Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

NOTE: The strains and materials used in this work are listed in Table 1 and the Table of Materials, respectively. In the transmission experiments, N315 and COL cfr-positive derivatives were used as donors of the cfr gene (N315-45 and COL-45). These strains were previously obtained by conjugation, using as the donor a clinical cfr-positive Staphyloccocus epidermidis strain (ST2), following the standard conjugation protocol (see below). This strain harbored the cfr gene on a pSCFS7-like plasmid⁷.

1. Conjugation Using the Filter-mating Method

NOTE: An S. aureus N315 strain carrying the cfr gene (N315-45)⁷ (Cm^R) was used as the donor. COL⁸ or Mu50⁹ strains (Tet^R, Cm^S) were used as the recipients. Double-resistant colonies (Tet^R, Cm^R) able to grow in the presence of 32 mg/L of chloramphenicol plus 8 mg/L of tetracycline were considered as putative transconjugants and were analyzed to determine the presence of cfr by colony PCR and to determine the recipient susceptibility profile.

1. Perform conjugation following a previously described protocol¹⁰ with a few modifications:
   1. Prepare 5 mL of overnight culture of the donor and recipient in Tryptic soy broth (TSB) medium (with and without 32 mg/L chloramphenicol, respectively), with shaking at 37 °C.
   2. Adjust the optical density (OD₆₀₀) of the overnight cultures to 1 (OD₆₀₀ = 1.0) by using fresh TSB medium. Mix 0.5 mL of the donor culture (N315-45) with 0.5 mL of the recipient culture (COL or Mu50). Add 1 ml of PBS to the mixture.
   3. Transfer the mixture of bacteria onto a 0.45 µm filter membrane using a vacuum pump system.
   4. Put the filter membranes on a sheep blood agar plate. Incubate at 37 °C for 1 day.
      NOTE: In this step, enriched medium is necessary to allow the growth of double-resistant cells.
   5. Take out the filter membrane from the plate and suspend in 10 ml of phosphate-buffered saline (PBS). Vortex well to collect all the bacteria attached on the membrane.
   6. Make a serial 10-fold dilution of the bacterial suspension by using fresh TSB. Plate 100 µl of the 0-10^-4 diluted samples on TSB agar (TSA) plates supplemented with 32 mg/L chloramphenicol and 8 mg/L tetracycline for the selection of transconjugants. Plate 100 µl of the diluted suspension (10^-5-10^-6) onto TSA plates with 8 mg/L tetracycline alone to count the total number of recipients. Incubate the plates at 37 °C for 18-24 hr.
   7. Analyze double-resistant colonies (putative transconjugants) for the presence of the cfr gene by colony PCR⁷ and their susceptibility profiles (antibiogram).
   8. Determine the antibiogram by measuring the minimum inhibitory concentrations (MICs) of appropriate antibiotics according to the standard microdilution method or disk diffusion method¹¹. The susceptibility profile of the transconjugants must be identical to the recipient, except for chloramphenicol (and other compounds affected by the cfr gene). This determination is essential to rule out tetracycline resistance developed by the donor strain.

2. Phage Transduction

NOTE: The bacteriophage MR83a belonging to the Siphoviridae family (laboratory stock) was used in the transduction experiments. N315-45 was used as the cfr donor for phage infection. N315, COL, or Mu50 strains were used as the recipients. The acquisition of cfr was determined by the ability of the recipient strain to grow in the presence of 32 mg/L chloramphenicol. Colonies growing under this condition were analyzed to determine the presence of cfr (by colony PCR) and to determine the susceptibility profile to rule out potential contamination.

