Name: Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus (Video) | JoVE Journal
Uploaded: 2017-03-10T12:30:00.000+00:00
Duration: 10 min 39 s
Description: Learn horizontal gene transfer methods in Staphylococcus aureus using conjugation, transduction, and natural transformation protocols. These techniques help researchers study antibiotic resistance spread in this pathogen.

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5 minutes

This work was partly supported by Takeda Science Foundation, Pfizer Academic Contribution and JSPS Postdoctoral Fellowship for Foreign Researchers (FC).

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 (CmR) was used as the donor. COL8 or Mu509 strains (TetR, CmS) were used as the recipients. Double-resistant colonies (TetR, CmR) 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.
<ol>
	<li>Perform conjugation following a previously described protocol10 with a few modifications:
	<ol>
		<li>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 &#176;C.</li>
		<li>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.</li>
		<li>Transfer the mixture of bacteria onto a 0.45 &#181;m filter membrane using a vacuum pump system.</li>
		<li>Put the filter membranes on a sheep blood agar plate. Incubate at 37 &#176;C for 1 day. 
		NOTE: In this step, enriched medium is necessary to allow the growth of double-resistant cells.</li>
		<li>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.</li>
		<li>Make a serial 10-fold dilution of the bacterial suspension by using fresh TSB. Plate 100 &#181;l 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 &#181;l 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 &#176;C for 18-24 hr.</li>
		<li>Analyze double-resistant colonies (putative transconjugants) for the presence of the cfr gene by colony PCR7 and their susceptibility profiles (antibiogram).</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
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.
<ol>
	<li>Phage amplification on the donor
	<ol>
		<li>Prepare an overnight culture of N315-45 in 5 ml of nutrient broth supplemented with 3.6 mM Ca2+ (NBCaCl2) at 37 &#176;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.</li>
		<li>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 &#176;C with shaking (180 rpm).</li>
		<li>Prepare a series of diluted phage MR83a in NBCaCl2 medium (10-2, 10-3, 10-4, 10-5, and 10-6). Add 20 &#181;l 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.</li>
		<li>Grow the cultures at 37 &#176;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.</li>
		<li>Select one or two cleared culture vials with the highest dilution of the added phage.</li>
		<li>Transfer the cultures into 15 ml centrifuge tubes. Add 250 &#181;l of chloroform and mix thoroughly by inverting the tubes.</li>
		<li>Centrifuge the tubes at 5,000 x g for 20 min at 4 &#176;C.</li>
		<li>Transfer the supernatants to fresh tubes and store them at 4 &#176;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.</li>
	</ol>
	</li>
	<li>Measuring the phage titer (phage plaque assay)
	<ol>
		<li>Prepare nutrient broth agar (1.5% agar) medium; keep it warm in a water bath at 55 &#176;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).</li>
		<li>Prepare an overnight culture of N315 in 5 ml of NBCaCl2 at 37 &#176;C with shaking; this can be substituted with the control culture described above (step 2.1.3).</li>
		<li>Add 10 &#181;l of the overnight culture into 200 &#181;l 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.</li>
		<li>Make a serial 1:10 dilution (from 10-5-10-10) of previously prepared phage (step 2.1.8) using NBCaCl2 medium.</li>
		<li>Spot 3 &#181;l of each phage dilution onto the plate covered with bacteria.</li>
		<li>Incubate the plate overnight at 30 &#176;C.</li>
		<li>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)</li>
	</ol>
	</li>
	<li>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.
	<ol>
		<li>Prepare the overnight culture of N315, COL, or Mu50 in 5 ml of NBCaCl2 at 37 &#176;C with shaking (180 rpm).</li>
		<li>Dilute the phage in NBCaCl2 to ~109 pfu/mL.</li>
		<li>In a 50 ml glass vial, mix 500 &#181;l of overnight culture, 500 &#181;l 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 &#176;C with gentle shaking (100 rpm) for 30 min. 
		NOTE: Heat treatment of recipient cells just prior to the addition of phage (52 &#176;C for 2 min to inactivate the endogeneous restriction enzymes) may increase the efficiency12.</li>
		<li>Add 50 &#181;l of 20% Na3-Citrate. Continue gentle shaking for 30 min at 37 &#176;C. 
		NOTE: Na3-Citrate acts as a moderate chelator of calcium.</li>
		<li>Prepare melted brain-heart infusion (BHI) agar (1.5% agar) medium and keep it in a water bath at 55 &#176;C.</li>
		<li>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).</li>
		<li>Incubate the plate at 37 &#176;C for 24-48 hr.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
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, N2-2.12,7, 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 (CmR) 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.
<ol>
	<li>Preparation of donor DNA
	<ol>
		<li>Cultivate COL-45 overnight with shaking at 37 &#176;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.</li>
		<li>Collect the cells by centrifugation (8,000 x g for 10 min at 4 &#176;C).</li>
		<li>Extract the plasmids using a plasmid DNA extraction kit or a conventional DNA purification method to purify the whole DNA.</li>
		<li>Quantify the purified DNA by spectrometer and keep it at 4 &#176;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.</li>
	</ol>
	</li>
	<li>Transformation assay
	<ol>
		<li>Culture the recipient cell (N2-2.1) overnight in 5 ml of TSB at 37 &#176;C with shaking.</li>
		<li>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 &#176;C).</li>
		<li>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 transformation2,13.</li>
		<li>Grow the bacteria at 37 &#176;C with shaking (180 rpm) until the late exponential phase (about 8 hr).</li>
		<li>Harvest the cells by centrifugation (5,000 x g for 5 min at 4 &#176;C).</li>
		<li>Resuspend the cells in 10 ml of fresh CS2 medium.</li>
		<li>Add 10 &#181;g of purified plasmid or genome DNA (step 3.1.4) to the cell suspension. Shake at 37 &#176;C and 180 rpm for 2.5 hr. 
		NOTE: Shorter incubation times with DNA (&#60;2.5 hr) result in lower transformation frequencies.</li>
		<li>Collect cells by centrifugation (5,000 x g for 5 min at 4 &#176;C).</li>
		<li>Resuspend the cells in 10 mL of BHI medium. Mix the cell suspension with 90 ml of melted BHI agar (55 &#176;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.</li>
		<li>Incubate the plates at 37 &#176;C for 2 days.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>

