This research was supported by U.S. Public Health Service grants AI69233 and AI073843 from the National Institute of Allergy and Infectious Diseases. E.P.S. is a Burroughs Wellcome Fellow in the Pathogenesis of Infectious Diseases. K.P.H. was funded by the Cellular and Molecular Microbiology Training grant Program 5 T32 A107611-10.

1. Purification of Hemoglobin from Fresh Blood
<ol>
	<li>Acquire fresh human blood supplemented with an anticoagulant. Keep blood on ice or at 4 &deg;C throughout the purification.</li>
	<li>Centrifuge blood for 20 min at 1,500 x g. The red blood cells (RBCs) will be at the bottom of the tube. Carefully aspirate the supernatant and gently resuspend the pellet in ice-cold 0.9% (w/v) NaCl solution. Repeat the centrifugation and wash 3 times.</li>
	<li>Resuspend the pellet in 1 volume of ice-cold 10 mM Tris-HCl (pH 8.0). This will induce lysis of the RBCs due to osmotic pressure. Add toluene to ~ 20% final volume.</li>
	<li>Incubate at 4 &deg;C on a rotisserie overnight.</li>
	<li>Centrifuge the lysate at 20,000 x g for 1 hr. Collect the middle hemolysate fraction leaving the toluene (floating on top) and pellet untouched. Use a long-neck pipette to collect the middle fraction.</li>
	<li>Pass through 0.44 &mu;m syringe filter. If the solution contains particulate matter and cannot be passed through the filter, repeat step 1.5.</li>
	<li>Purify hemoglobin (Hb) with a high-performance liquid chromatography (HPLC) anion exchange column (Varian, PL-SAX 1,000 &Aring; 8 &mu;m, 150 mm &times; 4.6 mm). The mobile phase A is 10 mM Tris-HCl (pH 8.0) and mobile phase B is 10 mM Tris-HCl (pH 8.0) + 0.5 M NaCl. A 0%-100% gradient of solvent B is run over 2 min at 2.0 ml/min flow rate. The elution is monitored based on absorption (&lambda;: 410 nm and 280 nm). Collect only the fraction characterized by a bright red color and a prominent absorption peak (Figure 1).</li>
	<li>Dialyze the elution against phosphate-buffered saline (PBS) overnight and then for a few hours again. Sterilize by passing through a 0.22 &mu;m syringe filter.</li>
	<li>To measure Hb concentration, prepare standard Hb solutions of known concentrations in PBS. Determine the concentration of Hb in the sample by mixing the standard solutions (see the table with reagents used) or the sample solution with 2x Drabkin's reagent (prepared from powder) at a 1:1 ratio. For example, mix 100 &mu;l Hb solution with 100 &mu;l 2x Drabkin's reagent in 96-well plates. Incubate for 15 min and measure absorbance at 540 nm. Plot a standard curve and determine the Hb concentration in the sample. Five to fifteen mg/ml yields are typical.</li>
	<li>Run 15-20 &mu;g purified hemoglobin on a 15% SDS-PAGE in duplicate. Stain one of the gels; transfer the proteins from another gel onto a nitrocellulose membrane and immunoblot for hemoglobin (Figure 2).</li>
	<li>Freeze and store 1 ml aliquots of hemoglobin in liquid nitrogen.</li>
</ol>
2. Preparation of Iron-deplete Growth Media
<ol>
	<li>Prepare Roswell Park Memorial Institute (RPMI) broth by dissolving the RPMI powder in water, add sodium bicarbonate as recommended by the manufacturer and 1% cassamino acids (CA) (w/v). Sterilize by passing through a 0.2 &mu;m filter and store refrigerated.</li>
	<li>Prepare metal-depleted RPMI (NRPMI) by adding 7% (w/v) Chelex 100 and mixing overnight on a stir plate. Remove Chelex 100 by passing through a 0.2 &mu;m filter and store refrigerated. Supplement media with essential non-iron metals: 25 &mu;M ZnCl2, 25 &mu;M MnCl2, 100 mM CaCl2 and 1 mM MgCl2 prepared in advance as a sterile 1,000x solution. Use disposable plastic containers for this step to avoid iron contamination from re-usable supplies.</li>
</ol>
3. Staphylococcus aureus Growth using Hemoglobin as a Sole Iron Source
<ol>
	<li>Streak S. aureus for isolation on tryptic soy agar (TSA) from a frozen stock. Incubate at 37 &deg;C for 20-24 hr.</li>
	<li>Resuspend ethylenediamine-N,N'-bis(2-hydroxyphenylacetic acid) (EDDHA) in anhydrous ethanol to 100 mM. EDDHA does not go into solution but is sterilized by ethanol.</li>
	<li>Add EDDHA to RPMI to a final concentration of 0.5 mM. Allow EDDHA to dissolve for at least 30 min before proceeding to the next step. Due to batch-to-batch variation, final EDDHA concentration may need to be lowered to 0.25 mM to allow bacterial growth.</li>
	<li>Inoculate single colonies of S. aureus into 5 ml of RPMI containing EDDHA in 15 ml screw-cap conical tubes. Incubate at 37 &deg;C with shaking at 180 rotations per minute (rpm) for 16-20 hr.</li>
	<li>Centrifuge the overnight cultures for 5 min at 7,500 x g and resuspend the pellet in NRPMI containing 0.5 mM EDDHA. Normalize OD600 to ~3.</li>
	<li>Prepare NRPMI containing 2.5 &mu;g per ml Hb and 0.1-1.0 mM EDDHA. Due to variation between the batches, the EDDHA concentration required to chelate free iron in NRPMI may vary.</li>
	<li>Subculture 10 &mu;l of bacterial suspension from step 3.5 into 1 ml NRPMI + EDDHA + Hb in a 15 ml screw-cap conical tube.</li>
	<li>Incubate the cultures at 37 &deg;C for up to 48 hr with shaking at 180 rpm or on a rolling drum.</li>
	<li>Every 6-12 hr take OD600 readings by removing 50 &mu;l of the culture and mixing it with 150 &mu;l of PBS in 96-well plates.</li>
</ol>

