How soon after you mixed the phage and bacteria together did you do the dilution? At an MOI of 100 (which is really really high), I would expect most of the bacteria to be killed and maybe only a few resistant survivors to grow. At the lower dilutions, if done quickly, you might have diluted out some cells prior to them being infected and so they survive.

I mixed them for only 10 min, then I started the dilution and spotting.

The purpose of the experiment is to see the possibility of having insensitive bacterial mutants that resist the phage.

There are phage resistant mutants for most phages, that is something that has been known for a very long time. It may also be that with an MOI of 100 you are getting a lot of indirect killing so that the resistant mutants are not surviving at very high phage concentrations. This is something that has also been shown previously for many bacteriophages.

Found that in my recent experiment..

I'd have some thoughts about the high phage concentration in the primary dilutions which during the overnight incubation time may killed some/all the bacteria, however, in the last dilutions the bacterial CFU extremely exceeded the phage PFU which would allow a secondary bacterial growth.

Michael J. Benedik

Sorry to disturbing you, can you explain the word indirect killing please. I'm done MOI assay and resistance bacteria appeared at high MOI with a lower time point compared to low MOI.

you can have resistant bacteria that can still be killed at very high MOI of phages because if you have lots of phage absorbing and injecting they can still create membrane disruption and cell killing even if they fail to replicate. It does depend on the nature of the resistance mutation and the phage as to whether it is a problem or not.

Michael J. Benedik thank you for answering my question. Can you explain the mechanism that how bacterial cell resist to phage at high MOI. I'm a bit confused about the resistance if the host cell burst as soon as the phage inject their genome, does it mean resistant mechanisms such as CRISPR-Cas9 of host cell doesn't work?.

If a cell is infected by one or two phages and those phages fail to replicated and lyse the cell, then the cell is resistant to the phage, regardless of the mechanism. It could be CRISPR-Cas, it could be some mutation that blocks gene expression or replication, it could be some mutation that blocks phage genome injection, it could be some mutation that changes the cell surface receptor where the phage binds. There are many ways a cell could be resistant.

Oftentimes these mutations are not absolute, say you have a mutation in the surface receptor that reduces phage adsorption by 100-fold. The cells would appear to be resistant because 99% of the time the phage doesn't infect and 99% of the cells survive. But if you now have an MOI of 100 instead of 1, then nearly every cell is going to have 1 phage actually get inside and most of the cells would die.

Another mechanism is say the phage absorb to the surface but fail to inject their DNA. If you have a cell with one phage on the surface its not a problem. But if you have 100 or 1000 on the surface you might block all molecules of that receptor on the surface so it no longer functions. This could be lethal.

You might also have a situation where a phage that fails to inject its DNA can still make a hole in the membrane. If you have one phage, the whole can readily be repaired, but if you have 1000 then it might not be able to repair them all quickly enough.

So the point I am making is that at really high multiplicities, 100 or 1000, you can begin to have direct and indirect effects that you would never observed at lower multiplicities just due to the really high numbers.

Michael J. Benedik Thank you for very clearly and appreciate for the answer.