E. coli producing strain of B-lactamase grow normally in presence of pencillin and also in presence of my unknown substance. But in combination between them the bacteria stop growing completely.
This means that my substance is not antibiotic but it inhibit B-lactamase. After extracting the enzyme and study the effect of substance on it, I found no inhibitory effect of the substance.
Does anyone know any substance that stop B-lactamase production "although in presence of pencillin"?
It depends on the B-lactamase, Each class is susceptible to different inhibitors. Once you have narrowed down which type of B-lactamase you are dealing with, it will make identification of the inhibitor easier. You may want to check out this reference on aptamers as metallo-beta-lactamase inhibitors.
http://www.ncbi.nlm.nih.gov/pubmed/19751419
Thanks Cynthe.. my B-lactamase is pencillinase which inhibited by clavulanic acid (class A). But my substance can't inhibit the enzyme "by using 0.5 ml in assay". it inhibit its production "by adding 20 microliter to 5 ml culture" not activity.
Do you know any substance that stop B-lactamase production "although in presence of pencillin"? or do you suggest other explanation?
Noha, reason genetically may be occur switch of for gene B-lactamase production due to excess concentration of pencillin
Thanks Doaa.. I used 10 microgram per ml pencillins in culture. not excess conc.
using pencillin only or my substance only gave equal growth to normal culture. However combination stop growth completely.
What genetically switch you mean??
Do you think conc. not suitable??
Switch off mean stop genes related to growth, try use concentration less
Dr noha omr 10 microgram per mill too excess of course this enough to stop growth genes
Dr Doaa this E.coli is resistant to pencillin even if I use 100 microgram per mill. growth stop only if I combined pencillin with my substance
also, my substance alone can't affect growth
The common B-lactamase inhibitors affect the activity and not the production. If your substance does not affect the activity, there is still several possible explanations: 1) transcription of B-lactamase is inhibited, 2) translation of B-lactamase is inhibited, 3) B-lactamase modifies your substance and activates it. Or something unrelated is also possible.
1 and 2 could be tested e.g. by using reporter constructs (lacZ, GFP, ...), 3 by overexpressing B-lactamase in the absence of the antibiotic.
Hi Noha,
Why do you assume your compound inhibits the beta-lactamase? It seems you made in vitro studies with purified lactamase and saw no effect, so it means that the target is not the lactamase. I personally don't think your problem is the lack of lactamase production, buta that your 2 compounds are acting sinergistically against the bacteria. Just to give you an example, if you make MICs for penicillin and clavulanic acid, you'll see a certain value. Now, if you combine both drugs, the MIC value will be much lower, because they are acting in sinergy. I know this 2 compounds share the same targets, but you'll see similar effects with different antimicrobial classes.
Now I have some questions that may help to explain your problem:
1- how do you see that your enzyme is not inhibited by your compound? did you make kinetics? did you use any reporter substrate?
2- what MIC ranges did you use to determine the effect of your compound? What was the highest level?
I would rather think on something easier and simpler to explain what you are seeing...
Thanks Nuno
I extract the lactamase of the same inhibitted E. coli and include my compound in the enzyme assay (as an inhibitor). It didn't affect the activity. I used pencillin G as substrate. My compound is crude metabolite (not pure) so MIC now isn't accurate.
What do you think is easier and simpler to explain what I am seeing??
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