Name: anti-virulent disruption of pathogenic biofilms using engineered quorum-quenching lactonases
Uploaded: 2016-01-01T13:00:00.000+00:00
Duration: 7 min 47 s
Description: Watch this Scientific Journal Video about Anti-virulent Disruption of Pathogenic Biofilms using Engineered Quorum-quenching Lactonases at JoVE.com

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 volume is 100 &#181;l.</li>
	<li>Prepare a control culture as well; this will not contain any enzyme. Repeat similar conditions to yield the desired number of replicates.</li>
	<li>Cover the plate with a lid and place it into a sealed 10 L plastic container. Incubate the plate at 30 &#176;C for 3 hr before gently removing the media.</li>
	<li>Add another 100 &#181;l of fresh LB medium to the well and incubate the plate for 21 hr at 30 &#176;C.</li>
	<li>After the second period of incubation, gently remove all the media. Wash the planktonic bacteria cell with 200 &#181;l sterile water. Ensure that there is only minimal disturbance to the cells during washing.</li>
	<li>Add 100 &#181;l of 1% crystal violet solution to each well and incubate for 15 min at RT. Remove the crystal violet solution by washing the well with 200 &#181;l sterile water. Repeat the wash for two more times.</li>
	<li>Add 100 &#181;l of 33% acetic acid to each well and incubate for 15 min with gentle shaking; this will dissolve the dye.</li>
	<li>Quantitate the amount of biofilm formed by measuring the absorbance of crystal violet at 600 nm. The amount of crystal violet is proportional to the amount of biofilm formed.</li>
</ol>
2. Confocal Laser Scanning Microscopy of A. baumannii S1 Biofilm
<ol>
	<li>Grow a 5 ml culture of A. baumannii S1 in LB 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 35 mm glass-bottomed &#181;-Dish, inoculate the bacteria culture (1:100 dilution) into fresh LB containing 30 &#181;l of purified GKL enzyme (40 mg/ml); the new culture&#39;s final volume is 1 ml.</li>
	<li>Cover the &#181;-Dish with a lid and place it in a sealed 10 L plastic container. Incubate the &#181;-Dish at 30 &#176;C for 3 hr before gently removing the media. Add 30 &#181;l of purified GKL enzyme and fresh medium, bringing it to a total volume of 1 ml. Incubate for another 21 hr at 30 &#176;C.</li>
	<li>Repeat step 2.3 and incubate the &#181;-Dish for another 24 hr at 30 &#176;C. Then, remove the media gently.</li>
	<li>Add 500 &#181;l of 5 &#181;g/ml Alex Fluo 488-conjugated wheat germ agglutinin (WGA) dissolved in Hank&#39;s balanced salt solution (HBSS) to the &#181;-Dish and incubate at 37 &#176;C for 30 min; this will stain the formed biofilm. Remove the staining solution and wash the &#181;-Dish with 2 ml of HBSS. Repeat the wash step one more time.</li>
	<li>Add 500 &#181;l of 3.7 % formaldehyde dissolved in HBSS and incubate at 37 &#176;C for 30 min; this will fix the biofilm onto the &#181;-Dish. Wash the &#181;-Dish once with 2 ml of HBSS and then remove the solution completely. The &#181;-Dish fixed with biofilm can be stored in the dark at 4 &#176;C prior to CLSM imaging.</li>
	<li>For CLSM imaging and analysis, use 63 times magnification to generate 97 stacks per image with an interval of 0.21 &#181;m per stack.</li>
</ol>

