The author would like to thank Li Zhang, Xian-Zhi Li, Aaron Hinz, and Clayton Hall for editorial help with this manuscript. This assay was initially developed in the lab of George O&#8217;Toole, Geisel School of Medicine at Dartmouth. Research in Dr. Mah&#8217;s lab is supported by grants from the Natural Sciences and Engineering Research Council of Canada and Cystic Fibrosis Canada.

1. MBC-B
<ol>
	<li>Growing a biofilm (adapted from O&#39;Toole9).
	<ol>
		<li>Grow a culture of the wild-type strain of interest and mutant strain for 16 hr in a rich medium at 37 &#176;C.</li>
		<li>Dilute the saturated overnight cultures 1:100 into fresh medium for antibiotic resistance assays. A standard medium for P. aeruginosa is M63 minimal medium supplemented with magnesium sulfate and arginine (see Table 1). This medium stimulates the formation of a more robust biofilm.</li>
		<li>Add 100 &#956;l of the dilution per well in a 96-well microtiter dish (see Table 1). Since these assays are typically performed in triplicate for each strain, there should be 24 wells of each strain.</li>
		<li>Incubate the microtiter plate for 24 hr at 37 &#176;C.</li>
	</ol>
	</li>
	<li>Exposing the preformed biofilm to a concentration gradient of antibiotic
	<ol>
		<li>Prepare a 10x&#160;dilution series of antibiotic for 7 wells. Example: for the antibiotic tobramycin, the final concentrations in the wells are 0.4, 0.2, 0.1, 0.05, 0.025, 0.0125, and 0.006 mg/ml 5-8 . From a stock of 25 mg/ml, prepare 10x&#160;dilutions of 4, 2, 1, 0.5, 0.25, 0.125, and 0.06 mg/ml. Leave on ice.</li>
		<li>Remove the spent supernatant (containing planktonic cells) using a multichannel pipette (see Table 1).</li>
		<li>Add 90 &#956;l M63 (Mg/Arg) to all of the wells.</li>
		<li>Add 10 &#956;l of each 10x&#160;antibiotic concentration in order to achieve the desired final concentrations. Add 10 &#956;l water (no antibiotic control) to the final well for each replicate and strain.</li>
		<li>Incubate the microtiter plate for 24 hr at 37 &#176;C.</li>
	</ol>
	</li>
	<li>Refreshing the media and allowing for the detachment of live cells.
	<ol>
		<li>Remove the spent supernatant (containing planktonic cells) using a multichannel pipette.</li>
		<li>Add 115 &#956;l M63 (Mg/Arg) to all of the wells.</li>
		<li>Incubate the microtiter plate for 24 hr at 37 &#176;C.</li>
	</ol>
	</li>
	<li>Assay for live cells.
	<ol>
		<li>Label two LB agar plates per 96-well microtiter plate.</li>
		<li>Sterilize the multiprong device (see Table 1) by dipping the prongs in 100% ethanol and passing the prongs across the open flame of a Bunsen burner. Repeat. Let the prongs cool slightly. Using the multiprong device, transfer ~3 &#956;l (amount that is typically retained on the tips of the prongs) of planktonic culture from each well of the microtiter&#160;plate to the surface of an LB agar plate.</li>
		<li>Incubate the LB agar plates for 16 hr at 37 &#176;C.</li>
		<li>Determine minimal bactericidal concentration of the antibiotic by identifying, by eye, the cut-off for bacterial growth (Figure 1).</li>
	</ol>
	</li>
</ol>
2. MBC-P&#160;
<ol>
	<li>Preparing bacterial strains
	<ol>
		<li>Grow a culture of the wild-type strain of interest and mutant strain for 16 hr in a rich medium at 37 &#176;C.</li>
		<li>Dilute the saturated overnight cultures 1:100 into fresh medium for antibiotic resistance assays. A standard medium for P. aeruginosa is M63 minimal medium supplemented with magnesium sulfate and arginine (see Table 1).</li>
		<li>Add 90 &#956;l of the dilution per well in a 96-well microtiter dish (see Table 1). Since these assays are typically performed in triplicate for each strain, there should be 24 wells of each strain.</li>
	</ol>
	</li>
	<li>Exposing planktonic cells to a concentration gradient of antibiotic
	<ol>
		<li>Prepare 10x&#160;dilution series of antibiotic for 7 wells. Example: for the antibiotic tobramycin, the final concentrations in the wells are 0.032, 0.016, 0.008, 0.004, 0.002, 0.001, and 0.0005 mg/ml. From a stock of 25 mg/ml, prepare dilutions of 0.32, 0.16, 0.08, 0.04, 0.02, 0.01, and 0.005 mg/ml. Leave on ice.</li>