1. Phage amplification on the donor
   1. Prepare an overnight culture of N315-45 in 5 ml of nutrient broth supplemented with 3.6 mM Ca²⁺ (NBCaCl₂) at 37 °C with shaking (180 rpm).
      NOTE: Calcium is required for phage infection. See the Materials Table. Nutrient broth from other companies might have issues, such as calcium precipitation.
   2. Prepare subcultures by diluting the overnight culture 1:1,000 in a final volume of 10 mL of NBCaCl₂ in 50 ml glass flasks or glass vials. Grow the bacteria for 1 hr at 37 °C with shaking (180 rpm).
   3. Prepare a series of diluted phage MR83a in NBCaCl₂ medium (10^-2, 10^-3, 10^-4, 10^-5, and 10^-6). Add 20 µl of the diluted phage into the bacterial culture. Also prepare a control culture without phage infection, which serves as the positive control for bacterial cell growth and can be used to determine the phage titer the next day.
   4. Grow the cultures at 37 °C with gentle shaking (100 rpm) overnight.
      NOTE: The cells will grow to some extent when the infecting phage is not in excess; then, they will enter into lysis phase due to the phage amplification through the lytic cycle. The culture without phage will show full growth, while cultures with phage will show distinct rates of transparency.
   5. Select one or two cleared culture vials with the highest dilution of the added phage.
   6. Transfer the cultures into 15 ml centrifuge tubes. Add 250 µl of chloroform and mix thoroughly by inverting the tubes.
   7. Centrifuge the tubes at 5,000 x g for 20 min at 4 °C.
   8. Transfer the supernatants to fresh tubes and store them at 4 °C until use.
      NOTE: The phage is stable for at least a few months, but storing the phage preparation for too long reduces the titer and transduction efficiency.
2. Measuring the phage titer (phage plaque assay)
   1. Prepare nutrient broth agar (1.5% agar) medium; keep it warm in a water bath at 55 °C. Add autoclaved 0.5 M CaCl₂ solution to the medium to a final concentration of 3.6 mM. Pour it into 90 mm Petri dishes (NBCaCl₂ plates).
   2. Prepare an overnight culture of N315 in 5 ml of NBCaCl₂ at 37 °C with shaking; this can be substituted with the control culture described above (step 2.1.3).
   3. Add 10 µl of the overnight culture into 200 µl of NBCaCl₂ and evenly spread it onto the NBCaCl₂ plate. Stop the spreading when the surface of the plate is covered with liquid. Let the plate dry.
   4. Make a serial 1:10 dilution (from 10^-5-10^-10) of previously prepared phage (step 2.1.8) using NBCaCl₂ medium.
   5. Spot 3 µl of each phage dilution onto the plate covered with bacteria.
   6. Incubate the plate overnight at 30 °C.
   7. Count the plaque numbers and calculate the phage titer using the following equation: Plaque forming unit (pfu)/ml = Number of plaques x Dilution factor/spotted volume (3 x 10^-3 ml)
3. Phage transduction
   NOTE: The phage pool prepared in the previous step (2.1.8) is used to test the transduction of the cfr gene into the S. aureus strains. In this experiment, strains N315, COL, or Mu50 are used as the recipients.
   1. Prepare the overnight culture of N315, COL, or Mu50 in 5 ml of NBCaCl₂ at 37 °C with shaking (180 rpm).
   2. Dilute the phage in NBCaCl₂ to ~10⁹ pfu/mL.
   3. In a 50 ml glass vial, mix 500 µl of overnight culture, 500 µl of fresh NBCaCl₂, and 1 ml of phage (~10⁹ pfu/ml); the expected multiplicity of infection (MOI) must not exceed 1. Incubate the mixture at 37 °C with gentle shaking (100 rpm) for 30 min.
      NOTE: Heat treatment of recipient cells just prior to the addition of phage (52 °C for 2 min to inactivate the endogeneous restriction enzymes) may increase the efficiency¹².
   4. Add 50 µl of 20% Na₃-Citrate. Continue gentle shaking for 30 min at 37 °C.
      NOTE: Na₃-Citrate acts as a moderate chelator of calcium.
   5. Prepare melted brain-heart infusion (BHI) agar (1.5% agar) medium and keep it in a water bath at 55 °C.
   6. Transfer the bacterium-phage mixture to a 100 ml flask, add 50 ml of warm BHI agar supplemented with 32 mg/L chloramphenicol, and mix well. Pour the mixture into the 90 mm Petri dishes.
      NOTE: This method enables the detection of a small number of growing colonies (putative transductants).
   7. Incubate the plate at 37 °C for 24-48 hr.
   8. Further test the generated colonies (transductants) for resistance by transferring the colonies onto new BHI agar plates supplemented with 32 mg/L chloramphenicol. Confirm the presence of the cfr gene by colony PCR⁷.

