I tried to do MIC tests on my two S. aureus strains, Methicillin-Susceptible S. aureus (MSSA) ATCC 25923 and Methicillin-Resistant S. aureus (MRSA) ATCC 43300. For the MIC test by broth macrodilution, I used Cation Adjusted MH Broth (supplemented with Ca and Mg), while for MIC test by disc diffusion, I used MH Agar. I tried to do the MIC test for 3 antibiotics: Oxacillin, Rifampin and Daptomycin. For Oxacillin and Rifampin, I didn't find any problem. Their MICs seems okay when compared to literature.
However, I do face problem when doing the broth macrodilution for Daptomycin. By broth macrodilution, I always get MIC of >15 mg/L on my MRSA and MSSA, which doesn’t make sense because my strains were purchased from ATCC and should not be resistant to daptomycin. I repeated the MIC tests several times, and always observed that the culture is turbid under high daptomycin concentration (>2 mg/L, which is the breakpoint of daptomycin resistance). I also centrifuged the culture and confirmed that the pellet is indeed growing S. aureus (distinct yellow pigment). My daptomycin was purchased from Sigma, and it is a ready-made solution (1mg/mL stock solution in DMSO). The antibiotic just arrived a week ago, so it should still be active. To ensure that the activity of the antibiotic, I tried to do disc diffusion and observe that under 10 microgram disc potency, the zone of inhibition is ~15 - 16mm in both MSSA and MRSA.
Any idea why my daptomycin is ineffective in killing MSSA and MRSA? Is it possible that the Calcium is degraded in my cation adjusted MH broth and cause the daptomycin to be inactivated?
There are many reasons for this phenomenon as follows:
1. Can you bring Daptomycin from another company.
2. Change MH by Brain Heart Infusion broth.
3. Check MIC by reproducible way such as agar well diffusion method.
4. Check pH of your culture.
All, thanks for the suggestions! I found the problem already.
As I said, it is not the antibiotic, because I tried Disc diffusion method and I observe a zone of inhibition that categorize them as susceptible. It is not the pH either as I used neutral pH water for the medium, and the bacteria are susceptible to other antibiotics except daptomycin in the same MH medium with the same pH.
The problem here is the calcium content. For daptomycin to be effective, the MH broth need to be supplemented with physiological Ca2+ level, that is 50 mg/L. I tried to supplement my MH broth with different Ca2+ level so that the final Ca2+ concentration is 25, 50, and 75 mg/L in the culture. It seems that daptomycin works better at higher Ca2+ concentration (50-75 mg/L), and it turns out that their mechanism of action is dependent on Ca2+ (Silverman, Perlmutter, Shapiro (2003) Antimicrobial Agents and Chemotherapy, 47, 2538-2544). Without Ca2+, they won’t kill the cells. Under 50 mg/L calcium content, the MIC of daptomycin is 0.5 mg/L for both MRSA and MSSA.
Note that the MH agar contain much more calcium content compared to MH broth, but MH agar have high lot-to-lot variability while MH broth don't (D'Amato, Thornsberry (1979) Current Microbiology, 2, 135-138). That's why I observed the zone of inhibition when doing disc diffusion assay.
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