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>

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	<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. 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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 enumeration of viable cells via counting of CFUs. This manufacturer-recommended procedure involves at least 5 additional manipulation steps, each of them error prone, labor intensive, and impractical to automate. The advantage of simplifying the read-out procedure of the peg plate assay increases its attractiveness for high throughput operations and applications.
Though a lengthy protocol by number of steps, this procedure is rather conceptually simple, with few critical steps. Most crucially, the ability to repeatedly grow biofilms of the same quality is essential for obtaining reproducible results. Furthermore, one must exercise care whenever removing or replacing the peg-lid. Contact at any point with a well&#39;s wall would disrupt the biofilm, skewing results. More so, adequate washing is crucial: failure to remove planktonic cells at any point removes any ability to measure biofilm-associated bacteria. Conversely, though, washing must not be too rigorous; additional wash steps may begin removing biofilm, producing inconsistent results.
The peg plate approach has multiple uses, depending on the desired resulting data. The assay is described here as an information-rich kinetic measurement of metabolic resazurin conversion into a fluorescent dye 11. This conversion is associated with a color shift from blue to pink (Figure 2C), which, in addition to the quantitative fluorescence read, allows for an easy initial qualitative readout simply by observing the conversion. After removing the peg lid, this conversion can also be quantified by reading the absorbance at 600 nm should fluorescence capabilities not be available. Although the fluorescence eventually plateaus upon maximum dye conversion in each well (with survivors), the onset of dye conversion could be used to estimate the metabolically active survivor population relative to the untreated control (Figure 3). In our hands we have found that each ~100 min delay in the onset of resazurin conversion corresponds to approximately a 10-fold decrease in the number of metabolically active cells in the starting inoculum relative to the untreated control (as determined on planktonic cell culture serially diluted in 10-fold increments using biofilm recovery medium (data not shown)).
Beyond kinetic readouts, though, the modified peg plate method is more amenable to high-throughput drug screen projects than the original protocol. By measuring resazurin conversion as an endpoint instead of a kinetic, a user can generate a binary analysis of the samples to track simple activity of compounds (i.e., dye conversion/no conversion), which removes the need for 24 hr in a fluorescence plate reader, permitting conventional incubations. This methodology allows for determining the minimal biofilm eradicating concentration without the need of sonification and plating. The kinetic read also tracks the metabolic state of the biofilm depending on the treatment condition and inhibitor concentration. The reduced number of manipulation steps, increased reproducibility, and quantitative readout method (as opposed to plating each well individually 12) allows users to screen potentially thousands of compounds per day in a single concentration screen. In fact, peg-style plates have been previously used in high-throughput drug screens for biofilm dispersants, though resazurin (as used here) is a much more economical choice than the proprietary luciferase reagent utilized 13.
While a general improvement over the existing manufacturer protocol, the assay featured here does have certain limitations one must keep in mind. Inherently, we are only measuring metabolic capability of biofilm-associated cells. This assay does not measure biofilm mass or otherwise weaken the biofilm architecture or integrity. Those effects would not necessarily be detected. Metabolically inactive, but still viable, persister cells, which have been shown to exist in biofilms 14,15 will also be undetected. Further, compounds that induce quiescence without actually sterilizing a biofilm might erroneously appear as false positives, depending on length of the induced state. Viability indicators are ultimately surrogates for more iron-clad measurement methods such as flow cytometry or CFU enumeration, and the end user must determine which method is appropriate. Finally, as the resazurin conversion relies upon endogenous NADH, depletion of intracellular stores could be detrimental for bacteria. While we have not observed deleterious effects against S. aureus, other bacterial species might react differently. Users must empirically test and validate this protocol for reliability and reproducibility given varying organisms and lab environments.

