Ordering Information:
ORDER NUMBER:  91223
DATE AVAILABLE: Summer 2008

Printed Report		PDF
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Non-Subscriber	Awwa Bookstore (November 2008)
	IWA Publishing (February 2009)

PRINCIPAL INVESTIGATORS:
Michael T. Collins, Alice S. Yuroff, Rebecca M. Hoffman, Gregory W. Harrington, and Andrew Jacque

OBJECTIVES:
The objectives of this project included the following:

1. Evaluate liquid culture methods for growth of finished water-habituated Mycobacterium avium (MAC)
2. Compare filtration vs. centrifugation for concentration of M. avium ss. avium (MAA)
3. Assess decontamination methods on MAC isolation
4. Identify anti-MAC antibodies for immunomagnetic separation (IMS) concentration
5. Evaluate newly-developed IMS bead constructs for selective MAC concentration
6. Establish and evaluate real time capacity for a validated multiplex MAC PCR
7. Evaluate methods under simulation for both surface associated and planktonic MAC

BACKGROUND:

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.

HIGHLIGHTS:

• The method providing the greatest sensitivity in screening finished water for residual MAC species couples liquid culture-based methods with a multiple-target PCR that identifies the presence of mycobacterial species in general as well as specific MAC subspecies.
• Filtration, followed by DNA extraction from the filter, is superior to centrifugation in detecting MAC in finished water samples. Since the hydrophobic properties of MAC have a significant impact on results with MAC cells adhering to processing containers, handling protocols should limit the number of sample transfers.
• Placing coupons directly into culture media provided more accurate counts than scraping or swabbing coupons.

APPROACH:
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.

RESULTS/FINDINGS:
The method providing the greatest sensitivity in screening water for residual MAC species after treatment coupled liquid culture-based methods with multiplex PCR. An algorithm was developed with the MGIT culture method to count the number of MAC cells in a sample. Quantification of MAC by placement of coupons directly into culture media was superior to quantification in after swabbing coupons. The triplex PCR method, functional in the presence of contaminating organisms, also provided a degree of quantification using real-time PCR technology.

Filtration, followed by DNA extraction from the filter, was superior to centrifugation as a method of concentration and identification of MAC species from finished water samples. Significant losses of MAC occurred as substantial numbers attached to collection vessel surfaces due to the organisms’ hydrophobic nature. Experiment protocols using single sample mini-chambers with direct-culture coupons for either static or moving water conditions permitted unperturbed sampling, limiting chances for contamination and minimizing organism loss through handling.

IMPACT:
Finished water distribution systems surveillance for MAC using methods available to date likely significantly undercounts planktonic and surface-associated MAC cells. To obtain the most valid assessment of the level of MAC present in finished water, it is important to rely on both culture and PCR of samples and focus on surface-associated organisms, that is, make the organisms’ hydrophobicity favorable by using coupons inserted at key points in the water distribution system. The coupons should be designed to be a size that can be easily removed and directly placed into culture media. Bulk samples should be collected for planktonic MAC immediately after flushing pipes.

RESEARCH PARTNER:
USEPA

PARTICIPANTS:
Pinellas County Utilities provided the project with water samples.

ISBN 978-1-60573-026-4