Detection of Horizontal Gene Transfer Mediated by Natural Conjugative Plasmids in E. coli

1. Method 1: Conjugation

1. Day 1: Streak the donors and recipient. Streak separately from glycerol stocks on media C (MacConkey agar with no antibiotics), and incubate overnight at 37 °C.
   NOTE: This step is necessary to ensure that the experiment is conducted with pure isolates and to confirm the lactose phenotype by the color of colonies.
2. Day 2: Label a 14.0 mL culture tube for each donor and recipient. Select a single colony of each donor and recipient, and grow them overnight (18 h) in separate 14.0 mL culture tubes containing 2 mL of Mueller Hinton broth at 37 °C and shaking at 200 rpm.
   NOTE: In the strains used, the stationary phase is reached after overnight culture of 18 h.
3. Day 3: Measure the optical density at 600 nm (OD600) of the overnight culture of each donor and recipient (no vortexing) by using a 1:10 dilution of 900 µL of saline solution and 100 µL of the overnight culture.
4. Adjust the optical density of each donor and recipient to 2.0 (OD₆₀₀ = 2.0) with sterilized saline solution (0.85% NaCl in water).
   NOTE: An overnight culture of E. coli with an OD₆₀₀ = 2.0 has 1.6 x 10⁹ CFU/mL.
5. Label a 1.5 mL microcentrifuge tube "mating tube", indicating the donor and recipient strains. Transfer 500 µL of the adjusted (OD₆₀₀ = 2.0) suspension of each donor and recipient, and place them in the mating tube (this mating tube would have 0.8 x 10⁹ CFU/mL of each donor and recipient). Gently mix the mating tube by inversion.
6. Centrifuge the mating tube for 10 min at 500 x g at room temperature.
7. Without disturbing the pellet, pipette out 800 µL of the conjugation tube. There should be 200 µL left in the conjugation tube.
   NOTE: Discard the supernatant to a 10% bleach container to inactivate bacteria in the suspension.
8. Incubate the mating tube for 18 h (overnight) at 37 °C in an incubator. Mating occurs during the incubation time.
   NOTE: This step must be done without shaking in order not to break the conjugative pili.
9. As a negative control, streak the overnight culture of donors on media B and recipients on media A. Incubate the plates overnight at 37 °C
10. Day 4: Add 800 µL of saline solution to the mating tube and vortex to resuspend (this reconstitutes the mating mix to 1 mL).
    NOTE: Vortexing breaks the mating bacteria apart and homogenizes the culture to quantify the CFU/mL.
11. Prepare 1:10 dilutions (from 1 x 10⁰ to 1 x 10^-7) of the mating mix. Plate 100 µL of dilutions 10^-5 to 10^-7 on media A and media B, and all dilutions on media AB.
    NOTE: Dlution 10⁰ refers to the neat tube.
12. Allow the plates to dry before placing them in the incubator upside down. Incubate the plates for 18 h (overnight) at 37 °C.
13. Day 5: Inspect the negative controls to ensure that the streaks of pure overnight cultures of donors did not grow on media B, and the pure overnight culture of recipients did not grow on media A.
14. Record the number of colonies and dilutions that can be counted:
    1. Count the colonies in media A; these are the donors. The colonies should be pink (Figure 3A,B).
    2. Count the colonies in media B; these are the recipients and transconjugants. The colonies should be pale yellow (Figure 3C).
    3. Count the colonies in media AB; these are the transconjugants. The colonies should be pale yellow.
       NOTE: Select a countable plate. A countable plate has between 30 and 300 colonies (more than 300 colonies would be difficult to count, and less than 30 colonies are considered too small a sample size to present an accurate representation of the original sample).
15. Calculate the frequency of conjugation per donor or per recipient
    Frequency of conjugation per donor = (CFU/mL of the transconjugant [media AB])/ (CFU/mL of donors [media A]) x 100
    Frequency of conjugation per recipient = (CFU/mL of the transconjugant [media AB])/ (CFU/mL of recipients and transconjugants [media B]) x 100
    ​NOTE: The conjugation frequency for this protocol was calculated as transconjugants divided by the number of the recipient, multiplied by 100. According to the literature, the resulting quantity of conjugation could be named as exconjugant frequency, gene transfer frequency, conjugation frequency, recombinant yield, plasmid transfer efficiency, conjugation frequency based on the total bacterial count, proportion of transconjugants, fraction of transconjugants in recipient population, transconjugant frequency, conjugation frequency, the logarithm of conjugation rate, transfer rate constant, conjugation rate per mating pair, conjugation coefficient, or conjugation efficiency²⁰. This protocol refers to the term conjugation efficiency.
16. Store the transconjugants in glycerol stocks at -80 °C. Follow the substeps for preparing glycerol stocks.
    1. Select a single colony of transconjugants and grow them overnight (18 h) in 5.0 mL culture tubes containing 2 mL of Mueller Hinton broth supplemented with carbenicillin (100 mg/L), at 37 °C and shaking at 200 rpm.
    2. Label cryogenic vials, indicating the name of transconjugant, as follows: Tc + name of donor + antibiotic of selection in media A (as they are transconjugants, it is known that their background is the recipient strain, which is already resistant to streptomycin and rifampin but additionally harbors the plasmid carrying a beta-lactam resistance gene); for example, TcU1Carb. Add continuous numbers in case more than one transconjugant from the same donor need to be stored (e.g., TcU1Carb1, TcU1Carb2, etc.).
    3. Transfer 1 mL of the overnight culture to 1 mL of 50% glycerol (v/v; the final glycerol concentration should be 25%) and gently mix by inversion. Put the cryogenic vials on dry ice for 15 min and store the cryovials at -80 °C for future experiments.
17. For DNA extraction, streak the trasnconjugants from glycerol stocks on Mueller Hinton agar supplemented with carbenicillin (100 mg/L), and incubate overnight at 37 °C; then, follow recommendations from step 2.1.

