Grantee Research Project Results
2000 Progress Report: Meaningful Detection of Known and Emerging Pathogens in Drinking Water
EPA Grant Number: R826828Title: Meaningful Detection of Known and Emerging Pathogens in Drinking Water
Investigators: Cangelosi, Gerard A.
Institution: University of Washington , Seattle Biomedical Research Institute
EPA Project Officer: Hahn, Intaek
Project Period: September 1, 1998 through August 31, 2001
Project Period Covered by this Report: September 1, 1999 through August 31, 2000
Project Amount: $360,609
RFA: Drinking Water (1998) RFA Text | Recipients Lists
Research Category: Drinking Water , Water
Objective:
Microbial contaminants of drinking water often are difficult to detect by laboratory cultivation, and polymerase chain reaction (PCR) detection of their genetic material is of uncertain significance because of the detection of dead cells or their remnants. We hypothesized that we could differentially detect viable (living) bacterial cells by using PCR assays targeted to non-standard molecules, namely pre-rRNA or BrdU-DNA. As intermediates in ribosomal RNA (rRNA) synthesis, pre-rRNA molecules are abundant in growing bacterial cells, but rare in non-growing or non-viable cells. BrdU is a "label" that is incorporated into DNA during DNA replication in viable cells but not in nonviable cells. Pre-rRNA and BrdU-DNA can both be detected in species-specific fashion. The feasibility of these approaches has been demonstrated in bacterial model systems including Escherichia coli, Klebsiella pneumoniae, and Mycobacterium tuberculosis. We proposed to develop similar assays for the suspected water-borne pathogens Mycobacterium avium and Helicobacter pylori. The assays would then be used to study the physiology of M. avium and H. pylori in drinking water, and the resistance of M. avium to chlorine.Progress Summary:
We characterized the maintenance of pre-rRNA pools in M. avium. The results showed that pre-rRNA will not be a useful analyte for detection of viable M. avium cells.We characterized the uptake and incorporation of BrdU into the DNA of M. avium and H. pylori. In contrast to closely-related organisms such as M. tuberculosis, M. avium did not take up and incorporate BrdU from the medium. This rules out BrdU-DNA as a useful method for detecting viable M. avium cells. However, H. pylori exhibited excellent uptake and incorporation of BrdU. Therefore, H. pylori will be amenable to detection by this method.
Our most significant findings relate to chlorine resistance by M. avium. This property makes it difficult to remove M. avium from drinking water systems. Using traditional methods (plating and colony counting), we identified a specific chlorine-resistant form of the pathogen. This form is termed red transparent (RT). Significantly, the most virulent form of the pathogen, termed white transparent or WT, is much less resistant to chlorine than the RT form. These conclusions are based on observations of two M. avium clinical isolates; we will test additional isolates to determine whether this pattern holds true of all M. avium strains. If so, it would mean that removing virulent M. avium from drinking water may require less chlorine than is generally assumed. Such a conclusion would be significant in light of the harmful byproducts associated with excessive chlorine treatment of water. Regardless of the outcome of our expanded investigation, our findings demonstrate that chlorine-resistant forms of M. avium are not always the most virulent forms. This means that simple detection of M. avium colonies on existing growth media does not tell the whole story. Reliable methods for specifically detecting the virulent forms of M. avium, for example by growing colonies on Congo red agar (Cangelosi et al., Microbiology, in press), may yield better indications of the efficacy of drinking water treatment.
We also found that RT and WT variants differ with regard to motility, a factor that is important to the survival of bacteria in nutrient-poor environments like water. RT variants are motile but WT variants are not, potentially putting the latter at a disadvantage in water.
Future Activities:
Anticipated Year 3 activities include the following: (1) we will expand our analysis of chlorine resistance in M. avium; (2) we will assess the impact of red-white variation (responsible for the formation of separate RT and WT forms) on other parameters important to the survival and recovery of M. avium from drinking water, including the effects of the "decontamination" procedures that are routinely used to detect M. avium in water, the growth of M. avium on point-of-entry and point-of-use water filtration systems, and the formation of biofilms; and (3) having demonstrated that H. pylori takes up BrdU from the medium and incorporates it into its DNA, we will continue to develop BrdU-DNA assays for viable H. pylori. The procedure will be streamlined, tested against multiple strains, and tested against killed vs. living cells. The optimized procedure then will be used to study the survival of H. pylori in water.Journal Articles on this Report : 1 Displayed | Download in RIS Format
Other project views: | All 6 publications | 3 publications in selected types | All 3 journal articles |
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Cangelosi GA, Palermo CO, Bermudez LE. Phenotypic consequences of red-white colony type variation in Mycobacterium avium. Microbiology 2001;147(3):527-533. |
R826828 (2000) R826828 (Final) |
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Supplemental Keywords:
human health, infectious disease, decision making, epidemiology, biology, genetics, probes, environmental testing, analytical, measurement, Washington, WA., RFA, Scientific Discipline, Water, Geographic Area, Health Risk Assessment, Environmental Chemistry, State, Environmental Monitoring, Drinking Water, monitoring, microbial contamination, pathogens, Safe Drinking Water, human health effects, microbiological organisms, detection, exposure and effects, disinfection byproducts (DPBs), exposure, mycobacterium avium complex, kinetics, community water system, Washington (WA), genotoxicity, infectious disease, treatment, microbial risk management, water quality, drinking water contaminants, heliocobacter pylori, infectivity, water treatmentProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.