there is something that i have any wondering thing when i was experimenting with salmonella infection.
as you know, An aim of this experiment is researching a phenotype after salmonella infects a macrophage.
then my senior reseacher told me that i have to add a high gentamicin after finishing an infection, then harvest a T0 sample, at the same time add a low gentamicin to T21 sample and incubate for 21 times, except for T0 sample.
i know as the fact that gentamicin works by stopping the growth of bacteria.
what is difference of role of low and high gentamicin?
is there any other side effect of high gentamicin?
why do i have to change a concentration of gentamicin from high to low?
i think that will be okay if T21 was treated by high gentamicin continuously....
For phagocytic cells infection, also called Pulse / Chase infection phases).
During the pulse phase, the infection must be without antibiotic unless if kiiling of your bacteria does not matter, or if your bacterial strain must be maintained Under antibiotic presure. during this step there is uptake of your bacteria. In general between 30 min and 4 hours depending on host cells, bacteria and pathways targeted for analysis.
To stop the pulse phase, adding antibiotic serves for killing and eliminating extracellular bacteria. this step can be also performed by intensive wash and deadsorbing agents to detach bacteria still attached on macrophage surface.
Gentaminic is one of antibiotic used, I don't know about the phenotype are you looking for neither the strain (if recombinant). But it's important to know that gentamicin does not kill intracellular bacteria at high concentration but have other physiologic effects depending on the phagolysozomal compartment.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2258524/
It's not a good idea to maintain high concentration of aminoglycoside in your media during the chase phase, since Sugars in your culture media increases proton motive force, which is a process by which ATP is generated via the transfer of hydrogen ions across the membrane. This proton force is well known to play a role in aminoglycoside uptake. And will kill your intracellular Salmonella.
Using low concentration of gentamicin during the chase phase may be to prevent contamination. or to reflect the in vivo exposure of macrophase during in vivo treatment. Your supervisor can answer this question.
It has been reported that GM penetrate through plasma membrane leading to the cause of death of salmonella. Therefore, high conc. of GM are not allowed to be employed. This will be ublished in Yosei Med. J. in March this year.
The reason is simple-the high concentration is the basis of the gentamicin protection assay. Aminoglycosides are poorly permeable into eucaryotic cells (some may get in but not enough) which allow the extracellular bacteria to be killed whilst the intracellular are protected and survive. So if you are only looking at invasion into epithelial cells or uptake (both invasion and phagoytosis) then great you are done. If you wish to look at persistence a low level of gentamicin is added prevent growth of any Salmonella released from cells either killed by the Salmonella or from cells naturally dying. It works OK even though gentamicin is not the best bacteriostatic drug and the concentration may be too low to have bactericidal effect. Have been doing these assays for 18 years and they are a little crude but easy and cheap.
Do I have to treat A549 cells infected with Legionella by gentamicin to eliminate extracellular bacteria and can I use anaother antibiotic instead of it?