We thank Saran Kupul for technical assistance. This work was supported in part by NIH grant R01-AI104952 to FW. Further support was provided by the University of Alabama at Birmingham (UAB) Center for AIDS Research (CFAR), an NIH funded program (P30 AI027767) that was made possible by the following NIH Institutes: NIAID, NIMH, NIDA, NICHD, NHLDI, NIA.

1. Biofilm Initiation
<ol>
	<li>Grow a culture of biofilm producing organism in a nutrient rich medium. Inoculate Staphylococcus aureus strain Newman in 10 ml of Mueller-Hinton medium from a glycerol stock. Perform all work involving handling of S. aureus with gloves and within a biosafety cabinet.</li>
	<li>Incubate for 16 hr at 37 &#176;C on a rotary shaker (100-200 rpm).</li>
	<li>Determine OD600 of the culture using a spectrophotometer and a standard cuvette (path length: 1cm).</li>
	<li>Calculate the volume (V) of culture needed to prepare 20 ml of inoculum with OD600(inoculum) of 0.1 in chelexed Roswell Park Memorial Institute media (CRPMI)9 using the formula: [V = 20 ml*0.1/OD600(culture)] 
	Note: Reference 9 provides details on CRPMI preparation.
	<ol>
		<li>Add the calculated culture volume (from above) to a centrifuge tube and pellet cells by spinning for 1 min at 10,000 x g. To prepare biofilm inoculum, aspirate the spent growth medium and resuspend cell pellet in 20 ml CRPMI.</li>
	</ol>
	</li>
	<li>Pour inoculum into a 25 ml reservoir.</li>
	<li>Carefully remove the lid from a sterile 96-well peg plate by pulling it straight up. 
	Note: Care must be taken not to contaminate the pegs hanging down from the lid. The lid can be placed upside down on a clean surface within a biosafety cabinet.</li>
	<li>Using a 200 &#181;l multi-channel pipette, add 125 &#181;l inoculum to each well of rows A to G of the bottom plate.</li>
	<li>Fill 125 &#181;l sterile CRPMI medium in each well of row H as a sterility control.</li>
	<li>Take the peg-lid and hold it above the plate bottom with the pegs facing downwards. Carefully align the individual pegs with their respective wells. Avoid physical contact between the plate and the pegs. Once aligned, lower the lid carefully down. Avoid misalignments as readjustments performed after the pegs have touched the bottom plate may contaminate sterility controls in row H.</li>
	<li>Seal the lid to the plate with paraffin film to prevent evaporation and place in a sealable plastic bag, sealing tightly. Keep the air volume in the bag as low as possible to minimize evaporation.</li>
	<li>Incubate without shaking at 37 &#176;C for 48 hr in a 5% CO2 incubator. 
	Note: 48 hr of growth has been optimal for S. aureus, but will vary based upon the organism used. 
	Note: Although static incubation is a good starting point to optimize the assay, gently shaking at 150 rpm on a microplate shaker is a possible variation that may enhance biofilm formation of some S. aureus isolates.</li>
</ol>
2. Preparation of the Biofilm Challenge Plate
<ol>
	<li>On the day of the biofilm challenge take a fresh 96-well plate and add 100 &#181;l of CRPMI, equilibrated to RT, to each well of rows B to H. To avoid inconsistent results, utilize a 96-well plate that can hold 200 &#181;l of media when the pegs are inside, and features wells with dimensions identical to those of the biofilm initiation plate.</li>
	<li>Dilute up to 4 test compounds separately in 1.0 ml of CRPMI to 2x the desired final concentration, in order to account for a final dilution in step 2.7. 
	Note: A good starting point is 8 fold above the inhibitory concentration of planktonic cells.</li>
	<li>Add 200 &#181;l of compound 1 in wells A1 to A3, compound 2 in wells A4 to A6, compound 3 in wells A7 to A9 and compound 4 in wells A10 to A12.</li>
	<li>Serially dilute the test compounds by using a multichannel pipette and transfer 100 &#181;l from row A to row B and mix.</li>
	<li>In that manner, continue to transfer 100 &#181;l well to well. Mix after each transfer and stop in row F.</li>
