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MICOBACTERIUM PARATUBERCULOSIS AND NONTUBERCULOUS MYCOBACTERIAL IN POTABLE WATER
Citation:
Pfaller, S L. AND T C. Covert. MICOBACTERIUM PARATUBERCULOSIS AND NONTUBERCULOUS MYCOBACTERIAL IN POTABLE WATER. Presented at U.S. EPA's Research on Micoorganisms in Drinking Water Workshop, Cincinnati, OH, August 5-7, 2003.
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) include Mycobacterium species that are not members of the Mycobacterium tuberculosis Complex. Members of the NTM group are important causes of disease in birds and mammals. Mycobacterium avium, Mycobacterium intracellulare and Mycobacterium paratuberculosis are NTM and members of the Mycobacterium avium Complex (MAC). These organisms are found in a variety of environments including soil and water and are included on the Contaminant Candidate List (CCL). Earlier exploratory occurrence studies suggest that NTM have widespread occurrence in potable water throughout the U.S. M. paratuberculosis is the causative agent for Johne's disease in cattle. In addition to well - documented evidence of M. paratuberculosis as the causative agent of Johne's disease in cattle, there has been evidence linking M. paratuberculosis with Crohn's disease, a chronic inflammatory disease in of the intestinal tract in humans. Transmission of M. paratuberculosis via water contaminated with cattle feces may be one route of infection.
Current NTM research focuses on three areas: (1) Development of an improved cultural method for isolation of NTM in drinking water. (2) Development of a rapid PCR multiplex method for detection of MAC organisms in drinking water and (3) development of a molecular method for detection of M. paratuberculosis in water.
Improved Cultural Method
Goals/Objectives: Current methods for isolating NTM from environmental samples require harsh decontamination techniques to reduce the levels of background organisms often leading to loss of 50 -70% of the target NTM. The goal of this research is to develop improved selective method(s) which do not use classical decontamination procedures.
Approach: The use of antibiotics, dyes, detergents and other growth inhibitors are being examined for their ability to reduce background organisms and permit growth of NTM. A membrane filter method approach has been selected. Screening studies with spiked drinking water samples comparing candidate methods to classical decontamination techniques have been initiated. Candidate methods which permit better recovery of NTM and better reduction of background organisms will be tested with additional recovery studies and analyses of drinking water samples.
Preliminary Findings: Various antibiotics, dyes, detergents have been examined using a membrane filter cultural method approach. Thus far an oxidizer has shown promise for better recovery (80%) and reduction of background organisms than the standard accepted cultural method.
Significance: A improved cultural method would lead to better estimates of the occurrence of NTM, better estimates of the numbers of NTM in positive samples, and the possibility of recovering NTM unusually sensitive to decontaminating agents.
Next Steps: Future research will entail additional NTM recovery studies followed by comparison studies with the standard cultural approach and the improved method with distribution samples.
PCR Multiplex Method
Goals/Objectives: Current methods for detection of MAC organisms in drinking water typically take 3 - 8 weeks for completion of analyses with additional time for identification of the organisms. The goal of this research is to develop a rapid PCR multiplex method for detection of M. avium and M. intracellulare.
Approach: Drinking water samples (500 ml) are membrane filtered and the filters placed in modified 7H9 broth for seven day enrichment. After enrichment the cells are centrifuged and lysed to harvest the genomic DNA. The DNA is amplified (PCR) using primers specific for M. avium and M. intracellulare and all Mycobacteria. The PCR product is visualized by gel electrophoresis.
Preliminary Findings: Sixty samples (reservoir and drinking water) have been analyzed by the standard culture method and the multiplex PCR method. Nine samples were positive by both methods, seven were positive only by multiplex PCR and three were positive only by the cultural method.
Significance: The use of multiplex PCR significantly decreases the time for analyses for these organisms, and is able to detect MAC organisms not detected by the culture method.
Next Steps: Completion of detection limit studies and additional comparison studies with the standard culture technique using drinking water samples.
Method for detection of M. paratuberculosis
Goals/Objectives: A new project in our lab involves the development of a molecular detection and quantification method for M. paratuberculosis (MAP) in water. The method will be an important step in determining the significance of exposure to MAP in contaminated water, and may help to establish the link between contaminated water and Crohn's disease.
Approach: Current methods of detection, which include culture-based methods, are inadequate. A sixteen to twenty week incubation time is required to grow the organism, during which other microorganisms overgrow the medium. Harsh decontamination procedures used to reduce background organisms also kill a portion of MAP. This work proposes to develop a rapid molecular method to detect and quantify MAP in environmental samples by targeting a genetic molecule specific to MAP. One potential target is the MAP-specific insertion sequence IS900. The element is found only in MAP, and is present in fourteen to eighteen copies per cell. Other possible targets include seven recently discovered MAP-specific gene segments. A quantitative PCR-based method would significantly reduce detection times from approximately sixteen weeks to a few hours.