<ol>
	<li>Alanis, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resistance+to+antibiotics:+are+we+in+the+post-antibiotic+era?.">Resistance to antibiotics: are we in the post-antibiotic era?.</a> Archives of medical research. 36, 697-705 (2005).</li><li>Cegelski, L., Marshall, G. R., Eldridge, G. R., Hultgren, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biology+and+future+prospects+of+antivirulence+therapies.">The biology and future prospects of antivirulence therapies.</a> Nature reviews. Microbiology. 6, 17-27 (2008).</li><li>LaSarre, B., Federle, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploiting+quorum+sensing+to+confuse+bacterial+pathogens.">Exploiting quorum sensing to confuse bacterial pathogens.</a> Microbiology and molecular biology reviews : MMBR. 77, 73-111 (2013).</li><li>Waters, C. M., Bassler, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quorum+sensing:+cell-to-cell+communication+in+bacteria.">Quorum sensing: cell-to-cell communication in bacteria.</a> Annual Review of Cell and Developmental Biology. 21, 319-346 (2005).</li><li>Rasko, D. A., Sperandio, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-virulence+strategies+to+combat+bacteria-mediated+disease.">Anti-virulence strategies to combat bacteria-mediated disease.</a> Nature reviews. Drug discovery. 9, 117-128 (2010).</li><li>Lazar, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quorum+sensing+in+biofilms--how+to+destroy+the+bacterial+citadels+or+their+cohesion/power?.">Quorum sensing in biofilms--how to destroy the bacterial citadels or their cohesion/power?.</a> Anaerobe. 17, 280-285 (2011).</li><li>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=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. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+quorum-based+anti-virulence+therapeutics+targeting+Gram-negative+bacterial+pathogens.">Development of quorum-based anti-virulence therapeutics targeting Gram-negative bacterial pathogens.</a> International journal of molecular sciences. 14, 16570-16599 (2013).</li><li>Igarashi, J., Suga, H., Rumbaugh, K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+19.">Ch. 19.</a> Quorum Sensing. 692, 265-274 (2011).</li><li>Ng, F. S., Wright, D. M., Seah, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+phosphotriesterase-like+lactonase+from+Sulfolobus+solfataricus+and+its+immobilization+for+disruption+of+quorum+sensing.">Characterization of a phosphotriesterase-like lactonase from Sulfolobus solfataricus and its immobilization for disruption of quorum sensing.</a> Applied and environmental microbiology. 77, 1181-1186 (2011).</li><li>Stacy, D. M., Welsh, M. A., Rather, P. N., Blackwell, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attenuation+of+quorum+sensing+in+the+pathogen+Acinetobacter+baumannii+using+non-native+N-Acyl+homoserine+lactones.">Attenuation of quorum sensing in the pathogen Acinetobacter baumannii using non-native N-Acyl homoserine lactones.</a> ACS chemical biology. 7, 1719-1728 (2012).</li><li>Chow, J. Y., 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=Directed+evolution+of+a+thermostable+quorum-quenching+lactonase+from+the+amidohydrolase+superfamily.">Directed evolution of a thermostable quorum-quenching lactonase from the amidohydrolase superfamily.</a> The Journal of biological chemistry. 285, 40911-40920 (2010).</li><li>Chow, J. Y., Yang, Y., Tay, S. B., Chua, K. L., Yew, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+biofilm+formation+by+the+human+pathogen+Acinetobacter+baumannii+using+engineered+quorum-quenching+lactonases.">Disruption of biofilm formation by the human pathogen Acinetobacter baumannii using engineered quorum-quenching lactonases.</a> Antimicrobial Agents and Chemotherapy. 58, 1802-1805 (2014).</li><li>Pour, N. 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The authors declare that they have no competing financial interests.

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 shows that even if inactive enzymes sequester quorum molecules, biofilm formation is not disrupted.
A. baumannii S1 forms a delicate ring-like biofilm structure between air and liquid interface in the well of the 96-well plate. Hence, it is crucial to avoid excessive agitation during media removal and washing steps to prevent incidental removal of bacterial biofilms. LB media was removed after each incubation to eliminate planktonic cells. In addition, the 96-well plate was placed in a 10 L plastic container to minimize perturbations in airflow and to create a micro-anaerobic environment that favors biofilm formation. Biofilm quantitation in a 96-well plate is also dependent on the amount of crystal violet added to each well. In the event that excess crystal violet is added into a well, the number of wash steps may be increased. The crystal violet staining experiment provides a relative comparison of quorum-quenching efficiencies in biofilm disruption. Although crystal violet staining is quantitative for measuring biofilm formation, it is only semi-quantitative in terms of the catalytic efficiency of quorum-quenching enzymes. For accurate statistical comparison, sufficient sample sizes are important for the different treatment groups. Outliers should also be removed to prevent misrepresentation of results.
Confocal imaging and analysis of bacterial biofilm is a useful tool for assessing the qualitative effects of quorum-quenching enzymes. However, the process of selecting biofilm for analysis may be a potential channel for bias if the user is aware of the type of quorum-quenching enzyme administered (active or inactive). To avoid the possibility of bias, the experiment can be designed to exclude enzyme information from the user or use a random approach for selection. An unbiased selection strategy also enables better quantitative comparison of biofilm morphologies by CLSM. Nevertheless, this is the first study that describes a method for evaluating the outcome of quorum-quenching enzymes on bacterial biofilms. Crystal violet staining has demonstrated the utility of quorum-quenching enzymes in biofilm disruption and revealed that enzymes&#39; modi operandorum are not limited to reducing cell numbers and virulence factor expression. Meanwhile, morphological changes determined by CLSM analysis can also provide insights into possible molecular targets of these enzymes. Although the current experiment was designed to investigate the effects of enzymes pretreatment on bacterial biofilms, the protocol may be modified to assess the enzyme&#39;s effect on preformed biofilm by adding the enzyme after bacterial growth or to examine its competency under physiological conditions through the use of serum-like conditions for culture. The protocol used to study GKL enzymes may be extended to other quorum-quenching enzymes and pathogens to investigate their relationship in biofilm disruption.