		<li>Add 10 &#956;l of each 10x&#160;antibiotic concentration in order to achieve the desired final concentrations. Add 10 &#956;l water (no antibiotic control) to the final well for each replicate and strain.</li>
		<li>Incubate the microtiter plate for 24 hr at 37 &#176;C.</li>
	</ol>
	</li>
	<li>Assay for live cells.
	<ol>
		<li>Label two LB agar plates per 96-well microtiter plate.</li>
		<li>Sterilize the multiprong device (see Table 1) by dipping the prongs in 100% ethanol and passing the prongs across the open flame of a Bunsen burner. Repeat. Let the prongs cool slightly. Using the multiprong device, transfer ~3 &#956;l (amount that is typically retained on the tips of the prongs) of planktonic culture from each well of the microtiter plate to the surface of an LB agar plate.</li>
		<li>Incubate the LB agar plates for 16 hr&#160;at 37 &#176;C.</li>
		<li>Determine minimal bactericidal concentration of the antibiotic by identifying, by eye, the cut off for bacterial growth (Figure 2).</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Hoiby, N., Bjarnsholt, T., Givskov, M., Molin, S., Ciofu, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibiotic+resistance+of+bacterial+biofilms.">Antibiotic resistance of bacterial biofilms.</a> Int. J. Antimicrob. Agents. 35 (4), 322-332 (2010).</li><li>Mah, T. F., O'Toole, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+biofilm+resistance+to+antimicrobial+agents.">Mechanisms of biofilm resistance to antimicrobial agents.</a> Trends Microbiol. 9 (1), 34-39 (2001).</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=Biofilm-specific+antibiotic+resistance.">Biofilm-specific antibiotic resistance.</a> Future Microbiol. 7 (9), 1061-1072 (2012).</li><li>Mah, T. F., Pitts, B., Pellock, B., Walker, G. C., Stewart, P. S., O'Toole, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genetic+basis+for+Pseudomonas+aeruginosa+biofilm+antibiotic+resistance.">A genetic basis for Pseudomonas aeruginosa biofilm antibiotic resistance.</a> Nature. 426 (6964), 306-310 (2003).</li><li>Zhang, L., 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=The+Involvement+of+a+Novel+Efflux+System+in+Biofilm-Specific+Resistance+to+Antibiotics.">The Involvement of a Novel Efflux System in Biofilm-Specific Resistance to Antibiotics.</a> J. Bacteriol. 190 (13), 4447-4452 (2008).</li><li>Zhang, L., Hinz, A. J., Nadeau, J. P., 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=Pseudomonas+aeruginosa+tssC1+Links+Type+VI+Secretion+and+Biofilm-specific+Antibiotic+Resistance.">Pseudomonas aeruginosa tssC1 Links Type VI Secretion and Biofilm-specific Antibiotic Resistance.</a> J. Bacteriol. 193 (19), 5510-5513 (2011).</li><li>Beaudoin, T., Zhang, L., Hinz, A. J., Parr, C. J., 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=The+Biofilm-Specific+Antibiotic+Resistance+Gene,+ndvB,+is+Important+for+Expression+of+Ethanol+Oxidation+Genes+in+Pseudomonas+aeruginosa+Biofilms.">The Biofilm-Specific Antibiotic Resistance Gene, ndvB, is Important for Expression of Ethanol Oxidation Genes in Pseudomonas aeruginosa Biofilms.</a> J. Bacteriol. 194 (12), 3128-3136 (2012).</li><li>O'Toole, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtiter+Dish+Biofilm+Formation+Assay.">Microtiter Dish Biofilm Formation Assay.</a> J. Vis. Exp. (47), e2437 (2011).</li><li>Lewis, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multidrug+tolerance+of+biofilms+and+persister+cells.">Multidrug tolerance of biofilms and persister cells.</a> Curr. Top. Microbiol. Immunol. 322, 107-131 (2008).</li><li>Merritt, J. H., Kadouri, D. E., O'Toole, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growing+and+Analyzing+Static+Biofilms.">Growing and Analyzing Static Biofilms.</a> Curr. Protoc. Microbiol. 1, 1B.1.1-1B.1.17 (2005).</li><li>Ramey, B. E., Parsek, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+1.+Growing+and+analyzing+biofilms+in+fermenters.">Chapter 1. Growing and analyzing biofilms in fermenters.</a> Curr. Protoc. Microbiol. 1, 1B.3.1-1B.3.14 (2005).</li></ol>

