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 harbo...

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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 border="0" cellpadding="0" cellspacing="0" width="737">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:284px;">Tryptic Soy Broth (TSB)&#160;</td>
			<td style="width:216px;">Becton Dickinson&#160;</td>
			<td style="width:121px;">211825</td>
			<td style="width:116px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Brain Heart Infusion (BHI)</td>
			<td>Becton Dickinson&#160;</td>
			<td>211059</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nutrient Broth No. 2</td>
			<td>Oxoid</td>
			<td>CM0067</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agar powder</td>
			<td>Wako Pure Chemical Industries</td>
			<td>010-08725</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium citrate (Trisodium citrate dihydrate)</td>
			<td>Wako Pure Chemical Industries</td>
			<td>191-01785</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cellulose Ester Gridded 0.45 &#956;L HAWG filter</td>
			<td>Merck Milipore</td>
			<td>HAWG 02500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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

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&#39;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.
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Watch this Scientific Journal Video about Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus at JoVE.com

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

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

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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16.3K 조회수.  University of Tsukuba. 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 ge...