Name: a platform of anti-biofilm assays suited to the exploration of natural compound libraries
Uploaded: 2016-12-27T12:00:00
Description: Watch this Scientific Journal Video about A Platform of Anti-biofilm Assays Suited to the Exploration of Natural Compound Libraries at JoVE.com

The authors thank professor Paul Cos and LMPH, University of Antwerp, Belgium for his support during the filming process in his laboratory. This work was funded by Academy of Finland projects (projects 272266 and 282981) and Svenska Tekniska Vetenskapsakademien i Finland. The technical contributions of MSc Janni Kujala are acknowledged.

1. Growing the Bacteria
<ol>
	<li>Pre-culture the bacteria overnight in a tryptic soy broth (TSB) at 37 &#176;C with 220 rpm shaking (16-18 hr).</li>
	<li>Dilute the pre-culture 100-1,000 times (depending on the growth rate of the bacteria; here, 1,000 times is used for S. aureus) in fresh TSB and let it grow at 37 &#176;C and 200 rpm to reach exponential growth (optical density at 595 nm (OD595) between 0.2 and 0.6). 
	NOTE: This step requires strain-specific optimization.</li>
</ol>
<p class=...

<ol>
	<li>Costerton, J. W., Stewart, P. S., Greenberg, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+biofilms:+a+common+cause+of+persistent+infections.">Bacterial biofilms: a common cause of persistent infections.</a> Science. 284, 1318-1322 (1999).</li><li>Dufour, D., Leung, V., L&#233;vesque, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+biofilm:+structure,+fuction,+and+antimicrobial+resistance.">Bacterial biofilm: structure, fuction, and antimicrobial resistance.</a> Endodontic Topics. 22, 2-16 (2012).</li><li>Donne, J., Dewilde, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Challenging+World+of+Biofilm+Physiology.">The Challenging World of Biofilm Physiology.</a> Adv. 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There is no single method that can simultaneously measure the effect of a compound on the viability, biomass, and biofilm matrix. Therefore, there is a need for combining assays in order to detect an effect on the three endpoints, preferably at a primary screening stage.
Resazurin is a very simple staining protocol consisting only of the addition of the redox probe. However, establishing the optimal incubation time of the biofilms with the resazurin is crucial to the success of this assay. In ...

