I have a problem interpreting my MIC (minimum inhibitory concentration) results determined by the the alamar blue assay. Briefly, I am investigating the MIC of a cationic peptide against M. tuberculosis (H37Ra ATCC 25177). I have investigated the MIC of my compound in three media (7H9 with OADC (oleic acid, albumin, dextrose, catalase) enrichment, 7H9 without OADC enrichment and Sauton media [cation adjusted]), for the reason that I believe OADC enrichment causes an interaction with my cationic peptide, falsely elevating it's MIC.
I performed the assay as published previously by Franzblau et al. with a starting inoculum of 5 x 10^5 CFU/ml. For 7H9 with OADC and 7H9 without OADC, alamar blue is added on day 5 with color change from blue to pink indicating sufficient cellular growth in control wells and my MIC results are read. For Sauton media, alamar blue is added on day 9 and results are read. I recorded the following trend in MICs: 7H9 with OADC = > 100 ug/ml, 7H9 without OADC = 25 - 50 ug/ml and Sauton media = 0.39 - 0.77 ug/ml.
It must be noted that all my controls check out for my experiment i.e., no contamination etc.
What could explain this trend of decreasing MIC results? Is the decrease in MICs observed due to different growth rates and longer lag phases or possibly due to less drug interactions with the media?
I have not investigated how the MICs of isoniazid and rifampicin change with the different media. I guess this is the most logical starting point since these MICs are known.
If there is a change in MIC results (including isoniazid and rifampicin) does this mean there is a problem with growth rates? If I determine that 7H9 and OADC are in fact causing an drug interaction, but Sauton is not, how would it be possible to increase my growth rate to get comparable MIC results for isoniazid and rifampicin so that I can determine the correct MIC for my cationic peptide?
Any suggestions and help will be greatly appreciated.
Thank you
The middlebrook media are well known to yield higher MICs for many drugs, most notably the aminoglycosides. See my attached review paper. Why do you use the Suton medium? It is not a frequently used medium for M. tuberculosis, particularly not for DST. For standardization/harmonization matters, using middlebrook (with all its oddities) may be preferable.
Article Resistance mechanisms and drug susceptibility testing of non...
I am using Sauton media because I believe that Albumin and oleic acid interact with my peptide, producing falsely elevated MICs. Furthermore, OADC contains NaCl and it is known to antagonize the effect of cationic peptides. I would like to determine the MIC of my peptide in vitro with the least amount of interactions with media and any antagonizing effects. I have planned equilibrium dialysis of my peptide with media to determine if my hypothesis is correct and I'll quantify this doing UPLC.
I believe that comparison of different media at different time points (5 days vs 9 days) is not fair. You can start with including a positive control like Isoniazid and then look for the MIC at same time point (I add Alamar Blue to 7H9 + OADC media after 7 days) in different media.
As for your hypothesis that OADC interacts with the peptide, I guess thats true as you see different MICs with and without OADC in 7H9 medium.
With Sauton, the MIC is really low but this could be due to differences in the bacterial growth. It has been shown that BCG grown in Sauton media was more persistent inside macrophages (Vaccine, 2012, Venkataswamy et al).
All the best for your future experiments.
Thanks very much,
The reason I observe MICs on day 5 for 7H9 + OADC and day 9 for Sauton, is that those are the minimum days of incubation in each respective media to reach the minimum required actively dividing CFU/ml that would change Alamar blue from blue to pink in 24 hours. Thus, I have to incubate for a minimum of 9 days in Sauton media and I think that incubating in 7H9 + OADC for that long will cause an overgrowth of bacteria.
I think it is sort of fair adding alamar on those respective days since the amount of CFU/ml growing in each control well for each media are comparable.
Hi Shane,
Although I ma not a specialist of MIC calculation in mycos, I suggest you have a look at two papers: Sirgel et al. 2009 Measuring minimum inhibitory concentrations in mycobacteria. Methods Mol Biol 465:173-86; and Martin et al. 2005 Multicenter study of MTT and resazurin assays for testing susceptibility to first-line anti-tuberculosis drugs. Int J Tuberc Lung Dis 9(8):901-6.
There are plenty of techniques for DST in mycos, I would readily go for a simple technique like turbidity measurement or even better plating on serial dilution. Including RIF of INH is a good idea, as their MIC is widely known. Also look at papers by Yin Zhang from Johns Hopkins University, Baltimore, for instance, he's a master in the field, you could even contact him on my behalf, [email protected].
Best luck!
Olivier
I have a other problem using 7H9 and homemade oadc with isoniazid. It doesn´t work sometimes. Sometimes the well with isoniazid and bacteria in all dilutions become pink (little bit), but sometimes not. Before, it never happened. I changed everything: isoniazid, media, plates and etc. Some can help? Have the same problem?....ahh...others pre-drugs like ethambutol i have the same ploblem.
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