Impact/Purpose:

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.

Description:

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.

Record Details:

Record Type:PROJECT

Start Date:09/01/1997

Projected Completion Date:09/01/2004

Record ID: 18289

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Project Information:

Progress :Research initiated in FY99 to develop improved methods for isolation and detection of nontuberculous mycobacteria included temperature gradient studies to determine optimum incubation temperatures and heat resistance of MAC organisms and selection of antibiotics to inhibit background organisms and permit better recovery of nontuberculous mycobacteria. Temperature gradient studies have established the mean minimum, optimum, and maximum survival temperatures of each of the MAC organisms. The optimum growth temperature for the entire group is 35 C. Only minimal growth of MAC organisms may occur in distribution lines with temperatures of 14 - 16 C. Ninety(90%) of the M. avium strains tested were capable of survival of temperatures of 49 C (the recommended temperature of hot water plumbing systems) or greater. Various antibiotics and dyes have been examined as a means to replace "harsh" decontamination procedures currently used to reduce background organisms. Thus far a oxidizer with L-serine has shown promise for better recovery and reduction of background organisms than the standard accepted cultural approach. Sixty (60) samples (reservoir and drinking water) have been analyzed by the standard cultural method and the multiplex PCR method. Nine(9) of the samples were positive by both methods, seven(7) were positive only by multiplex PCR and three(3) were positive only by the cultural method. The use of multiplex PCR significantly decreases the time for analysis for these organisms. Detection limit studies have been initiated with three strains of M. avium and M. intracellulare. Cell lysis and PCR amplification will be completed in the near future. Problems were experienced with cell lysis with the previous detection limit studies. Genomic DNA has been purified from 87 M. avium and 19 M. intracellulare isolates obtained from patients, as well as 30 M. avium and 20 M. intracellulare isolates obtained from environmental samples for DNA fingerprinting studies. DNA has also been obtained from 14 isolates designated MX by the GenProbe system. The genomic DNA from these isolates have been digested with restriction enzymes, ligated to adapters, and a subset of the genome has been amplified using specific primers. Fingerprints were generated for all 170 isolates using the Applied Biosystem's 310 Genetic Analyzer. The fingerprint data was converted to presence/ absence data in a Lotus 1-2-3 spreadsheet, and used to calculate a similarity matrix using the coefficient of Nei and Li. The resulting similarity matrix was used to generate phylogenetic trees showing the genetic relationships between isolates. All of the M. avium isolates are genetically distinct, and fall into seven clusters. The drinking water isolates are the least diverse, clustering more closely to each other than with any of the other isolates. The non-AIDS patients isolates are the most diverse, and are found throughout the seven clusters in the tree. The AFLP method easily distinguished between M. avium and M. intracellulare isolates. The results suggest that current methods for isolating NTMs from drinking water are very harsh, and select for a subset of the organisms present in the sample. The results also suggest that AFLP is very discriminatory for typing MAC organisms at the strain level, is rapid, and highly reproducible.



Relevance :Research performed under this task is a high priority under the CCL research plan. The methods to be developed in this task will allow us to get more accurate data on the occurrence of MAC organisms in potable water and to get a more reliable assessment of the significance of exposures via potable water. Improved DNA fingerprinting methods are needed to aid in establishing the link between exposure from environmental sources and clinical MAC disease. All of this information will be used by OGWDW to determine whether or not to regulate these organisms in potable water.

Clients :Dr. Paul Berger, OGWDW

Research Component :M/DBP (MICROBIAL)

Risk Paradigm :EXPOSURE

Progress :Research initiated in FY99 to develop improved methods for isolation and detection of nontuberculous mycobacteria included temperature gradient studies to determine optimum incubation temperatures and heat resistance of MAC organisms and selection of antibiotics to inhibit background organisms and permit better recovery of nontuberculous mycobacteria. Temperature gradient studies have established the mean minimum, optimum, and maximum survival temperatures of each of the MAC organisms. The optimum growth temperature for the entire group is 35 C. Only minimal growth of MAC organisms may occur in distribution lines with temperatures of 14 - 16 C. Ninety(90%) of the M. avium strains tested were capable of survival of temperatures of 49 C (the recommended temperature of hot water plumbing systems) or greater. Various antibiotics and dyes have been examined as a means to replace "harsh" decontamination procedures currently used to reduce background organisms. Thus far a oxidizer with L-serine has shown promise for better recovery and reduction of background organisms than the standard accepted cultural approach. Sixty (60) samples (reservoir and drinking water) have been analyzed by the standard cultural method and the multiplex PCR method. Nine(9) of the samples were positive by both methods, seven(7) were positive only by multiplex PCR and three(3) were positive only by the cultural method. The use of multiplex PCR significantly decreases the time for analysis for these organisms. Detection limit studies have been initiated with three strains of M. avium and M. intracellulare. Cell lysis and PCR amplification will be completed in the near future. Problems were experienced with cell lysis with the previous detection limit studies. Genomic DNA has been purified from 87 M. avium and 19 M. intracellulare isolates obtained from patients, as well as 30 M. avium and 20 M. intracellulare isolates obtained from environmental samples for DNA fingerprinting studies. DNA has also been obtained from 14 isolates designated MX by the GenProbe system. The genomic DNA from these isolates have been digested with restriction enzymes, ligated to adapters, and a subset of the genome has been amplified using specific primers. Fingerprints were generated for all 170 isolates using the Applied Biosystem's 310 Genetic Analyzer. The fingerprint data was converted to presence/ absence data in a Lotus 1-2-3 spreadsheet, and used to calculate a similarity matrix using the coefficient of Nei and Li. The resulting similarity matrix was used to generate phylogenetic trees showing the genetic relationships between isolates. All of the M. avium isolates are genetically distinct, and fall into seven clusters. The drinking water isolates are the least diverse, clustering more closely to each other than with any of the other isolates. The non-AIDS patients isolates are the most diverse, and are found throughout the seven clusters in the tree. The AFLP method easily distinguished between M. avium and M. intracellulare isolates. The results suggest that current methods for isolating NTMs from drinking water are very harsh, and select for a subset of the organisms present in the sample. The results also suggest that AFLP is very discriminatory for typing MAC organisms at the strain level, is rapid, and highly reproducible.



Relevance :Research performed under this task is a high priority under the CCL research plan. The methods to be developed in this task will allow us to get more accurate data on the occurrence of MAC organisms in potable water and to get a more reliable assessment of the significance of exposures via potable water. Improved DNA fingerprinting methods are needed to aid in establishing the link between exposure from environmental sources and clinical MAC disease. All of this information will be used by OGWDW to determine whether or not to regulate these organisms in potable water.

Clients :Dr. Paul Berger, OGWDW

Research Component :CCL (MICROBIAL)

Risk Paradigm :EXPOSURE

Project IDs:

ID Code :EX.M.22

Project type :ORD-DW Plan

ID Code :508

Project type :OMIS