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. cristallo viola Quantificazione di formazione di biofilm in A. baumannii S1 <ol><li> Grow una cultura 5 ml di A. baumannii S1 in lisogenia brodo (LB) (triptone 10 g / L, estratto di lievito 5 g / L) a 30 ° C in un incubatore scuotimento (220 rpm) per 16 ore. </li><li> Regolare la cultura della A. baumannii S1 ad una OD 600 di 0,8 desiderato. Utilizzando una piastra a 96 pozzetti, inoculare la coltura batterica (diluizione 1: 100) in fresco LB contenente 10 ml di enzima purificato GKL (40 mg / ml); volume finale della nuova cultura è 100 l. </li><li> Preparare una coltura di controllo pure; questo non conterrà alcun enzima. Ripetere condizioni simili a cedere il numero desiderato di repliche. </li><li> Coprire la piastra con un coperchio e metterlo in un contenitore di plastica 10 L sigillato. Incubare la piastra a 30 ° C per 3 ore prima di rimuovere delicatamente il supporto. </li><li> Aggiungere altri 100 ml di mezzo fresco LB al pozzetto ed incubare la piastra per 21 ore a 30 ° C. </li&gt; <li> Dopo il secondo periodo di incubazione, rimuovere delicatamente tutti i media. Lavare il cellulare dei batteri planctonici con 200 ml di acqua sterile. Assicurarsi che vi sia solo il minimo disturbo alle cellule durante il lavaggio. </li><li> Aggiungere 100 ml di soluzione al cristalvioletto 1% ad ogni pozzetto ed incubare per 15 minuti a RT. Rimuovere la soluzione cristalvioletto lavando il bene con 200 microlitri di acqua sterile. Ripetere il lavaggio per altre due volte. </li><li> Aggiungere 100 ml di 33% di acido acetico ad ogni pozzetto e incubare per 15 minuti agitando delicatamente; questo si dissolverà il colorante. </li><li> Quantificare la quantità di biofilm formata misurando l&#39;assorbanza di cristalvioletto a 600 nm. La quantità di cristalvioletto è proporzionale alla quantità di biofilm formato. </li></ol> 2. Microscopia confocale a scansione laser di A. baumannii S1 biofilm <ol><li> Grow una cultura 5 ml di A. baumannii S1 in LB a 30 ° C in un incubatore scuotimento (220 rpm) per 16 ore. </li><li> Anno Dominisolo la cultura di A. baumannii S1 ad una OD 600 di 0,8 desiderato. Utilizzando un fondo di vetro μ-Dish 35 mm, inoculare la coltura batterica (diluizione 1: 100) in fresco LB contenente 30 ml di purificata GKL enzima (40 mg / ml); volume finale della nuova cultura è di 1 ml. </li><li> Coprire il μ-Piatto con un coperchio e metterlo in un contenitore di plastica sigillato 10 L. Incubare la μ-Dish a 30 ° C per 3 ore prima di rimuovere delicatamente il supporto. Aggiungere 30 ml di enzima purificato GKL e mezzo fresco, portando ad un volume totale di 1 ml. Incubare per 21 ore a un&#39;altra 30 ° C. </li><li> Ripetere il passaggio 2.3 e incubare la μ-Dish per un altro 24 ore a 30 ° C. Quindi, rimuovere il supporto delicatamente. </li><li> Aggiungere 500 pl di 5 mg / ml Alex Fluo 488-coniugato agglutinina di germe di grano (WGA) disciolto in soluzione salina bilanciata di Hank (HBSS) a μ-piatto e incubare a 37 ° C per 30 min; questo si macchia il biofilm formato. Rimuovere la soluzione colorante e lavare tegli μ-Piatto con 2 ml di HBSS. Ripetere la fase di lavaggio ancora una volta. </li><li> Aggiungere 500 ml di 3,7% di formaldeide sciolto in HBSS e incubare a 37 ° C per 30 min; questo risolverà il biofilm sul μ-Dish. Lavare il μ-piatto una volta con 2 ml di HBSS e quindi rimuovere completamente la soluzione. La μ-Dish fissa con biofilm può essere conservato al buio a 4 ° C prima di imaging CLSM. </li><li> Per CLSM di imaging e di analisi, utilizzare 63 ingrandimenti per generare 97 pile per immagine con un intervallo di 0,21 micron 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. 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K., 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=Biofilm+formation+by+Acinetobacter+baumannii+strains+isolated+from+urinary+tract+infection+and+urinary+catheters.">Biofilm formation by Acinetobacter baumannii strains isolated from urinary tract infection and urinary catheters.</a> FEMS immunology and medical microbiology. 62, 328-338 (2011).</li></ol>

The authors declare that they have no competing financial interests.

In entrambe le serie di esperimenti, A. baumannii S1 è stato coltivato in LB media senza NaCl come concentrazione di sali può ridurre la quantità di biofilm formato dai batteri 15. La presenza di tale manufatto potrebbe sottovalutare la quantità di biofilm formato, nonché gli effetti degli enzimi-quorum tempra attraverso diverse condizioni di trattamento. L&#39;uso di un enzima cataliticamente inattiva è importante come controllo negativo per eliminare i possibili effetti di enzima sequestro. <...