	<li>After mixing, expel 100 &#181;l from row F into a waste container. Do not transfer any liquid to rows G and H.</li>
	<li>Add 100 &#181;l of CRPMI to each well of the plate using a multichannel pipette to bring the volume to 200 &#181;l. See Figure 1 for final plate layout.</li>
</ol>
3. Biofilm Challenge
<ol>
	<li>Add 200 &#181;l of phosphate buffered saline (PBS) to a fresh sterile 96-well plate that features wells with dimensions identical to those of the biofilm initiation plate.</li>
	<li>Remove the plastic bag and paraffin film from incubated biofilm initiation plate.</li>
	<li>Lift the lid straight up. Avoid contact between the pegs and the plate bottom as this may damage the biofilm. Keep bottom part for subsequent OD600 analysis (see 3.8).</li>
	<li>Rinse off planktonic cells by placing the peg-lid onto the plate containing PBS (from step 3.1). Submerge pegs for 5 sec, lift out of PBS, and submerge once more, being careful not to hit the pegs against the well sides.</li>
	<li>Place the rinsed peg-lid into the challenge plate. Avoid unnecessary contact between pegs and bottom plate as this will damage the biofilm leading to inconsistent results.</li>
	<li>Seal plate with paraffin film and place into a sealable plastic bag as described in step 1.10.</li>
	<li>Incubate the plate without shaking for 24 hr at 37 &#176;C in a 5% CO2 incubator.</li>
	<li>Take bottom of biofilm initiation plate from 3.3. Resuspend the bacteria in each well using a multichannel pipette and measure the OD600 with a suitable microplate reader to confirm growth occurring in all wells but the sterility controls in row H.</li>
</ol>
4. Biofilm Determination
<ol>
	<li>Use a plate reader equipped with an incubation chamber and capable of taking kinetic fluorescence readings for detecting resazurin conversion. Set up a 24 hr kinetic fluorescence measurement using a 530 nm excitation and 590 nm emission.</li>
	<li>Set the chamber temperature to 37 &#176;C and have the plate read every 20 min. Set the plate reader to measure from the bottom of the plate according to manufacturer&#39;s instructions.</li>
	<li>Prepare wash plate by adding 200 &#181;l of PBS with a multichannel pipette to a sterile 96 well plate.</li>
	<li>Prepare biofilm recovery medium by adding 400 &#181;l of 0.8 mg/ml resazurin stock solution to 20 ml of CRPMI medium to a final concentration of 16 &#181;g/ml resazurin. 
	Note: Resazurin is a known skin irritant. Use a lab coat, splash goggles, and gloves while handling. CRPMI as the biofilm recovery media was used because it provides the lowest background signal, but it can be replaced by Mueller Hinton media if appropriate.</li>
	<li>Add 150 &#181;l of biofilm recovery medium with a multichannel pipette to each well of a fresh 96-well plate.</li>
	<li>Transfer challenge plate from incubator into a biosafety cabinet and carefully remove plastic bag and sealing film.</li>
	<li>Remove the peg-lid from the challenge plate and rinse off planktonic cells in PBS as described in 3.4. Keep the bottom of the challenge plate for subsequent OD600 analysis (see 4.10).</li>
	<li>After rinsing, place the peg-lid into the plate bottom containing the biofilm recovery medium.</li>
	<li>Wrap the side of the plate with paraffin film, being careful not to obstruct the bottom of the perimeter wells. Immediately after wrapping the plate, place it on the plate reader and start a kinetic read over a 24 hr period by initiating the previously made resazurin kinetic protocol from step 4.1.</li>
	<li>To quantify growth of planktonic cells that may or may not occur during challenge, read the OD600 of the entire challenge plate bottom using a micro plate reader. Follow instructions given in step 3.8. 
	Note: Carefully resuspend the content of each well as cells shedding off the biofilm tend to grow in clumps at the bottom of the well. Any bubbles within the well will strongly interfere with OD600 readings and must be avoided or removed prior to the read.</li>
</ol>

