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METHODS FOR ISOLATION AND CHARACTERIZATION OF NONTUBERCULOUS MYCOBACTERIA IN POTABLE WATER, CCL
1)Develop an improved method(s) for isolating and/or detecting nontuberculous mycobacteria from potable water. 2)Determine the best DNA fingerprinting method to use to determine genetic relatedness with clinical and environmental isolates.
Nontuberculous mycobacteria (NTM) are opportunist pathogens that usually infect individuals with impaired immunity, such as Acquired Immune Deficiency Syndrome (AIDS) patients, the elderly or those undergoing immunosuppressive drugs or chemotherapy. The sources of infection are unknown. These organisms are found in a variety of environments including soil and water and are included on the current Contaminant Candidate List (CCL). The results of earlier exploratory occurrence studies suggest that NTM have a widespread occurrence in potable water throughout the U.S., for example, Mycobacterium avium complex strains isolated from Los Angeles potable water are frequently identical to or closely related to isolates from patients in Los Angeles. However, these results are not adequate for use in a comprehensive assessment of the risk NTM pose to the public. The techniques currently available for isolating NTM from environmental matrices, including potable water, require harsh decontamination techniques to reduce the levels of background organisms, and these techniques often cause the loss of at least 50 -70% of the target NTM from each sample. This leads to underestimations of the number of samples that will contain NTM, a significant underestimation of the numbers of NTM in each positive sample and possibly to inability to isolate critical strains that might be unusually sensitive to the decontaminating agents. Improved methods for recovery of NTM are critically needed before initiation of any additional occurrence or exposure studies. Selective growth method(s) will be developed which do not use classical conventional decontamination procedures. The use of antibiotics and other growth inhibitors will be explored for their ability to inhibit background organisms and permit growth of NTM. The method, thus improved, will then be available to the Office of Groundwater and Drinking Water (OGWDW) and others for use in generating more complete occurrence data. This occurrence data will be used by OGWDW to help determine the regulatory fate of NTM organisms in the Unregulated Contaminant Monitoring Rule (UCMR) process. A rapid multiplex polymerase chain reaction (PCR) method will be developed that detects M. avium complex (MAC) organisms. Temperature gradient growth studies will be performed to establish minimum, optimum, and maximum survival temperatures of MAC organisms. The results of the Los Angeles study revealed a need to determine the best DNA fingerprinting method to use with these organisms to examine the genetic relatedness between patient and environmental isolates. This is very important to establish the link between exposure from environmental sources and clinical disease. Amplified fragment polymorphism (AFLP) analysis will be compared to pulsed field gel electrophoresis (PFGE) methods to determine the best method for typing mycobacterial isolates. The ABI PRISM 310 Genetic Analyzer or similar genetic analyzers which can measure DNA fragments accurately and without the need for gels will be used with the developed AFLP method.