2. Method 2: Polymerase chain reaction (PCR) to amplify antibiotic resistance genes and plasmid replicons in E. coli transconjugants

NOTE: Polymerase chain reaction (PCR) was developed by Dr. Kary Mullis in 1983. The PCR depends on specific primers (a short piece of DNA complementary to a given DNA sequence that acts as a point at which replication can proceed, generally 20-40 nucleotides in length and ideally with a guanine-cytosine content of 40%-60%), that are annealed to opposite strands of a denatured, double-stranded DNA template and extended through a thermostable DNA polymerase, thus generating an additional template for the next cycle of reactions, leading to the exponential amplification of the original template²¹. In this protocol, the replicons and resistance genes present in the conjugative plasmids were amplified to confirm the transfer in transconjugant recipient cells.

1. DNA extraction
   1. To detect the transfer of resistance genes carried by conjugative plasmids, use transconjugant DNA as a template. Use simple boil prep extraction to lyse the bacterial cell walls.
      NOTE: Simple boil prep extraction is a simple approach to lyse the bacterial cell walls. This extraction contains plasmid DNA mixed with genomic DNA, but as primers are designed according to specific DNA sequences of interest, this template is also useful for PCR. Plasmids can also be specifically purified, although yields tend to drop with increasing plasmid size²². This is a convenient stopping point. DNA can be stored for several months at -20 °C.
2. PCR
   1. Given that the experiment has a negative and a positive control, it is beneficial to set up a pool for all the reactions in a 1.5 mL tube. Label a 1.5 mL tube as "pool" and the required PCR tubes with the name of the gene and sample (e.g., OXA-U1).
   2. Pipette the PCR reagents in the following order into a 1.5 mL tube: sterile water, 2x Master Mix, primers, and polymerase (except the template DNA) (see Table 1). Gently mix by pipetting up and down at least 10 times. Keep on ice the entire time.
      NOTE: Wear gloves to avoid contaminating the reaction mixture or reagents. Try to avoid opening and mixing reagents and handling samples in the same area to prevent reagent contamination. Place the reagents into the ice bucket to suppress nuclease activity and nonspecific priming. Let them thaw completely before setting up a reaction. Keep the reagents on ice throughout the experiment. Taq Polymerase is added at the end because it is sensitive to pH changes; it needs to be buffered to avoid degradation or misfolding. The sequence of the primers is listed in Table 2-antibiotic resistance genes-and Table 3-replicons^{23,24,25,26,27,28}.
   3. Add the template DNA to the corresponding tube. Avoid introducing bubbles and secure caps on the PCR tubes.
   4. Place the PCR tubes into the thermal cycler.
   5. Start the program. Refer to the program settings listed for each gene in Table 2 (resistance genes) and Table 3 (replicons).
      NOTE: Set PCR programs to hold the samples at 4 °C after the run.
      1. PCR conditions for replicons: one cycle of denaturation at 94 °C for 5 min, followed by 30 cycles of denaturation at 94 °C for 1 min, annealing at 60 °C for 30 s, elongation at 72 °C for 1 min, and a final extension of one cycle at 72 °C for 5 min.
         NOTE: These are the conditions standardized by Carattoli et al.²⁹.
   6. When the program finishes, store the PCR tubes at 4 °C.
      NOTE: This is a convenient stopping point. PCR products can be stored for several months at -20 °C
   7. Prepare a 1% agarose gel by weighing 0.6 g of agarose in a 250 mL flask and adding 60 mL of 1x Tris-acetate-EDTA (TAE) buffer (adjust reagents if a different gel size is needed). Melt the agarose carefully and slowly in a microwave to avoid agarose spilling over the flask, and let it cool down to approximately 55 °C (10-15 min).
      1. Prepare all the reagents using ultrapure deionized water and analytical grade reagents:
      2. TAE 50x Buffer (Tris base, acetic acid, and EDTA) (1 L): Mix 121.1 g of Tris base + 372.24 g of EDTA, and add 500 mL of distilled water. Dissolve with a magnetic stirrer. Add 57.1 mL of acetic acid, and add distilled water to a total volume of 1,000 mL.
      3. Autoclave at 15 psi, 121 °C for 15 min, and keep at room temperature until ready for up to 6 months.
      4. TAE 1x Buffer (1 L): Combine 20 mL of TAE 50x buffer with 980 mL of distilled water and mix.
   8. Under a fume hood, add 6 µL of SYBR Green to the melted agarose, mix, and pour out into the tray (and comb), previously sealed with adhesive tape to avoid spillage. Let the gel set for 15-25 min until turbidity appears.
      NOTE: Other alternative dyes are also available^30,31,32,33.
   9. Remove the adhesive tape and place the gel into the electrophoresis chamber, adding enough 1x TAE buffer to cover the gel.
   10. Mix 5 µL of 6x DNA gel loading dye with 25 µL of each reaction. Gently mix by pipetting up and down at least 10 times.
       NOTE: Not all the PCR product needs to be loaded into the gel to visualize the expected product (adjust the amount of dye as corresponds). The remaining PCR sample could be saved and stored at -20 °C for several months.
   11. Load the mix into wells (avoid introducing bubbles) and put the chamber lid down.
       NOTE: Load the first sample into the second well (reserve the first to the ladder), starting with the negative control.
   12. Load 4 µL of 1 kb DNA ladder into the first well.
   13. Run the electrophoresis at 120 V, 400 mA, and 60 min.
   14. Visualize the gel under UV light (with a UV illuminator) and record the image.
       NOTE: If a PCR product is present, DNA bands stained can be detected using a standard UV transilluminator, a visible-light transilluminator, or a laser-based scanner.

Table 1: PCR reagents and pool for three reactions (an example). 

Table 2: Primers and PCR programs used to amplify diagnostic resistance genes. 

Table 3: Primers and PCR program used to classify plasmids p134797 and p101718 by PCR-based replicon typing²⁹.