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><tbody><tr><td>Tryptone&amp;nbsp;</td><td>BD</td><td>211705</td><td></td></tr><tr><td>Yeast Extract</td><td>BD</td><td>212750</td><td></td></tr><tr><td>96-well plate</td><td>Costar</td><td>3596</td><td></td></tr><tr><td>Crystal Violet</td><td>Sigma-Aldrich</td><td>C6158</td><td></td></tr><tr><td>Acetic Acid</td><td>Lab-Scan</td><td>PLA00654X</td><td>Caution: Flammable</td></tr><tr><td>&amp;mu;-Dish</td><td>Ibidi</td><td>80136</td><td></td></tr><tr><td>Alex Fluo 488-conjugated WGA</td><td>Invitrogen</td><td>W11261</td><td></td></tr><tr><td>Hank&amp;rsquo;s balanced salt solution&amp;nbsp;</td><td>Invitrogen</td><td>141475095</td><td></td></tr><tr><td>Formaldehyde</td><td>Sigma-Aldrich</td><td>F8775</td><td>Caution: Corrosive&amp;nbsp;</td></tr><tr><td>Synergy HT Microplate Reader</td><td>BioTek</td><td></td><td></td></tr><tr><td>1X-81 Inverted Fluorescence Microscope</td><td>Olympus</td><td></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 catalytically inactive enzymes (previously shown to not sequester AHL ligands) were also included as immediate controls (GKL D266N mutant and GKL E101G/R230C/D266N mutant). Besides using wild-type A. baumannii S1, the biofilm forming ability of a mutant &#916;abaI strain (AHL synthase-deficient) was also tested. Both quorum-quenching enzymes, wild-type GKL and GKL E101G/R230C mutant were able to significantly reduce biofilm formation in pretreated A. baumannii S1 cultures (n = 10; p-value &#8804; 0.0001) (Figure 1). Nevertheless, the extent of biofilm disruption for both enzymes is not proportional to their efficacy against 3-OH-C10-HSL. The turnover rate (kcat) of the GKL E101G/R230C mutant is six times faster than wild-type GKL against 3-OH-C10-HSL14. However, the difference in the extent of biofilm disruption between the enzymes is only two-fold.
<img alt="Figure 1" src="/files/ftp_upload/53243/53243fig1.jpg" /> 
Figure 1. A. baumannii biofilm disruption assay. Biofilm formation was measured by crystal violet staining. Red columns represent the amount of biofilm formed by wild type A. baumannii and &#916;abaI mutant, without the addition of quorum-quenching lactonases. Blue columns represent the amount of biofilm formed by wild-type A. baumannii in the presence of four different GKL enzymes: inactive GKL D266N mutant, wild-type GKL, inactive GKL E101G/R230C/D266N mutant and GKL E101G/R230C mutant. ****, P-value of &#8804; 0.0001. Reproduced with permission from Antimicrobial Agents and Chemotherapy 58, 1802-1805 (2014). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53243/53243fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Confocal imaging of A. baumannii S1 biofilm formation was used to provide a qualitative and quantitative measure of quorum-quenching effects on the structural morphology of these sessile domains. The improved GKL E101G/R230C mutant and its catalytically inactive enzyme were used for comparison. The differential image contrast (DIC) image showed that treatment with the improved GKL E101G/R230C mutant resulted in a decrease in biofilm size (Figure 2). Analysis of the fluorescence images also revealed that there was reduction in surface area, biomass and average thickness of biofilm when treated with the improved GKL E101G/R230C mutant (Table 1).
<img alt="Figure 2" src="/files/ftp_upload/53243/53243fig2.jpg" /> 
Figure 2. Representative confocal laser scanning microscopy images of A. baumannii biofilms. A. baumannii biofilms were treated with inactive GKL E101G/R230C/D266N mutant (A) and GKL E101G/R230C mutant (B) and stained with Alexa Fluor 488-conjugated WGA. DIC images of the biofilms (left) and fluorescence images of the biofilms (right) are shown for representative xy (center), yz (right), and xz (bottom) sections. Reproduced with permission from Antimicrobial Agents and Chemotherapy 58, 1802-1805 (2014). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53243/53243fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td></td>
			<td colspan="3">Value &#177; SDa</td>
		</tr>
		<tr>
			<td>Characteristic</td>
			<td>No treatment</td>
			<td>Treatment with inactive mutant</td>
			<td>Treatment with E101G/R230C mutant</td>
		</tr>
		<tr>
			<td>Biomass (&#181;m3/&#181;m2)</td>
			<td>2.57 &#177; 1.65</td>
			<td>3.39 &#177; 1.33</td>
			<td>1.37** &#177; 0.20</td>
		</tr>
		<tr>
			<td>Avg thickness (&#181;m)</td>
			<td>3.68 &#177; 2.51</td>
			<td>3.41 &#177; 1.31</td>
			<td>1.21** &#177; 0.21</td>
		</tr>
		<tr>
			<td>Surface area (&#181;m)</td>
			<td>235,920.59 &#177; 79,456.46</td>
			<td>209,872.6 &#177; 115,094.7</td>
			<td>115,354.9* &#177; 7,630.3</td>
		</tr>
		<tr>
			<td colspan="4">a n = 10 image stacks. **, P &#8804; 0.001; *, P &#8804; 0.05, compared with treatment with inactive E101G/R230C/D266N mutant.</td>
		</tr>
	</tbody>
</table>
Table 1. A. baumannii biofilm structural quantitation.

