Dear All, help me with the interpretation of the results obtained by one of our students -in his Kirby Bauer disk diffusion test appeared peculiar zones. This is not my field, but it trigger my interest. What is the reason of this inner circle?
Please, see the link or the attached file:
http://s10.postimg.org/g04czc0pl/Disk_zones.jpg
The test MO is E.coli.
Thank You in advance!
Hello.
I don't have a didactical explanation, no-one have ever told me and it's quite not written anywhere. It looks like a sort of swarming at a certain antibiotic concentration, like it was over-incubated and had the time to develope some resistant cells at a low concentration (but probably not). Pretty strange. Are you sure that the bacterial strain was an isolated single one?
I can tell you that I've never seen something like that with an E.coli, it only appened to me with some Pseudomonas aeruginosa in difficult CF isolates. I thought it could be due to a double strain presence, or maybe a fago-lysation happened (which is common in P.aer).
The explanation I gave to myself at that time was: since that sample was from a muliple colonized patient, could be due to a double strain presence. Also, it comes to my mind it can depends on the incubation period or maybe (could be a crazy idea) some cells produce enzimes that at the specifi concentration of the "first halo" can degradate the antibiotic, allowing the cells right nex to grow but then, when the effects end the antibiotic inhibit again and you have the "second halo" ???
Too crazy?
I'd like to have the explanation of someone with much more experience than me.
Does this make any sense?
Which E.coli strain are you using?
What drugs?
Without knowing anything else, and it's been a bit since I've done one of these, I would guess that those bacteria in the outer ring are resistant in some way. This doesn't mean the whole strain is resistant - in fact, depending on your measurements, they may be susceptible overall.
You have a zone of clearing, then some bacteria, then another small ring of clearing. We only see the bacteria that can survive in the presence of whatever drug you've used. Keep in mind that the closer you get to the disc, the more concentrated the drug, so close to the disc susceptible strains won't survive. As you move further from the disc, the concentration decreases. Sometimes at the edges funny things happen.
What's this mean? It means if the concentration of pharmaceutical is low enough at the far edge of the zone of clearing it may not be toxic. For instance, if I remember correctly, E.Coli use efflux pumps to drive toxic compounds like Tet out of the cell - it is possible that those bacteria you see in the outer ring are doing this better, or that the drug has begun to break down, etc. What really matters is the measurement of that inner zone of clearing.
Hope this helps.
Message me if you want more on this and I'll dig through some references.
Dear Ramona and Shayne,
Thank You for detailed answers. As for the actual case – this results appeared in routine students exercise, guided by one of mine colleagues. So I will ask him additionally about the details for the strain characteristics. According your answers, I presume these possible explanations: First, (most probable) – contamination of the laboratory strain. Second, presence of some percent of resistant cells (I am almost sure that the paper discs are too old); Third, Ramona, you have crazy but very intriguing idea. Must be discussed further. Next, one of the above combined with over-incubation. And last, I like Shayne idea about efflux pumps plus drug break down. Anyway, this caused interesting and useful discussion among students.
Thank You both!
We observed similar phenomenon and tested if it was due to efflux of the antibiotics. We found that certain high level concentration of antibiotics can induce efflux pumps which enable them to survive at higher concentration of antibiotics. However, concentrations below that particular high level of antibiotics fail to induce the efflux pumps and hence are inhibited by the antibiotics, thus resulting in double zone of inhibition. I have added the figures to explain the phenomenon.
Here is the paper for reference:
https://doi.org/10.1080/26896583.2020.1781494
Will I Think the problem in antibiotic disc my be it expired or the dregs diffusion is abnormal at first diffuse well and then stope and at end start diffuse again
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