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Identification of Mycobacterium avium subsp. hominissuis Isolated From Drinking Water
KING, D. N., A. Beumer, AND S. L. PFALLER. Identification of Mycobacterium avium subsp. hominissuis Isolated From Drinking Water. Presented at Water Technology Conference, Charlotte, NC, November 04 - 08, 2007.
Purpose: 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. Objective: Develop a real-time PCR method for the rapid detection and quantification of Mycobacterium avium and M. intracellulare in drinking water. Develop a integrated method to collect, concentrate, purify (if needed), extract nucleic acid and real-time PCR for the quantification of Mycobacterium avium and M. intracellulare in drinking water. Determine the subspecies of M. avium complex bacteria in EPA culture collection.
Mycobacterium avium (MA) is divided into four subspecies based primarily on host-range and consists of MA subsp. avium (birds), MA subsp. silvaticum (wood pigeons), MA subsp. paratuberculosis (broad, poorly-defined host range), and the recently described MA subsp. hominissuis (humans and swine). MA are ubiquitous in the environment and evidence suggests water is a possible source of human exposure. Routes of exposure to waterborne pathogens include ingestion, inhalation of water vapor, and ingestion of produce irrigated or washed with contaminated water. The goal of this study was to determine how specific MA subspecies hominissuis is to humans by subspeciating human clinical isolates of MA and also to determine the proportion of MA isolated from food and water that belong to this subspecies. Understanding the occurrence of MA subsp. hominissuis in water and food will aid the U.S. Environmental Protection Agency in assessing the risk of human exposure to these sources. Temperature growth range, IS1245 copy number, sequencing of the 16S-23S internal transcribed spacer region or hsp65 gene and other methods have been used to differentiate subspecies hominissuis from the other subspecies. MA subsp. hominissuis can grow on Lowenstein Jensen (LJ) medium supplemented with pyruvate at 45 ۫C while bird type isolates cannot (Mijs et al., 2002). Clinical and environmental MA isolates from our collection were inoculated on this medium and allowed to incubate for up to 90 days. Ninety percent of the isolates grew at 25 ۫C and 67% grew at 45 ۫C. Isolates that grew at 45 ۫C also grew at 25 ۫C. Nearly 100% of the human isolates included in this study grew at 45 ۫C, suggesting they are strains of MA subsp. hominissuis. In addition, we performed PCR and sequence analysis on the 3′ end of the hsp65 gene as a means to confirm MA subsp. hominissuis identification. Human isolates had hsp65 sequences closely related to hsp65 sequences from MA subspecies hominissuis described previously and several hsp65 sequences from environmental isolates were identical or very closely related to those of hominissuis. None of the human or environmental isolates in our study had hsp65 sequences that clustered with MA subsp. avium or paratuberculosis. These data suggest treated drinking water and produce are potential sources of exposure to the type of MA that can infect humans.