Name: targeting biofilm associated staphylococcus aureus using resazurin based drug-susceptibility assay
Uploaded: 2016-05-05T13:00:00
Description: Watch this Scientific Journal Video about Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay at JoVE.com

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>
	<l...

<ol>
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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 ...

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