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<li>Morikawa, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Expression+of+a+cryptic+secondary+sigma+factor+gene+unveils+natural+competence+for+DNA+transformation+in+Staphylococcus+aureus%5BTitle%5D)+AND+%22PLoS+Pathog%22%5BJournal%5D)">Expression of a cryptic secondary sigma factor gene unveils natural competence for DNA transformation in Staphylococcus aureus.</a> PLoS Pathog. 8, e1003003(2012).</li>
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<li>Novick, R. P., Christie, G. E., Penades, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+phage-related+chromosomal+islands+of+Gram-positive+bacteria%5BTitle%5D)+AND+%22Nat.+Rev.+Microbiol%22%5BJournal%5D)">The phage-related chromosomal islands of Gram-positive bacteria.</a> Nat. Rev. Microbiol. 8, 541-551 (2010).</li>
<li>Dubey, G. P., Ben-Yehuda, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intercellular+nanotubes+mediate+bacterial+communication%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Intercellular nanotubes mediate bacterial communication.</a> Cell. 144, 590-600 (2011).</li>
<li>Dubey, G. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Architecture+and+Characteristics+of+Bacterial+Nanotubes%5BTitle%5D)+AND+%22Dev+cell%22%5BJournal%5D)">Architecture and Characteristics of Bacterial Nanotubes.</a> Dev cell. 36, 453-461 (2016).</li>
<li>Cafini, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Horizontal+gene+transmission+of+the+cfr+gene+to+MRSA+and+Enterococcus%3A+role+of+Staphylococcus+epidermidis+as+a+reservoir+and+alternative+pathway+for+the+spread+of+linezolid+resistance%5BTitle%5D)+AND+%22J.+Antimicrob.+Chemother%22%5BJournal%5D)">Horizontal gene transmission of the cfr gene to MRSA and Enterococcus: role of Staphylococcus epidermidis as a reservoir and alternative pathway for the spread of linezolid resistance.</a> J. Antimicrob. Chemother. 71, 587-592 (2016).</li>
<li>Dyke, K. G., Jevons, M. P., Parker, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Penicillinase+production+and+intrinsic+resistance+to+penicillins+in+Staphylococcus+aures%5BTitle%5D)+AND+%22Lancet%22%5BJournal%5D)">Penicillinase production and intrinsic resistance to penicillins in Staphylococcus aures.</a> Lancet. 1, 835-838 (1966).</li>
<li>Kuroda, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Whole+genome+sequencing+of+meticillin-resistant+Staphylococcus+aureus%5BTitle%5D)+AND+%22Lancet%22%5BJournal%5D)">Whole genome sequencing of meticillin-resistant Staphylococcus aureus.</a> Lancet. 357, 1225-1240 (2001).</li>
<li>Marraffini, L. A., Sontheimer, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CRISPR+interference+limits+horizontal+gene+transfer+in+staphylococci+by+targeting+DNA%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA.</a> Science. 322, 1843-1845 (2008).</li>
<li>Clinical Laboratory Standards Institute. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22Methods+for+Dilution+Antimicrobial+Susceptibility+Tests+for+Bacteria+that+Growth+Aerobically+-+Seventh+Edition%3A+Approved+Standard+M7-A7%22">Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Growth Aerobically - Seventh Edition: Approved Standard M7-A7</a>. , CLSI. Wayne, PA, USA. (2006).</li>
<li>Edwards, R. A., Helm, R. A., Maloy, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Increasing+DNA+transfer+efficiency+by+temporary+inactivation+of+host+restriction%5BTitle%5D)+AND+%22BioTechniques%22%5BJournal%5D)">Increasing DNA transfer efficiency by temporary inactivation of host restriction.</a> BioTechniques. 26, 892-894 (1999).</li>
<li>Thi, L. T., Romero, V. M., Morikawa, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell+wall-affecting+antibiotics+modulate+natural+transformation+in+SigH-expressing+Staphylococcus+aureus%5BTitle%5D)+AND+%22J.+Antibiot%22%5BJournal%5D)">Cell wall-affecting antibiotics modulate natural transformation in SigH-expressing Staphylococcus aureus.</a> J. Antibiot. , (2015).</li>
<li>Long, K. S., Poehlsgaard, J., Kehrenberg, C., Schwarz, S., Vester, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Cfr+rRNA+methyltransferase+confers+resistance+to+Phenicols%2C+Lincosamides%2C+Oxazolidinones%2C+Pleuromutilins%2C+and+Streptogramin+A+antibiotics%5BTitle%5D)+AND+%22Antimicrob.+Agents+Chemother%22%5BJournal%5D)">The Cfr rRNA methyltransferase confers resistance to Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins, and Streptogramin A antibiotics.</a> Antimicrob. Agents Chemother. 50, 2500-2505 (2006).</li>
<li>Ando, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Restriction-modification+system+differences+in+Helicobacter+pylori+are+a+barrier+to+interstrain+plasmid+transfer%5BTitle%5D)+AND+%22Mol+microbiol%22%5BJournal%5D)">Restriction-modification system differences in Helicobacter pylori are a barrier to interstrain plasmid transfer.</a> Mol microbiol. 37, 1052-1065 (2000).</li>
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<li>Wilson, D. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Variation+of+the+natural+transformation+frequency+of+Campylobacter+jejuni+in+liquid+shake+culture%5BTitle%5D)+AND+%22Microbiology%22%5BJournal%5D)">Variation of the natural transformation frequency of Campylobacter jejuni in liquid shake culture.</a> Microbiology. 149, 3603-3615 (2003).</li>
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</ol>

