Efficacy of calcium hypochlorite and ultraviolet irradiation against Mycobacterium fortuitum and Mycobacterium marinum.

BACKGROUND: Nontuberculous mycobacteria (NTM) cause opportunistic infections with increasing frequency in immunocompromised humans. Water is one of the natural sources for transmission of NTM and plays a major role in the epidemiology of NTM infections. This study evaluated the efficacy of calcium hypochlorite and ultraviolet irradiation (UV) to eliminate potentially zoonotic NTM species such as M. marinum and M. fortuitum. MATERIALS AND METHODS: Bacterial suspensions containing1-4 × 105 CFU/ml were exposed to 5, 50, 100, 1,000 and 10,000 mg/L of Ca (OCl)2for 1, 5, 10, 15, 20, 30 and 60 minutes, and 6,000 µW/cm2 UV dose for 5, 10, 20, 30, 60 and 120 seconds. RESULTS: Of the two methods tested, UV irradiation was more effective than chlorine in achieving three log reduction in viable bacterial count (UV dose 6,000 µW/cm2, exposure time 60 S) as well as in eliminating the organisms (UV dose 17,000 µW/cm2, exposure time: 30 S). When 10,000 mg/L of chlorine was used, 10 and 20 min contact times were required to achieve three log inactivation and complete elimination of M. fortuitum respectively. CONCLUSION: Our study suggest that initial disinfection of water by chlorine at the water treatment plant followed by UV irradiation at the household level would minimise the spread of NTM to the susceptible population via drinking water.

Transfer, stable maintenance and expression of the mycolactone polyketide megasynthase mls genes in a recombination-impaired Mycobacterium marinum.

The human pathogen Mycobacterium ulcerans produces a polyketide metabolite called mycolactone with potent immunomodulatory activity. M. ulcerans strain Agy99 has a 174 kb plasmid called pMUM001 with three large genes (mlsA1, 51 kb; mlsA2, 7.2 kb; mlsB, 43 kb) that encode type I polyketide synthases (PKS) required for the biosynthesis of mycolactone, as demonstrated by transposon mutagenesis. However, there have been no reports of transfer of the mls locus to another mycobacterium to demonstrate that these genes are sufficient for mycolactone production because in addition to their large size, the mls genes contain a high level of internal sequence repetition, such that the entire 102 kb locus is composed of only 9.5 kb of unique DNA. The combination of their large size and lack of stability during laboratory passage makes them a challenging prospect for transfer to a more rapidly growing and genetically tractable host. Here we describe the construction of two bacterial artificial chromosome Escherichia coli/Mycobacterium shuttle vectors, one based on the pMUM001 origin of replication bearing mlsB, and the other based on the mycobacteriophage L5 integrase, bearing mlsA1 and mlsA2. The combination of these two constructs permitted the two-step transfer of the entire 174 kb pMUM001 plasmid to Mycobacterium marinum, a rapidly growing non-mycolactone-producing mycobacterium that is a close genetic relative of M. ulcerans. To improve the stability of the mls locus in M. marinum, recA was inactivated by insertion of a hygromycin-resistance gene using double-crossover allelic exchange. As expected, the DeltarecA mutant displayed increased susceptibility to UV killing and a decreased frequency of homologous recombination. Southern hybridization and RT-PCR confirmed the stable transfer and expression of the mls genes in both wild-type M. marinum and the recA mutant. However, neither mycolactone nor its predicted precursor metabolites were detected in either strain. These experiments show that it is possible to successfully manipulate and stably transfer the large mls genes, but that other bacterial host factors appear to be required to facilitate mycolactone production.