Name: anti-virulent disruption of pathogenic biofilms using engineered quorum-quenching lactonases
Uploaded: 2016-01-01T13:00:00
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 v...

<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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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.

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	<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

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly constituted by proteins and exopolysaccharides, among other factors 7-10. The microbial community encased within the biofilm often shows the differentiation of distinct subpopulation of specialized cells 11-17. These subpopulations coexist and often show spatial and temporal organization within the biofilm 18-21.
Biofilm formation in the model organism Bacillus subtilis requires the differentiation of distinct subpopulations of specialized cells. Among them, the subpopulation of matrix producers, responsible to produce and secrete the extracellular matrix of the biofilm is essential for biofilm formation 11,19. Hence, differentiation of matrix producers is a hallmark of biofilm formation in B. subtilis.
We have used fluorescent reporters to visualize and quantify the subpopulation of matrix producers in biofilms of B. subtilis 15,19,22-24. Concretely, we have observed that the subpopulation of matrix producers differentiates in response to the presence of self-produced extracellular signal surfactin 25. Interestingly, surfactin is produced by a subpopulation of specialized cells different from the subpopulation of matrix producers 15.
We have detailed in this report the technical approach necessary to visualize and quantify the subpopulation of matrix producers and surfactin producers within the biofilms of B. subtilis. To do this, fluorescent reporters of genes required for matrix production and surfactin production are inserted into the chromosome of B. subtilis. Reporters are expressed only in a subpopulation of specialized cells. Then, the subpopulations can be monitored using fluorescence microscopy and flow cytometry (See Fig 1).
The fact that different subpopulations of specialized cells coexist within multicellular communities of bacteria gives us a different perspective about the regulation of gene expression in prokaryotes. This protocol addresses this phenomenon experimentally and it can be easily adapted to any other working model, to elucidate the molecular mechanisms underlying phenotypic heterogeneity within a microbial community.

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Microbial biofilms are generally constituted by distinct subpopulations of specialized cells. Single-cell analysis of these subpopulations requires the use of fluorescent reporters. Here we describe a protocol to visualize and monitor several subpopulationswithin B. subtilis biofilms using fluorescence microscopy and flow cytometry.

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly ...

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including antibiotics. Although stress tolerance of M. tuberculosis is one of the likely contributors to the 6-month long chemotherapy of tuberculosis 1, the molecular mechanisms underlying this characteristic phenotype of the pathogen remain unclear. Many microbial species have evolved to survive in stressful environments by self-assembling in highly organized, surface attached, and matrix encapsulated structures called biofilms 2-4. Growth in communities appears to be a preferred survival strategy of microbes, and is achieved through genetic components that regulate surface attachment, intercellular communications, and synthesis of extracellular polymeric substances (EPS) 5,6. The tolerance to environmental stress is likely facilitated by EPS, and perhaps by the physiological adaptation of individual bacilli to heterogeneous microenvironments within the complex architecture of biofilms 7.
In a series of recent papers we established that M. tuberculosis and Mycobacterium smegmatis have a strong propensity to grow in organized multicellular structures, called biofilms, which can tolerate more than 50 times the minimal inhibitory concentrations of the anti-tuberculosis drugs isoniazid and rifampicin 8-10. M. tuberculosis, however, intriguingly requires specific conditions to form mature biofilms, in particular 9:1 ratio of headspace: media as well as limited exchange of air with the atmosphere 9. Requirements of specialized environmental conditions could possibly be linked to the fact that M. tuberculosis is an obligate human pathogen and thus has adapted to tissue environments. In this publication we demonstrate methods for culturing M. tuberculosis biofilms in a bottle and a 12-well plate format, which is convenient for bacteriological as well as genetic studies. We have described the protocol for an attenuated strain of M. tuberculosis, mc27000, with deletion in the two loci, panCD and RD1, that are critical for in vivo growth of the pathogen 9. This strain can be safely used in a BSL-2 containment for understanding the basic biology of the tuberculosis pathogen thus avoiding the requirement of an expensive BSL-3 facility. The method can be extended, with appropriate modification in media, to grow biofilm of other culturable mycobacterial species.
Overall, a uniform protocol of culturing mycobacterial biofilms will help the investigators interested in studying the basic resilient characteristics of mycobacteria. In addition, a clear and concise method of growing mycobacterial biofilms will also help the clinical and pharmaceutical investigators to test the efficacy of a potential drug.