<ol>
	<li>Mazmanian, S., 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=Passage+of+heme-iron+across+the+envelope+of+Staphylococcus+aureus.">Passage of heme-iron across the envelope of Staphylococcus aureus.</a> Science. 299, 906-909 (2003).</li><li>Pishchany, G., Skaar, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taste+for+blood:+hemoglobin+as+a+nutrient+source+for+pathogens.">Taste for blood: hemoglobin as a nutrient source for pathogens.</a> PLOS Pathogens. 8, e1002535 (2012).</li><li>Haley, K. P., Skaar, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+battle+for+iron:+host+sequestration+and+Staphylococcus+aureus+acquisition.">A battle for iron: host sequestration and Staphylococcus aureus acquisition.</a> Microbes and infection. Institut Pasteur. 14, 217-227 (2012).</li><li>Krishna Kumar, K., 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=Structural+basis+for+hemoglobin+capture+by+Staphylococcus+aureus+cell-surface+protein.">Structural basis for hemoglobin capture by Staphylococcus aureus cell-surface protein.</a> IsdH. The Journal of biological chemistry. 286, 38439-38447 (2011).</li><li>Grigg, J. C., Mao, C. X., Murphy, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron-coordinating+tyrosine+is+a+key+determinant+of+NEAT+domain+heme+transfer.">Iron-coordinating tyrosine is a key determinant of NEAT domain heme transfer.</a> Journal of Molecular Biology. 413, 684-698 (2011).</li><li>Villareal, V. A., 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=Transient+weak+protein-protein+complexes+transfer+heme+across+the+cell+wall+of+Staphylococcus+aureus.">Transient weak protein-protein complexes transfer heme across the cell wall of Staphylococcus aureus.</a> Journal of the American Chemical Society. 133, 14176-14179 (2011).</li><li>Muryoi, N., 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=Demonstration+of+the+iron-regulated+surface+determinant+(Isd)+heme+transfer+pathway+in+Staphylococcus+aureus.">Demonstration of the iron-regulated surface determinant (Isd) heme transfer pathway in Staphylococcus aureus.</a> J. Biol. Chem. 283, 28125-28136 (2008).</li><li>Reniere, M. L., Skaar, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staphylococcus+aureus+haem+oxygenases+are+differentially+regulated+by+iron+and+haem.">Staphylococcus aureus haem oxygenases are differentially regulated by iron and haem.</a> Mol. Microbiol. 69, 1304-1315 (2008).</li><li>Liu, 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=Direct+hemin+transfer+from+IsdA+to+IsdC+in+the+iron-regulated+surface+determinant+(Isd)+heme+acquisition+system+of+Staphylococcus+aureus.">Direct hemin transfer from IsdA to IsdC in the iron-regulated surface determinant (Isd) heme acquisition system of Staphylococcus aureus.</a> J. Biol. Chem. 283, 6668-6676 (2008).</li><li>Pishchany, G., 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=Specificity+for+human+hemoglobin+enhances+Staphylococcus+aureus+infection.">Specificity for human hemoglobin enhances Staphylococcus aureus infection.</a> Cell Host Microbe. 8, 544-550 (2010).</li><li>Pishchany, G., Dickey, S. E., Skaar, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcellular+localization+of+the+Staphylococcus+aureus+heme+iron+transport+components+IsdA+and+IsdB.">Subcellular localization of the Staphylococcus aureus heme iron transport components IsdA and IsdB.</a> Infect. Immun. 77, 2624-2634 (2009).</li><li>Torres, V. J., Pishchany, G., Humayun, M., Schneewind, O., Skaar, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staphylococcus+aureus+IsdB+is+a+hemoglobin+receptor+required+for+heme+iron+utilization.">Staphylococcus aureus IsdB is a hemoglobin receptor required for heme iron utilization.</a> J. Bacteriol. 188, 8421-8429 (2006).</li><li>Kim, H. K., 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=IsdA+and+IsdB+antibodies+protect+mice+against+Staphylococcus+aureus+abscess+formation+and+lethal+challenge.">IsdA and IsdB antibodies protect mice against Staphylococcus aureus abscess formation and lethal challenge.</a> Vaccine. 28, 6382-6392 (2010).</li><li>Cheng, A. G., 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=Genetic+requirements+for+Staphylococcus+aureus+abscess+formation+and+persistence+in+host+tissues.">Genetic requirements for Staphylococcus aureus abscess formation and persistence in host tissues.