This work was supported by grants from the Academic Research Fund of the Ministry of Education, and the National Medical Research Council and the National Research Foundation, Singapore.

1. Crystal Violet Quantitation of Biofilm Formation in A. baumannii S1
<ol>
	<li>Grow a 5 ml culture of A. baumannii S1 in Lysogeny broth (LB) (tryptone 10 g/L, yeast extract 5 g/L) at 30 &#176;C in a shaking incubator (220 rpm) for 16 hr.</li>
	<li>Adjust the culture of A. baumannii S1 to a desired OD600 of 0.8. Using a 96-well plate, inoculate the bacteria culture (1:100 dilution) into fresh LB containing 10 &#181;l of purified GKL enzyme (40 mg/ml); the new culture&#39;s final v...

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W. <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>Perez, F., 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=Global+challenge+of+multidrug-resistant+Acinetobacter+baumannii.">Global challenge of multidrug-resistant Acinetobacter baumannii.</a> Antimicrob Agents Chemother. 51, 3471-3484 (2007).</li><li>Tay, S. B., Yew, W. 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In both sets of experiment, A. baumannii S1 was cultured in LB media without NaCl as a high salt concentration may reduce the amount of biofilm formed by the bacteria15. The presence of such artifact could underestimate the amount of biofilm formed, as well as the effects of quorum-quenching enzymes across different treatment conditions. The use of a catalytically inactive enzyme is important as a negative control to eliminate the possible effects of enzyme sequestration. Figure 1 sho...

Treatment options for infectious diseases have been complicated by the rapid increase in multidrug-resistant bacteria that are immune to a wide range of antibiotic drugs1. With high morbidity and mortality rates from resistant bacteria-mediated infections, there is a need to escalate drug development processes and/or explore better anti-bacterial alternatives to improve therapeutic options. Lately, the anti-virulence approach is gaining interest given its potential in preventing virulence via non-bactericidal methods, hence mitigating the risks of resistance mechanisms2.
Quorum-sensing is a &#39;master switch&#39; in bacterial virulence and disruption of this signaling phenomenon is a promising anti-virulence method against pathogenesis3. The onset of virulence requires the accumulation of quorum molecules in the extracellular environment after a critical bacterial population density is reached. As quorum molecules diffuse back into the intracellular matrix, binding with their cognate receptors leads to the activation of virulence factors as well as genes associated with antibiotic resistance and biofilm formation4. In general, quorum-sensing disruption involves inhibiting quorum molecule and receptor interaction without affecting primary metabolic pathways. Hence, it does not have any direct implication on cellular growth. Since fitness is not compromised, there is minimal selection pressure for bacteria to evolve and gain resistance against such treatments5. In addition, quorum-sensing disruption can interfere with inherent bacterial protective mechanisms, as in the case of biofilm formation, which provides protection from anti-bacterial agents and host immune responses.
It is estimated that 99% of microbes on Earth exist in complex biofilm-like matrices, conferring crucial survival advantages to the microorganisms living within these structures6. More importantly, formation of these sessile domains is the cause of most persistent and chronic hospital-acquired infections7. Acinetobacter baumannii is one of the major human pathogens that is associated with global hospital-acquired infections and its virulence is largely attributed to quorum-sensing-mediated biofilm formation8. Quorum-quenching enzymes have been used successfully in disrupting quorum-mediated signal transduction by targeting a group of compounds known as N-acyl homoserine lactones (AHLs) that are produced by Gram-negative bacteria9. Several studies have also expanded upon the use of these enzymes to block bacterial pathogenesis through the reduction of virulence factor expression and cell numbers in biofilms10,11. Unfortunately, there remains a lack of palpable demonstration of the effective use of quorum-quenching enzymes against biofilm formation by bacterial pathogens. There have been attempts to use quorum inhibitors (AHL analogues), instead of quorum-quenching enzymes, to disrupt A. baumannii biofilm formation12. Although this method of using small molecules inhibitors is a valid approach, sustaining its bioavailability in translational uses can be a challenge. On the contrary, the use of catalytic quorum-quenching enzymes could circumvent the bioavailability issue as enzymes are more amenable towards immobilization on surfaces of biomedical devices for therapeutic effects.
Here, we describe an assessment of the effects of engineered quorum-quenching lactonases from Geobacillus kaustophilus (GKL)13 on bacterial biofilm formation, using crystal violet staining and confocal laser scanning microscopy (CLSM). This study is the first successful demonstration of biofilm disruption in a clinically relevant A. baumannii S1 strain using quorum-quenching enzymes. The methods described in this study are useful for assessing the efficacy of other quorum-quenching enzymes in subsequent therapeutic development efforts against pathogenic Gram-negative bacteria.

