I am testing a range of natural products mostly herbal remedies on C. difficile. trying to assess the mechanism of action for products that have shown me antibacterial activity against C. difficile in vitro.
Would appreciate if you can suggest any assay/protocol
Hi Nilou, you could try several methods like agar-disk diffusion method, agar well diffusion method, bioautography , Time-kill test (time-kill curve), ATP bioluminescence assay or detection of apoptosis. All depends what kind of natural product you have; if you have a essential oil, you'd need to know and do probes; how to mix this product with culture medium. If you have hydroalcoholic extract, first you need to dry this extract, remove water and alcohol and then, try to dissolve it with Dimethylsulfoxide (DMSO ) (Doing stock of extract), do dilutions ant test different concentrations. I suggest a review, maybe it could help you!
Article Methods for in vitro evaluating antimicrobial activity: A review
In my point of view, if you are using crude extracts, you have to separate these products into fractions using silica column then determine the most effective fractions. these fractions should undergoes hplc, GC mass and NMR to identify the active ingredient(s) thus you can asses its mode of action online.
Try to analyse bacterial primary metabolites (Carbohydrate, protein and lipids) in both treated and untreated cultures. Also analysis of broth medium for secondary metabolites should help.
Hi Dr.Nilou Roshan
Warm greetings.
1. I understood from your question that you are trying to assess the mechanism of action for products that have shown me antibacterial activity against C. difficile in vitro.
2. It means you have product that have shown antibacterial activity against C.D. and you want to find its mechanism of action (MoA), which means how is it acting inside said bacteria and killing it or inhibiting its further growth.
3. Usually for MoA one can usually check effect of the bioactive extract on synthesis major macromolecule DNA/RNA/Protein/lipids or cell wall against strong positive and negative controls.
4. Once it shows some activity in any of these then we can investigate substeps in their synthesis. If not active in these then we can plan membrane related activity.
5. Sometime if we know the structure of active compound/s in the extract we can predict possible mechanisms and try to establish its proof for regulatory purpose.
6. Type of assay I mentioned you may get details easily if you google.
7. If you want any more details please let us know.
Hope this helped to some extent.
Good luck.
Best Regards
Girish
thank you Mr Mahajan for your answer, can you suggest some type of assays to assess effect on protein synthesis, DNA,RNA and cell wall inhibition.
thank you,
Hi Dr.Nilou Roshan,
About those type of assay one of the best reference I am sharing with you is the reference "A" pasted below.
I have used this assay several types. One such publication of mine may also be useful to you. Please refer reference "B" in the following paste.
To begin with you may have to check all simultaneously.
References:
A. Hilliard, J. J. et al. Mechanisms of action for inhibitors of histidine protein kinases from bacterial two component systems. Antimicrob. Agents Chemother. 43, 1693–1699
(1999). [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC89345/pdf/ac001693.pdf ]
B.In vitro antibacterial activities of a thiazolyl peptide antibiotic PM2409, The Journal of Antibiotics (2014), 1–4 [http://www.nature.com/ja/journal/v68/n1/full/ja201488a.html]. This is only abstract. I have attached the published paper for your personal reference.
Hope this helps you. Please feel to free to contact us if you need more details and specific queries in this area.
Good luck and best regards
Girish
- Badges
- Science topic
- Similar topics
- Medicine
- Disease
- Infectious Diseases