</a> Faseb J. 23, 3393-3404 (2009).</li><li>Andreini, C., Bertini, I., Cavallaro, G., Holliday, G. L., Thornton, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metal+ions+in+biological+catalysis:+from+enzyme+databases+to+general+principles.">Metal ions in biological catalysis: from enzyme databases to general principles.</a> J. Biol. Inorg. Chem. 13, 1205-1218 (2008).</li><li>Weinberg, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron+availability+and+infection.">Iron availability and infection.</a> Biochimica et Biophysica Acta (BBA) - General Subjects. 1790, 600-605 (2009).</li><li>Drabkin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolism+of+the+Hemin+Chromoproteins.">Metabolism of the Hemin Chromoproteins.</a> Physiological Reviews. 31, 345-431 (1951).</li><li>Graversen, J. H., Madsen, M., Moestrup, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD163:+a+signal+receptor+scavenging+haptoglobin-hemoglobin+complexes+from+plasma.">CD163: a signal receptor scavenging haptoglobin-hemoglobin complexes from plasma.</a> The international journal of biochemistry &amp; cell biology. 34, 309-314 (2002).</li><li>Torres, V. J., 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=Staphylococcus+aureus+Fur+regulates+the+expression+of+virulence+factors+that+contribute+to+the+pathogenesis+of+pneumonia.">Staphylococcus aureus Fur regulates the expression of virulence factors that contribute to the pathogenesis of pneumonia.</a> Infect. Immun. 78, 1618-1628 (2010).</li><li>Hammer, N. D., Skaar, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Mechanisms+of+Staphylococcus+aureus+Iron+Acquisition.">Molecular Mechanisms of Staphylococcus aureus Iron Acquisition.</a> Annu. Rev. Microbiol. , (2011).</li><li>Hurd, A. 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=The+iron-regulated+surface+proteins+IsdA,+IsdB,+and+IsdH+are+not+required+for+heme+iron+utilization+in+Staphylococcus+aureus.">The iron-regulated surface proteins IsdA, IsdB, and IsdH are not required for heme iron utilization in Staphylococcus aureus.</a> Fems. Microbiology Letters. 329, 93-100 (2012).</li><li>Boys, B. L., Kuprowski, M. C., Konermann, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Symmetric+behavior+of+hemoglobin+alpha-+and+beta-+subunits+during+acid-induced+denaturation+observed+by+electrospray+mass+spectrometry.">Symmetric behavior of hemoglobin alpha- and beta- subunits during acid-induced denaturation observed by electrospray mass spectrometry.</a> Biochemistry. 46, 10675-10684 (2007).</li><li>Williams, R. C., Tsay, K. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+convenient+chromatographic+method+for+the+preparation+of+human+hemoglobin.">A convenient chromatographic method for the preparation of human hemoglobin.</a> Analytical Biochemistry. 54, 137-145 (1973).</li><li>Shen, T. J., 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=Production+of+unmodified+human+adult+hemoglobin+in+Escherichia+coli.">Production of unmodified human adult hemoglobin in Escherichia coli.</a> Proc. Natl. Acad. Sci. U.S.A. 90, 8108-8112 (1993).</li><li>Manjula, B. N., Acharya, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+molecular+analysis+of+hemoglobin+by+high-performance+liquid+chromatography.">Purification and molecular analysis of hemoglobin by high-performance liquid chromatography.</a> Methods Mol. Med. 82, 31-47 (2003).</li><li>Neilands, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+envelope+proteins+related+to+iron.">Microbial envelope proteins related to iron.</a> Annual review of microbiology. 36, 285-309 (1982).</li><li>Chart, H., Buck, M., Stevenson, P., Griffiths, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron+regulated+outer+membrane+proteins+of+Escherichia+coli:+variations+in+expression+due+to+the+chelator+used+to+restrict+the+availability+of+iron.">Iron regulated outer membrane proteins of Escherichia coli: variations in expression due to the chelator used to restrict the availability of iron.</a> Journal of General Microbiology. 132, 1373-1378 (1986).</li><li>Rogers, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron-Binding+Catechols+and+Virulence+in+Escherichia+coli.">Iron-Binding Catechols and Virulence in Escherichia coli.</a> Infection and Immunity. 7, 445-456 (1973).</li></ol>

