I did some experiments infecting Salmonella Typhi with the lytic phage I isolated from the environment. It gives a nice clear plaques on agar overlay. However, in tube co-culture growth assay, the Salmonella are inhibited for at least 6 hrs (as measured by nephelometric readings) but showed uninhibited growth at 24 hrs. The isolates at 24 hrs were actually found to be resistant to the original phage. Is it possible that bacteria developed immunity to phages via CRISPR-Cas system? If true, can it develop phage resistance in less than 24 hrs?
OR
Is 24 hrs good enough time to develop mutation in the bacterial receptors that phage identifies?
I will very much appreciate if you can direct me to assays to characterize CRISPR loci to understand the phenomenon.
Thank you.
Jivan Shakya In cellular and bacterial culture, the effects of virus and other extracts، growth inhibitors should be checked at least 24 hours after culture. i think your phage doesn't really any effect on the bacterium because you have done the analysis early.(6h)
Dear Razieh Sadat Hosseini,
Thanks for your response.
But when I compare the growth of my treated bacteria with control untreated ones, there is a nice increase in nephelometric reading in control tube even at 6h. Does it not indicate that my viruses are affecting bacterial growth in some way?
Also my speculation of viral resistance is supported by inability of plaque formation on phage treated bacteria while the same phage gives plenty plaques on the control parental isolate which was not treated with the phage before.
There are some BIM (bacteriophage induced mutants) that are resistant to phages even in second and third co-culture/co-infection of phage and bacteria. Haven't been able to do any molecular tests to find out how that happens (receptor modification and or CRISPR mutants). But, I believe it's normal to have BIM. The only approach right now is to sequence the both the bacteria (that phage can infect and the one phage cannot) and compare their genome. I assume its more of a receptor modification than CRISPR modification.
Dear Roshan Nepal
By sequencing do you mean the whole genome sequencing? How would you know where to look for in cases where receptors for phage interactions haven't been characterized yet.
Yes. I can only think of whole-genome sequencing for now as we can compare genomes of bacteria (before being resistant and after being resistant). I'm working on the same approach and haven't been able to think of any other way as phage-bacteria infection model hasn't been characterized much.
Please keep sharing approaches you believe can work.
- Badges
- Science topic