Grantee Research Project Results
Final 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 Amount: $360,609
RFA: Drinking Water (1998) RFA Text | Recipients Lists
Research Category: Drinking Water , Water
Objective:
Microbial contaminants in drinking water can be 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 molecular assays (DNA probes and PCR) can differentially detect viable bacterial cells when the assays are targeted to novel nucleic acid analytes. One of these analytes is pre-ribosomal ribonucleic acid (rRNA). As intermediates in ribosome synthesis, pre-rRNA molecules are abundant in growing bacterial cells, but rare in dead or non-growing cells. An alternative analyte is bromodeoxyuridine (BrdU)-labeled DNA. The BrdU analyte is incorporated into DNA during DNA replication in viable cells. It can be detected by BrdU-specific immunocapture followed by PCR. Both approaches are species-specific, and both are diagnostic of cells capable of nucleic acid synthesis and growth. We hypothesized that assays for these analytes can prevent the false-positive detection of residual nucleic acid from dead cells or contaminants.
Summary/Accomplishments (Outputs/Outcomes):
We used bacterial model systems (Escherichia coli, Mycobacterium avium, and Helicobacter pylori), to test the feasibility and utility of measuring bacterial pre-rRNA and BrdU-DNA in water supplies. The objectives of this research project were to:
· Optimize pre-rRNA and BrdU-DNA assays using E. coli as a model system.
· Use pre-rRNA and BrdU-DNA to answer questions regarding the growth physiology of M. avium (also known as MAI or MAC) in drinking water and its resistance to disinfection.
· Test the hypothesis that assays for pre-rRNA and/or BrdU-DNA can distinguish replicative from non-replicative forms of H. pylori in water.
We also completed a detailed analysis of morphotypic switching by M. avium. We identified a new morphotypic switch in this pathogen and characterized the importance of the switch to the pathogen's survival in water environments, its pathogenicity, and its susceptibility to antimicrobial agents, including antibiotics and chlorine. Objective 1 was completed using Klebsiella pneumoniae as a model system. Both assays performed well in enteric organisms. Objective 2 yielded the most significant data, as outlined below.
M. avium maintains significant pre-rRNA pools in dead and stationary-phase cells. The pre-rRNA approach depends on the assumption that pre-rRNA pools are drained in non-growing target cells. Preliminary evidence indicated this for E. coli and Mycobacterium bovis (BCG), a bacterium that is closely related to M. avium. However, we found that M. avium maintains a large pre-rRNA pool even when growth is halted. This eliminated the use of pre-rRNA as an indicator of active growth and viability in M. avium.
M. avium does not incorporate BrdU or bromouracyl (BrU) into its DNA. With the abandonment of the pre-rRNA approach for M. avium, we concentrated our efforts on BrdU-DNA. As with the pre-rRNA approach, this approach worked well in preliminary experiments with E. coli, K. pneumoniae, and BCG. Unfortunately, M. avium was again an exception. Regardless of the culture medium used, M. avium cells did not incorporate significant amounts of BrdU into their DNA. Similar results were observed with an alternative thymidine analog, BrU. M. avium appears to lack uptake mechanisms for these compounds.
Morphotypic characteristics of M. avium in drinking water environments. When it became clear that our new detection technologies will not work well with M. avium, we turned to traditional methods (plating and colony counting) to answer questions related to the growth and survival of M. avium in drinking water. We gained significant data that are relevant to the detection and control of M. avium in drinking water.
Isolates of M. avium have been known to segregate into "colony types" (morphotypes) that vary with regard to clinically important parameters, including drug susceptibility and virulence. Until recently, the opaque-to-transparent (O-T) colony type switch was considered the most important of these switches. The O-T switch is reversible at frequencies of 10-4 to 10-6 per generation. Transparent variants predominate in patient samples, grow better in macrophage and animal models, and are more resistant than opaque variants to most antimycobacterial drugs. They survive well in water and are favored during incubation in deionized water (Cangelosi, et al., 2001). They also are generally considered to be the more chlorine-resistant forms of the pathogen.
We recently found a new type of phenotypic variation that was detected when opaque or transparent colonies of M. avium were grown on agar media containing the lipoprotein stain Congo red (CR). Most fresh clinical isolates form red and white colonies under these conditions. White opaque (WO) and white transparent (WT) variants were found to be more resistant to antibiotics than their red opaque (RO) and red transparent (RT) counterparts.
Within the transparent morphotype, red-white variation affects virulence, as shown by three lines of evidence. First, WT variants fared significantly better than RT variants when cultured within human macrophages (white blood cells). Second, when mice were infected intravenously with RT or WT variants, the latter grew significantly better over time in livers and spleens. Third, analysis of 32 fresh clinical isolates suggested that the white morphotype is predominant among M. avium cells living within human hosts. These data show that the transparent morphotype can no longer be considered a homogeneous group with regard to virulence. Rather, there are subgroups-red transparent and white transparent-and the latter appears to be a more significant threat to human health.