We have nothing to disclose.

Iron is an essential nutrient required by organisms from all kingdoms of life15. In vertebrates, iron is sequestered to avoid toxicity caused by this element. This sequestration also conceals iron from invading microbes in a process known as nutritional immunity16. In response, pathogens have evolved strategies that circumvent nutritional immunity. One such mechanism relies on hemoglobin, which is the most abundant source of iron within the host17. Hemoglobin is contained within r...

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>HPLC anion exchange column</td>
 <td>Varian</td>
 <td>PL1551-3802</td>
 <td></td>
 </tr>
 <tr>
 <td>Drabkin's reagent</td>
 <td>Sigma</td>
 <td>D5941-6VL</td>
 <td></td>
 </tr>
 <tr>
 <td>Hemoglobin standard</td>
 <td>Pointe Scientific</td>
 <td>H7506-STD</td>
 <td></td>
 </tr>
 <tr>
 <td>RPMI</td>
 <td> HyClone</td>
 <td>SH30011.02</td>
 <td></td>
 </tr>
 <tr>
 <td>Chelex 100 sodium form</td>
 <td>Sigma</td>
 <td>C7901</td>
 <td></td>
 </tr>
 <tr>
 <td>EDDHA</td>
 <td>LGC Standards GmbH</td>
 <td>ANC 001</td>
 <td></td>
 </tr>
 <tr>
 <td>Hemoglobin a antibody</td>
 <td>Santa Cruz Biotechnology, Inc</td>
 <td>SC-21005</td>
 <td></td>
 </tr>
 <tr>
 <td>Tryptic soy agar</td>
 <td>BD</td>
 <td>236920</td>
 <td></td>
 </tr>
 </tbody>
</table>

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.

We have purified human hemoglobin from hemolysate with HPLC (Protocol step 1.7). Figure 1 shows recorded absorbance of eluate at 280 and 410 nm wavelengths. Fraction 5 was collected and other fractions were discarded. Yields of five to fifteen milligrams of hemoglobin per milliliter of eluate are typically acquired. Purified hemoglobin was analyzed by SDS-PAGE in duplicate and the gels were either stained for proteins or transferred onto nitrocellulose and immunoblotted (Protocol step 1.10, Figur...