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis forms drug tolerant biofilms when cultured in certain conditions. Here we describe methods for culturing M. tuberculosis biofilms and determining the frequency of drug tolerant persisters. These protocols will be useful for further studies into the mechanisms of drug tolerance in M. tuberculosis.

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including ...

Concurrent Quantification of Cellular and Extracellular Components of Biofilms

Confocal laser scanning microscopy (CLSM) is a powerful tool for investigation of biofilms. Very few investigations have successfully quantified concurrent distribution of more than two components within biofilms because: 1)&#160;selection of fluorescent dyes having minimal spectral overlap is complicated, and 2)&#160;quantification of multiple fluorochromes poses a multifactorial problem. Objectives: Report a methodology to quantify and compare concurrent 3-dimensional distributions of three cellular/extracellular components of biofilms grown on relevant substrates. Methods: The method consists of distinct, interconnected steps involving biofilm growth, staining, CLSM imaging, biofilm structural analysis and visualization, and statistical analysis of structural parameters. Biofilms of Streptococcus mutans (strain UA159) were grown for 48 hr on sterile specimens of Point 4 and TPH3 resin composites. Specimens were subsequently immersed for 60 sec in either Biot&#232;ne PBF (BIO) or Listerine Total Care (LTO) mouthwashes, or water (control group; n=5/group). Biofilms were stained with fluorochromes for extracellular polymeric substances, proteins and nucleic acids before imaging with CLSM. Biofilm structural parameters calculated using ISA3D image analysis software were biovolume and mean biofilm thickness. Mixed models statistical analyses compared structural parameters between mouthwash and control groups (SAS software; &#945;=0.05). Volocity software permitted visualization of 3D distributions of overlaid biofilm components (fluorochromes). Results: Mouthwash BIO produced biofilm structures that differed significantly from the control (p&#60;0.05) on both resin composites, whereas LTO did not produce differences (p&#62;0.05) on either product. Conclusions: This methodology efficiently and successfully quantified and compared concurrent 3D distributions of three major components within S. mutans biofilms on relevant substrates, thus overcoming two challenges to simultaneous assessment of biofilm components. This method can also be used to determine the efficacy of antibacterial/antifouling agents against multiple biofilm components, as shown using mouthwashes. Furthermore, this method has broad application because it facilitates comparison of 3D structures/architecture of biofilms in a variety of disciplines.

A protocol for the concurrent quantification and comparison of three cellular and extracellular components within biofilms is presented. The methodology involves the use of confocal laser scanning microscopy, biofilm structural analysis and visualization software, and statistical analysis software.

Confocal laser scanning microscopy (CLSM) is a powerful tool for investigation of biofilms. Very few investigations have successfully quantified ...

Ratiometric Imaging of Extracellular pH in Dental Biofilms

The pH in bacterial biofilms on teeth is of central importance for dental caries, a disease with a high worldwide prevalence. Nutrients and metabolites are not distributed evenly in dental biofilms. A complex interplay of sorption to and reaction with organic matter in the biofilm reduces the diffusion paths of solutes and creates steep gradients of reactive molecules, including organic acids, across the biofilm. Quantitative fluorescent microscopic methods, such as fluorescence life time imaging or pH ratiometry, can be employed to visualize pH in different microenvironments of dental biofilms. pH ratiometry exploits a pH-dependent shift in the fluorescent emission of pH-sensitive dyes. Calculation of the emission ratio at two different wavelengths allows determining local pH in microscopic images, irrespective of the concentration of the dye. Contrary to microelectrodes the technique allows monitoring both vertical and horizontal pH gradients in real-time without mechanically disturbing the biofilm. However, care must be taken to differentiate accurately between extra- and intracellular compartments of the biofilm. Here, the ratiometric dye, seminaphthorhodafluor-4F 5-(and-6) carboxylic acid (C-SNARF-4) is employed to monitor extracellular pH in in vivo grown dental biofilms of unknown species composition. Upon exposure to glucose the dye is up-concentrated inside all bacterial cells in the biofilms; it is thus used both as a universal bacterial stain and as a marker of extracellular pH. After confocal microscopic image acquisition, the bacterial biomass is removed from all pictures using digital image analysis software, which permits to exclusively calculate extracellular pH. pH ratiometry with the ratiometric dye is well-suited to study extracellular pH in thin biofilms of up to 75 &#181;m thickness, but is limited to the pH range between 4.5 and 7.0.

A pH-sensitive ratiometric dye is used in combination with confocal laser scanning microscopy and digital image analysis to monitor extracellular pH in dental biofilms in real-time.