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><tbody><tr><td>&amp;nbsp;Mueller-Hinton Medium</td><td>Oxoid</td><td>CM0405</td><td>Follow recommendations of manufacturer</td></tr><tr><td>RPMI-medium</td><td>Corning&amp;nbsp;</td><td>17-105-CV</td><td></td></tr><tr><td>CRPMI</td><td>Ref 9</td><td></td><td>RPMI-1640 medium chelexed for 1 hr with Chelex 100 resin and then supplemented with 10% unchelexed RPMI-1640</td></tr><tr><td>Chelex 100 Resin</td><td>Bio Rad</td><td>142-2822</td><td></td></tr><tr><td>MBEC-plates</td><td>Innovotech</td><td>19111</td><td></td></tr><tr><td>Resazurin Sodium Salt</td><td>Sigma</td><td>R7017</td><td>800 &amp;micro;g/ml in DI water&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Filter sterile</td></tr><tr><td>Micro Plate Shaking Platform&amp;nbsp;</td><td>Heidolph Titramax 1000</td><td></td><td></td></tr><tr><td>Cytation 3 Plate Reader</td><td>Biotek</td><td></td><td></td></tr><tr><td>Gen5 software</td><td>Biotek</td><td></td><td>Recording and analysis of resazurin conversion</td></tr><tr><td>Neocuproine</td><td>Sigma</td><td>N1501&amp;nbsp;</td><td>prepare 10 mM stock in 100% Ethanol, store at -80 &amp;ordm;C</td></tr><tr><td>Copper sulfate</td><td>Acros Organics</td><td>7758-99-8</td><td>prepare a 100 mM stock solution in water, store at 4 &amp;ordm;C</td></tr><tr><td>Cu-Neocuproine</td><td>Self-Made</td><td></td><td>Generated by mixing equal molarities of neocuproine and copper sulfate. Mix was diluted in CRPMI medium to desired concentration.</td></tr><tr><td>Gentamicin</td><td>Sigma</td><td>G3632-1G</td><td></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 be quantified using a fluorescent micro plate reader. In this paper, neocuproine and Cu-neocuproine (neocuproine complexed with copper) were tested for their activity toward biofilm associated S. aureus applying a layout that is based on Figure 1. The data obtained during execution of the protocol are an OD600 reading of the biofilm initiation plate, which serves as a quality control (data not shown), an OD600 reading of the plate bottom after challenge has been completed (Figure 2A), and a kinetic recording of resazurin conversion into its fluorescent metabolite resorufin of the recovery plate (Figure 2B). Further, an endpoint image of the challenge plate can be used for visual inspection of resazurin conversion (Figure 2C) if a yes/no evaluation of resazurin conversion is sufficient for the purpose of the experiment. Alternatively, if kinetic recording capabilities are unavailable or multiple plates are run in parallel, fluorescence can be determined after 24 hr by a single endpoint read. As seen in Figure 2A, OD readings from the challenge plate indicate that neocuproine alone does not inhibit growth of planktonic cells; however, Cu-neocuproine inhibits growth at 1.3 &#181;M, as expected 10. A similar pattern is observed from the resazurin kinetic of the biofilm plate, indicating that only Cu-neocuproine is able to eliminate metabolically active biofilm-associated cells (Figure 2B).
In addition to providing biofilm eradication concentrations, the kinetic assay provides information on the metabolic state of the biofilm which is indicative of the number of viable bacteria. When comparing biofilms treated with gentamicin at increasing concentrations to the untreated controls, a lag of resazurin conversion can be seen (Figure 3). For 2.5 &#181;g/ml gentamicin, there is a 13 hr delay until the well reaches the same conversion of resazurin as the untreated control. This lag likely comes from a reduced number of viable cells present in the treated biofilm compared to the untreated.
<img alt="Figure 1" src="/files/ftp_upload/53925/53925fig1.jpg" /> 
Figure 1.&#160;Challenge plate layout. Example layout of challenge plate accommodating parallel testing of four compounds in triplicate at six distinct concentrations. Untreated and sterility controls are also included. The layout design supports convenient in-plate preparation of serial dilutions using a multichannel pipet. Concentrations are in &#181;M. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53925/53925fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53925/53925fig2.jpg" /> 
Figure 2.&#160;Determining minimum biofilm eradication concentration. Neocuproine and Cu-neocuproine were tested for biofilm eradication potential. (A) The OD600 was taken from the challenge plate after removal of the peg-lid to quantify the growth of biofilm dissociated cells. (B) Resazurin conversion was monitored kinetically in individual wells over a 24 hr period. Individual minigraphs show fluorescence (RFU) over time (24 hr). The lack of resazurin conversion indicates the absence of survivors. Resazurin conversion indicates the presence of viable cells shedding off the biofilm and growing in the recovery medium. (C) If a kinetic readout is not possible or not needed, then a simple yes/no answer for the presence of viable cells can be determined visually from the color conversion of the blue resazurin to its pink, reduced form. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53925/53925fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53925/53925fig3.jpg" /> 
Figure 3.&#160;Kinetic resazurin assay reveals inhibitory effects on bacteria in a biofilm. Compounds may not fully eradicate biofilm embedded cells, but can still reduce their viability. This can be seen when comparing the delay of resazurin conversion of wells treated with different concentrations of gentamicin. Wells that have a delay in resazurin conversion indicate a reduced number of viable cells. Black (only media), Orange (0 &#181;g/ml), Green (0.3 &#181;g/ml), Blue (0.6 &#181;g/ml), Purple (2.5 &#181;g/ml), and Gray (5 &#181;g/ml). &#916;t refers to the time differential of exponential resazurin conversion between untreated and gentamicin treated samples at the approximate half maximum fluorescence value. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53925/53925fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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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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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 ...

