I have screened a useful compound, which can inhibit the growth of Staphylococcus aureus and Streptococcus penumoniae at 5 ug/ml. And I have selected a reisistant Streptococcus pneumoniae mutant and this strain can grow at 80 ug/ml.
What seems weird is that: If I culture the mutant in broth without the inhibitor firstly, then add compound to final concentration 80 ug/ml at OD600 0.4, the bacteria will die. But if I pick colonies directly into broth with 80 ug/ml, the mutant can grow up.
I think this weird phenomenon suggests the unstability of the resistant mutant. Is there any possibilty the resistant mutant is due to the temporary overexpression of some efflux pump? Is there any method to test whether the reisistant mutant is due to the overexpressiong of efflux pump?
The most straight forward way to test for active efflux is by using carbonyl cyanide 3-chlorophenylhydrazone (CCCP), can be bought from Sigma Aldrich. This inhibits the activity of the pump, but it is quite toxic, so be careful using it. There are some papers with the methodology for this, check those from Laura Piddock as she used it to test for efflux of fluoroquinolones. The idea is that the MIC of the antibiotic will decrease with the CCCP, usually 3 or more doubling dilutions, indicating that the bacteria has become more susceptible to the antibiotic again. I think normally this is used in agar dilutions tests rather than broths, but it should work in both. Also, be sure to check that the CCCP doesn't inhibit the growth of the bacteria!
Dear Liu,
Observe the reduction of MIC of the strain using the same compound in a suitable medium after adding phe-arg-beta-naphthylamide (PAβN) at a concentration of 20mg/L. If you observe more than or equal to 4 fold reduction in MIC you can think of the possibility of role of efflux pump. But first check the effect of the PAβN alone over the desied strain of bacterium.
Dear Fiona,
Thanks for your nice advice.That is really useful. I will try that. I am also wondering whether the strains I got is not resistant but tolerance, so I want to try some test to distinguish tolerance and resistant firstly. Do you know something about that? Do you think that may be due to tolerance?
Best wishes,
Xue.
@Jose chlorpromazine may be a good inhibitor of some pumps (I think chiefly MDR/PDRtype ABC transporters) but not all within that family and not all other families, so it may not block Liu's pump. I like your PDF thanks for posting.
@Fiona I think CCCP is indeed more general in blocking all energy dependent pumps but indeed you have to check if the effect is synergistic rather than additive as it has some toxicity on it's own.
@Liu It may indeed be an inducible mutant. Do the mutant colonies you pick come from a control plate or a palte with the compound.
Try to induce liquid cultures of the WT and the mutant with about 1/5th of the MIC and after a few hours with the high concentration to see if you induced the resistance.
@Pradyot, I haven't worked with phe-arg-beta-naphthylamide myself but it may indeed be a good additional compound to block yet another family of transporters.
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