<ol>
	<li>Bjarnsholt, T., Ciofu, O., Molin, S., Givskov, M., Hoiby, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applying+insights+from+biofilm+biology+to+drug+development+-+can+a+new+approach+be+developed.">Applying insights from biofilm biology to drug development - can a new approach be developed.</a> Nat Rev Drug Discov. 12, 791-808 (2013).</li><li>Song, Z., 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=Prosthesis+infections+after+orthopedic+joint+replacement:+the+possible+role+of+bacterial+biofilms.">Prosthesis infections after orthopedic joint replacement: the possible role of bacterial biofilms.</a> Orthop Rev (Pavia). 5, 65-71 (2013).</li><li>Hall-Stoodley, L., Costerton, J. W., Stoodley, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+biofilms:+from+the+natural+environment+to+infectious+diseases).">Bacterial biofilms: from the natural environment to infectious diseases).</a> Nat Rev Microbiol. 2, 95-108 (2004).</li><li>Bordi, C., de Bentzmann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hacking+into+bacterial+biofilms:+a+new+therapeutic+challenge.">Hacking into bacterial biofilms: a new therapeutic challenge.</a> Ann Intensive Care. 1, 19 (2011).</li><li>Fux, C. A., Shirtliff, M., Stoodley, P., Costerton, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+laboratory+reference+strains+mirror+'real-world'+pathogenesis?.">Can laboratory reference strains mirror 'real-world' pathogenesis?.</a> Trends Microbiol. 13, 58-63 (2005).</li><li>Kwasny, S. M., Opperman, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Static+biofilm+cultures+of+Gram-positive+pathogens+grown+in+a+microtiter+format+used+for+anti-biofilm+drug+discovery.">Static biofilm cultures of Gram-positive pathogens grown in a microtiter format used for anti-biofilm drug discovery.</a> Curr Protoc Pharmacol. Chapter 13, Unit 13A 18 (2010).</li><li>Ceri, H., 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+MBEC+Assay+System:+multiple+equivalent+biofilms+for+antibiotic+and+biocide+susceptibility+testing.">The MBEC Assay System: multiple equivalent biofilms for antibiotic and biocide susceptibility testing.</a> Methods Enzymol. 337, 377-385 (2001).</li><li>Ceri, H., 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+Calgary+Biofilm+Device:+new+technology+for+rapid+determination+of+antibiotic+susceptibilities+of+bacterial+biofilms.">The Calgary Biofilm Device: new technology for rapid determination of antibiotic susceptibilities of bacterial biofilms.</a> J Clinical Microbiol. 37, 1771-1776 (1999).</li><li>Baker, 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=Copper+stress+induces+a+global+stress+response+in+Staphylococcus+aureus+and+represses+sae+and+agr+expression+and+biofilm+formation.">Copper stress induces a global stress response in Staphylococcus aureus and represses sae and agr expression and biofilm formation.</a> Appl Environ Microbiol. 76, 150-160 (2010).</li><li>Haeili, 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=Copper+complexation+screen+reveals+compounds+with+potent+antibiotic+properties+against+methicillin-resistant+Staphylococcus+aureus.">Copper complexation screen reveals compounds with potent antibiotic properties against methicillin-resistant Staphylococcus aureus.</a> Antimicrob Agents Chemother. 58, 3727-3736 (2014).</li><li>O'Brien, J., Wilson, I., Orton, T., Pognan, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+the+Alamar+Blue+(resazurin)+fluorescent+dye+for+the+assessment+of+mammalian+cell+cytotoxicity.">Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity.</a> Eur J Biochem. 267, 5421-5426 (2000).</li><li>Mah, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishing+the+minimal+bactericidal+concentration+of+an+antimicrobial+agent+for+planktonic+cells+(MBC-P)+and+biofilm+cells+(MBC-B).">Establishing the minimal bactericidal concentration of an antimicrobial agent for planktonic cells (MBC-P) and biofilm cells (MBC-B).</a> J Vis Exp. (83), e50854 (2014).</li><li>Junker, L. 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The authors have nothing to disclose.