Watch this Scientific Journal Video about Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source at JoVE.com

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.

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 ...

staphylococcus-aureus-growth-using-human-hemoglobin-as-an-iron-source

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Immunology and Infection

16.4K Views.  Vanderbilt University Medical School. 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.

Video: Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

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 ...

Cost-effective Method for Microbial Source Tracking Using Specific Human and Animal Viruses

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant limitations, viruses are more resistant to many inactivation processes and standard fecal indicators do not inform on the source of contamination. The development of cost-effective methods for the concentration of viruses from water and molecular assays facilitates the applicability of viruses as indicators of fecal contamination and as microbial source tracking (MST) tools. Adenoviruses and polyomaviruses are DNA viruses infecting specific vertebrate species including humans and are persistently excreted in feces and/or urine in all geographical areas studied. In previous studies, we suggested the quantification of human adenoviruses (HAdV) and JC polyomaviruses (JCPyV) by quantitative PCR (qPCR) as an index of human fecal contamination. Recently, we have developed qPCR assays for the specific quantification of porcine adenoviruses (PAdV) and bovine polyomaviruses (BPyV) as animal fecal markers of contamination with sensitivities of 1-10 genome copies per test tube. In this study, we present the procedure to be followed to identify the source of contamination in water samples using these tools. As example of representative results, analysis of viruses in ground water presenting high levels of nitrates is shown.
Detection of viruses in low or moderately polluted waters requires the concentration of the viruses from at least several liters of water into a much smaller volume, a procedure that usually includes two concentration steps in series. This somewhat cumbersome procedure and the variability observed in viral recoveries significantly hamper the simultaneous processing of a large number of water samples.
In order to eliminate the bottleneck caused by the two-step procedures we have applied a one-step protocol developed in previous studies and applicable to a diversity of water matrices. The procedure includes: acidification of ten-liter water samples, flocculation by skimmed milk, gravity sedimentation of the flocculated materials, collection of the precipitate and centrifugation, resuspension of the precipitate in 10 ml phosphate buffer. The viral concentrate is used for the extraction of viral nucleic acids and the specific adenoviruses and polyomaviruses of interest are quantified by qPCR. High number of samples may be simultaneously analyzed using this low-cost concentration method.
The procedure has been applied to the analysis of bathing waters, seawater and river water and in this study, we present results analyzing groundwater samples. This high-throughput quantitative method is reliable, straightforward, and cost-effective.

The study describes a cost-effective method for the identification of the source of fecal/urine contamination or contamination by nitrates in water using qPCR for the specific quantification of human/porcine/bovine DNA viruses, adenoviruses and polyomaviruses, proposed as MST tools.

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant ...

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 ...

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. ...

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, ...

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.

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.

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

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 ...

Quantifying the Cytotoxicity of Staphylococcus aureus Against Human Polymorphonuclear Leukocytes

Staphylococcus aureus is capable of secreting a wide range of leukocidins that target and disrupt the membrane integrity of polymorphonuclear leukocytes (PMNs or neutrophils). This protocol describes both the purification of human PMNs and the quantification of S. aureus cytotoxicity against PMNs in three different sections. Section 1 details the isolation of PMNs and serum from human blood using density centrifugation. Section 2 tests the cytotoxicity of extracellular proteins produced by S. aureus against these purified human PMNs. Section 3 measures the cytotoxicity against human PMNs following the phagocytosis of live S. aureus. These procedures measure disruption of PMN plasma membrane integrity by S. aureus leukocidins using flow cytometry analysis of PMNs treated with propidium iodide, a DNA binding fluorophore that is cell membrane impermeable. Collectively, these methods have the advantage of rapidly testing S. aureus cytotoxicity against primary human PMNs and can be easily adapted to study other aspects of host-pathogen interactions.

Quantifying the Cytotoxicity of Staphylococcus aureus Against Human Polymorphonuclear Leukocytes

This protocol describes a method for the purification of polymorphonuclear leukocytes from whole human blood and two distinct assays that quantify the cytotoxicity of Staphylococcus aureus against these important innate immune cells.

Staphylococcus aureus is capable of secreting a wide range of leukocidins that target and disrupt the membrane integrity of polymorphonuclear leukocytes ...