Bacteria like E.coli carry antibiotic resistant genes in conjugative plasmids. This bacteria can be found in hospitals, living as commensals in animals, and in aquatic environments. Natural conjugative plasmids horizontal transfer of genes between different bacterial lineages.
We present metals for the detection of conjugative plasmids and formation conjugation efficiency. This technique is simple and sensitive. It is sensitive because it uses markers for the plasmid, the donor, and the recipient.
These markers are selectable, which means that they can identify single events in very large populations. The protocols we present are useful to study the evolution of antibiotic resistance and for epidemiological surveillance, that is to monitor the flow of drug-resistant genes. Demonstrating the procedure will be Caison Warner, a PhD student from my laboratory, and Pepper St.Clair, an undergraduate student from CAMS lab.
On day one, separately streak the donors and recipient from glycerol stocks on media C which is MacConkey agar without antibiotics and incubate overnight at 37 degrees Celsius. On day two, label a 14 milliliter culture tube for each donor and recipient. Select a single colony of each donor and recipient and grow them in two milliliters of Mueller Hinton Broth at 200 RPM for 18 hours at 37 degrees Celsius.
On day three, measure the optical density of 10 times saline diluted overnight grown culture at 600 nanometers without vortexing. If required, use sterilized saline solution to adjust the optical density to two. Label a 1.5 milliliter microcentrifuge tube as a mating tube, indicating the donor and recipient strains.
Transfer 500 microliters of the adjusted suspension of each donor and recipient in the mating tube and gently mix the suspension by inversion. Centrifuge the mating tube at room temperature for 10 minutes at 500 G.Pipette out 800 microliters of supernatant from the mating tube without disturbing the pellet, leaving 200 microliters in the tube. Incubate the mating tube for 18 hours at 37 degrees Celsius.
Further, as a negative control, streak the overnight culture of donors on media B and recipients on media A and incubate the plates overnight at 37 degrees Celsius. After incubation, on day four, add 800 microliters of saline solution to the mating tube and resuspend by vortexing. Make serial dilutions of the mating suspension and add 100 microliters of each dilution on media plates.
Once the plates are dried, incubate them for 18 hours at 37 degrees Celsius and calculate the frequency of conjugation per donor or per recipient as described in the manuscript. After extracting DNA from the bacterial cell walls using a simple boil prep extraction method, label a 1.5 milliliter tube as a pool in the required PCR tubes with the name of the gene and sample. In the tube labeled as pool, add sterile water, PCR master mix, primers, and polymerase.
Mix gently by pipetting up and down at least 10 times in a chilled environment. Add the template DNA to the corresponding tube and secure the PCR tubes with caps. Place the PCR tubes into the thermocycler and start the program for resistance genes and replicons as described in the manuscript.
For amplicon confirmation, prepare a 1%agarose gel by adding agarose and TAE buffer in a 250 milliliter flask and melt the agarose using a microwave. Once the agarose solution has cooled to approximately 55 degrees Celsius, under a fume hood, add six microliters of SYBR Green and mix. Pour the gel into a tray sealed with adhesive tape to avoid spillage and allow the gel to set for 15 to 25 minutes until turbidity appears.
After removing the adhesive tape, place the gel into the electrophoresis chamber and add TAE buffer to cover the gel. Mix five microliters of six times DNA gel loading dye with 25 microliters of each reaction by pipetting up and down at least 10 times. Then load the mixture into wells by avoiding bubbles.
After adding four microliters of one kilobase pair DNA ladder into the first well, close the chamber lid and run the gel for 60 minutes at 120 volts and 400 milliamperes. Visualize the gel under UV light and record the image. MacConkey agar distinguished the lactose positive donors which produced large pink colonies from the recipient and transconjugants, which were lactose negative and produced smaller pale yellow colonies.
The five conjugation efficiencies of a donor strain SSW4955 were all within the same order of magnitude, indicating the mobilization of conjugative plasmid can be detected regardless of the selection used for transconjugant identification. Whereas the donor strain SSW7037 showed a conjugation efficiency three times lower, which allows the comparison of conjugation efficiencies of different donors and plasmid types with the same recipients. The gel electrophoresis of PCR products confirmed the presence of expected replicons in transconjugants.
This assay completely depends on selectable markers. Controls must be in place to confirm that each selection is working and transconjugants need to be confirmed colorimetrically or by PCR. The protocols presented here can be adapted for the study of conjugation in vivo or biofilm models, and for a studying variables modulating conjugation efficiency.
This protocol for detecting mobilizable plasmids at scale will greatly add to our understanding of the flow of genes across the environment, and to study the variables affecting the efficiency of conjugation.