Here, we have described a modified biofilm assay for determining activity of tested inhibitors on S. aureus biofilms focusing on the metabolic state of biofilm associated cells. While the described biofilm initiation and challenge procedures mostly mimicked manufacturer recommendations, the use of resazurin dye to detect and quantify biofilm-associated cells that survive a 24 hr inhibitor challenge dramatically simplifies the recommended procedure of cell recovery (via water bath sonification) and subsequent enu...

Pathogenic microbes can form biofilms in vivo leading to non-acute chronic infections 1. Biofilm-associated infection is a serious risk factor of medical procedures that involve implantation of foreign objects (e.g., artificial bone replacements, breast implants) or the installation of tracheal tubes or urinary catheters 2. In these contexts, anti-infective therapy is almost always necessary as biofilm-based infections are rarely cleared on their own, even in immunocompetent individuals. Staphylococcus aureus is one of the most frequently observed pathogens implicated in biofilm-related complications occurring during the use of invasive medical devices 3.
Unfortunately, the very nature of biofilms as a protective barrier makes them more resistant to treatment than planktonic cells 1,4, and evaluation of predicted clinical efficacy is a critical part of both initial drug development as well as drug resistance surveillance. There is increasing acknowledgement that laboratory conditions, focused on planktonic cultures, may not faithfully represent real-world disease 5. Further compounding the problem, replication of biofilm phenotypes is difficult, and existing biofilm models are tedious and suffer from high inter- and intra-assay variability 6. Thus, many researchers, through necessity, default to planktonic cell assays of drug susceptibility, thereby potentially neglecting an important aspect of bacterial virulence and disease.
Here we describe the protocol for assaying bacteria, specifically S. aureus, in pre-grown biofilms utilizing a 96-well based biofilm system 7,8. While the protocol for biofilm formation and challenge follows essentially the recommendation of the manufacturer, we present an alternative information-rich methodology for quantifying the viability of the biofilm after challenge. Briefly, bacteria are cultured in the peg plate, where biofilms form on the protruding pegs attached to the plate&#39;s lid. After biofilm formation, the pegs are gently dipped in wells of a fresh plate filled with PBS to remove planktonic cells. The peg-lid, with biofilms attached, is transferred to a new challenge plate, containing various concentrations of antibiotics to be assayed. After a second incubation, lids are again removed, washed, and transferred to a recovery plate containing resazurin dye, where they undergo a final incubation. Resazurin conversion can be recorded kinetically or taken as an endpoint reading after a defined recovery period. This dye-based method of quantifying the viability of biofilms differs considerably from the tedious CFU (colony forming units) count-based methodology described in the original protocol 7. OD600 measurements of the drug challenge plate and resazurin conversion kinetics serve as viability readouts of planktonic and biofilm cells, respectively, offering a fast, reliable, information-rich and technically simple assay for biofilm survival.

<table border="0" cellpadding="0" cellspacing="0" width="612">
	<tbody>
		<tr height="61">
			<td height="61" style="height:61px;width:227px;">&#160;Mueller-Hinton Medium</td>
			<td style="width:89px;">Oxoid</td>
			<td style="width:103px;">CM0405</td>
			<td style="width:195px;">Follow recommendations of manufacturer</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:227px;">RPMI-medium</td>
			<td style="width:89px;">Corning&#160;</td>
			<td style="width:103px;">17-105-CV</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="101">
			<td height="101" style="height:101px;width:227px;">CRPMI</td>
			<td style="width:89px;">Ref 9</td>
			<td style="width:103px;"></td>
			<td style="width:195px;">RPMI-1640 medium chelexed for 1h with Chelex 100 resin and then supplemented with 10% unchelexed RPMI-1640</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:227px;">Chelex 100 Resin</td>
			<td style="width:89px;">Bio Rad</td>
			<td style="width:103px;">142-2822</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:227px;">MBEC-plates</td>
			<td style="width:89px;">Innovotech</td>
			<td style="width:103px;">19111</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:227px;">Resazurin Sodium Salt</td>
			<td style="width:89px;">Sigma</td>
			<td style="width:103px;">R7017</td>
			<td style="width:195px;">800&#181;g/ml in DI water&#160;&#160;&#160;&#160; Filter sterile</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:227px;">Micro Plate Shaking Platform&#160;</td>
			<td style="width:89px;">Heidolph Titramax 1000</td>
			<td style="width:103px;"></td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:227px;">Cytation 3 Plate Reader</td>
			<td style="width:89px;">Biotek</td>
			<td style="width:103px;"></td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="57">
			<td height="57" style="height:58px;width:227px;">Gen5 software</td>
			<td style="width:89px;">Biotek</td>
			<td style="width:103px;"></td>
			<td style="width:195px;">Recording and analysis of resazurin conversion</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:227px;">Neocuproine</td>
			<td style="width:89px;">Sigma</td>
			<td style="width:103px;">N1501&#160;</td>
			<td style="width:195px;">prepare 10 mM stock in 100% Ethanol, store at -80&#186;C</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:227px;">Copper sulfate</td>
			<td style="width:89px;">Acros Organics</td>
			<td style="width:103px;">7758-99-8</td>
			<td style="width:195px;">prepare a 100 mM stock solution in water, store at 4&#186;C</td>
		</tr>
		<tr height="121">
			<td height="121" style="height:121px;width:227px;">Cu-Neocuproine</td>
			<td style="width:89px;">Self-Made</td>
			<td style="width:103px;"></td>
			<td style="width:195px;">Generated by mixing equal molarities of neocuproine and copper sulfate. Mix was diluted in CRPMI medium to desired concentration.</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:227px;">Gentamicin</td>
			<td style="width:89px;">Sigma</td>
			<td style="width:103px;">G3632-1G</td>
			<td style="width:195px;"></td>
		</tr>
	</tbody>
</table>