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 ...

anti-virulent-disruption-pathogenic-biofilms-using-engineered-quorum

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

10.7K Views.  Yong Loo Lin School of Medicine. 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.

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

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 ...

Engineering Adherent Bacteria by Creating a Single Synthetic Curli Operon

The method described here consists in redesigning E. coli adherence properties by assembling the minimum number of curli genes under the control of a strong and metal-overinducible promoter, and in visualizing and quantifying the resulting gain of bacterial adherence. This method applies appropriate engineering principles of abstraction and standardization of synthetic biology, and results in the BBa_K540000 Biobrick (Best new Biobrick device, engineered, iGEM 2011).
The first step consists in the design of the synthetic operon devoted to curli overproduction in response to metal, and therefore in increasing the adherence abilities of the wild type strain. The original curli operon was modified in silico in order to optimize transcriptional and translational signals and escape the "natural" regulation of curli. This approach allowed to test with success our current understanding of curli production. Moreover, simplifying the curli regulation by switching the endogenous complex promoter (more than 10 transcriptional regulators identified) to a simple metal-regulated promoter makes adherence much easier to control.
The second step includes qualitative and quantitative assessment of adherence abilities by implementation of simple methods. These methods are applicable to a large range of adherent bacteria regardless of biological structures involved in biofilm formation. Adherence test in 24-well polystyrene plates provides a quick preliminary visualization of the bacterial biofilm after crystal violet staining. This qualitative test can be sharpened by the quantification of the percentage of adherence. Such a method is very simple but more accurate than only crystal violet staining as described previously 1 with both a good repeatability and reproducibility. Visualization of GFP-tagged bacteria on glass slides by fluorescence or laser confocal microscopy allows to strengthen the results obtained with the 24-well plate test by direct observation of the phenomenon.

The design of a synthetic operon encoding both the secretory apparatus and the structural monomers of curli fibers is described. Overproduction of these amyloids and adherent polymers allows a measurable gain of adherence of the E. coli chassis1. Easy ways to visualize and quantify adherence are explained.

The  method described here consists in redesigning E. coli adherence properties by assembling the minimum number of  curli genes under the control of a ...

Bioengineering

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 ...

Use of the Soft-agar Overlay Technique to Screen for Bacterially Produced Inhibitory Compounds

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, including work with bacteriophages and bacteriocins, proteinaceous antibacterial agents. This approach is relatively inexpensive, with minimal resource requirements. This technique consists of spotting supernatant from a donor strain (potentially harboring a toxic compound(s)) onto a solidified soft agar overlay that is seeded with a bacterial test strain (potentially sensitive to the toxic compound(s)). We utilized this technique to screen a library of Pseudomonas syringae strains for intraspecific killing. By combining this approach with a precipitation step and targeted gene deletions, multiple toxic compounds produced by the same strain can be differentiated. The two antagonistic agents commonly recovered using this technique are bacteriophages and bacteriocins. These two agents can be differentiated using two simple additional tests. Performing a serial dilution on a supernatant containing bacteriophage will result in individual plaques becoming less in number with greater dilution, whereas serial dilution of a supernatant containing bacteriocin will result a clearing zone that becomes uniformly more turbid with greater dilution. Additionally, a bacteriophage will produce a clearing zone when spotted onto a fresh soft agar overlay seeded with the same strain, whereas a bacteriocin will not produce a clearing zone when transferred to a fresh soft agar lawn, owing to the dilution of the bacteriocin.

