Bicyclomycin ≠ bicyclomycin benzoate

A month ago I wrote a post about a planned experiment using the antibiotic bicyclomycin, to see if it induces H. influenzae cells to develop competence.  At the time I couldn't remember why this was a reasonable question, but a commenter pointed me to this paper, which describes the induction of competence by bicyclomycin in Legionella pneumophila.

Bicyclomycin is expensive, and we're close to broke, but a generous colleague had given us 4 mg of it to use in a trial experiment.  So I put our summer undergraduate to work on the project.  She began by testing H. influenzae's ability to grow in different concentrations of bicyclomycin, since we wanted to use a semi-inhibitory (but not lethal) concentration for our experiment.  We had found a paper that reported the minimum inhibitory concentration (MIC) for H. influenzae was 3.1 µg/ml, so she tested a wide range (up to 20 µg/ml).  But she saw no inhibition of growth at all.

That MIC had been for a clinical strain, not the lab workhorse KW20, so she repeated the test (this time using the neighbour-lab's BioScreen system) for both a clinical strain (86-028NP) and KW20, and for a couple of E. coli strains (the same paper reported MICs for E. coli  strains between 6 and 12 µg/ml), using bicyclomycin concentrations up to 50 µg/ml.  Still no evidence of growth inhibition!

But now I think I've solved the mystery.  Before making up our bicyclomycin stock we searched for solubility info.  We learned that it's reasonably soluble in water, but that there's a related antibiotic called bicyclomycin benzoate that needs to be made up in ethanol.  The colleague who gave us the 4 mg remimded me that she'd sent an email saying to dissolve it in ethanol.  I'd forgotten about this email, but reading it now reminded me of the solubility difference, and when I checked with her I found out that what she'd given us was bicyclomycin benzoate.

The same paper that gave us the H. influenzae MIC for bicyclomycin tested a wide range of derivatives, one of which was bicyclomycin benzoate.  It's MIC for H. influenzae was >100 µg/ml.  No wonder our cells didn't care about the concentrations we tested!

Bicyclomycin is about 10 times more expensive that bicyclomycin benzoate ($280/mg) so I don't think we'll be doing this experiment after all.