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Detection of Mycobacterium avium subsp. paratuberculosis in Drinking Water and Biofilms Using Quantitative PCR
Beumer, A., D. N. KING, AND S. L. PFALLER. Detection of Mycobacterium avium subsp. paratuberculosis in Drinking Water and Biofilms Using Quantitative PCR. Presented at American Society for Microbiology General Meeting, Boston, MA, June 01 - 05, 2008.
1) Develop a real-time PCR method for the rapid detection and quantification of Mycobacterium avium and M. intracellulare in drinking water. 2) 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. 3) Determine the subspecies of M. avium complex bacteria in EPA culture collection.
Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne’s disease in domestic animals and has been implicated in Crohn’s disease in humans. Cows infected with Johne’s disease shed large quantities of MAP into soil. Further, MAP has been isolated from surface water, is resistant to chlorine and able to form biofilms; therefore, it is likely to colonize drinking water distribution systems and be found in drinking water. Occurrence of MAP in drinking water has never been demonstrated. Because MAP is extremely difficult to isolate from water quantitative PCR (QPCR) assays have been developed to identify the presence and estimate the number of MAP. We used primer and probe sets designed to target IS900 sequences in the MAP genome, and we confirmed our IS900 results using Target 251 which is known to be specific to MAP. Drinking water was collected from 31 cold water faucets in southwestern Ohio. Two one liter samples were collected: a first pull sample, taken when the water was first turned on, and a standard method sample taken after running the water two minutes. The first pull is intended to collect cells from household pipes and potentially cells sloughed from biofilms; the standard method sample is intended to collect cells primarily from the water column. Biofilms were collected by swabbing the entire faucet grating, followed by vortexing in sterile water to remove attached biofilm. Water and biofilm samples were filtered, DNA was extracted and analyzed using QPCR (IS900 and Target 251 primers). Eighty four percent of first pull samples were positive for MAP, 92% of standard methods samples were positive, and 89% of biofilm samples were positive for MAP. Numbers of MAP (assuming 18 copies IS900 per organism) ranged from 0 to 29,000; however 82% of samples had less than 500 organisms per liter or faucet biofilm. There does not appear to be any statistical difference between presence in the drinking water distribution system and biofilm or first pull samples. This study shows that MAP is common in drinking water distribution systems, residential pipes and residential biofilms.