Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

Summary

We describe here three different protocols for the in vitro investigation of conjugation, transduction, and natural transformation in Staphylococcus aureus.

Abstract

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to antibiotics. Genomic studies reveal that S. aureus carries many virulence and resistance genes located in mobile genetic elements, suggesting that horizontal gene transfer (HGT) plays a critical role in S. aureus evolution. However, a full and detailed description of the methodology used to study HGT in S. aureus is still lacking, especially regarding natural transformation, which has been recently reported in this bacterium. This work describes three protocols that are useful for the in vitro investigation of HGT in S. aureus: conjugation, phage transduction, and natural transformation. To this aim, the cfr gene (chloramphenicol/florfenicol resistance), which confers the Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins, and Streptogramin A (PhLOPSA)-resistance phenotype, was used. Understanding the mechanisms through which S. aureus transfers genetic materials to other strains is essential to comprehending the rapid acquisition of resistance and helps to clarify the modes of dissemination reported in surveillance programs or to further predict the spreading mode in the future.

Introduction

Staphylococcus aureus is a commensal Gram-positive bacterium that naturally inhabits the skin and nasal cavity of human beings and animals. This bacterial species is the leading cause of nosocomial infections in hospitals and healthcare settings. Moreover, its ability to develop resistance to different antimicrobial compounds has made the management of the infections caused by this bacterium into a global concern.

Two main pathways involved in the spreading of resistance phenotypes are known: the clonal dissemination of resistant genotypes and the dissemination of genetic determinants among the bacterial pool. In the case of S. aureus, different antibiotic resistance genes (as well as virulence determinants) have been found to be associated with mobile genetic elements (MGEs)¹. The presence of these elements in the genome of S. aureus indicates that the acquisition and transfer of genetic material within the bacterial population could play an important role for S. aureus adaptation and evolution.

Genetic material can be exchanged through three well-known mechanisms of HGT in Gram-positive bacteria: transformation, conjugation, and phage transduction. Transformation involves the uptake of free DNA. To acquire foreign DNA, bacterial cells need to develop a special physiological phase: the competence stage. When this stage is reached, competent cells are capable of transporting DNA into the cytoplasm, acquiring new genetic determinants. In the case of S. aureus, the existence of natural transformation has been recently demonstrated². In line with this, our group has shed light on the relevance of the expression of the SigH factor (a cryptic secondary transcription sigma factor) in the competence stage of development and on how its constitutive expression renders S. aureus capable of reaching the competence stage, which allows for the acquisition of resistant phenotypes by natural transformation².

Conjugation is a process involving the transmission of DNA from one living cell (donor) to another (recipient). Both cells must be in direct contact, allowing the DNA to be exchanged while being protected by special structures, such as tubes or pores. The transfer of DNA by this method requires the conjugative machinery. In S. aureus, the prototype conjugative plasmid is PGO1, which harbors the conjugative operon TraA³.

Phage transduction involves the transfer of DNA from cell to cell through bacteriophage infection and implies the packing of bacterial DNA, instead of viral DNA, into the phage capsid. Most of S. aureus isolates are lysogenized by bacteriophages¹. Upon stress conditions, prophages can be excised from the bacterial genome and shift to the lytic cycle.

These are the three well known mechanisms for DNA transmission in S. aureus. There are some additional transfer mechanisms, such as "pseudo-transformation"² and phage-like systems in the transfer of pathogenicity islands⁴. Recently, one group reported that "nanotubes" are involved in the transfer of cellular materials (including plasmid DNA) between neighboring cells^5,6, but a follow-up study has not appeared from other groups so far.

This work provides the necessary methodology to study HGT in S. aureus by addressing the three main transfer pathways: conjugation, transduction, and natural transformation. The results obtained with these methodologies were used to study the transmission of the cfr gene (chloramphenicol/florfenicol resistance) among S. aureus strains⁷. These three techniques are versatile tools for the investigation of MGE transmission in S. aureus.

Protocol

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.

Results

The results represented here have been previously published (adapted from reference⁷ with the publisher's permission). We studied the potential transmission pathways of the cfr gene, which causes low-level linezolid resistance and the expression of the PhLOPSA-resistance phenotype^14,15 in S. aureus strains, by investigating three mechanisms of HGT.

Discussion

This work describes the three major methods to study the HGT of genetic determinants in S. aureus. Although transduction and conjugation have been studied for decades, the existence of natural transformation was only recently recognized². Thus, S. aureus is equipped with all of the three major modes of HGT, and testing all of them is required to clarify the possible dissemination pathways of genetic determinants. The aim of this work is to compile complete protocols and to provid...

Materials

Name	Company	Catalog Number	Comments
Tryptic Soy Broth (TSB)	Becton Dickinson	211825
Brain Heart Infusion (BHI)	Becton Dickinson	211059
Nutrient Broth No. 2	Oxoid	CM0067
Sheep blood agar	Eiken Chemical Co.,Ltd.	E-MR96	Tryptic soy agar added with 5% (v/v) sheep blood according to the manufacturer.
Agar powder	Wako Pure Chemical Industries	010-08725
Sodium citrate (Trisodium citrate dihydrate)	Wako Pure Chemical Industries	191-01785
Cellulose Ester Gridded 0.45 μL HAWG filter	Merck Milipore	HAWG 02500
QIAfilter Plasmid Midi kit	QIAGEN	12243