Bacteria can switch between two very different lifestyles, planktonic and sessile, of which a biofilm is the most common example. In biofilms, bacteria form structured communities embedded in a self-produced matrix1. This self-produced matrix is a barrier between the bacteria and their external environment, and it protects the microbial cells, keeping them in close proximity. The composition of the biofilm matrix varies between and even within species, but it mostly consists of a tight network of lipopolysaccharides, extracellular DNA, and proteins. The matrix serves as a physical barrier inhibiting the entrance of harmful agents, but it also protects the biofilm from dehydration and prevents nutrients from escaping the cell2.
Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are purportedly responsible for over 80% of antibiotic-tolerant infections3. They display an inherently high tolerance against external threats: humidity, osmotic pressure, mechanical stress4, heat, UV radiation5, disinfectants, antimicrobial agents, and the host immune system1. For example, the required antimicrobial agent concentration required to kill a biofilm has been shown to be up to 1,000 times higher in comparison to that required to kill planktonic bacteria. The explanation for this higher tolerance seems to be multifactorial. The matrix provides a physical barrier that decreases the penetration of antibiotics into the biofilm. Also, cells within the biofilms are phenotypically diverse; they transition between different metabolic states due to an existing gradient of oxygen, nutrients, and metabolites between the inner and outer parts of the biofilm6. Hence, in some biofilm regions, such as the core, bacteria are deprived of oxygen and nutrients and live in a metabolically less active or a completely dormant state7. The completely dormant cells are referred to as persister cells, and they are not susceptible to conventional antimicrobial treatments8. Thus, it is likely that biofilm resilience arises from a combination of the presently suggested and other, yet unknown, mechanisms. Staphylococcus spp. are still among the most problematic gram-positive bacteria, causing severe, often biofilm-related, infections4. It is suggested that up to 99% of all bacteria are associated within biofilms, making it the predominant bacterial lifestyle3. However, all of the currently existing antibiotics have been developed against single-cell (planktonic) bacteria. 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.
From a methodological perspective, additional challenges exist, as only a limited number of biofilm methods have been developed by standard-setting organizations, especially those applicable to the high-throughput screening of chemical libraries. All standardized assays (with only one exception) are based upon biofilm reactors, and these methods require large working volumes and large amounts of compounds to be tested, which are usually unavailable during the early investigational stage9-12. The only existing standardized screening-applicable assay is the so-called Calgary Biofilm Device, from which the commercially available minimum biofilm eliminating concentration (MBEC) system was developed13-15. However, the limitation of this assay is that the biofilms are grown on pegs, and not all bacterial species or even strains within the same species are able to form biofilms on this device. Moreover, methods that can be particularly applied to the exploration of natural compounds are needed. Natural products have been the major source for innovation in antimicrobial drug discovery over the past century16. They can provide novel anti-biofilm compounds with unique mechanisms of action that can also be effective against persister cells. Thus, the exploration of natural and naturally inspired libraries has high chances of producing promising and unique anti-biofilm leads.
Here, we present the experimental details of a platform of assays that was developed for the chemical screening of anti-biofilm compounds using three assays to measure the effects on the viability, total biomass, and matrix of Staphylococcus aureus biofilms. The first assay measures biofilm viability, and it is based on resazurin staining. Resazurin is a redox stain that is blue and non-fluorescent in its oxidized state and turns into pink, highly fluorescent resorufin when reduced by the metabolic activity of the bacteria. It is a very simple and fast method suitable for primary screenings17-20. The second assay, based on crystal violet staining, measures total biofilm mass. Crystal violet is a widely used stain for studying bacteria and bacteria in biofilms19,21-23. The assay is based on inexpensive reagents and has a simple absorbance endpoint reading. Finally, the third assay targets the extracellular polymeric substance (EPS)-matrix of the biofilm via wheat germ agglutinin (WGA), which binds specifically to poly-N-acetyl-glucosamine residues (PNAG) present in the matrix of staphylococcal biofilms24. The WGA is conjugated with a fluorophore that can be detected using fluorescence intensity readers25. We present here the rationale and details of the platform we developed, including examples of applications.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Resazurin</td>
			<td>Sigma Aldrich</td>
			<td>R7017</td>
			<td></td>
		</tr>
		<tr>
			<td>Crystal violet</td>
			<td>Sigma Aldrich</td>
			<td>HT90132&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Wheat Germ Agglutinin, Alexa Fluor 488 Conjugate</td>
			<td>Thermo Fisher Scientific</td>
			<td>W11261</td>
			<td></td>
		</tr>
		<tr>
			<td>LIVE/DEAD BacLight&#160;</td>
			<td>Molecular Probes</td>
			<td>L7012</td>
			<td>SYTO 9 for staining viable cells green and propidium iodide for staining dead cells red</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone soy agar</td>
			<td>Lab M, Neogen</td>
			<td>LAB011</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptine soy broth</td>
			<td>Lab M, Neogen</td>
			<td>LAB004</td>
			<td></td>
		</tr>
		<tr>
			<td>F96 Well Plate Polystyrene Sterile Clear Flat bottom</td>
			<td>Thermo Fisher Scientific</td>
			<td>161093</td>
			<td></td>
		</tr>
		<tr>
			<td>BRAND caps, strips of 8</td>
			<td>Sigma Aldrich</td>
			<td>BR781413-300EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Branson CPX series ultrasonic bath</td>
			<td>Sigma Aldrich</td>
			<td>Z769428-1EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Multipipette</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Multidrop dispenser</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Biomek 3000</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Varioskan Flash Multiplate reader</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Staphylococcus aureus</td>
			<td>ATCC&#160;</td>
			<td>25923</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

In the proposed platform, the effects on the viability, biomass, and biofilm matrix are quantified. In the working sequence (Figure 1), one sample plate is stained with resazurin and subsequently with crystal violet to simultaneously evaluate the effects on bacterial biofilm viability and on total biofilm biomass. Both assays can be performed consecutively in the same plate because it was demonstrated earlier that a first staining with resazurin had no statistically signi...

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

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 ...

