committee thus warrants that the necessary condition of technological feasibility also exists.
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FIGURE 6–3 Linkage between microbial virulence factors and human disease. to look for on a routine basis (e.g., rotavirus)
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the specific needs of EPA are likely to be recognized by others as being of crucial importance to shared problems. For example, the "identification" problem that exists for EPA -- identifying potentially pathogenic microbial water contaminants that are difficult or impossible to culture -- has already been recognized by other government agencies as a high-priority area for research (BRWG, 2000)
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related to the formation of such a working group within EPA, including how it should be administered and supported (e.g., logistically and financially) or where it could be located.
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