We describe a simple method for screening bacterial cultures for the production of compounds inhibitory towards other bacteria.

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, ...

Quantifying the Effects of Antimicrobials on In vitro Biofilm Architecture using COMSTAT Software

Biofilms are aggregates of microorganisms that rely on a self-produced matrix of extracellular polymeric substance for protection and structural integrity. The nosocomial pathogen, Pseudomonas aeruginosa, is known to adopt a biofilm mode of growth, causing chronic pulmonary infection in patients with cystic fibrosis (CF). The computer program, COMSTAT, is a useful tool for quantifying antimicrobial-induced changes in P. aeruginosa biofilm architecture by extracting data from three-dimensional confocal images. However, standardized operation of the software is less commonly addressed, which is important for optimal reporting of biofilm behavior and cross-center comparison. Thus, the aim of this protocol is to provide a simple and reproducible framework for quantifying in vitro biofilm structures under varying antimicrobial conditions via COMSTAT. The technique is modeled using a CF P. aeruginosa isolate, grown in the form of biofilm replicates, and exposed to tobramycin and the anti-Psl monoclonal antibody, Psl0096. The step-by-step approach aims to reduce user ambiguity and minimize the chance of overlooking crucial image-processing steps. Specifically, the protocol emphasizes the elimination of subjective variations associated with the manual operation of COMSTAT, including image segmentation and the selection of appropriate quantitative analysis functions. Although this method requires users to spend additional time processing confocal images prior to running COMSTAT, it helps minimize misrepresented biofilm heterogenicity in automated outputs.

Quantifying the Effects of Antimicrobials on In vitro Biofilm Architecture using COMSTAT Software

Antimicrobial-induced changes to Pseudomonas aeruginosa biofilm architecture differ among clinical isolates cultured from patients with cystic fibrosis and chronic pulmonary infection. Following confocal microscopy, COMSTAT software can be utilized to quantify variations in biofilm architecture (e.g., surface area, thickness, biomass) for individual isolates to assess the efficacy of anti-infective agents.

Biofilms are aggregates of microorganisms that rely on a self-produced matrix of extracellular polymeric substance for protection and structural ...

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 ...

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 ...

Biology

Biological Assessment of Thiophenesulfonamides in Quorum Sensing Inhibition

Synthesis and Assay of Vibrio Quorum Sensing Inhibitors

Bacteria detect local population numbers using quorum sensing, a method of cell-cell communication broadly utilized to control bacterial behaviors. In Vibrio species, the master quorum sensing regulators LuxR/HapR control hundreds of quorum sensing genes, many of which influence virulence, metabolism, motility, and more. Thiophenesulfonamides are potent inhibitors of LuxR/HapR that bind the ligand pocket in these transcription factors and block downstream quorum sensing gene expression. This class of compounds served as the basis for the development of a set of simple, robust, and educational procedures for college students to assimilate their chemistry and biology skills using a CURE model: course-based undergraduate research experience. Optimized protocols are described that comprise three learning stages in an iterative and multi-disciplinary platform to engage students in a year-long CURE: (1) design and synthesize new small molecule inhibitors based on the thiophenesulfonamide core, (2) use structural modeling to predict binding affinity to the target, and (3) assay the compounds for efficacy in microbiological assays against specific Vibrio LuxR/HapR proteins. The described reporter assay performed in E. coli successfully predicts the efficacy of the compounds against target proteins in the native Vibrio species.

Author Spotlight: Advancing Therapeutics to Treat Vibriosis in Humans and Aquatic Organisms

Thiophenesulfonamide compounds are potent and specific inhibitors of Vibrio quorum sensing regulators LuxR/HapR that block their activity in vivo, thus preventing transcription of genes for virulence, motility, and biofilms. This protocol details how these compounds are synthesized, modeled in silico, and assayed in vivo for activity against LuxR/HapR.

Bacteria detect local population numbers using quorum sensing, a method of cell-cell communication broadly utilized to control bacterial behaviors. In ...

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

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