The authors have nothing to disclose.

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...

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 &#34;pseudo-transformation&#34;2 and phage-like systems in the transfer of pathogenicity islands4. Recently, one group reported that &#34;nanotubes&#34; are involved in the transfer of cellular materials (including plasmid DNA) between neighboring cells5,6, 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.

<table><tbody><tr><td>Tryptic Soy Broth (TSB)&amp;nbsp;</td><td>Becton Dickinson&amp;nbsp;</td><td>211825</td><td></td></tr><tr><td>Brain Heart Infusion (BHI)</td><td>Becton Dickinson&amp;nbsp;</td><td>211059</td><td></td></tr><tr><td>Nutrient Broth No. 2</td><td>Oxoid</td><td>CM0067</td><td></td></tr><tr><td>Sheep blood agar</td><td>Eiken Chemical Co.,Ltd.</td><td>E-MR96</td><td>Tryptic soy agar added with 5% (v/v) sheep blood according to the manufacturer.&amp;nbsp;</td></tr><tr><td>Agar powder</td><td>Wako Pure Chemical Industries</td><td>010-08725</td><td></td></tr><tr><td>Sodium citrate (Trisodium citrate dihydrate)</td><td>Wako Pure Chemical Industries</td><td>191-01785</td><td></td></tr><tr><td>Cellulose Ester Gridded 0.45 &amp;mu;L HAWG filter</td><td>Merck Milipore</td><td>HAWG 02500</td><td></td></tr><tr><td>QIAfilter Plasmid Midi kit</td><td>QIAGEN</td><td>12243</td><td></td></tr></tbody></table>

methodology for the study of horizontal gene transfer in staphylococcus aureus

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

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.

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 phenotype14,15 in S. aureus strains, by investigating three mechanisms of HGT.
<p class="jove_content" fo:keep-toget...

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

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to ...

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.

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Immunology and Infection

Source: Cafini, F., et.al. Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus. J. Vis. Exp. (2017)This video demonstrates competence-induced DNA uptake in Staphylococcus aureus through natural transformation. A chloramphenicol-sensitive strain engineered to constitutively express a sigma factor is cultured under nutrient-limiting conditions to enhance competence, then incubated with donor DNA carrying a resistance gene. Successful gene transfer is confirmed by the...

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Learn horizontal gene transfer methods in Staphylococcus aureus using conjugation, transduction, and natural transformation protocols. These techniques help researchers study antibiotic resistance spread in this pathogen.

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

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