In Year 3 of the grant, we teamed up with a laboratory to assess the morphotypic characteristics of M. avium cells isolated from drinking water environments. Thus far, we have examined 11 clones isolated from water taps in Boston area institutional buildings. All were in the WT morphotype (Table 1). We also examined a naturally-occurring biofilm isolate grown in a unique "bypass" system; it too was WT. Based on this small sample, the clinically significant WT morphotype appears to be common in drinking water environments (unpublished results). This study will be expanded in the coming years.
A chlorine-resistant morphotypic variant of M. avium. Regarding our finding that white variants are more resistant than red variants to multiple antibiotics, we examined the susceptibility of these variants to chlorine in the form of sodium hypochlorite. The goal was to test the common assumption that disinfectant resistance and morphotypic multi-drug resistance are conferred by common mechanisms in M. avium. Two experimental formats were used. In one, isolated RT, WT, RO, and WO clones were exposed to varying concentrations of sodium hypochlorite for 4 hours in ultrapure water, and samples were plated to obtain total colony counts. The RT variant was significantly more resistant to this treatment than the other forms (Figure 1). A caveat associated with this approach is that chlorine availability may not have been uniform, due to differences in cell numbers, cell surface composition, and other factors. To address this issue, we also used a competition format, in which RT and WT cells were mixed and exposed to chlorine together, assuring that both cell types were exposed to identical environments. The RT variant also was significantly more resistant than the WT variant under these conditions as well, confirming that the results are attributed to differences in intrinsic chlorine resistance, rather than experimental artifacts (manuscript in preparation). However, examination of a second clinical isolate, M. avium strain HMC10, revealed similar levels of chlorine resistance in RT and WT variants.
In both strains examined (HMC02 and HMC10), WT variants were significantly more resistant to multiple antibiotics than are their RT counterparts. In contrast, WT variants were equally or more sensitive to chlorine than their RT counterparts. Thus, chlorine resistance did not correlate with multi-drug resistance in these strains, suggesting that the two phenomena are conferred by independent mechanisms. Cell-to-cell aggregation can increase chlorine resistance; however, that cannot explain our results because white variants are more flocculent than red variants, not the reverse. Among the clones we examined, RT variants of strain HMC02 exhibited a uniquely high level of chlorine resistance. These clones represent a distinct chlorine-resistant morphotype, the first to be identified in M. avium.
A new genotypic marker for M. avium. In the course of characterizing the genetic basis for the red-white switch in M. avium, we identified a novel mobile genetic element, IS999. This IS3-family insertion element is common in M. avium genomes. It is less stable than IS1245, the most commonly-used marker for molecular epidemiological analysis of M. avium. IS999 will be a useful tool for tracking the rapid genetic drift that M. avium strains undergo in nature and in the laboratory.
Table 1. Morphotype of Isolates From Institutional Water Supplies in the Boston Area
| Source # | Water Temp | Sample Type | Collection Address | Decontamination method | Species (rDNA PCR) | Morphotype |
| W1001 | 32.0 | Hot Water - Bathroom | Boston VA Research Lab, 151 S Huntington Avenue, Jamaica Plain, MA 02130 | CPC 0.04% | M. avium | WT |
| W1005 | N/C | Hot Water - Bathroom | Boston VA Research Lab, 151 S Huntington Ave, Jamaica Plain, MA 02130 | CPC 0.005% | M. avium | WT |
| W2001 | 34.5 | Hot Water - Laboratory | HSPH-1, G22, 665 Huntington Ave, Boston, MA 02115 | CPC 0.04% | M. avium | WT |
| W2008 | 38.2 | Hot Water - Bathroom | HMS Building D, Rm. 131, 210 Longwood Avenue, Boston, MA 02115 | CPC 0.04% | M. avium | WT |
| W2010 | 29.8 | Hot Water - Bathroom | Children's Hospital, Orthopedics Department Mensroom, 300 Longwood Avenue, Boston, MA 02115 | CPC 0.04% | M. avium | WT |
| W2011 | 25.8 | Hot Water - Bathroom | Brigham and Women's Hospital, Peter Bent Building, Boston, MA 02115 | CPC 0.04% | M. avium | WT |
| W2013 | 33.4 | Hot Water - Kitchen | HSPH Building 1, G4A, 665 Huntington Avenue, Boston, MA 02115 | CPC 0.04% | M. avium | WT |
| W2015 | 36.5 | Hot Water - Bathroom | Beth Israel-Deaconess, Carl J. Shapiro Building, Room CG107B, 330 Brookline Avenue, Boston, MA 02215 | CPC 0.04% | M. avium | WT |
| W2016 | 35.8 | Hot Water - Bathroom | Harvard Institutes of Medicine, Room 1C4, 4 Black Fan Circle, Boston, MA 02115 | CPC 0.04% | M. avium | WT |
| W2017 | 31.9 | Hot Water - Bathroom | Joslin Diabetes Center, Room 258, 1 Joslin Place, Boston, MA 02215 | CPC 0.04% | M. avium | WT |
| W2025 | 43.7 | Hot Water - Bathroom | HMS, Building, D, Room 131, 210 Longwood Avenue, Boston, MA 02115 | CPC 0.005% | M. avium | WT |
Additional Activities Related to Objective 2. Additional activities related to Objective 2 included the: (1) analysis of the effects of red-white variation on motility (white variants are non-motile); (2) analysis of the effects of cetylpyridnium chloride (CPC) and alkaline sample decontamination procedures on red and white variants (they did not differentially affect either morphotype); and (3) development of a transposon mutagenesis system for analysis of virulence and biofilm formation by M. avium. We hope to submit a description of the mutagenesis system for publication in late 2002.