The author declares that she has no competing financial interests.

Antibiotic resistance in planktonic cells is defined as an increase in the minimum inhibitory concentration (MIC) of an antibiotic due to a permanent change in the cells (e.g. a mutation). The mechanisms of biofilm-specific resistance or tolerance that have been identified to date are the result of the expression of wild type genes within biofilms. Thus, the classical definition of resistance does not apply to biofilms. However, another set of definitions has been presented: resistance mechanisms prevent the ant...

Antibiotic resistance assays were initially developed to assay resistance of planktonic (free-swimming) cultures of bacteria. Since many bacterial infections involve biofilms (surface-attached cells), we were interested in developing a method to assay biofilm-specific antibiotic resistance. However, most antibiotic resistance assays are poorly suited for measuring the resistance of biofilms. For example, determining the minimal inhibitory concentration (MIC) is the gold standard for determining antibiotic resistance of planktonic bacterial cultures 1. This assay entails mixing a diluted planktonic culture with a dilution series of antibiotic.&#160; The concentration of antibiotic that inhibits the visible growth of the planktonic cells is the MIC. Since this assay relies on inhibition of growth, by definition, it cannot work with biofilm cultures, which requires examining the antibiotic sensitivity of cells in a pregrown biofilm. Instead of measuring growth inhibition, the MBC-B assay described here determines the concentration of antibiotic that kills cells already existing in a biofilm. Thus, this assay aims to mimic antibiotic treatments of established biofilm infections, and provide a more relevant view of the bacterial antibiotic resistance in vivo.
Since biofilms are generally more antibiotic resistant than planktonic cultures 2-4 , it was necessary to devise a method that directly relates the antibiotic resistance of a biofilm to that of a planktonic culture. Thus another goal of this method is to be able to directly compare the level of antibiotic resistance between planktonic and biofilm cells. The MBC-P and MBC-B assays described here make this possible because cells are cultured under similar conditions. We have utilized this method to study several genes that are important for biofilm-specific antibiotic resistance 5-8 .