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

Biosensor for Detection of Antibiotic Resistant Staphylococcus Bacteria

A structurally transformed lytic bacteriophage having a broad host range of Staphylococcus aureus strains and a penicillin-binding protein (PBP 2a) antibody conjugated latex beads have been utilized to create a biosensor designed for discrimination of methicillin resistant (MRSA) and sensitive (MSSA) S. aureus species 1,2. The lytic phages have been converted into phage spheroids by contact with water-chloroform interface. Phage spheroid monolayers have been moved onto a biosensor surface by Langmuir-Blodgett (LB) technique 3. The created biosensors have been examined by a quartz crystal microbalance with dissipation tracking (QCM-D) to evaluate bacteria-phage interactions. Bacteria-spheroid interactions led to reduced resonance frequency and a rise in dissipation energy for both MRSA and MSSA strains. After the bacterial binding, these sensors have been further exposed to the penicillin-binding protein antibody latex beads. Sensors analyzed with MRSA responded to PBP 2a antibody beads; although sensors inspected with MSSA gave no response. This experimental distinction determines an unambiguous discrimination between methicillin resistant and sensitive S. aureus strains. Equally bound and unbound bacteriophages suppress bacterial growth on surfaces and in water suspensions. Once lytic phages are changed into spheroids, they retain their strong lytic activity and show high bacterial capture capability. The phage and phage spheroids can be utilized for testing and sterilization of antibiotic resistant microorganisms. Other applications may include use in bacteriophage therapy and antimicrobial surfaces.

Lytic phage biosensors and antibody beads are able to discriminate between methicillin resistant (MRSA) and sensitive staphylococcus bacteria. The phages were immobilized by a Langmuir-Blodgett method onto a surface of a quartz crystal microbalance sensor and worked as broad range staphylococcus probes. Antibody beads recognize MRSA.

A structurally transformed lytic bacteriophage having a broad host range of Staphylococcus aureus strains and a penicillin-binding protein (PBP 2a) ...

Bioengineering

Stress-induced Antibiotic Susceptibility Testing on a Chip

We have developed a rapid microfluidic method for antibiotic susceptibility testing in a stress-based environment. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria survive these stressful conditions. The hypothesis behind this method is new: stress activation of biochemical pathways, which are targets of antibiotics, can accelerate antibiotic susceptibility testing. As compared to standard antibiotic susceptibility testing methods, the rate-limiting step -&#160;bacterial growth -&#160;is omitted during antibiotic application. The technical implementation of the method is in a combination of standard techniques and innovative approaches. The standard parts of the method include bacterial culture protocols, defining microfluidic channels in polydimethylsiloxane&#160;(PDMS), cell viability monitoring with fluorescence, and batch image processing for bacteria counting. Innovative parts of the method are in the use of culture media flow for mechanical stress application, use of enzymes to damage but not kill the bacteria, and use of microarray substrates for bacterial attachment. The developed platform can be used in antibiotic and nonantibiotic related drug development and testing. As compared to the standard bacterial suspension experiments, the effect of the drug can be turned on and off repeatedly over controlled time periods. Repetitive observation of the same bacterial population is possible over the course of the same experiment.

We have developed a microfluidic platform for rapid antibiotic susceptibility testing. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria can survive these stressful conditions.

We have developed a rapid microfluidic method for antibiotic susceptibility testing in a stress-based environment. Fluid is passed at high speeds over ...

