Improved Mycobacterium avium Complex Detection Methods

Mycobacteria belonging to the M. avium complex (MAC) are opportunistic pathogens found in planktonic forms, in biofilms, and within protozoa. Members of this complex are commonly associated with human disease. While a respectable amount of information is available on the occurrence of these pathogens, current methods of detection limit conclusions about their true presence in water distribution systems. MAC species are thus prime targets for improved methods of detection in finished water.

The objectives of this project included the following: Evaluate liquid culture methods for growth of finished water-habituated Mycobacterium avium (MAC)Compare filtration vs. centrifugation for concentration of M. avium ss. avium (MAA) Assess decontamination methods on MAC isolation Identify anti-MAC antibodies for immunomagnetic separation (IMS) concentration Evaluate newly-developed IMS bead constructs for selective MAC concentration Establish and evaluate real time capacity for a validated multiplex MAC PCR Evaluate methods under simulation for both surface associated and planktonic MAC

Production of a highly accurate and reliable MAC detection technique entailed optimizing numerous method components of a multi-step process. These steps were conducted in the lab and included concentration of MAC-spiked water samples, decontamination of the concentrated samples, selective concentration of mycobacteria, and detection and enumeration of total MAC and identification of MAC species. Concentration approaches included centrifugation and filtration trials. Decontamination studies assessed the efficacy of chlorine and cetylpyridinum chloride (CPC) against common water bacterial contaminants as well as mycobacteria. Methods for accurate identification and quantification of MAC species using liquid culture and genetic analysis (PCR) were validated. The final multi-step method was tested with treated water.