<table><tbody><tr><td> &lt;p&gt;1x&amp;nbsp;M63&lt;/p&gt; </td><td></td><td>&lt;p align=&quot;center&quot;&gt;&lt;/p&gt;</td><td> &lt;p&gt;Prepare as a 5x&amp;nbsp;M63 stock by dissolving 15g KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, 35g K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt; and 10g (NH&lt;sub&gt;4&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt; in 1 L of water. This stock does not need to be autoclaved and can be stored at room temperature.&amp;nbsp; Dilute 5x&amp;nbsp;stock 1:5, autoclave, cool, then add the desired components.&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;&lt;/p&gt; </td><td>Fisher</td><td>&lt;p align=&quot;center&quot;&gt;P285-500&lt;/p&gt;</td><td> &lt;p&gt;&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&lt;/p&gt; </td><td>Fisher</td><td>&lt;p align=&quot;center&quot;&gt;P288-500&lt;/p&gt;</td><td> &lt;p&gt;&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;(NH&lt;sub&gt;4&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;&lt;/p&gt; </td><td>Sigma</td><td>&lt;p align=&quot;center&quot;&gt;A5132&lt;/p&gt;</td><td> &lt;p&gt;&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Magnesium sulfate&lt;/p&gt; </td><td>Fisher</td><td>&lt;p align=&quot;center&quot;&gt;M63-500&lt;/p&gt;</td><td> &lt;p&gt;Add to 1 mM final concentration.&amp;nbsp; Prepare as a 1 M stock in water and autoclave.&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Tobramycin&lt;/p&gt; </td><td>Sigma</td><td>&lt;p align=&quot;center&quot;&gt;&lt;/p&gt;</td><td> &lt;p&gt;Prepare 50 mg/m&amp;nbsp;stock. Aliquot and store at &lt;span style=&quot;font-size: 11.199999809265137px;&quot;&gt;-&lt;/span&gt;20&amp;deg;C.&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Arginine&lt;/p&gt; </td><td>Sigma</td><td>&lt;p align=&quot;center&quot;&gt;A5131&lt;/p&gt;</td><td> &lt;p&gt;Add to 0.4% final concentration.&amp;nbsp; Prepare as a 20% stock in water and filter sterilize.&amp;nbsp; This alternative carbon/energy source can replace glucose and casamino acids&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;96-well microtiter plates&lt;/p&gt; </td><td>Corning</td><td>&lt;p align=&quot;center&quot;&gt;3595&lt;/p&gt;</td><td> &lt;p&gt;Sterile, flat-bottom, low evaporation&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Tranferpette (multichannel pipette)&lt;/p&gt; </td><td>BrandTech</td><td>&lt;p align=&quot;center&quot;&gt;2703610&lt;/p&gt;</td><td> &lt;p&gt;8-channel, 20-200 &amp;mu;l&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Multiprong device&lt;/p&gt; </td><td>Dan-Kar</td><td>&lt;p align=&quot;center&quot;&gt;MC48&lt;/p&gt;</td><td> &lt;p&gt;48 prongs fit into &amp;frac12; of a 96-well microtiter plate&lt;/p&gt; </td></tr></tbody></table>

establishing the minimal bactericidal concentration of an antimicrobial agent for planktonic cells (mbc-p) and biofilm cells (mbc-b)

This protocol allows for a direct comparison between planktonic and biofilm resistance for a bacterial strain that can form a biofilm in vitro. Bacteria are inoculated into the wells of a 96-well microtiter plate. In the case of the planktonic assay, serial dilutions of the antimicrobial agent of choice are added to the bacterial suspensions. In the biofilm assay, once inoculated, the bacteria are left to form a biofilm over a set period of time. Unattached cells are removed from the wells, the media is replenished and serial dilutions of the antimicrobial agent of choice are added. After exposure to the antimicrobial agent, the planktonic cells are assayed for growth. For the biofilm assay, the media is refreshed with fresh media lacking the antimicrobial agent and the biofilm cells are left to recover. Biofilm cell viability is assayed after the recovery period. The MBC-P for the antimicrobial agent is defined as the lowest concentration of drug that kills the cells in the planktonic culture.&#160; In contrast, the MBC-B for a strain is determined by exposing preformed biofilms to increasing concentrations of antimicrobial agent for 24 hr. The MBC-B is defined as the lowest concentration of antimicrobial agent that kills the cells in the biofilm.