Le opzioni di trattamento per le malattie infettive sono state complicate dal rapido aumento di batteri multiresistenti che sono immuni a una vasta gamma di farmaci antibiotici 1. Con l&#39;alta morbilità e mortalità da infezioni batteri mediata resistenti, vi è la necessità di intensificare i processi di sviluppo di droga e / o esplorare alternative migliori antibatterici per migliorare le opzioni terapeutiche. Ultimamente, l&#39;approccio anti-virulenza sta guadagnando interesse dato il suo potenziale nella prevenzione virulenza attraverso media non battericida, quindi mitigare i rischi del meccanismi di resistenza 2. Quorum-sensing è un &#39;interruttore generale&#39; nella virulenza batterica e la rottura di questo fenomeno di segnalazione è un metodo anti-virulenza promettente contro patogenesi 3. L&#39;insorgenza della virulenza richiede l&#39;accumulo di molecole quorum nell&#39;ambiente extracellulare dopo aver raggiunto una densità di popolazione batterica critica. Come quomolecole rum diffondono nella matrice intracellulare, legandosi con i loro recettori cognate porta all&#39;attivazione di fattori di virulenza e geni associati con resistenza agli antibiotici e formazione di biofilm 4. In generale, l&#39;interruzione del quorum sensing comporta inibendo molecola quorum e recettore senza influenzare le principali vie metaboliche. Quindi, non ha alcuna implicazione diretta sulla crescita cellulare. Dal momento che il fitness non è compromessa, c&#39;è minima pressione selettiva per i batteri ad evolversi e acquisire resistenza contro tali trattamenti 5. Inoltre, l&#39;interruzione quorum-sensing può interferire con i meccanismi di protezione batterici intrinseci, come nel caso della formazione del biofilm, che fornisce protezione dagli agenti antibatterici e risposta immunitaria. Si stima che il 99% dei microbi sulla Terra esiste in matrici complesse come biofilm, le conferisce dei vantaggi di sopravvivenza fondamentali per i microrganismi che vivono all&#39;interno thstrutture ESE 6. Ancora più importante, la formazione di questi domini sessili è la causa della maggior parte delle infezioni persistenti e croniche nosocomiali 7. Acinetobacter baumannii è uno dei principali agenti patogeni umani che è associato a infezioni contratte in ospedale globali e la sua virulenza è in gran parte attribuito al quorum sensing formazione di biofilm mediata 8. Enzimi tempra quorum sono stati utilizzati con successo in interrompere quorum mediata trasduzione del segnale di mira un gruppo di composti noti come N -acyl lattoni Homoserine (AHLs) che sono prodotte da batteri Gram-negativi 9. Diversi studi hanno inoltre ampliato sull&#39;uso di questi enzimi per bloccare patogenesi batterica attraverso la riduzione dei numeri di espressione fattore di virulenza e di cella in biofilm 10,11. Purtroppo, permane una mancanza di manifestazione palpabile di un uso efficace di enzimi-quorum tempra contro la formazione di biofilm da batteri patogeni. Ilre sono stati tentativi di utilizzare gli inibitori del quorum (analoghi AHL), invece di enzimi quorum-tempra, di interrompere A. baumannii biofilm formazione 12. Anche se questo metodo di utilizzo di piccole molecole inibitori è un valido approccio, sostenendo la sua biodisponibilità negli usi traslazionali può essere una sfida. Al contrario, l&#39;utilizzo di enzimi quorum tempra catalitici potrebbe aggirare il problema biodisponibilità come enzimi sono più suscettibili verso l&#39;immobilizzazione su superfici di dispositivi biomedicali per effetti terapeutici. Qui, descriviamo una valutazione degli effetti della ingegnerizzati-quorum tempra lactonases da Geobacillus kaustophilus (GKL) 13 sulla formazione di biofilm batterico, utilizzando colorazione cristallo viola e microscopia confocale a scansione laser (CLSM). Questo studio è il primo dimostrazione riuscita di interruzione biofilm in una clinicamente rilevante A. ceppo baumannii S1 utilizzando enzimi quorum-tempra. I metodi descrittiin questo studio sono utili per valutare l&#39;efficacia di altri enzimi quorum tempra nei successivi sforzi di sviluppo terapeutici contro i batteri Gram-negativi patogeni.

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

Nell&#39;esperimento quantificazione cristallo viola, due enzimi quorum-tempra sono stati usati per dimostrare la fattibilità a interrompere la formazione di biofilm: wild-type GKL e una migliore doppio mutante GKL (E101G / R230C). Entrambi gli enzimi sono stati indicati per dimostrare l&#39;attività lactonase contro 3-idrossi-L-decanoil-omoserina lattone (3-OH-C 10 -HSL), la principale molecola quorum utilizzato da A. baumannii S1 14. Per valida la valut...

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.

Turbativa Anti-virulento di patogeni biofilm utilizzando Engineered Quorum-tempra Lactonases

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

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Immunology and Infection

10.5K 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

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.

