Our long-term goal is to exploit """"""""unculturable"""""""" soil microorganisms for production of broad-spectrum antibiotics against potential biowarfare agents. Soil microorganisms have served as the main source of antibiotics, but only 1% grow in vitro, and have been over mined. In this exploratory project, we will develop approaches to culture the bulk of this previously inaccessible biodiversity, based on a method we developed for growing uncultured marine organisms (Kaeberlein, T., Lewis, K., and Epstein, S.S. (2002) Isolating """"""""uncultivable"""""""" microorganisms in pure culture in a simulated natural environment. Science 296:1127-1129). We also aim to achieve proof-of-principle for obtaining antibiotics from uncultured soil microorganisms.
The specific aims are: 1. Growing uncultured microorganisms. Methods for growing soil microorganisms in situ will be optimized. This will form the basis for then developing in vitro culturing methods, based on our findings with marine organisms. The two approaches we will use are """"""""domestication"""""""", sequential subculture in a diffusion chamber and subsequent adaptation to growth in vitro; and co-culture with symbiotic organisms+ 2. Screening for antimicrobial activity against BW agents. Extracts from individual microorganisms will be obtained and tested for growth inhibition activity with an avirulent Bacillus anthracis* and LVG strain of Francisella tularensis. Hits will be verified against virulent strains of B. anthracis and F. tularensis (NIAID category A). 3. Purification and identification of antimicrobial compounds. Early-stage dereplication will indicate extracts containing antimicrobial compounds of chemical novelty, and these will be used to isolate a pure substance. A sufficiently pure compound will be used to determine MIC with the pathogen strains, and those with high potency will be studied further. Determination of chemical structure will be performed by a combination of MS and NMR methods.
| Gavrish, Ekaterina; Bollmann, Annette; Epstein, Slava et al. (2008) A trap for in situ cultivation of filamentous actinobacteria. J Microbiol Methods 72:257-62 |
| Bollmann, Annette; Lewis, Kim; Epstein, Slava S (2007) Incubation of environmental samples in a diffusion chamber increases the diversity of recovered isolates. Appl Environ Microbiol 73:6386-90 |