The, overall, goal of this assay platform is to provide a meaningful screening work flow combining three, relevant, end points, viability, biomass, and biofilm matrix, for identification and characterization of new, anti-biofilm compounds. This platform can help answers key questions in the field of antibiofilm agent discovery, by allowing an early distinction of short versus long term chemotherapeutic effects in a quick, explaining compatible manner. The main advantage of this developed platform is that it combines different assays to provide a more complete picture of the anti-biofilm effects of chemical libraries, especially of natural compounds.To begin the experiment, prepare untreated, control samples with 200 microliters of 10 to the sixth CFU per milliliter bacterial culture per well of a sterile, 96 microwell plate. Include media controls without bacterial culture. Next, on the same 96 well plates, prepare samples with four microliters of 50x antibiotic stock solution and 196 microliters of the bacteria culture per well, then, incubate the cultures.Continue with resazurin staining by using a multichannel pipette to transfer the entire planktonic solution, carefully, without touching the biofilms or creating air bubbles, to a separate, clean, 96 well plate. Again, using a multichannel pipette, wash the biofilms, once, with sterile PBS by adding 200 microliters per well and remove the solution carefully. Add 200 microliters of 20 micromolar resazurin solution, per well, of the biofilm plate and incubate the biofilm, in darkness, at 200 rpm for 20 minutes, until the untreated biofilm controls are evenly pink.Then, measure the fluorescence with a plate reader. Remove the resazurin stain carefully from the wells. Fix the biofilms with 200 microliters of methanol, for 15 minutes.After fixation, remove the methanol and let the plate air dry for 10 minutes. Next, use a multichannel pipette to carefully add 190 microliters of 0.02%crystal violet solution to the biofilm. Avoid touching the sides of the wells, with the stain, while pipetting, and prevent the formation of air bubbles, and do not press the pipette to complete blowout.Then, incubate the plate for five minutes at room temperature. With a multichannel pipette, remove the stain carefully. Wash the biofilm with 200 microliters of deionized water.Let the wells dry for five minutes at room temperature, and dissolve the remaining stain in 33%acetic acid. Incubate the plate for one hour at room temperature. After incubation, read the absorbance at 595 nanometers.Measure the turbidity at 595 nanometers of the prepared, planktonic solution plate. This can be done during the incubation of the crystal violet stained plate. Next, stain the planktonic bacteria, with resazurin, by adding 10 microliters of the resazurin stock, per well.Pipette the solution to mix it well. Incubate the plate, in darkness, at room temperature, 200 rpm, for approximately five minutes, until the untreated controls are evenly pink. Then, measure the fluorescence.Obtain one of the parallel sample plates for this staining, and use a multichannel pipette to remove the planktonic solution from the wells. Wash the wells, once, with steril PBS carefully, without touching the biofilms. Add 200 microliters of wheat germ agglutinin, or WGA solution, per well, to be stained.Then, incubate the plate. After incubation, wash the cells with PBS, three times, to remove the unbound stain. Let the plate dry for 15 minutes at room temperature.Visualize the samples with a fluorescence microscope and an FITC filter. Dissolve the bound stain in 200 microliters of 33%acetic acid per well. Seal the wells with strip caps and sonicate them using a water bath sonicator.After sonication, incubate the plate, for one hour, at 37 degrees Celsius. After one hour, repeat sonication while keeping the wells sealed between the sonication steps. Measure the fluorescence with a plate reader.Again, take one of the parallel sample plates for this staining, and using a multichannel pipette, remove the planktonic solution from the wells. Then, wash the wells once with sterile PBS. Add six microliters of the staining solution per well, and incubate the plate in darkness for 15 minutes.Before microscopy, remove the excess liquid, manually, using a multichannel pipette. Capture the images using a fluorescence microscope with a FITC filter, for green fluorescence, to visualize viable cells and a TRITC filter, for red fluorescence, to visualize dead cells. Use one of the parallel sample plates for this staining, and remove the planktonic solution, from the wells, and wash the wells once with sterile PBS, using a multichannel pipette.Next, add 200 microliters of the staining solution, per well, and incubate them, for 15 minutes, in darkness. Finally, measure the fluorescence with a plate reader. Two screenings were run to demonstrate the performance of the platform as a percent of the untreated biofilm control.The screening of a cinchona alkaloid derivative library identified one, active compound. The screening of a library of abietane-type diterpenoids and derivatives, identified five active compounds. Penicillin G and Ciprofloxacin both significantly decrease the viability, biomass, and biofilm matrix PNAG content prior to biofilm formation.However, this was not the case when post-exposure was used. The most prominent result was the increase, of the biofilm matrix, to over 200%when preformed biofilms were treated with high concentrations of Penicillin G.This fluorescent image confirms that Penicillin G kills about 50%of the preformed, Staph aureus, biofilm, bacterial population. The average of the green to red fluorescence ratios for the untreated biofilm wells indicate a predominance of live cells.While Penicillin treated wells have about 56%live cells. The cells remaining alive, after Penicillin treatment, produced a significantly higher amount of extracellular polymeric substance, or EPS, when compared with untreated biofilms. While attempting this procedure, it's important to remember to pay attention to the manual handling of the pipette to avoid contamination of bacteria and stain between wells.Following this platform, antibiofilm compounds effective against Staphylococcus aureus can be quickly identified and characterised. If other bacterial species are used, other optimization of the staining protocols will be needed, but the rational of the platform will remain the same.

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