MBC-P and MBC-B assays were carried out, comparing the sensitivity of PA14 wild type with PA14 &#8710;ndvB. Tobramycin was used as the antibiotic. Results corresponding to step 1.4.4 (Figure 1) and step 2.3.4 (Figure 2) are presented. PA14 and &#8710;ndvB were inoculated into the MBC-P and MBC-B assays in triplicate. After completing steps 1.0-1.4 of the MBC-B protocol and steps 2.0-2.3 of the MBC-P protocol, the viable cells were plated onto an LB agar plate. Concentra...

Watch this Scientific Journal Video about Establishing the Minimal Bactericidal Concentration of an Antimicrobial Agent for Planktonic Cells MBC-P and Biofilm Cells MBC-B at JoVE.com

Establishing the Minimal Bactericidal Concentration of an Antimicrobial Agent for Planktonic Cells MBC-P and Biofilm Cells MBC-B

This protocol allows for a direct comparison between planktonic and biofilm resistance for a bacterial strain that can form a biofilm in vitro using a 96-well microtiter plate. Planktonic or biofilm bacteria are exposed to serial dilutions of the antimicrobial agent of choice. Viability is assayed by growth on agar plates.

Establishing the Minimal Bactericidal Concentration of an Antimicrobial Agent for Planktonic Cells (MBC-P) and Biofilm Cells (MBC-B)

This protocol allows for a direct comparison between planktonic and biofilm resistance for a bacterial strain that can form a biofilm in vitro. Bacteria ...

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19.9K Views.  University of Ottawa. This protocol allows for a direct comparison between planktonic and biofilm resistance for a bacterial strain that can form a biofilm in vitro using a 96-well microtiter plate. Planktonic or biofilm bacteria are exposed to serial dilutions of the antimicrobial agent of choice. Viability is assayed by growth on agar plates.

Video: Establishing the Minimal Bactericidal Concentration of an Antimicrobial Agent for Planktonic Cells MBC-P and Biofilm Cells MBC-B

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 ...

Anti-virulent Disruption of Pathogenic Biofilms using Engineered Quorum-quenching Lactonases

The rapid emergence of multi-drug resistant bacteria has accelerated the need for novel therapeutic approaches to counter life-threatening infections. The persistence of bacterial infection is often associated with quorum-sensing-mediated biofilm formation. Thus, the disruption of this signaling circuit presents an attractive anti-virulence strategy. Quorum-quenching lactonases have been reported to be effective disrupters of quorum-sensing circuits. However, there have been very few reports of the effective use of these enzymes in disrupting bacterial biofilm formation. This protocol describes a method to disrupt biofilm formation in a clinically relevant A. baumannii S1 strain through the use of an engineered quorum-quenching lactonase. Acinetobacter baumannii is a major human pathogen implicated in serious hospital-acquired infections globally and its virulence is attributed predominantly to its biofilm's tenacity. The engineered lactonase treatment achieved significant A. baumannii S1 biofilm reduction. This study also showed the possibility of using engineered quorum-quenching enzymes in future treatment of biofilm-mediated bacterial diseases. Lastly, the method may be used to evaluate the competency of promising quorum-quenching enzymes.

Quorum-quenching enzymes are anti-virulent and anti-bacterial options that can mitigate pathogenesis without risk of incurring resistance, by preventing the expression of virulence factors and genes associated with antibiotic resistance and biofilm formation. In this study, we report a method that demonstrates the efficacy of quorum-quenching enzymes in bacterial biofilm disruption.

The rapid emergence of multi-drug resistant bacteria has accelerated the need for novel therapeutic approaches to counter life-threatening infections. The ...

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

Most pathogenic bacteria are able to form biofilms during infection, but due to the difficulty of manipulating and assessing biofilms, the vast majority of laboratory work is conducted with planktonic cells. Here, we describe a peg plate biofilm assay as performed with Staphylococcus aureus. Bacterial biofilms are grown on pegs attached to a 96-well microtiter plate lid, washed through gentle submersion in buffer, and placed in a drug challenge plate. After subsequent incubation they are again washed and moved to a final recovery plate, in which the fluorescent dye resazurin serves as a viability indicator. This assay offers greatly increased ease-of-use, reliability, and reproducibility, as well as a wealth of data when conducted as a kinetic read. Moreover, this assay can be adapted to a medium-throughput drug screening approach by which an endpoint fluorescent readout is taken instead, offering a path for drug discovery efforts.