A Platform of Anti-biofilm Assays Suited to the Exploration of Natural Compound Libraries

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of antibiotic-tolerant infections. Biofilms have displayed an exceptionally high tolerance for chemotherapy, which is thought to be multifactorial. For instance, the matrix provides a physical barrier that decreases the penetration of antibiotics into the biofilm. Also, cells within the biofilms are phenotypically diverse. Likely, biofilm resilience arises from a combination of these and other, yet unknown, mechanisms. All of the currently existing antibiotics have been developed against single-cells (planktonic) bacteria. Therefore, so far, a very limited repertoire of molecules exists that can selectively act on mature biofilms. This situation has driven a progressive paradigm shift in drug discovery, in which searching for anti-biofilms has been urged to occupy a more prominent place. An additional challenge is that there are a very limited number of standardized methods for biofilm research, especially those that can be used for large-throughput screening of chemical libraries. Here, an experimental anti-biofilm platform for chemical screening is presented. It uses three assays to measure biofilm viability (with resazurin staining), total biomass (with crystal violet staining), and biofilm matrix (using a wheat germ agglutinin, WGA-fluorescence-based staining of the poly-N-acetyl-glucosamine, PNAG, fraction). All the assays were developed using Staphylococcus aureus as the model bacteria. Examples of how the platform can be used for primary screening as well as for functional characterization of identified anti-biofilm hits are presented. This experimental sequence further allows for the classification of the hits based upon the measured end-points. It also provides information on their mode of action, especially on long-term versus short-term chemotherapeutic effects. Thus, it is very advantageous for the quick identification of high-quality hit compounds that can serve as starting points for various biomedical applications.

Biofilm infections show high tolerance towards chemotherapy. No single assay captures the complexity of biofilms. Instead, complementary assays are needed. We present a screening platform (developed for S. aureus) that combines assays for viability, biomass, and biofilm matrix. It allows anti-biofilm drug discovery, including the assessment of long-term chemotherapeutic effects.

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of ...

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

Standardized In vitro Assays to Visualize and Quantify Interactions between Human Neutrophils and Staphylococcus aureus Biofilms

Neutrophils are the first line of defense deployed by the immune system during microbial infection. In vivo, neutrophils are recruited to the site of infection where they use processes such as phagocytosis, production of reactive oxygen and nitrogen species (ROS, RNS, respectively), NETosis (neutrophil extracellular trap), and degranulation to kill microbes and resolve the infection. Interactions between neutrophils and planktonic microbes have been extensively studied. There have been emerging interests in studying infections caused by biofilms in recent years. Biofilms exhibit properties, including tolerance to killing by neutrophils, distinct from their planktonic-grown counterparts. With the successful establishment of both in vitro and in vivo biofilm models, interactions between these microbial communities with different immune cells can now be investigated. Here, techniques that use a combination of traditional biofilm models and well-established neutrophil activity assays are tailored specifically to study neutrophil and biofilm interactions. Wide-field fluorescence microscopy is used to monitor the localization of neutrophils in biofilms. These biofilms are grown in static conditions, followed by the addition of neutrophils derived from human peripheral blood. The samples are stained with appropriate dyes prior to visualization under the microscope. Additionally, the production of ROS, which is one of the many neutrophil responses against pathogens, is quantified in the presence of a biofilm. The addition of immune cells to this established system will expand the understanding of host-pathogen interactions while ensuring the use of standardized and optimized conditions to measure these processes accurately.

Standardized In vitro Assays to Visualize and Quantify Interactions between Human Neutrophils and Staphylococcus aureus Biofilms

The present protocol describes the study of neutrophil-biofilm interactions. Staphylococcus aureus biofilms are established in vitro and incubated with peripheral blood-derived human neutrophils. The oxidative burst response from neutrophils is quantified, and the neutrophil localization within the biofilm is determined by microscopy.

Neutrophils are the first line of defense deployed by the immune system during microbial infection. In vivo, neutrophils are recruited to the site of ...

A Soluble Tetrazolium-Based Reduction Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilms

Candida species are the fourth-most common cause of systemic nosocomial infections. Systemic or invasive candidiasis frequently involves biofilm formation on implanted devices or catheters, which is associated with increased virulence and mortality. Biofilms produced by different Candida species exhibit enhanced resistance against various antifungal drugs. Therefore, there is a need to develop effective immunotherapies or adjunctive treatments against Candida biofilms. While the role of cellular immunity is well established in anti-Candida protection, the role of humoral immunity has been studied less.
It has been hypothesized that inhibition of biofilm formation and maturation is one of the major functions of protective antibodies, and Candida albicans germ tube antibodies (CAGTA) have been shown to suppress in vitro growth and biofilm formation of C. albicans earlier. This paper outlines a detailed protocol for evaluating the role of antibodies on biofilms formed by C. tropicalis. The methodology for this protocol involves C. tropicalis biofilm formation in 96-well microtiter plates, which were then incubated in the presence or absence of antigen-specific antibodies, followed by a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-carboxanilide-2H-tetrazolium (XTT) assay for measuring the metabolic activity of fungal cells in the biofilm.
The specificity was confirmed by using appropriate serum controls, including Sap2-specific antibody-depleted serum. The results demonstrate that antibodies present in the serum of immunized animals can inhibit Candida biofilm maturation in vitro. In summary, this paper provides important insights regarding the potential of antibodies in developing novel immunotherapies and synergistic or adjunctive treatments against biofilms during invasive candidiasis. This in vitro protocol can be used to check the effect of potential new antifungal compounds on the metabolic activity of Candida species cells in biofilms.