The pH in bacterial biofilms on teeth is of central importance for dental caries, a disease with a high worldwide prevalence. Nutrients and metabolites ...

A New Method for Qualitative Multi-scale Analysis of Bacterial Biofilms on Filamentous Fungal Colonies Using Confocal and Electron Microscopy

Bacterial biofilms frequently form on fungal surfaces and can be involved in numerous bacterial-fungal interaction processes, such as metabolic cooperation, competition, or predation. The study of biofilms is important in many biological fields, including environmental science, food production, and medicine. However, few studies have focused on such bacterial biofilms, partially due to the difficulty of investigating them. Most of the methods for qualitative and quantitative biofilm analyses described in the literature are only suitable for biofilms forming on abiotic surfaces or on homogeneous and thin biotic surfaces, such as a monolayer of epithelial cells.
While laser scanning confocal microscopy (LSCM) is often used to analyze in situ and in vivo biofilms, this technology becomes very challenging when applied to bacterial biofilms on fungal hyphae, due to the thickness and the three dimensions of the hyphal networks. To overcome this shortcoming, we developed a protocol combining microscopy with a method to limit the accumulation of hyphal layers in fungal colonies. Using this method, we were able to investigate the development of bacterial biofilms on fungal hyphae at multiple scales using both LSCM and scanning electron microscopy (SEM). This report describes the protocol, including microorganism cultures, bacterial biofilm formation conditions, biofilm staining, and LSCM and SEM visualizations.

This protocol describes a new method to grow and qualitatively analyze bacterial biofilms on fungal hyphae by confocal and electron microscopy.

Bacterial biofilms frequently form on fungal surfaces and can be involved in numerous bacterial-fungal interaction processes, such as metabolic ...

Paraffin Embedding and Thin Sectioning of Microbial Colony Biofilms for Microscopic Analysis

Sectioning via paraffin embedding is a broadly established technique in eukaryotic systems. Here we provide a method for the fixation, embedding, and sectioning of intact microbial colony biofilms using perfused paraffin wax. To adapt this method for use on colony biofilms, we developed techniques for maintaining each sample on its growth substrate and laminating it with an agar overlayer, and added lysine to the fixative solution. These optimizations improve sample retention and preservation of micromorphological features. Samples prepared in this manner are amenable to thin sectioning and imaging by light, fluorescence, and transmission electron microscopy. We have applied this technique to colony biofilms of Pseudomonas aeruginosa, Pseudomonas synxantha, Bacillus subtilis, and Vibrio cholerae. The high level of detail visible in samples generated by this method, combined with reporter strain engineering or the use of specific dyes, can provide exciting insights into the physiology and development of microbial communities.

We describe fixation, paraffin embedding, and thin sectioning techniques for microbial colony biofilms. In prepared samples, biofilm substructure and reporter expression patterns can be visualized by microscopy.

Sectioning via paraffin embedding is a broadly established technique in eukaryotic systems. Here we provide a method for the fixation, embedding, and ...

Monitoring Extracellular pH in Cross-Kingdom Biofilms using Confocal Microscopy

Cross-kingdom biofilms consisting of both fungal and bacterial cells are involved in a variety of oral diseases, such as endodontic infections, periodontitis, mucosal infections and, most notably, early childhood caries. In all of these conditions, the pH in the biofilm matrix impacts microbe-host interactions and thus the disease progression. The present protocol describes a confocal microscopy-based method to monitor pH dynamics inside cross-kingdom biofilms comprising Candida albicans and Streptococcus mutans. The pH-dependent dual-emission spectrum and the staining properties of the ratiometric probe C-SNARF-4 are exploited to determine drops in pH in extracellular areas of the biofilms. Use of pH ratiometry with the probe requires a meticulous choice of imaging parameters, a thorough calibration of the dye, and careful, threshold-based post-processing of the image data. When used correctly, the technique allows for the rapid assessment of extracellular pH in different areas of a biofilm and thus the monitoring of both horizontal and vertical pH gradients over time. While the use of confocal microscopy limits Z-profiling to thin biofilms of 75 &#181;m or less, the use of pH ratiometry is ideally suited for the noninvasive study of an important virulence factor in cross-kingdom biofilms.

The protocol describes the cultivation of cross-kingdom biofilms consisting of Candida albicans and Streptococcus mutans and presents a confocal microscopy-based method for the monitoring of extracellular pH inside these biofilms.

Cross-kingdom biofilms consisting of both fungal and bacterial cells are involved in a variety of oral diseases, such as endodontic infections, ...