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

Most bacterial infections produce a biofilm. By virtue of their environment, biofilm associated bacteria are often phenotypically drug resistant. Novel antibacterial molecules that kill bacteria in biofilms are thus a high priority. We establish an assay to quickly screen for antimicrobial compounds that are effective at eradicating biofilms.

Most pathogenic bacteria are able to form biofilms during infection, but due to the difficulty of manipulating and assessing biofilms, the vast majority ...

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and resilience of Bacillus subtilis biofilms. First, methods are presented that select for small molecule inhibitors with biofilm-specific targets in order to separate the effect of the small molecule inhibitors on planktonic growth from their effect on biofilm formation. Next, we focus on how inoculation conditions affect the sensitivity of multicellular, floating B. subtilis cultures to small molecule inhibitors. The results suggest that discrepancies in the reported effects of such inhibitors such as D-amino acids are due to inconsistent pre-culture conditions. Furthermore, a recently developed protocol is described for evaluating the contribution of small molecule treatments towards biofilm resistance to antibacterial substances. Lastly, scanning electron microscopy (SEM) techniques are presented to analyze the three-dimensional spatial arrangement of cells and their surrounding extracellular matrix in a B. subtilis biofilm. SEM facilitates insight into the three-dimensional biofilm architecture and the matrix texture. A combination of the methods described here can greatly assist the study of biofilm development in the presence and absence of biofilm inhibitors, and shed light on the mechanism of action of these inhibitors.

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This study presents the development of reproducible methodologies to study biofilm inhibitors and their effects on Bacillus subtilis multicellularity.

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and ...

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 ...

Metabolic Profiling to Determine Bactericidal or Bacteriostatic Effects of New Natural Products using Isothermal Microcalorimetry

Due to the global threat of rising antimicrobial resistance, novel antibiotics are urgently needed. We investigate natural products from Myxobacteria as an&#160;innovative source of such new compounds. One bottleneck in the process is typically the elucidation of their mode-of-action. We recently established isothermal microcalorimetry as part of a routine profiling pipeline. This technology allows for investigating the effect of antibiotic exposure on the total bacterial metabolic response, including processes that are decoupled from biomass formation. Importantly, bacteriostatic and bactericidal effects are easily distinguishable without any user intervention during the measurements. However, isothermal microcalorimetry is a rather new approach and applying this method to different bacterial species usually requires pre-evaluation of suitable measurement conditions. There are some reference thermograms available of certain bacteria, greatly facilitating interpretation of results. As the pool of reference data is steadily growing, we expect the methodology to have increasing impact in the future and expect it to allow for in-depth fingerprint analyses enabling the differentiation of antibiotic classes.

The elucidation of the mode-of-action of a novel antibiotic is a challenging task in the drug discovery process. The goal of the method described here is the application of isothermal microcalorimetry using calScreener in antibacterial profiling to provide additional insight into drug-microbe interactions.

Due to the global threat of rising antimicrobial resistance, novel antibiotics are urgently needed. We investigate natural products from Myxobacteria as ...

