In vitro models of mycobacterial survival during transmission focusing on tuberculosis, desiccation and bacterial lipid bodies.

Background: Understanding how Mycobacterium tuberculosis (Mtb) survives stresses encountered during transmission could open up new ways to control tuberculosis. Lipid metabolism is central to Mtb survival during infection and may contribute to transmission. Bacterial lipid bodies (LBs), a key lipi product, increase the environmental survival of an Mtb relative, Rhodococcus spp. Using M. bovis BCG as a surrogate for Mtb and luciferase to report on viability, the aim of this work was to test whether LBs support survival during desiccation, a key stress encountered during transmission. Additional work was done with M. smegmatis and M. abscessus.

Methods: Following assessment of multiple alternate formats, desiccation survival assays were established separately using plate and filter systems. The former allowed assessment in droplets while the latter involved more diffuse distribution of the test organism. Only luminescence could be tested with the plates while additional colony forming (CFU) tests were enabled by filters. LB contributions were manipulated by defined media and use of a lipase inhibitor. Preliminary work was done on aerosol survival in Goldberg drum experiments.

Results: Luciferase assays on M. bovis BCG ffluc during desiccation indicated an initial 1-2 log10 fall in viability and a slower subsequent decline. In contrast, the filter desiccation assay showed slow decline in survival by luminescence and a rapid decline by CFU. Preliminary evidence for enhanced survival attributable to LBs was found but variation in results indicated that validation with higher replicate numbers might provide confirmation. A preliminary Goldberg drum experiment was also undertaken.

Conclusion: Despite preliminary indications, the hypothesis that high LB content promotes desiccation survival was not clearly supported. Multiple desiccation assay formats were explored and will provide a strong basis for future studies. Further work is needed to understand relationships between luminescence- and CFU-based estimations of mycobacterial viability.