Cultures of H. pylori were established by using the biphasic culture system as recommended by the American Type Culture Collection, the source of our H. pylori strains. Cells were grown with and without BrdU or BrU for 5 days at 37°C under microaerophilic conditions established by using the Campy Pak Plus system. Cells were lysed using a lysozyme-proteinase K method, and DNA was extracted using phenol-chloroform and ethanol precipitation. DNA was denatured, exposed to anti-BrdU or anti-BrU antibodies, and added to Protein A-coated tubes following the protocols established in Objective 1. The tubes were washed to remove unbound (unlabeled) DNA, then filled with a PCR reaction mixture containing the H. pylori-specific primers HPU1 and HPU2 (Clayton, et al., 1992), and PCR reactions were conducted. The BrU labeling did not give consistent results, but BrdU labeling worked well when the analog was provided at concentrations ± 0.5 µg/ml. Therefore, the BrdU labeling method shows promise as a means to detect viable H. pylori cells.
We explored the feasibility of two novel technologies for detecting viable cells of two pathogens, M. avium and H. pylori, in drinking water. We conclude that these technologies will most likely not be useful for M. avium; however, the BrdU-DNA method has some promise for detection of H. pylori.
The most significant and tangible results were of our analysis of the biology of M. avium in clinical and drinking water samples. M. avium is an important waterborne pathogen, but very little is known about how it lives in water supplies and how it can be controlled. Our research added a significant amount of new information, and it will continue to do so as we pursue the lines of investigation initiated under our Environmental Protection Agency (EPA) grant. Specifically, we learned that MAC occurs in multiple forms, not all of which are infectious. The most infectious form appears to be quite common among isolates from water supplies. The ability of MAC to resist killing by disinfectant treatments such as chlorine was shown to occur by mechanisms that are independent of the pathogen's intrinsic drug resistance. Moreover, the most chlorine-resistant form of the pathogen, the "red-transparent" type, is not the most virulent form. Finally we identified the genetic marker IS999; it will be useful for tracking the evolution and spread of the pathogen in the environment.
We expect to submit two additional manuscripts in 2002 or 2003. One describes the transposon mutagenesis system for M. avium, and the other presents the chlorine resistance results.
References:
Clayton CL, Kleanthous H, Coates PJ, Morgan DD, Tabaqchali S. Sensitive detection of Helicobacter pylor by using polymerase chain reaction. Journal of Clinical Microbiology 1992;30:192-200.
Journal Articles on this Report : 3 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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Laurent J-P, Faske S, Cangelosi GA. Characterization of IS999, an unstable genetic element in Mycobacterium avium. Gene 2002;294(1-2):249-257. |
R826828 (Final) |
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Mukherjee S, Petrofsky M, Yaraei K, Bermudez LE, Cangelosi GA. The white morphotype of Mycobacterium avium-intracellulare is common in infected humans and virulent in infection models. Journal of Infectious Diseases 2001;184(11):1480-1484. |
R826828 (Final) |
Exit Exit |
Supplemental Keywords:
human health, infectious disease, decisionmaking, epidemology, biology, genetics, probes, environmental testing, analytical, measurement, Washington State, drinking water, polymerase chain reaction, PCR, bromodeoxyuridine, BrdU, bromouracy, BrU., RFA, Scientific Discipline, Geographic Area, Water, Health Risk Assessment, State, Environmental Chemistry, Drinking Water, Environmental Monitoring, community water system, microbial contamination, Safe Drinking Water, treatment, water quality, microbiological organisms, Washington (WA), infectious disease, microbial risk management, monitoring, pathogens, kinetics, drinking water system, drinking water contaminants, exposure, infectivity, water treatment, genotoxicity, heliocobacter pylori, disinfection byproducts (DPBs), human health effects, mycobacterium avium complex, detectionProgress 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.