Bioengineering

Evaluation of Antimicrobial Activities of Nanoparticles and Nanostructured Surfaces In Vitro

The antimicrobial activities of nanoparticles and nanostructured surfaces, such as silver, zinc oxide, titanium dioxide, and magnesium oxide, have been explored previously in clinical and environmental settings and in consumable food products. However, a lack of consistency in the experimental methods and materials used has culminated in conflicting results, even amongst studies of the same nanostructure types and bacterial species. For researchers who wish to employ nanostructures as an additive or coating in a product design, these conflicting data limit their utilization in clinical settings. 
To confront this dilemma, in this article, we present four different methods to determine the antimicrobial activities of nanoparticles and nanostructured surfaces, and discuss their applicability in different scenarios. Adapting consistent methods is expected to lead to reproducible data that can be compared across studies and implemented for different nanostructure types and microbial species. We introduce two methods to determine the antimicrobial activities of nanoparticles and two methods for the antimicrobial activities of nanostructured surfaces. 
For nanoparticles, the direct co-culture method can be used to determine the minimum inhibitory and minimum bactericidal concentrations of nanoparticles, and the direct exposure culture method can be used to assess real-time bacteriostatic versus bactericidal activity resulting from nanoparticle exposure. For nanostructured surfaces, the direct culture method is used to determine the viability of bacteria indirectly and directly in contact with nanostructured surfaces, and the focused-contact exposure method is used to examine antimicrobial activity on a specific area of a nanostructured surface. We discuss key experimental variables to consider for in vitro study design when determining the antimicrobial properties of nanoparticles and nanostructured surfaces. All these methods are relatively low cost, employ techniques that are relatively easy to master and repeatable for consistency, and are applicable to a broad range of nanostructure types and microbial species.

Evaluation of Antimicrobial Activities of Nanoparticles and Nanostructured Surfaces In Vitro

We introduce four methods to evaluate the antimicrobial activities of nanoparticles and nanostructured surfaces using in vitro techniques. These methods can be adapted to study the interactions of different nanoparticles and nanostructured surfaces with a broad range of microbial species.

The antimicrobial activities of nanoparticles and nanostructured surfaces, such as silver, zinc oxide, titanium dioxide, and magnesium oxide, have been ...

Generation of Greater Bacterial Biofilm Biomass using PCR-Plate Deep Well Microplate Devices

Bacterial biofilms are difficult to eradicate from surfaces using conventional antimicrobial interventions. High-throughput 96-well microplate methods are frequently used to cultivate bacterial biofilms for rapid antimicrobial susceptibility testing to calculate minimal biofilm eradication concentration (MBEC) values. Standard biofilm devices consist of polystyrene pegged-lids fitted to 96-well microplates and are ideal for measuring biofilm biomass and MBEC values, but these devices are limited by available peg surface area for biomass accumulation and cost. Here, we outline a protocol to use self-assembled polypropylene 96-well deep well PCR-plate pegged-lid device to grow Escherichia coli BW25113 and Pseudomonas aeruginosa PAO1 biofilms. A comparison of 24-hour biofilms formed on standard and deep well devices by each species using crystal violet biomass staining and MBEC determination assays are described. The larger surface area of deep well devices expectedly increased overall biofilm formation by both species 2-4-fold. P. aeruginosa formed significantly greater biomass/mm2 on deep well pegs as compared to the standard device. E. coli had greater biomass/mm2 on standard polystyrene devices as compared the deep well device. Biofilm eradication assays with disinfectants such as sodium hypochlorite (bleach) or benzalkonium chloride (BZK) showed that both compounds could eliminate E. coli and P. aeruginosa biofilms from both devices but at different MBEC values. BZK biofilm eradication resulted in variable E. coli MBEC values between devices, however, bleach demonstrated reproducible MBEC values for both species and devices. This study provides a high throughput deep well method for growing larger quantities of biofilms on polypropylene devices for downstream studies requiring higher amounts of static biofilm.

This protocol presents methodology to perform biofilm growth and biomass measurements using self-assembled deep well PCR-plate devices for high-throughput 96-well pegged lid static biofilm screening.

Bacterial biofilms are difficult to eradicate from surfaces using conventional antimicrobial interventions. High-throughput 96-well microplate methods are ...