Clarifying and Imaging Candida albicans Biofilms

The microbial fungus Candida albicans can undergo a change from commensal colonization to virulence that is strongly correlated with its ability to switch from yeast-form growth to hyphal growth. Cells initiating this process become adherent to surfaces as well as to each other, with the resulting development of a biofilm colony. This commonly occurs not only on mucosal tissue surfaces in yeast infections, but also on medical implants such as catheters. It is well known that biofilm cells are resistant to antifungal drugs, and that cells that shed from the biofilm can lead to dangerous systemic infections. Biofilms range from heavily translucent to opaque due to refractive heterogeneity. Therefore, fungal biofilms are difficult to study by optical microscopy. To visualize internal structural, cellular, and subcellular features, we clarify fixed intact biofilms by stepwise solvent exchange to a point of optimal refractive index matching. For C. albicans biofilms, sufficient clarification is attained with methyl salicylate (n = 1.537) to enable confocal microscopy from apex to base in 600 &#181;m biofilms with little attenuation. In this visualization protocol we outline phase contrast refractometry, the growth of laboratory biofilms, fixation, staining, solvent exchange, the setup for confocal fluorescence microscopy, and representative results.

Clarifying and Imaging Candida albicans Biofilms

To view and quantify the internal features of Candida albicans biofilms, we prepare fixed intact specimens that are clarified by refractive index matching. Then, optical sectioning microscopy can be used to obtain three-dimensional image data though the full thickness of the biofilm.

The microbial fungus Candida albicans can undergo a change from commensal colonization to virulence that is strongly correlated with its ability to switch ...

Double Labeling Immunofluorescence using Antibodies from the Same Species to Study Host-Pathogen Interactions

Nowadays, it is possible to find a wide range of molecular tools available to study parasite-host cell interactions. However, some limitations exist to obtain commercial monoclonal or polyclonal antibodies that recognize specific cell structures and proteins in parasites. Besides, there are few commercial antibodies available to label trypanosomatids. Usually, polyclonal antibodies against parasites are prepared in-house and could be more challenging to use in combination with other antibodies produced in the same species. Here, the protocol demonstrates how to use polyclonal and monoclonal antibodies raised in the same species to perform double labeling immunofluorescence to study host cell and pathogen interactions. To achieve the double labeling immunofluorescence, it is crucial to incubate first the mouse polyclonal antibody and then follow the incubation with the secondary mouse IgG antibody conjugated to any fluorochrome. After that, an additional blocking step is necessary to prevent any trace of the primary antibody from being recognized by the next secondary antibody. Then, a mouse monoclonal antibody and its specific IgG subclass secondary antibody conjugated to a different fluorochrome are added to the sample at the appropriate times. Additionally, it is possible to perform triple labeling immunofluorescence using a third antibody raised in a different species. Also, structures such as nuclei and actin can be stained subsequently with their specific compounds or labels. Thus, these approaches presented here can be adjusted for any cell whose sources of primary antibodies are limited.

Here, the protocol describes how to perform double labeling immunofluorescence using primary antibodies raised in the same species to study host-pathogen interactions. Also, it can include the third antibody from a different host in this protocol. This approach can be made in any cell type and pathogens.

Nowadays, it is possible to find a wide range of molecular tools available to study parasite-host cell interactions. However, some limitations exist to ...

Production of Human CRISPR-Engineered CAR-T Cells

Adoptive cell therapies using chimeric antigen receptor T cells (CAR-T cells) have demonstrated remarkable clinical efficacy in patients with hematological malignancies and are currently being investigated for various solid tumors. CAR-T cells are generated by removing T cells from a patient's blood and engineering them to express a synthetic immune receptor that redirects the T-cells to recognize and eliminate target tumor cells. Gene editing of CAR-T cells has the potential to improve safety of current CAR-T cell therapies and further increase the efficacy of CAR-T cells. Here, we describe methods for the activation, expansion, and characterization of human CRISPR-engineered CD19 directed CAR-T cells. This comprises transduction of the CAR lentiviral vector and use of single guide RNA (sgRNA) and Cas9 endonuclease to target genes of interest in T cells. The methods described in this protocol can be universally applied to other CAR constructs and target genes beyond the ones used for this study. Furthermore, this protocol discusses strategies for gRNA design, lead gRNA selection and target gene knockout validation to reproducibly achieve high-efficiency, multiplex CRISPR-Cas9 engineering of clinical grade human T cells.

Here, we present a protocol for gene editing in primary human T cells using CRISPR Cas Technology to modify CAR-T cells.