Name: methodology for the study of horizontal gene transfer in staphylococcus aureus
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Description: Watch this Scientific Journal Video about Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus at JoVE.com

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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P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Architecture+and+Characteristics+of+Bacterial+Nanotubes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Penicillinase+production+and+intrinsic+resistance+to+penicillins+in+Staphylococcus+aures.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+genome+sequencing+of+meticillin-resistant+Staphylococcus+aureus.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR+interference+limits+horizontal+gene+transfer+in+staphylococci+by+targeting+DNA.">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://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Growth Aerobically - Seventh Edition: Approved Standard M7-A7. , (2006).</li><li>Edwards, R. A., Helm, R. A., Maloy, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increasing+DNA+transfer+efficiency+by+temporary+inactivation+of+host+restriction.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+wall-affecting+antibiotics+modulate+natural+transformation+in+SigH-expressing+Staphylococcus+aureus.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Cfr+rRNA+methyltransferase+confers+resistance+to+Phenicols,+Lincosamides,+Oxazolidinones,+Pleuromutilins,+and+Streptogramin+A+antibiotics.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restriction-modification+system+differences+in+Helicobacter+pylori+are+a+barrier+to+interstrain+plasmid+transfer.">Restriction-modification system differences in Helicobacter pylori are a barrier to interstrain plasmid transfer.</a> Mol microbiol. 37, 1052-1065 (2000).</li><li>Evans, B. 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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.

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			<td height="21" style="height:21px;width:284px;">Tryptic Soy Broth (TSB)&#160;</td>
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			<td height="21" style="height:21px;">Brain Heart Infusion (BHI)</td>
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			<td height="21" style="height:21px;">Nutrient Broth No. 2</td>
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			<td height="21" style="height:21px;">Sheep blood agar</td>
			<td>Eiken Chemical Co.,Ltd.</td>
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			<td>Tryptic soy agar added with 5% (v/v) sheep blood according to the manufacturer.&#160;</td>
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			<td height="21" style="height:21px;">Agar powder</td>
			<td>Wako Pure Chemical Industries</td>
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			<td height="21" style="height:21px;">Sodium citrate (Trisodium citrate dihydrate)</td>
			<td>Wako Pure Chemical Industries</td>
			<td>191-01785</td>
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			<td height="21" style="height:21px;">Cellulose Ester Gridded 0.45 &#956;L HAWG filter</td>
			<td>Merck Milipore</td>
			<td>HAWG 02500</td>
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			<td height="21" style="height:21px;">QIAfilter Plasmid Midi kit</td>
			<td>QIAGEN</td>
			<td>12243</td>
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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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JoVE Journal

Immunology and Infection

The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis

Most microbes in nature are thought to exist as surface-associated communities in biofilms.1 Bacterial biofilms are encased within a matrix and attached to a surface.2 Biofilm formation and development are commonly studied in the laboratory using batch systems such as microtiter plates or flow systems, such as flow-cells. These methodologies are useful for screening mutant and chemical libraries (microtiter plates)3 or growing biofilms for visualization (flow cells)4. Here we present detailed protocols for growing Staphylococcus aureus in two additional types of flow system biofilms: the drip flow biofilm reactor and the rotating disk biofilm reactor.
Drip flow biofilm reactors are designed for the study of biofilms grown under low shear conditions.5 The drip flow reactor consists of four parallel test channels, each capable of holding one standard glass microscope slide sized coupon, or a length of catheter or stint. The drip flow reactor is ideal for microsensor monitoring, general biofilm studies, biofilm cryosectioning samples, high biomass production, medical material evaluations, and indwelling medical device testing.6,7,8,9
The rotating disk reactor consists of a teflon disk containing recesses for removable coupons.10 The removable coupons can by made from any machinable material. The bottom of the rotating disk contains a bar magnet to allow disk rotation to create liquid surface shear across surface-flush coupons. The entire disk containing 18 coupons is placed in a 1000 mL glass side-arm reactor vessel. A liquid growth media is circulated through the vessel while the disk is rotated by a magnetic stirrer. The coupons are removed from the reactor vessel and then scraped to collect the biofilm sample for further study or microscopy imaging. Rotating disc reactors are designed for laboratory evaluations of biocide efficacy, biofilm removal, and performance of anti-fouling materials.9,11,12,13

The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis

Protocols for utilizing open system flow biofilms with drip flow reactors and rotating disk reactors are presented in detail.

Most microbes in nature are thought to exist as surface-associated communities in biofilms.1  Bacterial biofilms are encased within a matrix and attached ...

