Once again, with the real bicyclomycin!

Last month I wrote that we were abandoning our plan to test whether the antibiotic bicyclomycin induces competence in Haemophilus influenzae, as it does in Legionella pneumophila, because (i) the free 'bicyclomycin' we'd been given by a colleague turned out to be bicyclomycin benzoate, and (ii) the real bicyclomycin we wanted to test cost hundreds of dollars per milligram.

But last week I got the budget statement for our NSERC grant and discovered that we're not as broke as I thought.  The credit for this goes mainly to the PhD student, who has been earning a large fraction of his annual stipend by working as a teaching assistant!  So the summer undergrad and I worked out how much bicyclomycin we'd need to test its effect, and found that 1 mg should be enough to detect if there is any effect, and if there is to begin characterizing it.

I'm going to write out the plan and explain our calculations here, to check that we haven't made any dumb mistakes.

Step 1:  Confirm that the reported MIC is correct and determine the best concentrations to test.

We will use the lab next door's Bioscreen incubation system for this.  It can record density changes in two culture plates, each with 100 wells holding 0.3 ml of culture each.  We'll only use a single plate.

Muller et al (1979) tested a wide range of bicyclomycin derivatives, and reported that the MIC (minimum inhibitory concentration) of bicyclomycin for H. influenzae is 3.1 µg/ml.  (MICs are typically only tested in increments of 2-fold, so this is a rough estimate.)  We will evaluate the following µg/ml concentrations for growth effects: 0, 0.1, 0.2, 0.5, 1.0, 2.0, 3.5, 5.0, 10.0 and 20.0.  That's 10 wells (3 ml) of each concentration, which would need 126.9 µg.  We'll actually make up 3.5 ml to allow for measurement errors, so I'll round this up to 150 µg.  We could even omit the 20 µg/ml test, and would then only need about 80 µg.

We should start these cultures with a fairly high density of cells, already in log phase, rather than the usual very low density.  With the usual low density, the cells must double for at least several hours before the culture turbidity becomes high enough to detect, but these first doublings are where we would see the effect of bicyclomycin.  So we should start the Bioscreen cultures with cells that are in roughly the same state and density as the cells will be in the competence-test described below.  The actual density to use is discussed there.

Notes after setting up the actual experiment:  We decided to replace the [0.1 µg/ml] treatment with a no-cells treatment.  The culture we used was in log phase, at OD600 = 0.1 (about 3 x 10^8 cells/ml).  When we went to make up the bicyclomycin stock, the vial we had purchased appeared to be completely empty.  Looking at it with the dissecting microscope revealed a tiny amount of dust-like material in the vial; we were reassured when it dissolved rapidly in water., and I'm now optimistically assuming that the 1 mg of bicyclomycin in the vial had been added as a liquid and dried onto the bottom of the vial.  But if we don't see any effect of the bicyclomycin on cell growth, I'll begin to question whether the vial actually contained any.

Step 2:  Examine kinetics of growth and survival in different concentrations:

The Bioscreen only measures OD600; this is a good indicator of initial growth but not of long-term survival.  At the end of the run we will collect the cultures from individual wells, and dilute and plate the cells to measure overnight survival.  I think we should test two wells of several different concentrations: 0 (the control), the highest concentration that gave normal-looking growth, a concentration where culture growth was slowed but eventually reached normal density, and a concentration that drastically reduced growth.

Maybe a killing-curve experiment:

We'd be happy to do the competence-induction test with a concentration of bicyclomycin high enough to inhibit growth, but we don't want to use one that will actually kill the cells in the 30-90 minute incubation periods, since then we couldn't test whether any cells became competent.  The only way to find this out will be to design a killing-kinetics experiment after we have the Bioscreen results, where we give cells a high concentration and take samples every 15 minutes to measure survival.  But probably this can wait until after we have the results of the first competence-induction test described below.

Step 3:  Test effect of short-term exposure to bicyclomycin on competence induction:

The induction experiment:  (first draft)

This is intended to be a quick-and-dirty experiment.  We won't worry about getting all the conditions just right, but will quickly assess a range of plausible conditions to see if any induce competence.

1. Start with non-competent cells (in log-phase growth, at OD600 = 0.2?).
2. Transfer 5 ml of the culture to tubes or tiny flasks with three different concentrations of bicyclomycin: one that slows growth without killing the cells, one that more severely inhibits growth, and one that completely stops growth within 90 min.  Also a negative control culture with no bicyclomycin, and a positive control culture with 1 mM cAMP.
3. At three different times (30 min, 60 min, 90 min?), pellet 1 ml of cells and resuspend them in medium containing MAP7 DNA (no bicyclomycin).  Incubate for 1 hr (to allow continued development of competence and then DNA uptake), dilute and plate ± novobiocin to measure transformation (and survival).

Issues:

Timing and initial density: As planned above, total growth times will range from 90 min to 150 min; this is enough time for about 2.5 to 4 cell doublings under normal conditions.  Initially I planned to start with cultures at OD=0.2.  But from this initial density the negative control (no bicyclomycin) cells would go on to develop the usual low-level competence during the incubation with DNA, and transformation frequency would be 10^-5 - 10^-4 even for the shortest growth time. This would also mask the induction we expect in the positive control (+ cAMP).

What if we started at OD = 0.05 instead?  Negative-control samples using 90 min of total growth time would not give any transformants.  Samples that had longer incubations would, but we might still be able to detect an induction effect.

Or we could start even lower.  But this risks using so few cells that we can't detect low-level increases in transformation frequency (the limit of detection depends on the cell density), especially with bicyclomycin concentrations that inhibit growth.

We could use different initial densities for the different concentrations (OD=0.05 for 0 and low bicyclomycin and cAMP, and 0.1 for the higher concentrations).

Time management issues:

Another timing consideration is the need to be doing many things at once.  As set out above, we'd need to be pelleting and resuspending the t=90 samples at the same time as we're diluting and plating the t=30 samples.  Better to spread out the exposure times a bit more (say 20 min, 60 min and 100 min), so the different tasks are due at different times.  The initial choices were semi-arbitrary, so these new times should be just as good.

Semi-final plan:

1. Start with non-competent cells (in log-phase growth, at OD600 = 0.1?).
2. Transfer 5 ml of the culture (undiluted or diluted 1:1 with sBHI) to tubes or tiny flasks with no drug, cAMP (1 mM), and three different concentrations of bicyclomycin, chosen after consideration of the Bioscreen results.  Use the undiluted culture for flasks with high bicyclomycin.  
3. At three different times (20 min, 60 min, 100 min), pellet 1 ml of cells and resuspend them in sBHI medium containing 1 µg MAP7 DNA (no bicyclomycin).  Incubate for 1 hr, dilute and plate ± novobiocin to measure transformation and survival.