A Soluble Tetrazolium-Based Reduction Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilms

A 96-well microtiter plate-based protocol using a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-carboxanilide-2H-tetrazolium (XTT) reduction assay is described herein, to study antibodies' effects on biofilms formed by C. tropicalis. This in vitro protocol can be used to check the effect of potential new antifungal compounds on the metabolic activity of Candida species cells in biofilms.

Candida species are the fourth-most common cause of systemic nosocomial infections. Systemic or invasive candidiasis frequently involves biofilm formation ...

Improved Enzyme Protection Assay to Study Staphylococcus aureus Internalization and Intracellular Efficacy of Antimicrobial Compounds

Staphylococcus aureus expresses virulence factors to trigger its internalization into eukaryote cells and to survive inside different subcellular compartments. This paper describes an enzyme protection assay to study the extent of S. aureus internalization and its intracellular survival in adherent non-professional phagocytic cells (NPPCs) as well as the intracellular efficacy of antimicrobial compounds. NPPCs are grown in a multi-well plate until they reach 100% confluence. S. aureus cultures are grown overnight in cell culture medium. The bacterial suspension is diluted according to the number of cells per well to inoculate the cells at a controlled multiplicity of infection. Inoculated cells are incubated for 2 h to allow the bacteria to be internalized by the NPPCs, following which lysostaphin is added to the culture medium to selectively kill extracellular bacteria. Lysostaphin is present in the culture medium for the rest of the experiment. 
At this point, the infected cells could be incubated with antimicrobial compounds to assess their intracellular activities against S. aureus. Next, the cells are washed three times to remove the drugs, and intracellular S. aureus load is then quantified by culturing on agar plates. Alternatively, for studying staphylococcal virulence factors involved in intracellular survival and cell toxicity, lysostaphin could be inactivated with proteinase K to eliminate the need for washing steps. This tip improves the reliability of the intracellular bacterial load quantification, especially if cells tend to detach from the culture plate when they become heavily infected because of the multiplication of intracellular S. aureus. These protocols can be used with virtually all types of adherent NPPCs and with 3D cell culture models such as organoids.

Improved Enzyme Protection Assay to Study Staphylococcus aureus Internalization and Intracellular Efficacy of Antimicrobial Compounds

This protocol aims to describe how to study the extent of Staphylococcus aureus internalization and its ability to survive inside the human host cell, as well as the intracellular efficacy of antimicrobial compounds.

Staphylococcus aureus expresses virulence factors to trigger its internalization into eukaryote cells and to survive inside different subcellular ...

Biology

In Vitro Biofilm Synthesis by Staphylococcus aureus

Source: Rana, P. S. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/63773">Standardized In vitro Assays to Visualize and Quantify Interactions between Human Neutrophils and Staphylococcus aureus Biofilms.</a> J. Vis. Exp. (2022)
In this video, we demonstrate the preparation of in vitro biofilm by S. aureus in a poly-L-Lysine-coated multi-well plate. The biofilm-formed plate can be used for downstream experiments.

Source: Rana, P. S. et al., Standardized In vitro Assays to Visualize and Quantify Interactions between Human Neutrophils and Staphylococcus aureus ...

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

Summary

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Chapters in this video

Microtiter Dish Biofilm Formation Assay

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Biosensor for Detection of Antibiotic Resistant Staphylococcus Bacteria

Stress-induced Antibiotic Susceptibility Testing on a Chip

A Platform of Anti-biofilm Assays Suited to the Exploration of Natural Compound Libraries

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Standardized In vitro Assays to Visualize and Quantify Interactions between Human Neutrophils and Staphylococcus aureus Biofilms

A Soluble Tetrazolium-Based Reduction Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilms

Improved Enzyme Protection Assay to Study Staphylococcus aureus Internalization and Intracellular Efficacy of Antimicrobial Compounds

In Vitro Biofilm Synthesis by Staphylococcus aureus