Subcutaneous Infection of Methicillin Resistant Staphylococcus Aureus (MRSA)

MRSA is a worldwide threat to public health, and MRSA skin and soft-tissue infections now account for more than half of all soft-tissue infections in the United States. Among soft-tissue infections, myositis, pyomyositis, and necrotizing fasciitis have been increasingly reported in association with MRSA arising from the community. To understand the interplay between MRSA and host immunity leading to more severe infection, the availability of animal models is critical, permitting the study of host and bacterial factors. Several infection models have been introduced to assess the pathogenesis of S. aureus during superficial skin infection. Here, we describe a subcutaneous infection model that examines the skin, subcutaneous, and muscle pathologies.

Subcutaneous Infection of Methicillin Resistant Staphylococcus Aureus MRSA

Murine skin and soft tissue infection model is utilized for assessing the virulence function of methicillin resistant Staphylococcus aureus (MRSA) and the host immunological responses. Here, we presented a subcutaneous infection model for skin and soft tissue infection.

MRSA is a worldwide threat to public health, and MRSA skin and soft-tissue infections now account for more than half of all soft-tissue infections in the ...

Experimental Endocarditis Model of Methicillin Resistant Staphylococcus aureus (MRSA) in Rat

Endovascular infections, including endocarditis, are life-threatening infectious syndromes1-3. Staphylococcus aureus is the most common world-wide cause of such syndromes with unacceptably high morbidity and mortality even with appropriate antimicrobial agent treatments4-6. The increase in infections due to methicillin-resistant S. aureus (MRSA), the high rates of vancomycin clinical treatment failures and growing problems of linezolid and daptomycin resistance have all further complicated the management of patients with such infections, and led to high healthcare costs7, 8. In addition, it should be emphasized that most recent studies with antibiotic treatment outcomes have been based in clinical settings, and thus might well be influenced by host factors varying from patient-to-patient. Therefore, a relevant animal model of endovascular infection in which host factors are similar from animal-to-animal is more crucial to investigate microbial pathogenesis, as well as the efficacy of novel antimicrobial agents. Endocarditis in rat is a well-established experimental animal model that closely approximates human native valve endocarditis. This model has been used to examine the role of particular staphylococcal virulence factors and the efficacy of antibiotic treatment regimens for staphylococcal endocarditis. In this report, we describe the experimental endocarditis model due to MRSA that could be used to investigate bacterial pathogenesis and response to antibiotic treatment.

Experimental Endocarditis Model of Methicillin Resistant Staphylococcus aureus MRSA in Rat

Experimental rat endocarditis model due to methicillin-resistant S. aureus.

Endovascular infections, including endocarditis, are life-threatening infectious syndromes1-3. Staphylococcus aureus is the most common world-wide cause ...

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

S. aureus is a pathogenic bacterium that requires iron to carry out vital metabolic functions and cause disease. The most abundant reservoir of iron inside the human host is heme, which is the cofactor of hemoglobin. To acquire iron from hemoglobin, S. aureus utilizes an elaborate system known as the iron-regulated surface determinant (Isd) system1. Components of the Isd system first bind host hemoglobin, then extract and import heme, and finally liberate iron from heme in the bacterial cytoplasm2,3. This pathway has been dissected through numerous in vitro studies4-9. Further, the contribution of the Isd system to infection has been repeatedly demonstrated in mouse models8,10-14. Establishing the contribution of the Isd system to hemoglobin-derived iron acquisition and growth has proven to be more challenging. Growth assays using hemoglobin as a sole iron source are complicated by the instability of commercially available hemoglobin, contaminating free iron in the growth medium, and toxicity associated with iron chelators. Here we present a method that overcomes these limitations. High quality hemoglobin is prepared from fresh blood and is stored in liquid nitrogen. Purified hemoglobin is supplemented into iron-deplete medium mimicking the iron-poor environment encountered by pathogens inside the vertebrate host. By starving S. aureus of free iron and supplementing with a minimally manipulated form of hemoglobin we induce growth in a manner that is entirely dependent on the ability to bind hemoglobin, extract heme, pass heme through the bacterial cell envelope and degrade heme in the cytoplasm. This assay will be useful for researchers seeking to elucidate the mechanisms of hemoglobin-/heme-derived iron acquisition in S. aureus and possibly other bacterial pathogens.

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

Here we describe a growth assay for Staphylococcus aureus using hemoglobin as the sole source of available nutrient iron. This assay establishes the role of bacterial factors involved in hemoglobin-derived iron acquisition.

