Rapid, Quantitative Assessment of Antimycobacterial Water Disinfection based on the Firefly Luciferase Reporter Gene
Cowan, Heather Elizabeth
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Mycobacterium avium causes disseminated infection in humans with immunodeficiency, pulmonary infections in individuals with predisposing lung conditions (e.g., pneumoconiosis), and cervical lymphadenitis in children. Twenty-five to fifty percent of late stage AIDS patients are infected with M. avium. M. avium has been recovered from drinking water and strains from water share identical DNA fingerprints with isolates recovered from patients exposed to the water. Further, M. avium is resistant to chlorine, a disinfectant commonly used in municipal water supplies. Because of the slow growth of M. avium, measuring its susceptibility to disinfectants is laborious and reaction to a potential problem is delayed. Thus, there exists a need for a rapid test to measure the antimycobacterial disinfectant capability of chlorine containing water samples. The objective of this research was to develop a rapid and quantitative assay for the viability of mycobacteria using firefly luciferase as a reporter gene for disinfection survival studies. Derivatives of M. avium strains MD1 and A5, Mycobacterium smegmatis strain VT307 and Mycobacterium bovis BCG strain Pasteur carrying the firefly luciferase gene (pLUC10) were constructed. In pLUC10-carrying strains of M. avium strain A5 and M. smegmatis strain VT307, a direct correlation was shown between the quantity of light produced and the number of cells recovered as colony forming units. In disinfection studies of both pLUC10-carrying derivatives of M. avium strain A5 and M. smegmatis strain VT307, survival, as measured in colony forming units, correlated with survival in relative light units. Luciferase measurements appear to offer a method for rapid enumeration of mycobactericidal disinfection capacity of chlorinated water.
- Masters' Theses