3. Natural Transformation

NOTE: The natural transformation assay in S. aureus was carried out following the method described in our previous study². The N315 derivative, N2-2.1^2,7, was used as the recipient. In this strain, the sigH locus was duplicated so as to constitutively express SigH². For detailed procedures on how to isolate SigH-expressing competence variants, please see a previous description². If the resistance marker to be transferred is not chloramphenicol, pRIT-sigH (Cm^R) can be used to express SigH, as described previously². Purified plasmid or whole DNA extract from cfr-acquired S. aureus COL-45⁷ is used as the donor DNA for transformation.

1. Preparation of donor DNA
   1. Cultivate COL-45 overnight with shaking at 37 °C in a 300 ml flask containing 50 mL of TSB supplemented with 32 mg/L chloramphenicol. Culture E. coli HST04 carrying pHY300 plasmid^(TetR) to extract the plasmid for the control experiment.
      NOTE: HST04 (dam^–/dcm^–) lacks the DNA methylase genes¹³. Other conventional strains with DNA methylases can also be used to prepare the DNA donor, because the DNA methylation state does not affect the transformation efficiency. Alternatively, pT181 plasmid purified from the S. aureus COL strain can also be used as a positive control for the transformation assay².
   2. Collect the cells by centrifugation (8,000 x g for 10 min at 4 °C).
   3. Extract the plasmids using a plasmid DNA extraction kit or a conventional DNA purification method to purify the whole DNA.
   4. Quantify the purified DNA by spectrometer and keep it at 4 °C until use.
      NOTE: Use a fresh DNA preparation for the transformation assay, usually less than one week old. Old DNA preparations reduce the efficiency of transformation.
2. Transformation assay
   1. Culture the recipient cell (N2-2.1) overnight in 5 ml of TSB at 37 °C with shaking.
   2. Transfer 0.5 ml of overnight culture into a 1.5 ml tube. Precipitate the cells by centrifugation (10,000 x g for 1 min at 4 °C).
   3. Suspend the cells with 10 mL of CS2 medium in a 50 ml tube.
      NOTE: CS2 medium is a complete synthetic medium that induces competence for natural transformation in S. aureus² (see the medium composition in the Material Table). Other standard laboratory media, such as TSB or BHI, are not suitable for transformation^2,13.
   4. Grow the bacteria at 37 °C with shaking (180 rpm) until the late exponential phase (about 8 hr).
   5. Harvest the cells by centrifugation (5,000 x g for 5 min at 4 °C).
   6. Resuspend the cells in 10 ml of fresh CS2 medium.
   7. Add 10 µg of purified plasmid or genome DNA (step 3.1.4) to the cell suspension. Shake at 37 °C and 180 rpm for 2.5 hr.
      NOTE: Shorter incubation times with DNA (<2.5 hr) result in lower transformation frequencies.
   8. Collect cells by centrifugation (5,000 x g for 5 min at 4 °C).
   9. Resuspend the cells in 10 mL of BHI medium. Mix the cell suspension with 90 ml of melted BHI agar (55 °C) supplement with 32 mg/L chloramphenicol (or 5 mg/L tetracycline in the control experiment). Pour the mixture into the 90 mm Petri dishes. Swiftly cool and let the agar solidify.
   10. Incubate the plates at 37 °C for 2 days.
   11. Replicate generated colonies (transformants) by transferring the colonies (using toothpicks) to new BHI agar plates containing the appropriate antibiotics to confirm their resistance characteristics. Confirm the acquired resistance gene (cfr or tetM) by colony PCR⁷.