Impact/Purpose:

Develop a real-time PCR method for the rapid detection and quantification of Mycobacterium avium and M. intracellulare in drinking water.

Determine the subspecies of those M. avium complex bacteria in EPA culture collection.

Description:

Bacteria belonging to the Mycobacterium avium complex (MAC), including Mycobacterium avium and M. intracellulare, are clinically relevant and cause a myriad of opportunistic infections. Children, the elderly, and persons with previous lung conditions or immune system dysfunction due to HIV infection, chronic steroid use, and chemotherapy treatment are at risk for becoming seriously or fatally ill from MAC infections. As the numbers of at-risk populations continue to rise, so do the numbers of MAC infections. There is no evidence of person-to-person transmission of MAC organisms. The link between the environment and patient has been established, with drinking water being a major source of exposure. Current methods for detecting mycobacteria in drinking water utilize cultural techniques. Unfortunately, M. avium and M. intracellulare take an average of five weeks to isolate from environmental samples using cultural methods. Furthermore, the harsh decontamination procedures required to reduce background organisms also reduces the recovery of mycobacteria by 50 to 70%.

There is a critical need for rapid methods of detection of MAC organisms in diagnostic laboratories in order to prevent outbreaks of disease. Rapid methods are also essential in laboratories that process large numbers of drinking water samples for unregulated contaminant monitoring (UCMR). The proposed research will result in a real-time PCR method for detecting and quantifying MAC organisms in potable water. The method will require that organisms first be concentrated from drinking water samples using centrifugation or a membrane filtration technique. Cells will be physically disrupted to expose nucleic acids. Quantitative, real-time PCR will be performed on total nucleic acids using an Applied BioSystem's 7700 or 7900 Sequence Detection System, with MAC-specific primers and either M. avium and M. intracellulare-specific internal probes. The method will be developed using drinking water spiked with known concentrations of MAC organisms, and tested and validated with actual drinking water samples.

Recent genetic data for the Mycobacterium avium species has revealed that it is comprised of 4 distinct subspecies (with distinct animal hosts)- avium (birds only), paratuberculosis (host range unknown), hominisuis (humans and swine) and sylvatica (wood pigeons). Current culture methods likely allow the isolation of both avium and hominisuis subspecies if they are present in environmental samples. Work is needed to determine is both of these subspecies can be found in drinking water.

Record Details:

Record Type:PROJECT

Start Date:10/01/2003

Projected Completion Date:09/01/2007

OMB Category:Other

Record ID: 56117

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Project Information:

Progress :An extensive literature review was undertaken in order to evaluate a suitable DNA target for the real-time detection and quantification of M. avium and M. intracellulare in drinking water. The gene coding for the small subunit of the ribosome, or 16S gene, was chosen as a potential target based on its widespread use in bacterial taxonomy for differentiating species of bacteria. A sequence alignment was prepared using 16S sequences obtained from the Ribosomal Database Project and GenBank, which contained multiple strains of M. avium and M. intracellulare in addition to several other species of mycobacteria. From this alignment, primer sets and dual-labeled fluorogenic probes were designed for both species using OLIGO and PRIMER EXPRESS software programs. Multiple pure cultures of M. avium and M. intracellulare have been grown for use in generating standard curves on the Applied BioSystem's 7900 Sequence Detection System. An internal PCR control is being constructed to measure overall reaction efficiency and determine if negative water samples truly lack the organisms or are negatives due to the presence of PCR inhibitors.



Relevance :Development of methods to detect MAC organisms in drinking water are a priority as these organisms are on the current CCL. Furthermore, molecular methods that avoid culturing are critical for rapid detection, in order to prevent possible outbreaks of disease in at-risk populations. Currently, there is no rapid, quantitative method for measuring MAC organisms in drinking water. The resulting method can be used to collect occurrence data, and as a compliance monitoring tool. Occurrence data will aid EPA's Office of Ground Water and Drinking Water in determining if these organisms should be regulated.

Clients :Office of Water

Project IDs:

ID Code :15584

Project type :OMIS