<table border="0" cellpadding="0" cellspacing="0" width="780">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Tryptone&#160;</td>
			<td style="width:151px;">BD</td>
			<td style="width:132px;">211705</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Yeast Extract</td>
			<td style="width:151px;">BD</td>
			<td style="width:132px;">212750</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">96-well plate</td>
			<td style="width:151px;">Costar</td>
			<td style="width:132px;">3596</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Crystal Violet</td>
			<td style="width:151px;">Sigma-Aldrich</td>
			<td style="width:132px;">C6158</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Acetic Acid</td>
			<td style="width:151px;">Lab-Scan</td>
			<td style="width:132px;">PLA00654X</td>
			<td>Caution: Flammable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#956;-Dish</td>
			<td style="width:151px;">Ibidi</td>
			<td style="width:132px;">80136</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alex Fluo 488-conjugated WGA</td>
			<td style="width:151px;">Invitrogen</td>
			<td style="width:132px;">W11261</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hank&#8217;s balanced salt solution&#160;</td>
			<td style="width:151px;">Invitrogen</td>
			<td style="width:132px;">141475095</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Formaldehyde</td>
			<td style="width:151px;">Sigma-Aldrich</td>
			<td style="width:132px;">F8775</td>
			<td>Caution: Corrosive&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Synergy HT Microplate Reader</td>
			<td style="width:151px;">BioTek</td>
			<td style="width:132px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">1X-81 Inverted Fluorescence Microscope</td>
			<td style="width:151px;">Olympus</td>
			<td style="width:132px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

In the crystal violet quantitation experiment, two quorum-quenching enzymes were used to demonstrate feasibility in disrupting biofilm formation: wild-type GKL and an improved GKL double mutant (E101G/R230C). Both enzymes have been shown to demonstrate lactonase activity against 3-hydroxy-decanoyl-L-homoserine lactone (3-OH-C10-HSL), the major quorum molecule used by A. baumannii S114. For valid assessment of biofilm disruption, their respective catalyticall...

Watch this Scientific Journal Video about Anti-virulent Disruption of Pathogenic Biofilms using Engineered Quorum-quenching Lactonases at JoVE.com

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

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 ...