Adaptable CF Model for Antimicrobial Testing and Microbial Dynamics Analysis

Development of a Polymicrobial Colony Biofilm Model to Test Antimicrobials in Cystic Fibrosis

A range of bacteria biofilm models exist for the testing of antibiotics. However, many of these are limited to a single experimental output, such as colony-forming units or metabolic activity. Furthermore, many biofilm models do not reflect the biological and physiochemical properties of the human host environment. This is an important issue in many conditions, but most noticeably in cystic fibrosis (CF). A large proportion of people with CF suffer from both chronic and intermittent infections, and in vitro, antibiotic susceptibility testing poorly correlates with patient treatment outcomes. Some biofilm models incorporate CF lung-relevant media, including synthetic sputum mimics, but do not consider the polymicrobial nature of the environment, which alters biofilm architecture, physiology, and the way microbes respond to treatment. The solid-air interface colony biofilm model described here is highly adaptable and incorporates both CF-relevant media and a polymicrobial context. This model can also be used for mid-throughput screening of antimicrobials and to study their effect on polymicrobial dynamics. Output measurements from the model can be colony-forming units, metabolic activity, and confocal microscopy analysis. The model can easily be adapted to different microorganisms, media, temperatures, and variable oxygen conditions and can be used to test a wide range of chemical, biological, and physical treatments.

This methodology allows for the establishment of a polymicrobial biofilm model in cystic fibrosis for antimicrobial sensitivity testing in research and clinical laboratories. This model provides accurate and reliable results over a range of outputs.

A range of bacteria biofilm models exist for the testing of antibiotics. However, many of these are limited to a single experimental output, such as ...

Evaluation of the Efficacy of Organic Peroxyacids for Eradicating Dairy Biofilms Using an Approach Combining Static and Dynamic Methods

The presence of biofilms in the dairy industry is of major concern, as they may lead to the production of unsafe and altered dairy products due to their high resistance to most clean-in-place (CIP) procedures frequently used in processing plants. Therefore, it is imperative to develop new biofilm control strategies for the dairy industry. This protocol is aimed at evaluating the efficacy of organic peroxyacids (peracetic, perpropionic, and perlactic acids and a commercial peracetic acid-based disinfectant) for eradicating dairy biofilms using a combination of static and dynamic methods. All the disinfectants were tested on the strongest biofilm-producing bacteria in either a single or a mixed biofilm using the minimum biofilm eradication concentration (MBEC) assay, a static high-throughput screening method. A contact time of 5 min with the disinfectants at the recommended concentrations successfully eradicated both the single and mixed biofilms. Studies are currently ongoing to confirm these observations using the Center for Disease Control (CDC) biofilm reactor, a dynamic method to mimic in situ conditions. This type of bioreactor enables the use of a stainless-steel surface, which constitutes most industrial equipment and surfaces. The preliminary results from the reactor appear to confirm the efficacy of organic peroxyacids against biofilms. The combined approach described in this study may be used to develop and test new biological or chemical formulations for controlling biofilms and eradicating microorganisms.

This protocol describes an approach combining static and dynamic methods to evaluate the efficacy of organic peroxyacids for eradicating biofilms in the dairy industry. This approach may also be used to test the effectiveness of new biological or chemical formulations for controlling biofilms.

The presence of biofilms in the dairy industry is of major concern, as they may lead to the production of unsafe and altered dairy products due to their ...

Establishing the Minimal Bactericidal Concentration of an Antimicrobial Agent for Planktonic Cells (MBC-P) and Biofilm Cells (MBC-B)

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

Metabolic Profiling to Determine Bactericidal or Bacteriostatic Effects of New Natural Products using Isothermal Microcalorimetry

Evaluation of Antimicrobial Activities of Nanoparticles and Nanostructured Surfaces In Vitro

Generation of Greater Bacterial Biofilm Biomass using PCR-Plate Deep Well Microplate Devices

Development of a Polymicrobial Colony Biofilm Model to Test Antimicrobials in Cystic Fibrosis

Evaluation of the Efficacy of Organic Peroxyacids for Eradicating Dairy Biofilms Using an Approach Combining Static and Dynamic Methods