Disk diffusion whole cell microbiology assays

[BabcockHall]

Hello Everyone, I am making enzyme inhibitors and turning them over to a microbiology laboratory for testing against standard strains of bacteria and fungi.  They are performing disk diffusion assays, and seeing a few results that look positive.  I would like better to understand the meaning and limitations of this experiment.  I would hazard a guess that the compound's concentration falls off at greater distances from the disk, but I don't know how to interpret the diameter of the dead zone in a quantitative way, or even if that is possible.  Perhaps there is a good textbook treatment of this subject, for example.

[CharonY]

For the most part it is used semi-quantitatively by measuring and comparing inhibition zones. It is in principle possible to try to estimate concentration, but it is a bit tricky. As the process is diffusion, the concentration decreases with the square of the distance. But then you also need the diffusion coefficient of the compound and also consider that diffusion happens also in three dimensions. But often the theoretical values or simulation are going to bit off unless you spend a lot of time to make the assay very accurate. However that is counter the typical benefit of such assays, i.e. being quick. There are quite a few other issues with quantifying antimicrobial activities (e.g. factors that influence MICs) but that is not specific to disk diffusion assays.

[BabcockHall]

One other issue occurs to me.  As the compound diffuses, the microorganisms are growing.  So it is possible that the compound arrives at a certain place with a particular concentration only after growth has ceased for other reasons, such as buildup of waste or consumption of nutrients.

Edited May 29, 2021 by BabcockHall

[CharonY]

Somewhat possible, but the compound should still have some influence. In the assay you should have sufficient nutrient and cell density to create a lawn in absence of interfering compounds. However, nutrient levels do decline with growth and the ability to grow in presence of stressors is dependent on the cellular status. Another issue is that the assay cannot identify e.g. dormancy or tolerance. One way to check it is to remove the disk and replace it with one soaked with medium. If you see small colonies in the inhibition zone, or even a lawn developing, that would be indicative of growth inhibition instead of killing, for example.

[PhilGeis]

Disk diffusion (aka Kirby Bauer) does not give quantitiative results, and you should ot assume a "dead zone."   You're looking at inhibition of growth.  Your scenario of concentration dynamics after growth "has ceased" is not realistic - do not wate time on it.   If you stuff is that isoluble you need another test protocol. 

Quantitative estimates can be obtained by microbial inhiibtion testing - typically determining growth in context of serially  diluted the test compound in liquid (broth) growth medium inoclated with test micororganisms.   Here's on group's description - https://emerypharma.com/wp-content/uploads/2017/12/mic-guide.pdf

This kind of testing is largely academic - do you have a specific application in mind?

Edited June 1, 2021 by PhilGeis

[BabcockHall]

That is a helpful reference; thank you.  We synthesized a family of compounds that are good inhibitors of an essential microbial enzyme.  As a whole they are not very water-soluble, although there is variation in the group.  One of the more hydrophilic ones is one of two that showed growth inhibition.  Therefore, we are synthesizing the next generations of inhibitors that might be more water-soluble and thinking about what are the meanings of various test results.

Edited June 2, 2021 by BabcockHall

[PhilGeis]

How developed is this effort and to what eventual application are you thinking?  

Potential use as chemotherapeutic agents would be decades away but there are near term commercial applications that might find this of value.  There is so little research into the latter that even early efforts get attention.

[BabcockHall]

The enzyme has been shown to be essential in some pathogenic bacterial strains and also in Candida albicans.  We are in the process of revising our first manuscript on our inhibitors.  For the best compounds in this series, the ratios of binding energies to molecular weights are promising, among other possibly favorable properties.  We envision either an antifungal or antibacterial compound, although at the moment we are more fungal-focused.

What sort of near-term commercial applications do you mean?

Edited June 2, 2021 by BabcockHall

[PhilGeis]

Biocides - including preservatives.

any patents ?

[BabcockHall]

I asked my institution to file a provisional patent, but I have not yet heard back.

[CharonY]

7 hours ago, BabcockHall said:

As a whole they are not very water-soluble, although there is variation in the group.  One of the more hydrophilic ones is one of two that showed growth inhibition. 

If the compounds have different solubility the results in disk diffusion are difficult to interpret at best. While some folks use organic solvents as carriers, I am quite skeptical about the usefulness of such tests. MIC determination in broth is better but you would still need to see if solubility is an issue (and run the test with organic diluent and an appropriate control). Considering that you likely have a good idea of the mechanism of inhibition I would actually also check whether the medium composition has an impact- we found MICs to be a bit of a moving target and does depend on a number of factors (including medium manufacturer, but also growth state of the diluted microbe).

 

 

[BabcockHall]

Our collaborators used only volatile organic solvents to dissolve the compounds prior to the preparation of the disks.  I am not sure whether or not this information is useful.

Edited June 2, 2021 by BabcockHall

[PhilGeis]

The only interpretation is an observable zone of inhibition  +/-., please don't attempt additional interpretation.    Some use solvents like DMSO or ethanol in MIC testing of materials of limited aqueous solubility - generates numbers but introduces another variable.

II'm with Charon on the testing.  It's not hard find materials showing some degree of inhibition in such testing.  Patent and tehnical literature is full of such gee whiz reports.    What will you do with materials of some antimicrobial capacity?

[BabcockHall]

I don't know the answer to your question, but I am inclined to say that we will try to optimize the compounds.  With regard to these compounds, we have in vitro data against a validated target enzyme.  They are irreversible inhibitors with ligand binding efficiencies greater than 0.3.  The second generation inhibitors that we are currently synthesizing were designed with transport across membranes as a consideration.

Edited June 3, 2021 by BabcockHall

[PhilGeis]

Perhaps add aq. solubility as secondary dev objective

[BabcockHall]

We are synthesizing the next generation of inhibitors, and they have increased water solubility.

[PhilGeis]

Suggest you consider an application - relevant evaluation of efficacy in addition to MIC/MBC.    There's no shortage of compounds reported in literature/patents to have "broad spectrum" efficacy at low concentrations.

[BabcockHall]

Is there any point in repeating a successful disk diffusion assay at lower starting concentration of substance?  Or would it make more sense to move to some form of minimum inhibitory concentration assays?

[PhilGeis]

On 4/1/2022 at 6:26 PM, BabcockHall said:

Is there any point in repeating a successful disk diffusion assay at lower starting concentration of substance?  Or would it make more sense to move to some form of minimum inhibitory concentration assays?

No.  Just run an MIC.