S. aureus is a pathogenic bacterium that requires iron to  carry out vital metabolic functions and cause disease. The most abundant  reservoir of iron ...

Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus

Staphylococcal Cassette Chromosome mec (SCCmec) typing is a very important molecular tool for understanding the epidemiology and clonal strain relatedness of methicillin-resistant Staphylococcus aureus (MRSA), particularly with the emerging outbreaks of community-associated MRSA (CA-MRSA) occurring on a worldwide basis. Traditional PCR typing schemes classify SCCmec by targeting and identifying the individual mec and ccr gene complex types, but require the use of many primer sets and multiple individual PCR experiments. We designed and published a simple multiplex PCR assay for quick-screening of major SCCmec types and subtypes I to V, and later updated it as new sequence information became available. This simple assay targets individual SCCmec types in a single reaction, is easy to interpret and has been extensively used worldwide. However, due to the sophisticated nature of the assay and the large number of primers present in the reaction, there is the potential for difficulties while adapting this assay to individual laboratories. To facilitate the process of establishing a MRSA SCCmec assay, here we demonstrate how to set up our multiplex PCR assay, and discuss some of the vital steps and procedural nuances that make it successful.

Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus

We demonstrate a simple multiplex PCR assay for quick-screening and typing of Staphylococcal Cassette Chromosome mec (SCCmec) types I-V for methicillin-resistant Staphylococcus aureus, and provide some of the vital steps and procedural nuances that make it successful for adapting this assay to individual laboratories.

Staphylococcal Cassette Chromosome mec (SCCmec) typing  is a very important molecular tool for understanding the epidemiology and  clonal strain ...

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of phenol soluble modulins (PSMs) and other toxins produced and secreted by Staphylococcus aureus on neutrophils. To study the effects of the PSMs on neutrophils we isolate fresh neutrophils using density gradient centrifugation. These neutrophils are loaded with a dye that fluoresces upon calcium mobilization. The activation of neutrophils by PSMs initiates a rapid and transient increase in the free intracellular calcium concentration. In a flow cytometry experiment this rapid mobilization can be measured by monitoring the fluorescence of a pre-loaded dye that reacts to the increased concentration of free Ca2+. Using this method we can determine the PSM concentration necessary to activate the neutrophil, and measure the effects of specific and general inhibitors of the neutrophil activation.	
	To investigate the expression of the PSMs in the intracellular space, we have constructed reporter fusions of the promoter of the PSM&alpha; operon to GFP. When these reporter strains of S. aureus are phagocytosed by neutrophils, the induction of expression can be observed using fluorescence microscopy.

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of PSMs and other toxins secreted by Staphylococcus aureus on neutrophils using flow cytometry and fluorescence microscopy.

We present methods to study the effect of phenol soluble modulins (PSMs)  and other toxins produced and secreted by Staphylococcus  aureus on neutrophils. ...

Methodology for the Efficient Generation of Fluorescently Tagged Vaccinia Virus Proteins

Tagging of viral proteins with fluorescent proteins has proven an indispensable approach to furthering our understanding of virus-host interactions. Vaccinia virus (VACV), the live vaccine used in the eradication of smallpox, is particularly amenable to fluorescent live-cell microscopy owing to its large virion size and the ease with which it can be engineered at the genome level. We report here an optimized protocol for generating recombinant viruses. The minimal requirements for targeted homologous recombination during vaccinia replication were determined, which allows the simplification of construct generation. This enabled the alliance of transient dominant selection (TDS) with a fluorescent reporter and metabolic selection to provide a rapid and modular approach to fluorescently label viral proteins. By streamlining the generation of fluorescent recombinant viruses, we are able to facilitate downstream applications such as advanced imaging analysis of many aspects of the virus-host interplay that occurs during virus replication.

A rapid and modular protocol for the generation of recombinant vaccinia viruses expressing fluorescently&#160;tagged proteins simultaneously using the method of transient dominant selection is described here.

Tagging of viral proteins with fluorescent  proteins has proven an indispensable approach to furthering our understanding  of virus-host interactions. ...

Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

Most pathogenic bacteria are able to form biofilms during infection, but due to the difficulty of manipulating and assessing biofilms, the vast majority of laboratory work is conducted with planktonic cells. Here, we describe a peg plate biofilm assay as performed with Staphylococcus aureus. Bacterial biofilms are grown on pegs attached to a 96-well microtiter plate lid, washed through gentle submersion in buffer, and placed in a drug challenge plate. After subsequent incubation they are again washed and moved to a final recovery plate, in which the fluorescent dye resazurin serves as a viability indicator. This assay offers greatly increased ease-of-use, reliability, and reproducibility, as well as a wealth of data when conducted as a kinetic read. Moreover, this assay can be adapted to a medium-throughput drug screening approach by which an endpoint fluorescent readout is taken instead, offering a path for drug discovery efforts.

Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

Most bacterial infections produce a biofilm. By virtue of their environment, biofilm associated bacteria are often phenotypically drug resistant. Novel antibacterial molecules that kill bacteria in biofilms are thus a high priority. We establish an assay to quickly screen for antimicrobial compounds that are effective at eradicating biofilms.

Most pathogenic bacteria are able to form biofilms during infection, but due to the difficulty of manipulating and assessing biofilms, the vast majority ...

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR (RS-PCR)

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, therefore, suitable to be used for routine purposes. For best resolution, data evaluation and data management, a miniaturized electrophoresis system is required. Together with such an electrophoresis system and the in-house developed software (freely available here) assignment of the pattern of bands to a genotype is standardized and straight forward. DNA extraction is simple (boiling prep), setting-up of the reactions is easy and they can be run on any standard PCR machine. PCR cycling is common except prolonged ramping and elongation times. Compared to spa typing and Multi Locus Sequence Typing (MLST), RS-PCR does not require DNA sequencing what simplifies the analysis considerably and allows a high throughput. Furthermore, the resolution for bovine strains of S. aureus is at least as good as spa typing and better than MLST or pulsed-field gel electrophoresis (PFGE). The RS-PCR data base includes presently a total of 141 genotypes and variants. The method is highly associated with the virulence gene pattern, contagiosity and pathogenicity of S. aureus strains involved in bovine mastitis. S. aureus genotype B (GTB) is contagious and causes herds problems causing large costs in the Switzerland and other European countries. All the other genotypes observed in Switzerland infect individual cows and quarters. Genotyping by RS-PCR allows the reliable prediction of the epidemiological and the pathogenic potential of S. aureus involved in bovine intramammary infection (IMI), two key factors for clinical veterinary medicine. Because of these beneficial properties together with moderate costs and a high sample throughput the goal of this publication is to give a detailed, step-by-step protocol for easily establishing and running RS-PCR for genotyping S. aureus in other laboratories.

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR RS-PCR

Here, ribosomal spacer PCR (RS-PCR) is used together with a miniaturized electrophoresis system as a fast and high resolution method for genotyping S. aureus at moderate costs allowing a high throughput.

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, ...

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence rates of S. aureus infection climbing annually, there is a demand for additional research in its&#160;pathogenicity. Animal models of infectious disease advance our understanding of the host-pathogen response and lead to the development of effective therapeutics. Neutrophils play a primary role in the innate immune response that controls S. aureus infections by forming an abscess to wall off the infection and facilitate bacterial clearance; the number of neutrophils that infiltrate an S. aureus skin infection often correlates with disease outcome. LysM-EGFP mice, which possess the enhanced green fluorescent protein (EGFP) inserted in the Lysozyme M (LysM) promoter region (expressed primarily by neutrophils), when used in conjunction with in vivo whole animal fluorescence imaging (FLI) provide&#160;a means of quantifying neutrophil emigration noninvasively and longitudinally into wounded skin. When combined with a bioluminescent S. aureus strain and sequential in vivo whole animal bioluminescent imaging (BLI), it is possible to longitudinally monitor both the neutrophil recruitment dynamics and in vivo bacterial burden at the site of infection in anesthetized mice from onset of infection to resolution or death. Mice are more resistant to a number of virulence factors produced by S. aureus that facilitate effective colonization and infection in humans. Immunodeficient mice provide a more sensitive animal model to examine persistent S. aureus infections and the ability of therapeutics to boost innate immune responses. Herein, we characterize responses in LysM-EGFP mice that have been bred to MyD88-deficient mice (LysM-EGFP&#215;MyD88-/- mice) along with wild-type LysM-EGFP mice to investigate S. aureus skin wound infection. Multispectral simultaneous detection enabled study of neutrophil recruitment dynamics by using in vivo FLI, bacterial burden by using in vivo BLI, and wound healing longitudinally and noninvasively over time.

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

An approach is described for real-time detection of the innate immune response to cutaneous wounding and Staphylococcus aureus infection of mice. By comparing LysM-EGFP mice (which possess fluorescent neutrophils) with a LysM-EGFP crossbred immunodeficient mouse strain, we advance our understanding of infection and the development of approaches to combat infection.

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence ...