As a rule, Antimicrobials are most effective when tested against metabolically active cells. Your variations of MIC may be due to this fact

Yes, I second the above answer. Dont let them stay under unfavorable conditions before you start your inhibition assays. Leaving your Mtb cultures for 48 hrs out of incubator may compromise their overall metabolic fitness. This could be one of the likely reasons that you need lower amount of peptide to see same degree of inhibition.

Where are the cells kept for 48 h?

I am agree with Doctors Elmanama and Alam, for confirmate this the best way is to develop a viability test counting the ufc/mL in Middlebrook agar comparing the times out the incubator, also is possible perform a time kill study with the times out the incubator with 4,8,12,24, 36 and 48 hours for to observe the phenomenon of viability reduction. With best regards

You don't answer the question. In his exp, the metabolically cells are less susceptible to drugs, which usually they are.

You can try other non amino acid antibiotics to see if it only happens for peptides. And also try other bacteria to see if it is common.

A possible explanation could be growing cells are more likely to degrade oligo-peptides which make the them less susceptible.

Interesting explanation Dr Chen, until now we are focused in the technical problem of the MIC values that can change under the culture conditions. For observe your affirmation would be very interesting perform experiments with dormancy mycobacterial cells. Because with other compounds the MIC increase with dormancy cells, not decreased as in this case. With best regards

The cells are kept at room temperature for 48 hours. I have seen they remain variable for up to one month when kept like this. So that can't it explain it.

I believe it must be a dormancy issue and the peptide is active against dormant, non replicating bacteria rather than exponentially replicating.

What do you think? How could I report this?

*viable, not variable

I agree with Dr. Bueno's suggestion to perform a time course study and to quantify the exact cfu/mL.

I agree with Mary Marquart moreover what shae van suggest is also a good point because you are the one who is dealing with this micro-organisam and peptide. You should report them after taking sufficient experiments as suggested by all the paticipants.

Thanks to all

I would like to add that the latent bacilli is certainly a issue and ur compound is likely to be more active against latent mycobacteria and u can run expt to test mic against latent mycobacteria and if u get good mic values that will be an added value to our research. Moreover what method u r using may also be affecting ur mic values. Goog luck

I dont see bacteria getting into latent form in just 48 hrs, bcoz when people work with wayne's model where they deplete the oxygen levels it takes longer period. so i just dont believe that it becomes latent so fast.

regarding MIC issue, do the same exp with rifampicin and isoniazid and find out if u are getting the same variation. even if u get, u ought to design some basic experiment to prove them latent

Good luck!!

This seems interesting to me, but question for me is what strain of MTC you are using and do you see this with M. bovis, M. africanum, or Spoligotype Beijing either. Maybe this is some type of special room temperature associated behaviour. I would not interpret the results of one strain only

Alexander

I do not think they are going dormant within in 48 h especially at room temperature, unless they are subjected to other stresses. If you could induce dormancy by several different stresses, oxygen, pH etc and then you could determine whether your peptide if more effective against dormant cells rather than actively growing cells. Sorry... One last question are kept in the light or dark for the 48 h?

Dark, so if nit dormancy, what is happening?

It's H37Ra. I have seen the same with a MDR clinical isolate

I am using the MABA to determine MIC values. I have seen very slight variation in INH and RIF. Nothing like my peptide.

The way I see it is dormancy and slower growth are different things especially in mycobacteria. Dormancy is a mechanism to survive stressful environments whether within a host or in the environment. Being stored in media at room temperature is not really stressful so there would be not pressure to enter a dormant phase. Other things could be at play, for example the mycobacteria I work with clumps together depending on how it is cultured and changes my data all the time if I do not compensate for it. It also becomes pigmented when exposed to different stresses, which may or may not change something in the cells that make them more susceptible to your peptide. physically things change in bacteria when they reach stationary phase (on a growth curve not mycobacterial dormancy) so there may be something then that has changed.

It sounds really interesting what you have go though and if you can definitively induce the cells into a dormant state and then test you peptide I think you will be on to something.

Doing MICs with mycobacterial species is a bit more tricky. Do you perform a glass bead vortexing step before you run the MIC? Mycobacteria like to aggregate because of their lipophilic nature.

Do you know about mechanism of your peptide? It may be effect in a metabolite that increase in mid long cell or may be inhibit a mechanism that is more active in this state

That is why with the usual routine susceptibility testing against the classical bacterial pathogens as S aureus , E col, Klebsiella , pseudomonas spp ...etc.. Only single medium is used namely Muller Hinton agar or sensitivity agar medium so scientists can fairly compare their data as long as control strains are ok. Because variation in medium composition will indeed affect the growth curves(Lag, Logarithmic, stationary , and decline phase. and thereby the uptake of the drug will vary with each phase. But reproducible results are usually during the logarithmic growth.

With MTB because it is very slow growers generation time around 3.5hr (E coli 20 min) it is hard to control it and the medium must be specified (Middlebrooks 12) in parallel with the control as the resistance ratio response rather than absolute value.

What method of MIC did you use? There are at least 3 differeny methods that are known to be used. Absolute concentration method, Proprotion method, Resistance ratio method etc. M. tuberculosis have characteristic cord formation. The cells are in 'clusters' and have to be homoginised before the DST is done. Review the techniques you used and compare them if the results are reproducable.

As i mentioned before we got to measure the respnse of MTB to the tested peptide , in comparison to a known antimycobacterium drug as a control for the response and then measure the ratio of the test response to the refernce drug to bypass such variability tha coud be attributed to several reasons because the organism is very slow grower and thereby slow mtabolic activity that involes primarly the uptake of the drug to reach its target site

2- During this 48hrs out of the incubator where it was kept is it at room temperature

or at 4 C

3- Was washed of the medium or it was not ???

4- These variable enviromental condition will affect downstream results

5 some times due to starvation, others due temperature shift,or temperature inductions

6- these factor might facilitate the uptake of your test peptide or simple affect its lethal by pass

7- so to annulate these variable we got to run in parallel a known drug to show the similar flactuations

I think a possibility is how you harvest the microorganisms. The solution can´t be helpful to preserve the viability of the cells and suffer any damage in their structure and they turn more sensible. I recommend reviewing the book "Antibiotics in Laboratory Medicine" by Victor Lorian. I look at the 80´s and the 2005 editions. There is a lot of recommendations and technics that can be useful for your research

You should go for a routine MIC testing in both ways. Use  Muller Hinton agar or sensitivity agar medium for comparison of  data with  control strains. What happens during lag to log phase nutrient uptake increase but drug molecules are limited by permeability effect.   This is the main reason that variation in medium composition will indeed affect the growth curves and thereby the uptake of the drug  varies from medium to medium  You should only use results of logarithmic growth, a phase of active growth.