Grantee Research Project Results
2000 Progress Report: Mycobacterium avium Complex in Drinking Water: Detection, Distribution, and Routes of Exposure
EPA Grant Number: R828036Title: Mycobacterium avium Complex in Drinking Water: Detection, Distribution, and Routes of Exposure
Investigators: Ford, Timothy E.
Institution: Harvard University
Current Institution: Harvard University , Montana State University
EPA Project Officer: Hahn, Intaek
Project Period: (Extended to March 21, 2004)
Project Period Covered by this Report: January 1, 2000 through January 1, 2001
Project Amount: $516,679
RFA: Drinking Water (1999) RFA Text | Recipients Lists
Research Category: Drinking Water , Water
Objective:
The objectives of this project are to: (1) develop improved methods for detecting Mycobacterium avium complex (MAC) in biofilms; (2) explore and implement more sensitive techniques for the detection of MAC in drinking water samples; and (3) determine the prevalence of MAC in municipal drinking water distribution systems and at sites of end user exposure (drinking water, hot water, and toilet tank water).
Progress Summary:
We have conducted a series of experiments in the laboratory to establish the optimal conditions for the traditional water filtration procedure to isolate mycobacteria from water samples. This was a necessary first step due to variation in concentration of decontamination agent (cetylpyridinium chloride [CPC]) and decontamination times observed in a number of different studies in the literature.
Thus far, we have obtained 50 water samples from the various buildings in the Longwood Medical Area (LMA) in Boston. The area comprises several teaching hospitals and academic institutions, including HSPH. A special identification number has been assigned to each sample and to a data form that is filled with details on: exact collection site location; type of sample; water temperature; type of water system used in the building; and age of building. All of the water samples have been filtered and subcultured; up to nine colonies from each sample have been stored as frozen suspensions (species identification will be carried out by polymerase chain reaction [PCR] later this year). These LMA water samples were used for the CPC optimization experiments as well as to provide MAC colonization data to identify the best possible site for our hot water bypass system.
During the course of the year, we have designed a bypass system that is in the construction phase at the Harvard Medical School machine shop. In coordination with HSPH operations personnel, we have identified a readily accessible part of one of HSPH's hot water systems. Water samples from this system have tested positive for MAC, and HSPH plumbers currently are installing "T" junctions (with valves and pressure gauges) that will attach directly to the bypass loop. The bypass design will allow us to gain easy access to "biofilm cartridges." The biofilms from the system will provide specimens grown under realistic environmental conditions. These cartridges have been designed to allow us to collect biofilm samples easily and without risk of contamination. Inside the cartridge are "coupon stacks," on which biofilms will grow, enabling us to test various substrata (e.g., stainless steel, polycarbonate plastics, brass, and copper). The system has various valves, pressure gauges, and flowmeters. These will enable us to closely monitor precise flow conditions and to investigate the effect of different flow speeds on biofilm growth.
We are using an artificial laboratory recirculating system to generate mixed culture (Pseudomonas aeruginosa and Mycobacterium avium) biofilms to test our florescence in situ hybridization (FISH) probes for MAC. This is at the preliminary stage of optimization to specifically detect MAC. Insertion elements such as IS1245 and IS1311 initially were to be used as targets for the FISH probes, but recent literature reveals overlap with other non-tuberculous mycobacteria and problems when encountering strains with low copy numbers of these elements. We are investigating whether the 16S rRNA would be a more suitable target as it is highly conserved and is used widely for phylogenetic analysis of bacteria.
The biofilms obtained from the artificial system also have been used to test our upgraded epiflorescence microscope and image analysis software. This software will allow us to produce quantitative data on MAC spatial distribution within the biofilm. By double and triple florescence staining techniques, we potentially can examine spatial associations of MAC with other microorganisms.
Future Activities:
We have begun to work with the municipal drinking water authorities to identify an appropriate site for installing a duplicate bypass system. This should be located at, or close to, a municipal treatment plant. By comparing treatment plant and end user site biofilms, we can establish whether MAC prevalence is magnified in the distribution system.
We also are beginning to identify point-of-use-sites where residential water and biofilm samples can be collected from pipes, toilet tanks, and showerheads. The strains obtained from these sites will be typed and analyzed against isolates obtained from other geographic locations to assess whether they are genetically related. This fall, we will begin four-season collections from both these residential sites and a limited number of distribution system sites to look at seasonal variations. With the use of GIS software, we plan to map trends in MAC prevalence in the eastern Massachusetts drinking water distribution sites and residential areas.
In recent weeks, we also have started a collaboration with Dr. Jerry Cangelosi's group in Seattle, Washington, who are looking at morphotypic variation in MAC. Some MAC morphotypes appear to have increased virulence and also can undergo morphotype switching. We are investigating the ability of the various MAC morphotypes to produce biofilms. Our hypothesis would investigate whether more virulent MAC morphotypes dominate biofilms. We will investigate single as well as mixed culture biofilms grown in four identical artificial recirculating systems. These systems already have been set up and will be inoculated with four different MAC morphotype strains.
Journal Articles:
No journal articles submitted with this report: View all 6 publications for this projectSupplemental Keywords:
pathogens, survival, proliferation, new techniques., RFA, Scientific Discipline, Water, Environmental Chemistry, Health Risk Assessment, Environmental Microbiology, Environmental Monitoring, Ecological Risk Assessment, Drinking Water, alternative disinfection methods, monitoring, CCL, detection, water quality parameters, exposure and effects, mycobacterium avium complex, routes of exposure, exposure, community water system, treatment, emerging pathogens, microbial exposure, water quality, contaminant removal, drinking water contaminants, drinking water treatment, water treatment, contaminant candidate list, DNA microarraysRelevant Websites:
http://www.hsph.harvard.edu/water
Progress 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.