The overall goal of this experiment is to demonstrate the efficacy of quorum-quenching enzymes in bacterial biofilm disruption. This method will help answer key questions in the field of anti-bacterial therapeutics, and to help understand the effect of quorum-quenching lactonases on pathogenic bacteria within a biofilm environment. The main advantage of this technique is that quorum-quenching lactonases can be directly added in an in vitro environment.Thus, its effect on biofilm disruption are easily quantitated. Demonstrating this procedure is Terrence Tay a technician in our lab. To begin, grow a 5 mililiter culture of A.baumannii S1 in LB at 30 degrees Celsius in a shaking incubator for 16 hours.Adjust the culture to an OD 600 of 0.8. Then use fresh LB containing 4 miligrams per mililiter of purified GKL enzyme to dilute the culture one to 100. And add 100 microliters to the wells of a 96-well plate.Use the lids to cover the plates, and place them into a 10 liter plastic container. Seal the container and incubate at 30 degrees Celsius for three hours, before gently removing the medium. Add another 100 microliters of fresh LB medium to the wells, and incubate the plate at 30 degrees Celsius for 21 hours.After gently removing the medium, use 200 microliters of sterile water to gently wash the planktonic bacteria cells. Position the pipette tip at the bottom of the well and carefully remove the liquid medium to ensure minimal perturbations on the biofilm foreign between the air and liquid interface. Then add 100 microliters of 1%crystal violet solution to each well, and incubate for 15 minutes at room temperature.Remove the crystal violet solution by using 200 microliters of sterile water to wash the wells three times. Add 100 microliters of 33%acetic acid to each well and incubate with gentle shaking for 15 minutes to dissolve the dye. Quantitate the amount of biofilm by measuring the absorbance of crystal violet at 600 nanometers.The amount of crystal violet is proportional to the amount of biofilm formed. To carry out confocal laser scanning microscopy, or CLSM, of A.baumannii S1 biofilms, grow a five mililiter culture of A.baumannii S1 in LB at 30 degrees Celsius in a shaking incubator for 16 hours. Adjust the culture of A.baumannii S1 to an OD 600 of 0.8.Then use fresh LB containing 1.2 miligrams per milileter of purified GKL enzyme to dilute the culture, one to 100, and add one mililiter to a 35 mililiter glass-bottomed microdish. Cover the microdish with a lid and place it in a 10 liter plastic container and seal it before incubating the dish at 30 degrees Celsius for three hours. After gently removing the medium, add one mililter of fresh medium containing 30 microliters of purified GKL enzyme then incubate at 30 degrees Celsius for another 21 hours.Gently remove the medium a second time and replace with another one mililiter of GKL enzyme in fresh medium. Incubate 30 degrees Celsius for 24 hours. After the incubation, gently remove the medium and add 500 microliters of five micrograms per mililiter of Alexa Fluor 488 conjugated wheat germ agglutinin dissolved in HBSS.Incubate the microdish at 37 degrees Celsius for 30 minutes to stain the biofilms. To fix the biofilms, add 500 microliters of 3.7%formaldehyde dissolved in HBSS and incubate at 37 degrees Celsius for 30 minutes. Use two mililiters of HBSS to wash the microdish once, then remove the solution completely.Store the dish in the dark at four degrees Celsius prior to CLSM imaging. For CLSM imaging and analysis, use a 63X objective to generate 97 stacks per image with an interval of 0.21 microns per stack. In the crystal violet quantitation experiment, Wild-type GKL and an improved GKL double-mutant quorum-quenching enzyme were used for lactonase activity against three hydroxydecanoyl l homoserine lactone or 3OHC10HSL, the major quorum molecule used by A.baumanii S1.As shown here, both enzymes were able to significantly reduce biofilms formation in pre-treated Wild-type A.baumanii S1.The extent of biofilms disruption by both enzymes is not proportional to their efficacy against 3OHC10HSL.As seen in this table, the turnover rate of the mutant GKL is six times faster than Wild-type GKL against 3OHC10HSL. However, the difference in the extent of biofilms disruption between the enzymes is only twofold. In this experiment, the improved GKL mutant enzyme and its catecholy inactive enzyme were used for comparison.This DIC image shows that treatment with the improved GKL mutant enzyme resulted in a decrease in biofilm size compared to the inactive enzyme. Analysis of the fluorescence images also revealed that there was a reduction in surface area, biomass, and average thickness of biofilms, when treated with the improved GKL mutant enzyme. Once mastered, this technique can be done in three to four hours excluding incubation time if it is performed properly.While attempting this procedure, it is important to remember to minimize the removal and disruption of the biofilm during the washing steps. Following this procedure, other methods like cell counting and enzyme stability assays can be performed. This will answer additional questions like the effects of quorum-quenching lactonases on cell population within the biofilm as well as their duration of action.After its development, this technique paved the way for researchers in the field of infectious disease treatment to explore the effects of potential quorum-quenching lactonases in the biofilm produced by pathogenic bacteria. After watching this video, you should have a good understanding on how to quantify biofilm disruption by crystal violet staining, and to assess morphological changes in the biofilm by confocal imaging. Don't forget that working with pathogenic material can be extremely hazardous.Precautions, such as using proper biological risk-containment facilities should always be taken while performing this procedure.

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Research

JoVE Journal

Immunology and Infection

10.5K vistas.  Yong Loo Lin School of Medicine. The overall goal of this experiment is to demonstrate the efficacy of quorum-quenching enzymes in bacterial biofilm disruption. This method will help answer key questions in the field of anti-bacterial therapeutics, and to help understand the effect of quorum-quenching lactonases on pathogenic bacteria within a biofilm environment. The main advantage of this technique is that quorum-quenching lactonases can be directly added in an in vitro environment.Thus, its effect on biofilm disrup...