Hi all,
I'm conducting hydrogen peroxide MIC screens on various strains of Group A Streptococcus and thought to use catalase-positive Staphylococcus aureus strain Newman as a comparison microbe. While the positive controls of my S. aureus plates are growing, I note no growth of bacteria in any well containing hydrogen peroxide, even down to 5.8x10-4% peroxide. I have repeated this experiment twice with identical results. In contrast, the supposedly more ROS-sensitive Streptococcus are growing in the 5.8x10-4% peroxide wells. Any ideas on why my S. aureus is not growing in the presence of these (very low) concentrations of H2O2? Thanks!
I am suspecting the S. aureus culture, if you have already confirmed the S. aureus by specific test or PCR characterization. Then you should perform the qualitative (Slide) test for catalase activity, before proceeding to MIC assay. For slide test you can compare with the strep isolates as well.
Best
5.8x10-4% hyrogene peroxide are equal to around 0.2mM
In my experiments the MIC was 0.2mM for S. aureus and 0.5mM fo S. pyogenes (GAS) and therfore similar to your results.
1. These results are significant lower the published data, but most methods use complex media like THB, THY, LB, ...
2. Complex media contains keto acids and peptides with thiol groups which slowly deoxidize H2O2 over time. And after 24h incubation the final H2O2 concentration is lower then expected and therefore a lot of published data overestimates the MIC for H2O2. (e.g. THB buffers H2O2 up to a final concentration of 4.8mM)
3. Possible reasons why the MIC for catalase-negative GAS is higher then for catalase-positive S. aureus:
GAS also exhibit alot of detoxing enzymes, like peroxidases, iron binding proteins, repair proteins, ...
GAS possess a very potent NADH-Oxidase which can detoxify H2O2 and doesn´t require NADPH like most other detoxing enzymes. (NADPH production might be limited, e.g. pentose-phosphate pathway)
GAS have a glycolytic metabolism, whereas S. aureus has a respiratory metabolism. The latter contains more iron- and copper-dependent enzymes and therefore a higher risk for the Fenton-reaction.
4. I recommend the determination of MBC, which gives similar results for 24h incubation. But at a 30min incubation with a high concentration of H2O2 (80 - 200mM), which simulates the oxidative burst in human neutrophils, S. aureus shows a higher survival (MBC) compared to GAS.
A standardized protocol similar to antibotic MIC determinations together with my data are allready published:
https://www.ncbi.nlm.nih.gov/pubmed/30395910
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