Singola cella Analisi Bacillus subtilis I biofilm usando la microscopia a fluorescenza e citometria a flusso

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 ...

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Immunologia e infezioni

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.

Crescita Mycobacterium tuberculosis I biofilm

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

Use of Image Cytometry for Quantification of Pathogenic Fungi in Association with Host Cells

Studies of the cellular pathogenesis mechanisms of pathogenic yeasts such as Candida albicans, Histoplasma capsulatum, and Cryptococcus neoformans commonly employ infection of mammalian hosts or host cells (i.e. macrophages) followed by yeast quantification using colony forming unit analysis or flow cytometry. While colony forming unit enumeration has been the most commonly used method in the field, this technique has disadvantages and limitations, including slow growth of some fungal species on solid media and low and/or variable plating efficiencies, which is of particular concern when comparing growth of wild-type and mutant strains. Flow cytometry can provide rapid quantitative information regarding yeast viability, however, adoption of flow cytometric detection for pathogenic yeasts has been limited for a number of practical reasons including its high cost and biosafety considerations. Here, we demonstrate an image-based cytometric methodology using the Cellometer Vision (Nexcelom Bioscience, LLC) for the quantification of viable pathogenic yeasts in co-culture with macrophages. Our studies focus on detection of two human fungal pathogens: Histoplasma capsulatum and Candida albicans. H. capsulatum colonizes alveolar macrophages by replicating within the macrophage phagosome, and here, we quantitatively assess the growth of H. capsulatum yeasts in RAW 264.7 macrophages using acridine orange/propidium iodide staining in combination with image cytometry. Our method faithfully recapitulates growth trends as measured by traditional colony forming unit enumeration, but with significantly increased sensitivity. Additionally, we directly assess infection of live macrophages with a GFP-expressing strain of C. albicans. Our methodology offers a rapid, accurate, and economical means for detection and quantification of important human fungal pathogens in association with host cells.

Here, we demonstrate how image cytometry can be used for quantification of pathogenic fungi in association with host cells in culture. This technique can be used as an alternative to CFU enumeration.

L&#39;uso di Citometria immagine per la quantificazione dei funghi patogeni in associazione con cellule ospiti

Studies of the cellular pathogenesis mechanisms of pathogenic yeasts such as Candida albicans, Histoplasma capsulatum, and Cryptococcus neoformans ...

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.

Quantificazione concomitante di cellulare e extracellulare componenti del biofilm

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.

Imaging Raziometrico del pH extracellulare nel dentale biofilm

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.

Un nuovo metodo per l&#39;analisi qualitativa multi-scala batterica biofilm su filamentose Fungal Colonies Utilizzando confocale e microscopia elettronica

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.

Inclusione in paraffina e sezionamento sottile di biofilm di colonie microbiche per l'analisi al microscopio

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

Production of Pseudotyped Particles to Study Highly Pathogenic Coronaviruses in a Biosafety Level 2 Setting

The protocol aims to generate coronavirus (CoV) spike (S) fusion protein pseudotyped particles with a murine leukemia virus (MLV) core and luciferase reporter, using a simple transfection procedure of the widely available HEK-293T cell line. Once formed and released from producer cells, these pseudovirions incorporate a luciferase reporter gene. Since they only contain the heterologous coronavirus spike protein on their surface, the particles behave like their native coronavirus counterparts for entry steps. As such, they are the excellent surrogates of native virions for studying viral entry into host cells. Upon successful entry and infection into target cells, the luciferase reporter gets integrated into the host cell genome and is expressed. Using a simple luciferase assay, transduced cells can be easily quantified. An important advantage of the procedure is that it can be performed in biosafety level 2 (BSL-2) facilities instead of BSL-3 facilities required for work with highly pathogenic coronaviruses such as Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus (SARS-CoV). Another benefit comes from its versatility as it can be applied to envelope proteins belonging to all three classes of viral fusion proteins, such as the class I influenza hemagglutinin (HA) and Ebola virus glycoprotein (GP), the class II Semliki forest virus E1 protein, or the class III vesicular stomatitis virus G glycoprotein. A limitation of the methodology is that it can only recapitulate virus entry steps mediated by the envelope protein being investigated. For studying other viral life cycle steps, other methods are required. Examples of the many applications these pseudotype particles can be used in include investigation of host cell susceptibility and tropism and testing the effects of virus entry inhibitors to dissect viral entry pathways used.

Here, we present a protocol to generate pseudotyped particles in a BSL-2 setting incorporating the spike protein of the highly pathogenic viruses Middle East respiratory syndrome and severe acute respiratory syndrome coronaviruses. These pseudotyped particles contain a luciferase reporter gene allowing quantification of virus entry into target host cells.

Produzione di particelle pseudotipizzate per studiare la patogenicità coronavirus in un ambiente di livello 2 di biosicurezza

The protocol aims to generate coronavirus (CoV) spike (S) fusion protein pseudotyped particles with a murine leukemia virus (MLV) core and luciferase ...

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.

Monitoraggio del pH extracellulare nei biofilm tra regni incrociati utilizzando la microscopia Confocal

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.

Chiarire e 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 ...