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.

The resazurin assay is sensitive enough to reliably detect very few viable cells capable of replicating in drug free medium after challenge. In this methodology, we define the minimal biofilm eradicating concentration as the lowest concentration at which no resazurin conversion is seen within 24 hr. The resazurin assay relies on oxidative molecules found in metabolically active cells which convert the dye color from blue to pink. Dye conversion is thus an indicator of cell growth, and can...

Watch this Scientific Journal Video about Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay at JoVE.com

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.

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 ...

targeting-biofilm-associated-staphylococcus-aureus-using-resazurin

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Immunology and Infection

13.0K Views.  University of Alabama at Birmingham. 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. 

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

Microtiter Dish Biofilm Formation Assay

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings. In fact, life in a biofilm probably represents the predominate mode of growth for microbes in most environments. Mature biofilms have a few distinct characteristics. Biofilm microbes are typically surrounded by an extracellular matrix that provides structure and protection to the community. Microbes growing in a biofilm also have a characteristic architecture generally comprised of macrocolonies (containing thousands of cells) surrounded by fluid-filled channels. Biofilm-grown microbes are also notorious for their resistance to a range of antimicrobial agents including clinically relevant antibiotics.
The microtiter dish assay is an important tool for the study of the early stages in biofilm formation, and has been applied primarily for the study of bacterial biofilms, although this assay has also been used to study fungal biofilm formation. Because this assay uses static, batch-growth conditions, it does not allow for the formation of the mature biofilms typically associated with flow cell systems. However, the assay has been effective at identifying many factors required for initiation of biofilm formation (i.e, flagella, pili, adhesins, enzymes involved in cyclic-di-GMP binding and metabolism) and well as genes involved in extracellular polysaccharide production. Furthermore, published work indicates that biofilms grown in microtiter dishes do develop some properties of mature biofilms, such a antibiotic tolerance and resistance to immune system effectors.
This simple microtiter dish assay allows for the formation of a biofilm on the wall and/or bottom of a microtiter dish. The high throughput nature of the assay makes it useful for genetic screens, as well as testing biofilm formation by multiple strains under various growth conditions. Variants of this assay have been used to assess early biofilm formation for a wide variety of microbes, including but not limited to, pseudomonads, Vibrio cholerae, Escherichia coli, staphylocci, enterococci, mycobacteria and fungi.
In the protocol described here, we will focus on the use of this assay to study biofilm formation by the model organism Pseudomonas aeruginosa. In this assay, the extent of biofilm formation is measured using the dye crystal violet (CV). However, a number of other colorimetric and metabolic stains have been reported for the quantification of biofilm formation using the microtiter plate assay. The ease, low cost and flexibility of the microtiter plate assay has made it a critical tool for the study of biofilms.

The assay describes a rapid means to measure early biofilm formation in bacteria and fungi. This method uses a microtiter plate as the substratum for microbial biofilm formation, and the biofilm is visualized using crystal violet strain. The assay provides either a qualitative or quantitative assay for early biofilm formation.

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings.  In fact, life in a biofilm ...

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. ...

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 ...

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

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Microtiter Dish Biofilm Formation Assay

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

Subcutaneous Infection of Methicillin Resistant Staphylococcus Aureus (MRSA)

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

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

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

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

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

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

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