The overall goal of this procedure is to provide a detailed methodology to study the horizontal transmission of DNA in staphylococcus aureus by addressing the three main transfer pathways. Conjugation, transduction, and natural transformation. This method is about horizontal transfer of antibiotic resistant genes in human pathogen staph aureus.
The main advantage of the techniques introduced here is that we can test three major modes of transfer, including recently discovered natural genetic transformation Demonstrating the procedure will be Le Thuy, a PhD student from our lab. To begin the experiment, prepare five milliliters of overnight culture of the donor and the recipient bacteria in tryptic soy broth. Or TSB medium with and without 32 milligrams per liter chloramphenicol, respectively.
With shaking at 37 degrees Celsius. The next day, adjust the optical density of the overnight cultures to one with fresh TSB medium. Mix 0.5 milliliters of the donor culture with 0.5 milliliters of the recipient culture.
And add one milliliter of phosphate buffered saline, or PBS to the mixture. Then using a vacuum pump system, transfer the mixture of bacteria onto a 0.45 micrometer filter membrane. Place the filter membranes on a sheep blood agar plate.
Incubate the plates at 37 degrees celsius for one day. Take out the filter membrane from the plate and suspend it in 10 milliliters of PBS. Vortex the mixture for about one minute to collect all the bacteria attached on the membrane.
Make a serial 10 fold dilution of the bacterial suspension with fresh TSB. Plate 100 microliters of the 0, 10, 4 diluted samples on TSB agar. Or TSA plates supplemented with 32 milligrams per liter of chloramphenicol and eight milligrams per liter of tetracycline to select transconjugants.
Plate 100 microliters of the diluted suspension onto TSA plates with eight milligrams per liter tetracycline alone. To count the total number of recipients. After incubation, analyze double resistant colonies for the presence of the CFR gene by colony PCR and their susceptibility profiles.
Determine the antibiogram by measuring the minimum inhibitory concentrations, MICs, of appropriate antibiotics according to the standard microdilution method or disc diffusion method. The susceptibility profile of the transconjugants must be identical to the recipient. Except for chloramphenicol and other compounds affected by the CFR gene.
To rule out tetracycline resistance developed by the donor strain. Prepare an overnight culture of N315-45 in five milliliters of nutrient broth supplemented with 3.6 millimolar of calcium. Prepare subcultures by diluting the overnight culture one to 1, 000 in a final volume of 10 milliliters in 50 milliliter glass vials.
Grow the bacteria for one hour at 37 degrees celsius with shaking, 180 rotations per minute. Next prepare a series of diluted phage MR83a in NB calcium chloride medium. Add 20 microliters of the diluted phage into the bacterial culture.
Prepare a control culture without phage infection to serve as the positive control for bacterial cell growth. Then grow the cultures at 37 degrees celsius with gentle shaking at 100 rotations per minute overnight. The next day select one or two cleared culture vials with the highest dilution of the added phage.
Transfer the cultures into 15 milliliter centrifuge tubes. Add 250 microliters of chloroform to the tubes and mix the culture thoroughly by inverting them. Centrifuge the culture at 5, 000 times G for 20 minutes at 4 degrees celsius.
Transfer the supernatants to fresh tubes and store them at 4 degrees celsius until use. Prepare nutrient broth agar medium and keep it warm in a water bath at 55 degrees celsius. Add autoclaved 0.5 molar calcium chloride solution to the medium to a final concentration of 3.6 millimolar.
Pour it into 90 millimeter petri dishes NB calcium chloride plates. Prepare an overnight culture of N315 in five milliliters of NB calcium chloride at 37 degrees celsius with shaking. The next day add 10 microliters of the overnight culture into 200 microliters of NB calcium chloride and evenly spread it onto the NB calcium chloride plate.
Stop spreading when the surface of the plate is covered with liquid. Then allow the plate to dry for five minutes. Make a serial one to 10 dilution of the previously prepared phage using NB calcium chloride medium.
Spot three microliters of each phage dilution onto the plate covered with bacteria. Incubate the plate overnight at 30 degrees celsius. The following morning, count the plaque numbers and calculate the phage titer using the following equation.
Prepare the overnight culture of N315, COL, or MU50, in five milliliters of NB calcium chloride at 37 degrees celsius with shaking at 180 rotations per minute. After overnight culture, dilute the phage in NB calcium chloride to 109 PFU per milliliter. In a 50 milliliter glass vial, mix 500 microliters of overnight culture, 500 microliters of fresh NB calcium chloride, and one milliliter of phage 109 PFU per milliliter.
Incubate the mixture at 37 degrees celsius with a gentle shaking at 100 rotations per minute for 30 minutes. After incubation, add 50 microliters of 20%trisodium citrate to the cultures. Continue the gentle shaking for 30 minutes at 37 degrees celsius.
Prepare the melted brain heart infusion or BHI agar medium and keep it in a water bath at 55 degrees celsius, and treat the agar medium with 32 milligrams per liter of chloramphenicol. Then transfer the bacterium phage mixture to a 100 milliliter flask. Add 50 milliliters of warm BHI agar supplemented with 32 milligrams per liter chloramphenicol, and mix well.
Pour the mixture into the 90 millimeter petri dishes. Incubate the plate at 37 degrees celsius. Further test the generated colonies, the transductants, for resistance, by transferring the colonies onto new BHI agar plates supplemented with 32 milligrams per liter chloramphenicol.
Confirm the presence of the CFR gene by colony PCR. Culture the recipient cell overnight, in five milliliters of TSB at 37 degrees celsius with shaking. The following morning, transfer 0.5 milliliters of the overnight culture into a 1.5 milliliter tube.
Precipitate the cells by centrifugation. Then suspend the cells with 10 milliliters of CS2 medium in a 50 milliliter tube. Next grow the bacteria at 37 degrees celsius with shaking at 180 rotations per minute for eight hours until the late exponential phase.
Harvest the cells by centrifugation. Discard the supernatant, and resuspend the cells in 10 milliliters of fresh CS2 medium. Add 10 micrograms of purified plasmid or genomic DNA to the cell suspension.
Shake the culture at 180 rotations per minute. Then collect the cells by centrifugation. Resuspend the cells in 10 milliliters of BHI medium.
Mix the cell suspension with 90 milliliters of melted BHI agar supplement With 32 milligrams per liter chloramphenicol, And five micrograms per liter tetracycline in the control experiment. Pour the mixture into the 90 millimeter petri dishes and swiftly cool and allow the agar to solidify. Replicate colonies using toothpicks to plates containing appropriate antibiotics to confirm their resistance characteristics.
The conjugation protocol is useful for intra species transmission where staphylococcus epidermidis was used as the CFR donor. And the staphylococcus aureus N315 strain was used as the recipient. The protocol used for inter species transmission yields similar results using the same conjugative vector in different conjugative donors.
This is the first paper in which natural transformation details described. The methodology provided would be useful for the researcher to study the transmission and spreading of antibiotic resistance, as well as primary determinant in the human pathogen staph aureus.
